CN116615241A

CN116615241A - Methods and compositions for diabetes treatment and beta cell regeneration

Info

Publication number: CN116615241A
Application number: CN202180070362.6A
Authority: CN
Inventors: 斯拉维察·图达扎罗娃-特拉伊科夫斯卡
Original assignee: University of California
Current assignee: University of California
Priority date: 2020-08-18
Filing date: 2021-08-17
Publication date: 2023-08-18

Abstract

Aspects of the present disclosure relate to methods and compositions for promoting beta cell regeneration by inhibiting the hif1α -PFKFB3 pathway. Certain aspects describe methods for treating and preventing pre-diabetes and diabetes, including type 2 diabetes. Methods and compositions for enhancing beta cell regeneration are also disclosed.

Description

Methods and compositions for diabetes treatment and beta cell regeneration

The present application claims priority from U.S. provisional application Ser. Nos. 63/067,187 and 63/169,776, filed 8/18/2020, and 4/2021, which are incorporated herein by reference in their entireties.

Sequence listing

The present application contains a sequence listing that has been submitted in ASCII format and is incorporated by reference herein in its entirety. The ASCII copy was created at 2021, 8/16, named ucla_p0116wo_seq_listing.

Technical Field

Aspects of the application relate at least to the fields of molecular biology and medicine.

Background

In type 2 diabetes (T2D) patients, there is a gradual decline in beta cell function, which is related to some extent to the accumulation of toxic oligomers of Islet Amyloid Pancreatic Polypeptide (IAPP) [3-5 ] ]. Although there is sufficient evidence to suggest that beta cell stress (altered mitochondrial network and activity, ca ²⁺ Toxicity, oxidation and DNA damage) [6-9]However, the rate of beta cell consumption is surprisingly slow, and beta cell mass remains between 35% and 76% even decades after the onset of T2D [4 ]]. However, the relative maintenance of beta cell mass contrasts with the early loss of beta cell glucose responsiveness prior to the onset of T2D. This suggests that although most β cells in T2D patients are viable, these cells are dysfunctional.

After acute injury, in healthHypoxia-inducible factor-1-alpha (HIF1α) initiates a series of steps required for successful tissue repair in cells of the tissue [10,11]. First, hif1α target 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB 3) mediates significant metabolic remodeling from mitochondrial-dependent oxidative phosphorylation to high flux via aerobic glycolysis, producing ATP regardless of oxygen availability. Energy transfer to aerobic glycolysis allows DNA repair by increased nucleotide synthesis via the pentose phosphate pathway [12 ]]. High-throughput glycolysis also enables the mitochondrial network to adopt defensive fragmentation patterns around the nucleus, which protects mitochondria from damage-induced Ca ²⁺ Effect of toxicity [13 ]]. Second, cells that retain DNA damage are eliminated by apoptosis, while those that do not serve to regenerate lost tissue. Tissue regeneration is achieved by the expansion of progenitor stem cells, replication or transdifferentiation following dedifferentiation. Third, once tissue regeneration is complete, the impairment signal inducing hif1α -PFKFB3 pathway is attenuated and the cells will resume their functional metabolic state.

Unlike healthy tissue, beta cells in humans with T2D remain trapped in the pro-survival phase of the hif1α injury/repair response, where metabolism and mitochondrial networks are altered, which slows down the rate of cell consumption at the expense of losing beta cell function [2]. Despite extensive research, it is unclear why beta cells cannot be successfully regenerated. Methods and compositions for inducing damaged cell elimination and healthy cell (e.g., beta cell) regeneration to improve insulin sensitivity and better treat subjects suffering from diabetes are needed.

Disclosure of Invention

Aspects of the present disclosure provide methods for treating diabetes and related conditions, including type 2 diabetes, and compositions useful in such methods. Embodiments of the present disclosure address certain needs by providing methods and compositions for promoting healthy beta cell regeneration by inhibiting hif1α activity, in some cases in combination with inhibiting PFKFB3 activity. Certain aspects relate to methods for treating type 2 diabetes, comprising providing a hif1α inhibitor. Such methods may further comprise administering a PFKFB3 inhibitor. In some embodiments, pharmaceutical compositions comprising a hif1α inhibitor and a PFKFB3 inhibitor are also disclosed.

Embodiments of the present disclosure include: methods for treating a disorder associated with protein misfolding in a subject, methods for treating diabetes in a subject, methods for treating type 2 diabetes in a subject, methods for treating type 1 diabetes in a subject, methods for diagnosing type 2 diabetes, methods for determining sensitivity of a subject with type 2 diabetes to treatment with a hif1α inhibitor, methods for improving insulin sensitivity, methods for targeting a hif1α inhibitor to β -cells, methods for determining PFKFB3 expression levels, methods for killing damaged β -cells, methods for stimulating regeneration of healthy β -cells, compositions comprising one or more hif1α inhibitors, compositions comprising one or more PFKFB3 inhibitors, and compositions comprising a hif1α inhibitor and a PFKFB3 inhibitor. The disclosed methods may include at least 1, 2, 3, 4, 5, or more of the following steps: providing an effective amount of a hif1α inhibitor, providing an effective amount of a PFKFB3 inhibitor, diagnosing whether the subject has type 2 diabetes, diagnosing whether the subject has type 1 diabetes, identifying the subject as having type 2 diabetes, identifying the subject as being at risk for having type 2 diabetes, identifying the subject as having pre-diabetes, measuring the expression level of PFKFB3 in the subject, measuring the expression level of PFKFB3 in beta cells of the subject, determining that the subject has an increased expression level of PFKFB3 in beta cells of the subject, and providing one or more additional treatments for type 2 diabetes to the subject. One or more of the foregoing steps may be excluded from embodiments of the present disclosure. The compositions of the present disclosure may include 1, 2, 3, 4, or more of the following: HIF1 a inhibitors, PFKFB3 inhibitors, targeting molecules, GLP-1 receptor antibodies, metformin, GLP-1 receptor agonists, DPP-4 inhibitors, sulfonylureas, and one or more pharmaceutically acceptable excipients. One or more of the foregoing components may be excluded from embodiments of the present disclosure.

In some embodiments, disclosed herein is a method of treating type 2 diabetes in a subject, the method comprising administering to the subject an effective amount of a hif1α inhibitor. In some embodiments, also disclosed herein is a method of treating type 2 diabetes in a subject, comprising administering to the subject an effective amount of a hif1α inhibitor, the subject determined to have an increased level of PFKFB3 expression in beta cells from the subject relative to the level of PFKFB3 expression in beta cells from a healthy subject not suffering from type 2 diabetes. In some embodiments, a method of treating type 2 diabetes in a subject is disclosed, the method comprising (a) determining that the subject has an increased level of PFKFB3 expression in beta cells from the subject relative to the level of PFKFB3 expression in beta cells from a healthy subject not suffering from type 2 diabetes; and (b) administering to the subject an effective amount of a hif1α inhibitor. In some embodiments, a method of stimulating regeneration of healthy beta cells in a subject having type 2 diabetes, wherein the healthy beta cells do not express PFKFB3, is disclosed, the method comprising administering to the subject an effective amount of a hif1α inhibitor.

In some embodiments, the hif1α inhibitor promotes hif1α degradation. In some embodiments, the hif1α inhibitor inhibits hif1α/hif1β dimer formation. In some embodiments, the hif1α inhibitor decreases hif1α transcriptional activity. In some embodiments, the HIF 1. Alpha. Inhibitor is KC7F2, IDF-11774, aminoflavone, AJM290, AW464, tanspiramycin (tanespimycin), avilamycin (alvespimycin), PX-478, or FM19G11. In some embodiments, the hif1α inhibitor is a nucleic acid inhibitor. In some embodiments, the hif1α inhibitor is an antisense oligonucleotide. In some embodiments, the HIF 1. Alpha. Inhibitor is EZN-2698. In some embodiments, the hif1α inhibitor is an siRNA or a short hairpin RNA. In some embodiments, the HIF1 alpha inhibitor is resveratrol, rapamycin, everolimus, CCI779, silibinin, digoxin, YC-1, phenethyl isothiocyanate, black hair mycin, fraapine, bortezomib, amphotericin B, bay-2243, PX-478, or Gan Ni ganetastib. In some embodiments, the hif1α inhibitor is an anti-hif1α antibody or antibody-like molecule. In some embodiments, the hif1α inhibitor is a nanobody. In some embodiments, the hif1α inhibitor is administered intravenously, intramuscularly, intraperitoneally, subcutaneously, intra-articular, intrasynovially, intrathecally, orally, topically, via inhalation, or via a combination of two or more routes of administration.

In some embodiments, the method further comprises administering to the subject a PFKFB3 inhibitor. In some embodiments, the PFKFB3 inhibitor is 3- (3-pyridyl) -1- (4-pyridyl) -2-propen-1-one (3-PO) or an analog thereof. In some embodiments, the PFKFB3 inhibitor is an analog of 3-PO, wherein the analog is 1- (4-pyridyl) -3- (2-quinolyl) -2-propen-1-one (PFK 15). In some embodiments, the PFKFB3 inhibitor is BrAcNHEtOP, YN1, YZ9, PQP, PFK-158, compound 26, KAN0436151, or KAN0436067. In some embodiments, the PFKFB3 inhibitor is a nucleic acid inhibitor. In some embodiments, the PFKFB3 inhibitor is an antisense oligonucleotide. In some embodiments, the PFKFB3 inhibitor is an siRNA or a short hairpin RNA. In some embodiments, the PFKFB3 inhibitor is operably linked to a targeting molecule configured to bind to a β cell of the subject. In some embodiments, the hif1α inhibitor is operably linked to a targeting molecule configured to bind to β cells of the subject. In some embodiments, the targeting molecule is an antibody. In some embodiments, the targeting molecule is an antibody-like molecule. In some embodiments, the targeting molecule is configured to bind to a GLP-1 receptor. In some embodiments, the hif1α inhibitor and the PFKFB3 inhibitor are administered sequentially to the subject. In some embodiments, the hif1α inhibitor and the PFKFB3 inhibitor are administered to the subject substantially simultaneously.

In some embodiments, administering an effective amount of a hif1α inhibitor increases insulin sensitivity in the subject. In some embodiments, the subject has not been suffering from or has not been diagnosed with cancer. In some embodiments, the subject has been diagnosed with type 2 diabetes. In some embodiments, the method further comprises, prior to administration, diagnosing that the subject has type 2 diabetes. In some embodiments, the subject has previously received treatment for type 2 diabetes. In some embodiments, the subject is determined to be resistant to a previous treatment. In some embodiments, the subject has not been suffering from or has not been diagnosed with diabetic nephropathy or diabetic retinopathy. In some embodiments, the method further comprises measuring the expression level of PFKFB3 in a beta cell from the subject. In some embodiments, the expression level of PFKFB3 in beta cells from the subject is increased relative to the expression level of PFKFB3 in beta cells from a healthy subject not suffering from type 2 diabetes.

Embodiments of the present disclosure also include methods of increasing insulin sensitivity in a subject, the methods comprising administering to the subject an effective amount of a hif1α inhibitor. In some embodiments, the subject suffers from prediabetes. In some embodiments, the subject suffers from insulin resistance. In some embodiments, a method for stimulating cell death in a damaged beta cell expressing PFKFB3 is also disclosed, the method comprising providing a hif1α inhibitor to the beta cell. In some embodiments, a method for stimulating regeneration of a beta cell that does not express PFKFB3 is disclosed, the method comprising providing a hif1α inhibitor to the beta cell. In some embodiments, the hif1α inhibitor is provided to the β -cells in vitro. In some embodiments, the hif1α inhibitor is provided to a beta cell in vivo.

In certain aspects, disclosed herein is a method for killing damaged beta cells expressing PFKFB3 in a subject having diabetes, the method comprising administering to the subject an effective amount of a hif1α inhibitor. In some embodiments, a method of treating diabetes in a subject is also disclosed, the method comprising administering to the subject an effective amount of a hif1α inhibitor. In some embodiments, the diabetes is type 1 diabetes. In some embodiments, the diabetes is type 2 diabetes.

Certain embodiments relate to methods for diagnosing a disease or disorder. In some embodiments, a method for diagnosing a subject as having type 2 diabetes is disclosed, the method comprising: (a) Measuring the expression level of PFKFB3 in beta cells from the subject; (b) Comparing the expression level to the expression level of PFKFB3 in beta cells from a healthy subject not suffering from type 2 diabetes; and (c) determining that the expression level of PFKFB3 in the beta cells from the subject is increased relative to the expression level of PFKFB3 in the beta cells from a healthy subject, thereby diagnosing the subject as having type 2 diabetes.

In some embodiments, various pharmaceutical compositions are further disclosed herein. In some embodiments, pharmaceutical compositions comprising (a) a hif1α inhibitor and (b) a PFKFB3 inhibitor are disclosed. The hif1α inhibitor may be any hif1α inhibitor, examples of which are disclosed herein. The PFKFB3 inhibitor may be any PFKFB3 inhibitor, examples of which are disclosed herein.

In some embodiments, a method of depleting a bishormonal cell from a cell population is also disclosed, the method comprising administering to the cell population an effective amount of a PFKFB3 inhibitor. In some embodiments, the PFKFB3 inhibitor is administered outside the cell population. In some embodiments, the PFKFB3 inhibitor is administered into a cell population. The PFKFB3 inhibitor may be any PFKFB3 inhibitor, examples of which are disclosed herein. Further disclosed in some embodiments is a method of depleting a bishormonal cell from a cell population comprising administering to the cell population an effective amount of a hif1α inhibitor. The hif1α inhibitor may be any hif1α inhibitor, examples of which are disclosed herein. In some embodiments, the hif1α inhibitor is administered outside the cell population. In some embodiments, a hif1α inhibitor is administered into a cell population. In some embodiments, the cell population is an islet cell population. In some embodiments, the population of cells is a population of differentiated stem cells. In some embodiments, the differentiated stem cells are differentiation-induced pluripotent stem cells (ipscs). In some embodiments, the differentiated stem cell is an Embryonic Stem Cell (ESC).

Throughout this disclosure, the term "about" is used to indicate that a value includes inherent error variation of a measurement or quantification method.

The use of the terms "a" or "an" when used in conjunction with the term "comprising" may mean "one/one" but is also consistent with the meaning of "one/one or more/multiple", "at least one/one" and "one/one or more than one/one"

The phrase "and/or" means "and" or ". For illustration, A, B and/or C include: a alone, B alone, a combination of C, A and B alone, a combination of a and C, a combination of B and C, or a combination of A, B and C. In other words, "and/or" operates as a non-ubiquitous "or.

The words "comprise" (and any form of "comprising"), such as "comprises" and "comprising an item", "having" (and any form of "having", such as "having" and "having an item"), "include" (and any form of "including", such as "including" and "including an item") or "contain" (and any form of "containing", such as "contains" and "contains an item") are non-inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

The compositions and methods of use thereof may "comprise," consist essentially of, or consist of any of the ingredients or steps disclosed throughout the specification. Compositions and methods that "consist essentially of any of the ingredients or steps" disclosed "limit the scope of the claims to specific materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. As used in this specification and the claims, the terms "comprise" (and any form of "comprising," such as "comprises" and "comprising") are used in a non-exclusive or open-ended manner, and do not exclude additional, unrecited elements or method steps. It is contemplated that embodiments described herein in the context of the term "comprising" may also be practiced in the context of the term "consisting of … …" or "consisting essentially of … …".

It is specifically contemplated that any of the limitations discussed with respect to one embodiment of the present application may be applied to any other embodiment of the present application. Furthermore, any of the compositions of the present application can be used in any of the methods of the present application, and any of the methods of the present application can be used to produce or utilize any of the compositions of the present application. Aspects of the embodiments set forth in the examples are also embodiments that may be practiced in the context of embodiments that are discussed elsewhere in the different examples or elsewhere in the application (such as in the summary, detailed description, claims, and accompanying drawings).

Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may also be described in the language "using" the claimed subject matter, such as "using" any compound, composition, or agent discussed herein to achieve or perform the described therapeutic, diagnostic, or physiological purpose or effect.

Other objects, features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the application, are given by way of illustration only, since various changes and modifications within the spirit and scope of the application will become apparent to those skilled in the art from this detailed description.

Drawings

The following drawings form a part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Fig. 1A to 1D show results indicating the following: beta cells in T2D patients and diabetic rodent models (hIAPP transgenic rats (HIP) and mice (hTG)) showed reduced cell adaptation. Fig. 1A shows confocal images of T2D versus islet cells immunostained for the mitochondrial marker Tom20 in disease-free (ND), indicating reduced mitochondrial area and perinuclear fragmented mitochondrial distribution in islets from T2D donors. FIG. 1B shows Oxygen Consumption Rate (OCR) in basal respiration rate as measured by Seahorse, indicating a decrease in isolated islets from 4.5 months old HIP after stimulation with high glucose (16.7 mM) compared to WT rats. Fig. 1C shows cytosolic ca2+ as measured with FURA2 AM, indicating an increase in islets from hIAPP (hTG) compared to control rodent IAPP (rTG) transgenic mice. FIG. 1D shows immunoblot analysis of Whole Cell Extracts (WCE) and nuclear fractions from 3 non-diabetic patients (ND) and 3T 2D donor islets, revealing that increases in PFKFB3 and HIF1a were accompanied by increases in the following lesion markers: tumor suppressors p53, p21WAF1 and γh2a.x (genotoxic stress markers).

Fig. 2A to 2D show the following results: cells in humans with T2D show metabolic remodeling by the pro-survival hif1α -PFKFB3 pathway. Fig. 2A shows immunofluorescence images of islets (nPOD collection) from non-diabetic (ND) and T2D subjects immunostained for PFKFB3, insulin, and nuclei. FIG. 2B shows PFKFB3 controls Ca in stressed cells ²⁺ Schematic beta of homeostasis, mitochondria and metabolome. FIGS. 2C and 2D show quantification of cell death by TUNEL positive INS 832/13 cells following HIF1α inhibition (FIG. 2C) or PFKFB3 siRNA silencing (FIG. 2D) in the presence or absence of hiaPP expression.

FIGS. 3A and 3B show PFKFB3 ^βKO hIAPP ^+/- Experimental protocols and validation results for mice on a High Fat Diet (HFD). Fig. 3A shows an experimental timeline. FIG. 3B shows PFKFB3 immunostained against PFKFB3 (red), insulin (green) and nuclei (blue) from hIAPP +/-background and High Fat Diet (HFD) ^WT And PFKFB3 ^βKO Is an immunofluorescence image of islets of langerhans. PFKFB3 ^WT hIAPP ^+/+ Used as positive control.

Fig. 4A to 4D show results indicating the following: PFKFB3 ^βKO IAPP ^+/- Mice exhibit reduced abdominal blood glucose, increased insulin sensitivity, and PFKFB3 on a High Fat Diet (HFD) ^WT IAPP ^+/- Mice had impaired glucose tolerance and reduced plasma levels of C peptide. Fig. 4A shows fasting blood glucose. FIG. 4B shows the results of an intraperitoneal glucose tolerance test (IP-GTT). Plasma C peptide and glucagon measured at the end of the experimental protocol of fig. 4C and the insulin resistance test (ITT) of fig. 4D.

FIGS. 5A to 5E show a junction indicatingThe method comprises the following steps: PFKFB3 ^βKO IAPP ^+/- Mice and PFKFB3 ^WT IAPP ^+/- Mice exhibit increased β -cell replication compared to the mice. FIG. 5A shows PFKFB3 immunostained against MCM2, insulin and nuclei from hiaPP +/-or hiaPP-/-background and High Fat Diet (HFD) based ^WT And PFKFB3 ^βKO- Representative immunofluorescence image of islets of mice. Fig. 5B shows β -cell area fraction. FIG. 5C shows the beta-/alpha-cell ratio. Fig. 5D shows β -cell death as measured by TUNEL assay. Fig. 5E shows β -cell replication by minichromosome maintenance protein 2 (MCM 2) immunostaining and quantitative measurement (n=4, sem x p<0.05)。

Fig. 6A to 6C show results indicating the following: PFKFB3 ^βKO IAPP ^+/- Mice showed residual hif1α immunopositions. FIG. 6A shows PFKFB3 immunostained against HIF1α, insulin and nucleus from hIAPP +/-or hIAPP-/-background and High Fat Diet (HFD) based ^WT And PFKFB3 ^βKO- Representative immunofluorescence image of islets of mice. FIG. 6B shows quantification of HIF 1. Alpha. Positive beta. -cells after immunostaining with specific antibodies. Fig. 6C shows quantification of C-Myc positive β -cells after immunostaining with specific antibodies (n=4, sem p <0.05)。

Fig. 7A and 7B show results indicating the following: the LDHA-positive beta cell subset is enriched in insulin secretion-related genes in T2D. FIG. 7A shows UMAP cluster [1] identification of subpopulations of cluster 7 overlapping LDHA positive beta-cells from published RNA-Seq data on beta-cells. Fig. 7B shows a table of differentially expressed genes that were UP-regulated (UP) or DOWN-regulated (DOWN) in cluster 7 relative to 1 and LDHA positive versus negative beta cells.

Fig. 8A to 8D show results indicating the following: PFKFB3 of HIF1α under High Fat Diet (HFD) ^βKO hIAPP ^+/- Up-regulation in mice. FIG. 8A shows PFKFB3 immunostained against PFKFB3 (red), insulin (green) and nuclei (blue) from hIAPP +/-background and High Fat Diet (HFD) ^WT And PFKFB3 ^βKO Representative immunofluorescence images of islets of langerhans. Fig. 8B shows quantification of the image in fig. 8A. FIG. 8C shows that the cells are derived from hIAPP +/based materialsOr hIAPP-/-background and High Fat Diet (HFD) PFKFB3 immunostained against hif1α, insulin and nucleus ^WT And PFKFB3 ^βKO- Representative immunofluorescence image of islets of mice. Fig. 8D shows the quantification of the image in fig. 8A (n=3 for PFKFB3 ^βKO hIAPP+/-，n＝4，SEM*p<0.05)。

Fig. 9A to 9I show results indicating the following: relative to PFKFB3 ^WT IAPP ^+/- PFKFB3 on High Fat Diet (HFD) in mice ^βKO IAPP ^+/- Mice showed increased impaired glucose tolerance and similar insulin plasma levels but reduced glucagon plasma levels. Fig. 9A shows the results of an intraperitoneal glucose tolerance test (IP-GTT) 9 weeks after the start of High Fat Diet (HFD). FIG. 9B shows the quantification of area under the curve (AUC) in the experimental group of FIG. 9A as mg/dL x min. FIG. 9C shows the results of an intraperitoneal glucose tolerance test (IP-GTT) 12 weeks after the onset of HFD. FIG. 9D shows the quantification of area under the curve (AUC) in the experimental group of FIG. 9C as mg/dL x min. Fig. 9E shows the results of the insulin resistance test 9 weeks after the start of HFD. FIG. 9F shows the quantification of area under the curve (AUC) in the experimental group of FIG. 9E as mg/dL x min. Figures 9G and 9H show fasting plasma insulin (figure 9G) and C-peptide (figure 9H) presented relative to beta cell mass (12 weeks after HFD initiation). Fig. 9I shows fasting plasma glucagon (12 weeks after HFD onset) (n=3 for PFKFB3 ^βKO hIAPP+/-，n＝4，SEM*p<0.05)。

Fig. 10A-10F show results demonstrating the following: although relative to PFKFB3 ^WT IAPP ^+/- Mice, PFKFB3 ^βKO IAPP ^+/- Mice showed an increase in cell death, but an increase in the β -cell/a-cell ratio. FIG. 10A shows the quantification of the β -cell area fraction (%). FIG. 10B shows quantification of beta-cell mass (mg). Fig. 10C shows quantification of β -cell death as expressed relative to the β -cell area fraction as measured (%) by labeling with TUNEL assay. FIG. 10D shows the quantification of beta cells relative to the number of a-cells in the indicated experimental group. FIG. 10E shows PFKFB3 immunostained against lytic caspase-3, insulin and nucleus from hiaPP +/-or hiaPP-/-background and High Fat Diet (HFD) ^WT And PFKFB3 ^βKO- Representative immunofluorescence image of islets of mice. Fig. 10F shows quantification of the image from fig. 10E. (n=3 for PFKFB 3) ^βKO hIAPP+/-，n＝4，SEM*p<0.05)。

Fig. 11A to 11D show results indicating the following: PFKFB3 ^βKO IAPP ^+/- Mice and PFKFB3 ^WT IAPP ^+/- Mice exhibit increased healthy β -cell replication compared to the mice. FIG. 11A shows PFKFB3 immunostained against MCM2, insulin and nuclei from hiaPP +/-or hiaPP-/-background and High Fat Diet (HFD) based ^WT And PFKFB3 ^βKO- Representative immunofluorescence image of islets of mice. Fig. 11B shows the quantification of the image according to fig. 11A. FIG. 11C shows PFKFB3 immunostained against C-Myc, insulin and nucleus from hIAPP +/-or hIAPP-/-background and High Fat Diet (HFD) based ^WT And PFKFB3 ^βKO- Representative immunofluorescence image of islets of mice. FIG. 11D shows quantification of cytoplasmic C-Myc (Myc-nick) indicating that cells underwent hiaPP-induced calpain activation (injury) as revealed by immunostaining in FIG. 11C (n=3 for PFKFB3 ^βKO hIAPP+/-，n＝4，SEM*p<0.05)。

Fig. 12A to 12D show results indicating the following: UMAP clustering of beta-cells from published RNA-Seq data [26] cluster 7 subpopulations overlapping LDHA positive (beta-cells with HIF1 alpha signature) were identified. FIG. 12A shows UMAP-2 cluster distribution of pancreatic cells from healthy and T2D donors. Figure 12B shows UMAP-2 distribution of identity-based α -cells (α), β -cells (β), cells with low counts, cells with indeterminate identity, acinar cells, and ductal cells. FIG. 12C shows UMAP-2 distribution of pancreatic cells in 9 pancreatic subpopulations based on the expression level of an identity tag. FIG. 12D shows UMAP-2 distribution (LDHA, indicative of HIF 1. Alpha. Targets characteristic of HIF 1. Alpha.) of lactate dehydrogenase-positive and negative pancreatic cells.

Fig. 13A to 13D show results indicating the following: differential gene expression between β -cell subsets from ND-or T2D revealed that cluster 7 and LDHA positive β -cells had dual identities [ insulin (ins+) and glucagon (gcg+) ]. Differential gene expression between β -cell cluster 7 relative to cluster 1 was shown in non-diabetic patients (ND) (fig. 13A) and T2D (fig. 13B). Differential gene expression between LDHA positive and negative β -cell clusters was shown in non-diabetic patients (ND) (fig. 13C) and T2D (fig. 13D).

Fig. 14A to 14E show results indicating the following: PFKFB3 ^βKO IAPP ^+/- Mice showed a decrease in double positive insulin+/glucagon+ cells. Figure 14A shows quantification of the ratio (%) between single insulin positive β -cells relative to all single positive β -cells and α -cells. Figure 14B shows quantification of the ratio (%) between single glucagon-positive alpha-cells relative to all single positive beta-cells and alpha-cells. Figure 14C shows quantification of the ratio (%) between double insulin (ins+) and glucagon (gcg+) positive cells relative to all single insulin or glucagon positive β -cells and α -cells, respectively. Figure 14D shows the cell composition of single insulin positive β -cells, single glucagon positive α -cells, and dual insulin and glucagon positive cells in the indicated experimental groups. Use of HFD-free WT homozygote (hom) hIAPP ^+/+ Mice were compared to study groups (n=3 for PFKFB 3) with pre-diabetes (pre-DM) and Diabetes (DM) (WT, hom TG-pre-DM and hom TG-DM) ^βKO hIAPP+/-，n＝4，SEM*p<0.05). Fig. 14E shows the cell composition of single insulin positive β -cells, single glucagon positive α -cells, and dual insulin positive and glucagon positive cells in the indicated experimental group (n=3 for PFKFB3 ^βKO DS，n＝4，SEM*p<0.05)。

Fig. 15A to 15D show results indicating the following: the body weight of the experimental group during the course of the experiment was not affected. The body weights of the experimental groups at baseline (t=0) (fig. 15A), 1 week before HFD starts (fig. 15B), 4 weeks HFD (fig. 15C), and 13 weeks HFD (fig. 15D) were specified.

Fig. 16A to 16C show results indicating the following: organ weights of the experimental group during the course of the experiment were unaffected. Weights (g) of pancreas (fig. 16A), liver (fig. 16B), and spleen (fig. 16C) in the experimental group were specified.

Figures 17A to 17C show violin plots showing the distribution of gene numbers (figure 17A), transcript numbers (figure 17B) and percentage of mitochondrial expression (figure 17C) in cells from each donor.

Fig. 18A to 18C show violin plots showing the distribution of gene numbers (fig. 18A), transcript numbers (fig. 18B) and percentage of mitochondrial expression (fig. 18C) in cells from each cluster.

Figure 19 shows the relative contributions of nine annotated pancreatic cell types in healthy and T2D, expressed as percent (%).

Figure 20 shows the results of single cell RNA sequencing analysis of human islet cells from healthy and T2D donors. Marker genes were ranked by fold change in expression by comparing the designated cluster to all other clusters. The size of the dots represents the percentage of genes detected in the cell. Color scale indicates the proportional expression of genes.

FIG. 21 shows a dot plot showing the top marker gene for each cluster. Marker genes were ranked by fold change in expression by comparing the designated cluster to all other clusters. The size of the dots represents the percentage of genes detected in the cell. Color scale indicates the proportional expression of genes.

Fig. 22A and 22B show the results from the sting analysis, which present the relationship between cluster 7 relative to differentially expressed genes in cluster 1 in health (ND) (fig. 22A) and type 2 diabetes (T2D) (fig. 22B).

Fig. 23A and 23B show the results from the sting analysis, which present the relationship between differentially expressed genes in LDHA positive (cluster 7) and LDHA negative β -cells (cluster 1) in health (ND) (fig. 23A) and type 2 diabetes (T2D) (fig. 23B).

FIGS. 24A and 24B show the results from a STRING analysis, which presents the relationship between differentially expressed genes in cluster 1 (FIG. 24A) or LDHA negative (FIG. 24B) β -cells in healthy (ND) subjects.

FIG. 25 is a schematic representation of the model disclosed herein for the effect of beta-cell adaptation comparisons in beta-cell supplementation under stress.

Detailed Description

The present disclosure is based, at least in part, on the identification of beta cell dysfunction in T2D, which results from the inability of diseased beta cells to be purified by cell competition with the remaining healthy beta cells due to activation of hif1α -PFKFB3 injury/repair reactions. Without wishing to be bound by theory, it is believed that damaged beta cells with remodelling metabolism are trapped via high glycolysis that breaks away from the TCA cycle, which renders the beta cells unresponsive to glucose and chronic activation of the hif1α -PFKFB3 pathway prevents steady state cell competition required to clear the damaged beta cells. Given that survival of damaged beta cells in T2D is dependent on the hif1α -PFKFB3 pathway, hif1α -PFKFB3 damage/repair pathways are believed to aid in the selection of damaged beta cells to evade cell competition, and inhibition of cell competition by remodelling metabolism may hinder beta cell regeneration. In some embodiments, disclosed herein are methods and compositions for promoting beta cell regeneration by inhibiting hif1α -PFKFB3 pathway (including hif1α inhibition, PFKFB3 inhibition, and combinations thereof). Aspects of the present disclosure address various needs in the art by providing treatment of subjects suffering from diabetes (including type 1 diabetes and type 2 diabetes).

I. Therapeutic method

Aspects of the present disclosure relate to methods and compositions for treating certain diseases and disorders. In some embodiments, methods for treating a subject having a disorder associated with (e.g., characterized by) protein misfolding are disclosed. In some embodiments, the disorder associated with protein misfolding includes diabetes and related disorders (e.g., pre-diabetes, type 1 diabetes, type 2 diabetes). While often described with respect to the treatment or prevention of type 2 diabetes, it is to be understood that the disclosed methods and compositions may also be used to treat or prevent other conditions associated with protein misfolding, including type 1 diabetes and pre-diabetes.

In some embodiments, disclosed herein is a method for treating type 2 diabetes (T2D) in a subject and/or preventing the subject from developing T2D, the method comprising providing one or more agents capable of inhibiting hif1α -PFKFB3 pathway in a cell of the subject. In some embodiments, the subject has been diagnosed with T2D. In some embodiments, the subject is at risk of developing T2D. In some embodiments, the subject has prediabetes. In some embodiments, the subject has T2D subtype 1, subtype 2 or subtype 3. The T2D subtype is known in the art and is described, for example, in Li L, cheng WY, glicksberg BS, et al Sci Transl Med.2015;7 (311): 311ra174, which is incorporated herein by reference in its entirety. In some embodiments, the subject has T2D subtype 1. In some embodiments, the subject has T2D subtype 2. In some embodiments, the subject has T2D subtype 3. In some embodiments, the subject does not have T2D subtype 1. In some embodiments, the subject does not have T2D subtype 2. In some embodiments, the subject does not have T2D subtype 3.

As recognized herein, inhibition of the hif1α -PFKFB3 pathway enhances killing of unhealthy β cells and facilitates regeneration of healthy β cells in T2D. In some embodiments, unhealthy beta cells describe beta cells that express PFKFB3 (e.g., have a higher PFKFB3 expression level than beta cells from subjects not suffering from T2D). In some embodiments, healthy beta cells describe beta cells that do not express PFKFB3 or beta cells that do not have significant differences in PFKFB3 expression levels from beta cells from subjects not suffering from T2D. Treating T2D in a subject may include ameliorating a T2D symptom in the subject, e.g., increasing insulin sensitivity in the subject.

Methods of treating T2D in a subject may include providing an effective amount of a HIF1α inhibitor. In some embodiments, providing a hif1α inhibitor reduces PFKFB3 levels in the β cells of the subject. Methods of treating T2D in a subject may include providing an effective amount of a PFKFB3 inhibitor. In some embodiments, the disclosed methods comprise providing a hif1α inhibitor and a PFKFB3 inhibitor to a subject. Hif1α inhibitors and PFKFB3 inhibitors may be administered sequentially or substantially simultaneously. Hif1α inhibitors and PFKFB3 inhibitors may be provided in the same composition or in separate compositions.

In some embodiments, the subject treated as described herein does not have cancer or has not been diagnosed with cancer. In some embodiments, the subject treated as described herein has not had or has not been diagnosed with diabetic nephropathy or diabetic retinopathy.

In further embodiments, aspects of the disclosed methods include measuring the expression level of PFKFB3 in a subject. In some embodiments, PFKFB3 expression levels are measured in beta cells from a subject. In some embodiments, PFKFB3 expression levels are used to identify a subject as suffering from T2D. In some embodiments, PFKFB3 expression levels are used to determine whether a subject will be susceptible to a treatment comprising a hif1α and/or PFKFB3 inhibitor. In some embodiments, the beta cells from the subject are determined to have increased PFKFB3 expression levels relative to a healthy or control subject. In some embodiments, the healthy subject is a subject not suffering from diabetes or pre-diabetes. In some embodiments, the healthy subject is a subject not suffering from T2D. In some embodiments, the beta cells from the subject are determined to have increased PFKFB3 expression levels relative to control cells from the subject.

In some embodiments, the subject is treated with a hif1α inhibitor and/or a PFKFB3 inhibitor in combination with one or more additional therapies (e.g., type 2 diabetes therapy). In some embodiments, the subject is treated with a HIF1 alpha inhibitor and/or a PFKFB3 inhibitor in combination with a GLP-1 receptor agonist. In some embodiments, the subject is treated with a hif1α inhibitor and/or a PFKFB3 inhibitor in combination with metformin. In some embodiments, the subject is treated with a hif1α inhibitor and/or a PFKFB3 inhibitor in conjunction with insulin. In some embodiments, the subject is treated with a HIF1 alpha inhibitor and/or a PFKFB3 inhibitor in combination with a DPP-4 inhibitor.

II.HIF1α

Hypoxia-inducible factor 1-alpha (HIF 1 alpha; also referred to as HIF 1A) is a transcription factor involved in the transcriptional regulation of various genes, including those involved in hypoxia-adaptive response.

The following sequence illustrates HIF1α mRNA (SEQ ID NO: 1) in humans:

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the protein sequence is illustrated below (SEQ ID NO: 2):

HIF1α inhibitors

Hif1α inhibitors may refer to any member of the class of compounds or agents that have an IC concentration of 200 μm or less for hif1α activity ₅₀ For example, at least or up to or about 200, 100, 80, 50, 40, 20, 10, 5, 1 μm,100, 10, 1nM or less (or any range or value derivable therein). Hif1α inhibitors may refer to any compound or agent that inhibits hif1α expression. Examples of inhibitors of hif1α0 activity or function may include, but are not limited to, agents that prevent hif1α1/hif1β dimerization, agents that reduce or eliminate protein expression, agents that promote degradation of hif1α2 (e.g., proteasome degradation), agents that prevent hif1α3 interaction with DNA, and agents that inhibit hif1α4 transcriptional activity. In some embodiments, the hif1α inhibitor is an agent that binds directly to hif1α. In some embodiments, the hif1α inhibitor does not bind directly to hif1α. Exemplary HIF1α inhibitors are described, for example, in Onnis et al, J.cell.mol.Med.200913 (9 a): 2780-2786, which is incorporated herein by reference in its entirety. The methods and compositions of the present disclosure may comprise one or more hif1α inhibitors. It is specifically contemplated that one or more of the disclosed hif1α inhibitors may be excluded from certain embodiments of the present disclosure. Also herein Pharmaceutically acceptable salts and prodrugs of the described hif1α inhibitors are contemplated. Although certain exemplary hif1α inhibitors are described herein, it is contemplated that any hif1α inhibitor may be implemented in certain embodiments of the present disclosure.

In some embodiments, the hif1α inhibitor is operably linked (e.g., covalently linked, non-covalently linked, etc.) to the targeting molecule. Targeting molecules describe molecules designed to bind to a specific biological or cellular target. The targeting molecule can be used to specifically direct or target an agent (e.g., a therapeutic agent, such as a hif1α inhibitor) to a particular biological tissue or cell type (e.g., a β cell). In some embodiments, the targeting molecule is configured to bind to a β cell of the subject. In some embodiments, the targeting molecule is configured to bind to a glucagon-like peptide-1 (GLP-1) receptor, thereby targeting the hif1α inhibitor to the β -cells of the subject. In some embodiments, the targeting molecule is an antibody, antibody fragment, or antibody-like molecule.

HIF1α inhibitory nucleic acids

Inhibitory nucleic acids known in the art or any method of inhibiting hif1α gene expression are contemplated in certain embodiments. Examples of inhibitory nucleic acids include, but are not limited to, antisense nucleic acids, such as: siRNA (small interfering RNA), short hairpin RNA (shRNA), double stranded RNA, and any other antisense oligonucleotide. Also included are ribozymes or nucleic acids encoding any of the inhibitors described herein. Inhibitory nucleic acids can inhibit gene transcription or prevent translation of a gene transcript in a cell. The inhibitory nucleic acid may be 16 to 1000 nucleotides long, and in certain embodiments, may be 18 to 100 nucleotides long. The nucleic acid may have at least or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 50, 60, 70, 80, 90 nucleotides or any range derivable therein.

As used herein, "isolated" means altered or removed from a natural state via human intervention. For example, an siRNA naturally occurring in a living animal is not "isolated", but is synthesized, or partially or completely isolated from coexisting materials in its natural state. The isolated siRNA may be present in a substantially purified form, or may be present in a non-natural environment, such as, for example, in a cell into which the siRNA has been delivered.

Inhibitory nucleic acids are well known in the art. For example, siRNA and double stranded RNA have been described in U.S. Pat. Nos. 6,506,559 and 6,573,099 and U.S. patent publications 2003/0051263, 2003/0055020, 2004/0265839, 2002/0168707, 2003/0159161 and 2004/0064842, all of which are incorporated herein by reference in their entirety.

In particular, an inhibitory nucleic acid may be capable of reducing expression of hif1α by at least 10%, 20%, 30%, or 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95% or more, or any range or value derivable therein.

In further embodiments, a synthetic nucleic acid is present that is a hif1α inhibitor. Inhibitors may be between 17 and 25 nucleotides in length, and may comprise a 5 'to 3' sequence that is at least 90% complementary to any portion of the 5 'to 3' sequence of mature hif1α mRNA. In certain embodiments, the inhibitor molecule is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. In addition, the inhibitor molecule has a sequence (from 5 'to 3') that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100% complementary to any portion of the 5 'to 3' sequence of mature hif1α mRNA (particularly mature naturally occurring mRNA), or any range derivable therein. One skilled in the art can use a portion of the probe sequence that is complementary to the mature mRNA sequence as the sequence of the mRNA inhibitor. In addition, the portion of the probe sequence can be altered to still have 90% complementarity with the mature mRNA sequence.

Exemplary HIF 1. Alpha. Inhibitory nucleic acids include EZN-2698.

HIF1α inhibitory polypeptide

In certain embodiments, disclosed herein are hif1α inhibitor polypeptides. In some embodiments, the hif1α inhibitor polypeptide is a hif1α antibody. In some embodiments, the anti-hif1α antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody is a chimeric antibody, affinity matured antibody, humanized antibody, or human antibody. In some embodiments, the inhibitor polypeptide is an antibody-like molecule. In some embodiments, the antibody-like molecule is a nanobody. In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment comprises Fab, fab '-SH, F (ab') 2, or scFv. In one embodiment, the antibody is a chimeric antibody, e.g., an antibody comprising an antigen binding sequence grafted to a heterologous non-human, human or humanized sequence (e.g., a framework and/or constant domain sequence) from a non-human donor. In one embodiment, the non-human donor is a mouse. In one embodiment, the antigen binding sequence is synthetic, e.g., obtained by mutagenesis (e.g., phage display screening, etc.). In one embodiment, the chimeric antibody has a murine V region and a human C region. In one embodiment, the murine light chain V region is fused to a human kappa light chain or human IgG 1C region.

Examples of antibody fragments include, but are not limited to: (i) A Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) an "Fd" fragment consisting of VH and CH1 domains; (iii) An "Fv" fragment consisting of the VL and VH domains of a single antibody; (iv) a "dAb" fragment consisting of a VH domain; (v) an isolated CDR region; (vi) A F (ab') 2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) A single chain Fv molecule ("scFv") in which a VH domain and a VL domain are connected by a peptide linker that allows the two domains to bind to form a binding domain; (viii) Bispecific single chain Fv dimers (see U.S. Pat. No. 5,091,513) and (ix) diabodies, multivalent or multispecific fragments constructed by gene fusion (U.S. patent publication No. 2005/0214860). Fv, scFv or diabody molecules may be stabilised by incorporating disulphide bonds linking the VH and VL domains. Minibodies comprising scfvs conjugated to the CH3 domain can also be prepared (Hu et al, 1996).

In some embodiments, use of anti-hif1α nanobodies in, for example, treating diabetes is disclosed.

HIF1α inhibitory small molecules

As used herein, "small molecule" refers to an organic compound synthesized via conventional organic chemistry methods (e.g., in the laboratory) or found in nature. Typically, small molecules are characterized in that they contain several carbon-carbon bonds and have a molecular weight of less than about 1500 g/mole. In certain embodiments, the small molecule is less than about 1000 g/mole. In certain embodiments, the small molecule is less than about 550 grams/mole. In certain embodiments, the small molecule is between about 200 and about 550 grams/mole. In certain embodiments, the small molecule does not include a peptide (e.g., a compound comprising 2 or more amino acids joined by peptide bonds). In certain embodiments, the small molecule does not include a nucleic acid.

For example, a small molecule hif1α inhibitor may be any small molecule determined to inhibit hif1α function or activity. Such small molecules may be determined based on functional assays in vitro or in vivo. Certain hif1α -inhibiting molecules (i.e., hif1α inhibitors) are known in the art and include, for example, KC7F2, IDF-11774, aminoflavones, AJM290, AW464, tamspiramycin, aclacinomycin, PX-478, FM19G11, resveratrol, rapamycin, everolimus, CCI779, silybin, digoxin, YC-1, phenethyl isothiocyanate, black mucomycin, fraapine, bortezomib, amphotericin B, bay-2243, PX-478, and Gan Ni tamoxifen.

One or more hif1α inhibitors described herein may be excluded from certain embodiments of the present disclosure.

III.PFKFB3

PFKFB3 is also known as 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3, 6PF-2-K/Fru-2,6-P2 enzyme brain/placenta isozymes, renal cancer antigen NY-REN-56, 6PF-2-K/Fru-2,6-P2 enzyme 3, PFK/FBPase 3, IPFK-2, inducible 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3, fructose-6-phosphate, 2-kinase/fructose-2, 6-bisphosphatase 3, 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3, IPFK2 and PFK2.

The following sequence illustrates PFKFB3 mRNA (SEQ ID NO: 3) in humans:

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the protein sequence is illustrated below (SEQ ID NO: 4):

the above protein and mRNA sequences represent one isoform of the gene (isoform 2), but other isoforms are known in the art. For example, the following Genbank numbers represent additional isoforms. The sequences associated with these Genbank numbers are incorporated by reference for all purposes.

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The proteins encoded by this gene belong to a family of bifunctional proteins involved in the synthesis and degradation of fructose-2, 6-bisphosphate, a regulator molecule that controls glycolysis in eukaryotes. The encoded protein has fructose-2-phosphate kinase activity that catalyzes the synthesis of fructose-2, 6-bisphosphate (F2, 6 BP), and fructose-2, 6-bisphosphatase activity that catalyzes the degradation of F2,6 BP. Such proteins are required for cell cycle progression and prevention of apoptosis. It acts as a modulator of cyclin-dependent kinase 1, linking glucose metabolism with cell proliferation and survival of tumor cells.

PFKFB3 inhibitors

PFKFB3 inhibitors may refer to any member of the class of compounds or agents having an IC concentration of 200 μm or less for PFKFB3 activity ₅₀ For example, at least or up to or about 200, 100, 80, 50, 40, 20, 10, 5, 1 μm,100, 10, 1nM or less (or any range or value derivable therefrom), or may refer to any compound or agent that inhibits PFKFB3 expression. Examples of PFKFB3 activity or function may include, but are not limited to, glycolytic regulation, kinase activity, CDK1 regulation, fructose-2-phosphate kinase activity, fructose-2, 6-bisphosphate 2-phosphatase activity, ATP binding activity, and enzyme catalytic activity. In some embodiments, inhibition may be reduced compared to a control level or sample. In further embodiments, functional assays, such as MTT assays, cell proliferation assays, ki67 immunofluorescence, apoptosis assays, or glycolysis assays, may be used to test PFKFB3 inhibitors. The methods and compositions of the present disclosure may comprise one or more PFKFB3 inhibitors. It is specifically contemplated that one or more of the disclosed PFKFB3 inhibitors may be excluded from certain embodiments of the present disclosure. Pharmaceutically acceptable salts and prodrugs of the described PFKFB3 inhibitors are also contemplated herein. Although certain exemplary PFKFB3 inhibitors are described herein, it is contemplated that any PFKFB3 inhibitor may be implemented in certain embodiments of the present disclosure.

In some embodiments, the PFKFB3 inhibitor is operably linked (e.g., covalently linked, non-covalently linked, etc.) to the targeting molecule. Targeting molecules describe molecules designed to bind to a specific biological or cellular target. The targeting molecule can be used to specifically direct or target an agent (e.g., a therapeutic agent, such as a PFKFB3 inhibitor) to a particular biological tissue or cell type (e.g., beta cells). In some embodiments, the targeting molecule is configured to bind to a β cell of the subject. In some embodiments, the targeting molecule is configured to bind to a glucagon-like peptide-1 (GLP-1) receptor, thereby targeting the PFKFB3 inhibitor to the beta cells of the subject. In some embodiments, the targeting molecule is an antibody or antibody-like molecule.

PFKFB3 inhibitory nucleic acids

Inhibitory nucleic acids known in the art or any method of inhibiting PFKFB3 gene expression are contemplated in certain embodiments. Examples of inhibitory nucleic acids include, but are not limited to, antisense nucleic acids, such as: siRNA (small interfering RNA), short hairpin RNA (shRNA), double stranded RNA, and any other antisense oligonucleotide. Also included are ribozymes or nucleic acids encoding any of the inhibitors described herein. Inhibitory nucleic acids can inhibit gene transcription or prevent translation of a gene transcript in a cell. The inhibitory nucleic acid may be 16 to 1000 nucleotides long, and in certain embodiments, may be 18 to 100 nucleotides long. The nucleic acid may have at least or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 50, 60, 70, 80, 90 nucleotides or any range derivable therein.

In particular, the inhibitory nucleic acid may be capable of reducing the expression of PFKFB3 by at least 10%, 20%, 30%, or 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95% or more, or any range or value derivable therein.

In further embodiments, there is a synthetic nucleic acid that is an inhibitor of PFKFB 3. Inhibitors may be between 17 and 25 nucleotides in length and comprise a 5 'to 3' sequence that is at least 90% complementary to any portion of the 5 'to 3' sequence of mature PFKFB3 mRNA. In certain embodiments, the inhibitor molecule is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. In addition, the inhibitor molecule has a sequence (from 5 'to 3') that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary to any portion of the 5 'to 3' sequence of mature PFKFB3mRNA (particularly mature naturally occurring mRNA), or any range derivable therein. One skilled in the art can use a portion of the probe sequence that is complementary to the mature mRNA sequence as the sequence of the mRNA inhibitor. In addition, the portion of the probe sequence can be altered to still have 90% complementarity with the mature mRNA sequence.

Inhibitor nucleic acids for PFKFB3 are also commercially available. For example, the following mirnas may inhibit PFKFB3: the hsa-mir-26b-5p (MIRT 028775), hsa-mir-330-3p (MIRT 043840), hsa-mir-6779-5p (MIRT 454747), hsa-mir-6780a-5p (MIRT 454748), hsa-mir-3689c (MIRT 454749), hsa-mir-3689b-3p (MIRT 454750), hsa-mir-3689a-3p (MIRT 454751), hsa-mir-30b-3p (MIRT 454752), hsa-mir-1273h-5p (MIRT 454753), hsa-mir-6778-5p (MIRT 454754), hsa-mir-1233-5p (MIRT 454755), hsa-mir-6799-5p (MIRT 454756), hsa-mir-7106-5p (MIRT 454757), hsa-mir-3 p (MIRT 3775), hsa-mir-3 p (RT) and/or (MIRT 3135), hsa-mir-6780-673 h-5p (MIRT) and/or (MIRT 6835), hsa-mir-6778-5p (MIRT 6775), hsa-mir-673 h-5p (MIRT 6875), hsa-mir-679-5 p (MIRT 679-5 p) and (MIRT 6735).

siRNA and shRNA are also commercially available from, for example, santa Cruz biotechnology (sc-44011 and sc-44011-SH, respectively).

PFKFB3 inhibitory polypeptide

In certain embodiments, disclosed herein are PFKFB3 inhibitor peptides. In some embodiments, the PFKFB3 inhibitor polypeptide is a PFKFB3 antibody. In some embodiments, the anti-PFKFB 3 antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody is a chimeric antibody, affinity matured antibody, humanized antibody, or human antibody. In some embodiments, the antibody is an antibody-like molecule. In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment comprises Fab, fab '-SH, F (ab') 2, or scFv. In one embodiment, the antibody is a chimeric antibody, e.g., an antibody comprising an antigen binding sequence grafted to a heterologous non-human, human or humanized sequence (e.g., a framework and/or constant domain sequence) from a non-human donor. In one embodiment, the non-human donor is a mouse. In one embodiment, the antigen binding sequence is synthetic, e.g., obtained by mutagenesis (e.g., phage display screening, etc.). In one embodiment, the chimeric antibody has a murine V region and a human C region. In one embodiment, the murine light chain V region is fused to a human kappa light chain or human IgG 1C region.

Pfkfb3 inhibitory small molecules

For example, a small molecule PFKFB3 inhibitor may be any small molecule determined to inhibit PFKFB3 function or activity. Such small molecules may be determined based on functional assays in vitro or in vivo. In some embodiments, the PFKFB3 inhibitors of the present disclosure are PFKFB3 inhibitory molecules. PFKFB3 inhibitory molecules are known in the art and are described, for example, in U.S. patent publications 20130059879, 20120177749, 20100267815, 20100267815 and 20090074884, which are incorporated herein by reference.

Exemplary inhibitory compounds include: (1H-benzo [ g ] indol-2-yl) -phenyl-methanone; (3H-benzo [ e ] indol-2-yl) -phenyl-methanone; (3H-benzo [ e ] indol-2-yl) - (4-methoxy-phenyl) -methanone; (3H-benzo [ e ] indol-2-yl) -pyridin-4-yl-methanone; an HCl salt of (3H-benzo [ e ] indol-2-yl) -pyridin-4-yl-methanone; (3H-benzo [ e ] indol-2-yl) - (3-methoxy-phenyl) -methanone; (3H-benzo [ e ] indol-2-yl) -pyridin-3-yl-methanone; (3H-benzo [ e ] indol-2-yl) - (2-methoxy-phenyl) -methanone; (3H-benzo [ e ] indol-2-yl) - (2-hydroxy-phenyl) -methanone; (3H-benzo [ e ] indol-2-yl) - (4-hydroxy-phenyl) -methanone; (5-methyl-3H-benzo [ e ] indol-2-yl) -phenyl-methanone; phenyl- (7H-pyrrolo [2,3-H ] quinolin-8-yl) -methanone; (3H-benzo [ e ] indol-2-yl) - (3-hydroxy-phenyl) -methanone; (3H-benzo [ e ] indol-2-yl) - (2-chloro-pyridin-4-yl) -methanone; (3H-benzo [ e ] indol-2-yl) - (1-oxy-pyridin-4-yl) -methanone; phenyl- (6, 7,8, 9-tetrahydro-3H-benzo [ e ] indol-2-yl) -methanone; (3H-benzo [ e ] indol-2-yl) - (4-hydroxy-3-methoxythiophenylmethyl) -methanone; (3H-benzo [ e ] indol-2-yl) - (4-benzyloxy-3-methoxy-phenyl) -methanone; 4- (3H-benzo [ e ] indole-2-carbonyl) -benzoic acid methyl ester; 4- (3H-benzo [ e ] indole-2-carbonyl) -benzoic acid; (4-amino-phenyl) - (3H-benzo [ e ] indol-2-yl) -methanone; 5- (3H-benzo [ e ] indole-2-carbonyl) -2-benzyloxy-benzoic acid methyl; 5- (3H-benzo [ e ] indole-2-carbonyl) -2-benzyloxy-benzoic acid methanone; (3H-benzo [ e ] indol-2-yl) - (2-methoxy-pyridin-4-yl) -methanone; (5-fluoro-3H-benzo [ e ] indol-2-yl) - (3-methoxy-phenyl) -methanone; (5-fluoro-3H-benzo [ e ] indol-2-yl) -pyridin-4-yl-methanone; (4-benzyloxy-3-methoxy-phenyl) - (5-fluoro-3H-benzo [ e ] indol-2-yl) -methanone; (5-fluoro-3H-benzo [ e ] indol-2-yl) - (4-hydroxy-3-methoxy-phenyl) -methanone; (3H-benzo [ e ] indol-2-yl) - (3-hydroxymethyl-phenyl) -methanone; cyclohexyl- (5-fluoro-3H-benzo [ e ] indol-2-yl) -methanone; (5-fluoro-3H-benzo [ e ] indol-2-yl) - (3-fluoro-4-hydroxy-phenyl) -methanone; (3H-benzo [ e ] indol-2-yl) -p-tolyl-methanone; (3H-benzo [ e ] indol-2-yl) - (3-methoxy-phenyl) -methanol; (3H-benzo [ e ] indol-2-yl) -pyridin-4-yl-methanol; 3H-benzo [ e ] indole-2-carboxylic acid phenylamide; 3H-benzo [ e ] indole-2-carboxylic acid (3-methoxy-phenyl) -amide; (3H-benzo [ e ] indol-2-yl) - (4-dimethylamino-phenyl) -methanone; (4-amino-3-methoxy-phenyl) - (3H-benzo [ e ] indol-2-yl) -methanone; (4-amino-3-methoxy-phenyl) - (5-hydroxy-3H-benzo [ e ] indol-2-yl) -methanone; (4-amino-3-methoxy-phenyl) - (5-methoxy-3H-benzo [ e ] indol-2-yl) -methanone; n- [4- (3H-benzo [ e ] indole-2-carbonyl) -phenyl ] -methanesulfonamide; 3H-benzo [ e ] indole-2-carboxylic acid (4-amino-phenyl) -amide; (4-amino-phenyl) - (5-methoxy-3H-benzo [ e ] indol-2-yl) -methanone; (4-amino-2-fluoro-phenyl) - (5-methoxy-3H-benzo [ e ] indol-2-yl) -methanone; (4-amino-3-fluoro-phenyl) - (5-methoxy-3H-benzo [ e ] indol-2-yl) -methanone; (4-amino-2-methoxy-phenyl) - (5-methoxy-3H-benzo [ e ] indol-2-yl) -methanone; (4-amino-phenyl) - (9-methoxy-3H-benzo [ e ] indol-2-yl) -methanone; (4-amino-3-methoxy-phenyl) - (9-methoxy-3H-benzo [ e ] indol-2-yl) -methanone; (4-amino-2-methoxy-phenyl) - (9-methoxy-3H-benzo [ e ] indol-2-yl) -methanone; (4-amino-3-fluoro-phenyl) - (9-methoxy-3H-benzo [ e ] indol-2-yl) -methanone; (4-amino-2-fluoro-phenyl) - (9-methoxy-3H-benzo [ e ] indol-2-yl) -methanone; (4-amino-3-fluoro-phenyl) - (3H-benzo [ e ] indol-2-yl) -methanone; (4-amino-2-fluoro-phenyl) - (3H-benzo [ e ] indol-2-yl) -methanone; (4-amino-phenyl) - (7-methoxy-3H-benzo [ e ] indol-2-yl) -methanone; (4-amino-phenyl) - (5-hydroxy-3-methyl-3H-benzo [ e ] indol-2-yl) -methanone; (7-amino-5-fluoro-9-hydroxy-3H-benzo [ e ] indol-2-yl) - (3-methyl-pyridin-4-yl) -methanone; (5-amino-3H-pyrrolo [3,2-f ] isoquinolin-2-yl) - (3-methoxy-pyridin-4-yl) -methanone; (4-amino-2-methyl-phenyl) - (9-hydroxy-3H-pyrrolo [2,3-c ] quinolin-2-yl) -methanone; and (4-amino-phenyl) - (7-methanesulfonyl-3H-benzo [ e ] indol-2-yl) -methanone.

Other exemplary inhibitory compounds include: 1-pyridin-4-yl-3-quinolin-4-yl-propenone; 1-pyridin-4-yl-3-quinolin-3-yl-propenone; 1-pyridin-3-yl-3-quinolin-2-yl-propenone; 1-pyridin-3-yl-3-quinolin-4-yl-propenone; 1-pyridin-3-yl-3-quinolin-3-yl-propenone; 1-naphthalen-2-yl-3-quinolin-2-yl-propenone; 1-naphthalen-2-yl-3-quinolin-3-yl-propenone; 1-pyridin-4-yl-3-quinolin-3-yl-propenone; 3- (4-hydroxy-quinolin-2-yl) -1-pyridin-4-yl-propenone; 3- (8-hydroxy-quinolin-2-yl) -1-pyridin-3-yl-propenone; 3-quinolin-2-yl-1-p-tolyl-propenone; 3- (8-hydroxy-quinolin-2-yl) -1-pyridin-4-yl-propenone; 3- (8-hydroxy-quinolin-2-yl) -1-p-tolyl-propenone; 3- (4-hydroxy-quinolin-2-yl) -1-p-tolyl-propenone; 1-phenyl-3-quinolin-2-yl-propenone; 1-pyridin-2-yl-3-quinolin-2-yl-propenone; 1- (2-hydroxy-phenyl) -3-quinolin-2-yl-propenone; 1- (4-hydroxy-phenyl) -3-quinolin-2-yl-propenone; 1- (2-amino-phenyl) -3-quinolin-2-yl-propenone; 1- (4-amino-phenyl) -3-quinolin-2-yl-propenone; 4- (3-quinolin-2-yl-acryloyl) -benzamide; 4- (3-quinolin-2-yl-acryloyl) -benzoic acid; 3- (8-methyl-quinolin-2-yl) -1-pyridin-4-yl-propenone; 1- (2-fluoro-pyridin-4-yl) -3-quinolin-2-yl-propenone; 3- (8-fluoro-quinolin-2-yl) -1-pyridin-4-yl-propenone; 3- (6-hydroxy-quinolin-2-yl) -1-pyridin-4-yl-propenone; 3- (8-methylamino-quinolin-2-yl) -1-pyridin-4-yl-propenone; 3- (7-methyl-quinolin-2-yl) -1-pyridin-4-yl-propenone; and 1-methyl-4- [3- (8-methyl-quinolin-2-yl) -acryloyl ] -pyridinium.

Other exemplary inhibitory compounds include: PFK15 (1- (4-pyridyl) -3- (2-quinolyl) -2-propen-1-one); (2S) -N- [4- [ [ 3-cyano-1- (2-methylpropyl) -1H-indol-5-yl ] oxy ] phenyl ] -2-pyrrolidinecarboxamide 3PO (3- (3-pyridinyl) -1- (4-pyridinyl) -2-propen-1-one); (2S) -N- [4- [ [ 3-cyano-1- [ (3, 5-dimethyl-4-isoxazolyl) methyl ] -1H-indol-5-yl ] oxy ] phenyl ] -2-pyrrolidinecarboxamide; and 7-hydroxy-2-oxo-2H-1-benzopyran-3-carboxylic acid ethyl ester.

Other exemplary inhibitory compounds include: n-bromoacetyl ethanolamine phosphate (BrAcNHETOP), 7, 8-dihydroxy-3- (4-hydroxyphenyl) benzopyran-4-one (YN 1), 7-hydroxy-2-oxo benzopyran-3-carboxylic acid ethyl ester (YZ 9), 1- (3-pyridyl) -3- (2-quinolyl) -2-propen-1-one (PQP), PFK-158, compound 26 (Boyd et al J.Med Chem 2015), KAN0436151 and KAN0436067.

One or more PFKFB3 inhibitory molecules described herein may be excluded from certain embodiments of the present disclosure.

Elimination of Dihormonal cells

Aspects of the present disclosure relate to methods of eliminating a bishormonal cell from a cell population, and compositions for use thereof. As used herein, the term "bi-hormonal cell" (also referred to as "multi-hormonal cell") refers to the production of insulin (i.e., "insulin) ⁺ ") and glucagon (i.e.," glucagon ⁺ ") are both cells. Such bi-hormonal cells are well known in the art and are described, for example, in JE, erener s. Et al, stem Cell res.2014, month 1; 12 (1) 194-208 and Alvarez-domiiguez JR, et al Cell Stem Cell 2020, 1 month and 2 days; 26 (1) 108-122.e10, each of which is incorporated herein by reference.

As disclosed herein, inhibition of PFKFB3 and/or hif1α may be used to eliminate bishormonal cells from, for example, an islet cell population. Accordingly, aspects of the present disclosure relate to methods for eliminating a bi-hormonal cell from a cell population comprising providing a PFKFB3 inhibitor and/or a hif1α inhibitor. In some embodiments, the inhibitor is provided outside the cell population. In some embodiments, the inhibitor is provided into the cell population. In some embodiments, the population of cells comprises differentiated stem cells. Such differentiated stem cells include, for example, islet cells derived from differentiated stem cells and bishormonal cells derived from differentiated stem cells. In some embodiments, the stem cells are induced pluripotent stem cells (ipscs). In some embodiments, the stem cell is an embryonic stem cell. In some embodiments, the stem cells are obtained from a patient suffering from, at risk of suffering from, or suspected of suffering from type 1 diabetes or type 2 diabetes. The disclosed methods may further comprise, after providing the PFKFB3 inhibitor and/or the hif1α inhibitor, administering the cell population to the subject. In some embodiments, the subject has, is at risk of having, or is suspected of having type 1 diabetes or type 2 diabetes. In some embodiments, the population of cells is autologous to the subject. In some embodiments, the population of cells is not autologous to the subject.

V. pharmaceutical composition

Embodiments include methods of treating diabetes with a composition comprising a hif1α inhibitor and/or a PFKFB3 inhibitor. In some embodiments, the disclosed compositions comprise a hif1α inhibitor. In some embodiments, the disclosed compositions comprise PFKFB3 inhibitors. In some embodiments, the disclosed compositions comprise a hif1α inhibitor and a PFKFB3 inhibitor. The administration of the composition will generally be via any common route. This includes, but is not limited to, oral, parenteral, in situ, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, intratumoral or intravenous injection. Oral formulations include commonly used excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain from about 10% to about 95% of the active ingredient or from about 25% to about 70%. In some embodiments, the composition is administered orally.

Typically, the compositions are administered in a manner compatible with the dosage formulation, and in such amounts as will be therapeutically effective and subject to immune modification. The number to be administered depends on the subject to be treated. The exact amount of active ingredient to be administered will depend on the discretion of the practitioner.

The manner of application may vary widely. Any conventional method for administering a pharmaceutical composition is suitable. These are considered to include oral administration based on a physiologically acceptable solid matrix or in a physiologically acceptable dispersion, parenterally, by injection, or the like. The dosage of the pharmaceutical composition will depend on the route of administration and will vary depending on the size and health of the subject.

In many cases, it is desirable to have multiple administrations up to about or at least about 3, 4, 5, 6, 7, 8, 9, 10 or more times. The administration may be in the range of 2 days to twelve weeks apart, more typically one to two weeks apart. The administration process may be followed by a determination of hif1α and/or PFKFB3 activity.

The term "pharmaceutically" or "pharmacologically acceptable" means that the molecular entities and compositions do not produce adverse, allergic or other untoward reactions when administered to an animal or human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and medicaments for pharmaceutically active substances is well known in the art. Except insofar as any conventional medium or agent is incompatible with the active ingredient, use of the medium or agent in immunogenic and pharmaceutical compositions is contemplated.

By "pharmaceutically acceptable salt" is meant a salt of a compound of the invention (e.g., hif1α inhibitor, PFKFB3 inhibitor), which is pharmaceutically acceptable as defined above, and which has the desired pharmacological activity. Such salts include acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or acid addition salts with organic acids such as 1, 2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4' -methylenebis (3-hydroxy-2-en-1-carboxylic acid), 4-methylbicyclo [2.2.2] oct-2-en-1-carboxylic acid, acetic acid, aliphatic mono-and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, caproic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o- (4-hydroxybenzoyl) benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acid, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, t-butylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which are formed when the acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. It should be appreciated that the particular anion or cation forming part of any salt of the invention is not critical, so long as the salt as a whole is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and methods of making and using them are presented in Handbook of Pharmaceutical Salts: properties, and Use (p.H.Stahl and C.G.Wermuth et al, verlag Helvetica Chimica Acta, 2002).

The disclosed compositions may include a prodrug. "prodrug" means a compound which can be metabolically converted in vivo into an inhibitor according to the invention. Prodrugs may or may not themselves have activity against a given target protein. For example, compounds comprising hydroxyl groups may be administered as esters that are converted to hydroxyl compounds by in vivo hydrolysis. Suitable esters that can be converted to hydroxyl compounds in vivo include acetates, citrates, lactates, phosphates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene-bis-beta-hydroxynaphthoates, gentisates, isethionates, di-p-toluyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates, quiniates, amino acid esters, and the like. Similarly, compounds comprising amine groups may be administered as amides that are converted to amine compounds by in vivo hydrolysis.

Hif1α inhibitors and/or PFKFB3 inhibitors may be formulated for parenteral administration, for example, formulated for injection via intravenous, intradermal, intramuscular, subcutaneous, or even intraperitoneal routes. In some embodiments, the composition is administered by intravenous injection. The preparation of aqueous compositions containing active ingredients will be known to those skilled in the art in light of the present disclosure. Typically, such compositions may be prepared as injectables, either as liquid solutions or suspensions; it is also possible to prepare solid forms suitable for use in preparing solutions or suspensions by adding liquids prior to injection; and, these formulations may also be emulsified.

Pharmaceutical dosage forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid so that it can be easily injected. It should also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

The composition may be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (acid addition salts formed with free amino groups of proteins) and acid addition salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like.

The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), suitable mixtures thereof, and vegetable oils. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents which delay absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active ingredient in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by sterile filtration. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

An effective amount of the therapeutic or prophylactic composition is determined based on the intended target. The term "unit dose" or "dose" refers to physically discrete units suitable for a subject, each unit containing a predetermined amount of a composition calculated to produce the desired response discussed above in connection with its administration (i.e., the appropriate route and regimen). Depending on the number of treatments and unit dose, the amount to be administered depends on the desired outcome and/or protection. The precise amount of the composition will also depend on the discretion of the practitioner and will vary from individual to individual. Factors that affect the dosage include the physical and clinical state of the subject, the route of administration, the intended target of the treatment (relief of symptoms and cure), and the efficacy, stability, and toxicity of the particular composition. After formulation, the solution will be administered in a manner compatible with the dosage of the formulation and in an amount such as therapeutically or prophylactically effective. These formulations are readily administered in a variety of dosage forms, such as the injectable solution types described above.

After formulation, the solution will be administered in a manner compatible with the dosage of the formulation and in an amount such as therapeutically or prophylactically effective. These formulations are readily administered in a variety of dosage forms, such as the injectable solution types described above.

As an example, for an adult (body weight of about 70 kg), about 0.1mg to about 3000mg (including all values and ranges therebetween), or about 5mg to about 1000mg (including all values and ranges therebetween), or about 10mg to about 100mg (including all values and ranges therebetween) of the compound is administered. It will be appreciated that these dosage ranges are by way of example only, and that administration may be adjusted depending on factors known to the skilled artisan.

In some embodiments of the present invention, in some embodiments, about, at least about, or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4.4.4% 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0 and 12.0. 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 185. 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430 440, 441, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 milligrams (mg) or micrograms (mcg) or μg/kg/min or mg/kg/min or micrograms/kg/hr or mg/kg/hr, or any range derivable therefrom.

The dose may be administered as needed or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 hours (or any range derivable therefrom) or 1, 2, 3, 4, 5, 6, 7, 8, 9 or more times per day (or any range derivable therefrom). The dose may be administered prior to or after the symptoms of the disorder. In some embodiments, the first dose of the regimen is administered to the patient 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours (or any range derivable therefrom) or 1, 2, 3, 4, or 5 days after the patient experiences or exhibits the sign or symptom of the disorder (or any range derivable therefrom). The patient may be treated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days (or any range derivable therefrom), or until the symptoms of the disorder disappear or are reduced, or after 6, 12, 18 or 24 hours or 1, 2, 3, 4 or 5 days after the symptoms of the infection disappear or are reduced.

Administration of therapeutic compositions and combinations

Therapies provided herein can include administering a combination of therapeutic agents, such as a first treatment (e.g., a hif1α inhibitor) and a second treatment (e.g., a PFKFB3 inhibitor). The therapy may be administered in any suitable manner known in the art. For example, the first treatment and the second treatment are administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the first treatment and the second treatment are administered as separate compositions. In some embodiments, the first treatment and the second treatment are in the same composition.

Embodiments of the present disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of agents may be employed.

The therapeutic agents of the present disclosure may be administered by the same route of administration or by different routes of administration. In some embodiments, the therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, the severity and course of the disease, the clinical condition of the individual, the clinical history and response of the individual to treatment, and the decision of the attending physician.

Treatment may include various "unit doses". A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount to be administered, as well as the particular route and formulation, are within the skill of one of skill in the clinical arts in determining. The unit dose need not be administered as a single injection, but may include continuous infusion over a set period of time. In some embodiments, the unit dose comprises a single administrable dose.

Depending on the number of treatments and unit dose, the amount to be administered depends on the desired therapeutic effect. An effective dose is understood to mean the amount required to achieve a particular effect. In the practice of certain embodiments, it is contemplated that dosages in the range of 10mg/kg to 200mg/kg may affect the protective capabilities of these agents. Thus, contemplated dosages include dosages of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 and 200, 300, 400, 500, 1000 μg/kg, mg/kg, μg/day, or any range derivable therein. Furthermore, such doses may be administered multiple times during a day, and/or over multiple days, weeks, or months.

In certain embodiments, an effective dose of the pharmaceutical composition is a dose that can provide a blood level of about 1 μm to 150 μm. In another embodiment, the effective dose provides the following blood levels: about 4 μm to 100 μm; or about 1 μm to 100 μm; or about 1 μm to 50 μm; or about 1 μm to 40 μm; or about 1 μm to 30 μm; or about 1 μm to 20 μm; or about 1 μm to 10 μm; or about 10 μm to 150 μm; or about 10 μm to 100 μm; or about 10 μm to 50 μm; or about 25 μm to 150 μm; or about 25 μm to 100 μm; or about 25 μm to 50 μm; or about 50 μm to 150 μm; or about 50 μm to 100 μm (or any range derivable therein). In other embodiments, the dose may provide the following agent blood levels resulting from administration of a therapeutic agent to a subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM, or any range derivable therein. In certain embodiments, a therapeutic agent administered to a subject is metabolized in vivo to a metabolized therapeutic agent, in which case blood levels may refer to the amount of the agent. Alternatively, to the extent that the therapeutic agent is not metabolized by the subject, the blood levels discussed herein may refer to the non-metabolized therapeutic agent.

The precise amount of therapeutic composition will also depend on the discretion of the practitioner and will vary from individual to individual. Factors that affect the dosage include the physical and clinical state of the patient, the route of administration, the intended target of treatment (relief of symptoms and cure), and the efficacy, stability, and toxicity of the particular therapeutic substance or other treatment that the subject may be receiving.

Those skilled in the art will understand and appreciate that dosage units of μg/kg or mg/kg body weight can be converted and expressed as comparable μg/ml or mM concentration units (blood levels), such as 4 μM to 100 μM. It should also be understood that uptake depends on the species and the organ/tissue. Suitable scaling factors and physiological assumptions regarding uptake and concentration measurements are well known and will allow one skilled in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the dosage, efficacy, and results described herein.

Testing genetic characteristics

Particular embodiments relate to methods of detecting genetic characteristics of an individual. In some embodiments, methods for detecting genetic features may include, for example, selective oligonucleotide probes, arrays, allele-specific hybridization, molecular beacons, restriction fragment length polymorphism assays, enzymatic chain reactions, flap endonuclease assays, primer extension, 5' -nuclease assays, oligonucleotide ligation assays, single-strand conformation polymorphism assays, temperature gradient gel electrophoresis, denaturing high performance liquid chromatography, high resolution melting, DNA mismatch binding protein assays, survivin nuclease assays, sequencing, or combinations thereof. Methods for detecting genetic features may include, for example, fluorescent in situ hybridization, comparative genomic hybridization, arrays, polymerase chain reaction, sequencing, or combinations thereof. Detection of a genetic feature may involve using a particular method to detect one feature in the genetic feature and additionally using the same method or a different method to detect a different feature in the genetic feature. The same feature or features may be detected using a variety of different methods, either individually or in combination. In some embodiments, the disclosed methods comprise detecting the expression level of PFKFB3 in a cell (e.g., a beta cell) from a subject.

A.DNA sequencing

In some embodiments, DNA may be analyzed by sequencing. The DNA may be prepared for sequencing by any method known in the art, such as library preparation, hybridization capture, sample quality control, library preparation based on ligation of the products used, or a combination thereof. DNA can be prepared for any sequencing technique. In some embodiments, sequencing can be performed to cover about 70%, 75%, 80%, 85%, 90%, 95%, 99% or greater percent of the target under a coverage of more than 20x, 25x, 30x, 35x, 40x, 45x, 50x, or greater than 50 x. In some embodiments, DNA sequencing is used to determine the expression level of PFKFB3 in cells (e.g., beta cells) from a subject.

RNA sequencing

In some embodiments, RNA can be analyzed by sequencing. RNA may be prepared for sequencing by any method known in the art, such as polyadenylation selection, cDNA synthesis, chain or non-chain library preparation, or a combination thereof. RNA can be prepared for use in any type of RNA sequencing technique, including chain-specific RNA sequencing. In some embodiments, sequencing can be performed to generate about 10M, 15M, 20M, 25M, 30M, 35M, 40M or more reads, including paired reads. The sequencing may be performed at read lengths of about 50bp, 55bp, 60bp, 65bp, 70bp, 75bp, 80bp, 85bp, 90bp, 95bp, 100bp, 105bp, 110bp, or longer. In some embodiments, raw sequencing data may be converted to estimated read counts (RSEM), fragments per kilobase transcript per million mapped reads (FPKM), and/or reads per kilobase transcript per million mapped Reads (RPKM). In some embodiments, RNA sequencing is used to determine the expression level of PFKFB3 in cells (e.g., beta cells) from a subject.

C. Proteomics

In some embodiments, the protein may be analyzed by mass spectrometry. The protein may be prepared for mass spectrometry using any method known in the art. Proteins, including any isolated proteins encompassed herein, can be treated with DTT followed by iodoacetamide. The protein may be incubated with at least one peptidase, including endopeptidase, protease or any enzyme that cleaves the protein. In some embodiments, the protein is incubated with endopeptidase, lysC, and/or trypsin. The protein may be incubated with one or more protein cleaving enzymes at any ratio, including a ratio of enzyme μg to protein μg of about 1:1000, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:1, or any range therebetween. In some embodiments, the cleaved protein may be purified, such as by column purification. In certain embodiments, the purified peptide may be quick frozen and/or dried, such as under vacuum. In some embodiments, the purified peptide may be fractionated, such as by reverse phase chromatography or basic reverse phase chromatography. Fractions may be combined to practice the methods of the present disclosure. In some embodiments, phosphopeptide enrichment is performed on one or more fractions, including pooled fractions, including phosphate enrichment by affinity chromatography and/or binding, ion exchange chromatography, chemical derivatization, immunoprecipitation, co-precipitation, or a combination thereof. Mass spectrometry can be performed on all or part of one or more fractions, including the pooled fraction and/or the phosphoric acid-rich fraction. In some embodiments, the raw mass spectral data may be processed and normalized using at least one associated bioinformatics tool. In some embodiments, proteomics is used to determine the amount of PFKFB3 in cells (e.g., beta cells) from a subject.

VIII kit

Certain aspects of the invention also relate to kits containing the compositions of the present disclosure or compositions for practicing the methods of the present disclosure. In some embodiments, the kit may be used to evaluate one or more biomarkers. In certain embodiments, the kit contains, at least contains, or at most contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more probes, primers or primer sets, synthetic molecules or inhibitors, or any value or range and combination derivable therein. In some embodiments, there is a kit for assessing biomarker activity in a cell. In some embodiments, kits for assessing the expression level of one or more biomarker activities are disclosed. In some embodiments, a kit for assessing PFKFB3 expression levels in beta cells from a subject is disclosed.

The kit may comprise components, which may be packaged separately or placed in containers, such as tubes, bottles, vials, syringes, or other suitable container means.

The individual components may also be provided in the kit in concentrated amounts; in some embodiments, the components are provided separately at the same concentration as the other components in the solution. The concentration of the components may be provided as 1x, 2x, 5x, 10x, or 20x or higher.

Kits for using probes, synthetic nucleic acids, non-synthetic nucleic acids, and/or inhibitors of the present disclosure for prognostic or diagnostic applications are included as part of the present disclosure. Of particular concern are any such molecules corresponding to any of the biomarkers identified herein, including nucleic acid primers/primer sets and probes that are identical or complementary to all or part of the biomarker, which may include non-coding sequences of the biomarker, as well as coding sequences of the biomarker. In certain aspects, some kit embodiments include negative and/or positive control nucleic acids, probes, and inhibitors.

Any embodiment of the disclosure that relates to a particular biomarker by name is contemplated to also encompass embodiments that relate to a biomarker whose sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the mature sequence of the specified nucleic acid.

Embodiments of the present disclosure include kits for analyzing a pathological sample by assessing a biomarker profile of the sample comprising two or more biomarker probes in a suitable container device, wherein the biomarker probes detect one or more biomarkers identified herein. The kit may further comprise reagents for labeling nucleic acids in the sample. The kit may further comprise a labeling reagent comprising at least one of an amine modified nucleotide, a polyadenylation polymerase, and a polyadenylation polymerase buffer. The labeling reagent may include an amine reactive dye.

Examples

The following examples are included to demonstrate preferred embodiments of the present invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1-analysis of beta cells in type 2 diabetes

Method

Histological evaluation

After removal of the smaller fragments, the pancreas was fixed overnight in 4% paraformaldehyde (Electron Microscopy Sciences 19202, hatfield, pa, usa) at 4 ℃, paraffin embedded and sectioned at 4 μm thickness. For the beta cell region, deparaffinized sections were subjected to peroxidase and hematoxylin staining, and these sections were sequentially stained with rabbit anti-insulin antibody (Cell Signaling Technology C C9, danvers, MA, USA, 1:400) followed by F (ab') ₂ Incubation with conjugates of biotin-SP (Jackson ImmunoResearch 711-066-152,West Grove,PA,USA,1:100 for IHC), after these steps, the VECTASTAIN ABC kit (HRP) (Vector Laboratories PK-4000, burlingame, CA, USA), DAB substrate kit (HRP) (Vector Laboratories SK-4100, burlingame, CA, USA) and Harris hematoxylin (Harris Hematoxylin) were applied before sealing with Permount (Fisher SP15-100, hampton, NH, USA). Morphometric analysis was performed on an Olympus IX70 inverted tissue culture microscope (Olympus, center Valley, PA, USA) using Image-Pro Plus 5.1 software. The genotype of the experimental mice for each slice was imaged and data analyzed blindly by two observers. Islet edges are defined manually using multi-channel images. The insulin and hematoxylin positive areas of each islet are determined using pixel thresholds. The beta cell area was then calculated as insulin positive area/hematoxylin positive area x 100%.

Immunofluorescence analysis was performed in Openlab 5.5.0 software on a Leica DM 6000B study microscope. The following antibodies were used: rabbit anti-PFKFB 3 (origin)ne AP15137PU-N, rockville, md., USA, 1:100); mouse anti-MCM 2 (BD Transduction Laboratories610700, san Diego, calif., USA, 1:100); rabbit anti-lytic caspase-3 (Cell Signaling Technology 9664S, danvers, mass., USA, 1:400); guinea pig insulin resistance (Abcam ab195956, cambridge, mass., USA, 1:400); mouse anti-glucagon (Sigma-Aldrich G2654, st.Louis, MO, USA,1:1000 for IF), mouse anti-c-Myc (Santa Cruz Biotechnology Inc E10 sc-40, dallas, texas, USA, 1:100); mice were resistant to HIF1α (Novus Biologicals NB-105, centennial, CO, USA, 1:50). The secondary antibodies were: f (ab') ₂ Conjugates (Jackson ImmunoResearch-706-096-148,West Grove,PA,USA,1:200 for IF); f (ab') with Cy3 ₂ Conjugates (Jackson ImmunoResearch 711-166-152,West Grove,PA,USA,1:200 for IF) and F (ab') with Alexa 647 ₂ Conjugates (Jackson ImmunoResearch 715-606-150,West Grove,PA,USA,1:100 for IF). Cell death was determined by TUNEL assay using In Situ Cell Death Detection Kit (Roche Diagnostics Corporation 12156792910, indianapolis, in, usa). A Vectashield with DAPI (Vector Laboratories H, 1200, burlingame, CA, USA) patch was used.

Results

Beta cell function in T2D gradually declines, this part being associated with hIAPP [3-5 ]]Is associated with the accumulation of toxic oligomers. hIAPP forms membrane permeable toxic oligomers that are associated with misfolded protein stress in T2D. In response to misfolded protein stress by hIAPP, mitochondria in β cells from humans with T2D acquire a defensive posture via mitochondrial network cleavage (fig. 1A), which results in 30% attenuation of mitochondrial respiration (fig. 1B). Alterations in mitochondrial form and function and exposure to Ca ²⁺ The toxic neurons are very similar and thus reflect high cytosolic Ca ²⁺ Is adapted to the application of (1). Indeed, islets from human IAPP transgenic mice (hTG) exhibited higher cytosolic Ca than rodent IAPP (rTG) control littermates ²⁺ Horizontal (fig. 1C). Oxidative and DNA damage in subcellular fractions of islets from donors with T2D relative to non-diabetic (ND) donors is clearObvious, e.g. DNA damage response proteins p53, p21 ^WAF1 And increased expression of γh2a.x (fig. 1D).

DNA damage response (accumulation of p53/p21WAF1 axis and γh2a.x) recorded the damage of β cells in T2D and T2D rodent models. Stress and cytosolic Ca caused by protein misfolding ²⁺ The proliferation of (c) triggered protective metabolic reprogramming of approximately one third of all beta cells in T2D by a conserved hif1α -PFKFB3 injury/repair program (fig. 2A). PFKFB3 was identified as responsible for Ca in beta cells under stress ²⁺ Homeostasis, mitochondrial remodeling, and metabolomic changes (fig. 2B), ultimately result in survival of damaged beta cells. This was confirmed by in vitro analysis using adenovirus transfected INS 832/13 cells to express hIAPP in the presence or absence of hif1α inhibition or PFKFB 3. Hif1α inhibition (fig. 2C) or PFKFB3 post-transcriptional silencing (fig. 2D) resulted in increased cell death in hIAPP-expressing cells, as demonstrated by TUNEL assay.

Example 2-PFKFB3 ^βKO hIAPP ^+/- Analysis of +HFD mice

Method

Animals

Homozygous hIAPP ^+/+ Mice were given from Peter Butler doctor laboratory and have been described previously [15 ]]. By hybridizing mice carrying the floxed PFKFB3 gene (JAX laboratory) with mice expressing Cre recombinase under the control of the rat insulin promoter (RIP-CreERT), a beta cell-specific inducible PFKFB3 knockout mouse model (RIP-CreERT: PFKFB3 was developed ^fl/fl ). Will be based on homozygous hIAPP ^+/+ Background mice and PFKFB3 ^fl/fl Or RIP-Creet mice, followed by PFKFB3 ^fl/fl hIAPP ^+/- And PFKFB3 ^fl/fl RIP-CreERT mice were hybridized together to generate three experimental genotypes: RIP-CreERT PFKFB3 ^wt/wt hIAPP ^-/- 、RIP-CreERT PFKFB3 ^wt/wt hIAPP ^+/- And RIP-CreERT PFKFB3 ^fl/fl hIAPP ^+/- Herein referred to as PFKFB3 ^WT hIAPP ^-/- 、PFKFB3 ^WT hIAPP ^+/- And PFKFB3 ^βKO hIAPP ^+/- . All experimental groups wereReceives a high fat diet. Cre-loxP recombination at the floxed site in Pfkfb3 was induced by intraperitoneal injection of tamoxifen at 20-27 weeks of age. Following tamoxifen injection, mice were fed with feed for 10 weeks and then all mice were exposed to a high fat diet for an additional 13 weeks (HFD, research Diets Inc, new Brunswick, NJ, USA) to induce as p hIAPP ^+/- And HFD responsive diabetes, because for hIAPP (hIAPP only ^+/+ ) Homozygous male mice spontaneously develop diabetes [15 ]]. Mice were kept on a 12 hour day/night cycle in a university of california, los angeles division institution animal care and use committee (UCLA Institutional Animal Care and Use Committee) (ARC) approved mouse community facility. At 30-37 weeks of age, all mice were allocated to receive diets containing high amounts of fat (35% w/w or 60% calories from fat; number D12492). The fat composition of the high fat diet was 32.2% saturated fat, 35.9% monounsaturated fat, and 31.9% polyunsaturated fat. During the duration of the study, the mice were allowed to eat and drink ad libitum. Body weight and fasting blood glucose levels were assessed weekly and additional measurements were made over several days, including glucose and insulin resistance tests (IP-GTT and ITT, respectively).

Insulin and glucose tolerance test

Intraperitoneal glucose tolerance test (IP-GTT) was performed at 9 and 12 weeks after HFD (19 and 22 weeks after tamoxifen injection). Tail vein blood glucose was collected 15, 30, 60, 90, 120 minutes before and after glucose bolus injection. After 30 minutes before and after glucose bolus, retroorbital blood collection was used to collect blood for the second IP-GTT. Mice were anesthetized by brief exposure to isoflurane (10 seconds). Blood was collected in EDTA-coated microcentrifuge tubes and plasma was obtained by centrifuging the sample for 10 minutes (5000 rcf,10min,4 ℃).

Glucose and insulin assays

Plasma glucose was measured using the glucose oxidase method and analyzed using the YSI 2300STAT PLUS Glucose&L-Lactate analyzer.

Fasting blood glucose was measured weekly from blood drawn from the tail using a fresh glucometer (Abbott Diabetes Care Inc, alameda, CA, USA) after 18 hours of fasting (after periodic replacement of cages and pads and cessation of feeding while water was provided ad libitum).

Plasma insulin, c-peptide and glucagon levels were determined using Ultrasensitive ELISA for mouse insulin (Mercodia 10-1247-01, uppsala, sweden), mouse c-peptide (Crystal Chem 90050, il, usa) and mouse glucagon (Mercodia 10-1281-01, uppsala, sweden).

Awake mice, 6 hours fasted, were subjected to an intraperitoneal insulin resistance test (0.75 IU/kg) 10 weeks after HFD (19 weeks after tamoxifen injection) (Lilly insulin Lispro, LLC, indianapolis, USA). Tail vein blood was collected for glucose measurement before and 0, 20, 40, 60 minutes after insulin administration.

Pancreatic perfusion and separation

One week after IP-GTT and ITT, mice were euthanized by cervical dislocation. Medial incision was used to open the abdominal and thoracic cavities while the right ventricle was incised and then the left ventricle was needled into to slowly infuse 10ml of cold Phosphate Buffered Saline (PBS) for pancreatic infusion. After perfusion, the pancreas was placed in cold PBS and separated from other tissues including surrounding fat. The pancreas was then weighed after absorbing additional PBS with tissue.

Histological evaluation

After removal of the smaller fragments, the pancreas was fixed overnight in 4% paraformaldehyde (Electron Microscopy Sciences 19202, hatfield, pa, usa) at 4 ℃, paraffin embedded and sectioned at 4 μm thickness. For the beta cell region, deparaffinized sections were subjected to peroxidase and hematoxylin staining, and these sections were sequentially stained with rabbit anti-insulin antibody (Cell Signaling Technology C C9, danvers, MA, USA, 1:400) followed by F (ab') ₂ Incubation with conjugates of biotin-SP (Jackson ImmunoResearch 711-066-152,West Grove,PA,USA,1:100 for IHC), after these steps, the VECTASTAIN ABC kit (HRP) (Vector Labora) was applied before sealing with Permount (Fisher SP15-100, hampton, NH, USA)tories PK-4000, burlingame, calif., USA), DAB substrate kit (HRP) (Vector Laboratories SK-4100, burlingame, calif., USA) and Harris hematoxylin. Morphometric analysis was performed on an Olympus IX70 inverted tissue culture microscope (Olympus, center Valley, PA, USA) using Image-Pro Plus 5.1 software. The genotype of the experimental mice for each slice was imaged and data analyzed blindly by two observers. Islet edges are defined manually using multi-channel images. The insulin and hematoxylin positive areas of each islet are determined using pixel thresholds. The beta cell area was then calculated as insulin positive area/hematoxylin positive area x 100%.

Immunofluorescence analysis was performed in Openlab 5.5.0 software on a Leica DM 6000B study microscope. The following antibodies were used: rabbit anti-PFKFB 3 (origin AP15137PU-N, rockville, md., USA, 1:100); mouse anti-MCM 2 (BD Transduction Laboratories610700, san Diego, calif., USA, 1:100); rabbit anti-lytic caspase-3 (Cell Signaling Technology 9664S, danvers, mass., USA, 1:400); guinea pig insulin resistance (Abcam ab195956, cambridge, mass., USA, 1:400); mouse anti-glucagon (Sigma-Aldrich G2654, st.Louis, MO, USA,1:1000 for IF), mouse anti-c-Myc (Santa Cruz Biotechnology Inc E10 sc-40, dallas, texas, USA, 1:100); mice were resistant to HIF1α (Novus Biologicals NB-105, centennial, CO, USA, 1:50). The secondary antibodies were: f (ab') ₂ Conjugates (Jackson ImmunoResearch-706-096-148,West Grove,PA,USA,1:200 for IF); f (ab') with Cy3 ₂ Conjugates (Jackson ImmunoResearch 711-166-152,West Grove,PA,USA,1:200 for IF) and F (ab') with Alexa 647 ₂ Conjugates (Jackson ImmunoResearch 715-606-150,West Grove,PA,USA,1:100 for IF). Cell death was determined by TUNEL assay using In Situ Cell Death Detection Kit (Roche Diagnostics Corporation 12156792910, indianapolis, in, usa). A Vectashield with DAPI (Vector Laboratories H, 1200, burlingame, CA, USA) patch was used.

Statistical analysis

Data are shown as mean error (standard error, SEM) for the indicated number of mice. For IP-GTT and ITT, the area under the curve (AUC) for glucose, insulin, C-peptide and glucagon was calculated using the trapezoidal rule. Average data between groups were compared by analysis using student's t-test. P values less than 0.05 are considered statistically significant.

Results

Mice were based on hIAPP in order to mimic the effects of insulin resistance, misfolded protein stress and senile as a cumulative risk factor for diabetes and to investigate the effect of PFKFB3 on preservation of damaged beta cells under hyperdiabetic stress ^+/- Background beta cell-specific conditional disruption of the Pfkfb3 gene (see methods section), 13 weeks exposure to high fat diet (Pfkfb 3 ^βKO hIAPP ^+/- +HFD) and with PFKFB3 ^WT hIAPP ^+/- +HFD and PFKFB3 ^WT hIAPP ^-/- The +hfd control was compared (experimental timelines presented in fig. 3A). By immunostaining of pancreatic sections from mice of the indicated experimental group with PFKFB3 and using PFKFB3 ^WT hIAPP ^+/+ As a positive control, effective disruption of PFKFB3 expression was confirmed (fig. 3B).

PFKFB3 ^βKO hIAPP ^+/- Analysis of metabolic performance in +HFD mice revealed that relative to PFKFB3 ^WT hIAPP ^+/- +hfd, lower fasting blood glucose levels (fig. 4A), increased insulin sensitivity (fig. 4D) and a comparable increase in glucose intolerance (fig. 4B). When combined with PFKFB3 ^WT hIAPP ^-/- PFKFB3 when compared to +hfd control ^βKO hIAPP ^+/- +HFD and PFKFB3 ^WT hIAPP ^+/- +hfd mice all had reduced C peptide levels (fig. 4C). These results, together with increased insulin sensitivity, indicate PFKFB3 ^βKO hIAPP ^+/- Insulin secretion in +hfd mice is impaired, probably due to failure of β -cell mass to expand under diabetogenic stress in the absence of PFKFB 3. However, the β -cell area fraction and mass were unchanged among the experimental groups (fig. 5B).

To investigate the final result in PFKFB3 ^βKO hIAPP ^+/- +HFD-and PFKFB3 ^WT hIAPP ^+/- Growth kinetics equivalent to beta cell mass between +HFD mice were performed TUNEL staining to determine beta cell death and MCM2 immunostaining to determine beta cell replication. According to TUNEL analysis, relative to PFKFB3 ^WT hIAPP ^-/- +HFD control, PFKFB3 ^βKO hIAPP ^+/- Increased beta cell death in +HFD mice, but not PFKFB3 ^WT hIAPP ^+/- +hfd mice were equivalent (fig. 5D). Cell death is mainly due to beta cells, because of the presence of PFKFB3 ^βKO hIAPP ^+/- +HFD-and PFKFB3 ^WT hIAPP ^+/- In +HFD mice, the β -/α cell ratio tended to decrease (FIG. 5C). From PFKFB3 ^βKO hIAPP ^+/- Beta cells of +hfd mice showed a three-fold increase in MCM2 labeling (.p)<0.05 Indicating that with hIAPP ^+/- Beta cell replication was increased compared to +HFD mice, and was similar to PFKFB3 ^WT IAPP ^-/- +hfd control (fig. 5A and 5E). Although in PFKFB3 ^βKO hIAPP ^+/- Cell death and beta cell replication were both increased in +hfd mice, but the beta cell area fraction was comparable for all three groups (fig. 5B).

To demonstrate that beta cell function failed to recover despite recovery of beta cell mass, in PFKFB3 ^βKO hIAPP ^+/- Hif1α immunostaining was performed in pancreatic sections of +hfd mice and it was found that about 10% of all β cells remained positive for hif1α, indicating that a fraction of hif1α positive but PFKFB3 negative β cells remained after PFKFB3 gene depletion (fig. 6A and 6B). Residual hif1α immunostaining suggests that sustained injury (stress) is responsible for the inability of β -cells to resume function. To determine whether there is sustained damage to beta cells, a marker of beta cell damage, the truncated c-Myc [14 ] called Myc-nick, is used ]. Although reduced by half, myc-Nick is expressed in PFKFB3 ^βKO hIAPP ^+/- Still persisted in +hfd mice (fig. 6C). Together with the sustained hif1α positive β cell fraction, myc-nick upregulation suggests that despite PFKFB3 knockout, a portion of PFKFB3 is present ^βKO The cells have not restored their complete function. This suggests that hif1α would play a role in β -cell loss of function even in the absence of PFKFB 3.

EXAMPLE 3 analysis of RNA-seq data from type 2 diabetes patients

To characterize the potential contribution of hif1α -positive β cells to loss of function, published RNA Seq data from human T2D was analyzed [1 ]]. Beta cells from healthy and T2D donors were reclustered (umap_cluster) and annotated as specific cell types based on genetic markers such as Insulin (INS) for beta cells (umap_celltype, fig. 7A). The beta cells are then separated into healthy and T2D conditioned beta cells (umap_disease, not shown). For each condition and each gene (e.g., LDHA as an example), cells were divided into gene (e.g., LDHA) positive and gene (e.g., LDHA) negative cells, and differential expression analysis was performed between the two groups. LDHA expression is used to distinguish healthy beta cells from stressed beta cells because LDHA is the transcriptional target of hif1α, which leads to the last step of aerobic glycolysis and metabolic remodeling of stressed beta cells. LDHA positive cells overlap with the beta cell subset marked by cluster 7 and are metabolized, ca ²⁺ Steady state and ion channels and insulin secretion regulation related genes co-aggregate (fig. 7A and 7B). These results clearly indicate that, similar to the diabetic mouse model, in humans with T2D, a fraction of beta cells (LDHA positive cells within cluster 7) show genetic features that can partially explain the loss of beta cell function.

Example 4-analysis of the role of HIF 1. Alpha. -PFKFB3 signaling in diabetic stress

Method

Animals

Homozygous hIAPP ^+/+ Mice were given from Peter Butler doctor laboratory and have been previously described (Janson et al, 1996). By hybridizing mice carrying the floxed Pfkfb3 gene (JAX laboratories) with mice expressing Cre recombinase under the control of the rat insulin promoter (RIP-CreERT), a beta cell-specific inducible Pfkfb3 knockout mouse model (RIP-CreERT: pfkfb3 was generated ^fl/fl ). Will be based on homozygous hIAPP ^+/+ Background mice and PFKFB3 ^fl/f Or RIP-Creet mice, followed by PFKFB3 ^fl/fl hIAPP ^+/- And PFKFB3 ^fl/fl RIP-CreERT mice were hybridized together to generate three experimental genotypes: RIP-CreERT PFKFB3 ^fl/fl hIAPP ^-/- 、RIP-CreERT PFKFB3 ^wt/wt hIAPP ^+/- And RIP-CreERT PFKFB3 ^fl/fl hIAPP ^+/- Herein referred to as PFKFB3, respectively ^WT hIAPP ^-/- 、PFKFB3 ^WT hIAPP ^+/- (PFKFB3 ^WT Diabetes stress, or PFKFB3 ^WT DS) and PFKFB3 ^βKO hIAPP ^+/- (PFKFB3 ^βKO DS). Cre-loxP recombination at the floxed site in Pfkfb3 was induced by intraperitoneal injection of tamoxifen at 20-27 weeks of age. Following tamoxifen injection, mice were given feed for 10 weeks and then a high fat diet for 13 weeks (HFD, research Diets Inc, new Brunswick, NJ, USA) to induce as p-hIAPP ^+/- And HFD responsive diabetes, because for hIAPP (hIAPP only ^+/+ ) Homozygous male mice spontaneously develop diabetes (Janson et al, 1996). Mice were kept on a 12 hour day/night cycle in a university of california, los angeles division institution animal care and use committee (UCLA Institutional Animal Care and Use Committee) (ARC) approved mouse community facility. At 30-37 weeks of age, all mice were allocated to receive a diet containing high fat (35% w/w or 60% calories from fat; D12492). The fat composition of the high fat diet was 32.2% saturated fat, 35.9% monounsaturated fat, and 31.9% polyunsaturated fat. During the duration of the study, the mice were allowed to eat and drink ad libitum. Body weight and fasting blood glucose levels were assessed weekly and additional measurements were made over several days, including glucose and insulin resistance tests (IP-GTT and ITT, respectively).

Insulin and glucose tolerance test

Glucose and insulin assays

Fasting blood glucose was measured weekly from blood drawn from the tail using a fresh glucometer (Abbott Diabetes Care Inc, alameda, CA, USA) after 18 hours of fasting (after periodic replacement of cages and pads and cessation of feeding while water was provided ad libitum). When blood glucose exceeded the detection range of the blood glucose meter, plasma glucose was measured using glucose oxidase and analyzed using YSI 2300STAT PLUS Glucose&L-Lactate analyzer.

Plasma insulin, C-peptide and glucagon levels were determined using Ultrasensitive ELISA for mouse insulin (Merodia 10-1247-01, uppsala, sweden), mouse C-peptide (Crystal Chem 90050, IL, USA) and mouse glucagon (Merodia 10-1281-01, uppsala, sweden).

Pancreatic perfusion and separation

One week after IP-GTT and ITT, mice were euthanized by cervical dislocation. Medial incision was used to open the abdominal and thoracic cavities while the right ventricle was incised and then the left ventricle was needled into to slowly infuse 10ml of cold Phosphate Buffered Saline (PBS) for pancreatic infusion. Following perfusion, the pancreas was placed in cold PBS and separated from other tissues including surrounding fat. The pancreas was then weighed after absorbing additional PBS with tissue.

Histological evaluation

After excision of the smaller fragments, the pancreas was fixed overnight in 4% paraformaldehyde (Electron Microscopy Sciences 19202, hatfield, pa, usa) at 4 ℃, paraffin embedded, and sectioned at 4 μm thickness. For the beta cell region, deparaffinized sections were subjected to peroxidase and hematoxylin staining, and these sections were sequentially stained with rabbit anti-insulin antibody (Cell Signaling Technology C C9, danvers, MA, USA, 1:400), followed by F (ab') ₂ Incubation with conjugates of biotin-SP (Jackson ImmunoResearch 711-066-152,West Grove,PA,USA,1:100 for IHC), after these steps, the VECTASTAIN ABC kit (HRP) (Vector Laboratories PK-4000, burlingame, CA, USA), DAB substrate kit (HRP) (Vector Laboratories SK-4100, burlingame, CA, USA) and Harris hematoxylin were applied before sealing with Permount (Fisher SP15-100, hampton, NH, USA). Morphometric analysis was performed on an Olympus IX70 inverted tissue culture microscope (Olympus, center Valley, PA, USA) using Image-Pro Plus 5.1 software. The genotype of the experimental mice for each slice was imaged and data analyzed blindly by two observers. Islet edges are defined manually using multi-channel images. The insulin and hematoxylin positive areas of each islet are determined using pixel thresholds. The beta cell area was then calculated as insulin positive area/hematoxylin positive area by 100%.

Immunofluorescence analysis was performed in Openlab 5.5.0 software on a Leica DM 6000B study microscope. The following antibodies were used: rabbit anti-PFKFB 3 (Abcam ab181861, cambridge, MA, USA, 1:100); mouse anti-MCM 2 (BD Transduction Laboratories 610700,San Diego,CA,USA,1:100); rabbit anti-lytic caspase-3 (Cell Signaling Technology 9664S, danvers, mass., USA, 1:400); guinea pig insulin resistance (Abcam ab195956, cambridge, mass., USA, 1:400); mouse anti-glucagon (Sigma-Aldrich G2654, st.Louis, MO, USA, 1:1000), mouse anti-c-Myc (Santa Cruz Biotechnology Inc E10 sc-40, dallas, texas, USA, 1:100); mice were resistant to HIF1α (Novus Biologicals NB-105, centennial, CO, USA, 1:50). The secondary antibodies were: f (ab') 2 conjugate with FITC donkey anti-guinea pig IgG (H+L) (Jackson ImmunoResearch 706-096-148,West Grove,PA,USA,1:200 for IF); f (ab') 2 conjugate with Cy3 donkey anti-rabbit IgG (H+L) (Jackson ImmunoResearch 711-166-152,West Grove,PA,USA,1:200 for IF); f (ab ') 2 conjugates with Cy3 donkey anti-mouse IgG (H+L) (Jackson ImmunoResearch 711-165-151,West Grove,PA,USA,1:200 for IF) and F (ab') 2 conjugates with Alexa 647 donkey anti-mouse IgG (H+L) (Jackson ImmunoResearch 715-606-150,West Grove,PA,USA,1:100 for IF). Cell death was determined by TUNEL assay using In Situ Cell Death Detection Kit (Roche Diagnostics Corporation 12156792910, indianapolis, in, usa). A Vectashield with DAPI (Vector Laboratories H, 1200, burlingame, CA, USA) patch was used.

Single cell RNA sequencing data analysis

The single cell RNA-seq dataset was obtained from GEO accession No. GSE124742, where healthy cells and type 2 diabetes cells were used. To identify the different cell types and to find the signature genes for each cell type, the expression matrix was analyzed using R-package setup (version 3.1.2). Cells with less than 100 genes and 500 UMIs detected were removed from further analysis. The semat function normazedata was used to normalize the raw counts. Variable region genes were identified using the findbariablegenes function. The expression assignment in the dataset is scaled and centered for dimension reduction using the setup ScaleData function. Default parameters are used in the aforementioned setup function. Principal Component Analysis (PCA) and Unified Manifold Approximation and Projection (UMAP) are used to reduce the dimensionality of the data, and the first two dimensions are used for mapping. The cells were later clustered using the findcclusts function. The marker genes for each cluster were determined using the findalmarkers function and then used to define cell types. Differential expression analysis was performed between two groups of cells using findmarks function. Wilcoxon rank sum test was performed in differential analysis and the error discovery rate was adjusted using the Benjamini-Hochberg program.

Statistical analysis

Data are shown as mean error (standard error, SEM) for the indicated number of mice. For IP-GTT and ITT, the area under the curve (AUC) for glucose, insulin, C-peptide and glucagon was calculated using the trapezoidal rule. Average data between groups were compared by analysis using student's t-test. P values less than 0.05 were considered significant.

Results

To study and dissect the role of PFKFB3 from hif1α in impaired beta cell survival under in vivo diabetic stress, mice were based on hIAPP ^+/- Background beta cell-specific conditional disruption of the Pfkfb3 gene was generated and exposed to a high fat diet for 13 weeks (Pfkfb 3 ^βKO hIAPP ^+/- +hfd). The diabetogenic stress is considered high, as it relates to insulin resistance (obesity) and via hIAPP ^+/- Expression of proteins exposed to misfolding, both of which are affected by high fat diets and the elderly, all of which together are known as cumulative risk factors for diabetes [16-18 ]]。

PFKFB3 ^fl/fl hIAPP ^+/- Mice were born at the expected mendelian ratio. There was no difference in body weight between the different experimental groups from one week before monitoring the mice until the end of the experiment (fig. 15A-15D). No difference in pancreas weight was observed, but with PFKFB3 ^WT hIAPP ^-/- PFKFB3 compared to +hfd control ^WT hIAPP ^+/- +HFD mice and PFKFB3 ^βKO hIAPP ^+/- Both the spleen and liver of +hfd mice showed less weight, but did not reach significant differences (fig. 16A-16C).

PFKFB3 ^βKO hIAPP ^+/- +HFD mice and PFKFB3 ^WT hIAPP ^+/- +HFD and PFKFB3 ^WT hIAPP ^-/- The +hfd control was compared. By using PFKFB3 ^WT hIAPP ^+/+ Pair PFKFB3 as positive control ^βKO hIAPP ^+/- Pancreatic sections of +hfd mice were PFKFB3 immunostained, confirming efficient disruption of PFKFB3 expression (fig. 8A). Diabetes stress leading to PFKFB3 ^WT hIAPP ^+/- 33.9+ -6.4% PFKFB3 immunolabeling of beta cells in +HFD mice (p < 0.05), similar to that previously reported for humans with T2D [18 ]]. From PFKFB3 ^WT PFKFB3 immunolabeling of beta cells of hIAPP-/- + HFD treated mice was 3.7±1.9% while in PFKFB3 ^βKO hIAPP ^+/- In +hfd mice, they were successfully eliminated and accounted for 1.0±0.8% (fig. 8A and 8B).

To determine if PFKFB3 in this model was associated with hif1α expression, pancreatic sections from all experimental groups were immunostained with hif1α antibodies. And PFKFB3 ^WT HIAPP-/- +HFD control from PFKFB3 ^WT hIAPP ^+/- HIF1α in beta cells of +HFD miceExpression increased to 18.4±4.2% (p)<0.05). In PFKFB3 ^βKO hIAPP ^+/- In +HFD mice, 14.2+ -3.8% of all beta cells (FIGS. 8C and 8D) continued to show HIF1α immunoposity cytoplasm and nucleus. This clearly shows that PFKFB3 knockout triggered compensatory hif1α expression in response to stress.

For PFKFB3 ^βKO hIAPP ^+/- Analysis of the metabolic performance of +hfd mice revealed that 9 weeks after the start of high fat diet (×p)<Glucose intolerance increased at both 0.05, n=4) and 12 weeks (fig. 9A-9D). Insulin resistance test shows that PFKFB3 ^βKO hIAPP ^+/- The insulin sensitivity of +hfd mice was higher and, although not significant, fasting blood glucose levels were lower, this difference gradually decreasing in the experimental group at a later time point. (FIGS. 9C and 9D). Plasma insulin levels reflect C peptide levels and are associated with PFKFB3 ^WT hIAPP ^-/- In PFKFB3 compared to +HFD control ^βKO hIAPP ^+/- +HFD and PFKFB3 ^WT hIAPP ^+/- Lower in +HFD (p < 0.05) (FIGS. 9G-2H). Interestingly, although PFKFB3 ^βKO hIAPP ^+/- +HFD and PFKFB3 ^WT hIAPP ^+/- Plasma insulin levels were lower in +HFD mice, but their later plasma glucagon levels were much higher than in PFKFB3 ^WT hIAPP ^+/- Compared with +HFD, PFKFB3 ^βKO hIAPP ^+/- +HFD exhibits a sharp decrease, and is associated with PFKFB3 ^WT hIAPP ^-/- The same levels seen in the +hfd control (fig. 9I). These results, together with increased insulin sensitivity, indicate PFKFB3 ^βKO hIAPP ^+/- Insulin secretion was impaired in +hfd mice. Next, the inventors interrogate PFKFB3 ^βKO hIAPP ^+/- Whether impaired insulin secretion in +HFD mice is due to failure of the beta cell mass to expand under diabetogenic stress in the absence of PFKFB 3.

Therefore, the beta cell area fraction and mass must be compared. The beta cell area fraction and mass were unchanged in the experimental group (fig. 10A and 10B). To investigate the final result in PFKFB3 ^βKO hIAPP ^+/- +HFD-and PFKFB3 ^WT hIAPP ^+/- Beta cell mass between +HFD miceComparable growth kinetics TUNEL staining was performed as a measure of past cell death (fig. 10C) and lytic caspase 3 immunostaining as a measure of active beta cell death (fig. 10E and 10F). And PFKFB3 ^WT hIAPP ^+/- Compared with +HFD mice, PFKFB3 ^βKO hIAPP ^+/- Beta cell death was increased in +hfd mice (fig. 10C). Surprisingly, relative to PFKFB3 ^WT hIAPP ^+/- +HFD mice, PFKFB3 ^βKO hIAPP ^+/- The beta-/alpha cell ratio in +HFD-was increased (FIG. 10D). No difference in active cell death was observed based on the lytic caspase 3 immunostaining of all experimental groups of mice. Thus, compared to the other groups, from PFKFB3 ^βKO hIAPP ^+/- Beta cells of +HFD mice are characterized by increased cell death in the past, while ongoing cell death is not differentiated.

To further elucidate whether the increase in beta-/alpha cell ratio is dependent on an increase in beta cell production, immunolabeling with early replication initiation marker minichromosome maintenance protein 2 (MCM 2) [19,20 ]]. The results showed that from PFKFB3 ^βKO hIAPP ^+/- Beta cells of +hfd mice showed a three-fold increase (5.3% ± 0.8%,. P) in MCM2 labeling<0.05 Indicating that with PFKFB3 ^WT hIAPP ^+/- Beta cell replication was increased (1.9.+ -. 0.04%) and was similar to PFKFB3 in +HFD mice ^WT hIAPP ^-/- +hfd control (7.0±1.3%, fig. 11A and 11B). Although in PFKFB3 ^βKO hIAPP ^+/- Cell death and beta cell replication were both increased in +hfd mice, but the beta cell area fraction was comparable for all three groups (fig. 10A). Thus, PFKFB3 ^βKO hIAPP ^+/- The increase in beta cell replication in +hfd mice appears to maintain beta cell mass despite cell death and an increase in initial loss of damaged beta cells (measured by TUNEL assay) in the absence of PFKFB3 protection and pro-survival.

To elucidate whether replicating beta cells have any residual damage, the inventors utilized the specific marker of beta cell damage caused by hIAPP-calpain mediated cytoplasmic accumulation of protein c-Myc truncation, termed Myc-nick [14]. c-Myc staining analysisRevealing that PFKFB3 under diabetes mellitus stress ^WT hIAPP ^+/- In Myc-nick expression was increased (3.3.+ -. 0.5%,. P)<0.05 But in PFKFB3 ^βKO hIAPP ^+/- In +HFD mice, reversion to PFKFB3 ^WT hIAPP ^-/- +HFD control mice levels (0.8+ -0.4% and 0.7+ -0.6%, respectively, FIGS. 11C and 11D). These results indicate that healthy β -cells contribute to increased replication, possibly after reduced misfolding stress of hIAPP protein.

PFKFB3 ^βKO hIAPP ^+/- Hif1α immunostaining in pancreatic sections of +hfd affected approximately 14% of all β cells, and although it was consistent with a decrease in misfolded protein stress as measured by cytoplasmic C-Myc, this may indicate the cause of unrecoverable metabolic function in replicating β cells (fig. 8C and 8D). To characterize the potential contribution of these hif1α -positive β cells to loss of function, published single cell RNA Seq (scRNA Seq) data from humans with T2D were used to compare to non-diabetic patients [1 ]. First, the inventors analyzed the quality and validated the scRNA Seq data (FIGS. 11A-12D). Pancreatic cells from healthy and T2D donors were reclustered (umap_cluster) and annotated to specific cell types (umap_celltype, fig. 12A-12D) based on genetic markers such as insulin for beta cells (INS). Nine different cell types were identified and their differential gene expression is depicted in figures 12C, 20 and 21. Two beta 0 cell clusters, cluster 1 and cluster 7, were identified using INS as beta cell markers, while clusters 2-6, 8 and 9 refer to other pancreatic cell types (fig. 12B and 12C). The composition in clusters 6 (alpha cell subpopulations), 7 (beta cell subpopulations) and 8 (delta cells) differed most between healthy and T2D donors (fig. 19). Since hif1α is regulated primarily in a posttranslational manner, β cells were also differentiated based on the expression or absence of lactate dehydrogenase a (LDHA), a hif1α transcription target from aerobic glycolysis (fig. 12D). For each case and based on LDHA expression, cells were divided into LDHA positive and LDHA negative cells and differential expression analysis was performed between the two groups. LDHA positive beta cells overlap with the beta cell subpopulation depicted by cluster 7 and are not associated with disease states, but are associated with the following genes: genes associated with metabolism, such as LDHA, aromatic nucleus Translocator 2 (ARNT 2, i.e. hif1α), glucokinase (GK), phosphofructokinase 1 platelets (PFKFP), pyruvate dehydrogenase kinase 4 (PDK 4), or genes related to insulin secretion, such as FBP1 via phosphoenolpyruvate pool, or genes related to identity, such as glucagon (GCG, progenitor α cell identity) and awnless associated homeobox (Aristaless Related Homeobox) (ARX, progenitor α cell identity) and INS (lower expression, α0 cell identity). LDHA positive or clustered 7β cells show an immature phenotype, consistent with up-regulation of genes such as aldehyde dehydrogenase 1A1 (ALDH 1 A1). These results indicate that in humans with T2D, a fraction of beta cells (LDHA positive cells overlapping with cluster 7 beta cells) have genetic characteristics of reduced INS expression and increased GCG and ARX expression. Ingenuity Pathway Analysis (IPA) enrichment data revealed that the differences between the significantly altered genes in cluster 1 and cluster 7, and between LDHA positive and negative cells, are summarized by LXR/RXR retinoid receptors, suggesting that this upstream regulator is part of the upper HIF1α -PFKFB3 non-classical metabolic pathway. Furthermore, string analysis clearly shows that while the differences between cluster 1 and 7 and LDHA negative and positive beta cells remain intact in the healthy state, these differences are greatly reduced in T2D. These data indicate that in T2D, β -cell clusters started to be similar to each other and the differences decreased under stress (fig. 22A-23B). Genes differentially expressed in cluster 1 or LDHA negative beta cells showed significant overlap when compared between T2D and healthy individuals.

To find the complement of the β -cell population in cluster 7 or LDHA positive cells, the inventors double stained pancreatic sections from the experimental mouse group with specific insulin and glucagon antibodies. And PFKFB3 ^WT hIAPP ^-/- Diabetic stress caused PFKFB3 compared to +hfd control ^WT hIAPP ^+/- The number of double positive cells in +hfd increased by a factor of two (5.7±2.8% relative to 2.7±1.8%, respectively). When combined with PFKFB3 ^WT PFKFB3 knockout resulted in a decrease in cells accompanying insulin and glucagon immunopositions when DS was compared (0.8±0.3% versus 5.7±2.8%, fig. 13A). After elimination of PFKFB 3-positive damaged beta cells, the bishormone (insulin ⁺ Pancreatic hypertensionSugar element ⁺ ) Not only is part of the cell eliminated, but it is also associated with a significant increase in beta cells (.p)<0.05). This suggests that PFKFB3 disruption may result in specific depletion of beta cells with dual insulin and glucagon identities and/or stimulation of replication by insulin-positive beta cells alone. The situation was exactly reversed for alpha cells relative to all alpha cells, beta cells and bi-hormonal cells together (fig. 13B). And PFKFB3 ^WT hIAPP ^+/- The ratio reached the control level and was found to be in PFKFB3 in comparison to +hfd mice ^βKO hIAPP ^+/- Reduction in +HFD (.p)<0.05). Double insulin and glucagon stained cells were clearly present in experimental mice exposed to high fat diet, but not in WT control or hIAPP ^+/+ Is detected-whether in prediabetes or diabetic stages (fig. 13C and 13D).

Thus, PFKFB3 knockout resulted in the disappearance of β cells with dual insulin and glucagon identities, suggesting that these cells are not only dependent on surviving PFKFB 3-mediated hIAPP damage, but also by dual identity adaptation to surviving cells, both likely contributing to their impaired function.

Interestingly, these data indicate that PFKFB3 ^βKO hIAPP ^+/- Hif1α positive cells in +hfd do not have dual β -and α -identities, indicating that survival of cells with dual β -and α -identities is dependent on PFKFB3 instead of hif1α.

These analyses also indicate that while beta cell mass is restored after PFKFB3 gene depletion, the accumulation of hif1α -positive cells in the diabetic mouse model may be responsible for the loss of beta cell function. PFKFB3 depletion appears to trigger the rejection of damaged beta cells with beta and alpha-cell identity, which is sufficient and necessary for the supplementation of healthy beta cells by replication. However, β -cell insulin secretion is further compromised by independent immune labeling of hif1α.

These studies strongly suggest that targeting (i.e., inhibiting) hif1α, with or without co-targeting PFKFB3, will result in the restoration of a functional beta cell population and reversal of diabetes.

Discussion of the invention

In these studies, the inventors demonstrated that specific beta cell disruption of the Pfkfb3 gene in adult mice resulted in partial islet regeneration under high diabetogenic stress. This is achieved by rejecting damaged and di-hormonal (insulin and glucagon positive) cells and replicating the remaining healthy beta cells.

Rejection may affect a large number of beta cells. The significance of PFKFB3 in remodelling metabolism explains its effect on survival [13]However, problems associated with preservation of beta cell function have also been raised. Thus, metabolic remodeling through the HIF1α -PFKFB3 pathway in misfolded protein stress (hIAPP) summarizes the consequences of HIF1α expression following conditional inactivation of the von Hippel Lindau gene (Vhl) [13,31 ]]. HIF 1. Alpha. Activation in the presence or absence of diabetes-induced stress results in cytoplasmic Ca ²⁺ Glucose-stimulated changes in concentration, electrical activity and insulin secretion are reduced, ultimately leading to impaired systemic glucose tolerance of pancreatic beta cells [13,31]。

HIF1α is derived from PFKFB3 ^βKO Continuous expression (-14%) in a large number of beta cells in DS mice. This suggests that in this diabetic mouse model, the associated hif1α response is not associated with PFKFB 3. PFKFB3 compared to the WT control ^βKO DS and PFKFB3 ^WT Hif1α expression levels (14% and 18%, respectively) in DS mice were consistent with glucose intolerance and lower plasma insulin and C peptide levels (p < 0.05).

To investigate the role of hif1α in the molecular basis of β -cell dysfunction, sc RNA Seq data from people with obese T2D and non-diabetic (ND) were analyzed [27]. The inventors utilized the distinction of LDHA positive beta cells relative to LDHA negative beta cells because LDHA is a true target and thus serves as a surrogate marker α for HIF 1. Hif1α is regulated primarily posttranslationally, with no change in transcript levels. LDHA positive beta cells overlap with the cluster 7β cell subpopulations and are represented by HIF1a- (ARNT 2, GK, PFKFP, PDK) and the characteristics of the bishormonal (a-cell and beta-cell identities) (GCG, ARX and INS) and some immature markers (ALDH 1 A1). Double insulin and glucagon positive cells in the mouse model resemble LDHA positive or clustered 7β cells and may be derived from dedifferentiated or transdifferentiated β cells [32].

Ingenuity Pathway Analysis (IPA) was used to make a comparison between LDHA-positive (Cluster 7) and LDHA-negative (Cluster 1) beta cells in healthy and T2D, indicating the presence of a major upstream regulator, the liver X receptor, a retinoid receptor (LXR/RXR) [33], possibly part of the up-regulated HIF1α metabolic pathway. Activation of this receptor may increase aerobic glycolysis independently via transcriptional upregulation of hexokinase 1 and 3 (HK 1 and 3) and SLC2A1 (GLUT 1) and interaction with hif1α in response to a high fat diet [34].

Interestingly, the subpopulation of di-hormonal beta cells was present in obese non-diabetic patients and WT controls under HFD, suggesting that the formation of di-hormonal cells is likely to be an adaptive response to increased adipogenesis and high fat diet. Thus, in the absence of HFD (used as a negative control) in WT control or hIAPP ^+/+ Dual insulin and glucagon positive cells were not detected, whether in pre-diabetes or diabetic stages. The dual hormone beta cell cluster 7 (LDHA positive beta cells) can be distinguished from cluster 1 by the effect of LXR/RXR, both in healthy state and in T2D. Previous reports indicate that unlike acute activation of adaptive responses to increased insulin secretion demands, chronic activation of LXR may lead to beta cell dysfunction through accumulation of free fatty acids and triglycerides [33]. Furthermore, sting analysis showed that while the differences between cluster 1 and 7 and LDHA negative and positive beta cells remained intact in the healthy state, these differences were reduced in T2D. The retention characteristics of the bi-hormonal cells may be relevant in the context of cell adaptive competition, where we believe they may underlie the recognition and homeostatic control of the bi-hormonal cells.

Cell adaptive competition is an important external cell quality control based on the differentiation of cell populations with poorly adapted (survival) characteristics relative to cell populations with well adapted (survival) characteristics. This distinction is critical to triggering the selective elimination of cell populations with poorly adapted characteristics ("losers") and to driving the expansion of cell populations with well adapted characteristics ("winners"). Replacement of the "loser" with the "winner" allows maintenance of steady state organization. In a sense, reversal of cell-competing tissue organization (cluster 1 and cluster 7 similarity), as we see in T2D, may result in inhibition of cell competition, followed by tissue dysfunction over time.

In the diabetic mouse model, PFKFB3 knockout in adult beta cells resulted in a large decrease in injured beta cells and bi-hormonal (insulin and glucagon positive) cells and a concomitant increase in healthy beta cell replication. The inventors monitored damaged beta cells by measuring the extent of calpain (hIAPP) -mediated cytoplasmic c-Myc truncation [26 ]]. It has been previously reported that calpain can directly reflect the toxicity of the hIAPP misfolded protein [35,36 ]]. In PFKFB3 ^βKO In DS mice, cytoplasmic c-Myc was reversed to almost undetectable levels (0.8.+ -. 0.4% and 0.7.+ -. 0.6%, respectively) as measured in the WT control.

These results indicate that the increase in replication is contributed by healthy beta cells, and it is conceivable that with continued calpain activation and thus hIAPP damage, a massive loss of beta cells promotes an increase in replication. Thus, the results indicate that cell-competition-dependent beta cell regeneration can be performed by rejecting damaged beta cells after PFKFB3 knockout. Thus, further in PFKFB3 ^βKO In DS mice, with PFKFB3 ^WT An increase in the beta/a ratio and a decrease in glucagon levels compared to DS may be responsible for the observed trend of higher insulin sensitivity [37-39 ]]。

In summary, PFKFB3 ^βKO Preservation of beta cell mass and increase in beta-/a ratio in DS mice originate cumulatively from beta cell replication, which overcomes the initial loss of damaged beta cells and the reduction of bi-hormonal cells.

Although regenerative growth under diabetogenic stress is largely dependent on PFKFB3, the mismatched metabolic function may account for the part of hif1α positive cells that are independent of PFKFB 3. Thus, hif1α may be responsible for the loss of β -cell function in a diabetic mouse model, despite the restoration of β -cell mass after PFKFB3 gene depletion.

These studies strongly suggest that targeting hif1α would result in restoration of functional beta cell mass and reversal of diabetes with/without co-targeting PFKFB 3.

Example 5-Generation of a model of action for comparison of beta cell fitness in beta cell supplementation under stress

Based on the studies described in examples 1-4, a model was generated for the comparison of beta cell fitness in beta cell replication under stress. Fig. 25 shows a schematic diagram of the generated model. The upper panel shows how suboptimal cells (dark) are eliminated from tissue by competition with healthy beta-cells (light) (replicating them to regenerate lost tissue) after metabolic stress such as High Fat Diet (HFD) in healthy non-diabetic patients. The middle panel shows how damaged suboptimal β -cells (dark) survive with reduced fitness in cell competition for sustained damage (T1D and T2D) and cannot be cleared from tissue due to metabolic remodeling by hif1α -PFKFB3 pathway. These damaged beta cells may prevent the replenishment (replication) of healthy beta cells. The bottom panel shows how targeting of pro-survival PFKFB3 and/or hif1α pathways results in activation of cell competition and elimination of suboptimal (damaged) β -cells (dark) under stress and injury conditions. Elimination of suboptimal (damaged) beta cells results in replication of remaining healthy beta cells (MCM 2 positive cells).

***

In accordance with the present disclosure, all methods disclosed and claimed herein can be performed and executed without undue experimentation. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Farber, J.L., the role of calcium ions in toxic cell injury.Environ Health Perspin, 1990.84, pages 107-11.

Conacci-Sorrell, M., C.Ngouenet and R.N.Eisenman, myc-nick: acytoplasmic cleavage product of Myc that promotes alpha-tubulin acetylation and cell differentiation. Cell,2010.142 (3): pages 480-93.

15.Janson J,Soeller WC,Roche PC et al Spontaneous diabetes mellitus in transgenic mice expressing human islet amyloid polypeptide. Proc Natl Acad Sci U S A.1996.93 (14): pages 7283-7288.

Bellou, V.et al Risk factors for type 2diabetes mellitus:An exposure-wide umbrella review of meta-analysis, PLoS One,2018.13 (3): page e 0194127.

Fletcher, B., M.Gulanick and C.Lamendola, risk factors for type 2diabetes mellitus.J Cardiovasc Nurs,2002.16 (2): pages 17-23.

Wu, Y et al Risk factors contributing to type 2diabetes and recent advances in the treatment and prevention.Int J Med Sci,2014.11 (11): pages 1185-200.

Bleichert, F., mechanisms ofreplication origin licensing: a structural hyperspective. Curr Opin structural Biol,2019.59: pages 195-204.

Shetty, A. Et al DNA replication licensing and cell cycle kinetics of normal and neoplastic break.Br J Cancer 2005.93 (11): pages 1295-300.

Ortiz-Barahona, A. Et al Genome-wide identification of hypoxia-inducible factor binding sites and target genes by a probabilistic model integrating transcription-profiling data and in silico binding site predictions nucleic Acids Res 2010.38 (7): pages 2332-45.

Valvona, C.J. et al The Regulation and Function of Lactate Dehydrogenase A: therapeutic Potential in Brain Tumor. Brain Pathol,2016.26 (1): pages 3-17.

Zehetner, J. Et al PVHL is a regulator of glucose metabolism and insulin secretion in pancreatic beta cells. Genes Dev,2008.22 (22): pages 3135-46.

Schuis, F.C. et al Glucose sensing in pancreatic beta-cells a model for the study ofother glucose-regulated cells in gut, pancrees, and hypothamus diabetes 2001.50 (1): pages 1-11.

Adeva-Andany, M.M. et al Metabolic effects of glucagon in humans.JClin Transl Endocrinol,2019.15, pages 45-53.

Faerch, K et al Insulin Resistance Is Accompanied by Increased Fasting Glucagon and Delayed Glucagon Suppression in Individuals With Normal and Impaired Glucose regulation.diabetes,2016.65 (11): pages 3473-3481.

Ahren, B. And O.Thorsson, increased insulin sensitivity is associated with reduced insulin and glucagon secretion and increased insulin clearance in man J Clin Endocrinol Metab,2003.88 (3): pages 1264-70.

Choe, S.S. et al Chronic activation of liver X receptor induces beta-cell apoptosis through hyperactivation of lipogenesis:river X receptor-mediated lipotoxicity in pancreatic beta-cells. Diabetes,2007.56 (6): pages 1534-43.

Miyazaki, S.et al Nuclear hormone Retinoid X Receptor (RXR) negatively regulates the glucose-stimulated insulin secretion of pancreatic ss-cells, diabetes,2010.59 (11): pages 2854-61.

30.Dusachalcy, R.et al, high-fat diet impacts more changes in beta-cell compared to alpha-cell trans-script.PLoS One,2019.14 (3): page e 0213299.

Talchai, C. Et al Pancreatic beta cell dedifferentiation as a mechanism of diabetic beta cell failure. Cell,2012.150 (6): pages 1223-34.

Coelho, D.S. et al Culling Less Fit Neurons Protects against Amyloid-beta-Induced Brain Damage and Cognitive and Motor decline. Cell Rep,2018.25 (13): pages 3661-3673, e3.

Sequence listing

<110> board of university of california (The Regents of the University of California)

<120> for diabetes treatment and beta

Methods and compositions for cell regeneration

<130> UCLA.P0116WO (1001177507)

<140>

<141> 2021-08-17

<150> 63/067,187

<151> 2020-08-18

<150> 63/169,776

<151> 2021-04-01

<160> 4

<170> patent in version 3.5

<210> 1

<211> 3946

<212> DNA

<213> Chile person

<400> 1

agtgcacagt gctgcctcgt ctgaggggac aggaggatca ccctcttcgt cgcttcggcc 60

agtgtgtcgg gctgggccct gacaagccac ctgaggagag gctcggagcc gggcccggac 120

cccggcgatt gccgcccgct tctctctagt ctcacgaggg gtttcccgcc tcgcaccccc 180

acctctggac ttgcctttcc ttctcttctc cgcgtgtgga gggagccagc gcttaggccg 240

gagcgagcct gggggccgcc cgccgtgaag acatcgcggg gaccgattca ccatggaggg 300

cgccggcggc gcgaacgaca agaaaaagat aagttctgaa cgtcgaaaag aaaagtctcg 360

agatgcagcc agatctcggc gaagtaaaga atctgaagtt ttttatgagc ttgctcatca 420

gttgccactt ccacataatg tgagttcgca tcttgataag gcctctgtga tgaggcttac 480

catcagctat ttgcgtgtga ggaaacttct ggatgctggt gatttggata ttgaagatga 540

catgaaagca cagatgaatt gcttttattt gaaagccttg gatggttttg ttatggttct 600

cacagatgat ggtgacatga tttacatttc tgataatgtg aacaaataca tgggattaac 660

tcagtttgaa ctaactggac acagtgtgtt tgattttact catccatgtg accatgagga 720

aatgagagaa atgcttacac acagaaatgg ccttgtgaaa aagggtaaag aacaaaacac 780

acagcgaagc ttttttctca gaatgaagtg taccctaact agccgaggaa gaactatgaa 840

cataaagtct gcaacatgga aggtattgca ctgcacaggc cacattcacg tatatgatac 900

caacagtaac caacctcagt gtgggtataa gaaaccacct atgacctgct tggtgctgat 960

ttgtgaaccc attcctcacc catcaaatat tgaaattcct ttagatagca agactttcct 1020

cagtcgacac agcctggata tgaaattttc ttattgtgat gaaagaatta ccgaattgat 1080

gggatatgag ccagaagaac ttttaggccg ctcaatttat gaatattatc atgctttgga 1140

ctctgatcat ctgaccaaaa ctcatcatga tatgtttact aaaggacaag tcaccacagg 1200

acagtacagg atgcttgcca aaagaggtgg atatgtctgg gttgaaactc aagcaactgt 1260

catatataac accaagaatt ctcaaccaca gtgcattgta tgtgtgaatt acgttgtgag 1320

tggtattatt cagcacgact tgattttctc ccttcaacaa acagaatgtg tccttaaacc 1380

ggttgaatct tcagatatga aaatgactca gctattcacc aaagttgaat cagaagatac 1440

aagtagcctc tttgacaaac ttaagaagga acctgatgct ttaactttgc tggccccagc 1500

cgctggagac acaatcatat ctttagattt tggcagcaac gacacagaaa ctgatgacca 1560

gcaacttgag gaagtaccat tatataatga tgtaatgctc ccctcaccca acgaaaaatt 1620

acagaatata aatttggcaa tgtctccatt acccaccgct gaaacgccaa agccacttcg 1680

aagtagtgct gaccctgcac tcaatcaaga agttgcatta aaattagaac caaatccaga 1740

gtcactggaa ctttctttta ccatgcccca gattcaggat cagacaccta gtccttccga 1800

tggaagcact agacaaagtt cacctgagcc taatagtccc agtgaatatt gtttttatgt 1860

ggatagtgat atggtcaatg aattcaagtt ggaattggta gaaaaacttt ttgctgaaga 1920

cacagaagca aagaacccat tttctactca ggacacagat ttagacttgg agatgttagc 1980

tccctatatc ccaatggatg atgacttcca gttacgttcc ttcgatcagt tgtcaccatt 2040

agaaagcagt tccgcaagcc ctgaaagcgc aagtcctcaa agcacagtta cagtattcca 2100

gcagactcaa atacaagaac ctactgctaa tgccaccact accactgcca ccactgatga 2160

attaaaaaca gtgacaaaag accgtatgga agacattaaa atattgattg catctccatc 2220

tcctacccac atacataaag aaactactag tgccacatca tcaccatata gagatactca 2280

aagtcggaca gcctcaccaa acagagcagg aaaaggagtc atagaacaga cagaaaaatc 2340

tcatccaaga agccctaacg tgttatctgt cgctttgagt caaagaacta cagttcctga 2400

ggaagaacta aatccaaaga tactagcttt gcagaatgct cagagaaagc gaaaaatgga 2460

acatgatggt tcactttttc aagcagtagg aattggaaca ttattacagc agccagacga 2520

tcatgcagct actacatcac tttcttggaa acgtgtaaaa ggatgcaaat ctagtgaaca 2580

gaatggaatg gagcaaaaga caattatttt aataccctct gatttagcat gtagactgct 2640

ggggcaatca atggatgaaa gtggattacc acagctgacc agttatgatt gtgaagttaa 2700

tgctcctata caaggcagca gaaacctact gcagggtgaa gaattactca gagctttgga 2760

tcaagttaac tgagcttttt cttaatttca ttcctttttt tggacactgg tggctcatta 2820

cctaaagcag tctatttata ttttctacat ctaattttag aagcctggct acaatactgc 2880

acaaacttgg ttagttcaat tttgatcccc tttctactta atttacatta atgctctttt 2940

ttagtatgtt ctttaatgct ggatcacaga cagctcattt tctcagtttt ttggtattta 3000

aaccattgca ttgcagtagc atcattttaa aaaatgcacc tttttattta tttatttttg 3060

gctagggagt ttatcccttt ttcgaattat ttttaagaag atgccaatat aatttttgta 3120

agaaggcagt aacctttcat catgatcata ggcagttgaa aaatttttac accttttttt 3180

tcacatttta cataaataat aatgctttgc cagcagtacg tggtagccac aattgcacaa 3240

tatattttct taaaaaatac cagcagttac tcatggaata tattctgcgt ttataaaact 3300

agtttttaag aagaaatttt ttttggccta tgaaattgtt aaacctggaa catgacattg 3360

ttaatcatat aataatgatt cttaaatgct gtatggttta ttatttaaat gggtaaagcc 3420

atttacataa tatagaaaga tatgcatata tctagaaggt atgtggcatt tatttggata 3480

aaattctcaa ttcagagaaa tcatctgatg tttctatagt cactttgcca gctcaaaaga 3540

aaacaatacc ctatgtagtt gtggaagttt atgctaatat tgtgtaactg atattaaacc 3600

taaatgttct gcctaccctg ttggtataaa gatattttga gcagactgta aacaagaaaa 3660

aaaaaatcat gcattcttag caaaattgcc tagtatgtta atttgctcaa aatacaatgt 3720

ttgattttat gcactttgtc gctattaaca tccttttttt catgtagatt tcaataattg 3780

agtaatttta gaagcattat tttaggaata tatagttgtc acagtaaata tcttgttttt 3840

tctatgtaca ttgtacaaat ttttcattcc ttttgctctt tgtggttgga tctaacacta 3900

actgtattgt tttgttacat caaataaaca tcttctgtgg accagg 3946

<210> 2

<211> 826

<212> PRT

<213> Chile person

<400> 2

Met Glu Gly Ala Gly Gly Ala Asn Asp Lys Lys Lys Ile Ser Ser Glu

1 5 10 15

Arg Arg Lys Glu Lys Ser Arg Asp Ala Ala Arg Ser Arg Arg Ser Lys

20 25 30

Glu Ser Glu Val Phe Tyr Glu Leu Ala His Gln Leu Pro Leu Pro His

35 40 45

Asn Val Ser Ser His Leu Asp Lys Ala Ser Val Met Arg Leu Thr Ile

50 55 60

Ser Tyr Leu Arg Val Arg Lys Leu Leu Asp Ala Gly Asp Leu Asp Ile

65 70 75 80

Glu Asp Asp Met Lys Ala Gln Met Asn Cys Phe Tyr Leu Lys Ala Leu

85 90 95

Asp Gly Phe Val Met Val Leu Thr Asp Asp Gly Asp Met Ile Tyr Ile

100 105 110

Ser Asp Asn Val Asn Lys Tyr Met Gly Leu Thr Gln Phe Glu Leu Thr

115 120 125

Gly His Ser Val Phe Asp Phe Thr His Pro Cys Asp His Glu Glu Met

130 135 140

Arg Glu Met Leu Thr His Arg Asn Gly Leu Val Lys Lys Gly Lys Glu

145 150 155 160

Gln Asn Thr Gln Arg Ser Phe Phe Leu Arg Met Lys Cys Thr Leu Thr

165 170 175

Ser Arg Gly Arg Thr Met Asn Ile Lys Ser Ala Thr Trp Lys Val Leu

180 185 190

His Cys Thr Gly His Ile His Val Tyr Asp Thr Asn Ser Asn Gln Pro

195 200 205

Gln Cys Gly Tyr Lys Lys Pro Pro Met Thr Cys Leu Val Leu Ile Cys

210 215 220

Glu Pro Ile Pro His Pro Ser Asn Ile Glu Ile Pro Leu Asp Ser Lys

225 230 235 240

Thr Phe Leu Ser Arg His Ser Leu Asp Met Lys Phe Ser Tyr Cys Asp

245 250 255

Glu Arg Ile Thr Glu Leu Met Gly Tyr Glu Pro Glu Glu Leu Leu Gly

260 265 270

Arg Ser Ile Tyr Glu Tyr Tyr His Ala Leu Asp Ser Asp His Leu Thr

275 280 285

Lys Thr His His Asp Met Phe Thr Lys Gly Gln Val Thr Thr Gly Gln

290 295 300

Tyr Arg Met Leu Ala Lys Arg Gly Gly Tyr Val Trp Val Glu Thr Gln

305 310 315 320

Ala Thr Val Ile Tyr Asn Thr Lys Asn Ser Gln Pro Gln Cys Ile Val

325 330 335

Cys Val Asn Tyr Val Val Ser Gly Ile Ile Gln His Asp Leu Ile Phe

340 345 350

Ser Leu Gln Gln Thr Glu Cys Val Leu Lys Pro Val Glu Ser Ser Asp

355 360 365

Met Lys Met Thr Gln Leu Phe Thr Lys Val Glu Ser Glu Asp Thr Ser

370 375 380

Ser Leu Phe Asp Lys Leu Lys Lys Glu Pro Asp Ala Leu Thr Leu Leu

385 390 395 400

Ala Pro Ala Ala Gly Asp Thr Ile Ile Ser Leu Asp Phe Gly Ser Asn

405 410 415

Asp Thr Glu Thr Asp Asp Gln Gln Leu Glu Glu Val Pro Leu Tyr Asn

420 425 430

Asp Val Met Leu Pro Ser Pro Asn Glu Lys Leu Gln Asn Ile Asn Leu

435 440 445

Ala Met Ser Pro Leu Pro Thr Ala Glu Thr Pro Lys Pro Leu Arg Ser

450 455 460

Ser Ala Asp Pro Ala Leu Asn Gln Glu Val Ala Leu Lys Leu Glu Pro

465 470 475 480

Asn Pro Glu Ser Leu Glu Leu Ser Phe Thr Met Pro Gln Ile Gln Asp

485 490 495

Gln Thr Pro Ser Pro Ser Asp Gly Ser Thr Arg Gln Ser Ser Pro Glu

500 505 510

Pro Asn Ser Pro Ser Glu Tyr Cys Phe Tyr Val Asp Ser Asp Met Val

515 520 525

Asn Glu Phe Lys Leu Glu Leu Val Glu Lys Leu Phe Ala Glu Asp Thr

530 535 540

Glu Ala Lys Asn Pro Phe Ser Thr Gln Asp Thr Asp Leu Asp Leu Glu

545 550 555 560

Met Leu Ala Pro Tyr Ile Pro Met Asp Asp Asp Phe Gln Leu Arg Ser

565 570 575

Phe Asp Gln Leu Ser Pro Leu Glu Ser Ser Ser Ala Ser Pro Glu Ser

580 585 590

Ala Ser Pro Gln Ser Thr Val Thr Val Phe Gln Gln Thr Gln Ile Gln

595 600 605

Glu Pro Thr Ala Asn Ala Thr Thr Thr Thr Ala Thr Thr Asp Glu Leu

610 615 620

Lys Thr Val Thr Lys Asp Arg Met Glu Asp Ile Lys Ile Leu Ile Ala

625 630 635 640

Ser Pro Ser Pro Thr His Ile His Lys Glu Thr Thr Ser Ala Thr Ser

645 650 655

Ser Pro Tyr Arg Asp Thr Gln Ser Arg Thr Ala Ser Pro Asn Arg Ala

660 665 670

Gly Lys Gly Val Ile Glu Gln Thr Glu Lys Ser His Pro Arg Ser Pro

675 680 685

Asn Val Leu Ser Val Ala Leu Ser Gln Arg Thr Thr Val Pro Glu Glu

690 695 700

Glu Leu Asn Pro Lys Ile Leu Ala Leu Gln Asn Ala Gln Arg Lys Arg

705 710 715 720

Lys Met Glu His Asp Gly Ser Leu Phe Gln Ala Val Gly Ile Gly Thr

725 730 735

Leu Leu Gln Gln Pro Asp Asp His Ala Ala Thr Thr Ser Leu Ser Trp

740 745 750

Lys Arg Val Lys Gly Cys Lys Ser Ser Glu Gln Asn Gly Met Glu Gln

755 760 765

Lys Thr Ile Ile Leu Ile Pro Ser Asp Leu Ala Cys Arg Leu Leu Gly

770 775 780

Gln Ser Met Asp Glu Ser Gly Leu Pro Gln Leu Thr Ser Tyr Asp Cys

785 790 795 800

Glu Val Asn Ala Pro Ile Gln Gly Ser Arg Asn Leu Leu Gln Gly Glu

805 810 815

Glu Leu Leu Arg Ala Leu Asp Gln Val Asn

820 825

<210> 3

<211> 4226

<212> DNA

<213> Chile person

<400> 3

ccctttcccc tccctcgccc gccccgccgc ccgcaggcgc cccgagtcgc ggggctgccg 60

cttggacgtc gtcctgtctg ggtgtcgcgg gccggccccg cggggagcgc ccccggcgcg 120

atgcccttca ggaaagcctg tgggccaaag ctgaccaact cccccaccgt catcgtcatg 180

gtgggcctcc ccgcccgggg caagacctac atctccaaga agctgactcg ctacctcaac 240

tggattggcg tccccacaaa agtgttcaac gtcggggagt atcgccggga ggctgtgaag 300

cagtacagct cctacaactt cttccgcccc gacaatgagg aagccatgaa agtccggaag 360

caatgtgcct tagctgcctt gagagatgtc aaaagctacc tggcgaaaga agggggacaa 420

attgcggttt tcgatgccac caatactact agagagagga gacacatgat ccttcatttt 480

gccaaagaaa atgactttaa ggcgtttttc atcgagtcgg tgtgcgacga ccctacagtt 540

gtggcctcca atatcatgga agttaaaatc tccagcccgg attacaaaga ctgcaactcg 600

gcagaagcca tggacgactt catgaagagg atcagttgct atgaagccag ctaccagccc 660

ctcgaccccg acaaatgcga cagggacttg tcgctgatca aggtgattga cgtgggccgg 720

aggttcctgg tgaaccgggt gcaggaccac atccagagcc gcatcgtgta ctacctgatg 780

aacatccacg tgcagccgcg taccatctac ctgtgccggc acggcgagaa cgagcacaac 840

ctccagggcc gcatcggggg cgactcaggc ctgtccagcc ggggcaagaa gtttgccagt 900

gctctgagca agttcgtgga ggagcagaac ctgaaggacc tgcgcgtgtg gaccagccag 960

ctgaagagca ccatccagac ggccgaggcg ctgcggctgc cctacgagca gtggaaggcg 1020

ctcaatgaga tcgacgcggg cgtctgtgag gagctgacct acgaggagat cagggacacc 1080

taccctgagg agtatgcgct gcgggagcag gacaagtact attaccgcta ccccaccggg 1140

gagtcctacc aggacctggt ccagcgcttg gagccagtga tcatggagct ggagcggcag 1200

gagaatgtgc tggtcatctg ccaccaggcc gtcctgcgct gcctgcttgc ctacttcctg 1260

gataagagtg cagaggagat gccctacctg aaatgccctc ttcacaccgt cctgaaactg 1320

acgcctgtcg cttatggctg ccgtgtggaa tccatctacc tgaacgtgga gtccgtctgc 1380

acacaccggg agaggtcaga ggatgcaaag aagggaccta acccgctcat gagacgcaat 1440

agtgtcaccc cgctagccag ccccgaaccc accaaaaagc ctcgcatcaa cagctttgag 1500

gagcatgtgg cctccacctc ggccgccctg cccagctgcc tgcccccgga ggtgcccacg 1560

cagctgcctg gacaaaacat gaaaggctcc cggagcagcg ctgactcctc caggaaacac 1620

tgaggcagac gtgtcggttc cattccattt ccatttctgc agcttagctt gtgtcctgcc 1680

ctccgcccga ggcaaaacgt atcctgagga cttcttccgg agagggtggg gtggagcagc 1740

gggggagcct tggccgaaga gaaccatgct tggcaccgtc tgtgtcccct cggccgctgg 1800

acaccagaaa gccacgtggg tccctggcgc cctgccttta gccgtggggc ccccacctcc 1860

actctctggg tttcctagga atgtccagcc tcggagacct tcacaaagcc ttgggagggt 1920

gatgagtgct ggtcctgaca ggaggccgct ggggacactg tgctgttttg tttcgtttct 1980

gtgatctccc ggcacgtttg gagctgggaa gaccacactg gtggcagaat cctaaaatta 2040

aaggaggcag gctcctagtt gctgaaagtt aaggaatgtg taaaacctcc acgtgactgt 2100

ttggtgcatc ttgacctggg aagacgcctc atgggaacga acttggacag gtgttgggtt 2160

gaggcctctt ctgcaggaag tccctgagct gagacgcaag ttggctgggt ggtccgcacc 2220

ctggctctcc tgcaggtcca cacaccttcc aggcctgtgg cctgcctcca aagatgtgca 2280

agggcaggct ggctgcacgg ggagagggaa gtattttgcc gaaatatgag aactggggcc 2340

tcctgctccc agggagctcc agggcccctc tctcctccca cctggacttg gggggaactg 2400

agaaacactt tcctggagct gctggctttt gcactttttt gatggcagaa gtgtgacctg 2460

agagtcccac cttctcttca ggaacgtaga tgttggggtg tcttgccctg gggggcttgg 2520

aacctctgaa ggtggggagc ggaacacctg gcatccttcc ccagcacttg cattaccgtc 2580

cctgctcttc ccaggtgggg acagtggccc aagcaaggcc tcactcgcag ccacttcttc 2640

aagagctgcc tgcacactgt cttggagcat ctgccttgtg cctggcactc tgccggtgcc 2700

ttgggaaggt cggaagagtg gactttgtcc tggccttccc ttcatggcgt ctatgacact 2760

tttgtggtga tggaaagcat gggacctgtc gtctcagcct gttggtttct cctcattgcc 2820

tcaaaccctg gggtaggtgg gacggggggt ctcgtgccca gatgaaacca tttggaaact 2880

cggcagcaga gtttgtccaa atgacccttt tcaggatgtc tcaaagcttg tgccaaaggt 2940

cacttttctt tcctgccttc tgctgtgagc cctgagatcc tcctcccagc tcaagggaca 3000

ggtcctgggt gagggtggga gatttagaca cctgaaactg ggcgtggaga gaagagccgt 3060

tgctgtttgt tttttgggaa gagcttttaa agaatgcatg tttttttcct ggttggaatt 3120

gagtaggaac tgaggctgtg cttcaggtat ggtacaatca agtgggggat tttcatgctg 3180

aaccattcaa gccctccccg cccgttgcac ccactttggc tggcgtctgc tggagaggat 3240

gtctctgtcc gcattcccgt gcagctccag gctcgcgcag ttttctctct ctccctggat 3300

gttgagtctc atcagaatat gtgggtaggg ggtggacgtg cacgggtgca tgattgtgct 3360

taacttggtt gtatttttcg atttgacatg gaaggcctgt tgctttgctc ttgagaatag 3420

tttctcgtgt ccccctcgca ggcctcattc tttgaacatc gactctgaag tttgatacag 3480

ataggggctt gatagctgtg gtcccctctc ccctctgact acctaaaatc aatacctaaa 3540

tacagaagcc ttggtctaac acgggacttt tagtttgcga agggcctaga tagggagaga 3600

ggtaacatga atctggacag ggagggagat actatagaaa ggagaacact gcctactttg 3660

caagccagtg acctgccttt tgaggggaca ttggacgggg gccgggggcg ggggttgggt 3720

ttgagctaca gtcatgaact tttggcgtct actgattcct ccaactctcc accccacaaa 3780

ataacgggga ccaatatttt taactttgcc tatttgtttt tgggtgagtt tcccccctcc 3840

ttattctgtc ctgagaccac gggcaaagct cttcattttg agagagaaga aaaactgttt 3900

ggaaccacac caatgatatt tttctttgta atacttgaaa tttatttttt tattattttg 3960

atagcagatg tgctatttat ttatttaata tgtataagga gcctaaacaa tagaaagctg 4020

tagagattgg gtttcattgt taattggttt gggagcctcc tatgtgtgac ttatgacttc 4080

tctgtgttct gtgtatttgt ctgaattaat gacctgggat ataaagctat gctagctttc 4140

aaacaggaga tgcctttcag aaatttgtat attttgcagt tgccagacca ataaaatacc 4200

tggttgaaat acatggacga agtaaa 4226

<210> 4

<211> 500

<212> PRT

<213> Chile person

<400> 4

Met Pro Phe Arg Lys Ala Cys Gly Pro Lys Leu Thr Asn Ser Pro Thr

1 5 10 15

Val Ile Val Met Val Gly Leu Pro Ala Arg Gly Lys Thr Tyr Ile Ser

20 25 30

Lys Lys Leu Thr Arg Tyr Leu Asn Trp Ile Gly Val Pro Thr Lys Val

35 40 45

Phe Asn Val Gly Glu Tyr Arg Arg Glu Ala Val Lys Gln Tyr Ser Ser

50 55 60

Tyr Asn Phe Phe Arg Pro Asp Asn Glu Glu Ala Met Lys Val Arg Lys

65 70 75 80

Gln Cys Ala Leu Ala Ala Leu Arg Asp Val Lys Ser Tyr Leu Ala Lys

85 90 95

Glu Gly Gly Gln Ile Ala Val Phe Asp Ala Thr Asn Thr Thr Arg Glu

100 105 110

Arg Arg His Met Ile Leu His Phe Ala Lys Glu Asn Asp Phe Lys Ala

115 120 125

Phe Phe Ile Glu Ser Val Cys Asp Asp Pro Thr Val Val Ala Ser Asn

130 135 140

Ile Met Glu Val Lys Ile Ser Ser Pro Asp Tyr Lys Asp Cys Asn Ser

145 150 155 160

Ala Glu Ala Met Asp Asp Phe Met Lys Arg Ile Ser Cys Tyr Glu Ala

165 170 175

Ser Tyr Gln Pro Leu Asp Pro Asp Lys Cys Asp Arg Asp Leu Ser Leu

180 185 190

Ile Lys Val Ile Asp Val Gly Arg Arg Phe Leu Val Asn Arg Val Gln

195 200 205

Asp His Ile Gln Ser Arg Ile Val Tyr Tyr Leu Met Asn Ile His Val

210 215 220

Gln Pro Arg Thr Ile Tyr Leu Cys Arg His Gly Glu Asn Glu His Asn

225 230 235 240

Leu Gln Gly Arg Ile Gly Gly Asp Ser Gly Leu Ser Ser Arg Gly Lys

245 250 255

Lys Phe Ala Ser Ala Leu Ser Lys Phe Val Glu Glu Gln Asn Leu Lys

260 265 270

Asp Leu Arg Val Trp Thr Ser Gln Leu Lys Ser Thr Ile Gln Thr Ala

275 280 285

Glu Ala Leu Arg Leu Pro Tyr Glu Gln Trp Lys Ala Leu Asn Glu Ile

290 295 300

Asp Ala Gly Val Cys Glu Glu Leu Thr Tyr Glu Glu Ile Arg Asp Thr

305 310 315 320

Tyr Pro Glu Glu Tyr Ala Leu Arg Glu Gln Asp Lys Tyr Tyr Tyr Arg

325 330 335

Tyr Pro Thr Gly Glu Ser Tyr Gln Asp Leu Val Gln Arg Leu Glu Pro

340 345 350

Val Ile Met Glu Leu Glu Arg Gln Glu Asn Val Leu Val Ile Cys His

355 360 365

Gln Ala Val Leu Arg Cys Leu Leu Ala Tyr Phe Leu Asp Lys Ser Ala

370 375 380

Glu Glu Met Pro Tyr Leu Lys Cys Pro Leu His Thr Val Leu Lys Leu

385 390 395 400

Thr Pro Val Ala Tyr Gly Cys Arg Val Glu Ser Ile Tyr Leu Asn Val

405 410 415

Glu Ser Val Cys Thr His Arg Glu Arg Ser Glu Asp Ala Lys Lys Gly

420 425 430

Pro Asn Pro Leu Met Arg Arg Asn Ser Val Thr Pro Leu Ala Ser Pro

435 440 445

Glu Pro Thr Lys Lys Pro Arg Ile Asn Ser Phe Glu Glu His Val Ala

450 455 460

Ser Thr Ser Ala Ala Leu Pro Ser Cys Leu Pro Pro Glu Val Pro Thr

465 470 475 480

Gln Leu Pro Gly Gln Asn Met Lys Gly Ser Arg Ser Ser Ala Asp Ser

485 490 495

Ser Arg Lys His

500

Claims

1. A method of treating type 2 diabetes in a subject, the method comprising administering to the subject an effective amount of a hif1α inhibitor.

2. The method of claim 1, wherein the hif1α inhibitor promotes hif1α degradation.

3. The method of claim 1, wherein the hif1α inhibitor inhibits

Hif1α/hif1β dimer formation.

4. The method of claim 1, wherein the hif1α inhibitor decreases hif1α transcriptional activity.

5. The method of any one of claims 1-4, wherein the hif1α inhibitor is KC7F2, IDF-11774, aminoflavone, AJM290, AW464, tanspirmycin (tanespimycin), acleidamycin (alvespimycin), PX-478, or FM19G11.

6. The method of any one of claims 1-4, wherein the hif1α inhibitor is a nucleic acid inhibitor.

7. The method of any one of claims 1-4, wherein the hif1α inhibitor is an antisense oligonucleotide.

8. The method of claim 7, wherein the hif1α inhibitor is EZN-2698.

9. The method of any one of claims 1-4, wherein the hif1α inhibitor is an siRNA or a short hairpin RNA.

10. The method of any one of claims 1-4, wherein the hif1α inhibitor is resveratrol, rapamycin, everolimus, CCI779, silibinin, digoxin, YC-1, phenethyl isothiocyanate, black hair mycin, fraping, bortezomib, amphotericin B, bay-2243, PX-478, or Gan Ni ganetascib.

11. The method of any one of claims 1-4, wherein the hif1α inhibitor is an anti-hif1α antibody or antibody-like molecule.

12. The method of claim 11, wherein the hif1α inhibitor is a nanobody.

13. The method of any one of claims 1-12, wherein the hif1α inhibitor is administered intravenously, intramuscularly, intraperitoneally, subcutaneously, intra-articular, intra-synovially, intrathecally, orally, topically, via inhalation, or via a combination of two or more routes of administration.

14. The method of any one of claims 1 to 13, further comprising administering a PFKFB3 inhibitor to the subject.

15. The method of claim 14, wherein the PFKFB3 inhibitor is 3- (3-pyridyl) -1- (4-pyridyl) -2-propen-1-one (3-PO) or an analog thereof.

16. The method of claim 15, wherein the PFKFB3 inhibitor is an analog of 3-PO, wherein the analog is 1- (4-pyridyl) -3- (2-quinolyl) -2-propen-1-one (PFK 15).

17. The method of claim 14, wherein the PFKFB3 inhibitor is BrAcNHEtOP, YN, YZ9, PQP, PFK-158, compound 26,

KAN0436151 or KAN0436067.

18. The method of claim 14, wherein the PFKFB3 inhibitor is a nucleic acid inhibitor.

19. The method of claim 14, wherein the PFKFB3 inhibitor is an antisense oligonucleotide.

20. The method of claim 14, wherein the PFKFB3 inhibitor is siRNA or short hairpin RNA.

21. The method of any one of claims 14 to 20, wherein the PFKFB3 inhibitor is operably linked to a targeting molecule configured to bind to a beta cell of the subject.

22. The method of claim 21, wherein the targeting molecule is an antibody.

23. The method of claim 22, wherein the targeting molecule is an antibody-like molecule.

24. The method of any one of claims 21 to 23, wherein the targeting molecule is configured to bind to a GLP-1 receptor.

25. The method of any one of claims 14-24, wherein the hif1α inhibitor and the PFKFB3 inhibitor are administered sequentially to the subject.

26. The method of any one of claims 14-24, wherein the hif1α inhibitor and the PFKFB3 inhibitor are administered to the subject substantially simultaneously.

27. The method of any one of claims 1-26, wherein the hif1α inhibitor is operably linked to a targeting molecule configured to bind to β -cells of the subject.

28. The method of claim 27, wherein the targeting molecule is an antibody.

29. The method of claim 27, wherein the targeting molecule is an antibody-like molecule.

30. The method of any one of claims 27 to 29, wherein the targeting molecule is configured to bind to a GLP-1 receptor.

31. The method of any one of claims 1-30, wherein administering the effective amount of the hif1α inhibitor increases insulin sensitivity in the subject.

32. The method of any one of claims 1-31, wherein the subject has not been suffering from or has not been diagnosed with cancer.

33. The method of any one of claims 1-32, wherein the subject has had type 2 diabetes.

34. The method of any one of claims 1-32, further comprising, prior to the administering, diagnosing the subject as having the type 2 diabetes.

35. The method of claim 33 or 34, wherein the subject has previously received treatment for type 2 diabetes.

36. The method of claim 35, wherein the subject is determined to be resistant to a previous treatment.

37. The method of any one of claims 1-36, wherein the subject has not been suffering from or has not been diagnosed with diabetic nephropathy or diabetic retinopathy.

38. The method of any one of claims 1 to 37, further comprising measuring the expression level of PFKFB3 in beta cells from the subject.

39. The method of claim 38, wherein the expression level of PFKFB3 in the beta cells from the subject is increased relative to the expression level of PFKFB3 in beta cells from a healthy subject not suffering from type 2 diabetes.

40. A method of treating type 2 diabetes in a subject, comprising administering to the subject an effective amount of a hif1α inhibitor, the subject determined to have an increased level of PFKFB3 expression in beta cells from the subject relative to the level of PFKFB3 expression in beta cells from a healthy subject not suffering from type 2 diabetes.

41. The method of claim 40, wherein the hif1α inhibitor promotes hif1α degradation.

42. The method of claim 40, wherein the hif1α inhibitor inhibits

Hif1α/hif1β dimer formation.

43. The method of claim 40, wherein the hif1α inhibitor decreases hif1α transcriptional activity.

44. The method of any one of claims 40-43, wherein the hif1α inhibitor is KC7F2, IDF-11774, aminoflavone, AJM290, AW464, tamsulosin, aclacinomycin, histone deacetylase inhibitor, PX-478, FM19G11.

45. The method of any one of claims 40-43, wherein the hif1α inhibitor is a nucleic acid inhibitor.

46. The method of any one of claims 40-43, wherein the hif1α inhibitor is an antisense oligonucleotide.

47. The method of claim 46, wherein the HIF1a inhibitor is EZN-2698.

48. The method of any one of claims 40-43, wherein the hif1α inhibitor is an siRNA or a short hairpin RNA.

49. The method of any one of claims 40-43, wherein the hif1α inhibitor is resveratrol, rapamycin, everolimus, CCI779, silibinin, digoxin, YC-1, phenethyl isothiocyanate, black hair mycin, fraping, bortezomib, amphotericin B, bay-2243, PX-478, or Gan Ni tacxib.

50. The method of any of claims 40-43, wherein the hif1α inhibitor is an anti-hif1α antibody or antibody-like molecule.

51. The method of claim 50, wherein the hif1α inhibitor is a nanobody.

52. The method of any one of claims 40-51, wherein the inhibitor is administered intravenously, intramuscularly, intraperitoneally, subcutaneously, intra-articular, intrasynovially, intrathecally, orally, topically, via inhalation, or via a combination of two or more routes of administration.

53. The method of any one of claims 40 to 52, further comprising administering a PFKFB3 inhibitor to the subject.

54. The method of claim 53, wherein the PFKFB3 inhibitor is 3- (3-pyridyl) -1- (4-pyridyl) -2-propen-1-one (3-PO) or an analog thereof.

55. The method of claim 54, wherein the PFKFB3 inhibitor is an analog of 3-PO, wherein the analog is 1- (4-pyridyl) -3- (2-quinolyl) -2-propen-1-one (PFK 15).

56. The method of claim 53, wherein the PFKFB3 inhibitor is a nucleic acid inhibitor.

57. The method of claim 53, wherein the PFKFB3 inhibitor is an antisense oligonucleotide.

58. The method of claim 53, wherein the PFKFB3 inhibitor is siRNA or short hairpin RNA.

59. The method of any one of claims 53 to 58, wherein the PFKFB3 inhibitor is operably linked to a targeting molecule, the targeting molecule being configured to bind to a beta cell of the subject.

60. The method of claim 59, wherein the targeting molecule is an antibody.

61. The method of claim 60, wherein the targeting molecule is an antibody-like molecule.

62. The method of any one of claims 59 to 61, wherein the targeting molecule is configured to bind to a GLP-1 receptor.

63. The method of any one of claims 53-62, wherein the hif1α inhibitor and the PFKFB3 inhibitor are administered sequentially to the subject.

64. The method of any one of claims 53-62, wherein the hif1α inhibitor and the PFKFB3 inhibitor are administered to the subject substantially simultaneously.

65. The method of any one of claims 40-64, wherein the hif1α inhibitor is operably linked to a targeting molecule configured to bind to β cells of the subject.

66. The method of claim 65, wherein the targeting molecule is an antibody.

67. The method of claim 65, wherein the targeting molecule is an antibody-like molecule.

68. The method of any one of claims 65 to 67, wherein the targeting molecule is configured to bind to a GLP-1 receptor.

69. The method of any one of claims 40-68, wherein administering the effective amount of the hif1α inhibitor increases insulin sensitivity in the subject.

70. A method of treating type 2 diabetes in a subject, the method comprising:

(a) Determining that the subject has an increased level of PFKFB3 expression in beta cells from the subject relative to the level of PFKFB3 expression in beta cells from a healthy subject not suffering from type 2 diabetes; and

(b) Administering to the subject an effective amount of a hif1α inhibitor.

71. A method of increasing insulin sensitivity in a subject, the method comprising administering to the subject an effective amount of a hif1α inhibitor.

72. The method of claim 71, wherein the subject is suffering from prediabetes.

73. The method of claim 71, wherein the subject is suffering from insulin resistance.

74. A method for stimulating cell death in a damaged beta cell expressing PFKFB3, the method comprising providing a hif1α inhibitor to the damaged beta cell.

75. The method of claim 74, wherein the hif1α inhibitor is provided to the injured β cell in vitro.

76. The method of claim 74, wherein the hif1α inhibitor is provided in vivo to the damaged β -cells.

77. A method for stimulating regeneration of a beta cell that does not express PFKFB3, the method comprising providing a hif1α inhibitor to the beta cell.

78. The method of claim 77, wherein said beta cells are provided in vitro

Hif1α inhibitors.

79. The method of claim 77, wherein said beta cells are provided in vivo

Hif1α inhibitors.

80. A method of stimulating β cell regeneration in a subject having type 2 diabetes, wherein the β cells do not express PFKFB3, the method comprising administering to the subject an effective amount of a hif1α inhibitor.

81. A method for killing damaged beta cells expressing PFKFB3 in a subject having diabetes, the method comprising administering to the subject an effective amount of a hif1α inhibitor.

82. A method of treating diabetes in a subject, the method comprising administering to the subject an effective amount of a hif1α inhibitor.

83. The method of claim 82, wherein the diabetes is type 1 diabetes.

84. The method of claim 82, wherein the diabetes is type 2 diabetes.

85. The method of any one of claims 82-84, wherein the subject is determined to have an increased level of expression of PFKFB3 in beta cells from the subject relative to the level of expression of PFKFB3 in beta cells from a healthy subject not suffering from type 2 diabetes.

86. A method for diagnosing a subject having type 2 diabetes, the method comprising:

(a) Measuring the expression level of PFKFB3 in beta cells from the subject;

(b) Comparing the expression level to the expression level of PFKFB3 in beta cells from a healthy subject not suffering from type 2 diabetes; and

(c) Determining that the expression level of PFKFB3 in beta cells from the subject is increased relative to the expression level of PFKFB3 in beta cells from the healthy subject, thereby diagnosing the subject with type 2 diabetes.

87. A pharmaceutical composition comprising (a) a hif1α inhibitor and (b) a PFKFB3 inhibitor.

88. The pharmaceutical composition of claim 87, wherein the hif1α inhibitor promotes hif1α degradation.

89. The pharmaceutical composition of claim 87, wherein the hif1α inhibitor inhibits

Hif1α/hif1β dimer formation.

90. The pharmaceutical composition of claim 87, wherein the hif1α inhibitor is decreased

Hif1α transcriptional activity.

91. The pharmaceutical composition of any one of claims 87-90, wherein the

The HIF1 alpha inhibitor is KC7F2, IDF-11774, aminoflavone, AJM290,

AW464, tamspiramycin, doxorubicine, PX-478 or FM19G11.

92. The pharmaceutical composition of any one of claims 87-90, wherein the

Hif1α inhibitors are nucleic acid inhibitors.

93. The pharmaceutical composition of claim 92, wherein the hif1α inhibitor is an antisense oligonucleotide.

94. The pharmaceutical composition of claim 93, wherein the hif1α inhibitor is EZN-2698.

95. The pharmaceutical composition of claim 92, wherein the hif1α inhibitor is an siRNA or a short hairpin RNA.

96. The pharmaceutical composition of any one of claims 87-90, wherein the

Hif1α inhibitors are resveratrol, rapamycin, everolimus, CCI779, silibinin, digoxin, YC-1, phenethyl isothiocyanate, black hair mycin, fraping, bortezomib, amphotericin B, bay-2243, PX-478, or Gan Ni tamoxib.

97. The pharmaceutical composition of any one of claims 87-90, wherein the

Hif1α inhibitors are anti-hif1α antibodies or antibody-like molecules.

98. The pharmaceutical composition of claim 97, wherein the hif1α inhibitor is a nanobody.

99. The pharmaceutical composition of any one of claims 87-98, wherein the

The PFKFB3 inhibitor is 3- (3-pyridyl) -1- (4-pyridyl) -2-propen-1-one (3-PO) or an analogue thereof.

100. The pharmaceutical composition of any one of claims 87-100, wherein the

The PFKFB3 inhibitor is an analog of 3-PO, wherein the analog is 1- (4-pyridyl) -3- (2-quinolyl) -2-propen-1-one (PFK 15).

101. The pharmaceutical composition of any one of claims 87-100, wherein the

The PFKFB3 inhibitor is BrAcNHEtOP, YN1, YZ9, PQP, PFK-158, compound 26, KAN0436151 or KAN0436067.

102. The pharmaceutical composition of any one of claims 87-100, wherein the

PFKFB3 inhibitors are nucleic acid inhibitors.

103. The pharmaceutical composition of claim 102, wherein the PFKFB3 inhibitor is an antisense oligonucleotide.

104. The pharmaceutical composition of claim 102, wherein the PFKFB3 inhibitor is siRNA or short hairpin RNA.

105. The pharmaceutical composition of any one of claims 87-104, wherein the PFKFB3 inhibitor is operably linked to a targeting molecule configured to bind to beta cells of the subject.

106. The pharmaceutical composition of claim 105, wherein the targeting molecule is an antibody.

107. The pharmaceutical composition of claim 105, wherein the targeting molecule is an antibody-like molecule.

108. The pharmaceutical composition of claim 105, wherein the targeting molecule is configured to bind to a GLP-1 receptor.

109. A method of treating type 2 diabetes in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 87-108.

110. A method of eliminating a bi-hormonal cell from a cell population comprising administering to the cell population an effective amount of a PFKFB3 inhibitor.

111. The method of claim 110, wherein the population of cells is a population of islet cells.

112. The method of claim 110, wherein the population of cells is a population of differentiated stem cells.

113. The method of claim 112, wherein the differentiated stem cell is a differentiation-Induced Pluripotent Stem Cell (iPSC).

114. The method of claim 112, wherein the differentiated stem cell is a differentiated Embryonic Stem Cell (ESC).

115. The method of any one of claims 110-114, wherein the PFKFB3 inhibitor is administered to the cell population in vitro.

116. The method of any one of claims 110-114, wherein the PFKFB3 inhibitor is administered in vivo to the cell population.

117. The method of any one of claims 110-116, wherein the PFKFB3 inhibitor is 3- (3-pyridyl) -1- (4-pyridyl) -2-propen-1-one (3-PO) or an analogue thereof.

118. The method of any one of claims 110-117, wherein the PFKFB3 inhibitor is an analog of 3-PO, wherein the analog is 1- (4-pyridyl) -3- (2-quinolyl) -2-propen-1-one (PFK 15).

119. The method of any one of claims 110-117, wherein the PFKFB3 inhibitor is BrAcNHEtOP, YN, YZ9, PQP, PFK-158, compound 26, KAN0436151 or KAN0436067.

120. The method of any one of claims 110-116, wherein the PFKFB3 inhibitor is a nucleic acid inhibitor.

121. The method of claim 120, wherein the PFKFB3 inhibitor is an antisense oligonucleotide.

122. The method of claim 120, wherein the PFKFB3 inhibitor is siRNA or short hairpin RNA.

123. The method of any one of claims 110-122, further comprising administering to the population of cells an effective amount of a hif1α inhibitor.

124. A method of depleting a di-hormonal cell from a cell population, the method comprising administering an effective amount of a hif1α inhibitor to the cell population.

125. The method of claim 124, wherein the population of cells is a population of islet cells.

126. The method of claim 124, wherein the population of cells is a population of differentiated stem cells.

127. The method of claim 126, wherein the differentiated stem cell is a differentiated iPSC.

128. The method of claim 112, wherein the differentiated stem cell is a differentiated ESC.

129. The method of any one of claims 124-128, wherein the hif1α inhibitor is administered to the population of cells in vitro.

130. The method of any one of claims 124-128, wherein the hif1α inhibitor is administered into the cell population.

131. The method of any one of claims 124-130, wherein the hif1α inhibitor is KC7F2, IDF-11774, aminoflavone, AJM290, AW464, tamsulosin, aclacinomycin, PX-478, or FM19G11.

132. The method of any one of claims 124-130, wherein the hif1α inhibitor is a nucleic acid inhibitor.

133. The method of claim 132, wherein the hif1α inhibitor is an antisense oligonucleotide.

134. The method of claim 133, wherein the hif1α inhibitor is EZN-2698.

135. The method of claim 132, wherein the hif1α inhibitor is an siRNA or a short hairpin RNA.

136. The method of any one of claims 124-130, wherein the hif1α inhibitor is resveratrol, rapamycin, everolimus, CCI779, silibinin, digoxin, YC-1, phenethyl isothiocyanate, glaucomycin, frataxine, bortezomib, amphotericin B, bay-2243, PX-478, or Gan Ni tacxib.

137. The method of any one of claims 124-130, wherein the hif1α inhibitor is an anti-hif1α antibody or antibody-like molecule.

138. The method of claim 137, wherein the hif1α inhibitor is a nanobody.

139. The method of any one of claims 124-138, further comprising administering to the population of cells an effective amount of a PFKFB3 inhibitor.