CN112618703A

CN112618703A - Application of Acsl4 in preparation of type 2 diabetes mellitus medicines

Info

Publication number: CN112618703A
Application number: CN202011549440.4A
Authority: CN
Inventors: 顾燕云; 崔灿琦; 李婷婷; 傅晨阳; 倪启程; 谢运; 宁光
Original assignee: SHANGHAI INSTITUTE OF ENDOCRINE AND METABOLIC DISEASES; Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
Current assignee: SHANGHAI INSTITUTE OF ENDOCRINE AND METABOLIC DISEASES; Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
Priority date: 2020-12-24
Filing date: 2020-12-24
Publication date: 2021-04-09

Abstract

The invention relates to an application of Acsl4 in preparation of a medicament for treating type 2 diabetes. The invention finds that if the lipid metabolism disorder can be corrected or prevented as soon as possible, or the beta cells can be prevented from metabolic remodeling and then dedifferentiation, the function decline of the beta cells is protected, and the occurrence of type 2 diabetes is delayed or prevented.

Description

Application of Acsl4 in preparation of type 2 diabetes mellitus medicines

Technical Field

The invention belongs to the field of type 2 diabetes mellitus medicines, and particularly relates to application of Acsl4 in preparation of a type 2 diabetes mellitus medicine.

Background

Type 2 diabetes is a complex, multi-organ-involved disease that has become the major chronic non-infectious disease worldwide, causing a huge economic burden with global costs of up to $ 1.31 trillion. Although the world health organization ranks diabetes as the sixth leading cause of death worldwide, the contribution of diabetes to heart disease (the first leading cause of death) and stroke (the second leading cause of death) has increased dramatically. In China, the prevalence of diabetes has increased dramatically over the last 40 years, from 0.67% to 10.9% in adults in 1980, and recent data shows that Chinese diabetics reach 1.164 million, predominantly type 2 diabetes. In the face of such huge medical health and economic burden, the research on the prevention and treatment of type 2 diabetes and the corresponding pathogenesis mechanism is still not satisfactory. Islet beta cell dysfunction is critical to the progression and prognosis of type 2 diabetes. In the progressive stage of type 2 diabetes, the pancreatic islets will have adaptive mass increase of beta cells and compensatory stage of insulin secretion increase, but with the progress of disease course, the beta cells finally fail to cause insulin secretion to meet the needs of the body. Whether the failing islets are caused by reduced beta cell numbers or impaired beta cell function is always a hotspot discussed by academia, and is not yet determined. In recent years, in addition to the apoptotic, functionally deficient or immature state of beta cells, beta cell dedifferentiation has been found to be another important pathophysiological process of beta cell failure during the progression of diabetes. Beta cell dedifferentiation is a beta cell with an altered phenotype that loses some of the important beta cell properties that lead to impaired function including insulin secretion. By cell lineage tracing techniques, researchers have found that beta cell loss may be due to dedifferentiation of beta cells, which no longer express insulin, rather than apoptosis or necrosis of the cells. The dedifferentiation means that the beta cells start to express the markers of Neurogenin3, Oct4, precursors such as Nanog and L-Myc and even progenitor cells, and lose the expression of the markers of the identity of mature cells, including MafA, Ucn3, Nkx6.1, FoxO1, PDX-1, Nkx6.1, Pax6, HNF3b, HNF4a, HNF1a and the like. These dedifferentiated cells also no longer have a perfect function of incretin secretion and gradually progress to a severe dysfunction of insulin secretion.

Knocking Nkx6.1 out to convert beta cells into delta cells, knocking Pdx1 out to prevent pancreas from developing ducts and secretory gland tissues; the insulin positive cells in the early stage of differentiation express both MafA and MafB, and the MafB expression is gradually turned off as the beta cells develop. If MafA is knocked out, both beta cell quality and function are significantly affected. Of particular note are FoxO1, FoxO1 which on the one hand are highly expressed to inhibit beta cell proliferation, and on the other hand are under-expressed to render beta cells undifferentiated, increasing susceptibility to nutritional stress or stress. Accili laboratories confirmed that FoxO1 deletion led to cell dedifferentiation and ultimately to hyperglycemia by diabetes models in different mice. In addition, the abnormally high expression of impermissible genes of some so-called islet beta cells (including Slc16a1, Ldha, Acot7, etc.) also has an effect on beta cell function and development, which in turn leads to abnormal proliferation and function.

When these molecular mechanisms are roughly elucidated, the scholars have raised another problem: how do these transcription factors change during the progression of type 2 diabetes? External self-environmental stresses, including overnutrition, including insulin resistance, are how to drive the de-differentiation process by affecting key transcription factors. It has been found that the main cause of dedifferentiation may be glucose toxicity and that lowering glucose levels by therapy may lead to recovery of certain functions. Unfolded protein accumulation and ER stress of pancreatic islets may be the mechanisms leading to dedifferentiation of beta cells and are associated with abnormal expression of key beta cell genes (e.g., Pdx 1). Likewise, chronic oxidative stress conditions may promote loss of beta cell identity by inhibiting beta cell transcription factor activity (e.g., Glut2, Pdx1, and MafA).

The rapamycin theoretical target protein is a highly conserved serine/threonine kinase, comprising complexes mTORC1 and mTORC2, which integrate stimulation from upstream nutrients, growth factor signals and hormone signals, participate in regulating cell growth, proliferation and function, etc., and the roles are multifaceted. mTORC1 is relatively sensitive to rapamycin action, mTORC2 is insensitive to rapamycin inhibition, the two complexes share mTOR, mLST8, DEPTOR and Tti1/Tel2 proteins, mTORC1 contains specific proteins Raptor and PRAS40, and mTORC2 contains specific proteins Rictor and mSin 1. mTORC1 and mTORC2, which are key cell nutrient sensors, play an important role in the maintenance of the normal functional quality of islet β cells, mTORC1 in regulating the physiological processes of protein processing, autophagy, and lipid synthesis of cells. Specific deletion of mTORC1/Raptor in islet beta cells results in severe hyperglycemia, and mTORC1 inhibits disallowed gene expression by affecting cellular DNA methylation, promoting beta cell functional maturation. mTORC2 plays an essential role in maintaining the function and activity of islet β cells. Slight hyperglycemia and a reduced rate of islet beta cell proliferation caused by islet-specific deletion of mTORC2/Rictor resulted in a more severe phenotype under nutritional metabolic stress, such as a high fat diet. Recent studies based on human islet transcriptome analysis found that there was a link between mTORC2/Rictor and beta cells with a de-differentiated state or a decline in function, but the specific mechanism was not elucidated. These suggest that mTORC2/Rictor is more likely to play a role in the pathogenesis of T2D relative to mTORC1 to regulate postnatal beta cell functional compensatory adaptation in the event of overnutrition. However, the mechanism of mTORC2/Rictor in islet beta cells and the function of the complex in other cells are not as clear as mTORC1, and further research is needed.

Acsl4 is a member of the long-chain fatty acid coenzyme a synthetase (Acsl) family, the major biological function of which is to mobilize the activation of long-chain fatty acids in an ATP-dependent manner, activate the long-chain fatty acids (12-20 carbons) to the corresponding fatty acyl-coas via an ATP-dependent pathway, and allow them to enter lipolysis (β -oxidation) or (phospholipid) lipid synthesis, and thus Acsl is a key rate-limiting enzyme that controls the metabolism of various lipids.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an application of Acsl4 in preparing a type 2 diabetes mellitus drug, and in the insulin resistant period, if the lipid metabolism disorder can be corrected or prevented as soon as possible or the beta cells can be prevented from metabolic remodeling and then dedifferentiation, the function decline of the beta cells is protected, and the occurrence of type 2 diabetes mellitus is delayed or prevented.

The invention provides application of Acsl4 in preparation of a diabetes drug.

The invention also provides an application of Acsl4 in preparation of a medicament for treating type 2 diabetes.

The application comprises correcting or preventing the lipid metabolism disorder through the expression of Ascl 4; or, preventing metabolic remodeling of beta cells followed by dedifferentiation; alternatively, the effect is obtained by protecting the function of the beta cells from decline.

Acsl4 is used as an active component and is matched with pharmaceutically acceptable auxiliary materials or auxiliary components to prepare the preparation for use.

The preparation is selected from one of tablets, powder, granules, capsules, oral liquid and sustained release agents.

The invention also provides a medicinal preparation for treating type 2 diabetes, which takes the component for reducing the expression of Acsl4 as an active component.

The invention also provides a 2-type diabetes mellitus medicinal preparation which takes the component for promoting the expression of Acsl4 as an active component.

The invention also provides a type 2 diabetes pharmaceutical preparation which takes a virus vector containing an Acsl4 related sequence as an active component.

The viral vector is an adenovirus vector.

Advantageous effects

Healthy beta cells may need to undergo dual insults of insulin resistance and glycolipid metabolic remodeling to actually enter a state of cellular dedifferentiation. It is suggested that in the insulin resistance period, if the lipid metabolism disorder can be corrected or prevented as soon as possible or the beta cells can be prevented from metabolic remodeling and then dedifferentiation, the function of the beta cells is protected from being declined, and the occurrence of type 2 diabetes is delayed or prevented.

Drawings

Fig. 1 is a graph of the relative mRNA expression levels of genes involved in maintaining beta cell identity and immaturity in islets of langerhans of the invention (. p <0.05, n ═ 3).

FIG. 2 is a graph showing the relative mRNA levels of the relevant genes in the Min6 cells of the invention involved in maintaining beta cell identity and immaturity (. p.sub.0.05, n: 3-4).

A in FIG. 3 is a representative Western Blot showing results of Aldh1a3 and NKX6.1 proteins in mouse islets; b is a WB Aldh1a3 and NKX6.1 gray scale analysis histogram (./p <0.05, n ═ 3).

FIG. 4 shows on the left the immunofluorescence of the primary islet patch of the present invention; the right is a MafA nuclear fluorescence intensity statistical histogram (. p <0.01, n ═ 3-4).

FIG. 5 is a histogram of the invention showing fold change in Min6 cells compared to control group H for each group₂O₂Horizontal results.

Fig. 6, a is a graph showing the results of MafA expression in Min6 cells after GSH treatment using representative Western blots of the invention, and B is a histogram showing the corresponding intensity of MafA bands (× p <0.01, n ═ 3).

FIG. 7 is a graph showing the results of Western Blot showing protein levels of MafA after Min6 cells were treated with a dose gradient of rotenone/antimycin A for 8 hours.

FIG. 8 is a graph showing the results of protein expression of FoxO1 in the islet tissue of Western Blot (A) and in Min6 cells of the present invention.

FIG. 9 is a graph showing the results of Western Blot of the present invention showing the expression of Sirt1 protein level in (A) mouse islets and (B) Min6 cells.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

1. Principal reagents and materials

SPF (specific pathogen free) grade Rictor^lox/loxThe C57BL/6N mice in (A) were Vanderbils in the United statesSupplied by doctor Mark.A. Magnuson of t University Medical Center, mated RIP-Cre mice, control mouse genotype Rictor^lox/lox。

Cell ROS (644/665nm) flow kit was purchased from Invitrogen, usa; mito Tracker was purchased from Thermo corporation, USA;

flow cytometry kits were purchased from Thermo corporation, usa; hydrogen peroxide H₂O₂The kit is purchased from Promega corporation, USA; antibody: PS6, IRS2 were purchased from Cell signaling technology, USA; anti-Aldh1a3 antibody was purchased from Novus, USA;

virus: lentiviruses of Acsl4 siRNA oligos (5'-GCAAUUUGAUUGCUGGAAUTT-3') were constructed with the assistance of shanghai gimar biotechnology limited; the overexpression adenovirus Acsl4-FLAG (NM _001033600.1) and the GFP control adenovirus were constructed with the assistance of Taicold Biotech Ltd.

2. Experimental methods

Cell dosing and virus:

1) min6 cell line cultured in 5% CO₂Gas mixed 37 ℃ incubator, logarithmic growth phase cells for experiments.

The culture solution comprises the following components: (50ml)

Glutamax	1:100	500ul
			PenStrep	1:100	500ul
Beta-mercaptoethanol	1:1000	50ul
			Gibco serum	15％	7.5ml
DMEM	The volume is fixed to 50ml	41.5ml

2) Rictor siRNA oligos (5'-AUAUUCUUGAUGAAGCUUGUTT-3') was constructed with the assistance of Shanghai Jima Biotechnology Inc.; MOI 50 titer, transfection for 72h,

3) and adenovirus transfection:

1) after the cells are paved on a twelve-well plate, an overexpression adenovirus with FLAG-tag and a control adenovirus with a GFP-tag sequence are added according to the MOI (molar equivalent of identity) of 5;

2) after 24 hours, replacing a new culture solution according to green fluorescence expression under an observation mirror;

3) after 48 hours, plates were harvested by washing three times with ice-precooled PBS.

4) Rotenone/antimycin a: PBS (poly (ethylene succinate)) is used for preparing rotenone/antimycin A to 5 mu M, different concentration gradients are prepared according to 10nM, 50nM and 100nM, cells are added with medicine for 8 hours, and plates are collected after observation under a mirror.

5、NT-157

a) The cells on the previous day are plated, after being plated in a twelve-well plate, Acsl4 overexpression adenovirus with a FLAG label and control virus with a GFP sequence are added according to MOI (molar equivalent to 5);

b) treating overnight with Fasiting in DMEM medium containing 0.2% BSA and low sugar (2.8 mM);

c) dissolving NT-157 with DMSO in advance, subpackaging and storing in a refrigerator of-80 deg.C;

d) diluting NT-157 to 2uM, adding medicine for 4 hr, changing to 0.2% BSA culture solution with low sugar or high sugar, observing cell morphology under the mirror, washing with ice-cold PBS for three times, and collecting the plate.

An Acsl4 transient high expression cell model was prepared by overexpressing the adenovirus-packaged Flag-tagged Acsl4 sequence (Ad-Acsl4) on the Min6 cell line.

Mice with specific Rictor knockout (. beta.RicKO) on islet beta cells were isolated from primary islet tissue and isolated islets were transfected with the adenovirus-packaged Acsl4 overexpression vector.

Western Blot experiment:

1. sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE)

1) Preparation of protein samples: adding 5 x protein sample loading buffer solution into 0.6ml centrifuge tube for each processed histone sample, mixing uniformly, heating to denature at 99 ℃ for 10min, cooling to room temperature at 4 ℃, and slightly centrifuging;

2) preparing separation gel and concentrated gel: dipping a little of liquid detergent to clean the glass plate and the comb, and airing for later use;

3) mounting a glass plate;

4) preparing separation gel: the concentration of the gel is selected according to the desired molecular weight, and if the gel is 10%, 30% Acry-Bis 4.95ml and ddH are added to a 50ml centrifuge tube₂O6.15 ml, 1.5mol/L Tris (pH8.8)3.75ml, 10% SDS 150. mu.l, finally adding 10% APS 75. mu.l and TEMED 7.5. mu.l, and mixing;

5) uniformly pouring glue, sealing with absolute ethyl alcohol, standing at room temperature for about 20min, pouring out the absolute ethyl alcohol, and slightly sucking residual liquid by using filter paper;

6) preparing concentrated glue: to a 50ml centrifuge tube, 30% Acry-Bis0.7ml and ddH were added in that order₂O3.1 ml, 1.0mol/L Tris (pH6.8)1.3ml, 10% SDS 50. mu.l, finally adding 10% APS 25. mu.l and TEMED 5. mu.l, and mixing well;

7) slowly adding concentrated gel on the gel, immediately inserting comb on the gel to prevent generation of bubbles, standing at room temperature for about 20min, and completing gel; removing the gel preparation clamp, fixing in an electrophoresis tank, pouring 1 × Tris-glycine electrophoresis solution, infiltrating gel, carefully extracting a comb, if not used in the day, infiltrating in triple distilled water, preserving at 4 deg.C, and using within one week;

8) loading: calculating the volume of the protein sample according to the concentration of the protein, wherein about 3-5ul of Thermo pre-staining protein Marker is added into the leftmost lane, and about 3-5ul of Thermo pre-staining protein Marker is added into the rightmost lane;

9) adjusting to a constant voltage mode, carrying out electrophoresis at the voltage of 180V for about 40 minutes, observing that the Marker runs to the smallest strip to the position close to the edge of the bottom of the gel, and turning off the power supply;

2. rotary film

1) Preparing a membrane transferring solution and preparing a PVDF membrane: diluting 5 × rotating membrane buffer solution by using triple distilled water, adding isopropanol, and specifically, measuring 400ml of pre-prepared 5 × rotating membrane buffer solution, adding 3.2L of filtered triple distilled water, finally adding 400ml of isopropanol, uniformly mixing, firstly soaking a PVDF membrane in methanol for about 30s-1min, then placing in an eBlot PVDF balanced solution for balancing for 3-5min, simultaneously placing glue in triple distilled water, and removing SDS foam;

2) prying open the glass plate gently, cutting off and inserting the comb part, and on rotating the membrane clamp, sequentially according to the following steps: assembling a sandwich clamp in the order of anode, filter paper, PVDF membrane, glue, filter paper and cathode, turning on the rotary die clamp and inserting the rotary die clamp into a Cassette after paying attention to the fact that air bubbles cannot exist between the glue and the membrane and the glue cannot be reversed;

3) the power is switched on, the Start A or B of the screen is pressed, and the mode switching mode and the time length are selected according to the molecular weight of the target protein (if the target protein is large, the time length is generally 16.5 minutes; if the protein is small, generally 11 minutes), disconnecting the power supply, taking out the PVDF membrane, dyeing the PVDF membrane with ponceau red, and quickly washing the PVDF membrane for 3 times within five minutes by using TBS to rinse and remove the dyeing;

4) 1X TBST is mixed with 10 percent of sealing liquid of skimmed milk, the mixture is gently shaken at room temperature and sealed for 1 hour, and a cutting film is marked according to a pre-dyed protein Marker;

3. hetero-antibody

1) Preparing 5% BSA antibody dilution with 1XTBST, according to the antibody specification or experience 1; dilution 1000 primary antibody, internal reference antibody Tubulin, GAPDH or HSP90 was performed according to 1: 10000 diluting primary antibody;

2) pouring the antibody and the PVDF membrane for incubation, and gently shaking at 4 ℃ in a refrigeration house overnight;

4. hybrid secondary antibody

1) Rinsing with TBST solution for 10min × 3 times, and washing to remove primary antibody;

2) diluting a secondary antibody (rabbit or mouse) marked by horseradish peroxidase dye with 5% BSA prepared by 1X TBST at a ratio of 1:2000, pouring the secondary antibody diluent into a corresponding box, slightly shaking for 1h at room temperature, and rinsing with TBST solution for 10min multiplied by 3 times;

5. development

1) According to the following steps of 1: 1, uniformly covering the mixed developing A, B liquid on a film, exposing by using an LAS-4000 fluorescence image analyzer, and storing;

2) and (3) data analysis: relative protein quantification was performed on the corresponding protein bands with the software Image J.

Statistical analysis:

statistical analysis was performed using SPSS11.0, and the data in the graphs are all expressed as mean. + -. standard deviation (X. + -.s). All experimental results were performed in triplicate and more. If the data which are not in accordance with the normal distribution are compared, the Two groups of data are compared by a One-factor ANOVA method, and the difference significance level is 0.05.

(1) Acsl4 overexpression promotes the dedifferentiation of mTORC2/Rictor specific knockout beta cells

After isolating mouse islets for 16 weeks and overexpressing the Acsl4 virus in vitro for 48 hours, islet extracted RNA was collected, reverse transcribed, and real-time quantitative PCR amplified. The sequences of primers used for reverse transcription of total RNA into cDNA are shown in Table 1, the mRNA sequence number of candidate gene was searched using the Genome search engine (UCSC Genome Browser) of Santa Cruz university, California, and was designed and verified on-line using Primer-Blast website, and the primers were synthesized by Shanghai Biotechnology, Inc.

TABLE 1

Primer: primer stock solutions (stored at-20 ℃ C. for long periods) were prepared at 100. mu.M concentration as described in the specification, and working solutions (stored at-20 ℃ C.) at 10. mu.M concentration were prepared by adding triple distilled water as shown in the following table.

Forward	10μl
		Reverse	10μl
3dH₂O	80μl

Sample dilution: 10-fold dilution, the dilution is as follows:

stock solution	20μl
		3dH₂O	180μl

Mix system:

reagent	Volume (μ l)
		SYBR Premix Ex TaqTM(2×)	5
Forward Primer(10μM)	0.2
		Reverse Primer(10μM)	0.2
cDNA template	2
		RNAse-free Water	2.6
Total	10

Adding 384-well plate, adding mix, centrifuging at 2000g and 4 ℃ for 2min, and adding cDNA.

Pasting the film, centrifuging at 2000g and 4 ℃ for 2min, and then loading on a machine

Amplification was performed in a Real Time PCR thermal cycler.

Reaction conditions are as follows:

as a result of the experiment, as shown in FIG. 1, we found that the expression of UCN3 gene was significantly down-regulated by Ad-Acsl4, while that of Aldh1a was significantly increased, and in addition, the mRNA levels of Acc1 and MafB were also up-regulated in β RicKO + Ad-Acsl4 pancreatic islets. FIG. 2 shows that in Min6 cells, sifB and Ngn3 were increased in siRic + Ad-GFP, Ucn3 was decreased, and Acc1 was increased and Nkx6.1 was decreased after Acsl4 was overexpressed.

After isolating mouse islets for 16 weeks and overexpressing Acsl4 virus in vitro for 48 hours, Western Blot and primary islet cell patch immunofluorescence staining revealed that Nkx6.1 and MafA expression in beta RicKO + Ad-Acsl4 islets were both significantly reduced, and the dedifferentiation marker Aldh1a3 was significantly increased, indicating the presence of hyperglycemia or the dedifferentiation state of beta cells (FIG. 3, FIG. 4).

Taken together, the changes in the expression of these key factors that maintain the state of beta cell differentiation, both at the transcriptional and translational levels, and in particular the increase in Aldh1a3, the decrease in FoxO1 and MafA, all agreed to suggest that beta cell dedifferentiation may occur in cells of beta RicKO + Ad Acsl 4.

(2) The improvement of the oxidative stress state of beta RicKO + Ad-Acsl4 can correct the expression of MafA and the functional differentiation state of beta cells, and whether the oxidative stress state is corrected or not is detected by using Glutathione (GSH) which is a reduction product. The method specifically comprises the following steps:

1) after cell plating treatment, siRictor lentivirus was infected with Ad-Acsl4 adenovirus for 72 hours,

2) on the day of the experiment, GSH was added for 2 hours.

3) Measurement of H₂O₂With prior addition of H₂O₂(50nM) substrate incubation for 6 hours, followed by addition of Ultra-Glo recombinant luciferase and d-cysteine to generate H₂O₂Proportional luminescence signal, protected from light for 20 minutes at room temperature. Measured using a microplate reader.

4) Cellular proteins were collected, and concentrations were measured as calibration and calculated. By H₂O₂As can be seen, after 2 hours of GSH addition, H was observed for either the control group plus drug or the β RicKO + Ad-Acsl4 group₂O₂All levels of (a) were allowed to reach normal levels, with a significant decrease in ROS production (fig. 5).

At the protein level, Western Blot showed that after 2 hours of GSH, MafA levels recovered, reaching essentially the same level as in the normal group (fig. 6).

Addition of rotenone/antimycin a inhibitor of mitochondrial respiratory chain complexes 1 and 3 to Min6 cell line overexpressing Acsl4 for 8 hours mimicked the mitochondrial respiratory impairment state in β RicKO islet cells. Western Blot showed that as the concentration of mitochondrial respiratory inhibitor treatment was increased, not only did MafA no longer increase due to Acsl4 overexpression, but rather, the expression levels were significantly reduced stepwise. Caspase3 suggested that this treatment did not significantly increase apoptosis (FIG. 7). Therefore, we believe that overexpression of Acsl4, in the case of a slightly impaired mitochondrial function, such as mTORC2 signaling inhibition, may induce mobilization of lipids and drive lipid oxidation to exacerbate oxidative stress states in beta cells, thereby reducing MafA levels.

(3) Overexpression of Acsl4 corrected for elevated Sirt1 levels caused by Rictor knockout

FoxO1 plasmid with HA-tag and Acsl4 adenovirus were overexpressed on Min6 cell line, cells were harvested after 48 hours, FoxO1 was enriched with HA antibody, and the degree of acetylation of FoxO1 was detected by co-IP. In Rictor knockout islets and siRic Min6 cells, we observed a significant increase in Ad-Acsl4 in FoxO1 and its downstream P27, still significantly in mTORC 2/Rictor-inhibiting beta cells (fig. 8). More interestingly, we found that the classical downstream phosphorylation regulatory signal pAKTS473 of mTORC2 was not affected by Acsl4 overexpression. Rictor/pAKTS473 is the major regulatory factor that facilitates the regulation of FoxO1 phosphorylation and ubiquitination modifications.

Sirt1 is a deacetylase for FoxO1 in liver, and we found that Sirt1 levels were elevated in β RicKO islets and siRic Min6 cells infected with Ad-GFP, suggesting that knockout Rictor might help deacetylate FoxO1 by raising Sirt 1. However, Western Blot showed Sirt1 protein level expression in mouse islets and Min6 cells: sirt1 levels were significantly suppressed after overexpression of Acsl4, synchronized with FoxO1 acetylation, and were not affected by Rictor knockdown (fig. 9). It was suggested that direct regulation of Sirt1 by Acsl4 might also act downstream of Rictor, thereby increasing protein acetylation, especially of FoxO 1. At the same time, we also see that the catalytic protein acetylates the substrate, acetyl coenzyme is obviously increased after over-expression of Acsl4, and further support the over-acetylation of FoxO1 in Ad Acsl4+ beta RicKO. Acetylated FoxO1, more prone to stay in the cytoplasm rather than entering the nucleus, is more susceptible to degradation by ubiquitination. Acetylated FoxO1 is also a key contributor to the promotion of lipid beta oxidation in beta cells and may therefore also contribute to increased lipid oxidation, oxidative stress and dedifferentiation of beta cells in beta RicKO + Ad-Acsl 4.

In conclusion, expression changes of a series of key beta cell transcription factors caused by activation and protein modification of Acsl4 long-chain fatty acid in islet cells are found, and the important role of mTORC2/Acsl4 in maintaining the differentiation state of mature beta cells is highlighted. It can be seen that the beta cell dysfunction of diabetes may be in the conditions of excessive lipid intake, metabolic disturbance and insulin resistance, two type 2 diabetes risk factors, perhaps under metabolic stress or the slow proliferation of Rictor knockout cells, and the low expression of Acsl4 plays a role in protecting beta cells and delaying or preventing the occurrence of type 2 diabetes.

Sequence listing

<110> Renjin Hospital affiliated to Shanghai university of transportation medical school

SHANGHAI INSTITUTE OF ENDOCRINE AND METABOLIC DISEASES

Application of <120> Acsl4 in preparation of type 2 diabetes mellitus medicines

<130> 1

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 1

gcaauuugau ugcuggaaut t 21

<210> 2

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 2

auauucuuga ugaagcuugu tt 22

<210> 3

<211> 41

<212> DNA

<213> Artificial sequence ()

<400> 3

tgacagactg atcgcagaga aagtggagag ccccacacac a 41

<210> 4

<211> 41

<212> DNA

<213> Artificial sequence ()

<400> 4

aggctgtatt aagaccttca gggaagttcc atggtgtaat g 41

<210> 5

<211> 40

<212> DNA

<213> Artificial sequence ()

<400> 5

cccaggccgg agtttaaccg ttgctcataa agtcggtgct 40

<210> 6

<211> 42

<212> DNA

<213> Artificial sequence ()

<400> 6

tagtgaccag ctataatcag agacgccaag gtctgaaggt cc 42

<210> 7

<211> 39

<212> DNA

<213> Artificial sequence ()

<400> 7

ccctgctggc cctgctctta ggtctgaagg tcacctgct 39

<210> 8

<211> 40

<212> DNA

<213> Artificial sequence ()

<400> 8

ttcagcaagg aggaggtcat ctctggagct ggcacttctc 40

<210> 9

<211> 43

<212> DNA

<213> Artificial sequence ()

<400> 9

ctgcgcccct agccctggac tcggcggccc tggcactcac aaa 43

<210> 10

<211> 39

<212> DNA

<213> Artificial sequence ()

<400> 10

gtcgggagaa ctaggatggc ggagcagtcc ctaggtatg 39

<210> 11

<211> 38

<212> DNA

<213> Artificial sequence ()

<400> 11

gaagcgtggt gttgagatga gggcccttcc aaacaagt 38

<210> 12

<211> 34

<212> DNA

<213> Artificial sequence ()

<400> 12

gctgtgcccc tcgaccttgg gcatcagcat cgct 34

Claims

Use of Acsl4 in the preparation of a medicament for the treatment of diabetes.
Use of Acsl4 in the preparation of a medicament for the treatment of type 2 diabetes.
3. Use according to claim 2, comprising correcting or preventing a disorder of lipid metabolism by expression of Ascl 4; or, preventing metabolic remodeling of beta cells followed by dedifferentiation; alternatively, the effect is obtained by protecting the function of the beta cells from decline.
4. The type-2 diabetes mellitus medicinal preparation is characterized by being prepared from Acsl4 serving as an active component and pharmaceutically acceptable auxiliary materials or auxiliary components.
5. The pharmaceutical preparation according to claim 4, wherein the preparation is selected from one of tablets, powders, granules, capsules, oral liquids and sustained release agents.
6. A pharmaceutical preparation for type 2 diabetes mellitus, which is characterized in that a component for reducing the expression of Acsl4 is used as an active component.
7. A pharmaceutical preparation for type 2 diabetes mellitus, which is characterized by comprising a component for promoting expression of Acsl4 as an active ingredient.
8. A medicinal preparation for treating type 2 diabetes is characterized in that a viral vector containing an Acsl4 related sequence is used as an active component.
9. The pharmaceutical formulation of claim 8, wherein the viral vector is an adenoviral vector.