WO2019118407A1 - Combination therapies for treatment of type ii diabetes - Google Patents

Abstract

Provided herein, in some embodiments, are combination therapies for treating type II diabetes and/or symptoms associated with type II diabetes.

Description

COMBINATION

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 62/596,987, filed December 11, 2017, which is incorporated by reference herein in its entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos. 5 U01 AG022308- 15, 5 R01 AG038560-05, and 5 R01 AG032333-05 awarded by National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Type 2 diabetes (T2D) is a chronic condition that affects sugar (glucose) metabolism. With T2D, the body either resists the effects of insulin or does not produce enough insulin to maintain a normal glucose level. Metformin (Glucophage^®), often used in combination with sulfonylurea or with insulin, is used to treat high blood sugar levels caused by T2D; however, long-term effects of metformin include increased kidney toxicity.

SUMMARY

The present disclosure provide, in some embodiments, combination therapies that increase (e.g., normalize) insulin sensitivity. The therapies provided herein use both metformin and rapamycin, the combination of which unexpectedly tempers the negative effects of metformin and rapamycin while exploiting their benefits. As shown herein, metformin administered alone, reduced hyperinsulinemia but exacerbated nephropathy. Rapamycin administered alone reduced weight gain and inflammation and prevented hyperinsulinemia and hepatic steatosis but exacerbated hyperglycemia, hypertriglyceridemia, and pancreatic islet degranulation. Surprisingly, however, metformin and rapamycin, when combined, work synergistically to retain the benefits of both and to prevent deleterious effects.

Starting at 12 weeks of age, mice were fed a control diet or diets supplemented with rapamycin, metformin, or a combination of both drugs. The combination of rapamycin and metformin normalized insulin sensitivity in the inherently insulin resistant NONcNZOlO/LtJ (NcZlO) males. Gene expression differences between treatment groups identified potential molecular mechanisms. In adipose tissue, rapamycin attenuated expression of genes associated with adipose tissue expansion ( Mest , Gpam ) and inflammation ( Itgam , Itgax, Hmoxl, Lbp, Serpinel). In liver, metformin counteracted rapamycin-induced alterations of Ppara, G6pc, and Ldlr expression that promote hyperglycemia and hypertriglyceridemia. Both rapamycin and metformin reduced hepatic Fasti consistent with increased insulin sensitivity and prevention of steatosis. These results produce a state of“insulin restriction” that, via multiple physiologic feedback loops, withdraws endocrine support for further adipogenesis, progression of the metabolic syndrome, and development of its co-morbidities. The results described herein are relevant for treating T2D.

Thus, some aspects of the present disclosure provide methods that include administering to a subject (e.g., a human subject) having T2D a therapeutically effective amount of rapamycin and metformin. In some embodiments, the therapeutically effective amount is sufficient to increase insulin sensitivity (e.g., by decreasing plasma insulin and/or plasma glucose) in the subject. In some embodiments, the therapeutically effective amount is sufficient to normalize insulin sensitivity in the subject.

In some embodiments, the type 2 diabetes is a genetic form of type 2 diabetes.

In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to increase insulin sensitivity in the subject, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of metformin without rapamycin.

In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to normalize insulin sensitivity in the subject.

In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to decrease (or prevent an increase of) plasma glucose levels (hyperglycemia) and/or HbAlc levels in the subject by at least 20%, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of rapamycin without metformin.

In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to decrease (or prevent an increase of) plasma insulin levels (hyperinsulinemia) in the subject by at least 20%, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of metformin without rapamycin.

In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to decrease (or prevent an increase of) pancreatic insulin content (PIC) in the subject by at least 20%, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of metformin without rapamycin. In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to decrease (or prevent an increase of) pancreatic islet degranulation in the subject, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of rapamycin without metformin.

In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to decrease (or prevent an increase of) weight gain in the subject by at least 20%, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of metformin without rapamycin.

In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to prevent weight gain in the subject.

In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to decrease (or prevent an increase of) adiposity (% fat) in the subject by at least 20% .

In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to decrease (or prevent an increase of) obesity-associated inflammation in the subject, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of metformin without rapamycin.

In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to decrease (or prevent an increase of) hepatic steatosis in the subject by at least 50%, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of metformin without rapamycin.

In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to prevent hepatic steatosis in the subject, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of metformin without rapamycin.

In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to decrease (or prevent an increase of) nephropathy in the subject, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of metformin without rapamycin.

In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to decrease (or prevent an increase of) a proportion of glomeruli with hyaline thrombi in the subject.

In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to decrease (or prevent an increase of) the albumin/creatinine ratio (ACR) in the subject. In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to decrease (or prevent an increase of) hypertriglyceridemia in the subject, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of rapamycin without metformin.

In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to decrease (or prevent an increase of) HLDL cholesterol in the subject by at least 10% .

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D: RAPA- and RAPA/MET-treatments reduced obesity. (FIG. 1A, Body Weight (g)) RAPA- and RAPA/MET-treatments prevented adult weight gain typical of NcZlO mice (P < 0.0001, repeated measures MANOVA for both RAPA and RAPA/MET vs. UNT). MET-treatment had no effect. (FIG. IB, % Fat (left), EPI Fat Wt. (middle), ING Fat Wt (right)) RAPA and RAPA/MET-treatments significantly reduced body fat, determined by DXA, with reduction in inguinal (ING) fat pad weight but not in the epididymal (EPI) fat pad weight. (FIG. 1C, Food Intake (kcal/d/kg lean wt) Treatments had no effect on food consumption. (FIG. ID, Adipose Gene Expression (AU/Tbp)) While RAPA- and RAPA/MET -treatment reduced Mest and Gpam expression in both fat pads, they increased Slc2a4 in EPI, but not ING, fat. Treatment effects on Lep expression mirrored effects on fat pad weight in both fat pads. Ucpl expression was unaffected by any treatment in either fat pad, and RAPA- and RAPA/MET-treatment reduced Dio2 in both, indicating that increased thermogenesis was not involved in decreased adiposity in any treatment. Mean ± SE. N = 9-10 mice per treatment except N = 4 mice per treatment for % Fat, ING Fat Wt., Food Intake, and ING fat gene expression. Within each histogram, treatment groups not annotated by the same superscript letter are significantly different at P < 0.05 (Tukey-Kramer HSD). Untreated = UNT and U, RAPA-treated = RAPA and R, MET-treated = MET and M, RAP A/MET -treated = RAPA/MET and R/M.

FIGS. 2A-2B: RAPA/MET-treatment prevents RAPA-mediated elevation of

triglycerides. (FIG. 2A, Triglycerides (mg/dl)) RAPA-treatment increased plasma triglyceride level, but combination with MET blocked this effect. (FIG. 2B, Hepatic Gene Expression (AU/Tbp)) RAPA-treatment reduced hepatic Ldlr expression; co-treatment with MET blocked the reduction of Ldlr expression and increased Ppara expression consistent with triglyceride level. Mean ± SE. N = 6-8 per treatment for triglycerides and 9-10 per treatment for gene expression. Significance annotations and treatment abbreviations as in FIGS. 1A-1D.

FIGS. 3A-3C: RAPA-treatment prevents hepatic steatosis. (FIG. 3A, % Steatosis) Reduction of steatosis was suggestive in MET-treated mice; steatosis was completely prevented in RAPA- and RAPA/MET -treated mice (nominal logistic fit, likelihood ratio test for presence of steatosis: overall, P < 0.0001; post hoc pairwise tests: UNT vs MET, P = 0.06; UNT vs RAPA or RAPA/MET, P < 0.0001). (FIG. 3B, Hepatic Gene Expression (AU/Tbp)) Ah 3 treatments reduced expression of Fasn, a key determinant of fatty acid synthesis. (FIG. 3C, Hepatic

Pathology) Representative histology of moderate steatosis in UNT mice, mild steatosis in MET- treated mice, and absence of steatosis in RAPA- and RAPA/MET-treated mice. Mean ± SE. N = 9-10 mice per treatment. Significance annotations and treatment abbreviations as in FIGS. 1A- 1D.

FIGS. 4A-4E: RAPA-treatment reduced extent of inflammation. Each mouse was scored (0-4) for number of tissues (pancreas, liver, kidney, spleen) with histologic evidence of inflammation. For analysis, numbers of mice with 3 or 4 affected tissues were combined. (FIG. 4A) Treatment significantly affected the extent of inflammation (P = 0.0006, ordinal logistic fit, likelihood ratio test). MET-treated mice had the greatest extent of inflammation. RAPA- and RAPA/MET-treatment significantly reduced the extent of inflammation compared to MET- treatment (post hoc pairwise analysis, ordinal logistic fit). (FIG. 4B, Gene Expression

(AU/Tbp)) RAPA- and RAPA/MET-treatment reduced Itgam and Itgax expression in

epididymal fat tissue and Itgax expression in inguinal fat tissue, indicative of diminished myeloid cell infiltration possibly mediated in part by reduced Hmoxlexpression. Serpinel, a key component of the senescent cell“secretome”, was reduced by both RAPA- and RAPA/MET- treatment in both fat pads, and Lbp was reduced by both treatments in epididymal fat pads. (FIG. 4C) Extent of inflammation was unrelated to plasma glucose levels at 28 weeks. Mice with more extensive inflammation had significantly (FIG. 4D) greater body weight at 28 weeks and (FIG. 4E) higher plasma insulin at 24 weeks. Mean ± SE. N = 9-10 mice per treatment except N = 4 per treatment for ING fat gene expression. Significance annotations and treatment abbreviations as in FIGS. 1A-1D.

FIGS. 5A-5E: RAPA/MET-treatment normalized insulin sensitivity. (FIG. 5A, Plasma Insulin (ng/ml)) UNT mice progressed toward hyperinsulinemia. MET-treatment reduced this hyperinsulinemia ( P = 0.01 vs. UNT after 12 weeks of treatment, Tukey-Kramer HSD). RAPA- and RAPA/MET-treatment prevented hyperinsulinemia ( P < 0.0001 for both vs. UNT after 12 weeks of treatment, Tukey-Kramer HSD). (FIG. 5B, Insulin Sensitivity (%basal value)) UNT mice were insulin resistant, indicated by the insulin tolerance test (ITT). RAPA-treatment for 17 weeks improved insulin sensitivity (repeated measures MANOVA, P = 0.03 vs. UNT; one RAPA-treated mouse with glucose values >600 mg/dl at 45 and 60 minutes after insulin injection was censored as an outlier). MET-treatment had no effect. RAPA/MET-treatment normalized insulin sensitivity to a level equivalent to that of untreated C57BL/6J males (repeated measures MANOVA, P = 0.005 vs. UNT). (FIG. 5C, Plasma Glucose (mg/dl)) UNT mice developed hyperglycemia as expected (15, 19). RAPA-treatment further elevated this hyperglycemia by 12 weeks of treatment ( P = 0.03, Tukey-Kramer HSD). MET-treatment had no effect. After treatment was initiated, RAPA/MET-treatment reduced hyperglycemia compared to RAPA-treatment alone (repeated measures MANOVA, 16-28 weeks of age, interaction of treatment with time, P = 0.03). Glucose levels in RAPA/MET -treated mice were comparable to levels in UNT mice after 16 weeks of treatment. (FIG. 5D, HbAlc (%IFCC Units))

RAPA/MET-treatment prevented RAPA-driven elevation of HbAlc, measured 18 weeks after treatment was initiated. (FIG. 5E, Hepatic Gene Expression (AU/Tbp)) Reduced hepatic Ppargcla expression in all treated mice, in the context of diminished circulating insulin, is indicative of increased hepatic insulin sensitivity and consistent with reduced gluconeogenesis. Reduced hepatic Gck and increased G6pc expression in RAPA-treated mice is associated with elevated hyperglycemia. RAPA/MET-treatment counteracts this effect likely by preventing the rapamycin-associated elevation of G6pc. Mean ± SE. N = 9-10 mice per treatment (a, c, e); N = 5-6 mice per treatment (b, d). Significance annotations and treatment abbreviations as in FIGS. 1A-1D.

FIGS. 6A-6C: Co-treatment with metformin prevented negative effects of RAPA- treatment on pancreatic islet morphology. All islets from 3 separate sections (per pancreas) were scored for granulation status (insulin storage): F = fully-granulated; P = partially-degranulated; or M = mostly-to-completely-degranulated. (FIG. 6A, Islet Morphology) UNT mice exhibited a pattern of -20% of islets fully granulated, -60% of islets showing partial degranulation, and -20% showing mostly-to-completely-degranulated. Treatment affected the islet degranulation profile (P = 0.0006, MANOVA, Wilk’s l). RAPA-treatment significantly exacerbated the phenotype (post hoc pairwise comparisons, P = 0.02, MANOVA, Wilk’s l). MET-treatment had no effect vs. UNT. RAPA/MET-treatment prevented the effect of RAPA-treatment on islet morphology (P = 0.03, MANOVA, Wilk’s l). (FIG. 6B, Pancreatic Insulin Content (ng/mg)) RAPA-, and RAPA/MET-treatment reduce pancreatic insulin content (FIG. 6C, Representative Islets) Representative islets illustrate effects of treatment on islet morphology (aldehyde fuchsin staining). The islet from an UNT mouse illustrates partial degranulation. The islet from a RAPA- treated mouse illustrates the mostly degranulated condition of the majority of islets in RAPA- treated mice. Islets from the MET- and RAPA/MET-treated mice illustrate the partial degranulation typical of islets from these mice. All histologic pictures are at the same

magnification. Mean ± SE. N = 9-10 mice per treatment. Significance annotations and treatment abbreviations as in FIGS. 1A-1D. FIGS. 7A-7C: RAPA- and RAPA/MET-treatment reduced development of nephropathy. (FIG. 7A, Glomerular Nephritis) MET-treatment increased the percentage of glomeruli showing nephritis; RAPA/MET-treatment prevented the effect of metformin on nephritis. (FIG. 7B, Hyaline Thrombi) RAPA- and RAPA/MET-treatment reduced the percentage of glomeruli exhibiting hyaline thrombi. (FIG. 7C, Albumin Creatine Ratio (ACR)) MET-treatment produced the highest urinary ACR level; RAPA/MET-treatment prevented the effect of metformin on ACR level. Mean ± SE. N = 9-10 mice per treatment. Significance annotations and treatment abbreviations as in FIGS. 1A-1D.

FIG. 8: Summary of treatment effects in the T2D NONcNZOlO/LtJ strain. RAPA/MET- treatment maintained positive effects of RAPA-treatment on various aspects of T2D while ameliorating negative side effects of RAPA-treatment and MET-treatment alone. *RAPA- treatment synergized with MET-treatment to completely normalize insulin sensitivity in

RAPA/MET-treated mice.†While MET-treatment reduced hyperinsulinemia, RAPA- and RAPA/MET-treatments both completely prevented hyperinsulinemia. Untreated = UNT, RAPA- treated = RAPA, MET-treated = MET, RAPA/MET-treated = RAPA/MET.

FIG. 9: Insulin restriction due to RAPA/MET combination treatment in this T2D mouse model generates multiple overlapping positive feedback loops leading to reduced co-morbidities and normalized insulin sensitivity.

DETAILED DESCRIPTION

In healthy adult mice, rapamycin treatment typically produces delayed glucose clearance, often, but not always, in conjunction with insulin resistance, depending on strain and

experimental protocol (10-12). Yet in a number of mouse models of type 2 diabetes (T2D), rapamycin treatment promotes insulin sensitivity and ameliorates multiple T2D-driven impairments, even while exacerbating hyperglycemia (13-17). Resolution of the physiological and genetic mechanisms that foster this paradox can identify key regulatory junctures that govern the outcome of rapamycin treatment. We investigated these mechanisms using the

NONcNZOlO/LtJ (NcZlO) mouse strain, which reflects attributes of human age-related T2D, including polygenic adult-onset hyperglycemia driven by moderate obesity with insulin resistance (18, 19). Rapamycin improves insulin sensitivity and reduces diabetic nephropathy in this model, despite elevating diabetic hyperglycemia (14, 15). To modulate the glycemic influence of rapamycin, we studied combined rapamycin-metformin treatment in NcZlO males. We employed long-term dietary treatment in a mammalian model of maturity-onset T2D that generates co-morbidities relevant to human T2D. This strategy identified key physiological and genetic regulatory mechanisms affected by the combination treatment that resulted in improved health outcomes.

The results provided herein demonstrate that it is possible to modulate the hyperglycemic effect of RAPA-treatment without diminishing its beneficial effect on the diabetic

pathophysiology or creating significant additional impairment, even in a model system that is inherently sensitive to the promotion of hyperglycemia, the NcZlO mouse. Addition of MET not only blocked the RAPA-elevated hyperglycemia, but also elevated hyperlipidemia and exacerbated pancreatic islet degranulation. The beneficial drug interaction was reciprocal: RAPA blocked the nephrotoxic effect of MET. Furthermore, the inhibition, by either drug, of the hepatic insulin resistance inherent to NcZlO mice was sustained during combined treatment. Importantly, both drugs synergized to generate completely normal systemic insulin sensitivity in the innately insulin resistant NcZlO model (FIG. 8).

The effects of RAPA/MET-treatment on the physiological systems are integrated through multiple feedback networks operating in concert (FIG. 9). Because insulin level is a key signal in the metabolic syndrome, we examined its regulation to understand the mechanisms by which RAPA- and RAPA/MET-treatments modulate diabetes and its co-morbidities.

RAPA-treatment dramatically prevented the weight gain associated with development of adiposity. Effects on adipose tissue and gene expression that could increase insulin sensitivity and reduce circulating insulin include diminished adipose tissue expansion (e.g., \Mest, [Gpcim), enhanced adipose tissue glucose uptake (e.g., Slc2a4) and diminished adipose inflammation (e.g., Hmox, \Lbp, jPAI- 1 ). Effects on gene expression that could reduce insulin sensitivity include elevation of circulating triglycerides (e.g., \Ldlr). Combination of MET with RAPA countered the negative effects of RAPA-treatment alone without diminishing its positive effects. The combined RAPA/MET-treatment prevented the progressive elevation of circulating insulin and normalized insulin tolerance to a level comparable to that of young, insulin sensitive, C57BL/6J males.

MET-treatment, like RAPA-treatment, reduced expression of a key transcription factor governing gluconeogenesis, Ppargcla. But unlike RAPA-treatment, MET-treatment did not shift the ratio of genes that regulate glucose phosphorylation ( Gck and G6pc ) to promote hepatic glucose export. MET, without being bound by theory, apparently overrides the hyperglycemic effect of RAPA-treatment on hepatic glucose transport genes to enable expression of the hypoglycemic effect of MET and RAPA on Ppargcla expression. Consequently, the

combination treatment prevented RAPA-induced hyperglycemia. An additional benefit of co treatment with MET was the prevention of RAPA-induced pancreatic islet degranulation. While MET had no effect on the RAPA-mediated loss of PIC and islet number that may reduce functional insulin reserves, MET-associated suppression of RAPA-driven hyperglycemia confers a protective effect on pancreatic b-cells by reducing hyperglycemic stress and thus reducing islet degranulation.

The retention of low-normal circulating insulin levels in RAPA/MET-treated mice cannot be completely explained by increased insulin sensitivity and reduced hyperglycemia. Plasma glucose was diminished to the level in untreated diabetic NcZlO mice, whereas circulating insulin was maintained at a level comparable to that of young pre-diabetic mice. Thus, an additional mechanism likely restricts insulin from reaching levels that would reduce plasma glucose to normal. Possibilities include reduced islet number, advanced islet degranulation status, and diminished pancreatic sensitivity to glucose (8). Without being bound by theory, we propose that, in the context of a T2D model, the metabolic paradox of diminished circulating insulin in the presence of exaggerated hyperglycemia that is created by RAPA-treatment is a consequence of an“insulin restriction” state, constraining the pancreatic response to glucose activation. Reduced PIC (14) and circulating insulin (73-76) are consistently associated with improved metabolic outcomes and reduction of co-morbidities, and in genetically heterogeneous mice of both sexes, RAPA- and RAPA/MET-treatments identical to that used in the present study consistently increased lifespan (1, 2, 20, 77-80).

The relevance of insulin restriction for optimization of treatments highlights the need to develop strategies that resolve the trade-off between the benefits of insulin restriction and the existent risks of hypoinsulinemia. Our findings indicate that RAPA/MET-treatment can be used to identify key regulatory elements that govern this trade-off. RAPA-treatment alone promotes insulin sensitivity through multiple mechanisms. In addition, RAPA-treatment alters glucose stimulated insulin secretion (GSIS) by diminishing b-cell sensitivity to glucose activation as well as islet number and PIC (8, 62). These effects produce an insulin restriction that could contribute to the protection from numerous degenerative conditions associated with the metabolic syndrome. However, a concern is that RAPA could create a hypoinsulinemic state in vulnerable individuals. Metformin provides protection from this vulnerability by limiting the hyperglycemic challenge to b-cells that creates islet degranulation and promotes reduction of PIC, while not limiting the insulin restriction generated by RAPA-treatment. GSIS is still suppressed, without jeopardizing future health of individual islets. Thus, RAPA, when combined with a selective anti-hyperglycemic treatment such as MET, provides the foundation for development of a new class of therapeutic regimens for treatment of diabetes and its co-morbidities.

The RAPA-driven decoupling of hyperglycemia from hyperinsulinemia in the NcZlO model of the metabolic syndrome also has clinical relevance as T2D-associated co-morbidities were reduced or prevented by RAPA despite elevation of circulating glucose in the context of normalized circulating insulin. Hyperinsulinemia is recognized as a correlate of multiple aspects of diabetes in humans (63, 73-76). The differential effects of hyperglycemia and

hyperinsulinemia on the pathogenic profile of the diabetic syndrome may account for the recently recognized sub-pattems of co-morbidity expressed among T2D patients (63). The prevention of hyperinsulinemia, without hypoinsulinemia, in the context of hyperglycemia (RAPA/MET-treatment) or exacerbated hyperglycemia (RAPA-treatment alone) in the NcZlO model system should be valuable to resolve the specific pathogenic roles of glucose and insulin and their interaction in the metabolic syndrome. In addition, diagnosis of T2D and the monitoring of its management would greatly benefit from the routine assessment of circulating insulin.

Further, multiple pre-clinical studies indicate that rapamycin, and its analogues, can have positive effects on chronic age-related impairments, including cardiomyopathy (16, 17, 81), nephropathy (15, 16, 66-71, 82-84), neurodegeneration (including Alzheimer’s disease [85,

86]), osteoporosis (87), and diabetic co-morbidities (13-15). Although broad use of rapamycin as an anti-ageing treatment has been contraindicated by its immunosuppressive and hyperglycemic effects, recent promising research indicates that immunosuppressive effects can be attenuated by alternate treatment schedules and lower doses than used for organ transplantation, and by use of various rapalogues (82, 88-92). The present study demonstrates that the hyperglycemic effects of RAPA can be managed with anti-hyperglycemic co-treatments such as MET and that the benefits of RAPA can even be enhanced by such co-treatment.

The present disclosure provides a method comprising administering to a subject having type 2 diabetes a therapeutically effective amount of rapamycin and metformin.

Type 2 Diabetes

Approximately 30.5 million Americans have diabetes, and of these approximately 29 million suffer from type 2 diabetes (T2D). T2D is characterized by elevated plasma glucose levels ( e.g ., 126 mg/dL after an overnight fast) and resistance to insulin. Risk factors for developing T2D include, but are not limited to: being overweight or obese, fat distribution, inactivity, family history, race, having elevated plasma glucose levels (also referred to as blood glucose levels) (e.g., prediabetes), gestational diabetes, and polycystic ovarian syndrome.

Prediabetes is a condition in which the fasting plasma glucose level of a subject is elevated above normal levels (e.g., less than 100 mg/dL), but is less than diabetic levels (e.g., greater than or equal to 126 mg/dL). Non-limiting examples of common symptoms of T2D include high plasma glucose (hyperglycemia), increased thirst, frequent urination, increased hunger, weight loss, fatigue, blurred vision, slowly-healing sores, frequent infections, and areas of darkened skin.

Plasma glucose levels are regulated by production and release of the hormone insulin from the beta cells in the Islet of Langerhans in the pancreas. Insulin is released by the pancreas into the bloodstream, where it lowers the plasma glucose level by stimulating cells to take up glucose from the blood stream. In patients with T2D, glucose uptake by cells is impaired, leading to hyperglycemia, which stimulates the beta cells to release more insulin. Over time, the production of insulin becomes impaired and the cells become resistant to insulin, leading to chronic hyperglycemia.

Complications of T2D can be disabling or even life-threatening. Non-limiting examples of complications of T2D include heart and blood vessel disease ( e.g ., heart attack, coronary artery disease, angina, stroke, narrowing of the arteries, high blood pressure), nerve damage (e.g., tingling, numbness, or burning in the extremities that slowly spreads upward; loss of feeling in the limbs; digestion side-effects including nausea, vomiting, diarrhea, and

constipation; and erectile dysfunction), kidney damage (e.g., end-stage kidney disease requiring dialysis or transplant), eye damage (diabetic retinopathy, blindness, cataracts, and glaucoma), foot damage, hearing impairment, skin conditions (e.g., bacterial and fungal infections), and Alzheimer’s disease.

In some embodiments, a subject having T2D is a mammal, optionally, a human, a mouse, a rat, a dog, a cat, a chicken, a pig, a rabbit, or a non-human primate.

In some embodiments the T2D is a genetic form of T2D. A genetic form of T2D refers to development of T2D, at least in part, due to genetic changes in a subject rather, whereas spontaneous T2D refers to development of T2D as a result of environmental factors. Non limiting examples of genes associated with developing T2D include NOTCH2, PROXJ /RSI, THADA, RBMS1/ITGB6, BCL11A, GCKR, IGF2BP2, PPARG, ADCY5, ADAMTS9, ZBED3, CDALK1, JAZF1, GCK, KLF14, DGKB/TMEM195, SLC30A8, TP53INPI, CDKN2AJB, TLE4, TCF7L2, HHEX, CDC123/CMK1 D, KCNQ1, KCNJ11/ABCC8, CENTD2, MTRN1B, KCNQ1, HMGA2, TSPAN8/LGR5, OASUHFN1A, PRd, ZFAND6, FTO, HNF1B, and DUSP9B.

Combination Therapies

The present disclosure is based on experimental data showing that administration of a therapeutically effective amount of rapamycin and metformin alleviates or prevents symptoms associated with T2D while preventing drug-associated side effects. Rapamycin, when given alone to subjects that have undergone or will undergo transplantation, can cause increased plasma glucose levels, even to the point of hyperglycemia and diabetes (e.g., greater than or equal to 126 mg/dL after an overnight fast). Metformin, while currently utilized as a treatment for hyperglycemia in T2D subjects, can cause kidney damage ( e.g ., diabetic nephropathy, lactic acidosis) with long term use. The present disclosure is based on data showing that administration of both rapamycin and metformin as a combination therapy to a subject having T2D does not induce hyperglycemia or kidney damage in the subject. Moreover, administration of the combination of drugs increases insulin sensitivity in subject relative to control subject.

A therapeutically effective amount of rapamycin and metformin refers to an amount of the drugs sufficient to produce a desired response (e.g., increasing insulin sensitivity by lowering plasma glucose levels and/or lowering plasma insulin levels). Therapeutically effective amounts of a drug, or a combination of drugs, may depend, at least in part, on the age, weight, height, sex, and genetic disposition of the subject.

Assessing the desired effect resulting from administration of a therapeutically effective amount of a combination of rapamycin and metformin includes, in some embodiments, comparing the desired effect to a control. A control may be a subject without T2D, an untreated subject with T2D (e.g., a subject not undergoing drug treatment), or a subject with T2D who is undergoing treatment with only rapamycin or with only metformin. An untreated subject may be the same subject having T2D, prior to receiving treatment. Thus, a control for insulin sensitivity (or for any other T2D-associated symptom provided herein) may be a baseline level (e.g., a baseline level of plasma insulin and/or plasma glucose), for example, insulin sensitivity measured within the 1-3 months prior to the subject receiving treatment for T2D.

Metformin and/or rapamycin may be administered by any route necessary. In some embodiments, metformin and/or rapamycin are administered orally (e.g., oral tablet or suspension). In embodiments, metformin and/or rapamycin are administered intravenously. In embodiments, metformin and/or rapamycin are administered intraperitoneally. In embodiments, metformin and/or rapamycin are administered intramuscularly.

Metformin. Metformin (Fortamet^®, Glucophage^®, Glucophage XR^®, Glumetza^®, or Riomet^®) is a drug administered to patients having T2D to reduce hepatic glucose production (e.g., indirectly lowers plasma glucose) (see, e.g., Song, R., 2016, Mechanism of Metformin: A Tale of Two Sites, Diabetes Care, 39(2): 187-189; Viollet, B., et ah, 2012, Cellular and molecular mechanisms of metformin: an overview, Clin. Sci. (Lond.), 122(6): 253-270; Rojas, et ah, 2013, Metformin: an old but still the best treatment for type 2 diabetes, Diabetology and Metabolic Syndrome, 5(6): 1-15). Some of the side effects associated with metformin

administration in T2D subjects, including nephropathy (e.g., kidney damage), can be

counteracted by co-administration with rapamycin, as described herein. In some embodiments, nephropathy is assessed before, during, and/or after metformin administration, for example, by measuring glomerular filtration rate (GFR). GFR may be measured by any number of methods, including but not limited to, creatinine clearance ( e.g Cockcroft- Gault equation), nonradioactive iohexol (plasma) versus insulin (urinary) clearance, ¹²⁵-I iothalamate clearance, and serum cy statin C. Other side effects of metformin that may be assessed/monitored include other forms of liver impairment and polycystic ovary syndrome.

In some embodiments, a therapeutically effective amount of metformin is 250 mg - 3000 mg per day or every 12 hours. For example, a therapeutically effective amount of metformin may be 250-2500 mg, 250-2000 mg, 250-1500 mg, 250-1000 mg, 250-500 mg, 500-3000 mg, 500- 2500 mg, 500-2000 mg, 500-1500 mg, 500-1000 mg, 750-3000 mg, 750-2500 mg, 750-2000 mg, 750-1500 mg, or 750-1000 mg per day or every 12 hours. In some embodiments, a

therapeutically effective amount of metformin is 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450,

1500, 1750, 2000, 2250, 2500, 2750, or 3000 mg per day or every 12 hours.

Rapamycin. Rapamycin (sirolimus or Rapamune^®) binds and inhibits the mammalian target of rapamycin (mTOR) protein, which is a master regulator of cell growth and metabolism. Rapamycin is administered to subjects undergoing or that will undergo a transplant to prevent an immune response to the transplant, as well as patients having the progressive lung disease lymphangioleimyomatosis (LAM). Some of the side effects associated with rapamycin administration in T2D subjects, including hyperglycemia, hypertriglyceridemia, and pancreatic islet degranulation, can be counteracted by co-administration with metformin, as described herein. Other side effects of rapamycin that may be assessed/monitored include immunologic risk) and liver impairment.

In some embodiments, a therapeutically effective amount of metformin is 0.25 mg - 3 mg per day or every 12 hours. For example, a therapeutically effective amount of metformin may be 0.25-2.5 mg, 0.25-2 mg, 0.25-1.5 mg, 0.25-1 mg, 0.25-0.5 mg, 0.5-3 mg, 0.5-2.5 mg, 0.5-2.0 mg, 0.5-1.5 mg, 0.5-1 mg, 0.75-3 mg, 0.75-2.5 mg, 0.75-2 mg, 0.75-1.5 mg, 0.75-1 mg 1-3 mg, 1-2.5 mg, 1-2 mg, 1.5-3 mg, or 2-3 mg per day or every 12 hours. In some embodiments, a therapeutically effective amount of metformin is 0.25, 1, 1.5, 2, 2.5, or 3 per day or every 12 hours.

In some embodiments, the molar ratio of rapamycin to metformin administered to a subject having T2D is 1:1 - 1:1000. In some embodiments, the molar ratio of rapamycin to metformin administered to a subject having T2D is 1:20 - 1:1000. In some embodiments, the molar ratio of rapamycin to metformin administered to a subject having T2D is 1:100 - 1:500. In some embodiments, the molar ratio of rapamycin to metformin administered to a subject having T2D is at least 1:1. In some embodiments, the molar ratio of rapamycin to metformin administered to a subject having T2D is less than or equal to 1:1000. In some embodiments, the molar ratio of rapamycin to metformin administered to a subject having T2D is 1:1, 1:2, 1:10, 1:25, 1:50, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000.

In some embodiments, rapamycin and metformin are administered to a subject have T2D simultaneously. In some embodiments, rapamycin and metformin that are administered simultaneously are in the same formulation ( e.g drug dose). In some embodiments, rapamycin and metformin that are administered simultaneously are not in the same formulation. In some embodiments, rapamycin and metformin are administered to a subject having T2D sequentially. In some embodiments, rapamycin is administered to a subject having T2D before metformin. In some embodiments, metformin is administered to a subject having T2D before rapamycin.

Insulin sensitivity

Subjects having T2D (a T2D subject) have decreased insulin sensitivity and thus have increased plasma glucose levels (hyperglycemia) and increased plasma insulin levels

(hyperinsulinemia). Insulin sensitivity is a measurement of how responsive cells are to insulin, which stimulates cells to take up glucose from the blood. In some embodiments administering a therapeutically effective amount of rapamycin and metformin is sufficient to increase insulin sensitivity in the subject, relative to a control. In some embodiments, insulin sensitivity is normalized in a T2D subject following administration of a therapeutically effective amount of rapamycin and metformin. Methods of measuring insulin sensitivity (e.g., measuring insulin levels) are described, for example, by Ferrannini E. et al. J Hypertens. 1998 Jul;l6(7):895-906, incorporated herein by reference in its entirety. Non-limiting methods for measuring insulin sensitivity in a subject include the insulin tolerance test (measures whether plasma glucose levels decrease following administration of insulin), the oral glucose tolerance test (OGTT), and the hyperinsulinemic-euglycemic clamp. In an insulin tolerance test (see, e.g., Ayala, et al., 2010, Standard operating procedures for describing and performing metabolic tests of glucose homeostasis in mice, Dis Model Mech., 3(9-10): 525-534, a known amount of insulin is injected into a subject’s vein, after which plasma glucose is measured at regular intervals over a fixed time period. The degree to which blood glucose falls after administration of the insulin is indicative of insulin sensitivity. In an OGTT (see, e.g., Andrikopoulos, et al., 2008, Evaluating the glucose tolerance test in mice, Am J Physiol Endrocrinol Metab., 295: E1323-E1332), a known amount of glucose is administered orally to a subject, after which plasma glucose is measured at regular intervals over a fixed time period. The degree to which blood glucose falls over time after administration of the glucose is also indicative of insulin sensitivity of the subject. In a hyperinsulinemic-euglycemic clamp (see, e.g., Pacini, et al., 2013, Methods and Models for Metabolic Assessment in Mice, J. Diabetes Res. 2013: 1-8), radioactively-labeled glucose (e.g., 3-3H glucose) is continuously administered to a subject to maintain blood glucose levels at a set concentration. The more glucose is needed, the higher the insulin sensitivity in the subject.

In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to increase insulin sensitivity in a T2D subject by at least 20% relative to a control subject. For example, a therapeutically effective amount of rapamycin and metformin is sufficient to increase insulin sensitivity in a T2D subject by at least 25%, 35, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control subject.

In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to increase insulin sensitivity in a T2D subject by 20-100%, 20-75%, 20-50%, 25- 100%, 25-75%, 25-50%, 50-100%, or 50-75% relative to a control subject.

Plasma Glucose

Plasma glucose (also referred to as blood glucose or blood sugar) is the amount of glucose present in the blood of a subject and is regulated by insulin. In a T2D subject, reduced insulin sensitivity leads to the requirement for excessive amounts of insulin that can in turn still not achieve normal glucose levels, at which point hyperglycemia (diabetes) results. Thus, a combination of rapamycin and metformin may be administered to a T2D subject in a

therapeutically effective amount sufficient to decrease plasma glucose levels in a subject.

Normal plasma glucose value ranges may vary. Many factors affect a subject’s plasma glucose level. Glucose homeostasis, when operating normally (e.g., in a non-T2D subject) restores the plasma glucose level to a narrow range of about 4.4 to 6.1 mmol/L (79 to 110 mg/dL) (as measured by a fasting blood glucose test). The normal blood glucose level (tested while fasting) for non-T2D subjects should be between 3.9 and 7.1 mmol/L (70 to 130 mg/dL). The mean normal blood glucose level in humans is about 5.5 mmol/L (100 mg/dL); however, this level fluctuates throughout the day. Plasma glucose levels for those without diabetes and who are not fasting should be below 6.9 mmol/L (125 mg/dL). The blood glucose target range for diabetics, according to the American Diabetes Association, should be 5.0-7.2 mmol/l (90- 130 mg/dL) before meals, and less than 10 mmol/L (180 mg/dL) after meals (as measured by a blood glucose monitor).

Hyperglycemia is a condition in which plasma glucose levels of a subject are above 180— 200 mg/dL, for example, after an overnight fast. In some embodiments, administration of a therapeutically effective amount of rapamycin and metformin prevents hyperglycemia in T2Ds subject. Non-limiting examples of methods for assessing plasma glucose levels include measuring the total plasma glucose levels or measuring glycated hemoglobin (HbAlc). Total plasma glucose may either be measured after an overnight fast or as part of a glucose tolerance test, in which a known amount of glucose is administered to a subject and plasma samples are taken from the subject at time intervals (see, e.g., Togashi, et ah, 2016, Evaluation of the

appropriateness of using glucometers for measuring the blood glucose levels in mice, Sci Rep. 6: 25465). The HbAlc glucose test (see, e.g., Han, et ah, 2008, Markers of glycemic control in the mouse: comparisons of 6-h- and overnight-fasted blood glucoses to HbAlC, Am J Physiol Endocrinol Metab, 295(4): E981-986) measures the percentage glycated hemoglobin and is thus an estimate of the average plasma glucose level over the previous two to three months.

In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease plasma glucose in a T2D subject by at least 20% relative to a control subject. For example, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease plasma glucose in a T2D subject by at least 25%, 35, 35%, 40%, 45%,

50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control subject.

In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease plasma glucose in a T2D subject by 20-100%, 20-75%, 20-50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75% relative to a control subject.

In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease HbAlc levels in a T2D subject by at least 20% relative to a control subject. For example, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease HbAlc levels in a T2D subject by at least 25%, 35, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease HbAlc levels in a T2D subject by 20-100%, 20-75%, 20-50%, 25-100%, 25-75%, 25- 50%, 50-100%, or 50-75% relative to a control subject.

Plasma Insulin

Plasma insulin is the amount of insulin present in the blood of a subject. Insulin is the main hormone involved in blood glucose homeostasis. In some embodiments administering a therapeutically effective amount of rapamycin and metformin is sufficient to prevent

hyperinsulinemia by reducing plasma insulin to normal levels (e.g., -57-79 pmol/L). In some embodiments administering a therapeutically effective amount of rapamycin and metformin is sufficient to decrease plasma insulin (e.g., via decreasing plasma glucose and increasing insulin sensitivity) in a subject relative to a control. Table 1 provides a reference range of insulin levels. Table 1, Reference Range of Insulin Levels (Melmed S, Polonsky KS, Larsen PR, Kronenberg HM. Williams Textbook of Endocrinology. 12th ed. Philadelphia: Elsevier Saunders; 2011)

Insulin Level Insulin Level (SI Units*)

*SI unit: conversional units x 6.945

Insulin levels are normally less than 25 mlU/L (174 pmol/L) after fasting, but are increased in subjects having T2D. Non-limiting methods for measuring plasma insulin in a subject include enzyme-linked immunosorbent assay (ELISA) and competitive ¹²⁵I-insulin antibody binding assay. In an ELISA (see, e.g., MacDonald, et al., 1989, A rapid ELISA for measuring insulin in a large number of research samples, Metabolism, 38(5): 450-452), insulin is detected by an insulin-specific antibody and the amount of insulin present is quantified with a fluorophore conjugated to the antibody. In a competitive ¹²⁵I-insulin binding assay (see, e.g., Human Insulin Specific Radioimmunoassay Kit, 2017, HI-14K, www.emdmillipore.com), radiolabeled l25I-insulin is incubated with an increasing concentration of plasma that contains endogenous insulin and antibody against insulin. The higher the concentration of plasma insulin, the less radiolabeled l25I-insulin will be bound to the insulin antibody.

In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease plasma insulin in a T2D subject by at least 20% relative to a control subject. For example, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease plasma insulin in a T2D subject by at least 25%, 35, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease plasma insulin in a T2D subject by 20-100%, 20-75%, 20-50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75% relative to a control subject.

In some embodiments, the fasting insulin level in a T2D subject following administration of a therapeutically effective amount of rapamycin and metformin is less than or equal to 25 mlU/L (174 pmol/L). For example, the fasting insulin level in a T2D subject following administration of a therapeutically effective amount of rapamycin and metformin may be 10-25, 10-20, 10-15, 15-25, 15-20, 20-25 mlU/L. In some embodiments, the fasting insulin level in a T2D subject following administration of a therapeutically effective amount of rapamycin and metformin is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mlU/L. In some embodiments, the fasting insulin level in a T2D subject following administration of a

therapeutically effective amount of rapamycin and metformin is less than 50 mlU/L, less than 40 mlU/L, or less than 30 mlU/L.

Pancreatic Insulin Content

Pancreatic insulin content (PIC) is the amount of insulin stored in the pancreas of a subject. Subjects having T2D produce and store increased levels of insulin in the pancreas in response to increased plasma glucose levels (hyperglycemia). This increased insulin production and storage can eventually lead to destruction (e.g., degranulation) of the beta-cells (b-cells) in the Islets of Langerhans, which function in the synthesis and release of insulin. In some embodiments, the therapeutically effective amount of rapamycin and metformin is sufficient to decrease pancreatic insulin content (PIC) in the subject, relative to a control

In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease PIC in a T2D subject by at least 20% relative to a control subject. For example, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease PIC in a T2D subject by at least 25%, 35, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease PIC in a T2D subject by 20-100%, 20-75%, 20-50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75% relative to a control subject.

Non-limiting methods for measuring pancreatic insulin content (PIC) include acid- ethanol extraction followed by ELISA detection and immunohistochemistry to stain for insulin. In acid-ethanol extraction (see, e.g., Huang, et al., 2011, Low insulin content of large islet population is present in situ and in isolated islets, Islets, 3(1): 6-13), a sample of the pancreas (e.g., Islets of Langerhans) is extracted and homogenized, followed by ELISA detection using an insulin-specific antibody. In immunohistochemistry (see, e.g., Campbell-Thompson, et al., 2012, Staining Protocols for Human Pancreatic Islets, J Vis Exp (63): 4068), sections of the pancreas (e.g., Islets of Langerhans) are stained with an insulin-specific antibody and the intensity of the staining is quantified as a measure of total PIC.

Pancreatic Islet Degranulation As a result of prolonged increased insulin production, subjects have T2D are susceptible to pancreatic islet degranulation. Pancreatic islet degranulation refers to the breakdown of pancreatic b-cell granules that contain insulin produced by the b-cells.

In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease pancreatic islet degradation in a T2D subject by at least 20% relative to a control subject. For example, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease pancreatic islet degradation in a T2D subject by at least 25%, 35, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease pancreatic islet degradation in a T2D subject by 20-100%, 20-75%, 20-50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75% relative to a control subject.

Non-limiting methods of measuring pancreatic islet degranulation include tissue biopsy and hematoxylin and eosin (H & E) staining, light-scattering analysis, and fluorescence imaging. H & E staining (see, e.g., Fischer, et al., 2014, Hematoxylin and Eosin Staining of Tissue and Cell Sections, Cold Spring Harbor Protocols) produces dark, round staining of the pancreatic b- cell granules. In light-scattering analysis (see, e.g., Ilegems, et al., 2015, Light scattering as an intrinsic indicator for pancreatic islet cell mass and secretion, Scientific Reports, 10740), more regular b-cell granules will scatter light more uniformly than degranulated pancreatic b-cells. In fluorescence imaging, an antibody specific for insulin that is conjugated with a fluorophore can be used to visualize insulin in the pancreatic b-cell granules.

Obesity, Liver Function, and Kidney Function

Subjects having T2D are susceptible to weight gain and obesity as a result of prolonged hyperglycemia and administration of exogenous insulin to compensate for insulin resistance. Liver function and kidney function may also, and often are, adversely affected in T2D subjects.

In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease weight gain in a T2D subject by at least 20% relative to a control subject. For example, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease weight gain in a T2D subject by at least 25%, 35, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease weight gain in a T2D subject by 20-100%, 20-75%, 20-50%, 25-100%, 25-75%, 25- 50%, 50-100%, or 50-75% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to prevent weight gain in a T2D subject (e.g., the subject does not gain a significant amount of weight following treatment with a therapeutically effective amount of rapamycin and metformin). It should be understood that prevention of weight gain refers to weight gain associated with T2D or with drugs used to treat T2D (e.g., as a direct result of increased plasma glucose and/or plasma insulin levels).

Adiposity. In some embodiments, treatment with a therapeutically effective amount of rapamycin and metformin decreases adiposity in a subject. Adiposity refers to fat or fatty tissue and is typically associated with being severely overweight or obese. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease adiposity in a T2D subject by at least 20% relative to a control subject. For example, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease adiposity in a T2D subject by at least 25%, 35, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease adiposity in a T2D subject by 20-100%, 20- 75%, 20-50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75% relative to a control subject. Non-limiting examples of methods for measuring adiposity include dual-energy X-ray absorptiometry (DXA), body adiposity index (BAI), and body mass index (BMI). In DXA (see, e.g., Pandit, et al., 2009, Body Fat Percentages by Dual-energy X-ray Absorptiometry

Corresponding to Body Mass Index Cutoffs for Overweight and Obesity in Indian Children, Clin Med Pediatr, 3: 55-61), two X-ray beams of different energy levels are aimed at a subject and create a low resolution“fat shadow” outside of the bone minerals and lean soft tissues. The fat mass is calculated by subtracting the bone mineral and lean soft tissues from the total body weight. In BAI (see, e.g., Geliebter, et al., 2013, Comparison of Body Adiposity Index (BAI) and Body Mass Index (BMI) with Estimations of % Body Fat in Clinically Severe Obese Women, Obesity (Silver Spring), 21(3): 493-498), the circumference of the hips, which may be correlated with body weight, is compared to the subject’s height while BMI is a correlation of a subject’s weight relative to their height.

Obesity-Associated Inflammation. Subjects with T2D are susceptible to obesity, which can lead to chronic, obesity-associated inflammation. Obesity-associated inflammation occurs when compounds such as monocyte chemoattractant protein 1 (MCP-l) and macrophage migration inhibitor factor (MIF) and the cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-a), and interleukin-l beta ( I L- 1 b) recruit immune cells into adipose tissue, resulting in an inflammatory response in the adipose tissue (Reilly, S.M. et al. Nature Reviews

Endocrinology 13, 633-643 (2017); Palmer, A.K. et al. Exp. Gerontol. 86, 97-105 (2016);

Frasca, D.et al. Front. Immunol. 8, 1745 (2017)). In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease obesity-associated inflammation in a T2D subject by at least 20% relative to a control subject. For example, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease obesity- associated inflammation in a T2D subject by at least 25%, 35, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease obesity-associated inflammation in a T2D subject by 20-100%, 20-75%, 20-50%, 25- 100%, 25-75%, 25-50%, 50-100%, or 50-75% relative to a control subject. In some

embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to prevent obesity-associated inflammation in a T2D subject (e.g., the subject does not exhibit signs of obesity-associated inflammation following treatment with a therapeutically effective amount of rapamycin and metformin). Non-limiting methods of measuring obesity-induced inflammation include staining tissue samples with hematoxylin and eosin (H & E staining) and measuring the expression of proteins associated with inflammation in adipose tissues. Tissue samples may be obtained from several organs (e.g., pancreas, liver, kidney) and stained with H & E, wherein inflammation is visualized by enlargement of the organ or tissue (hyperplasia) or the presence of foci containing lymphocytes (e.g., plasma cells, natural killer cells, dendritic cells). Non-limiting examples of proteins whose expression can be measured to quantify obesity-associated inflammation include: IL-l, IL-6, TNF-a, C-reactive protein (CRP), and adiponectin, whose expression can be measured by techniques such as real-time polymerase chain reaction (RT- PCR) and ELISA or immunohistochemistry with antibodies specific to the proteins associated with inflammation (see, e.g., Amsen, et ah, 2009, Approaches to Determine Expression of Inflammatory Cytokines, Methods Mol Biol, 511: 107-142)..

Hepatic Steatosis. Subjects with T2D are susceptible to developing hepatic steatosis due to increased weight gain and chronic obesity, for example. Hepatic steatosis refers to the buildup of fat tissue in the liver. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease hepatic steatosis in a T2D subject by at least 20% relative to a control subject. For example, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease hepatic steatosis in a T2D subject by at least 25%, 35, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease hepatic steatosis in a T2D subject by 20-100%, 20-75%, 20- 50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to prevent hepatic steatosis in a T2D subject (e.g., the subject does not exhibit signs of hepatic steatosis following treatment with a therapeutically effective amount of rapamycin and metformin). Non-limiting methods of measuring hepatic steatosis in the liver include computed tomography (CT), controlled attenuation parameter (CAP) ultrasound, and performing a liver biopsy and staining the liver with hematoxylin and eosin (H & E staining). CT scans (see, e.g., Wells, et ah, 2016, Computed Tomography Measurement of Hepatic Steatosis: Prevalence of Hepatic Steatosis in a Canadian Population) allows the direct imaging of the liver, wherein adipose tissue is opaque on the resulting radiograph. In CAP ultrasound (see, e.g., 2018,

Measurement of Controlled Attenuation Parameter: A Surrogate Marker of Hepatic Steatosis in Patients of Non-Alcoholic Fatty Liver Disease on Lifestyle Modification - A Prospective Follow-Up Study, Arg Gastroenterol, 55(1): 7-13), a vibrator generates low frequency waves through the liver which are then transmitted to an ultrasound receiver. The velocity of the waves is dependent on the tissue composition and elasticity, and the rate of wave propagation through the liver can be used as a measure of hepatic steatosis. In biopsy and H & E staining, adiposity is visualized by white cells (adipocytes) and can be quantified as a total of the liver tissue.

Hypertriglyceridemia/Hyperlipidemia. Subjects having T2D are susceptible to developing increased triglyceride levels in the blood (hypertriglyceridemia) as a result of obesity, chronic inflammation in adipose tissue, and hyperglycemia. The normal level of triglycerides in the blood (e.g., in a non-T2D subject) is less than 150 mg/dL (1.7 mmol/L) and high levels of triglycerides in the blood (e.g., in a T2D subject) are greater than or equal to 150 mg/dL. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease triglyceride levels in a T2D subject by at least 20% relative to a control subject. For example, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease triglyceride levels in a T2D subject by at least 25%, 35, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease

triglyceride levels in a T2D subject by 20-100%, 20-75%, 20-50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to prevent hypertriglyceridemia in a T2D subject (e.g., the subject does not exhibit signs of hepatic steatosis following treatment with a therapeutically effective amount of rapamycin and metformin). Non-limiting methods for measuring blood triglycerides include coupled enzymatic assays, total lipid profiles, and analytical ultracentrifugation. The coupled enzymatic assay for measuring triglycerides (see, e.g., Total Cholesterol, Direct HDL, Precipitated HDL, Triglycerides, and LDL, 2003-2004, Centers for Disease Control) in a blood sample involves conversion of triglycerides to H₂0₂ and glycerol, after which the H₂0₂ reacts with peroxidase and produces a color whose absorbance at 500 nm is proportional to triglyceride concentration. Total lipids are measured in a lipid panel, and triglycerides are the major component of total lipids in the bloodstream. Analytical ultracentrifugation (see, e.g., Schaefer, et al., 2018, The Measurement of Lipids, Lipoproteins, Apolipoproteins, Fatty Acids, and Sterols, and Next Generation Sequence for the Diagnosis and Treatment of Lipid Disorders, Endotext) can also be utilized to separate triglycerides from lipoproteins in blood samples because triglycerides have a density of 0.9 g/mL.

Subjects having T2D are also susceptible to developing increased high-density lipoprotein cholesterol levels (HLDL) in the blood due to obesity, hypertriglyceridemia, and chronic inflammation in the adipose tissue. HLDL removes cholesterol from the blood, and normal HLDL levels are 40-59 mg/dL. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease HLDL levels in a T2D subject by at least 10% relative to a control subject. For example, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease HLDL levels in a T2D subject by at least 10%, 15%, 20%, 25%, 35, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease HLDL levels in a T2D subject by 10-100%, 10-75%, 10-50%, 15-100%, 15-75%, 15-50%, 20-100%, 20-75%, 20-50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75% relative to a control subject. Non-limiting methods of measuring cholesterol (e.g., HLDL) in a blood sample include coupled enzymatic assays, total lipid profiles, and analytical ultracentrifugation. The coupled enzymatic assay for measuring HLDL in a blood sample involves conversion of HLDL to H₂0₂, after which the H₂0₂ reacts with peroxidase and produces a color whose absorbance at 500 nm is proportional to HLDL cholesterol

concentration. Total lipids are measured in a lipid panel, and triglycerides are the major component of total lipids in the bloodstream. Analytical ultracentrifugation can also be utilized to separate HLDL from low density lipoprotein (LDL) in blood samples because HLDL has a density of 1.063 - 1.21 g/mL, while LDL has a density of 1.019 - 1.063 g/mL.

Nephropathy. Subjects having T2D are susceptible to diabetic nephropathy. Diabetic nephropathy is kidney damage as a result of high glucose levels in T2D. Up to 40% of diabetes patients will eventually develop diabetic nephropathy. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease nephropathy in a T2D subject by at least 20% relative to a control subject. For example, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease nephropathy in a T2D subject by at least 20%, 25%, 35, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease nephropathy in a T2D subject by 20-100%, 20-75%, 20-50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to prevent nephropathy in a T2D subject. Non-limiting methods of measuring diabetic nephropathy include urinary albumin excretion (UAE), urine albumin/creatinine ratio (UACR), and the glomerular filtration rate (GFR). The UAE (see, e.g., Fagerstrom, et ah, 2015, Urinary albumin excretion in healthy adults: a cross sectional study of 24-hour versus timed overnight samples and impact of GFR and other personal characteristics, BMC Nephrology, 16:8) may be measured by collecting urine samples from a subject at times over a 24-hour period using an albumin-detection stick test. The UACR (see, e.g., Narva, et al., 2015, Faboratory Assessment of Diabetic Kidney Disease, Diabetes Spectrum, 28(3): 162-166) may be measured by collecting urine samples from a subject at times over a 24-hour period using a detection stick which measures the ratio of albumin/creatinine in urine. The GFR (see, e.g., Seegmiller, et ah, 2018, Challenges in Measuring Glomerular Filtration Rate: A Clinical Faboratory Perspective, Advances in Chronic Kidney Disease, 25(1): 84-92) may be measured by the creatinine clearance, wherein the concentration of creatinine in the urine is divided by the concentration of creatinine in the blood serum of a subject.

Subjects having T2D may be more susceptible to developing hyaline thrombi than subjects that do not have T2D. Hyaline thrombi are blood clots that form in small blood vessels (e.g., capillaries of the glomerulus in the kidney). These clots may form as a result of the obesity- associated inflammation, high blood pressure, hyperglycemia, and/or high blood pressure that characterize T2D. Hyaline thrombi may be detected by biopsying the kidney and staining with H & E.

Treatment with a therapeutically effective amount of metformin alone does not decrease hyaline thrombi in a subject having T2D. In some embodiments administering a therapeutically effective amount of rapamycin and metformin decreases the proportion of glomeruli with hyaline thrombi in the subject by at least 20%. For example, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease the proportion of glomeruli with hyaline thrombi in a T2D subject by at least 20%, 25%, 35, 35%, 40%, 45%, 50%, 55%, 60%, 65%,

70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease the proportion of glomeruli with hyaline thrombi in a T2D subject by 20-100%, 20-75%, 20-50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75% relative to a control subject.

Subjects having T2D may be more susceptible to decreased kidney function compared with subjects that do not have T2D. Kidney function may be measured by an albumin/creatinine ratio (ACR). Kidneys that are functioning properly do not allow protein to be excreted in the urine. Albumin is a globular protein found in the blood and represents protein excretion by the kidneys. Creatinine is a metabolite of creatine metabolism in the muscle and represents the excretion of waste products by the kidneys. Normal levels of ACR (e.g., in a non-T2D subject) are less than 30 pg/mg. In some embodiments, administering a therapeutically effective amount of rapamycin and metformin decreases ACR in the subject by at least 20%. For example, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease ACR in a T2D subject by at least 20%, 25%, 35, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control subject. In some embodiments, a therapeutically effective amount of rapamycin and metformin is sufficient to decrease ACR in a T2D subject by 20-100%, 20-75%, 20-50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75% relative to a control subject. Non-limiting methods of measuring an ACR in a subject include collecting urine samples from a subject at times over a 24-hour period using a detection stick which measures the ratio of albumin/creatinine in urine

Further embodiments

The present disclosure further provides embodiments encompassed by the following numbered paragraphs:

1. A method of treating type 2 diabetes in a subject comprising administering a

therapeutically effective amount of a combination of rapamycin and metformin to the subject, wherein the molar ratio of rapamycin to metformin is in the range of 1:1000 to 1:20.

2. The method of paragraph 1, wherein the molar ratio of rapamycin to metformin is in the range of 1:500 to 1:100.

3. A method of improving renal function in a subject having type 2 diabetes comprising administering a therapeutically effective amount of a combination of rapamycin and metformin to the subject, wherein the molar ratio of rapamycin to metformin is in the range of 1:1000 to 1:20.

4. The method of paragraph 3, wherein the therapeutically effective amount of the combination is an amount sufficient to provide a blood urea nitrogen (BUN) level in the subject that is significantly less than the BUN level in the subject before the combination was administered to the subject.

5. The method of paragraph 3, wherein the therapeutically effective amount of the combination is an amount sufficient to provide a BUN level in the subject of from 6 mg/ dL to 20 mg/ dL.

6. The method of paragraph 3, wherein the therapeutically effective amount of the combination is an amount sufficient to provide a creatine clearance rate in the subject that is significantly greater than the creatine clearance rate in the subject before the combination was administered to the subject. 7. The method of paragraph 3, wherein the therapeutically effective amount of the combination is an amount sufficient to provide a creatine clearance rate in subject of from 85 to 140 mL/ minute.

8. A method of improving insulin sensitivity in a subject having Type 2 diabetes, comprising administering a therapeutically effective amount of a combination of rapamycin and metformin to the subject, wherein the molar ratio of rapamycin to metformin is in the range of 1:1000 to 1:20.

9. The method of paragraph 9, wherein the therapeutically effective amount is an amount sufficient to significantly reduce plasma glucose level in the subject approximately 3 hours after the subject is injected with 1.0 mg/ kg insulin relative to the subject’s plasma glucose level approximately 3 hours after the subject is injected with 1.0 mg/ kg insulin before the

combination was administered to the subject.

10. The method of paragraph 9, wherein the therapeutically effective amount is an amount sufficient to produce a plasma glucose level of 60 mg/ mL to 105 mg/ ml in the subject approximately 3 hours after the subject is injected with 1.0 mg/ kg insulin.

11. A method of preventing or reducing insulin resistance in a subject having Type 2 diabetes, comprising administering a therapeutically effective amount of a combination of rapamycin and metformin to the subject, wherein the molar ratio of rapamycin to metformin is in the range of 1:1000 to 1:20.

12. The method of paragraph 11, wherein the therapeutically effective amount of the combination is an amount sufficient to provide a fasting plasma glucose level in the subject of 60 mg/ dL to 100 mg/ dl, a plasma glucose level in the subject of less than 200 mg/ dL

approximately 1 hour after the subject drinks a liquid comprising 75 grams of glucose, and a plasma glucose level in the subject of less than 140 mg/ mL approximately 2 hours after the subject drinks a liquid comprising 75 grams glucose.

13. The method of paragraph 1, wherein the therapeutically effective amount of the combination comprises an amount of metformin sufficient to prevent or reduce an adverse effect of rapamycin administration.

14. The method of paragraph 13, wherein the adverse effect of rapamycin administration that is prevented or reduced is any one or more of pancreatic islet degranulation, hyperglycemia, and hyperlipidemia.

15. The method of paragraph 1, wherein the therapeutically effective amount of the combination comprises an amount of rapamycin sufficient to prevent or reduce the adverse effects of metformin administration. 16. The method of paragraph 15, wherein the adverse effects of metformin administration that are prevented or reduced are any one or more of nephropathy and splenic hyperplasia.

17. The method of any one of paragraphs 1 to 16, wherein the therapeutically effective amount of the combination comprises a daily dose of 0.1 mg to 5.0 mg rapamycin.

18. The method of any one of paragraphs 1 to 17, wherein the therapeutically effective amount of the combination comprises a daily dose of 100 mg to 2500 mg metformin.

19. A combination oral dosage form comprising rapamycin and metformin, wherein the molar ratio of rapamycin to metformin in the dosage form is in the range of 1:1000 to 1:20.

EXAMPLES

The progression of diabetogenic hyperglycemia and associated pathogenic phenotypes was examined in male NONcNZOlO/LtJ (NcZlO) mice maintained on either 10% fat chow diet (untreated) or the same diet supplemented with 14 ppm rapamycin (RAPA), 0.1% metformin (MET) or a combination of both drugs (RAPA/MET) from 12 to 30 weeks of age. Results were evaluated for 10 individual phenotypic categories that interact to govern the development and pathogenesis of type 2 diabetes (T2D). Obesity, a key initiating factor in the development of T2D, is associated with elevated circulating lipids and the initiation of adipose-derived inflammatory processes, which are primary drivers of peripheral insulin resistance (25-27). This cascade of events leads to chronic hyperinsulinemia and hyperglycemia, which promote the pathogenic consequences of T2D, including steatosis, nephropathy, pancreatic impairment, and neuropathy.

Example 1. Obesity

RAPA- and RAPA/MET-treatment halted the normal body weight gain seen in untreated mice (FIG. 1A), primarily by preventing fat mass expansion (FIG. IB, Table 1). By the end of the study, RAPA- and RAPA/MET-treated mice showed reduced adiposity (% FAT; DXA) typified by reduced subcutaneous inguinal (ING) adipose tissue (FIG. IB (left), Table 1). MET- treatment showed no differences vs. untreated for these measurements. Visceral epididymal (EPI) adipose tissue, longitudinal growth (anus to nose length), bone mineral density (Table 1), and food intake (at 29 weeks of age, FIG. 1C) did not differ among treatment groups.

Gene expression analyses of both EPI and ING adipose tissue indicated robust RAPA- and RAPA/MET -mediated down-regulation of Mest (mesoderm specific transcript) and Gpam (glycerol-3-phosphate acyltransferase 1; mitochondrial), genes associated with adipose tissue function and growth (FIG. ID). The marked RAPA-mediated down-regulation of maternally- imprinted Mest was noteworthy because adipose tissue Mest mRNA and protein levels are positively associated with adipose tissue expansion in mice (28-32), whereas GPAM regulation of triglyceride synthesis can mediate adipocyte hypertrophy and adipose tissue response to insulin (33-36). Thermogenesis-associated genes (e.g. Ucpl and Dio2) were unaffected or downregulated by RAPA treatment (FIG. ID) consistent with reports showing RAPA-mediated suppression of browning of white adipose tissue (37, 38).

Reduced ING fat, but not EPI fat, in RAPA- and RAPA/MET-treated mice was reflected by Lep (leptin) expression, which was reduced 60-70% in the ING fat of RAPA- and

RAPA/MET-treated mice but unaffected in EPI fat (FIG. ID). Slc2a4 (solute carrier family 2, facilitated glucose transporter member 4; Glut4) was upregulated in EPI fat but not in ING fat

(FIG. ID). Elevated SLC2A4 would stimulate adipose hypertrophy by promotion of glucose uptake and lipogenesis and thus compensate for RAPA- and RAPA/MET downregulation of Mest and Gpam. MET-treatment alone showed no significant effects vs. untreated on gene expression in adipose tissue (FIG. ID).

Table 1. Effects of rapamycin, metformin, and rapamycin/metformin combination on body composition and food intake

Group UNT RAPA MET RAPA/MET

BW at Sacrifice (g) 42.9 + 0.8 36.0 + 1.0 *** 44.5 + 1.2 34.6 + 0.7 *** ABW 12-28 wks (g) +8.3 + 1.0 -1 3 + 1 1 * * * +5.9 + 0.7 -2.3 + 0.6 *** EPI Fat Wt. (g) 1.18 + 0.10 1.18 + 0.09 1.25 + 0.10 1.06 + 0.07

EPI Fat Wt./BW 0.027 + 0.002 0.033 + 0.002 0.028 + 0.003 0.031 + 0.001 ING Fat Wt. (g) 0.61 + 0.09 0.27 + 0.03 ** 0.64 + 0.06 0.24 + 0.03 ** ING Fat Wt./BW 0.015 + 0.002 0.008 + 0.001 ** 0.014 + 0.001 0.007 + 0.001 **

Fat Mass (g) by DXA 15.5 + 1.2 10.1 + 0.9 ** 14.8 + 0.7 8.9 + 0.5 ** % Fat by DXA 34.4 + 1.8 27.1 + 1.6 * 33.0 + 1.3 24.9 + 0.6 ** Lean Mass (g) by DXA 29.5 + 1.0 26.9 + 0.5 30.0 + 0.8 26.8 + 0.6 % Lean by DXA 65.6 + 1.8 72.9 + 1.6 * 67.0 + 1.3 75.0 + 0.6 ** Anus/Nose Length (cm) 9.7 + 0.1 9.8 + 0.1 9.9 + 0.1 9.6 + 0.1 BMD (g/cm²) by DXA 0.055 + 0.001 0.056 + 0.001 0.057 + 0.001 0.055 + 0.001 IENF (profiles/mm) 12.2 + 1.9 12.3 + 1.0 11.4 + 1.2 11.9 + 1.4

Mean + SE. Significant difference, treatment vs. untreated, ANOVA followed by Tukey-Kramer

HSD, ***P < 0.0001, ** P < 0.01, * P < 0.05, vs. UNT. N = 9-10 for BW, ABW, EPI Wt, EPI Wt./BW, IENF, and Anus/Nose Length; N = 3-4 for ING Wt., ING Wt./BW, and DXA data.

***P < 0.0001, ** P < 0.01, * P < 0.05, vs. UNT. Untreated = UNT, RAPA-treated = RAPA, MET-treated = MET, RAPA/MET-treated = RAPA/MET.

Example 2. Circulating lipids Hyperlipidemia is a side effect in patients undergoing treatments that inhibit mTOR (9, 39). In untreated NcZlO male mice, RAPA-treatment for 18 weeks elevated the level of circulating triglycerides above the pre-existing hypertriglyceridemia (Table 2). Importantly, although MET-treatment had no effect on circulating triglycerides vs. untreated, combination RAPA/MET-treatment prevented the RAPA-mediated elevation of circulating triglycerides

(FIG. 2A, Table 2). Total circulating cholesterol, HLDL cholesterol, and non-esterified fatty acids were unaffected by RAPA- or MET-treatment whereas combination RAPA/MET-treatment significantly lowered HLDL cholesterol (Table 2).

MET counteracts RAPA-driven hyperlipidemia by preventing RAPA-mediated downregulation of hepatic Ldlr (low-density lipoprotein receptor) expression. Reduced expression of Ldlr (FIG. 2B) mRNA and protein levels (40) in RAPA-treated mice, and the inhibition of this effect with RAPA/MET treatment (FIG. 2B), correspond with the predicted effects on circulating triglycerides due to the important role for LDLR in the clearance of APOB- and APOE-containing lipoproteins. Since PPARA (peroxisome proliferator- activated receptor a) has been suggested to mediate Ldlr expression via activation of SREBP2 (sterol regulatory element binding protein 2) (41), increased hepatic Ppara expression in combination

RAPA/MET-treatment is consistent with elevated Ldlr expression and reduced circulating triglyceride levels (FIG. 2B, 42-44).

Table 2. Effects of rapamycin, metformin, and rapamycin/metformin combination in type 2 diabetics: health and functional test results at end of study

Group UNT RAPA MET RAPA/MET

TG (mg/dL) 336 ± 22 445 + 13 ** 344 + 38 319 + 28

Total Chol. (mg/dL) 144 + 7 160 + 5 132 + 6 135 + 10

HDLD Chol. (mg/dL) 114 + 5 126 + 3 106 + 5 97 + 4 *

NEFA (mEq/L) 1.8 + 0.2 2.3 + 0.1 1.5 + 0.1 2.0 + 0.1

Steatosis

Mean + SE. Significant difference, treatment vs. untreated, ANOVA followed by Tukey-Kramer HSD, ***P < 0.0001, ** P < 0.01, * P < 0.05, vs. UNT. N = 6-10. $ Presence/absence of steatosis evaluated by nominal logistic regression, overall effect, P < 0.0001. Untreated = UNT, RAPA-treated = RAPA, MET-treated = MET, RAPA/MET-treated = RAPA/MET, TG = triglycerides; Chol. = cholesterol; NEFA = non-esterified fatty acids.

Example 3. Steatosis

Hepatic steatosis is one of the most common co-morbidities of T2D and we observed mild to moderate hepatic steatosis in 9/10 untreated NcZlO males. While MET-treatment alone had a modest effect, steatosis was completely prevented by RAPA- and RAPA/MET -treatment (FIG. 3A, Table 2). Protection from steatosis parallels the effect of these treatments on circulating insulin, which can promote hepatic steatosis through regulation of Fasti (hepatic fatty acid synthase) expression (45, 46). Fasti is a rate-limiting enzyme for hepatic fatty acid synthesis (47) and its elevated expression is closely associated with the development of hepatic steatosis in humans and mice (48). The suppression of Fasti by RAPA-, MET-, and RAPA/MET-treatment (FIG. 3B) suggests a mechanism through which diminished circulating insulin in treated mice prevents steatosis.

Example 4. Inflammation

The development of obesity can generate progressive inflammation, which promotes insulin resistance and aggravation of the metabolic syndrome (49). We evaluated systemic inflammation histologically by identifying hyperplasia of the spleen and foci of lymphocytes in the pancreas, liver, and kidney. As expected in mice with T2D, pancreatitis (inflammation of the stromal portion of the gland) occurred frequently (31/39, 79% of all NcZlO mice). Inflammation incidence among other tissues in NcZlO mice ranged from 14-26%. Overall, MET-treated mice had the greatest extent of inflammation, whereas RAPA- and RAPA/MET-treatment

significantly diminished the extent of inflammation compared to MET-treatment alone (FIG. 4A).

In adipose tissue, selective gene expression analysis was used to evaluate inflammation in more detail. Itgam (integrin alpha M) and Itgax (integrin alpha X) are genes for cell surface proteins on myeloid cells, including macrophages, monocytes, and granulocytes. RAPA- and RAPA/MET-treatment diminished Itgax and Itgam expression in EPI fat and Itgax expression in ING fat (FIG. 4B), indicating diminished myeloid cell infiltration of adipose tissue in NcZlO mice. This effect could be mediated in part by suppression of Hmoxl (heme oxygenase 1), as observed in EPI fat (FIG. 4B). Hmoxl is activated in response to oxidative stress and is critical for adipose myeloid infiltration and obesity-associated insulin resistance (50).

RAPA- and RAPA/MET-treatments diminished Lbp expression in EPI fat and PAI-l mRNA expression in both fat depots (FIG. 4B). LBP is an acute phase protein that facilitates presentation of lipopolysaccharide to toll-like receptors and CD- 14, whereas PAI-l contributes to the dissolution of blood clots and is a key component of the senescent cell secretome (51). Both PAI-l and LBP link adipose-derived inflammation with insulin resistance (52, 53). PAI-l mRNA expression is positively regulated by insulin, and promotes inflammation and insulin resistance, which, in turn, elevates circulating insulin (25, 27, 54). Lbp expression is up-regulated by insulin resistance, which enhances inflammation (55) and further intensifies insulin resistance (11, 56, 57). The reduction of myeloid infiltration and expression of genes for inflammatory stress-responsive factors in adipose tissue demonstrate that RAPA- and RAPA/MET-treatment suppress obesity-associated inflammation. Interestingly, the lack of an effect of RAPA- or RAPA/MET-treatment on EPI fat mass did not prevent beneficial RAPA-mediated effects on inflammatory gene expression.

While there was no correlation between hyperglycemia and inflammation (FIG. 4C), the positive association of body weight and plasma insulin concentrations with more extensive systemic inflammation (obesity-associated) manifests a pathogenic, hyperin sulinemic“vicious cycle”. Elevated insulin in pre-diabetic and diabetic mice advances adipose tissue hypertrophy, which fosters both adipose tissue expansion and systemic inflammation, promoting insulin resistance that, in turn, further elevates circulating insulin. Both RAPA- and RAPA/MET- treatment interrupt that cycle by: a) downregulating adipogenic gene expression, which prevents adipose tissue hypertrophy; b) suppressing hypertrophy- associated adipose infiltration and activation of inflammatory myeloid cells; and c) reducing expression of adipose-derived inflammatory stress-responsive factors.

Example 5. Insulin tolerance and circulating insulin

Plasma insulin increased progressively in untreated males (FIG. 5A) as observed previously (15). While MET-treatment mitigated this increase in circulating insulin, both RAPA- and RAPA/MET-treatment completely prevented it. Because plasma insulin level is partly determined by insulin sensitivity (49), we tested insulin tolerance (ITT) at 17 weeks of treatment. Untreated males were highly insulin resistant, whereas RAPA-treatment improved insulin sensitivity (FIG. 5B), as observed in other T2D strains of mice (13, 14). While MET-treatment had no effect, RAPA/MET-treatment completely normalized insulin tolerance to levels comparable to that of the young, insulin sensitive, C57BL/6J positive control males (FIG. 5B).

Example 6. Plasma glucose (PG) and gluconeogenesis

Untreated NcZlO males transited to diabetes (glucose > 250 mg/dL), as previously reported (15, 19). RAPA-treatment did not alter diabetes-associated PG elevation during the first 8 weeks of treatment but significantly increased PG elevation after 12 weeks of treatment (FIG. 5C) and circulating HbAlc level by the end of the study (FIG. 5D). MET-treatment had no effect on PG or HbAlc levels compared to untreated mice (FIG. 5C, 5D). While RAPA/MET- treatment for 8 weeks elevated PG levels, by 16 weeks of treatment, PG decreased to levels observed in untreated mice (FIG. 5C). In addition, RAPA/MET-treatment prevented the RAPA- associated increase in HbAlc (FIG. 5D). Thus, MET prevented the RAPA-mediated

exacerbation of hyperglycemia. Blood for glucose-determination was sampled during a short-term fast, when plasma glucose level is determined by the rate of hepatic release of glucose, circulating levels of insulin, and insulin sensitivity. Gluconeogenesis is regulated in part by Ppargcla (peroxisome proliferator-activated receptor gamma coactivator l-alpha; PGC-la, 58), and is suppressed by insulin, therefore elevated hepatic Ppargcla level in mice with T2D reflects hepatic insulin resistance (59). The downregulation of hepatic Ppargcla by RAPA-, MET- and RAPA/MET - treatment (FIG. 5E) demonstrates improved hepatic insulin sensitivity, which should reduce circulating insulin, as observed for RAPA-, MET- and RAPA/MET-treatments. Paradoxically, despite the downregulation of Ppargcla in RAPA-treated mice, circulating glucose was elevated, indicating that other factors were involved.

Reduced hepatic glucokinase ( Gck ) and increased glucose-6-phosphatase ( G6pc ) expression by RAPA-treatment would indicate increased release of glucose into circulation (60, 61) as shown in NcZlO males. While co-treatment with MET showed no effect on Gck expression, G6pc was suppressed (FIG. 5E). Thus, the contrary effect of RAPA- and

RAPA/MET-treatment on hyperglycemia may result, in part, from counterbalancing effects on genes that regulate intercellular glucose phosphorylation.

Example 7. Pancreas and pancreatic insulin content (PIC)

Untreated male NcZlO mice exhibited islet degranulation (FIG. 6A) indicative of diminished insulin storage that is characteristic of an early, hyperinsulinemic phase of T2D

(FIG. 5A). Treatments produced significant differences in degree of islet degranulation (FIG. 6A, MANOVA, P = 0.0006, Wilk’s l): Compared to untreated mice, RAPA-treatment produced a more severe degranulation (MANOVA, P = 0.02), and MET-treatment produced more fully granulated islets (MANOVA, P = 0.05). RAPA/MET-treatment prevented the RAPA-associated degranulation ( P = 0.03), maintaining a pattern comparable to that of untreated mice (FIG. 6A). Proportions of variably sized islets (small, medium, or large) were not affected by any of the treatments despite reduced islet numbers in RAPA- and RAPA/MET -treated mice (data not shown). RAPA- and RAPA/MET-treatment reduced PIC by 77% and 59% (P = 0.0004, P = 0.04, respectively, ANOVA), whereas MET had no effect versus untreated and did not significantly alter the effect of RAPA-treatment (FIG. 6B). An association of PIC with the extent of islet degranulation was due almost entirely to a negative correlation of PIC with the proportion of mostly degranulated islets (>50% degranulated; see supplementary material).

While loss of PIC may reduce functional insulin reserves in the pancreatic b-cells, it is possible that RAPA-suppression of hyperinsulinemia confers a protective effect on pancreatic b- cells by reducing proinsulin translation and preventing the toxic accumulation of misfolded proinsulin aggregates (62). However, PIC-depletion can reach a critical threshold below which a diabetic /nywinsulincmic hyperglycemia results (14, 63). It is doubtful that such a threshold is reached for non-diabetic mice in which long-term dietary treatment with encapsulated RAPA (14 ppm) increases lifespan (1-3). However, diabetic mice may be more vulnerable; in RAPA-treated TALLYHO/JngJ mice (14) and in the RAPA-treated NcZlO mice of the present study, PIC was very low, and circulating non-fasting glucose was elevated above the level of diabetic untreated mice b-cell depletion may even have contributed to the shortened lifespan of RAPA-treated diabetic C57BLKS/J-Lepr^a¾ mice (64), although, in a study using a smaller cohort of

C57 B LKS/ ]-Lepr'^lh mice, we observed no detrimental effect of RAPA on survivorship (16). It is possible that, for patients vulnerable to adverse effects of RAPA-treatment, co-treatment with MET can protect b-cells from excessive glycemic stress and preserve pancreatic islet integrity.

Example 8. Diabetic nephropathy and neuropathy

Nephritis was observed in -75% of the glomeruli in untreated males (FIG. 7A), with hyaline thrombi observed in -15% of the glomeruli (FIG. 7B). Surprisingly, the

albumin/creatinine ratio (ACR) was not elevated in untreated mice (FIG. 7C) as seen previously (15). RAPA-treatment had no significant effect on ACR, whereas MET-treatment increased the proportion of glomeruli with hyaline thrombi and doubled the ACR (FIG. 7B, 7C), consistent with a nephrotoxic potential for MET (65). Combination RAPA/MET -treatment completely blocked the nephrotoxic effect of MET (FIG. 7A, 7B, 7C).

In a previous study from our laboratory, RAPA-treatment of NcZlO males prevented the development of elevated ACR and the generation of hyaline thrombi as well as

glomerulonephritis (15). Similarly, RAPA-treatment for 16 weeks reduced ACR in diabetic female C 57 B LK S /i-Lp r^dh mice by 50% (16), and it delayed the development of diabetic nephropathy in streptozotocin-treated Sprague-Dawley rats (66-68). In the present study, addition of RAPA to MET-treatment prevented the advancement of glomerulonephritis, the occurrence of hyaline thrombi, and the elevation of ACR generated by the MET-treatment.

While the suppression of diabetic nephropathy by inhibition of mTOR in the podocyte of the nephron (69) provides a direct mechanism for the RAPA-mediated protection, reduction of circulating insulin is likely a contributing factor as, in humans, insulin resistance and

hyperinsulinemia are risk factors for elevated ACR and kidney disease (70, 71). Similarly, in the present study, RAPA/MET-treatment prevented both the nephropathy caused by MET-treatment alone and diabetic elevation of circulating plasma insulin. Thus, RAPA suppresses diabetic nephropathy through at least two mechanisms: direct inhibition of mTOR- mediated nephropathy and suppression of a mechanism linked to circulating insulin elevation.

Intra-epidermal small sensory nerve fiber density (IENF) in foot pad skin, identified by immunocytochemistry (72), is a standard histologic metric for neuropathy. IENF density did not differ among the 4 groups (Table 1). Evidence of peripheral neuropathy more typically appears in mice with T2D as insulinopenia develops (72), which may occur at a later stage of diabetes in NcZlO mice.

Methods

Analysis of relation of islet degranulation to pancreatic insulin content (PIC). For analysis of islet degranulation each islet was graded as either fully granulated, 10-50% degranulated (partially), or 50-100% degranulated (mostly). PIC was affected by the degree of islet degranulation even after covariation due to treatment effects (UNT, RAPA-treated, MET- treated, RAPA/MET-treated) was accounted for (MANOVA, P = 0.02); the effect of MET- treatment differed from the other three treatment groups (paired comparisons by MANOVA)

(Fig. 6a). To determine if the proportion of islets within a degranulation grade correlated with PIC, analysis of the MET-treated group was separated from the other three groups because the incidence of mostly degranulated islets was so low in MET-treated mice. Among UNT, RAPA- treated and RAPA/MET-treated mice, the proportion of mostly degranulated islets was correlated with PIC (ANCOVA, P = 0.03; the greater the proportion of mostly degranulated islets, the lower the PIC). There was no relationship of the proportion of fully granulated or of partially granulated islets to PIC among these three treatment groups. For MET-treated mice, there was no overall relation of PIC with relative degree of islet degranulation, probably because there were so few mostly degranulated islets among MET-treated mice (Fig. 6a). We observe comparable relationships of PIC with degranulation if the number, rather than the proportion, of islets in each granulation grade is analyzed; PIC correlated with the number of mostly degranulated islets among the UNT, RAPA-, and RAPA/MET-treated mice (ANCOVA, P = 0.01).

Animals, Diets, and Caging. Forty NONcNZOlO/LtJ (NcZlO; JAX® Stock Number 4456) males at eight weeks of age were transferred from The Jackson Laboratory (Bar Harbor, Maine) production facility to the investigator’s mouse room (room Dl) at The Jackson

Laboratory. Records of pathogens tested are available in health status reports for room Dl at the website: jaxmice.jax.org/genetichealth/index.html. The mouse room is maintained on a light/dark cycle of 12 hours, -25 ^°C, and 40-50% humidity. All mice were housed in weaning pens (10 mice per 30 cm x 30 cm pen) with pine shaving bedding, given acidified water (pH = 2.5-3.1), and maintained with ad lib access to chow diet containing 11% fat (5LA0; all diets prepared by Test Diet, Inc., a division of Purina Mills, Richmond, IN, USA) until 12 weeks of age. At 12 weeks of age groups of 10 mice each were either maintained on the chow diet (5LAO) or switched to 5LAO containing 14 ppm encapsulated rapamycin (RAPA), 0.1% metformin (MET), or both rapamycin (14 ppm) and metformin (0.1%) (RAPA/MET). A -40 gram NcZlO male eats -3 g of diet per day, thus the treated mice would be consuming roughly 1.1 mg/kg rapamycin and/or 75 mg/kg metformin per day. It is standard protocol for the NcZlO model to use at least a 10% fat chow diet in order to drive weight gain over the body weight threshold of 36 g to induce diabetes onset (http://jaxmice.jax.org/strain/004456.html).

Data Collection. Mice were weighed every 2 weeks and bled every 4 weeks from the retro-orbital sinus. Plasma glucose (PG) values were measured by glucometer (OneTouch, LifeScan, Inc., USA). Plasma insulin (PI) values were measured at 8, 16, and 24 weeks of age by ELISA (Meso Scale Discovery, Gaithersburg, MD, USA). On days that mice were bled, food was removed at 7:00 am (1 hr after lights on) and mice were bled at 10:00-11:00 a.m. Urine samples were collected at 25 weeks of age, and albumin/creatinine ratios (ACRs) were determined using the UniCel DxC 600 Synchron clinical system (Beckman Coulter, Inc., Brea, CA, USA). The insulin tolerance test (ITT) was performed at 29 weeks of age in a sub-set of each group (N = 5-6). Food was removed from the mice at 7:00 a.m. At -10:00-10:30, mice were weighed and bled from the retro-orbital sinus, and glucose was measured (OneTouch Ultra, Lifescan). Mice were then injected i.p. with 1.0 U/kg insulin (Humulin, Eli Lilly) in PBS.

Glucose was measured additionally at 15, 30, 45, and 60 minutes post injection. At 29 weeks, the subset of mice (N = 4) not tested by the ITT was measured for body composition by dual x-ray absorptiometry (DXA, Piximus) while under sedation with tribromoethanol. These mice were then singly housed in metabolic cages (CCMS) and acclimatized for three days prior to measurement of food consumption during the subsequent four days. One rapa-treated mouse died at 17 weeks of age and was removed from the study, and one untreated and one metformin- treated mouse died at -25 weeks of age. Mice were euthanized by C0₂ at 29-30 weeks of age. HbAlc was determined from whole blood taken at termination using the DxC 600. Total and HDL cholesterol, glucose, triglycerides, and non-esterified fatty acids (NEFA) were determined from termination serum using the DxC 600. A handling error precluded the analyses of 2-3 serum samples per group.

Histology. Pancreas, kidney, liver, spleen and paw skin were collected at termination for histology. Pancreas was fixed in Bouin’s solution, and three separate sections were stained with aldehyde fuchsin. Kidney, liver, and spleen were fixed in 10% neutral buffered formalin, and separate sections were stained with H & E and PAS. Kidney histology was assessed by scoring 100 glomeruli from each sample for evidence of nephritis and/or hyaline thrombi. Pancreatic histology was assessed by scoring all islets in three separate sections for degree of granulation: fully-granulated, partially-degranulated (10-50%), mostly-degranulated-to-completely- degranulated (50-100%). Skin was fixed in 4% buffered paraformaldehyde, processed into paraffin blocks, cut, and immunostained with anti-PGP9.5 antibody to visualize IENF and quantified exactly as described elsewhere (72). All scoring was assessed blind to treatment.

Tissue Collection and Processing. Liver, kidney, epididymal (EPI) fat, and inguinal (ING) fat pads were weighed at termination and quickly frozen in liquid nitrogen prior to storage at -80 °C. ING fat pads were taken only from the 16 mice, four per treatment group, that were subjected to DXA and CCMS analyses. RNA was extracted from liver, epididymal fat, and inguinal fat using TRI Reagent (Molecular Research Center Inc. Cincinnati, Ohio), with modifications to remove DNA using the Qiagen RNAeasy columns and DNasel (Qiagen, Valencia, CA) as described by Koza et al. (93). RNA was stored at -70 °C in nuclease-free water supplemented with the RNase inhibitor Superasin (Ambion, Austin, TX). Quality and quantity of RNA was determined using UV spectrophotometry (NanoDrop). Quantitative RT-PCR (qRT- PCR) using TaqMan probes and primers was performed essentially as described (94), except standard curves were generated using pooled RNA from the individual liver or adipose tissue samples used in the study. Gene expression data (arbitrary units; AU) based on the standard curve was normalized to TATA-box binding protein ( Tbp ) for each sample. Probe and primer sequences used to perform qRT-PCR mRNA analyses are available upon request.

References

1. Harrison, D. E. et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature

460(7253), 392-395 (2009).

2. Miller, R.A. et al. Rapamycin, but not resveratrol or simvastatin, extends life span of genetically

heterogeneous mice. J. Gerontol. A Biol. Sci. Med. Sci. 66(2), 191-201 (2011).

3. Wilkinson, J.E. et al. Rapamycin slows aging in mice. Aging Cell 11, 675-682 (2012).

4. Kaeberlein, M. et al. Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients.

Science 310, 1193-1196 (2005).

5. Powers, R.W. 3rd, Kaeberlein, M., Caldwell, S.D., Kennedy, B.K. & Fields, S. Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev. 20, 174—184 (2006).

6. Kapahi, P. et al. Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway.

Curr. Biol. 14, 885-890 (2004).

7. Vellai, T. et al. Genetics: influence of TOR kinase on lifespan in C. elegans. Nature 426, 620 (2003).

8. Barlow AD, Nicholson ML & Herbert TP. Evidence for rapamycin toxicity in pancreatic beta cells and a review of the underlying molecular mechanisms. Diabetes 62, 2674-2682 (2013).

9. Houde, V.P. et al. Chronic rapamycin treatment causes glucose intolerance and hyperlipidemia by

upregulating hepatic gluconeogenesis and impairing lipid deposition in adipose tissue. Diabetes 59(6), 1338— 1348 (2010).

10. Yang, S.B. et al. Rapamycin induces glucose intolerance in mice by reducing islet mass, insulin content, and insulin sensitivity. J. Mol. Med. (Berl). 90(5), 575-585 (2012).

11. Lamming, D.W. et al. Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity. Science 335, 1638-1643 (2012).

12. Lamming, D.W. et al. Young and old genetically heterogeneous HET3 mice on a rapamycin diet are glucose intolerant but insulin sensitive. Aging Cell 12(4), 712-718 (2013).

13. Deepa, S.S. et al. Rapamycin modulates markers of mitochondrial biogenesis and fatty acid oxidation in the adipose tissue of db/db mice. J. Biochem. Pharmacol. Res. 1(2), 114-123 (2013).

14. Reifsnyder, P.C., Flurkey, K. & Harrison, D.E. Rapamycin treatment benefits glucose metabolism in mouse models of type 2 diabetes. Aging 8(11), 3120-3130 (2016).

15. Reifsnyder, P.C., Doty, R. & Harrison, D.E. Rapamycin ameliorates nephropathy despite elevating

hyperglycemia in a polygenic mouse model of type 2 diabetes, NONcNZOlO/ LtJ. PLoS One 9(12), el 14324 (2014).

16. Reifsnyder, P.C. et al. Cardioprotective effects of dietary rapamycin on adult female C57BLKS/J-Lepr^db mice.

Ann. NY Acad. Sci. 1418(1), 106-117, doi:10.111 l/nyas.13557, Epub 2018 Jan 29, PMID:29377150 (2018).

17. Das, A.D. et al. Mammalian target of rapamycin (mTOR) inhibition with rapamycin improves cardiac function in type 2 diabetic mice: potential role of attenuated oxidative stress and altered contractile protein expression.

J. Biol. Chem. 289, 4145-4160 (2014).

18. Reifsnyder, P.C. & Letter, E.H. Deconstructing and reconstructing obesity-induced diabetes (diabesity) in mice. Diabetes 51, 825-832 (2002).

19. Letter, E.H. et al. Comparison of two new mouse models of polygenic type 2 diabetes at the Jackson

Laboratory, NONcNZOlOLt/J and T ALL YHO/J ng J . J. Diabetes Res. 2013, 165327 doi:10.1155/2013/165327 (2013).

20. Strong, R. et al. Longer lifespan in male mice treated with a weakly estrogenic agonist, an antioxidant, an a- glucosidase inhibitor or a Nrf2-inducer. Aging Cell 15, 872-884 (2016).

21. Shivaswamy, V., Bennett, R., Clure, C.C., Larsen, J.L. & Hamel, L.G. Metformin improves

immunosuppressant induced hyperglycemia and exocrine apoptosis in rats. Transplantation 95, 280-284 (2013).

22. Sun, I.O. et al. The effects of addition of coenzyme Q10 to metformin on sirolimus -induced diabetes mellitus.

Korean J. Intern Med. Dec 13, 1-10, doi: 10.3904/kjim.2017.004 [Epub ahead of print] (2017).

23. Jin, J. et al. Effects of metformin on hyperglycemia in an experimental model of tacrolimus- and sirolimus- induced diabetic rats. Korean J. Intern Med. 32(2), 314-322, doi: 10.3904/kjim.2015.394, [Epub 2016 Sep 30], (2017).

24. Weiss, R., Lernandez, E., Liu, Y., Strong, R. & Salmon, A.B. Metformin reduces glucose intolerance caused by rapamycin treatment in genetically heterogeneous female mice. Aging (Albany NY).

doi:10.18632/aging.101401 [Epub ahead of print] (2018).

25. Reilly, S.M. & Saltiel, A.R. Adapting to obesity with adipose tissue inflammation. Nature Reviews

Endocrinology 13, 633-643 doi: 10.1038/nrendo.2017.90 (2017).

26. Palmer, A.K. & Kirkland, J.L. Aging and adipose tissue: potential interventions for diabetes and regenerative medicine. Exp. Gerontol. 86, 97-105 (2016).

27. Frasca, D., Blomberg, B.B. & Paganelli, R. Aging, obesity, and inflammatory age-related diseases. Front.

Immunol. 8, 1745 doi: 10.3389/fimmu.2017.01745 (2017).

28. Nikonova, L. et al. Mesoderm-specific transcript is associated with fat mass expansion in response to a

positive energy balance. FASEB J. 22(11), 3925-3937 (2008).

29. Voigt, A., Agnew, K., van Schothorst, E.M., Keijer, J. & Klaus, S. Short-term, high fat feeding-induced

changes in white adipose tissue gene expression are highly predictive for long-term changes. Mol. Nutr. Food Res. 57(8), 1423-1434 (2013).

30. Anunciado-Koza, R.P., Manuel, J. & Koza, R.A. Molecular correlates of fat mass expansion in C57BL/6J mice after short-term exposure to dietary fat. Ann. N Y Acad. Sci. 1363, 50-58 (2016).

31. Jura, M., Jaroslawska, J., Chu, D.T. & Kozak, L.P. Mest and Sfrp5 are biomarkers for healthy adipose tissue.

Biochemie. 124, 124-133 (2016).

32. Anunciado-Koza, R.P. et al. Diet-induced adipose tissue expansion is mitigated in mice with a targeted

inactivation of mesoderm specific transcript (Mest). PFoS One 12(6), e0179879 (2017).

33. Yu, H. et al. Bovine lipid metabolism related gene GPAM: Molecular characterization, function identification, and association analysis with fat deposition traits. Gene 609, 9-18 doi: 10.1016/j . gene.2017.01.031,

Epub 2017, Jan 26 PMID:28131819 (2017).

34. Morgan-Bathke, M., Chen, L., Oberschneider, E., Harteneck, D. & Jensen, M.D. Sex and depot differences in ex vivo adipose tissue fatty acid storage and glycerol-3 -phosphate acyltransferase activity. Am. J. Physiol. Endocrinol. Metab. 308(9), E830-846 doi: 10.1152/ajpendo.00424.2014, Epub 2015 Mar 3, PMID:25738782 (2015).

35. Gimeno, R.E. & Cao, J. Thematic review series: glycero lipids. Mammalian glycerol-3-phosphate

acyltransferases: new genes for an old activity. J. Fipid Res. 49(10), 2079-2088 doi: 10.1194/jlr.R800013- JLR200, Epub 2008 Jul 24, PMID:18658143 (2008).

36. Wendel, A.A., Lewin, T.M. & Coleman, R.A. Glycerol-3-phosphate acyltransferases: rate limiting enzymes of triacylglycerol biosynthesis. Biochim. Biophys. Acta. 1791(6), 501-506 doi:10.1016/j.bbalip.2008.10.010,

Epub 2008 Nov 7, PMID: 19038363 (2009).

37. Liu, D. et al. Activation of mTORCl is essential for B-adrenergic stimulation of adipose browning. J. Clin.

Invest. 126(5), 1704-1716 (2016).

38. Tran, C.M. et al. Rapamycin blocks induction of the thermogenic program in white adipose tissue. Diabetes 65(4), 927-941 (2016).

39. Morrisett, J.D. et al. Effects of sirolimus on plasma lipids, lipoprotein levels, and fatty acid metabolism in renal transplant patients. J. Lipid Res. 43(8), 1170-1180 (2002).

40. Ai, D. et al. Regulation of hepatic LDL receptors by mTORCl and PCSK9 in mice. J. Clin. Invest. 122(4),

1262-70 doi: 10.1172/JCI61919, Epub 2012 Mar 19, PMID:22426206 (2012).

41. Huang, Z. et al. Activation of peroxisome proliferator-activated receptor-alpha in mice induces expression of the hepatic low-density lipoprotein receptor. Br. J. Pharmacol. 155(4), 596-605 doi:10.1038/bjp.2008.331, Epub 2008 Aug 18, PMID: 18852694 (2008).

42. Choi, S.Y., Fong, L.G., Kirven, M.J. & Cooper, A.D. Use of an anti-low density lipoprotein receptor antibody to quantify the role of the LDL receptor in the removal of chylomicron remnants in the mouse in vivo. J. Clin. Invest. 88(4), 1173-1181 (1991).

43. Linden, D., Alsterholm, M., Wennbo, H. & Oscarsson, J. PPARalpha deficiency increases secretion and serum levels of apolipoprotein B-containing lipoproteins. J. Lipid. Res. 42(11), 1831-1840 (2001).

44. De Souza, A.T. et al. Transcriptional and phenotypic comparisons of Ppara knockout and siRNA knockdown mice. Nucleic Acids Res. 34(16), 4486-4494 (2006).

45. Latasa, M.J., Griffin, M.J., Moon, Y.S., Kang, C. & Sul, H.S. Occupancy and function of the -150 sterol regulatory element and -65 E-box in nutritional regulation of the fatty acid synthase gene in living animals. Mol. Cell Biol. 23(16), 5896-5907 PMID:12897158 (2003).

46. Paulauskis, J.D. & Sul, H.S. Hormonal regulation of mouse fatty acid synthase gene transcription in liver. J.

Biol. Chem. 264(1), 574-577 PMID:2535847 (1989).

47. Wu, M. et al. Antidiabetic and antisteatotic effects of the selective fatty acid synthase (FAS) inhibitor

platensimycin in mouse models of diabetes. Proc. Natl. Acad. Sci. USA 108(13), 5378-5383

doi: 10.1073/pnas.1002588108, Epub 2011 Mar 9, PMID:21389266 (2011).

48. Dorn, C. et al. Expression of fatty acid synthtase in nonalcoholic fatty liver disease. Int J Clin Exp Pathol. 2010

Mar 25;3(5):505-14.

49. Paniagua, J.A. Nutrition, insulin resistance and dysfunctional adipose tissue determine the different

components of metabolic syndrome. World J. Diabetes 7(19), 483-514 (2016).

50. Huang, J.Y., Chiang, M.T., Yet, S.F. & Chau, L.Y. Myeloid heme oxygenase -1 haploinsufficiency reduces high fat diet-induced insulin resistance by affecting adipose macrophage infiltration in mice. PLoS One 7(6), e38626 doi: 10.1371/journal.pone.0038626. Epub 2012 Jun 21. PMID: 22761690 (2012).

51. Eren, M., Boe, A.E., Klyachko, E.A. & Vaughan, D.E. Role of plasminogen activator inhibitor-1 in senescence and aging. Semin. Thromb. Hemost. 40(6), 645-651 doi:10.1055/s-0034-1387883, Epub 2014 Aug 31, PMID:25173500 (2014).

52. De Taeye, B., Smith, L.H. & Vaughan, D.E. Plasminogen activator inhibitor-1: a common denominator in obesity, diabetes and cardiovascular disease. Curr. Opin. Pharmacol. 5, 149-154

doi:10.1016/j.coph.2005.01.007, PMID: 15780823 (2005).

53. Alessi, M.C., Poggi, M. & Juhan-Vague, I. Plasminogen activator inhibitor-1, adipose tissue and insulin

resistance. Curr. Opin. Pharmacol. 18(3), 240-245 doi: 10.1097/MOL.0b013e32814e6d29, PMID:17495595 (2007).

54. Atsumi, T. et al. Inflammation amplifier, a new paradigm in cancer biology. Cancer Res. 74(1), 8-14

doi: 10.1158/0008-5472. CAN- 13-2322, Epub 2013 Dec 20, PMID:24362915 (2013).

55. Ge, Q., Gerard, J., Noel, L., Scroyen, I. & Brichard, S.M. MicroRNAs regulated by adiponectin as novel targets for controlling adipose tissue inflammation. Endocrinology. 153(11), 5285-5296 doi: 10.1210/en.2012- 1623, Epub 2012 Sep 26, PMID:23015294 (2012).

56. Moreno-Navarrete, J.M. et al. Lipopolysaccharide binding protein is an adipokine involved in the resilience of the mouse adipocyte to inflammation. Diabetologia 58(10), 2424—2434 doi: 10.1007/s00125-015-3692-7, Epub 2015 Jul 23, PMID: 26201685 (2015).

57. Moreno-Navarrete, J.M. et al. A role for adipocyte-derived lipopolysaccharide-binding protein in

inflammation- and obesity-associated adipose tissue dysfunction. Diabetologia 56(11), 2524-2537 doi: 10.1007/s00125-013-3015-9, Epub 2013 Aug 21, PMID:23963324 (2013).

58. Yoon, J.C. et al. Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1. Nature

413(6852), 131-138, PMID:11557972 (2001).

59. Puigserver, P. et al. Insulin-regulated hepatic gluconeogenesis through FOXOl -PGC-1 alpha interaction.

Nature 423(6939), 550-555, Epub 2003 May 18, PMID:12754525 (2003).

60. Iynedjian, P. B. (January 2009). Molecular physiology of mammalian glucokinase" . Cellular and Molecular Life

Sciences. 66 (1): 27^12. doi: 10.1007/s00018-008-8322-9. PMC 2780631. PMID 18726182.

61. van Schaftingen, E. & Gerin, I. (March 2002). "The glucose-6-phosphatase system". The Biochemical Journal.

362 (Pt 3): 513-32. doi: 10.1042/0264-6021:3620513. PMC 1222414. PMID 11879177.

62. Arunagiri A., et al. Misfolded proinsulin in the endoplasmic reticulum during development of beta cell failure in diabetes. Ann. N.Y. Acad. Sci. Apr;1418(l):5-19, 2018.

63. Stidsen, J.V. et al. Pathophysiology-based phenotyping in type 2 diabetes: A clinical classification tool.

Diabetes Metab. Res. Rev. 34(5), e3005, doi:10.1002/dmrr.3005, Epub 2018 Apr 26 (2018). 64. Sataranatarajan, K. et al. Rapamycin increases mortality in db/db mice, a mouse model of type 2 diabetes. J. Gerontol A Bio. I Sci. Med. Sci. 71(7), 850-857 (2016).

65. Thomas C. C., Bakris G. (2014) Metformin nephrotoxicity insights: Will they change clinical management?

Journal of Diabetes. 6 (2014) 111-112. Doi: 10.1111/1753-0407.12113

66. Lloberas, N. et al. Mammalian target of rapamycin pathway blockade slows progression of diabetic kidney disease in rats. J. Am. Soc. Nephrol. 17, 1395-1404 (2006).

67. Yang, Y. et al. Rapamycin prevents early steps of the development of diabetic nephropathy in rats. Am. J.

Nephrol. 27, 495-502 (2007).

68. Ma, M. K. M., Yung, S. & Chan, T.M. mTOR inhibition and kidney disease. Transplantation 102, S32-S40.

(2018).

69. Inoki, K. et al. mTORCl activation in podocytes is a critical step in the development of diabetic nephropathy in mice. J. Clin. Invest. 121(6), 2181 (2011).

70. El-Atat, F.A., Stas, S.N., McFarlane, S.I. & Sowers, J.R. The relationship between hyperinsulinemia,

hypertension and progressive renal disease. J. Am. Soc. Nephrol. 15(11), 2816-2827, PMID: 15504934 (2004).

71. Whaley-Connell, A., Pavey, B.S, Afroze, A. & Bakris, G.F. Obesity and insulin resistance as risk factors for chronic kidney disease. J. Cardiometab. Synd. 1(3), 209-214; quiz 215-216, PMID: 17679819 (2006).

72. Jolivalt, C.G. et al. Peripheral Neuropathy in Mouse Models of Diabetes. Curr. Protoc. Mouse. Biol. 26(3),

223-255, doi : 10.1002/cpmo .11 (2016).

73. Reaven, G.M. Insulin resistance and compensatory hyperinsulinemia: role in hypertension, dyslipidemia, and coronary heart disease. Am Heart J. 121(4 Pt 2), 1283-1288 (1991).

74. Blagosklonny, M.V. Once again on rapamycin-induced insulin resistance and longevity: despite of or owing to. Aging (Albany NY) 4(5), 350-358 (2012).

75. Corkey, B.E. Banting lecture 2011: hyperinsulinemia: cause or consequence? Diabetes 61(1), 4—13,

doi:10.2337/dbl 1-1483, PMID:22187369 (2012).

76. Crofts, C.A.P., Zinn, C., Wheldon, M.C. & Schofield, G.M. Hyperinsulinemia: A unifying concept of chronic disease? Diabesity 1, 34-43, doi:10.15562/diabesity.2015.19 (2015).

77. Miller R.A., et al. Rapamycin-mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. Aging Cell. 13:468-77 (2014) Doi: 10.1111/acel.12194

78. Fok W. C. et al. Mice fed rapamycin have an increase in lifespan associated with major changes in the liver transcriptome. PLoS One 9, e92346 (2014).

79. Christy B. et al. p53 and rapamycin are additive. Oncotarget 6, 15802-132015 (2015).

80. Anisimov, V.N. et al. Rapamycin increases lifespan and inhibits spontaneous tumorigenesis in inbred female mice. Cell Cycle 10, 4230-4236 (2011).

81. Flynn, J.M. et al. Fate-life rapamycin treatment reverses age-related heart dysfunction. Aging Cell 12, 851- 862 (2013).

82. Walters, H.E. & Cox, F.S. mTORC inhibitors as broad-spectrum therapeutics for age-related diseases hit. J. of Mol. Sciences 19, 2325-2358, doi:10.3390/ijmsl9082325 (2018).

83. Zschiedrich, S. et al. Targeting mTOR signaling can prevent the progression of FSGS. J. Am. Soc. Nephrol.

28(7), 2144-2157, doi: 10.1681/ASN.2016050519, Epub 2017 Mar 7 (2017).

84. Inoki, K., Mori, H. & Wang, J. mTORCl activation in podocytes is a critical step in the development of diabetic nephropathy in mice. J. Clin. Invest. 121(6), 2181-2196 (2011).

85. Caccamo, A., Majumder, S., Richardson, A., Strong, R. & Oddo, S. Molecular interplay between mammalian target of rapamycin (mTOR), amyloid, and Tau: effects on cognitive impairments. J. Biol. Chem. 285, 13107— 13120 (2010).

86. Spilman, P. et al. Inhibition of mTOR by rapamycin abolishes cognitive deficits and reduces amyloid levels in a mouse model of Alzheimer’s disease. PLoS ONE 5, e9979 (2010).

87. Kneissel, M. et al. Everolimus suppresses cancellous bone loss, bone resorption, and cathepsin K expression by osteoclasts. Bone 35, 1144-1156 (2004).

88. Mannick, J.B. et al. TORC1 inhibition enhances immune function and reduces infections in the elderly. Sci.

Transl. Med. 10, 449, pii:eaaql564, doiTO.l 126/scitranslmed.aaql564 (2018).

89. Walters, H.E., Deneka-Hannemann, S. & Cox, F.S. Reversal of phenotypes of cellular senescence by pan- mTOR inhibition. Aging 8, 231-244 (2016).

90. Arriola Apelo, S.I. et al. Alternative rapamycin treatment regimens mitigate the impact of rapamycin on

glucose homeostasis and the immune system. Aging Cell 15, 28-38 (2016).

91. Hurez, V. et al. Chronic mTOR inhibition in mice with rapamycin alters T, B, myeloid, and innate lymphoid cells and gut flora and prolongs life of immune-deficient mice. Aging Cell 14, 945-956 (2015).

92. Chen, C., Fiu, Y., Fiu, Y. & Zheng, P. mTOR regulation and therapeutic rejuvenation of aging hematopoietic stem cells. Sci. Signal. 2(98), ra75. doi: 10.1126/scisignal.2000559, PMID: 19934433 (2009).

93. Koza, R.A. et al. Changes in gene expression foreshadow diet-induced obesity in genetically identical mice.

PLoS Genet. 2(5), e81 (2006).

94. Koza, R.A., Hohmann, S.M., Guerra, C., Rossmeisl, M. & Kozak, F.P. Synergistic gene interactions control the induction of the mitochondrial uncoupling protein (Ucpl) gene in white fat tissue. J. Biol. Chem. 275(44), 34486-34492 (2000).

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles“a” and“an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean“at least one.”

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,”“including,”“carrying,”“having,”“containing,”“involving,”“holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases“consisting of’ and“consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

The terms“about” and“substantially” preceding a numerical value mean ±10% of the recited numerical value.

Where a range of values is provided, each value between the upper and lower ends of the range are specifically contemplated and described herein.

Claims

What is claimed is: CLAIMS

1. A method comprising administering to a subject having type 2 diabetes a therapeutically effective amount of rapamycin and metformin.

2. The method of claim 1, wherein the therapeutically effective amount of rapamycin and metformin is sufficient to increase insulin sensitivity in the subject, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of metformin without rapamycin.

3. The method of claim 1 or 2, wherein the therapeutically effective amount of rapamycin and metformin is sufficient to normalize insulin sensitivity in the subject.

4. The method of any one of claims 1-3, wherein the therapeutically effective amount of rapamycin and metformin is sufficient to decrease plasma glucose levels (hyperglycemia) and/or HbAlc levels in the subject by at least 20%, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of rapamycin without metformin.

5. The method of claim 4, wherein the therapeutically effective amount of rapamycin and metformin is sufficient to decrease plasma insulin levels (hyperinsulinemia) in the subject by at least 20%, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of metformin without rapamycin.

6. The method of any one of claims 1-5, wherein the therapeutically effective amount of rapamycin and metformin is sufficient to decrease pancreatic insulin content (PIC) in the subject by at least 20%, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of metformin without rapamycin.

7. The method of any one of claims 1-6, wherein the therapeutically effective amount of rapamycin and metformin is sufficient to decrease pancreatic islet degranulation in the subject, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of rapamycin without metformin.

8. The method of any one of claims 1-7, wherein the therapeutically effective amount of rapamycin and metformin is sufficient to decrease weight gain in the subject by at least 20%, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of metformin without rapamycin.

9. The method of claim 8, wherein the wherein the therapeutically effective amount of rapamycin and metformin is sufficient to reduce adiposity (% fat) in the subject by at least 20%.

10. The method of any one of claims 1-7, wherein the therapeutically effective amount of rapamycin and metformin is sufficient to prevent weight gain in the subject.

11. The method of any one of claims 1-10, wherein the therapeutically effective amount of rapamycin and metformin is sufficient to decrease obesity-associated inflammation in the subject, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of metformin without rapamycin.

12 The method of any one of claims 1-11, wherein the therapeutically effective amount of rapamycin and metformin is sufficient to decrease hepatic steatosis in the subject by at least 50%, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of metformin without rapamycin.

13. The method of any one of claims 1-11, wherein the therapeutically effective amount of rapamycin and metformin is sufficient to prevent hepatic steatosis in the subject, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of metformin without rapamycin.

14. The method of any one of claims 1-13, wherein the therapeutically effective amount of rapamycin and metformin is sufficient to decrease nephropathy in the subject, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of metformin without rapamycin.

15. The method of claim 14, wherein the therapeutically effective amount of rapamycin and metformin is sufficient to decrease a proportion of glomeruli with hyaline thrombi in the subject.

16. The method of claim 14 or 15, wherein the therapeutically effective amount of rapamycin and metformin is sufficient to decrease the albumin/creatinine ratio (ACR) in the subject.

17. The method of any one of claims 1-16, wherein the therapeutically effective amount of rapamycin and metformin is sufficient to decrease hypertriglyceridemia in the subject, relative to a control, optionally wherein the control is an untreated subject or a subject administered an equivalent amount of amount of rapamycin without metformin.

18. The method of claim 17, wherein the therapeutically effective amount of rapamycin and metformin is sufficient to decrease HLDL cholesterol in the subject by at least 10%.

19 The method of any one of claims 1-18, wherein the subject is a human subject.

20. The method of any one of claims 1-19, wherein the type 2 diabetes is a genetic form of type 2 diabetes.