The study was undertaken to explore the effect of rapamycin, an anti-inflammatory agent, on the metabolic profile of type 2 diabetic mice. Seven-month-old diabetic db/db mice and their lean littermate non-diabetic controls (db/m) were randomized to receive control chow or chow mixed with rapamycin (2.24 mg/kg/day) (each group n =20, males and females) for 4 months and sacrificed. Serum samples were analyzed for the measurement of glucose, creatinine, blood urea nitrogen (BUN), alkaline phosphatase (ALP), alanine aminotransferase (ALT), total cholesterol, total triglyceride, and total protein, using the automated dry chemistry analysis. Rapamycin elevated serum glucose in female diabetic mice. Serum creatinine tended to be higher in diabetic mice but was not affected by rapamycin; there was no difference in BUN levels among the groups. Serum ALP was elevated in diabetic mice and rapamycin lowered it only in female diabetic mice; serum ALT levels were increased in female diabetic mice, unaffected by rapamycin. Serum total protein was elevated in diabetic mice of both genders but was not affected by rapamycin. Diabetic mice from both genders had elevated serum cholesterol and triglycerides; rapamycin did not affect serum cholesterol but decreased serum total triglycerides in male diabetic mice. We conclude that rapamycin elicits complex metabolic responses in aging diabetic mice, worsening hyperglycemia in females but improving ALP in female diabetic and total triglycerides in male diabetic mice, respectively. The metabolic effects of rapamycin should be considered while performing studies with rapamycin in mice.

The inhibition of mTOR (mechanistic target of rapamycin) by the macrolide rapamycin has many beneficial effects in mice, including extension of lifespan and reduction or prevention of several age-related diseases. At the same time, chronic rapamycin treatment causes impairments in glucose metabolism including hyperglycemia, glucose intolerance and insulin resistance. It is unknown whether these metabolic effects of rapamycin are permanent or whether they can be alleviated. Here, we confirmed that rapamycin causes glucose intolerance and insulin resistance in both inbred and genetically heterogeneous mice fed either low fat or high fat diets, suggesting that these effects of rapamycin are independent of genetic background. Importantly, we also found that these effects were almost completely lost within a few weeks of cessation of treatment, showing that chronic rapamycin treatment does not induce permanent impairment of glucose metabolism. Somewhat surprisingly, chronic rapamycin also promoted increased accumulation of adipose tissue in high fat fed mice. However, this effect too was lost when rapamycin treatment was ended suggesting that this effect of rapamycin is also not permanent. The reversible nature of rapamycin's alterations of metabolic function suggests that these potentially detrimental side-effects might be managed through alternative dosing strategies or concurrent treatment options.

Calorie restriction (CR), which lengthens lifespan in many species, is associated with moderate hyperadrenocorticism and attenuated inflammation. Given the anti-inflammatory action of glucocorticoids, we tested the hypothesis that the hyperadrenocorticism of CR contributes to its attenuated inflammatory response. We used a corticotropin-releasing-hormone knockout (CRHKO) mouse, which is glucocorticoid insufficient. There were four controls groups: CRHKO mice and wild-type (WT) littermates fed either ad libitum (AL) or CR (60% of AL food intake), and three experimental groups: (a) AL-fed CRHKO mice given corticosterone (CORT) in their drinking water titrated to match the integrated 24-hr plasma CORT levels of AL-fed WT mice, (b) CR-fed CRHKO mice given CORT to match the 24-hr CORT levels of AL-fed WT mice, and (c) CR-fed CHRKO mice given CORT to match the 24-hr CORT levels of CR-fed WT mice. Inflammation was measured volumetrically as footpad edema induced by carrageenan injection. As previously observed, CR attenuated footpad edema in WT mice. This attenuation was significantly blocked in CORT-deficient CR-fed CRHKO mice. Replacement of CORT in CR-fed CRHKO mice to the elevated levels observed in CR-fed WT mice, but not to the levels observed in AL-fed WT mice, restored the anti-inflammatory effect of CR. These results indicate that the hyperadrenocorticism of CR contributes to the anti-inflammatory action of CR, which may in turn contribute to its life-extending actions.

Loss of SURF1, a Complex IV assembly protein, was reported to increase lifespan in mice despite dramatically lower cytochrome oxidase (COX) activity. Consistent with this, our previous studies found advantageous changes in metabolism (reduced adiposity, increased insulin sensitivity, and mitochondrial biogenesis) in Surf1-/- mice. The lack of deleterious phenotypes in Surf1-/- mice is contrary to the hypothesis that mitochondrial dysfunction contributes to aging. We found only a modest (nonsignificant) extension of lifespan (7% median, 16% maximum) and no change in healthspan indices in Surf1-/- vs. Surf1+/+ mice despite substantial decreases in COX activity (22%-87% across tissues). Dietary restriction (DR) increased median lifespan in both Surf1+/+ and Surf1-/- mice (36% and 19%, respectively). We measured gene expression, metabolites, and targeted expression of key metabolic proteins in adipose tissue, liver, and brain in Surf1+/+ and Surf1-/- mice. Gene expression was differentially regulated in a tissue-specific manner. Many proteins and metabolites are downregulated in Surf1-/- adipose tissue and reversed by DR, while in brain, most metabolites that changed were elevated in Surf1-/- mice. Finally, mitochondrial unfolded protein response (UPRmt )-associated proteins were not uniformly altered by age or genotype, suggesting the UPRmt is not a key player in aging or in response to reduced COX activity. While the changes in gene expression and metabolism may represent compensatory responses to mitochondrial stress, the important outcome of this study is that lifespan and healthspan are not compromised in Surf1-/- mice, suggesting that not all mitochondrial deficiencies are a critical determinant of lifespan.

Rapamycin was found to increase (11% to 16%) the lifespan of male and female C57BL/6J mice most likely by reducing the increase in the hazard for mortality (i.e., the rate of aging) term in the Gompertz mortality analysis. To identify the pathways that could be responsible for rapamycin's longevity effect, we analyzed the transcriptome of liver from 25-month-old male and female mice fed rapamycin starting at 4 months of age. Few changes (<300 transcripts) were observed in transcriptome of rapamycin-fed males; however, a large number of transcripts (>4,500) changed significantly in females. Using multidimensional scaling and heatmap analyses, the male mice fed rapamycin were found to segregate into two groups: one group that is almost identical to control males (Rapa-1) and a second group (Rapa-2) that shows a change in gene expression (>4,000 transcripts) with more than 60% of the genes shared with female mice fed Rapa. Using ingenuity pathway analysis, 13 pathways were significantly altered in both Rapa-2 males and rapamycin-fed females with mitochondrial function as the most significantly changed pathway. Our findings show that rapamycin has a major effect on the transcriptome and point to several pathways that would likely impact the longevity.

Canagliflozin (Cana) is an FDA-approved diabetes drug that protects against cardiovascular and kidney diseases. It also inhibits the sodium glucose transporter 2 by blocking renal reuptake and intestinal absorption of glucose. In the context of the mouse Interventions Testing Program, genetically heterogeneous mice were given chow containing Cana at 180 ppm at 7 months of age until their death. Cana extended median survival of male mice by 14%. Cana also increased by 9% the age for 90th percentile survival, with parallel effects seen at each of 3 test sites. Neither the distribution of inferred cause of death nor incidental pathology findings at end-of-life necropsies were altered by Cana. Moreover, although no life span benefits were seen in female mice, Cana led to lower fasting glucose and improved glucose tolerance in both sexes, diminishing fat mass in females only. Therefore, the life span benefit of Cana is likely to reflect blunting of peak glucose levels, because similar longevity effects are seen in male mice given acarbose, a diabetes drug that blocks glucose surges through a distinct mechanism, i.e., slowing breakdown of carbohydrate in the intestine. Interventions that control daily peak glucose levels deserve attention as possible preventive medicines to protect from a wide range of late-life neoplastic and degenerative diseases.