Effects of mTOR inhibition on cardiac and adipose tissue pathology and glucose metabolism in rats with metabolic syndrome

Abstract

The mammalian target of rapamycin (mTOR) is a regulator of metabolism and is implicated in pathological conditions such as obesity and diabetes. We aimed to investigate the role of mTOR in obesity. A new animal model of metabolic syndrome (MetS), named DahlS.Z-Lepr^fa /Lepr^fa (DS/obese) rats was established previously in our laboratory. In this study, we used this model to evaluate the effects of mTOR inhibition on cardiac and adipose tissue pathology and glucose metabolism. DS/obese rats were treated with the mTOR inhibitor, everolimus, (0.83 mg/kg per day, per os) for 4 weeks at 9 weeks of age. Age-matched homozygous lean (DahlS.Z-Lepr⁺ /Lepr⁺ or DS/lean) littermates of DS/obese rats were used as controls. Treatment with everolimus ameliorated hypertension, left ventricular (LV) hypertrophy and fibrosis, and LV diastolic dysfunction, and attenuated cardiac oxidative stress and inflammation in DS/obese rats, but had no effect on these parameters in DS/lean rats. Treatment with everolimus reduced Akt Thr308 phosphorylation in the heart of DS/obese rats. It also alleviated obesity, hyperphagia, adipocyte hypertrophy, and adipose tissue inflammation in DS/obese rats. Everolimus treatment exacerbated glucose intolerance, but did not affect Akt phosphorylation levels in the fat or liver in these rats. Pancreatic β-cell mass was increased in DS/obese rats compared with that in DS/lean rats and this effect was attenuated by everolimus. Activation of mTOR signaling contributes to the pathophysiology of MetS and its associated complications. And mTOR inhibition with everolimus ameliorated obesity as well as cardiac and adipose tissue pathology, but exacerbated glucose metabolism in rats with MetS.

Keywords: Adipose tissue; cardiac remodeling; glucose metabolism; mammalian target of rapamycin; metabolic syndrome.

Figure 1

Chronic effects of everolimus on body weight, food intake, systolic blood pressure, and heart rate in DS/obese and control rats from 9 to 13 weeks of age. (A, B) Body weight and food intake were measured weekly. (C, D) Systolic blood pressure and heart rate were measured once every week by the tail‐cuff method. Values are presented as means ± SEM for animals. [n = 8, 10, 7, and 12 for Cont, Cont + Eve, MetS, and MetS + Eve]. *P < 0.05 versus Cont, †P < 0.05 versus Cont+Eve, ‡P < 0.05 versus MetS.

Figure 2

Cardiomyocyte hypertrophy, fibrosis and inflammatory in the left ventricle of rats from the four experimental groups at 13 weeks of age. (A) Representative sections (hematoxylin‐eosin stain) of the left ventricular (LV) myocardium. Scale bars show100 μm. (B) Quantitative measurements of cross‐sectional area of cardiac myocytes. (C, D) Expression of markers of cardiac hypertrophic response. Atrial natriuretic peptide (ANP, C) and brain natriuretic peptide (BNP, D) mRNA levels measured by RT‐PCR in LV tissue samples. Data were normalized to the amount of Gapdh mRNA and then expressed relative to the mean value for DS/lean rats treated with vehicle. (E, F) Histological analysis of fibrosis by Azan‐Mallory staining in perivascular (upper panels) or interstitial (lower panels) regions in the left ventricular myocardium. Scale bars, 100 μm. (G, H) Percentage of interstitial and perivascular fibrosis in the left ventricular (LV) myocardium. (I–K) Relative mRNA expression of collagen I, III, and transforming growth factor (TGF)‐β1 by quantitative PCR. (L) Immunohistochemistry using anti‐CD68 in the LV myocardium. Scale bars. 100 μm. (M) Quantification of immunohistochemical CD68 levels. (N–P) mRNA expression levels of the inflammatory markers osteopontin, monocyte chemoattractant protein‐1 (MCP‐1), and cyclooxygenase (COX)‐2 in the left ventricle. Values in (B) through (D), (G) through (K), and (M) through (P) are expressed as means ± SEM for animals. [n = 8, 10, 7, and 12 for Cont, Cont + Eve, MetS, and MetS + Eve]. *P < 0.05 versus CONT, †P < 0.05 versus Cont + Eve, ‡P < 0.05 versus MetS.

Figure 3

Oxidative stress, and insulin signaling in the left ventricle of rats from the four experimental groups at 13 weeks of age. (A) Impact of everolimus on cardiac oxidative stress level detected by fluorescence dye dihydroethidium (DHE; red spots) Scale bars, 100 μm. (B) DHE‐positive area was quantified as a percentage in each total high power field area. (C) Myocardial NADPH oxidase activity (D, E) Quantitative RT‐PCR analysis of p22^phox and gp91^phox mRNAs. (F–H) Evaluation of Akt/mTOR/p70S6K signal activity by western blotting analysis. Levels of phospho‐p70S6K (F), phospho‐Thr308‐Akt (G), and phospho‐Ser473‐Akt (H). All quantitative data represent means ± SEM. [n = 8, 10, 7, and 12 for Cont, Cont + Eve, MetS, and MetS + Eve (A–C) or n = 6 for each group (D–H)] *P < 0.05 versus CONT, †P < 0.05 versus Cont + Eve, ‡P < 0.05 versus MetS.

Figure 4

Visceral adipose tissue pathology. (A, B) Histology and mean cross‐sectional area of visceral adipocytes. (C) Representative photomicrographs showing immunohistochemical analysis using an anti‐CD68 antibody in visceral adipose tissue. Scale bars, 100 μm. (D) Quantification of anti‐CD68 positive macrophages as shown in (C). (E, F) mRNA expression levels of the inflammatory markers, osteopontin and monocyte chemoattractant protein‐1 (MCP‐1). (G–J) Evaluation of insulin signal Akt/mTOR/p70S6K and AMPK activity. Western blot analysis of the phosphorylation of p70S6K (G), Thr308‐Akt (H), Ser473‐Akt (I), and AMPK (J). All quantitative data represent means ± SEM. [n = 8, 10, 7, and 12 for Cont, Cont+Eve, MetS, and MetS+Eve (B and D–F) or n = 6 for each group (G–J)] *P < 0.05 versus CONT, †P < 0.05 versus Cont + Eve, ‡P < 0.05 versus MetS.

Figure 5

Hepatic inflammation, glycometabolism‐related gene expression, and insulin signal activity in rats from the four experimental groups at 13 weeks of age. (A) Representative pictures of CD68 ‐stained liver sections. Scale bars, 100 μm. (B) Morphometrical analysis of CD68 positive cells from (A). (C, D) Gene expression of osteopontin and monocyte chemoattractant protein‐1 (MCP‐1) in the liver. (E) Representative pictures of hematoxylin and eosin‐stained liver tissue sections. (F–I) mRNA levels of gluconeogenic factors, GR, G6Pase, PEPCK, and11β‐HSD1, in the liver. (J) mRNA expression levels of the glycolytic factor GK in the liver. (K) mRNA expression levels of the lipogenesis‐related factor, SREBP‐1c, in the liver. Results in (F–K) are normalized to the levels observed in Cont rats. (L–N) Evaluation of insulin signal Akt/mTOR/p70S6K activity. Western blot analysis of the phosphorylation of p70S6K (L), Thr308‐Akt (M), and Ser473‐Akt (N). Values in (B) through (J), and (L) through (N) are expressed as means ± SEM for animals. (n = 8, 10, 7, and 12 for Cont, Cont + Eve, MetS, and MetS + Eve) *P < 0.05 versus CONT, †P < 0.05 versus Cont + Eve, ‡P < 0.05 versus MetS.

Figure 6

Glucose metabolism. (A–G) Effects on the oral glucose tolerance test (OGTT), the insulin tolerance test (ITT), the fasting serum glucose, the fasting serum insulin, HOMA‐β, and HOMA‐IR. (H) Immunohistochemistry of pancreatic sections stained for insulin and (I, J) its‐associated morphological parameters. Scale bars, 500 μm. All quantitative data represent means ± SEM. [n = 5, 6, 6, and 7 (A–G) or n = 8, 10, 7, and 12 (I, J) for Cont, Cont + Eve, MetS, and MetS + Eve] *P < 0.05 versus Cont, †P < 0.05 versus Cont + Eve, ‡P < 0.05 versus MetS.