Kerstin Bellmann

Chronic Rapamycin Treatment Causes Glucose Intolerance and Hyperlipidemia by Upregulating Hepatic Gluconeogenesis and Impairing Lipid Deposition in Adipose Tissue

OBJECTIVE The mammalian target of rapamycin (mTOR)/p70 S6 kinase 1 (S6K1) pathway is a critical signaling component in the development of obesity-linked insulin resistance and operates a nutrient-sensing negative feedback loop toward the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway. Whereas acute treatment of insulin target cells with the mTOR complex 1 (mTORC1) inhibitor rapamycin prevents nutrient-induced insulin resistance, the chronic effect of rapamycin on insulin sensitivity and glucose metabolism in vivo remains elusive. RESEARCH DESIGN AND METHODS To assess the metabolic effects of chronic inhibition of the mTORC1/S6K1 pathway, rats were treated with rapamycin (2 mg/kg/day) or vehicle for 15 days before metabolic phenotyping. RESULTS Chronic rapamycin treatment reduced adiposity and fat cell number, which was associated with a coordinated downregulation of genes involved in both lipid uptake and output. Rapamycin treatment also promoted insulin resistance, severe glucose intolerance, and increased gluconeogenesis. The latter was associated with elevated expression of hepatic gluconeogenic master genes, PEPCK and G6Pase, and increased expression of the transcriptional coactivator peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α) as well as enhanced nuclear recruitment of FoxO1, CRTC2, and CREB. These changes were observed despite normal activation of the insulin receptor substrate/PI 3-kinase/Akt axis in liver of rapamycin-treated rats, as expected from the blockade of the mTORC1/S6K1 negative feedback loop. CONCLUSIONS These findings unravel a novel mechanism by which mTORC1/S6K1 controls gluconeogenesis through modulation of several key transcriptional factors. The robust induction of the gluconeogenic program in liver of rapamycin-treated rats underlies the development of severe glucose intolerance even in the face of preserved hepatic insulin signaling to Akt and despite a modest reduction in adiposity.

Heat shock protein hsp70 overexpression confers resistance against nitric oxide

Heat stress is known to render rat islet cells resistant against the toxic effects of nitric oxide, reactive oxygen intermediates and the islet cell toxin streptozotocin. We report here for the first time that protection against nitric oxide is mediated by the major heat shock protein, hsp70, even in the absence of heat stress. The human hsp70 gene was stably transfected into the rat insulinoma cell line RINm5F. Constitutive expression of hsp70 caused protection from NO‐induced cell lysis which was of the same extent as seen after heat stressing cells. Our results identify hsp70 as a defence molecule against nitric oxide.

Potential risk of oral insulin with adjuvant for the prevention of Type I diabetes: a protocol effective in NOD mice may exacerbate disease in BB rats

Summary The impact of oral treatment with insulin on disease development was studied in diabetes prone BB rats. Because of the positive outcome of a prior study in non obese diabetic (NOD) mice, BB rats received insulin in combination with a bacterial adjuvant. Porcine insulin was given orally twice weekly from 35–100 days of age, the E. coli preparation OM-89 was fed on alternate days. Other groups received vehicle, the bacterial adjuvant, or insulin alone. Both insulin containing oral dosing regimens induced a transient non significant delay in diabetes onset. Insulin alone, however did not decrease the final diabetes incidence. Oral dosing with insulin plus adjuvant caused exacerbation of disease development as judged from the decreased survival rate in comparison with the insulin treated group (p < 0.05). Intra-islet infiltration also increased (p < 0.005) compared with the insulin or vehicle treated groups. The effect correlated with enhanced interferon gamma (IFNγ) and decreased interleukin 10 (IL-10) gene expression in the gut suggesting a shift towards proinflammatory T helper 1 (Th1) reactivity (p < 0.01). Although treatment with adjuvant alone also increased the degree of insulitis, an enhanced incidence of diabetes and a shift in cytokine expression was only seen in the group receiving insulin plus adjuvant. Taken together, the data suggest that treatment with a bacterial adjuvant and oral insulin may alter the gut immunoregulatory state such that disease promoting rather than protective immune responses are induced. [Diabetologia (1998) 41: 844–847]

Major involvement of mTOR in the PPARγ-induced stimulation of adipose tissue lipid uptake and fat accretion

Evidence points to a role of the mammalian target of rapamycin (mTOR) signaling pathway as a regulator of adiposity, yet its involvement as a mediator of the positive actions of peroxisome proliferator-activated receptor (PPAR)γ agonism on lipemia, fat accretion, lipid uptake, and its major determinant lipoprotein lipase (LPL) remains to be elucidated. Herein we evaluated the plasma lipid profile, triacylglycerol (TAG) secretion rates, and adipose tissue LPL-dependent lipid uptake, LPL expression/activity, and expression profile of other lipid metabolism genes in rats treated with the PPARγ agonist rosiglitazone (15 mg/kg/day) in combination or not with the mTOR inhibitor rapamycin (2 mg/kg/day) for 15 days. Rosiglitazone stimulated adipose tissue mTOR complex 1 and AMPK and induced TAG-derived lipid uptake (136%), LPL mRNA/activity (2- to 6-fold), and fat accretion in subcutaneous (but not visceral) white adipose tissue (WAT; 50%) and in brown adipose tissue (BAT; 266%). Chronic mTOR inhibition attenuated the upregulation of lipid uptake, LPL expression/activity, and fat accretion induced by PPARγ activation in both subcutaneous WAT and BAT, which resulted in hyperlipidemia. In contrast, rapamycin did not affect most of the other WAT lipogenic genes upregulated by rosiglitazone. Together these findings demonstrate that mTOR is a major regulator of adipose tissue LPL-mediated lipid uptake and a critical mediator of the hypolipidemic and lipogenic actions of PPARγ activation.

Suppression of nitric oxide toxicity in islet cells by α-tocopherol

We show here that preincubation of pancreatic islet cells with α‐tocopherol significantly improves their resistance to toxic doses of nitric oxide (NO). No protection was afforded by other antioxidants such as vitamin C or glutathione‐monoethyl ester. The pathway of NO induced islet cell death involves DNA damage and excessive activation of poly(ADP‐ribose)polymerase leading to irreversible depletion of intracellular NAD+. α‐Tocopherol was found to interfere at early steps of this pathway, by preventing the occurrence of DNA strand breaks. This indicates that α‐tocopherol directly interacts with NO or its reactive intermediates. We conclude that α‐tocopherol is not only part of the cellular defence system against oxygen radicals but also protects eukaryotic cells from NO toxicity.

Oral insulin for diabetes prevention in NOD mice: potentiation by enhancing Th2 cytokine expression in the gut through bacterial adjuvant

Summary Oral administration of insulin suppresses the development of diabetes in nonobese diabetic (NOD) mice and deviates the cytokine balance in the islets of Langerhans from a Th1 to a Th2 type cytokine pattern. However, the effect of oral insulin is limited and disease suppression is limited to a narrow dose range. Therefore we tried to improve the outcome of suboptimal insulin dosing by bacterial adjuvant. Mice treated with a suboptimal dose of oral insulin showed no change in diabetes incidence although a shift from Th1 towards Th2 cytokine expression occurred in inflamed islets. Significant suppression of diabetes development was only seen in NOD mice receiving both, insulin and the bacterial preparation OM-89 as adjuvant. OM-89 is a protein extract of Escherichia coli, with nonspecific immunostimulatory properties. Potentiation of the effect of oral insulin by the adjuvant was associated with upregulation of interleukin (IL)-4 Th2 cells in infiltrated islets and sustained local IL-2 gene expression. RT PCR analyses of cytokine expression in the gut showed a clear deviation to Th2 type reactivity and downregulation of inducible nitric oxide (NO) synthase (iNOS) expression by the bacterial adjuvant but not by oral insulin alone. Since macrophages are the primary target cells of adjuvant action we tested its effect on mouse macrophages in vitro. Treatment with OM-89 induced transient release of tumour necrosis factor alpha and nitrite but rendered macrophages refractory to restimulation by the potent macrophage activator lipopolysaccharide. In conclusion, the protective effect of oral insulin can be potentiated by pretreatment with the bacterial adjuvant OM-89. This effect correlates with enhanced Th2 cytokine and decreased iNOS gene expression in the gut, probably due to the downregulation of proinflammatory mediators by exposure to the adjuvant. [Diabetologia (1997) 40: 902–909]

Low Stress Response Enhances Vulnerability of Islet Cells in Diabetes-Prone BB Rats

In islet cells isolated from normal outbred Wistar rats, the known high vulnerability of islet cells toward oxygen radicals or nitric oxide can be abolished by inducing a stress response, such as by heat shock. We show here that islet cells from diabetes-prone BB rats are unable to mount such a protective response. Islet cells from diabetes-prone BB rats without recognizable insulitis were heat stressed. Subsequently, cells were exposed to nitric oxide, to oxygen radicals, or to the β-cell toxin streptozotocin. While prior heat shock substantially increased the survival of toxin-treated Wistar rat islet cells, no protective stress response was noted for islet cells from diabetes-prone BB rats. Islet cells from diabetes-resistant BB rats were protected by heat stress to the same extent as Wistar rats. A survey of four additional major histocompatibility complex (MHC)-disparate rat strains confirmed the existence of a low and high responder type to stress. Parallel analysis of heat shock protein (hsp)70 induction by Western blot showed a low and high hsp70 response phenotype. A high hsp70 response coincided with a protective stress response. The presence (or absence) of a protective stress response correlated with the preservation (or loss) of intracellular NAD+ in toxin-treated islet cells. The lack of a protective stress response in islet cells from diabetes-prone BB rats, but not in diabetes-resistant BB rats, may promote β-cell lysis and autoantigen release, and hence could be important for initiation or propagation of the disease process.

Hepatocyte-Specific Ptpn6 Deletion Protects From Obesity-Linked Hepatic Insulin Resistance

The protein-tyrosine phosphatase Shp1 negatively regulates insulin action on glucose homeostasis in liver and muscle, but its potential role in obesity-linked insulin resistance has not been examined. To investigate the role of Shp1 in hepatic insulin resistance, we generated hepatocyte-specific Shp1 knockout mice (Ptpn6H-KO), which were subjected to extensive metabolic monitoring throughout an 8-week standard chow diet (SD) or high-fat diet (HFD) feeding. We report for the first time that Shp1 expression is upregulated in metabolic tissues of HFD-fed obese mice. When compared with their Shp1-expressing Ptpn6f/f littermates, Ptpn6H-KO mice exhibited significantly lowered fasting glycemia and heightened hepatic insulin sensitivity. After HFD feeding, Ptpn6H-KO mice developed comparable levels of obesity as Ptpn6f/f mice, but they were remarkably protected from liver insulin resistance, as revealed by euglycemic clamps and hepatic insulin signaling determinations. Although Ptpn6H-KO mice still acquired diet-induced peripheral insulin resistance, they were less hyperinsulinemic during a glucose tolerance test because of reduced insulin secretion. Ptpn6H-KO mice also exhibited increased insulin clearance in line with enhanced CC1 tyrosine phosphorylation in liver. These results show that hepatocyte Shp1 plays a critical role in the development of hepatic insulin resistance and represents a novel therapeutic target for obesity-linked diabetes.

Hepatocyte‐specific Ptpn6 deletion promotes hepatic lipid accretion, but reduces NAFLD in diet‐induced obesity: Potential role of PPARγ

Hepatocyte‐specific Shp1 knockout mice (Ptpn6H‐KO) are protected from hepatic insulin resistance evoked by high‐fat diet (HFD) feeding for 8 weeks. Unexpectedly, we report herein that Ptpn6H‐KO mice fed an HFD for up to 16 weeks are still protected from insulin resistance, but are more prone to hepatic steatosis, as compared with their HFD‐fed Ptpn6f/f counterparts. The livers from HFD‐fed Ptpn6H‐KO mice displayed 1) augmented lipogenesis, marked by increased expression of several hepatic genes involved in fatty acid biosynthesis, 2) elevated postprandial fatty acid uptake, and 3) significantly reduced lipid export with enhanced degradation of apolipoprotein B (ApoB). Despite more extensive hepatic steatosis, the inflammatory profile of the HFD‐fed Ptpn6H‐KO liver was similar (8 weeks) or even improved (16 weeks) as compared to their HFD‐fed Ptpn6f/f littermates, along with reduced hepatocellular damage as revealed by serum levels of hepatic enzymes. Interestingly, comparative microarray analysis revealed a significant up‐regulation of peroxisome proliferator‐activated receptor gamma (PPARγ) gene expression, confirmed by quantitative polymerase chain reaction. Elevated PPARγ nuclear activity also was observed and found to be directly regulated by Shp1 in a cell‐autonomous manner. Conclusion: These findings highlight a novel role for hepatocyte Shp1 in the regulation of PPARγ and hepatic lipid metabolism. Shp1 deficiency prevents the development of severe hepatic inflammation and hepatocellular damage in steatotic livers, presenting hepatocyte Shp1 as a potential novel mediator of nonalcoholic fatty liver diseases in obesity. (Hepatology 2014;59:1803–1815)

Deficient heat shock protein 70 response to stress in leukocytes at onset of type 1 diabetes

Type 1 diabetes is caused by the immune-mediated destruction of pancreatic beta cells. Animal models of the disease demonstrate an increased susceptibility of beta cells to immunological attacks due to their defective stress-responsiveness. To investigate the stress-responsiveness in human type 1 diabetes we analyzed the heat-inducibility of the dominant stress protein heat shock protein (Hsp)70 in diabetic patients at different disease stages. At diabetes-manifestation heat-induced Hsp70 levels in peripheral blood mononuclear cells (PBMC) reached only about 25% of the levels expressed by heat-treated PBMC from non-diabetic subjects (p<0.05). Heat-responsiveness improved with disease duration and was re-established at more than eight months after disease-manifestation. Hyperthermia-induced Hsp70 expression was decreased by the T-helper 1-associated cytokine interferon-gamma and increased by the T-helper 2-associated transforming growth factor-beta. We conclude that impaired cellular stress-responsiveness, aggravated by the inflammatory milieu at the onset of type 1 diabetes, contributes to disease manifestation.