Manuel Blandino-Rosano

Natural history of β-cell adaptation and failure in type 2 diabetes

Type 2 diabetes mellitus (T2D) is a complex disease characterized by β-cell failure in the setting of insulin resistance. The current evidence suggests that genetic predisposition, and environmental factors can impair the capacity of the β-cells to respond to insulin resistance and ultimately lead to their failure. However, genetic studies have demonstrated that known variants account for less than 10% of the overall estimated T2D risk, suggesting that additional unidentified factors contribute to susceptibility of this disease. In this review, we will discuss the different stages that contribute to the development of β-cell failure in T2D. We divide the natural history of this process in three major stages: susceptibility, β-cell adaptation and β-cell failure, and provide an overview of the molecular mechanisms involved. Further research into mechanisms will reveal key modulators of β-cell failure and thus identify possible novel therapeutic targets and potential interventions to protect against β-cell failure.

Decreased IRS Signaling Impairs β-Cell Cycle Progression and Survival in Transgenic Mice Overexpressing S6K in β-Cells

OBJECTIVE The purpose of this study was to evaluate the role of the S6K arm of mammalian target of rapamycin complex 1 (mTORC1) signaling in regulation of β-cell mass and function. Additionally, we aimed to delineate the importance of in vivo S6K activation in the regulation of insulin signaling and the extent to which alteration of insulin receptor substrate (IRS) signaling modulates β-cell mass and function. RESEARCH DESIGN AND METHODS The current experiments describe the phenotype of transgenic mice overexpressing a constitutively active form of S6K under the control of the rat insulin promoter. RESULTS Activation of S6K signaling in these mice improved insulin secretion in the absence of changes in β-cell mass. The lack of β-cell mass expansion resulted from decreased G1-S progression and increased apoptosis. This phenotype was associated with increased p16 and p27 and decreased Cdk2 levels. The changes in cell cycle were accompanied by diminished survival signals because of impaired IRS/Akt signaling. CONCLUSIONS This work defines the importance of S6K in regulation of β-cell cycle, cell size, function, and survival. These experiments also demonstrate that in vivo downregulation of IRS signaling by TORC1/S6K induces β-cell insulin resistance, and that this mechanism could explain some of the abnormalities that ultimately result in β-cell failure and diabetes in conditions of nutrient overload.

mTORC1 signaling and regulation of pancreatic β-cell mass

The capacity of β cells to expand in response to insulin resistance is a critical factor in the development of type 2 diabetes. Proliferation of β cells is a major component for these adaptive responses in animal models. The extracellular signals responsible for β-cell expansion include growth factors, such as insulin, and nutrients, such as glucose and amino acids. AKT activation is one of the important components linking growth signals to the regulation of β-cell expansion. Downstream of AKT, tuberous sclerosis complex 1 and 2 (TSC1/2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling have emerged as prime candidates in this process, because they integrate signals from growth factors and nutrients. Recent studies demonstrate the importance of mTORC1 signaling in β cells. This review will discuss recent advances in the understanding of how this pathway regulates β-cell mass and present data on the role of TSC1 in modulation of β-cell mass. Herein, we also demonstrate that deletion of Tsc1 in pancreatic β cells results in improved glucose tolerance, hyperinsulinemia and expansion of β-cell mass that persists with aging.

Long-lived crowded-litter mice exhibit lasting effects on insulin sensitivity and energy homeostasis

The action of nutrients on early postnatal growth can influence mammalian aging and longevity. Recent work has demonstrated that limiting nutrient availability in the first 3 wk of life [by increasing the number of pups in the crowded-litter (CL) model] leads to extension of mean and maximal lifespan in genetically normal mice. In this study, we aimed to characterize the impact of early-life nutrient intervention on glucose metabolism and energy homeostasis in CL mice. In our study, we used mice from litters supplemented to 12 or 15 pups and compared those to control litters limited to eight pups. At weaning and then throughout adult life, CL mice are significantly leaner and consume more oxygen relative to control mice. At 6 mo of age, CL mice had low fasting leptin concentrations, and low-dose leptin injections reduced body weight and food intake more in CL female mice than in controls. At 22 mo, CL female mice also have smaller adipocytes compared with controls. Glucose and insulin tolerance tests show an increase in insulin sensitivity in 6 mo old CL male mice, and females become more insulin sensitive later in life. Furthermore, β-cell mass was significantly reduced in the CL male mice and was associated with reduction in β-cell proliferation rate in these mice. Together, these data show that early-life nutrient intervention has a significant lifelong effect on metabolic characteristics that may contribute to the increased lifespan of CL mice.

Enhanced beta cell proliferation in mice overexpressing a constitutively active form of Akt and one allele of p21 Cip

Aims/hypothesisThe ability of pancreatic beta cells to proliferate is critical both for normal tissue maintenance and in conditions where there is an increased demand for insulin. Protein kinase B (Akt) plays a major role in promoting proliferation in many cell types, including the insulin-producing beta cells. We have previously reported that mice overexpressing a constitutively active form of Akt (caAktTg) show enhanced beta cell proliferation that is associated with increased protein levels of cyclin D1, cyclin D2 and cyclin-dependent kinase inhibitor 1A (p21Cip). In the present study, we sought to assess the mechanisms responsible for augmented p21Cip levels in caAktTg mice and test the role of p21Cip in the proliferative responses induced by activation of Akt signalling.MethodsTo gain a greater understanding of the relationship between Akt and p21Cip, we evaluated the mechanisms involved in the modulation of p21Cip by Akt and the in vivo role of reduced p21Cip in proliferative responses induced by Akt.ResultsOur experiments showed that Akt signalling regulates p21Cip transcription and protein stability. caAktTg/p21Cip+/− mice exhibited fasting and fed hypoglycaemia as well as hyperinsulinaemia when compared with caAktTg mice. Glucose tolerance tests revealed improved glucose tolerance in caAktTg/p21Cip+/− mice compared with caAktTg. These changes resulted from increased proliferation, survival and beta cell mass in caAktTg/p21Cip+/− compared with caAktTg mice.Conclusions/interpretationOur data indicate that increased p21Cip levels in caAktTg mice act as a compensatory brake, protecting beta cells from unrestrained proliferation. These studies imply that p21Cip could play important roles in the adaptive responses of beta cells to proliferate in conditions such as in insulin resistance.

Overexpression of kinase-dead mTOR impairs glucose homeostasis by regulating insulin secretion and not β-cell mass

Regulation of glucose homeostasis by insulin depends on β-cell growth and function. Nutrients and growth factor stimuli converge on the conserved protein kinase mechanistic target of rapamycin (mTOR), existing in two complexes, mTORC1 and mTORC2. To understand the functional relevance of mTOR enzymatic activity in β-cell development and glucose homeostasis, we generated mice overexpressing either one or two copies of a kinase-dead mTOR mutant (KD-mTOR) transgene exclusively in β-cells. We examined glucose homeostasis and β-cell function of these mice fed a control chow or high-fat diet. Mice with two copies of the transgene [RIPCre;KD-mTOR (Homozygous)] develop glucose intolerance due to a defect in β-cell function without alterations in β-cell mass with control chow. Islets from RIPCre;KD-mTOR (Homozygous) mice showed reduced mTORC1 and mTORC2 signaling along with transcripts and protein levels of Pdx-1. Islets with reduced mTORC2 signaling in their β-cells (RIPCre;Rictorfl/fl) also showed reduced Pdx-1. When challenged with a high-fat diet, mice carrying one copy of KD-mTOR mutant transgene developed glucose intolerance and β-cell insulin secretion defect but showed no changes in β-cell mass. These findings suggest that the mTOR-mediated signaling pathway is not essential to β-cell growth but is involved in regulating β-cell function in normal and diabetogenic conditions.

Exposure of mouse embryonic pancreas to metformin enhances the number of pancreatic progenitors

Aims/hypothesisDeveloping beta cells are vulnerable to nutrient environmental signals. Early developmental processes that alter the number of pancreatic progenitors can determine the number of beta cells present at birth. Metformin, the most widely used oral agent for treating diabetes, alters intracellular energy status in part by increasing AMP-activated protein kinase (AMPK) signalling. This study examined the effect of metformin on developing pancreas and beta cells.MethodsPancreatic rudiments from CD-1 mice at embryonic day 13.0 (E13.0) were cultured with metformin, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR, an AMPK activator) or vehicle control in vitro. In another set of studies, pregnant C57BL/6 mice were treated with metformin throughout gestation. Embryonic (E14.0) and neonatal pancreases were then analysed for their morphometry.ResultsIn vitro metformin treatment led to an increase in the proliferation and number of pancreatic duodenal homeobox 1-positive (PDX1+) progenitors. These results were reproduced by in vitro culture of embryonic pancreas rudiments with AICAR, suggesting that AMPK activation was involved. Similarly, metformin administration to pregnant dams induced an increase in both PDX1+ and neurogenin 3-positive progenitors in the embryonic pancreas at E14.0 and these changes resulted in an increased beta cell fraction in neonates.Conclusions/interpretationThese results indicate that exposure to metformin during gestation modulates the early steps of beta cell development (prior to E14.0) towards an increase in the number of pancreatic and endocrine progenitors. These changes ultimately result in a higher beta cell fraction at birth. These findings are of clinical importance given that metformin is currently used for the treatment of gestational diabetes.

Loss of mTORC1 signaling alters pancreatic α cell mass and impairs glucagon secretion

Glucagon plays a major role in the regulation of glucose homeostasis during fed and fasting states. However, the mechanisms responsible for the regulation of pancreatic &agr; cell mass and function are not completely understood. In the current study, we identified mTOR complex 1 (mTORC1) as a major regulator of &agr; cell mass and glucagon secretion. Using mice with tissue-specific deletion of the mTORC1 regulator Raptor in &agr; cells (&agr;RaptorKO), we showed that mTORC1 signaling is dispensable for &agr; cell development, but essential for &agr; cell maturation during the transition from a milk-based diet to a chow-based diet after weaning. Moreover, inhibition of mTORC1 signaling in &agr;RaptorKO mice and in WT animals exposed to chronic rapamycin administration decreased glucagon content and glucagon secretion. In &agr;RaptorKO mice, impaired glucagon secretion occurred in response to different secretagogues and was mediated by alterations in KATP channel subunit expression and activity. Additionally, our data identify the mTORC1/FoxA2 axis as a link between mTORC1 and transcriptional regulation of key genes responsible for &agr; cell function. Thus, our results reveal a potential function of mTORC1 in nutrient-dependent regulation of glucagon secretion and identify a role for mTORC1 in controlling &agr; cell–mass maintenance.

4E-BP2/SH2B1/IRS2 Are Part of a Novel Feedback Loop That Controls β-Cell Mass.

The mammalian target of rapamycin complex 1 (mTORC1) regulates several biological processes, although the key downstream mechanisms responsible for these effects are poorly defined. Using mice with deletion of eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2), we determine that this downstream target is a major regulator of glucose homeostasis and β-cell mass, proliferation, and survival by increasing insulin receptor substrate 2 (IRS2) levels and identify a novel feedback mechanism by which mTORC1 signaling increases IRS2 levels. In this feedback loop, we show that 4E-BP2 deletion induces translation of the adaptor protein SH2B1 and promotes the formation of a complex with IRS2 and Janus kinase 2, preventing IRS2 ubiquitination. The changes in IRS2 levels result in increases in cell cycle progression, cell survival, and β-cell mass by increasing Akt signaling and reducing p27 levels. Importantly, 4E-BP2 deletion confers resistance to cytokine treatment in vitro. Our data identify SH2B1 as a major regulator of IRS2 stability, demonstrate a novel feedback mechanism linking mTORC1 signaling with IRS2, and identify 4E-BP2 as a major regulator of proliferation and survival of β-cells.

Role of nutrients and mTOR signaling in the regulation of pancreatic progenitors development

Objective Poor fetal nutrition increases the risk of type 2 diabetes in the offspring at least in part by reduced embryonic β-cell growth and impaired function. However, it is not entirely clear how fetal nutrients and growth factors impact β-cells during development to alter glucose homeostasis and metabolism later in life. The current experiments aimed to test the impact of fetal nutrients and growth factors on endocrine development and how these signals acting on mTOR signaling regulate β-cell mass and glucose homeostasis. Method Pancreatic rudiments in culture were used to study the role of glucose, growth factors, and amino acids on β-cell development. The number and proliferation of pancreatic and endocrine progenitor were assessed in the presence or absence of rapamycin. The impact of mTOR signaling in vivo on pancreas development and glucose homeostasis was assessed in models deficient for mTOR or Raptor in Pdx1 expressing pancreatic progenitors. Results We found that amino acid concentrations, and leucine in particular, enhance the number of pancreatic and endocrine progenitors and are essential for growth factor induced proliferation. Rapamycin, an mTORC1 complex inhibitor, reduced the number and proliferation of pancreatic and endocrine progenitors. Mice lacking mTOR in pancreatic progenitors exhibited hyperglycemia in neonates, hypoinsulinemia and pancreatic agenesis/hypoplasia with pancreas rudiments containing ductal structures lacking differentiated acinar and endocrine cells. In addition, loss of mTORC1 by deletion of raptor in pancreatic progenitors reduced pancreas size with reduced number of β-cells. Conclusion Together, these results suggest that amino acids concentrations and in particular leucine modulates growth responses of pancreatic and endocrine progenitors and that mTOR signaling is critical for these responses. Inactivation of mTOR and raptor in pancreatic progenitors suggested that alterations in some of the components of this pathway during development could be a cause of pancreatic agenesis/hypoplasia and hyperglycemia.