Deletion of pancreatic β‐cell adenosine kinase improves glucose homeostasis in young mice and ameliorates streptozotocin‐induced hyperglycaemia

Severe reduction in the β‐cell number (collectively known as the β‐cell mass) contributes to the development of both type 1 and type 2 diabetes. Recent pharmacological studies have suggested that increased pancreatic β‐cell proliferation could be due to specific inhibition of adenosine kinase (ADK). However, genetic evidence for the function of pancreatic β‐cell ADK under physiological conditions or in a pathological context is still lacking. In this study, we crossed mice carrying LoxP‐flanked Adk gene with Ins2‐Cre mice to acquire pancreatic β ‐cell ADK deficiency (Ins2‐Cre^±Adk^fl/fl) mice. Our results revealed that Ins2‐Cre^+/‐Adk^fl/fl mice showed improved glucose metabolism and β‐cell mass in younger mice, but showed normal activity in adult mice. Moreover, Ins2‐Cre^±Adk^fl/fl mice were more resistant to streptozotocin (STZ) induced hyperglycaemia and pancreatic β‐cell damage in adult mice. In conclusion, we found that ADK negatively regulates β‐cell replication in young mice as well as under pathological conditions, such as STZ induced pancreatic β‐cell damage. Our study provided genetic evidence that specific inhibition of pancreatic β‐cell ADK has potential for anti‐diabetic therapy.     

Characterization of β‐Cell Mass and Insulin Resistance in Diet‐induced Obese and Diet‐resistant Rats

The selectively bred diet‐induced obese (DIO) and diet‐resistant (DR) rats represent a polygenetic animal model mimicking most clinical variables characterizing the human metabolic syndrome. When fed a high‐energy (HE) diet DIO rats develop visceral obesity, dyslipidemia, hyperinsulinemia, and insulin resistance but never frank diabetes. To improve our understanding of the underlying cause for the deteriorating glucose and insulin parameters, we have investigated possible adaptive responses in DIO and DR rats at the level of the insulin‐producing β‐cells. At the time of weaning, DR rats were found to have a higher body weight and β‐cell mass compared to DIO rats, and elevated insulin and glucose responses to an oral glucose load. However, at 2.5 months of age, and for the remaining study period, the effect of genotype became evident: the chow‐fed DIO rats steadily increased their body weight and β‐cell mass, as well as insulin and glucose levels compared to the DR rats. HE feeding affected both DIO and DR rats leading to an increased body weight and an increased β‐cell mass. Interestingly, although the β‐cell mass in DR rats and chow‐fed DIO rats appeared to constantly increase with age, the β‐cell mass in the HE‐fed DIO rats did not continue to do so. This might constitute part of an explanation for their reduced glucose tolerance. Collectively, the data support the use of HE‐fed DIO rats as a model of human obesity and insulin resistance, and accentuate its relevance for studies examining the benefit of pharmaceutical compounds targeting this disease complex.     

Targeted proteomics reveals strain‐specific changes in the mouse insulin and central metabolic pathways after a sustained high‐fat diet

The metabolic syndrome is a collection of risk factors including obesity, insulin resistance and hepatic steatosis, which occur together and increase the risk of diseases such as diabetes, cardiovascular disease and cancer. In spite of intense research, the complex etiology of insulin resistance and its association with the accumulation of triacylglycerides in the liver and with hepatic steatosis remains not completely understood. Here, we performed quantitative measurements of 144 proteins involved in the insulin‐signaling pathway and central metabolism in liver homogenates of two genetically well‐defined mouse strains C57BL/6J and 129Sv that were subjected to a sustained high‐fat diet. We used targeted mass spectrometry by selected reaction monitoring (SRM) to generate accurate and reproducible quantitation of the targeted proteins across 36 different samples (12 conditions and 3 biological replicates), generating one of the largest quantitative targeted proteomics data sets in mammalian tissues. Our results revealed rapid response to high‐fat diet that diverged early in the feeding regimen, and evidenced a response to high‐fat diet dominated by the activation of peroxisomal β‐oxidation in C57BL/6J and by lipogenesis in 129Sv mice.     

Role and impact of the extracellular matrix on integrin‐mediated pancreatic β‐cell functions

Understanding the organisation and role of the extracellular matrix (ECM) in islets of Langerhans is critical for maintaining pancreatic β‐cells, and to recognise and revert the physiopathology of diabetes. Indeed, integrin‐mediated adhesion signalling in response to the pancreatic ECM plays crucial roles in β‐cell survival and insulin secretion, two major functions, which are affected in diabetes. Here, we would like to present an update on the major components of the pancreatic ECM, their role during integrin‐mediated cell‐matrix adhesions and how they are affected during diabetes. To treat diabetes, a promising approach consists in replacing β‐cells by transplantation. However, efficiency is low, because β‐cells suffer of anoikis, due to enzymatic digestion of the pancreatic ECM, which affects the survival of insulin‐secreting β‐cells. The strategy of adding ECM components during transplantation, to reproduce the pancreatic microenvironment, is a challenging task, as many of the regulatory mechanisms that control ECM deposition and turnover are not sufficiently understood. A better comprehension of the impact of the ECM on the adhesion and integrin‐dependent signalling in β‐cells is primordial to improve the healthy state of islets to prevent the onset of diabetes as well as for enhancing the efficiency of the islet transplantation therapy.     

Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells

Cannabinoid 1 receptors (CB1Rs) are expressed in peripheral tissues, including islets of Langerhans, where their function(s) is under scrutiny. Using mouse β‐cell lines, human islets and CB1R‐null (CB1R^?/?) mice, we have now investigated the role of CB1Rs in modulating β‐cell function and glucose responsiveness. Synthetic CB1R agonists diminished GLP‐1‐mediated cAMP accumulation and insulin secretion as well as glucose‐stimulated insulin secretion in mouse β‐cell lines and human islets. In addition, silencing CB1R in mouse β cells resulted in an increased expression of pro‐insulin, glucokinase (GCK) and glucose transporter 2 (GLUT2), but this increase was lost in β cells lacking insulin receptor. Furthermore, CB1R^?/? mice had increased pro‐insulin, GCK and GLUT2 expression in β cells. Our results suggest that CB1R signalling in pancreatic islets may be harnessed to improve β‐cell glucose responsiveness and preserve their function. Thus, our findings further support that blocking peripheral CB1Rs would be beneficial to β‐cell function in type 2 diabetes.     

Excess cholesterol inhibits glucose‐stimulated fusion pore dynamics in insulin exocytosis

Type 2 diabetes is caused by defects in both insulin sensitivity and insulin secretion. Glucose triggers insulin secretion by causing exocytosis of insulin granules from pancreatic β‐cells. High circulating cholesterol levels and a diminished capacity of serum to remove cholesterol from β‐cells are observed in diabetic individuals. Both of these effects can lead to cholesterol accumulation in β‐cells and contribute to β‐cell dysfunction. However, the molecular mechanisms by which cholesterol accumulation impairs β‐cell function remain largely unknown. Here, we used total internal reflection fluorescence microscopy to address, at the single‐granule level, the role of cholesterol in regulating fusion pore dynamics during insulin exocytosis. We focused particularly on the effects of cholesterol overload, which is relevant to type 2 diabetes. We show that excess cholesterol reduced the number of glucose‐stimulated fusion events, and modulated the proportion of full fusion and kiss‐and‐run fusion events. Analysis of single exocytic events revealed distinct fusion kinetics, with more clustered and compound exocytosis observed in cholesterol‐overloaded β‐cells. We provide evidence for the involvement of the GTPase dynamin, which is regulated in part by cholesterol‐induced phosphatidylinositol 4,5‐bisphosphate enrichment in the plasma membrane, in the switch between full fusion and kiss‐and‐run fusion. Characterization of insulin exocytosis offers insights into the role that elevated cholesterol may play in the development of type 2 diabetes.     

A Spatial Model of Insulin‐Granule Dynamics in Pancreatic β‐Cells

Insulin secretion from pancreatic β‐cells in response to sudden glucose stimulation is biphasic. Prolonged secretion in vivo requires synthesis, delivery to the plasma membrane (PM) and exocytosis of insulin secretory granules (SGs). Here, we provide the first agent‐based space‐resolved model for SG dynamics in pancreatic β‐cells. Using recent experimental data, we consider a single β‐cell with identical SGs moving on a phenomenologically represented cytoskeleton network. A single exocytotic machinery mediates SG exocytosis on the PM. This novel model reproduces the measured spatial organization of SGs and insulin secretion patterns under different stimulation protocols. It proposes that the insulin potentiation effect and the rising second‐phase secretion are mainly due to the increasing number of docking sites on the PM. Furthermore, it shows that 6 min after glucose stimulation, the ‘newcomer’ SGs are recruited from a region within less than 600 nm from the PM.      

Thymus‐specific deletion of insulin induces autoimmune diabetes

Insulin expression in the thymus has been implicated in regulating the negative selection of autoreactive T cells and in mediating the central immune tolerance towards pancreatic β‐cells. To further explore the function of this ectopic insulin expression, we knocked out the mouse Ins2 gene specifically in the Aire‐expressing medullary thymic epithelial cells (mTECs), without affecting its expression in the β‐cells. When further crossed to the Ins1 knockout background, both male and female pups (designated as ID‐TEC mice for insulin‐deleted mTEC) developed diabetes spontaneously around 3 weeks after birth. β‐cell‐specific autoimmune destruction was observed, as well as islet‐specific T cell infiltration. The presence of insulin‐specific effector T cells was shown using ELISPOT assays and adoptive T cell transfer experiments. Results from thymus transplantation experiments proved further that depletion of Ins2 expression in mTECs was sufficient to break central tolerance and induce anti‐insulin autoimmunity. Our observations may explain the rare cases of type 1 diabetes onset in very young children carrying diabetes‐resistant HLA class II alleles. ID‐TEC mice could serve as a new model for studying this pathology.     

Chronic high glucose induced INS‐1β cell mitochondrial dysfunction: A comparative mitochondrial proteome with SILAC

As glucose‐stimulated insulin secretion of pancreatic β cell is triggered and promoted by the metabolic messengers derived from mitochondria, mitochondria take a central stage in the normal function of β cells. β cells in diabetics were chronically exposed to hyperglycemia stimulation, which have been reported to exert deleterious effects on β‐cell mitochondria. However, the mechanism of the toxic effects of hyperglycemia on β‐cell mitochondria was not clear. In this study, we characterized the biological functional changes of rat INS‐1β cells and their mitochondria with chronic exposure to hyperglycemia and created a research model of chronic hyperglycemia‐induced dysfunctional β cells with damaged mitochondria. Then, SILAC‐based quantitative proteomic approach was used to compare the mitochondrial protein expression from high glucose treated INS‐1β cells and control cells. The expression of some mitochondrial proteins was found with significant changes. Functional classification revealed most of these proteins were related with oxidative phosphorylation, mitochondrial protein biosynthesis, substances metabolism, transport, and cell death. These results presented some useful information about the effect of glucotoxicity on the β‐cell mitochondria.     

Glucose intolerance in young TallyHo mice is induced by leptin-mediated inhibition of insulin secretion

The pathophysiology of TallyHo mouse, a recently established animal model for type 2 diabetes mellitus, was analyzed at prediabetic state to examine the inherent defects which contribute to the development of diabetes. At 4 weeks of age, the TallyHo mice already revealed glucose intolerance while their peripheral tissues exhibited normal insulin sensitivity. On the other hand, decreased plasma insulin concentration was observed with little differences in pancreatic insulin contents, indicating the impaired insulin secretion. Such defect, however, was not found in the isolated islets, which suggests a role of endocrine factor in impaired insulin secretion of TallyHo mice. Treatment of leptin inhibited the glucose-stimulated insulin secretion from the isolated islets of TallyHo mice, while in vivo administration of anti-leptin antibody lowered plasma glucose concentration with increased insulin level in TallyHo mice. Expression of glucokinase mRNA was decreased both in whole pancreas and leptin treated islets of TallyHo mice compared with whole pancreas in C57BL/6 mice and untreated islets of TallyHo mice, respectively. These results suggest that elevated plasma leptin can, through the inhibition of insulin secretion, induce glucose intolerance in TallyHo mice.     

N‐cadherin is dispensable for pancreas development but required for β‐cell granule turnover

The cadherin family of cell adhesion molecules mediates adhesive interactions that are required for the formation and maintenance of tissues. Previously, we demonstrated that N‐cadherin, which is required for numerous morphogenetic processes, is expressed in the pancreatic epithelium at E9.5, but later becomes restricted to endocrine aggregates in mice. To study the role of N‐cadherin during pancreas formation and function we generated a tissue‐specific knockout of N‐cadherin in the early pancreatic epithelium by inter‐crossing N‐cadherin‐floxed mice with Pdx1Cre mice. Analysis of pancreas‐specific ablation of N‐cadherin demonstrates that N‐cadherin is dispensable for pancreatic development, but required for β‐cell granule turnover. The number of insulin secretory granules is significantly reduced in N‐cadherin‐deficient β‐cells, and as a consequence insulin secretion is decreased. genesis 48:374–381, 2010. © 2010 Wiley‐Liss, Inc.     

Dihydromyricetin enhances glucose uptake by inhibition of MEK/ERK pathway and consequent down‐regulation of phosphorylation of PPARγ in 3T3‐L1 cells

Accumulating evidence suggests that inhibition of mitogen‐activated protein kinase signalling can reduce phosphorylation of peroxisome proliferator‐activated receptor γ (PPARγ) at serine 273, which mitigates obesity‐associated insulin resistance and might be a promising treatment for type 2 diabetes. Dihydromyricetin (DHM) is a flavonoid that has many beneficial pharmacological properties. In this study, mouse fibroblast 3T3‐L1 cells were used to investigate whether DHM alleviates insulin resistance by inhibiting PPARγ phosphorylation at serine 273 via the MEK/ERK pathway. 3T3‐L1 pre‐adipocytes were differentiated, and the effects of DHM on adipogenesis and glucose uptake in the resulting adipocytes were examined. DHM was found to dose dependently increase glucose uptake and decrease adipogenesis. Insulin resistance was then induced in adipocytes using dexamethasone, and DHM was shown to dose and time dependently promote glucose uptake in the dexamethasone‐treated adipocytes. DHM also inhibited phosphorylation of PPARγ and ERK. Inhibition of PPARγ activity with GW9662 potently blocked DHM‐induced glucose uptake and adiponectin secretion. Interestingly, DHM showed similar effects to PD98059, an inhibitor of the MEK/ERK pathway. DHM acted synergistically with PD98059 to improve glucose uptake and adiponectin secretion in dexamethasone‐treated adipocytes. In conclusion, our findings indicate that DHM improves glucose uptake in adipocytes by inhibiting ERK‐induced phosphorylation of PPARγ at serine 273.     

A new paradigm in cell therapy for diabetes: Turning pancreatic α‐cells into β‐cells

Cell therapy means treating diseases with the body's own cells. One of the cell types most in demand for therapeutic purposes is the pancreatic β‐cell. This is because diabetes is one of the major healthcare problems in the world. Diabetes can be treated by islet transplantation but the major limitation is the shortage of organ donors. To overcome the shortfall in donors, alternative sources of pancreatic β‐cells must be found. Potential sources include embryonic or adult stem cells or, from existing β‐cells. There is now a startling new addition to this list of therapies: the pancreatic α‐cell. Thorel and colleagues recently showed that under circumstances of extreme pancreatic β‐cell loss, α‐cells may serve to replenish the insulin‐producing compartment. This conversion of α‐cells to β‐cells represents an example of transdifferentiation. Understanding the molecular basis for transdifferentiation may help to enhance the generation of β‐cells for the treatment of diabetes.     

Extract of Ocimum canum lowers blood glucose and facilitates insulin release by isolated pancreatic β-islet cells

Aqueous extract of Ocimum canum Sim, (Lamiaceae) is used by some Ghanaians to manage diabetes mellitus. In vivo modulation of levels of fasting blood glucose by 0. canum extract was evaluated in type-II diabetes mellitus using the C57BL/KsJ db/db genetically diabetic animal model, and its effects on glucose-stimulated insulin release in vitro were monitored using isolated rat pancreatic beta-islet cells. The results showed that fasting blood glucose levels and body weight decreased significantly (p < 0.05) in diabetic and non-diabetic C57BL/KsJ mice, which were administered aqueous extract of 0. canum. In vitro, the 0. canum extract significantly enhanced insulin release from isolated rat pancreatic beta-islet cells. Insulin release was found to be dependent on glucose concentration and increased with increasing O. canum concentration in the incubation medium up to an optimum extract concentration of 0.03 mg/ml. Release of the hormone decreased beyond this concentration of extract in the medium. Addition to the medium of Desmodium adscendens, a plant preparation used to manage inflammatory disorders, did not increase but rather inhibited insulin secretion by the pancreatic beta-islet cells. These results could explain the use of 0. canum in Ghanaian folk medicine to manage diabetes mellitus.     

Apolipoprotein E predisposes to obesity and related metabolic dysfunctions in mice

Obesity is a central feature of the metabolic syndrome and is associated with increased risk for insulin resistance and typeII diabetes. Here, we investigated the contribution of human apoliproteinE3 and mouse apoliproteinE to the development of diet-induced obesity in response to western-type diet. Our data show that apolipoproteinE contributes to the development of obesity and other related metabolic disorders, and that human apolipoproteinE3 is more potent than mouse apolipoproteinE in promoting obesity in response to western-type diet. Specifically, we found that apolipoproteinE3 knock-in mice fed western-type diet for 24 weeks became obese and developed hyperglycemia, hyperinsulinemia, hyperleptinemia, glucose intolerance and insulin resistance that were more severe than in C57BL/6 mice. In contrast, apolipoproteinE-deficient mice fed western-type diet for the same period were resistant to diet-induced obesity, had normal plasma glucose, leptin and insulin levels, and exhibited normal responses to glucose tolerance and insulin resistance tests. Furthermore, low-density lipoprotein receptor-deficient mice were more sensitive to the development of diet-induced obesity and insulin resistance than apolipoprotein E-deficient mice, but were still more resistant than C57BL/6 mice, raising the possibility that low-density lipoprotein receptor mediates, at least in part, the effects of apolipoproteinE on obesity. Taken together, our findings suggest that, in addition to other previously identified mechanisms of obesity, apolipoproteinE and possibly the chylomicron pathway are also important contributors to the development of obesity and related metabolic dysfunctions in mice.