Effect of chronic rosiglitazone, metformin and glyburide treatment on beta-cell mass, function and insulin sensitivity in mZDF rats

Here we investigate the effect of rosiglitazone (RSG), metformin (MET) and glyburide (GLIB) on plasma glucose levels, β-cell mass, function and insulin sensitivity in 10-week-old diabetic male Zucker diabetic fatty (mZDF) rats using quantitative morphometry and a mathematical model β-cell mass, insulin and glucose kinetics (βIG). At treatment start, 10-week-old diabetic mZDF rats were severely hyperglycaemic and had very low β-cell function (insulin secretory capacity). RSG treatment significantly lowered plasma glucose levels in 67% of the mZDF rats. MET was effective at lowering plasma glucose levels in 33% of the mZDF rats, while GLIB was completely ineffective at lowering blood glucose levels in 10-week-old mZDF rats. RSG treatment prevented the fall in β-cell mass after 6–8 weeks of treatment accompanied by a significant decrease in β-cell death while MET treatment had no effect on β-cell mass. RSG treatment increased insulin sensitivity 10-fold, increased β-cell function fivefold and modestly increased β-cell mass 1.4-fold. MET treatment increased insulin sensitivity fourfold, with no significant effect on β-cell function or mass. Although RSG treatment was highly successful in lowering plasma glucose levels, the 33% of mZDF rats that did not respond to the treatment had significantly lower β-cell function prior to treatment start compared with the responder group. Thus, the low level of β-cell function at treatment start may explain why none of these agents were completely effective at lowering blood glucose levels in 10-week-old diabetic mZDF rats. Nevertheless, these data suggest that the preservation of β-cell mass and improvement in β-cell function play a role in the overall beneficial effect of RSG in 10-week-old diabetic mZDF rats.     

Evaluation of β-cell mass and function in the Göttingen minipig

Increased knowledge about β-cell mass and function is important for our understanding of the pathophysiology of type 2 diabetes (T2DM). The relationship between the two is difficult to study in humans, whereas animal models allow studies of consequences of, for example, reduction of β-cell mass and induction of obesity and procurement of the pancreas for histological examination. An overview of results obtained in the Göttingen minipig in relation to β-cell function, and mass is provided here. Effects of a primary reduction of β-cell mass have indicated that not all of the defects of pulsatile insulin secretion in human T2DM can be explained by reduced β-cell mass. Furthermore, induction of obesity has shown deterioration of β-cell function and morphological changes in the pancreas. As in humans, obesity leads to an increased β-cell volume in the minipig, and based on the increased number of islets, neogenesis of islets is an important factor in expansion of β-cell mass in this species. Measurement of β-cell function as an estimate of β-cell mass is, at present, the only method possible in humans, and this approach has been validated using lean and obese minipigs with a range of β-cell mass. The effects on β-cell function and mass of obesity of longer duration and/or more pronounced hyperglycaemia remains to be determined, but the models developed so far represent a valuable tool for such investigations.     

Islet gene expression and function in type 2 diabetes; studies in the Goto-Kakizaki rat and humans

Defective β-cell function with resulting impairment of glucose-stimulated insulin release is a critical factor in the pathogenesis of type 2 diabetes. Accumulated studies in pancreatic islets of the spontaneously diabetic Goto-Kakizaki (GK) rat suggest that this is a useful animal model of type 2 diabetes. The GK rat is non-obese, and abnormal glucose regulation develops early in life in association with impaired insulin secretion. There are some differences in islet morphology and function reported between different GK rat colonies. In addition to reduction of β-cell mass, a number of β-cell defects have been described with possible relevance for the reduced insulin secretion. Interestingly, some of these defects have also been shown in isolated islets from type 2 diabetic humans. The polygenic nature of diabetes heredity in the GK rat may well resemble the genetic basis in the majority of patients with type 2 diabetes. Here, we review studies concerning β-cell function and islet gene expression in the GK rat and compare it with the limited number of investigations on similar topics in isolated islets from patients with type 2 diabetes.     

Diabetes mellitus and impaired pancreatic β-cell proliferation

The factors that normally regulate the proliferation of the insulin-producing pancreatic β-cell largely remain elusive although several factors have been identified that influence β-cell growth in vitro. The adult β-cell is normally virtually quiescent, but its replicatory activity can be enhanced in vitro by certain nutrients and growth factors, and long-term alterations in β-cell mass constitute an important means to accommodate an increased demand for insulin. Likewise, expansion of the β-cell mass by recruitment of β-cells to proliferate may constitute a means by which the organism can compensate for the loss or dysfunction of β-cells occurring in diabetes. However, neither in human or animal models for type-1 diabetes, nor in type-2 diabetes, is β-cell regeneration a noteworthy feature. Thus, if β-cells could be induced to replicate at a higher rate, this may prove beneficial in maintaining normoglycaemia, since the β-cell mass is a major determinant of the total amount of insulin that can be secreted by the pancreas. The present review will focus on the normal regulation of β-cell mitogenesis and hormones production in vitro and in vivo, and furthermore, will present evidence for an insufficient extent of β-cell regeneration in different forms of diabetes mellitus. Additionally, the possibility of manipulating β-cell proliferation by peptides and genetic engineering, and the significance of β-cell mitogenesis in islet transplantation will be discussed in relation to treatments of diabetes mellitus.     

Adult human β-cell neogenesis?

Prospects for inducing endogenous β-cell regeneration in the pancreas, one of the most attractive approaches to reverse type 1 and type 2 diabetes, have gained substantially from recent evidence that cells in the adult pancreas exhibit more plasticity than previously recognized. There are two major pathways to β-cell regeneration, β-cell replication and β-cell neogenesis. Substantial evidence for a role for both processes exists in different models. While β-cell replication clearly occurs during development and early in life, the potential for replication appears to decline substantially with age. In contrast, we have demonstrated that the exocrine compartment of the adult human pancreas contains a facultative stem cell that can differentiate into β-cells under specific circumstances. We have favoured the idea that, similar to models described in liver regeneration, β-cell mass can be increased either by neogenesis or replication, depending on the intensity of different stimuli or stressors. Understanding the nature of endocrine stem/progenitor cells and the mechanism by which external stimuli mobilize them to exhibit endocrine differentiation is central for success in therapeutic approaches to induce meaningful endogenous β-cell neogenesis .     

A transgenic model to study the pathogenesis of somatic mtDNA mutation accumulation in β-cells

Low levels of somatic mutations accumulate in mitochondrial DNA (mtDNA) as we age; however, the pathogenic nature of these mutations is unknown. In contrast, mutational loads of >30% of mtDNA are associated with electron transport chain defects that result in mitochondrial diseases such as mitochondrial encephalopathy lactic acidosis and stroke-like episodes. Pancreatic β-cells may be extremely sensitive to the accumulation of mtDNA mutations, as insulin secretion requires the mitochondrial oxidation of glucose to CO₂. Type 2 diabetes arises when β-cells fail to compensate for the increased demand for insulin, and many type 2 diabetics progress to insulin dependence because of a loss of β-cell function or β-cell death. This loss of β-cell function/β-cell death has been attributed to the toxic effects of elevated levels of lipids and glucose resulting in the enhanced production of free radicals in β-cells. mtDNA, localized in close proximity to one of the major cellular sites of free radical production, comprises more than 95% coding sequences such that mutations result in changes in the coding sequence. It has long been known that mtDNA mutations accumulate with age; however, only recently have studies examined the influence of somatic mtDNA mutation accumulation on disease pathogenesis. This article will focus on the effects of low-level somatic mtDNA mutation accumulation on ageing, cardiovascular disease and diabetes.     

Balancing needs and means: the dilemma of the β-cell in the modern world

The insulin resistance of type 2 diabetes mellitus (T2DM), although important for its pathophysiology, is not sufficient to establish the disease unless major deficiency of β-cell function coexists. This is demonstrated by the fact that near-physiological administration of insulin (CSII) achieved excellent blood glucose control with doses similar to those used in insulin-deficient type 1 diabetics. The normal β-cell adapts well to the demands of insulin resistance. Also in hyperglycaemic states some degree of adaptation does exist and helps limit the severity of disease. We demonstrate here that the mammalian target of rapamycin (mTOR) system might play an important role in this adaptation, because blocking mTORC1 (complex 1) by rapamycin in the nutritional diabetes model Psammomys obesus caused severe impairment of β-cell function, increased β-cell apoptosis and progression of diabetes. On the other hand, under exposure to high glucose and FFA (gluco-lipotoxicity), blocking mTORC1 in vitro reduced endoplasmic reticulum (ER) stress and β-cell death. Thus, according to the conditions of stress, mTOR may have beneficial or deleterious effects on the β-cell. β-Cell function in man can be reduced without T2DM/impaired glucose tolerance (IGT). Prospective studies have shown subjects with reduced insulin response to present, several decades later, an increased incidence of IGT/T2DM. From these and other studies we conclude that T2DM develops on the grounds of β-cells whose adaptation capacity to increased nutrient intake and/or insulin resistance is in the lower end of the normal variation. Inborn and acquired factors that limit β-cell function are diabetogenic only in a nutritional/metabolic environment that requires high functional capabilities from the β-cell.     

Type 2 diabetes – a matter of failing β-cell neogenesis? Clues from the GK rat model

Now that reduction in β-cell mass has been clearly established in humans with type 2 diabetes mellitus (T2D), the debate focuses on the possible mechanisms responsible for decreased β-cell number. Appropriate inbred rodent models are essential tools for this purpose. The information available from the Goto-Kakizaki (GK) rat, one of the best characterized animal models of spontaneous T2D, is reviewed in such a perspective. We propose that the defective β-cell mass in the GK model reflects mostly a persistently decreased β-cell neogenesis. The data discussed in this review are consistent with the notion that poor proliferation and/or survival of the endocrine precursor cells during GK foetal life will result in a decreased pool of endocrine precursors in the pancreas, and hence an impaired capacity of β-cell neogenesis (either primary in the foetus or compensatory in the newborn and the adult). As we also demonstrated that β-cell neogenesis can be pharmacologically reactivated in the GK model, our work supports, on a more prospective basis, the concept that facilitation of T2D treatment may be obtained through β-cell mass expansion after stimulation of β-cell regeneration/neogenesis in diabetic patients.     

Glucagon-like peptide-1-based therapies: new developments and emerging data

Type 2 diabetes is a chronic, progressive disease characterized by an inexorable decline in β-cell function. Current treatments fail to manage this continued β-cell failure, and eventually patients with type 2 diabetes will require insulin therapy. The 'ideal' therapy for type 2 diabetes needs to provide not only effective glycaemic control with no weight gain and preferably, no hypoglycaemia, but also address continued β-cell deterioration. The development of glucagon-like peptide-1 (GLP-1) based therapies provides an alternative to traditional oral antidiabetic drugs. Here, we present the evidence base showing that the incretin mimetics (exenatide, exenatide long-acting release and liraglutide) and the incretin enhancers (sitagliptin and vildagliptin) offer significant glycaemic improvements over existing treatments, with low rates of hypoglycaemia. In addition, we present evidence to show that incretin mimetics are associated with significant weight loss, in contrast to incretin enhancers, which are weight neutral. Finally, we summarize and discuss the key features of the incretin enhancers and incretin mimetics in terms of glycaemic control, side effects and flexibility of treatment regimen.     

The scientific evidence: vildagliptin and the benefits of islet enhancement

Vildagliptin is an oral incretin enhancer that acts to increase active levels of the incretin hormone glucagon-like peptide-1 (GLP-1) by inhibiting the dipeptidyl peptidase-4 enzyme responsible for the rapid deactivation of GLP-1 in vivo . This activity results in improved glucose-dependent functioning of pancreatic islet β and α cells, addressing two central deficits in type 2 diabetes mellitus (T2DM). Vildagliptin treatment improves β-cell sensitivity to glucose, producing increased insulin secretory rate relative to glucose in both postprandial and fasting states. Improved α-cell function is shown as restoration of appropriate glucose-related suppression of glucagon and, therefore, reduced endogenous glucose production during both postprandial and fasting periods. There is evidence that long-term vildagliptin treatment may slow underlying deterioration of β-cell function in T2DM. There is also a potential synergistic effect of vildagliptin and metformin in increasing active GLP-1 levels, and this activity may contribute to the long-term improvements in β-cell function observed in patients with T2DM who have vildagliptin added to ongoing metformin therapy. Vildagliptin treatment has also been associated with beneficial extrapancreatic effects, including improved peripheral insulin sensitivity and improved postprandial triglyceride-rich lipoprotein metabolism. Improvement of β- and α-cell function through incretin enhancement with vildagliptin results in more physiologic meal-related and fasting glycaemia profiles.     

Regulation of beta-cell mass and function by the Akt/protein kinase B signalling pathway

The insulin receptor substrate-2/phosphoinositide 3-kinase (PI3K) pathway plays a critical role in the regulation of beta-cell mass and function, demonstrated both in vitro and in vivo. The serine threonine kinase Akt is one of the promising downstream molecules of this pathway that has been identified as a potential target to regulate function and induce proliferation and survival of beta cells. Here we summarize some of the molecular mechanisms, downstream signalling pathways and critical components involved in the regulation of beta-cell mass and function by Akt.     

Towards better understanding of the contributions of overwork and glucotoxicity to the β-cell inadequacy of type 2 diabetes

Type 2 diabetes (T2D) is characterized by reduction of β-cell mass and dysfunctional insulin secretion. Understanding β-cell phenotype changes as T2D progresses should help explain these abnormalities. The normal phenotype should differ from the state of overwork when β-cells compensate for insulin resistance to keep glucose levels normal. When only mild hyperglycaemia develops, β-cells are subjected to glucotoxicity. As hyperglycaemia becomes more severe, so does glucotoxicity. β-Cells in all four of these situations should have separate phenotypes. When assessing phenotype with gene expression, isolated islets have artefacts resulting from the trauma of isolation and hypoxia of islet cores. An advantage comes from laser capture microdissection (LCM), which obtains β-cell-rich tissue from pancreatic frozen sections. Valuable data can be obtained from animal models, but the real goal is human β-cells. Our experience with LCM and gene arrays on frozen pancreatic sections from cadaver donors with T2D and controls is described. Although valuable data was obtained, we predict that the approach of taking fresh samples at the time of surgery is an even greater opportunity to markedly advance our understanding of how β-cell phenotype evolves as T2D develops and progresses.     

Intrauterine programming of the endocrine pancreas

Epidemiological studies have revealed strong relationships between poor foetal growth and subsequent development of the metabolic syndrome. Persisting effects of early malnutrition become translated into pathology, thereby determine chronic risk for developing glucose intolerance and diabetes. These epidemiological observations identify the phenomena of foetal programming without explaining the underlying mechanisms that establish the causal link. Animal models have been established and studies have demonstrated that reduction in the availability of nutrients during foetal development programs the endocrine pancreas and insulin-sensitive tissues. Whatever the type of foetal malnutrition, whether there are not enough calories or protein in food or after placental deficiency, malnourished pups are born with a defect in their β-cell population that will never completely recover, and insulin-sensitive tissues will be definitively altered. Despite the similar endpoint, different cellular and physiological mechanisms are proposed. Hormones operative during foetal life like insulin itself, insulin-like growth factors and glucocorticoids, as well as specific molecules like taurine, or islet vascularization were implicated as possible factors amplifying the defect. The molecular mechanisms responsible for intrauterine programming of the β cells are still elusive, but two hypotheses recently emerged: the first one implies programming of mitochondria and the second, epigenetic regulation.     

Melatonin prevents nitric oxide-induced apoptosis by increasing the interaction between 14-3-3β and p-Bad in SK-N-MC cells

Abstract:  The anti-apoptotic effect of melatonin has been described in vivo and in vitro. A previous report has revealed that melatonin suppresses nitric oxide (NO)-induced apoptosis via the induction of Bcl-2 expression in PGT-β pineal cells. To investigate the protective mechanism of melatonin on NO donor S -nitroso- N -acetyl-penicillamine (SNAP)-induced apoptosis, we examined the anti-apoptotic upstream signaling pathway of Bcl-2 in the human neuroblastoma cell line SK-N-MC. The flow cytometry results revealed that apoptosis occurred in NO-treated cells, while cell death was inhibited by pretreatment with melatonin (100 μ m ). In addition, decreased Bax expression, increased Bcl-2 expression and a decreased release of cytochrome c into the cytosol were observed in the melatonin-pretreated SK-N-MC cells. We also found that melatonin treatment induced the activation of Akt/PKB and the phosphorylation of GSK3α/β and Bad. Furthermore, melatonin treatment not only increased the protein–protein interactions between 14-3-3β and p-Bad, but also decreased the release of cytochrome c from mitochondria into the cytosol. In summary, the protective effect of melatonin against NO-induced apoptosis was mediated by the inhibition of Bad translocation from the cytosol to the mitochondria by the induction of protein–protein interactions between 14-3-3β and p-Bad.     

Beta cells in type 2 diabetes - a crucial contribution to pathogenesis

The healthy β-cell has an enormous capacity to adapt to conditions of higher insulin demand (e.g. in obesity, pregnancy, cortisol excess) to maintain normoglycaemia with an increase in its functional β-cell mass. This compensates in 80–90% of individuals for insulin resistance. However, in 10–20% of individuals, the β-cells are unable to match the demands of insulin resistance and insulin levels are relatively insufficient to maintain normal glycaemic control. This eventually leads to glucose intolerance and type 2 diabetes (T2DM). Accordingly, preservation of functional β-cell mass has become central in the treatment of type 1 diabetes as well as T2DM. The purpose of this review is to summarize the recently described mechanisms of β-cell death in T2DM and to postulate possible new targets for treatment.     

Role of the M3 muscarinic acetylcholine receptor in β-cell function and glucose homeostasis

The release of insufficient amounts of insulin in the presence of elevated blood glucose levels is one of the key features of type 2 diabetes. Various lines of evidence indicate that acetylcholine (ACh), the major neurotransmitter of the parasympathetic nervous system, can enhance glucose-stimulated insulin secretion from pancreatic β-cells. Studies with isolated islets prepared from whole body M₃ muscarinic ACh receptor knockout mice showed that cholinergic amplification of glucose-dependent insulin secretion is exclusively mediated by the M₃ muscarinic receptor subtype. To investigate the physiological relevance of this muscarinic pathway, we used Cre/loxP technology to generate mutant mice that lack M₃ receptors only in pancreatic β-cells. These mutant mice displayed impaired glucose tolerance and significantly reduced insulin secretion. In contrast, transgenic mice overexpressing M₃ receptors in pancreatic β-cells showed a pronounced increase in glucose tolerance and insulin secretion and were resistant to diet-induced glucose intolerance and hyperglycaemia. These findings indicate that β-cell M₃ muscarinic receptors are essential for maintaining proper insulin secretion and glucose homeostasis. Moreover, our data suggest that enhancing signalling through β-cell M₃ muscarinic receptors may represent a new avenue in the treatment of glucose intolerance and type 2 diabetes.