Temporal characterization of ß cell-adaptive and -maladaptive mechanisms during chronic high-fat feeding in C57BL/6NTac mice.

The onset of type 2 diabetes is characterized by transition from successful to failed insulin secretory compensation to obesity-related insulin resistance and dysmetabolism. Energy-rich diets in rodents are commonly studied models of compensatory increases in both insulin secretion and ß cell mass. However, the mechanisms of these adaptive responses are incompletely understood, and it is also unclear why these responses eventually fail. We measured the temporal trends of glucose homeostasis, insulin secretion, ß cell morphometry, and islet gene expression in C57BL/6NTac mice fed a 60% high-fat diet (HFD) or control diet for up to 16 weeks. A 2-fold increased hyperinsulinemia was maintained for the first 4 weeks of HFD feeding and then further increased through 16 weeks. ß cell mass increased progressively starting at 4 weeks, principally through nonproliferative growth. Insulin sensitivity was not significantly perturbed until 11 weeks of HFD feeding. Over the first 8 weeks, we observed two distinct waves of increased expression of ß cell functional and prodifferentiation genes. This was followed by activation of the unfolded protein response at 8 weeks and overt ß cell endoplasmic reticulum stress at 12-16 weeks. In summary, ß cell adaptation to an HFD in C57BL/6NTac mice entails early insulin hypersecretion and a robust growth phase along with hyperexpression of related genes that begin well before the onset of observed insulin resistance. However, continued HFD exposure results in cessation of gene hyperexpression, ß cell functional failure, and endoplasmic reticulum stress. These data point to a complex but not sustainable integration of ß cell-adaptive responses to nutrient overabundance, obesity development, and insulin resistance.

Peroxisome proliferator-activated receptor γ (PPARγ) and its target genes are downstream effectors of FoxO1 protein in islet ß-cells: mechanism of ß-cell compensation and failure.

The molecular mechanisms and signaling pathways that drive islet ß-cell compensation and failure are not fully resolved. We have used in vitro and in vivo systems to show that FoxO1, an integrator of metabolic stimuli, inhibits PPARγ expression in ß-cells, thus transcription of its target genes (Pdx1, glucose-dependent insulinotropic polypeptide (GIP) receptor, and pyruvate carboxylase) that are important regulators of ß-cell function, survival, and compensation. FoxO1 inhibition of target gene transcription is normally relieved when upstream activation induces its translocation from the nucleus to the cytoplasm. Attesting to the central importance of this pathway, islet expression of PPARγ and its target genes was enhanced in nondiabetic insulin-resistant rats and markedly reduced with diabetes induction. Insight into the impaired PPARγ signaling with hyperglycemia was obtained with confocal microscopy of pancreas sections that showed an intense nuclear FoxO1 immunostaining pattern in the ß-cells of diabetic rats in contrast to the nuclear and cytoplasmic FoxO1 in nondiabetic rats. These findings suggest a FoxO1/PPARγ-mediated network acting as a core component of ß-cell adaptation to metabolic stress, with failure of this response from impaired FoxO1 activation causing or exacerbating diabetes.

Thiazolidinediones in prediabetes and early type 2 diabetes: what can be learned about that disease's pathogenesis.

Several clinical trials have shown a high success rate of thiazolidinediones (TZDs) in prediabetes and early type 2 diabetes. The presumed mechanism of this effect has shifted from the best known effect of these agents to improve insulin sensitivity, to preservation of beta-cell function. The common explanation for this effect is unloading of the islet beta cell from the insulin resistance-induced hyperstimulation that eventually leads to beta-cell failure, so-called beta-cell rest. However, a recent finding is powerful biological effects of peroxisome proliferator-activated receptor (PPAR)gamma signaling in islet beta cells. This article reviews this topic by first describing the TZD intervention studies. Then it provides an overview of the current concepts regarding the beta-cell overwork and rest hypotheses, and the recent information about PPARgamma signaling effects in beta cells.

Peroxisome proliferator-activated receptor-gamma regulates expression of PDX-1 and NKX6.1 in INS-1 cells.

In the 60% pancreatectomy (Px) rat model of beta-cell adaptation, normoglycemia is maintained by an initial week of beta-cell hyperplasia that ceases and is followed by enhanced beta-cell function. It is unknown how this complex series of events is regulated. We studied isolated islets and pancreas sections from 14-day post-Px versus sham-operated rats and observed a doubling of beta-cell nuclear peroxisome proliferator-activated receptor (PPAR)-gamma protein, along with a 2-fold increase in nuclear pancreatic duodenal homeobox (Pdx)-1 protein and a 1.4-fold increase in beta-cell nuclear Nkx6.1 immunostaining. As PPAR-gamma activation is known to both lower proliferation and have prodifferentiation effects in many tissues, we studied PPAR-gamma actions in INS-1 cells. A 3-day incubation with the PPAR-gamma agonist troglitazone reduced proliferation and increased Pdx-1 and Nkx6.1 immunostaining, along with glucokinase and GLUT2. Also, a 75% knockdown of PPAR-gamma using RNA interference lowered the mRNA levels of Pdx-1, glucokinase, GLUT2, and proinsulin II by more than half. Our results show a dual effect of PPAR-gamma in INS-1 cells: to curtail proliferation and promote maturation, the latter via enhanced expression of Pdx-1 and Nkx6.1. Additional studies are needed to determine whether there is a regulatory role for PPAR-gamma signaling in the beta-cell adaptation following a 60% Px in rats.

Regulation of pancreatic beta-cell regeneration in the normoglycemic 60% partial-pancreatectomy mouse.

beta-Cell mass is determined by a dynamic balance of proliferation, neogenesis, and apoptosis. The precise mechanisms underlying compensatory beta-cell mass (BCM) homeostasis are not fully understood. To evaluate the processes that maintain normoglycemia and regulate BCM during pancreatic regeneration, C57BL/6 mice were analyzed for 15 days following 60% partial pancreatectomy (Px). BCM increased in Px mice from 2 days onwards and was approximately 68% of the shams by 15 days, partly due to enhanced beta-cell proliferation. A transient approximately 2.8-fold increase in the prevalence of beta-cell clusters/small islets at 2 days post-Px contributed substantially to BCM augmentation, followed by an increase in the number of larger islets at 15 days. To evaluate the signaling mechanisms that may regulate this compensatory growth, we examined key intermediates of the insulin signaling pathway. We found insulin receptor substrate (IRS)2 and enhanced-activated Akt immunoreactivity in islets and ducts that correlated with increased pancreatic duodenal homeobox (PDX)1 expression. In contrast, forkhead box O1 expression was decreased in islets but increased in ducts, suggesting distinct PDX1 regulatory mechanisms in these tissues. Px animals acutely administered insulin exhibited further enhancement in insulin signaling activity. These data suggest that the IRS2-Akt pathway mediates compensatory beta-cell growth by activating beta-cell proliferation with an increase in the number of beta-cell clusters/small islets.

Mechanisms of compensatory beta-cell growth in insulin-resistant rats: roles of Akt kinase.

The physiological mechanisms underlying the compensatory growth of beta-cell mass in insulin-resistant states are poorly understood. Using the insulin-resistant Zucker fatty (fa/fa) (ZF) rat and the corresponding Zucker lean control (ZLC) rat, we investigated the factors contributing to the age-/obesity-related enhancement of beta-cell mass. A 3.8-fold beta-cell mass increase was observed in ZF rats as early as 5 weeks of age, an age that precedes severe insulin resistance by several weeks. Closer investigation showed that ZF rat pups were not born with heightened beta-cell mass but developed a modest increase over ZLC rats by 20 days that preceded weight gain or hyperinsulinemia that first developed at 24 days of age. In these ZF pups, an augmented survival potential of beta-cells of ZF pups was observed by enhanced activated (phospho-) Akt, phospho-BAD, and Bcl-2 immunoreactivity in the postweaning period. However, increased beta-cell proliferation in the ZF rats was only detected at 31 days of age, a period preceding massive beta-cell growth. During this phase, we also detected an increase in the numbers of small beta-cell clusters among ducts and acini, increased duct pancreatic/duodenal homeobox-1 (PDX-1) immunoreactivity, and an increase in islet number in the ZF rats suggesting duct- and acini-mediated heightened beta-cell neogenesis. Interestingly, in young ZF rats, specific cells associated with ducts, acini, and islets exhibited an increased frequency of PDX-1+/phospho-Akt+ staining, indicating a potential role for Akt in beta-cell differentiation. Thus, several adaptive mechanisms account for the compensatory growth of beta-cells in ZF rats, a combination of enhanced survival and neogenesis with a transient rise in proliferation before 5 weeks of age, with Akt serving as a potential mediator in these processes.

Insulin management of diabetic patients on general medical and surgical floors.

OBJECTIVE: To review the standardized subcutaneous insulin protocols for diabetic patients on general medical and surgical floors at the University of Vermont. METHOD: Insulin protocols were developed for inpatients eating regular meals, and those receiving continuous tube feedings or total parental nutrition. RESULTS: The recommended starting subcutaneous insulin protocol for patients receiving meals is a basal-bolus approach using 0.5 U/kg basal insulin (glargine once daily, or neutral protamine Hagedorn [NPH] twice daily) and 0.1 U/kg rapid analog at each meal (lispro, aspart, or glulisine) for the average patient. The dose of basal is lowered by 0.2 U/kg for medical conditions with a high sensitivity to insulin or that have an added risk for hypoglycemia (renal or hepatic impairment, thin or normal weight, elderly, frail, hypothyroidism, adrenal insuffi ciency, etc.). Alternatively, an extra 0.2 U/kg basal is given for states of presumed high insulin resistance such as marked obesity with metabolic syndrome, open wounds, infections, etc. In turn, patients receiving continuous tube feedings receive the same 24-hour insulin doses (0.6-1.0 U/kg) in divided doses of premixed 70/30 (NPH/regular) insulin every 8 hours. This program allows lowering or eliminating 1 of the doses for the increasingly common tube-feeding programs in rehabilitation centers or patients' homes that entail discontinuation of tube feeding for 6 to 8 hours. A variation on this approach is used with total parenteral nutrition (TPN), with a portion of the insulin placed in the TPN bag and the remainder given as "q 8 hour 70/30" insulin. Starting insulin doses for all of the programs are adjusted daily to attain inpatient blood glucose goals of <110 mg/dL fasting and 110 to 180 mg/dL throughout the day. CONCLUSION: Standardized protocols have been developed and implemented at the University of Vermont for patients receiving regular meals and continuous tube feedings or TPN.

Basal-prandial insulin therapy: Scientific concept review and application.

The majority of patients with type 2 diabetes mellitus (T2DM) eventually require the addition of basal insulin to existing oral therapy to achieve the glycemic goals set forth by the American Diabetes Association (A1C, <7.0%). In many patients with T2DM, insulin is the only option for achieving glycemic control and may be used successfully to attain glycemic targets in regimens that combine basal insulin with oral antidiabetic agents, or in regimens that combine basal insulin with mealtime (prandial) insulin. Basal-prandial insulin regimens that use a long-acting insulin analogue to control the fasting plasma glucose level and a short-acting insulin analogue for post-meal glucose excursions replace insulin in a manner that most closely approximates normal physiologic patterns. The current body of evidence demonstrates that such regimens will prove to be the optimal strategy for achieving glycemic control in patients with T2DM who require both basal and prandial insulin replacement. Here, we review current findings in the published literature on the efficacy of basal-prandial insulin, with a focus on practical information that might help to provide an evidence-based guide for progressing to basal-prandial insulin therapy in appropriate patients with T2DM.

Characterization of a novel metabolic strategy used by drug-resistant tumor cells.

Acquired or inherent drug resistance is the major problem in achieving successful cancer treatment. However, the mechanism(s) of pleiotropic drug resistance remains obscure. We have identified and characterized a cellular metabolic strategy that differentiates drug-resistant cells from drug-sensitive cells. This strategy may serve to protect drug-resistant cells from damage caused by chemotherapeutic agents and radiation. We show that drug-resistant cells have low mitochondrial membrane potential, use nonglucose carbon sources (fatty acids) for mitochondrial oxygen consumption when glucose becomes limited, and are protected from exogenous stress such as radiation. In addition, drug-resistant cells express high levels of mitochondrial uncoupling protein 2 (UCP2). The discovery of this metabolic strategy potentially facilitates the design of novel therapeutic approaches to drug resistance.

Pathogenesis of type 2 diabetes mellitus.

The pathological sequence for type 2 diabetes is complex and entails many different elements that act in concert to cause that disease. This review proposes a sequence of events and how they interact by a careful analysis of the human and animal model literature. A genetic predisposition must exist, although to date very little is known about specific genetic defects in this disease. Whether the diabetes phenotype will occur depends on many environmental factors that share an ability to stress the glucose homeostasis system, with the current explosion of obesity and sedentary lifestyle being a major cause of the worldwide diabetes epidemic. We also propose that a lowered beta-cell mass either through genetic and/or beta-cell cytotoxic factors predisposes for glucose intolerance. As the blood glucose level rises even a small amount above normal, then acquired defects in the glucose homeostasis system occur--initially to impair the beta cell's glucose responsiveness to meals by impairing the first phase insulin response--and cause the blood glucose level to rise into the range of impaired glucose tolerance (IGT). This rise in blood glucose, now perhaps in concert with the excess fatty acids that are a typical feature of obesity and insulin resistance, cause additional deterioration in beta-cell function along with further insulin resistance, and the blood glucose levels rise to full-blown diabetes. This sequence also provides insight into how to better prevent or treat type 2 diabetes, by studying the molecular basis for the early defects, and developing targeted therapies against them.

Islet amyloid and type 2 diabetes: overproduction or inadequate clearance and detoxification?

A hallmark of type 2 diabetes is the reduction of pancreatic islet ß cell mass through induction of apoptosis and lack of regeneration. In most patients, ß cell dysfunction is associated with the presence of extracellular amyloid plaques adjacent to ß cells and intracellular toxic oligomers that are comprised of islet amyloid polypeptide (IAPP). In this issue of the JCI, three independent research groups reveal that a functional autophagy system normally prevents the accumulation of toxic IAPP oligomers in human IAPP-expressing murine models. Furthermore, mice expressing human IAPP but deficient for ß cell autophagy through genetic deletion of the autophagy initiator ATG7 developed ß cell apoptosis and overt diabetes. Together, these studies indicate that autophagy protects ß cells from the accumulation of toxic IAPP oligomers and suggest that enhancing autophagy may be a novel target for prevention of type 2 diabetes.

Insulin therapy in type 2 diabetes mellitus.

Health care providers and patients have lots of choice to treat type 2 diabetes, but the blood glucose improvement is limited. The one therapy with unlimited potential (at least theoretically) is insulin. Many studies show that glucose control is achievable with insulin safely in most patients with type 2 diabetes. Effective diabetes management at the primary care or specialty level requires a belief in the importance of insulin therapy in uncontrolled patients with type 2 diabetes. This review details the theories, observed outcomes, and how-tos regarding insulin use in type 2 diabetes.

Targeting beta-cell function early in the course of therapy for type 2 diabetes mellitus.

OBJECTIVE: This report examines current perspectives regarding likely mechanisms of beta-cell failure in type 2 diabetes and their clinical implications for protecting or sparing beta-cells early in the disease progression. In addition, it considers translation strategies to incorporate relevant scientific findings into educational initiatives targeting clinical practice behavior. PARTICIPANTS: On January 10, 2009, a working group of basic researchers, clinical endocrinologists, and primary care physicians met to consider whether current knowledge regarding pancreatic beta-cell defects justifies retargeting and retiming treatment for clinical practice. Based on this meeting, a writing group comprised of four meeting participants subsequently prepared this consensus statement. The conference was convened by The Endocrine Society and funded by an unrestricted educational grant from Novo Nordisk. EVIDENCE: Participants reviewed and discussed published literature, plus their own unpublished data. CONSENSUS PROCESS: The summary and recommendations were supported unanimously by the writing group as representing the consensus opinions of the working group. CONCLUSIONS: Workshop participants strongly advocated developing new systems to address common barriers to glycemic control and recommended several initial steps toward this goal. These recommendations included further studies to establish the clinical value of pharmacological therapies, continuing basic research to elucidate the nature and mechanisms of beta-cell failure in type 2 diabetes mellitus, and exploring new educational approaches to promote pathophysiology-based clinical practices. The Endocrine Society has launched a new website to continue the discussion between endocrinologists and primary care physicians on beta-cell pathophysiology in type 2 diabetes and its clinical implications. Join the conversation at http://www.betacellsindiabetes.org

Physiologic and pharmacologic modulation of glucose-dependent insulinotropic polypeptide (GIP) receptor expression in beta-cells by peroxisome proliferator-activated receptor (PPAR)-gamma signaling: possible mechanism for the GIP resistance in type 2 diabetes.

OBJECTIVE: We previously showed that peroxisome proliferator-activated receptor (PPAR)-gamma in beta-cells regulates pdx-1 transcription through a functional PPAR response element (PPRE). Gene Bank blast for a homologous nucleotide sequence revealed the same PPRE within the rat glucose-dependent insulinotropic polypeptide receptor (GIP-R) promoter sequence. We investigated the role of PPARgamma in GIP-R transcription. RESEARCH DESIGN AND METHODS: Chromatin immunoprecipitation assay, siRNA, and luciferase gene transcription assay in INS-1 cells were performed. Islet GIP-R expression and immunohistochemistry studies were performed in pancreas-specific PPARgamma knockout mice (PANC PPARgamma(-/-)), normoglycemic 60% pancreatectomy rats (Px), normoglycemic and hyperglycemic Zucker fatty (ZF) rats, and mouse islets incubated with troglitazone. RESULTS: In vitro studies of INS-1 cells confirmed that PPAR-gamma binds to the putative PPRE sequence and regulates GIP-R transcription. In vivo verification was shown by a 70% reduction in GIP-R protein expression in islets from PANC PPARgamma(-/-) mice and a twofold increase in islets of 14-day post-60% Px Sprague-Dawley rats that hyperexpress beta-cell PPARgamma. Thiazolidinedione activation (72 h) of this pathway in normal mouse islets caused a threefold increase of GIP-R protein and a doubling of insulin secretion to 16.7 mmol/l glucose/10 nmol/l GIP. Islets from obese normoglycemic ZF rats had twofold increased PPARgamma and GIP-R protein levels versus lean rats, with both lowered by two-thirds in ZF rats made hyperglycemic by 60% Px. CONCLUSIONS: Our studies have shown physiologic and pharmacologic regulation of GIP-R expression in beta-cells by PPARgamma signaling. Also disruption of this signaling pathway may account for the lowered beta-cell GIP-R expression and resulting GIP resistance in type 2 diabetes.

In vivo and in vitro studies of a functional peroxisome proliferator-activated receptor gamma response element in the mouse pdx-1 promoter.

We reported that peroxisome proliferator-activated receptor gamma (PPARgamma) transcriptionally regulates the beta-cell differentiation factor pancreatic duodenal homeobox (PDX)-1 based on in vitro RNA interference studies. We have now studied mice depleted of PPARgamma within the pancreas (PANC PPARgamma(-/-)) created by a Cre/loxP recombinase system, with Cre driven by the pdx-1 promoter. Male PANC PPARgamma(-/-) mice were hyperglycemic at 8 weeks of age (8.1+/-0.2 mM versus 6.4+/-0.3 mM, p=0.009) with islet cytoarchitecture and pancreatic mass of islet beta-cells that were indistinguishable from the controls. Islet PDX-1 mRNA (p=0.001) and protein levels (p=0.003) were lowered 60 and 40%, respectively, in tandem with impaired glucose-induced insulin secretion and loss of thiazolidinedione-induced increase in PDX-1 expression. We next identified a putative PPAR-response element (PPRE) in the mouse pdx-1 promoter with substantial homology to the corresponding region of the human PDX-1 promoter. Electrophoretic mobility supershift assays with nuclear extracts from beta-cell lines and mouse islets, also in vitro translated PPARgamma and retinoid X receptor, and chromatin immunoprecipitation analysis demonstrated specific binding of PPARgamma and retinoid X receptor to the human and mouse pdx-1 x PPREs. Transient transfection assays of beta-cells with reporter constructs of mutated PPREs showed dramatically reduced pdx-1 promoter activity. In summary, we have presented in vivo and in vitro evidence showing PPARgamma regulation of pdx-1 transcription in beta-cells, plus our results support an important regulatory role for PPARgamma in beta-cell physiology and thiazolidinedione pharmacology of type 2 diabetes.

Enhanced beta-cell mass without increased proliferation following chronic mild glucose infusion.

The physiological mechanisms underlying pancreatic beta-cell mass (BCM) homeostasis are complex and not fully resolved. Here we examined the factors contributing to the increased BCM following a mild glucose infusion (GI) whereby normoglycemia was maintained through 96 h. We used morphometric and immunochemical methods to investigate enhanced beta-cell growth and survival in Sprague-Dawley rats. BCM was elevated >2.5-fold over saline-infused control rats by 48 h and increased modestly thereafter. Unexpectedly, increases in beta-cell proliferation were not observed at any time point through 4 days. Instead, enhanced numbers of insulin(+) cell clusters and small islets (400-12,000 microm(2); approximately 23- to 124-microm diameter), mostly scattered among the acini, were observed in the GI rats by 48 h despite no difference in the numbers of medium to large islets. We previously showed that increased beta-cell growth in rodent models of insulin resistance and pancreatic regeneration involves increased activated Akt/PKB, a key beta-cell signaling intermediate, in both islets and endocrine cell clusters. GI in normal rats also leads to increased Akt activation in islet beta-cells, as well as in insulin(+) and insulin(-) cells in the common duct epithelium and endocrine clusters. This correlated with strong Pdx1 expression in these same cells. These results suggest that mechanisms other than proliferation underlie the rapid beta-cell growth response following a mild GI in the normal rat and involve Akt-regulated enhanced beta-cell survival potential and neogenesis from epithelial precursors.