Glucagon receptor inactivation leads to α-cell hyperplasia in zebrafish.

Glucagon antagonism is a potential treatment for diabetes. One potential side effect is α-cell hyperplasia, which has been noted in several approaches to antagonize glucagon action. To investigate the molecular mechanism of the α-cell hyperplasia and to identify the responsible factor, we created a zebrafish model in which glucagon receptor (gcgr) signaling has been interrupted. The genetically and chemically tractable zebrafish, which provides a robust discovery platform, has two gcgr genes (gcgra and gcgrb) in its genome. Sequence, phylogenetic, and synteny analyses suggest that these are co-orthologs of the human GCGR. Similar to its mammalian counterparts, gcgra and gcgrb are mainly expressed in the liver. We inactivated the zebrafish gcgra and gcgrb using transcription activator-like effector nuclease (TALEN) first individually and then both genes, and assessed the number of α-cells using an α-cell reporter line, Tg(gcga:GFP). Compared to WT fish at 7 days postfertilization, there were more α-cells in gcgra-/-, gcgrb-/-, and gcgra-/-;gcgrb-/- fish and there was an increased rate of α-cell proliferation in the gcgra-/-;gcgrb-/- fish. Glucagon levels were higher but free glucose levels were lower in gcgra-/-, gcgrb-/-, and gcgra-/-;gcgrb-/- fish, similar to Gcgr-/- mice. These results indicate that the compensatory α-cell hyperplasia in response to interruption of glucagon signaling is conserved in zebrafish. The robust α-cell hyperplasia in gcgra-/-;gcgrb-/- larvae provides a platform to screen for chemical and genetic suppressors, and ultimately to identify the stimulus of α-cell hyperplasia and its signaling mechanism.

Human islet preparations distributed for research exhibit a variety of insulin-secretory profiles.

Human islet research is providing new insights into human islet biology and diabetes, using islets isolated at multiple US centers from donors with varying characteristics. This creates challenges for understanding, interpreting, and integrating research findings from the many laboratories that use these islets. In what is, to our knowledge, the first standardized assessment of human islet preparations from multiple isolation centers, we measured insulin secretion from 202 preparations isolated at 15 centers over 11 years and noted five distinct patterns of insulin secretion. Approximately three quarters were appropriately responsive to stimuli, but one quarter were dysfunctional, with unstable basal insulin secretion and/or an impairment in stimulated insulin secretion. Importantly, the patterns of insulin secretion by responsive human islet preparations (stable Baseline and Fold stimulation of insulin secretion) isolated at different centers were similar and improved slightly over the years studied. When all preparations studied were considered, basal and stimulated insulin secretion did not correlate with isolation center, biological differences of the islet donor, or differences in isolation, such as Cold Ischemia Time. Dysfunctional islet preparations could not be predicted from the information provided by the isolation center and had altered expression of genes encoding components of the glucose-sensing pathway, but not of insulin production or cell death. These results indicate that insulin secretion by most preparations from multiple centers is similar but that in vitro responsiveness of human islets cannot be predicted, necessitating preexperimental human islet assessment. These results should be considered when one is designing, interpreting, and integrating experiments using human islets.

Comparison of the physiological relevance of systemic vs. portal insulin delivery to evaluate whole body glucose flux during an insulin clamp.

To understand the underlying pathology of metabolic diseases, such as diabetes, an accurate determination of whole body glucose flux needs to be made by a method that maintains key physiological features. One such feature is a positive differential in insulin concentration between the portal venous and systemic arterial circulation (P/S-IG). P/S-IG during the determination of the relative contribution of liver and extra-liver tissues/organs to whole body glucose flux during an insulin clamp with either systemic (SID) or portal (PID) insulin delivery was examined with insulin infusion rates of 1, 2, and 5 mU·kg(-1)·min(-1) under either euglycemic or hyperglycemic conditions in 6-h-fasted conscious normal rats. A P/S-IG was initially determined with endogenous insulin secretion to exist with a value of 2.07. During an insulin clamp, while inhibiting endogenous insulin secretion by somatostatin, P/S-IG remained at 2.2 with PID, whereas, P/S-IG disappeared completely with SID, which exhibited higher arterial and lower portal insulin levels compared with PID. Consequently, glucose disappearance rates and muscle glycogen synthetic rates were higher, but suppression of endogenous glucose production and liver glycogen synthetic rates were lower with SID compared with PID. When the insulin clamp was performed with SID at 2 and 5 mU·kg(-1)·min(-1) without managing endogenous insulin secretion under euglycemic but not hyperglycemic conditions, endogenous insulin secretion was completely suppressed with SID, and the P/S-IG disappeared. Thus, compared with PID, an insulin clamp with SID underestimates the contribution of liver in response to insulin to whole body glucose flux.

Dose-response effects of insulin glargine in type 2 diabetes.

OBJECTIVE: To determine the pharmacokinetic and pharmacodynamic dose-response effects of insulin glargine administered subcutaneously in individuals with type 2 diabetes. RESEARCH DESIGN AND METHODS: Twenty obese type 2 diabetic individuals (10 male and 10 female, aged 50 +/- 3 years, with BMI 36 +/- 2 kg/m(2) and A1C 8.3 +/- 0.6%) were studied in this single-center, placebo-controlled, randomized, double-blind study. Five subcutaneous doses of insulin glargine (0, 0.5, 1.0, 1.5, and 2.0 units/kg) were investigated on separate occasions using the 24-h euglycemic clamp technique. RESULTS Glargine duration of action to reduce glucose, nonessential fatty acid (NEFA), and beta-hydroxybutyrate levels was close to or >24 h for all four doses. Increases in glucose flux revealed no discernible peak and were modest with maximal glucose infusion rates of 9.4, 6.6, 5.5, and 2.8 mumol/kg/min for the 2.0, 1.5, 1.0, and 0.5 units/kg doses, respectively. Glargine exhibited a relatively hepatospecific action with greater suppression (P < 0.05) of endogenous glucose production (EGP) compared with little or no increases in glucose disposal. CONCLUSION: A single subcutaneous injection of glargine at a dose of >or=0.5 units/kg can acutely reduce glucose, NEFA, and ketone body levels for 24 h in obese insulin-resistant type 2 diabetic individuals. Glargine lowers blood glucose by mainly inhibiting EGP with limited effects on stimulating glucose disposal. Large doses of glargine have minimal effects on glucose flux and retain a relatively hepatospecific action in type 2 diabetes.

Pancreatic islet production of vascular endothelial growth factor--a is essential for islet vascularization, revascularization, and function.

To investigate molecular mechanisms controlling islet vascularization and revascularization after transplantation, we examined pancreatic expression of three families of angiogenic factors and their receptors in differentiating endocrine cells and adult islets. Using intravital lectin labeling, we demonstrated that development of islet microvasculature and establishment of islet blood flow occur concomitantly with islet morphogenesis. Our genetic data indicate that vascular endothelial growth factor (VEGF)-A is a major regulator of islet vascularization and revascularization of transplanted islets. In spite of normal pancreatic insulin content and beta-cell mass, mice with beta-cell-reduced VEGF-A expression had impaired glucose-stimulated insulin secretion. By vascular or diffusion delivery of beta-cell secretagogues to islets, we showed that reduced insulin output is not a result of beta-cell dysfunction but rather caused by vascular alterations in islets. Taken together, our data indicate that the microvasculature plays an integral role in islet function. Factors modulating VEGF-A expression may influence islet vascularity and, consequently, the amount of insulin delivered into the systemic circulation.

Proteomic exploration of pancreatic islets in mice null for the alpha2A adrenergic receptor.

The present studies extend recent findings that mice null for the alpha(2A) adrenergic receptor (alpha(2A) AR KO mice) lack suppression of exogenous secretagogue-stimulated insulin secretion in response to alpha(2) AR agonists by evaluating the endogenous secretagogue, glucose, ex vivo, and providing in vivo data that baseline insulin levels are elevated and baseline glucose levels are decreased in alpha(2A) AR KO mice. These latter findings reveal that the alpha(2A) AR subtype regulates glucose-stimulated insulin release in response to endogenous catecholamines in vivo. The changes in alpha(2A) AR responsiveness and resultant changes in insulin/glucose homeostasis encouraged us to utilize proteomics strategies to identify possible alpha(2A) AR downstream signaling molecules or other resultant changes due to perturbation of alpha(2A) AR expression. Although agonist stimulation of islets from wild type (WT) mice did not significantly alter islet protein profiles, several proteins were enriched in islets from alpha(2A) AR KO mice when compared with those from WT mice, including an enzyme participating in insulin protein processing. The present studies document the important role of the alpha(2A) AR subtype in tonic suppression of insulin release in response to endogenous catecholamines as well as exogenous alpha(2) agonists and provide insights into pleiotropic changes that result from loss of alpha(2A) AR expression and tonic suppression of insulin release.

Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy.

The recent success of pancreatic islet transplantation has generated considerable enthusiasm. To better understand the quality and characteristics of human islets used for transplantation, we performed detailed analysis of islet architecture and composition using confocal laser scanning microscopy. Human islets from six separate isolations provided by three different islet isolation centers were compared with isolated mouse and non-human primate islets. As expected from histological sections of murine pancreas, in isolated murine islets alpha and delta cells resided at the periphery of the beta-cell core. However, human islets were markedly different in that alpha, beta, and delta cells were dispersed throughout the islet. This pattern of cell distribution was present in all human islet preparations and islets of various sizes and was also seen in histological sections of human pancreas. The architecture of isolated non-human primate islets was very similar to that of human islets. Using an image analysis program, we calculated the volume of alpha, beta, and delta cells. In contrast to murine islets, we found that populations of islet cell types varied considerably in human islets. The results indicate that human islets not only are quite heterogeneous in terms of cell composition but also have a substantially different architecture from widely studied murine islets.

Assessment of pancreatic islet mass after islet transplantation using in vivo bioluminescence imaging.

BACKGROUND: Pancreatic islet transplantation is an emerging therapy for type 1 diabetes, but it is difficult to assess islets after transplantation and thus to design interventions to improve islet survival. METHODS: To image and quantify islets, the authors transplanted luciferase-expressing murine or human islets (by adenovirus-mediated gene transfer) into the liver or beneath the renal capsule of immunodeficient mice and quantified the in vivo bioluminescence imaging (BLI) of mice using a cooled charge-coupled device camera and digital photon-counting image analysis. To account for variables that are independent of islet mass such as transplant site, animal positioning, and wound healing, the BLI of transplanted islets was calibrated against measurement of luminescence of an implanted bead emitting a constant light intensity. RESULTS: BLI of mice bearing islet transplants was seen in the expected anatomic location, was stable for more than 8 weeks after transplantation, and correlated with the number of islets transplanted into the liver or kidney. BLI of the luminescent bead and of transplanted islets in the kidney was approximately four times greater than when transplanted in the liver, indicating that photon emission is dependent on optical absorption of generated light and thus light source location. CONCLUSION: In vivo BLI allows for quantitative, serial measurements of pancreatic islet mass after transplantation and should be useful in assessing interventions to sustain or increase islet survival of transplanted islets.

Reduced PDX-1 expression impairs islet response to insulin resistance and worsens glucose homeostasis.

In type 2 diabetes mellitus, insulin resistance and an inadequate pancreatic beta-cell response to the demands of insulin resistance lead to impaired insulin secretion and hyperglycemia. Pancreatic duodenal homeodomain-1 (PDX-1), a transcription factor required for normal pancreatic development, also plays a key role in normal insulin secretion by islets. To investigate the role of PDX-1 in islet compensation for insulin resistance, we examined glucose disposal, insulin secretion, and islet cell mass in mice of four different genotypes: wild-type mice, mice with one PDX-1 allele inactivated (PDX-1+/-, resulting in impaired insulin secretion), mice with one GLUT4 allele inactivated (GLUT4+/-, resulting in insulin resistance), and mice heterozygous for both PDX-1 and GLUT4 (GLUT4+/-;PDX-1+/-). The combination of PDX-1 and GLUT4 heterozygosity markedly prolonged glucose clearance. GLUT4+/-;PDX-1+/- mice developed beta-cell hyperplasia but failed to increase their beta-cell insulin content. These results indicate that PDX-1 heterozygosity (approximately 60% of normal protein levels) abrogates the beta-cell's compensatory response to insulin resistance, impairs glucose homeostasis, and may contribute to the pathogenesis of type 2 diabetes.

Gene expression phenotyping of an ACTH-producing small cell lung cancer line.

DNA microarray techniques were used to compare gene expression in an adrenocorticotropin (ACTH)-producing human small cell lung carcinoma line (DMS-79) with six other small cell lung cancer (SCLC) lines that do not produce ACTH. Twelve genes were expressed at more than five-fold higher levels in DMS-79 cells. Two transcription factors were the genes that exhibited the most remarkable over-expression: T-box 3 mRNA was detected at levels 19.37 +/- 3.78 times those observed in the SCLCs. Thyroid transcription factor (TTF-1, T/ebp, Nkx2.1) was expressed at 14.24 +/- 3.41-fold higher in DMS-79 cells. Seven genes were identified whose expression levels were at least five-fold lower in the ACTH-producing cell line. Variation in culture medium formulation did not significantly affect the gene expression profile of DMS-79 cells and expression data observed in microarray experiments were corroborated by northern blot analysis of RNA from the same cell lines. These experiments reveal new candidate genes that could be involved in the dysregulation of POMC gene expression manifested by ACTH-producing nonpituitary tumors.

A novel mutation in the preprovasopressin gene identified in a kindred with autosomal dominant neurohypophyseal diabetes insipidus.

Autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) is a defect in free water conservation caused by mutations in the single gene that encodes both vasopressin (VP) and its binding protein, neurophysin II (NP II). Most of the human mutations in this gene have been in the portion encoding the NP molecule; the resultant abnormal gene products are believed to cause cellular toxicity as improperly folded precursor molecules accumulate in the endoplasmic reticulum. We identified a new American kindred with ADNDI and found a novel mutation in the VP molecule. A 78-yr-old man was noted to have hypotonic polyuria and plasma hyperosmolarity; the urinary concentration defect was reversed by administration of VP. His symptomatology dated to childhood, and his family history was consistent with autosomal transmission of the polyuric syndrome, with affected members in three generations, including several females. Affected individuals were found to be heterozygous for a 3-bp deletion in exon 1 of arginine VP (AVP)-NP II, predicting a deletion of phenylalanine 3 (known to be critical for receptor binding) in the VP nonapeptide. Neuro 2A cells stably transfected with the mutant AVP-NP construct showed increased rates of apoptosis as assessed by flow cytometric methods. These observations support the concept that cellular toxicity of abnormal AVP-NP gene products underlies the development of ADNDI, and the data further demonstrate that mutations affecting the AVP moiety can result in initiation of these pathological processes.

Oxidative stress is a mediator of glucose toxicity in insulin-secreting pancreatic islet cell lines.

Pancreatic beta cells secrete insulin in response to changes in the extracellular glucose. However, prolonged exposure to elevated glucose exerts toxic effects on beta cells and results in beta cell dysfunction and ultimately beta cell death (glucose toxicity). To investigate the mechanism of how increased extracellular glucose is toxic to beta cells, we used two model systems where glucose metabolism was increased in beta cell lines by enhancing glucokinase (GK) activity and exposing cells to physiologically relevant increases in extracellular glucose (3.3-20 mm). Exposure of cells with enhanced GK activity to 20 mm glucose accelerated glycolysis, but reduced cellular NAD(P)H and ATP, caused accumulation of intracellular reactive oxygen species (ROS) and oxidative damage to mitochondria and DNA, and promoted apoptotic cell death. These changes required both enhanced GK activity and exposure to elevated extracellular glucose. A ROS scavenger partially prevented the toxic effects of increased glucose metabolism. These results indicate that increased glucose metabolism in beta cells generates oxidative stress and impairs cell function and survival; this may be a mechanism of glucose toxicity in beta cells. The level of beta cell GK may also be critical in this process.

Epidermal growth factor increases cortisol production and type II 3 beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase expression in human adrenocortical carcinoma cells: evidence for a Stat5-dependent mechanism.

We tested the ability of epidermal growth factor (EGF) to regulate a key enzyme in the adrenal synthesis of glucocorticoids: human type II 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3 beta HSD). EGF treatment (25 ng/ml) of human adrenocortical carcinoma cells (H295R) resulted in a 5-fold increase in cortisol production and a corresponding 2-fold increase in 3 beta HSD mRNA. Experiments were performed to determine whether EGF is acting through a previously identified signal transducer and activator of transcription 5 (Stat5)-responsive element located from -110 to -118 in the human type II 3 beta HSD promoter. A Stat5 expression construct was cotransfected with a 3 beta HSD-chloramphenol acetyltransferase (CAT) reporter construct comprised of nucleotides -301-->+45 of the human type II 3 beta HSD promoter linked to the CAT reporter gene sequence. The addition of EGF at doses as low as 10 ng/ml resulted in an 11- to 15-fold increase in CAT activity. The introduction of 3-bp point mutations into critical nucleotides in the Stat5 response element obviated the EGF response. Either Stat5a or Stat5b isoforms induced CAT reporter expression upon treatment with EGF. These results demonstrate the ability of EGF to regulate the expression of a critical enzyme (3 beta HSD) in the production of cortisol and suggest a molecular mechanism by which this regulation occurs.

Glucocorticoids enhance activation of the human type II 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase gene.

Glucocorticoids indirectly alter adrenocortical steroid output through the inhibition of ACTH secretion by the anterior pituitary. However, previous studies suggest that glucocorticoids can directly affect adrenocortical steroid production. Therefore, we have investigated the ability of glucocorticoids to affect transcription of adrenocortical steroid biosynthetic enzymes. One potential target of glucocorticoid action in the adrenal is an enzyme critical for adrenocortical steroid production: 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD). Treatment of the adrenocortical cell line (H295R) with the glucocorticoid agonist dexamethasone (DEX) increased cortisol production and 3beta-HSD mRNA levels alone or in conjunction with phorbol ester. This increase in 3beta-HSD mRNA was paralleled by increases in Steroidogenic Acute Regulatory Protein (StAR) mRNA levels. The human type II 3beta-HSD promoter lacks a consensus palindromic glucocorticoid response element (GRE) but does contain a Stat5 response element (Stat5RE) suggesting that glucocorticoids could affect type II 3beta-HSD transcription via interaction with Stat5. Transfection experiments show enhancement of human type II 3beta-HSD promoter activity by coexpression of the glucocorticoid receptor (GR) and Stat5A and treatment with 100nM dexamethasone. Furthermore, removal of the Stat5RE either by truncation of the 5' flanking sequence in the promoter or introduction of point mutations to the Stat5RE abolished the ability of DEX to enhance 3beta-HSD promoter activity. These studies demonstrate the ability of glucocorticoids to directly enhance the expression of an adrenal steroidogenic enzyme gene albeit independent of a consensus palindromic glucocorticoid response element.

Pancreatic islet beta-cells transiently metabolize pyruvate.

Pancreatic beta-cell metabolism was followed during glucose and pyruvate stimulation of pancreatic islets using quantitative two-photon NAD(P)H imaging. The observed redox changes, spatially separated between the cytoplasm and mitochondria, were compared with whole islet insulin secretion. As expected, both NAD(P)H and insulin secretion showed sustained increases in response to glucose stimulation. In contrast, pyruvate caused a much lower NAD(P)H response and did not generate insulin secretion. Low pyruvate concentrations decreased cytoplasmic NAD(P)H without affecting mitochondrial NAD(P)H, whereas higher concentrations increased cytoplasmic and mitochondrial levels. However, the pyruvate-stimulated mitochondrial increase was transient and equilibrated to near-base-line levels. Inhibitors of the mitochondrial pyruvate-transporter and malate-aspartate shuttle were utilized to resolve the glucose- and pyruvate-stimulated NAD(P)H response mechanisms. These data showed that glucose-stimulated mitochondrial NAD(P)H and insulin secretion are independent of pyruvate transport but dependent on NAD(P)H shuttling. In contrast, the pyruvate-stimulated cytoplasmic NAD(P)H response was enhanced by both inhibitors. Surprisingly the malate-aspartate shuttle inhibitor enabled pyruvate-stimulated insulin secretion. These data support a model in which glycolysis plays a dominant role in glucose-stimulated insulin secretion. Based on these data, we propose a mechanism for glucose-stimulated insulin secretion that includes allosteric inhibition of tricarboxylic acid cycle enzymes and pH dependence of mitochondrial pyruvate transport.

Reduction in pancreatic transcription factor PDX-1 impairs glucose-stimulated insulin secretion.

Complete lack of transcription factor PDX-1 leads to pancreatic agenesis, whereas heterozygosity for PDX-1 mutations has been recently noted in some individuals with maturity-onset diabetes of the young (MODY) and in some individuals with type 2 diabetes. To determine how alterations in PDX-1 affect islet function, we examined insulin secretion and islet physiology in mice with one PDX-1 allele inactivated. PDX-1(+/-) mice had a normal fasting blood glucose and pancreatic insulin content but had impaired glucose tolerance and secreted less insulin during glucose tolerance testing. The expression of PDX-1 and glucose transporter 2 in islets from PDX-1(+/-) mice was reduced to 68 and 55%, respectively, whereas glucokinase expression was not significantly altered. NAD(P)H generation in response to glucose was reduced by 30% in PDX-1(+/-) mice. The in situ perfused pancreas of PDX-1(+/-) mice secreted about 45% less insulin when stimulated with 16.7 mm glucose. The K(m) for insulin release was similar in wild type and PDX-1(+/-) mice. Insulin secretion in response to 20 mm arginine was unchanged; the response to 10 nm glucagon-like peptide-1 was slightly increased. However, insulin secretory responses to 10 mm 2-ketoisocaproate and 20 mm KCl were significantly reduced (by 61 and 66%, respectively). These results indicate that a modest reduction in PDX-1 impairs several events in glucose-stimulated insulin secretion (such as NAD(P)H generation, mitochondrial function, and/or mobilization of intracellular Ca(2+)) and that PDX-1 is important for normal function of adult pancreatic islets.