Thomas L. Jetton

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 β-cell mass, mice with β-cell–reduced VEGF-A expression had impaired glucose-stimulated insulin secretion. By vascular or diffusion delivery of β-cell secretagogues to islets, we showed that reduced insulin output is not a result of β-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.

GPR40 Is Necessary but Not Sufficient for Fatty Acid Stimulation of Insulin Secretion In Vivo

Long-chain fatty acids amplify insulin secretion from the pancreatic β-cell. The G-protein–coupled receptor GPR40 is specifically expressed in β-cells and is activated by fatty acids; however, its role in acute regulation of insulin secretion in vivo remains unclear. To this aim, we generated GPR40 knockout (KO) mice and examined glucose homeostasis, insulin secretion in response to glucose and Intralipid in vivo, and insulin secretion in vitro after short- and long-term exposure to fatty acids. Our results show that GPR40 KO mice have essentially normal glucose tolerance and insulin secretion in response to glucose. Insulin secretion in response to Intralipid was reduced by ∼50%. In isolated islets, insulin secretion in response to glucose and other secretagogues was unaltered, but fatty acid potentiation of insulin release was markedly reduced. The Gαq/11 inhibitor YM-254890 dose-dependently reduced palmitate potentiation of glucose-induced insulin secretion. Islets from GPR40 KO mice were as sensitive to fatty acid inhibition of insulin secretion upon prolonged exposure as islets from wild-type animals. We conclude that GPR40 contributes approximately half of the full acute insulin secretory response to fatty acids in mice but does not play a role in the mechanisms by which fatty acids chronically impair insulin secretion.

Regulation of Pancreatic β-Cell Regeneration in the Normoglycemic 60% Partial-Pancreatectomy Mouse

β-Cell mass is determined by a dynamic balance of proliferation, neogenesis, and apoptosis. The precise mechanisms underlying compensatory β-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 ∼68% of the shams by 15 days, partly due to enhanced β-cell proliferation. A transient ∼2.8-fold increase in the prevalence of β-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 β-cell growth by activating β-cell proliferation with an increase in the number of β-cell clusters/small islets.

β-Cell Failure in Diet-Induced Obese Mice Stratified According to Body Weight Gain: Secretory Dysfunction and Altered Islet Lipid Metabolism Without Steatosis or Reduced β-Cell Mass

OBJECTIVE C57Bl/6 mice develop obesity and mild hyperglycemia when fed a high-fat diet (HFD). Although diet-induced obesity (DIO) is a widely studied model of type 2 diabetes, little is known about β-cell failure in these mice. RESEARCH DESIGN AND METHODS DIO mice were separated in two groups according to body weight gain: low- and high-HFD responders (LDR and HDR). We examined whether mild hyperglycemia in HDR mice is due to reduced β-cell mass or function and studied islet metabolism and signaling. RESULTS HDR mice were more obese, hyperinsulinemic, insulin resistant, and hyperglycemic and showed a more altered plasma lipid profile than LDR. LDR mice largely compensated insulin resistance, whereas HDR showed perturbed glucose homeostasis. Neither LDR nor HDR mice showed reduced β-cell mass, altered islet glucose metabolism, and triglyceride deposition. Insulin secretion in response to glucose, KCl, and arginine was impaired in LDR and almost abolished in HDR islets. Palmitate partially restored glucose- and KCl-stimulated secretion. The glucose-induced rise in ATP was reduced in both DIO groups, and the glucose-induced rise in Ca2+ was reduced in HDR islets relatively to LDR. Glucose-stimulated lipolysis was decreased in LDR and HDR islets, whereas fat oxidation was increased in HDR islets only. Fatty acid esterification processes were markedly diminished, and free cholesterol accumulated in HDR islets. CONCLUSIONS β-Cell failure in HDR mice is not due to reduced β-cell mass and glucose metabolism or steatosis but to a secretory dysfunction that is possibly due to altered ATP/Ca2+ and lipid signaling, as well as free cholesterol deposition.

Mice lacking alpha-calcitonin gene-related peptide exhibit normal cardiovascular regulation and neuromuscular development.

alpha-Calcitonin gene-related peptide (alphaCGRP) is a pleiotropic peptide neuromodulator that is widely expressed throughout the Central and peripheral nervous systems. CGRP has been implicated in a variety of physiological processes including peripheral vasodilation, cardiac acceleration nicotinic acetylcholine receptor (AChR) synthesis and function, testicular descent, nociception, carbohydrate metabolism, gastrointestinal motility, neurogenic inflammation, and gastric acid secretion. To provide a better understanding of the physiological role(s) mediated by this peptide neurotransmitter, we have generated alphaCGRP-null mice by targeted modification in embryonic stem cells. Mice lacking alpha CGRP expression demonstrate no obvious phenotypic differences from their wild-type littermates. Detailed analysis of systemic cardiovascular function revealed no differences between control and mutant mice regarding heart rate and blood pressure under basal or exercise-induced conditions and subsequent to pharmacological manipulation. Characterization of neuromuscular junction in morphology including nicotinic receptor localization, terminal sprouting in response to denervation, developmental regulation of AChR subunit expression, and synapse elimination also revealed no differences in alphaCGRP-deficient animals. These results suggest that alphaCGRP is not required for the systemic regulation of cardiovascular hemodynamics or development of the neuromuscular junction.

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

In the 60% pancreatectomy (Px) rat model of β-cell adaptation, normoglycemia is maintained by an initial week of β-cell hyperplasia that ceases and is followed by enhanced β-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 β-cell nuclear peroxisome proliferator–activated receptor (PPAR)-γ protein, along with a 2-fold increase in nuclear pancreatic duodenal homeobox (Pdx)-1 protein and a 1.4-fold increase in β-cell nuclear Nkx6.1 immunostaining. As PPAR-γ activation is known to both lower proliferation and have prodifferentiation effects in many tissues, we studied PPAR-γ actions in INS-1 cells. A 3-day incubation with the PPAR-γ agonist troglitazone reduced proliferation and increased Pdx-1 and Nkx6.1 immunostaining, along with glucokinase and GLUT2. Also, a 75% knockdown of PPAR-γ 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-γ 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-γ signaling in the β-cell adaptation following a 60% Px in rats.

Cell-specific Expression and Regulation of a Glucokinase Gene Locus Transgene

Transgenic mice containing one or more extra copies of the entire glucokinase (GK) gene locus were generated and characterized. The GK transgene, an 83-kilobase pair mouse genomic DNA fragment containing both promoter regions, was expressed and regulated in a cell-specific manner, and rescued GK null lethality when crossed into mice bearing a targeted mutation of the endogenous GK gene. Livers from the transgenic mice had elevated GK mRNA, protein, and activity levels, compared with controls, and the transgene was regulated in liver by dietary manipulations. The amount of GK immunoreactivity in hepatocyte nuclei, where GK binds to the GK regulatory protein, was also increased. Pancreatic islets displayed increased GK immunoreactivity and NAD(P)H responses to glucose, but only when isolated and cultured in 20 mm glucose, as a result of the hypoglycemic phenotype of these mice (Niswender, K. D., Shiota, M., Postic, C., Cherrington, A. D., and Magnuson, M. A. (1997) J. Biol. Chem. 272, 22604–22609). Together, these results indicate that the region of the gene from −55 to +28 kilobase pairs (relative to the liver GK transcription start site) contains all the regulatory sequences necessary for expression of both GK isoforms, thereby placing an upper limit on the size of the GK gene locus.

Transamination Is Required for α-Ketoisocaproate but Not Leucine to Stimulate Insulin Secretion

It remains unclear how α-ketoisocaproate (KIC) and leucine are metabolized to stimulate insulin secretion. Mitochondrial BCATm (branched-chain aminotransferase) catalyzes reversible transamination of leucine and α-ketoglutarate to KIC and glutamate, the first step of leucine catabolism. We investigated the biochemical mechanisms of KIC and leucine-stimulated insulin secretion (KICSIS and LSIS, respectively) using BCATm−/− mice. In static incubation, BCATm disruption abolished insulin secretion by KIC, d,l-α-keto-β-methylvalerate, and α-ketocaproate without altering stimulation by glucose, leucine, or α-ketoglutarate. Similarly, during pancreas perfusions in BCATm−/− mice, glucose and arginine stimulated insulin release, whereas KICSIS was largely abolished. During islet perifusions, KIC and 2 mm glutamine caused robust dose-dependent insulin secretion in BCATm+/+ not BCATm−/− islets, whereas LSIS was unaffected. Consistently, in contrast to BCATm+/+ islets, the increases of the ATP concentration and NADPH/NADP+ ratio in response to KIC were largely blunted in BCATm−/− islets. Compared with nontreated islets, the combination of KIC/glutamine (10/2 mm) did not influence α-ketoglutarate concentrations but caused 120 and 33% increases in malate in BCATm+/+ and BCATm−/− islets, respectively. Although leucine oxidation and KIC transamination were blocked in BCATm−/− islets, KIC oxidation was unaltered. These data indicate that KICSIS requires transamination of KIC and glutamate to leucine and α-ketoglutarate, respectively. LSIS does not require leucine catabolism and may be through leucine activation of glutamate dehydrogenase. Thus, KICSIS and LSIS occur by enhancing the metabolism of glutamine/glutamate to α-ketoglutarate, which, in turn, is metabolized to produce the intracellular signals such as ATP and NADPH for insulin secretion.

Physiologic and Pharmacologic Modulation of Glucose-Dependent Insulinotropic Polypeptide (GIP) Receptor Expression in β-Cells by Peroxisome Proliferator–Activated Receptor (PPAR)-γ Signaling Possible Mechanism for the GIP Resistance in Type 2 Diabetes

OBJECTIVE We previously showed that peroxisome proliferator–activated receptor (PPAR)-γ in β-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 PPARγ 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 PPARγ knockout mice (PANC PPARγ−/−), 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-γ 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 PPARγ−/− mice and a twofold increase in islets of 14-day post-60% Px Sprague-Dawley rats that hyperexpress β-cell PPARγ. 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 PPARγ 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 β-cells by PPARγ signaling. Also disruption of this signaling pathway may account for the lowered β-cell GIP-R expression and resulting GIP resistance in type 2 diabetes.

Vagal control of pancreatic β-cell proliferation

The physiological mechanisms that preserve pancreatic β-cell mass (BCM) are not fully understood. Although the regulation of islet function by the autonomic nervous system (ANS) is well established, its potential roles in BCM homeostasis and compensatory growth have not been adequately explored. The parasympathetic vagal branch of the ANS serves to facilitate gastrointestinal function, metabolism, and pancreatic islet regulation of glucose homeostasis, including insulin secretion. Given the functional importance of the vagus nerve and its branches to the liver, gut, and pancreas in control of digestion, motility, feeding behavior, and glucose metabolism, it may also play a role in BCM regulation. We have begun to examine the potential roles of the parasympathetic nervous system in short-term BCM maintenance by performing a selective bilateral celiac branch-vagus nerve transection (CVX) in normal Sprague-Dawley rats. CVX resulted in no detectable effects on basic metabolic parameters or food intake through 1 wk postsurgery. Although there were no differences in BCM or apoptosis in this 1-wk time frame, β-cell proliferation was reduced 50% in the CVX rats, correlating with a marked reduction in activated protein kinase B/Akt. Unexpectedly, acinar proliferation was increased 50% in these rats. These data suggest that the ANS, via the vagus nerve, contributes to the regulation of BCM maintenance at the level of cell proliferation and may also mediate the drive for enhanced growth under physiological conditions when insulin requirements have increased. Furthermore, the disparate effects of CVX on β-cell and acinar cells suggest that the endocrine and exocrine pancreas respond to different neural signals in regard to mass homeostasis.