Woo Jin Song

Glucose and Weight Control in Mice with a Designed Ghrelin O-Acyltransferase Inhibitor

Metabolism Without Modification Obesity-associated metabolic disease has rapidly become a public health priority in the developed world and is being addressed through prevention strategies aimed at lifestyle changes and through pharmacological approaches. Barnett et al. (p. 1689, published online 18 November) designed a drug that inhibits the action of ghrelin, a circulating peptide hormone that increases fat mass and food intake. The drug, a bisubstrate analog called GO-CoA-Tat, is a selective antagonist of ghrelin O-acyltransferase (GOAT), an enzyme that catalyzes a posttranslational modification that is essential for ghrelin activity. Injection of GO-CoA-Tat into wild-type mice on a high-fat diet improved glucose tolerance and reduced weight gain, probably through changes in metabolic activity. Because GO-CoA-Tat is a peptide-based drug that requires repeated injection, it is unsuitable for clinical use, but GOAT does represent a potentially valuable target for future drug development efforts in metabolic disease. A drug inhibiting the activation of ghrelin, a peptide that promotes weight gain, has beneficial metabolic effects in mice. Ghrelin is a gastric peptide hormone that stimulates weight gain in vertebrates. The biological activities of ghrelin require octanoylation of the peptide on Ser3, an unusual posttranslational modification that is catalyzed by the enzyme ghrelin O-acyltransferase (GOAT). Here, we describe the design, synthesis, and characterization of GO-CoA-Tat, a peptide-based bisubstrate analog that antagonizes GOAT. GO-CoA-Tat potently inhibits GOAT in vitro, in cultured cells, and in mice. Intraperitoneal administration of GO-CoA-Tat improves glucose tolerance and reduces weight gain in wild-type mice but not in ghrelin-deficient mice, supporting the concept that its beneficial metabolic effects are due specifically to GOAT inhibition. In addition to serving as a research tool for mapping ghrelin actions, GO-CoA-Tat may help pave the way for clinical targeting of GOAT in metabolic diseases.

Increased Pancreatic β-Cell Proliferation Mediated by CREB Binding Protein Gene Activation

ABSTRACT The cyclic AMP (cAMP) signaling pathway is central in β-cell gene expression and function. In the nucleus, protein kinase A (PKA) phosphorylates CREB, resulting in recruitment of the transcriptional coactivators p300 and CREB binding protein (CBP). CBP, but not p300, is phosphorylated at serine 436 in response to insulin action. CBP phosphorylation disrupts CREB-CBP interaction and thus reduces nuclear cAMP action. To elucidate the importance of the cAMP-PKA-CREB-CBP pathway in pancreatic β cells specifically at the nuclear level, we have examined mutant mice lacking the insulin-dependent phosphorylation site of CBP. In these mice, the CREB-CBP interaction is enhanced in both the absence and presence of cAMP stimulation. We found that islet and β-cell masses were increased twofold, while pancreas weights were not different from the weights of wild-type littermates. β-Cell proliferation was increased both in vivo and in vitro in isolated islet cultures. Surprisingly, glucose-stimulated insulin secretion from perfused, isolated mutant islets was reduced. However, β-cell depolarization with KCl induced similar levels of insulin release from mutant and wild-type islets, indicating normal insulin synthesis and storage. In addition, transcripts of pgc1a, which disrupts glucose-stimulated insulin secretion, were also markedly elevated. In conclusion, sustained activation of CBP-responsive genes results in increased β-cell proliferation. In these β cells, however, glucose-stimulated insulin secretion was diminished, resulting from concomitant CREB-CBP-mediated pgc1a gene activation.

Glucagon Regulates Hepatic Kisspeptin to Impair Insulin Secretion

Early in the pathogenesis of type 2 diabetes mellitus (T2DM), dysregulated glucagon secretion from pancreatic α cells occurs prior to impaired glucose-stimulated insulin secretion (GSIS) from β cells. However, whether hyperglucagonemia is causally linked to β cell dysfunction remains unclear. Here we show that glucagon stimulates via cAMP-PKA-CREB signaling hepatic production of the neuropeptide kisspeptin1, which acts on β cells to suppress GSIS. Synthetic kisspeptin suppresses GSIS in vivo in mice and from isolated islets in a kisspeptin1 receptor-dependent manner. Kisspeptin1 is increased in livers and in serum from humans with T2DM and from mouse models of diabetes mellitus. Importantly, liver Kiss1 knockdown in hyperglucagonemic, glucose-intolerant, high-fat-diet fed, and Lepr(db/db) mice augments GSIS and improves glucose tolerance. These observations indicate a hormonal circuit between the liver and the endocrine pancreas in glycemia regulation and suggest in T2DM a sequential link between hyperglucagonemia via hepatic kisspeptin1 to impaired insulin secretion.

Snapin Mediates Incretin Action and Augments Glucose-Dependent Insulin Secretion

Impaired insulin secretion contributes to the pathogenesis of type 2 diabetes mellitus (T2DM). Treatment with the incretin hormone glucagon-like peptide-1 (GLP-1) potentiates insulin secretion and improves metabolic control in humans with T2DM. GLP-1 receptor-mediated signaling leading to insulin secretion occurs via cyclic AMP stimulated protein kinase A (PKA)- as well as guanine nucleotide exchange factor-mediated pathways. However, how these two pathways integrate and coordinate insulin secretion remains poorly understood. Here we show that these incretin-stimulated pathways converge at the level of snapin, and that PKA-dependent phosphorylation of snapin increases interaction among insulin secretory vesicle-associated proteins, thereby potentiating glucose-stimulated insulin secretion (GSIS). In diabetic islets with impaired GSIS, snapin phosphorylation is reduced, and expression of a snapin mutant, which mimics site-specific phosphorylation, restores GSIS. Thus, snapin is a critical node in GSIS regulation and provides a potential therapeutic target to improve β cell function in T2DM.

Exendin-4 Stimulation of Cyclin A2 in β-Cell Proliferation

OBJECTIVE—β-Cell proliferation is an important mechanism underlying β-cell mass adaptation to metabolic demands. We have examined effects, in particular those mediated through intracellular cAMP signaling, of the incretin hormone analog exendin-4 on cell cycle regulation in β-cells. RESEARCH DESIGN AND METHODS—Changes in islet protein levels of cyclins and of two critical cell cycle regulators cyclin kinase inhibitor p27 and S-phase kinase–associated protein 2 (Skp2) were assessed in mice treated with exendin-4 and in a mouse model with specific upregulation of nuclear cAMP signaling exhibiting increased β-cell proliferation (CBP-S436A mouse). Because cyclin A2 was stimulated by cAMP, we assessed the role of cylcin A2 in cell cycle progression in Min6 and isolated islet β-cells. RESULTS—Mice treated with exendin-4 showed increased β-cell proliferation, elevated islet protein levels of cyclin A2 with unchanged D-type cyclins, elevated PDX-1 and Skp2 levels, and reduced p27 levels. Exendin-4 stimulated cyclin A2 promoter activity via the cAMP–cAMP response element binding protein pathway. CBP-S436A islets exhibited elevated cyclin A2, reduced p27, and no changes in D-type cyclins, PDX-1, or Skp2. In cultured islets, exendin-4 increased cyclin A2 and Skp2 and reduced p27. Cyclin A2 overexpression in primary islets increased proliferation and reduced p27. In Min6 cells, cyclin A2 knockdown prevented exendin-4–stimulated proliferation. PDX-1 knockdown reduced exendin-4–stimulated cAMP synthesis and cyclin A2 transcription. CONCLUSIONS—Cyclin A2 is required for β-cell proliferation, exendin-4 stimulates cyclin A2 expression via the cAMP pathway, and exendin-4 stimulation of cAMP requires PDX-1.

Pancreatic β-Cell Response to Increased Metabolic Demand and to Pharmacologic Secretagogues Requires EPAC2A

Incretin hormone action on β-cells stimulates in parallel two different intracellular cyclic AMP-dependent signaling branches mediated by protein kinase A and exchange protein activated by cAMP islet/brain isoform 2A (EPAC2A). Both pathways contribute toward potentiation of glucose-stimulated insulin secretion (GSIS). However, the overall functional role of EPAC2A in β-cells as it relates to in vivo glucose homeostasis remains incompletely understood. Therefore, we have examined in vivo GSIS in global EPAC2A knockout mice. Additionally, we have conducted in vitro studies of GSIS and calcium dynamics in isolated EPAC2A-deficient islets. EPAC2A deficiency does not impact GSIS in mice under basal conditions. However, when mice are exposed to diet-induced insulin resistance, pharmacologic secretagogue stimulation of β-cells with an incretin hormone glucagon-like peptide-1 analog or with a fatty acid receptor 1/G protein–coupled receptor 40 selective activator, EPAC2A is required for the increased β-cell response to secretory demand. Under these circumstances, EPAC2A is required for potentiating the early dynamic increase in islet calcium levels after glucose stimulation, which is reflected in potentiated first-phase insulin secretion. These studies broaden our understanding of EPAC2A function and highlight its significance during increased secretory demand or drive on β-cells. Our findings advance the rationale for developing EPAC2A-selective pharmacologic activators for β-cell–targeted pharmacotherapy in type 2 diabetes.

There is Kisspeptin – And Then There is Kisspeptin

While kisspeptin was initially found to function as a metastasis suppressor, after identification of its receptor KISS1R and their expression profiles in tissues such as the hypothalamus and adrenals, kisspeptin and KISS1R were predominantly assigned endocrine functions, including regulating puberty and fertility through their actions on hypothalamic gonadotropin releasing hormone production. More recently, an alter ego for kisspeptin has emerged, with a significant role in regulating glucose homeostasis, insulin secretion, as well as food intake and body composition, and deficient kisspeptin signaling results in reduced locomotor activity and increased adiposity. This review highlights these recent observations on the role of kisspeptin in metabolism as well as several key questions that need to be addressed in the future.

Inter-organ communication and regulation of beta cell function.

The physiologically predominant signal for pancreatic beta cells to secrete insulin is glucose. While circulating glucose levels and beta cell glucose metabolism regulate the amount of released insulin, additional signals emanating from other tissues and from neighbouring islet endocrine cells modulate beta cell function. To this end, each individual beta cell can be viewed as a sensor of a multitude of stimuli that are integrated to determine the extent of glucose-dependent insulin release. This review discusses recent advances in our understanding of inter-organ communications that regulate beta cell insulin release in response to elevated glucose levels.

Increasing β-Cell Mass Requires Additional Stimulation for Adaptation to Secretory Demand

Type 2 diabetes mellitus (T2DM) is caused by relative insulin deficiency, subsequent to both reduced β-cell mass and insufficient insulin secretion, and both augmenting β-cell mass and β-cell function are therapeutic strategies for treating T2DM. However, the relative significance of increasing β-cell mass vs improving β-cell stimulus secretion coupling remains unclear. We have developed a mouse model that allows proliferation of β-cells in adult mice without affecting β-cell function by inducible expression of the positive cell cycle regulator cyclin A2 specifically in β-cells. In these mice, when kept on a standard diet, doubling of β-cell mass does not result in altered glucose tolerance or glucose-stimulated circulating insulin levels. Notably, a doubling of β-cell mass also does not confer improved glycemic control and ability of β-cells to respond to diabetogenic high-fat diet-induced glucose intolerance. However, in high-fat diet-exposed mice, an increase in endogenous β-cell mass confers increased potentiation of in vivo glucose-stimulated rise in circulating insulin in response to acute pharmacologic treatment with the incretin glucagon-like peptide-1 receptor agonist exendin-4. These observations indicate that increasing endogenous β-cell mass may not be sufficient to improve glycemic control in T2DM without additional strategies to increase β-cell stimulus secretion coupling.

Prkar1a gene knockout in the pancreas leads to neuroendocrine tumorigenesis

Carney complex (CNC) is a rare disease associated with multiple neoplasias, including a predisposition to pancreatic tumors; it is caused most frequently by the inactivation of the PRKAR1A gene, a regulator of the cyclic AMP (cAMP)-dependent kinase (PKA). The method used was to create null alleles of prkar1a in mouse cells expressing pdx1 (Δ-Prkar1a). We found that these mice developed endocrine or mixed endocrine/acinar cell carcinomas with 100% penetrance by the age of 4-5 months. Malignant behavior of the tumors was seen as evidenced by stromal invasion and metastasis to locoregional lymph nodes. Histologically, most tumors exhibited an organoid pattern as seen in the islet-cell tumors. Biochemically, the lesions exhibited high PKA activity, as one would expect from deleting prkar1a The primary neuroendocrine nature of these tumor cells was confirmed by immunohistochemical staining and electron microscopy, the latter revealing the characteristic granules. Although the Δ-Prkar1a mice developed hypoglycemia after overnight fasting, insulin and glucagon levels in the plasma were normal. Negative immunohistochemical staining for the most commonly produced peptides (insulin, c-peptide, glucagon, gastrin and somatostatin) suggested that these tumors were non-functioning. We hypothesize that the recently identified multipotent pdx1+/insulin- cell in adult pancreas, gives rise to endocrine or mixed endocrine/acinar pancreatic malignancies with complete prkar1a deficiency. In conclusion, this mouse model supports the role of prkar1a as a tumor suppressor gene in the pancreas and points to the PKA pathway as a possible therapeutic target for these lesions.