Kohtaro Minami

Dynamics of insulin secretion and the clinical implications for obesity and diabetes.

Insulin secretion is a highly dynamic process regulated by various factors including nutrients, hormones, and neuronal inputs. The dynamics of insulin secretion can be studied at different levels: the single β cell, pancreatic islet, whole pancreas, and the intact organism. Studies have begun to analyze cellular and molecular mechanisms underlying dynamics of insulin secretion. This review focuses on our current understanding of the dynamics of insulin secretion in vitro and in vivo and discusses their clinical relevance.

The cAMP Sensor Epac2 Is a Direct Target of Antidiabetic Sulfonylurea Drugs

Expanding Sulfonylurea Mechanisms Sulfonylureas are important drugs used for treatment of diabetes that act through adenosine triphosphate–sensitive potassium channels to promote secretion of insulin from the pancreas. Zhang et al. (p. 607) present another mechanism by which the beneficial effects of sulfonylureas may also be obtained. Sulfonylureas were identified in a screen for substances that modify the activity of Epac2, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rap1. Mice lacking Epac2 were less responsive to sulfonylureas, which may suggest that Epac2 would be a useful target for development of drugs for treatment of diabetes. A drug used to enhance insulin secretion in diabetes has a previously unrecognized protein target. Epac2, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rap1, is activated by adenosine 3′,5′-monophosphate. Fluorescence resonance energy transfer and binding experiments revealed that sulfonylureas, widely used antidiabetic drugs, interact directly with Epac2. Sulfonylureas activated Rap1 specifically through Epac2. Sulfonylurea-stimulated insulin secretion was reduced both in vitro and in vivo in mice lacking Epac2, and the glucose-lowering effect of the sulfonylurea tolbutamide was decreased in these mice. Epac2 thus contributes to the effect of sulfonylureas to promote insulin secretion. Because Epac2 is also required for the action of incretins, gut hormones crucial for potentiating insulin secretion, it may be a promising target for antidiabetic drug development.

PGRN is a Key Adipokine Mediating High Fat Diet-Induced Insulin Resistance and Obesity through IL-6 in Adipose Tissue

Adipose tissue secretes adipokines that mediate insulin resistance, a characteristic feature of obesity and type 2 diabetes. By differential proteome analysis of cellular models of insulin resistance, we identified progranulin (PGRN) as an adipokine induced by TNF-α and dexamethasone. PGRN in blood and adipose tissues was markedly increased in obese mouse models and was normalized with treatment of pioglitazone, an insulin-sensitizing agent. Ablation of PGRN (Grn(-/-)) prevented mice from high fat diet (HFD)-induced insulin resistance, adipocyte hypertrophy, and obesity. Grn deficiency blocked elevation of IL-6, an inflammatory cytokine, induced by HFD in blood and adipose tissues. Insulin resistance induced by chronic administration of PGRN was suppressed by neutralizing IL-6 in vivo. Thus, PGRN is a key adipokine that mediates HFD-induced insulin resistance and obesity through production of IL-6 in adipose tissue, and may be a promising therapeutic target for obesity.

Rim2α Determines Docking and Priming States in Insulin Granule Exocytosis

Insulin secretion is essential for maintenance of glucose homeostasis, but the mechanism of insulin granule exocytosis, the final step of insulin secretion, is largely unknown. Here, we investigated the role of Rim2alpha in insulin granule exocytosis, including the docking, priming, and fusion steps. We found that interaction of Rim2alpha and Rab3A is required for docking, which is considered a brake on fusion events, and that docking is necessary for K(+)-induced exocytosis, but not for glucose-induced exocytosis. Furthermore, we found that dissociation of the Rim2alpha/Munc13-1 complex by glucose stimulation activates Syntaxin1 by Munc13-1, indicating that Rim2alpha primes insulin granules for fusion. Thus, Rim2alpha determines docking and priming states in insulin granule exocytosis depending on its interacting partner, Rab3A or Munc13-1, respectively. Because Rim2alpha(-/-) mice exhibit impaired secretion of various hormones stored as dense-core granules, including glucose-dependent insulinotropic polypeptide, growth hormone, and epinephrine, Rim2alpha plays a critical role in exocytosis of these dense-core granules.

Establishment of new clonal pancreatic β‐cell lines (MIN6‐K) useful for study of incretin/cyclic adenosine monophosphate signaling

Incretin/cyclic adenosine monophosphate (cAMP) signaling is critical for potentiation of insulin secretion. Although several cell lines of pancreatic β‐cells are currently available, there are no cell lines suitable for investigation of incretin/cAMP signaling. In the present study, we have newly established pancreatic β‐cell lines (named MIN6‐K) from the IT6 mouse, which develops insulinoma. MIN6‐K8 cells respond to both glucose and incretins, such as glucagon‐like peptide‐1 (GLP‐1) and glucose‐dependent insulinotropic polypeptide (GIP), as is the case in pancreatic islets, whereas MIN6‐K20 cells respond to glucose, but not to incretins. Despite the difference in incretin‐potentiated insulin secretion between these two cell lines, the accumulation of cAMP after stimulation of GLP‐1 is comparable in these cells. Interestingly, we also found that incretin responsiveness is drastically induced by the formation of pseudoislets from MIN6‐K20 cells to a level comparable to that of pancreatic islets. Thus, these cell lines are useful for studying incretin/cAMP signaling in β‐cells. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2010.00026.x, 2010)

Pancreatic β-cell signaling: toward better understanding of diabetes and its treatment

Pancreatic β-cells play a central role in the maintenance glucose homeostasis by secreting insulin, a key hormone that regulates blood glucose levels. Dysfunction of the β-cells and/or a decrease in the β-cell mass are associated closely with the pathogenesis and pathophysiology of diabetes mellitus, a major metabolic disease that is rapidly increasing worldwide. Clarification of the mechanisms of insulin secretion and β-cell fate provides a basis for the understanding of diabetes and its better treatment. In this review, we discuss cell signaling critical for the insulin secretory function based on our recent studies.

Conditional Hypovascularization and Hypoxia in Islets Do Not Overtly Influence Adult β-Cell Mass or Function

It is generally accepted that vascularization and oxygenation of pancreatic islets are essential for the maintenance of an optimal β-cell mass and function and that signaling by vascular endothelial growth factor (VEGF) is crucial for pancreas development, insulin gene expression/secretion, and (compensatory) β-cell proliferation. A novel mouse model was designed to allow conditional production of human sFlt1 by β-cells in order to trap VEGF and study the effect of time-dependent inhibition of VEGF signaling on adult β-cell fate and metabolism. Secretion of sFlt1 by adult β-cells resulted in a rapid regression of blood vessels and hypoxia within the islets. Besides blunted insulin release, β-cells displayed a remarkable capacity for coping with these presumed unfavorable conditions: even after prolonged periods of blood vessel ablation, basal and stimulated blood glucose levels were only slightly increased, while β-cell proliferation and mass remained unaffected. Moreover, ablation of blood vessels did not prevent β-cell generation after severe pancreas injury by partial pancreatic duct ligation or partial pancreatectomy. Our data thus argue against a major role of blood vessels to preserve adult β-cell generation and function, restricting their importance to facilitating rapid and adequate insulin delivery.

Mature acinar cells are refractory to carcinoma development by targeted activation of Ras oncogene in adult rats

Pancreatic ductal adenocarcinoma (PDA) is one of the most debilitating malignancies in humans. A thorough understanding of the cytogenesis of this disease will aid in establishing successful treatments. We have developed an animal model which uses adult HrasG12V and KrasG12V transgenic rats in which oncogene expression is regulated by the Cre/loxP system and neoplastic lesions are induced by injection of adenovirus‐expressing Cre recombinase. When adenovirus with Cre recombinase under the control of the CMV enhancer/chicken β‐actin (CAG) promoter (Ad‐CAG‐Cre) is injected into the pancreatic duct of these animals, pancreatic neoplasias develop. Pathologically, the origin of these lesions is duct, intercalated duct, and centroacinar cells, but not acinar cells. The present study was undertaken to test the effect of acinar cell‐specific oncogenic ras expression. Adult transgenic rats were injected with adenovirus with Cre recombinase under the control of the acinar cell‐specific promoters amylase (Ad‐Amy‐Cre) and elastase‐1 (Ad‐Ela‐Cre) or under the control of the non‐specific CAG promoter. Injection of either Ad‐Amy‐Cre or Ad‐Ela‐Cre into the pancreatic ducts of transgenic animals in which oncogenic Kras is tagged with hemagglutinin (HA), HA‐KrasG12V rats resulted in expression of oncogenic ras in acinar cells but not in duct, intercalated duct, or centroacinar cells. Notably, injected animals did not develop any observable proliferative or neoplastic lesions. In marked contrast, injection of Ad‐CAG‐Cre resulted in pancreatic cancer development within 4 weeks. These results indicate that adult acinar cells are refractory to Ras oncogene activation and do not develop neoplasia in this model. (Cancer Sci2009)

Current status of regeneration of pancreatic β-cells.

Newly generated insulin‐secreting cells for use in cell therapy for insulin‐deficient diabetes mellitus require properties similar to those of native pancreatic β‐cells. Pancreatic β‐cells are highly specialized cells that produce a large amount of insulin, and secrete insulin in a regulated manner in response to glucose and other stimuli. It is not yet explained how the β‐cells acquire this complex function during normal differentiation. So far, in vitro generation of insulin‐secreting cells from embryonic stem cells, induced‐pluripotent stem cells and adult stem/progenitor‐like cells has been reported. However, most of these cells are functionally immature and show poor glucose‐responsive insulin secretion compared to that of native pancreatic β‐cells (or islets). Strategies to generate functional β‐cells or a whole organ in vivo have also recently been proposed. Establishing a protocol to generate fully functional insulin‐secreting cells that closely resemble native β‐cells is a critical matter in regenerative medicine for diabetes. Understanding the physiological processes of differentiation, proliferation and regeneration of pancreatic β‐cells might open the path to cell therapy to cure patients with absolute insulin deficiency.

In vitro generation of insulin-secreting cells from human pancreatic exocrine cells

Transplantation of surrogate β‐cells is a promising option for the treatment of insulin‐deficient diabetes mellitus in the future. Although pancreatic exocrine cells of rodents have been shown to transdifferentiate into insulin‐secreting cells, no studies are reported on human exocrine cells. Here, we report the generation of insulin‐secreting cells from exocrine cells of the human pancreas. When cultured in suspension with epidermal growth factor, human pancreatic exocrine cells readily formed spherical cell clusters. Expression of Pdx1 was induced in all 19 cases in which we successfully isolated exocrine cells, and insulin expression was induced in 11 cases. In addition, insulin secretion was evaluated in four cases, and the newly‐made cells were found to secrete insulin in response to various stimuli. Although further studies are required to improve both the quality and quantity of such insulin‐secreting cells, our data suggest that pancreatic exocrine cells represent a potential source of insulin‐secreting cells for treatment of type 1 diabetes. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2010.00095.x, 2011)