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Aims/hypothesis Endothelial cells are considered to be essential for normal pancreatic beta cell function. However, there have been no reports showing their importance for beta cell function. Materials and methods Using mice with disrupted vascular endothelial growth factor-A gene specifically in beta cells, we investigated the relation between islet vascular structure and beta cell function. Results Mice with disrupted vascular endothelial growth factor-A gene specifically in beta cells had reduced islet vascular density with impaired formation of endothelial fenestration. While their fasting glucose and body weight were comparable with control mice, their glucose-and tolbutamide-induced rapid insulin release were impaired, thus resulting in glucose intolerance. On the other hand, glucose and KCl enhanced the levels of insulin secreted from islets isolated from these mice. In addition, the production of soluble N-ethylmaleimide-sensitive factor attachment protein receptors in the islets was increased. Insulin content and expression of insulin I and pancreas duodenum homeobox 1 mRNA in the islets were also increased. Conclusions/interpretation Our results indicate that an abnormal quality and quantity of blood vessels due to reduced expression of vascular endothelial growth factor-A in beta cells could be a cause of impaired insulin secretion without impairment of beta cell function.

Protein kinase C (PKC) is considered to modulate glucosestimulated insulin secretion. Pancreatic ␤ cells express multiple isoforms of PKCs; however, the role of each isoform in glucosestimulated insulin secretion remains controversial. In this study we investigated the role of PKC␦, a major isoform expressed in pancreatic ␤ cells on ␤ cell function. Here, we showed that PKC␦ null mice manifested glucose intolerance with impaired insulin secretion. Insulin tolerance test showed no decrease in insulin sensitivity in PKC␦ null mice. Studies using islets isolated from these mice demonstrated decreased glucose-and KCl-stimulated insulin secretion. Perifusion studies indicated that mainly the second phase of insulin secretion was decreased. On the other hand, glucose-induced influx of Ca 2؉ into ␤ cells was not altered. Immunohistochemistry using total internal reflection fluorescence microscopy and electron microscopic analysis showed an increased number of insulin granules close to the plasma membrane in ␤ cells of PKC␦ null mice. Although PKC is thought to phosphorylate Munc18-1 and facilitate soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors complex formation, the phosphorylation of Munc18-1 by glucose stimulation was decreased in islets of PKC␦ null mice. We conclude that PKC␦ plays a non-redundant role in glucosestimulated insulin secretion. The impaired insulin secretion in PKC␦ null mice is associated with reduced phosphorylation of Munc18-1.Altered regulation of insulin secretion is a common feature of type 2 diabetes mellitus, although the underlying mechanism is not fully understood. The mechanism of glucose-stimulated insulin secretion involves closure of ATP-sensitive potassium channels by increased levels of glucose metabolites followed by depolarization of the plasma membrane and increased influx of Ca 2ϩ via voltage-dependent gating of Ca 2ϩ channels. The resultant elevation of intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) triggers transport of insulin into the plasma membrane and exocytosis (1). Although the events involved in initiating glucose-stimulated insulin secretion are well studied, those of regulated exocytosis are considerably less clear, although it is reported that several molecules expressed in ␤ cells play a crucial role in exocytosis (2).Various protein kinases are activated by downstream signals of glucose metabolism; however, their precise contribution to insulin secretion is not clear yet (3). Protein kinase C (PKC) 2 is one such protein kinase. Previous studies revealed that phorbol 12-myristate 13-acetate, which activates PKC, could trigger insulin secretion (4 -10). PKC is a serine/threonine kinase characterized by molecular structure and activation requirement. The isoforms consist of the conventional PKCs (␣, ␤, and ␥), which are sensitive to Ca 2ϩ and diacylglycerol (DAG), novel PKCs (␦, ⑀, , and ), which are sensitive to DAG only, and atypical PKCs (, ), which do not respond to either Ca 2ϩ or DAG (11,12). The ␤ cells express PKC␣ and -␦ as...

OBJECTIVE -To evaluate the effect of RANTES gene promoter polymorphism and RANTES receptor (CCR5 gene) promoter polymorphism on diabetic nephropathy in Japanese type 2 diabetic subjects. RESEARCH DESIGN AND METHODS -A total 616Japanese subjects with type 2 diabetes were recruited. Polymorphisms of Ϫ28 C/G and Ϫ403 G/A in the RANTES gene promoter region, and of 59029 G/A in the CCR5 gene promoter region were detected by PCR-RFLP (restriction fragment length polymorphism). The association of these genotypes with nephropathy was analyzed.RESULTS -While the RANTES -403 genotype showed no association with nephropathy, the frequency of the -28G allele was significantly higher in the DN2 group (urinary albuminuriato-creatinine ratio [ACR] м300 mg/g creatinine, serum creatinine Ͻ2.0 mg/dl) than in the DN0 (ACR Ͻ30 mg/g creatinine) and DN1 (ACR Ն30 mg/g creatinine and Ͻ300 mg/g creatinine) groups. The frequency of a RANTES -28G-positive genotype (C/G or G/G) was higher in the DN2 group than in the DN0 and DN1 groups (34% vs. 25 and 20%, P ϭ 0.0268, 2 ϭ 4.905), and the frequency of a CCR5 59029 A-positive genotype (G/A or A/A) was higher in the DN1 and DN2 groups than in the DN0 group (84 and 85% vs. 76%, P ϭ 0.0123, 2 ϭ 6.269). Discriminant analysis showed that the RANTES -28G-positive genotype and CCR5 59029A-positive genotype were independently associated with nephropathy. The percentage of macroalbuminuria was twofold higher in the subjects having Ϫ28G or 59029A and threefold higher in the subjects having Ϫ28G and 59029A than in the subjects without Ϫ28G and 59029A.CONCLUSIONS -The RANTES promoter -28G genotype and CCR5 promoter 59029A genotype may be independent risk factors for diabetic nephropathy in patients with type 2 diabetes and may have an additive effect on nephropathy. Diabetes Care 26:892-898, 2003D iabetic nephropathy is a serious complication in individuals with type 2 diabetes because of premature mortality due to coronary heart disease or chronic renal failure (1). It has previously been reported that monocyte/ macrophage infiltration was detected in the glomeruli of rats with streptozocininduced diabetes and in renal biopsy specimens from patients with diabetic nephropathy. These data suggest that chemokine signals are upregulated in diabetes and that monocyte recruitment to the kidneys and differentiation into macrophages may be associated with the development or progression of diabetic nephropathy (2-5). A hyperglycemic state is thought to increase the secretion of cytokines, such as tumor necrosis factor-␣ (TNF-␣) or interleukin-1␤ (IL-1␤), probably through activation of protein kinase C, oxidative stress, and formation of advanced glycation end products (6 -11). In turn, TNF-␣ and IL-1␤ stimulate the expression of a chemokine, known as regulated upon activation, normal T-cell expressed and secreted (RANTES), by human mesangial cells (12,13). Since the major receptor for RANTES expressed by monocyte/macrophages in renal tissue is chemotactic cytokine receptor 5 (CCR5), RANTES and CCR5-mediated sign...