Rho-associated kinase (ROCK) regulates reorganization of actin cytoskeleton. During adipogenesis, the structure of filamentous actin is converted from long stress fibers to cortical actin, suggesting that the ROCK is involved in adipogenesis. Two ROCK isoforms have been identified: ROCK-I and ROCK-II. However, pharmacological inhibitors of ROCK cannot distinguish two ROCK isoforms. In the present study, we examined the role of ROCK in adipogenesis and actin cytoskeleton using genetic and pharmacological approaches. Y-27632, which inhibits the activity of both ROCK isoforms, enhanced adipogenesis through the up-regulation of adipogenic transcription factors in 3T3-L1 cells.

To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas.

a b s t r a c tInduced pluripotent stem (iPS) cells were recently established from human fibroblasts. In the present study we investigated the adipogenic differentiation properties of four human iPS cell lines and compared them with those of two human embryonic stem (ES) cell lines. After 12 days of embryoid body formation and an additional 10 days of differentiation on Poly-L-ornithine and fibronectincoated dishes with adipogenic differentiation medium, human iPS cells exhibited lipid accumulation and transcription of adipogenesis-related molecules such as C/EBPa, PPARc2, leptin and aP2. These results demonstrate that human iPS cells have an adipogenic potential comparable to human ES cells.

Aims/hypothesis G protein-coupled receptor 40 (GPR40) is abundantly expressed in pancreatic beta cells in rodents, where it facilitates glucose-induced insulin secretion in response to mid-to long-chain fatty acids in vitro. However, GPR40 gene expression in humans has not been fully investigated, and little is known about the physiological and pathophysiological roles of GPR40 in humans. The aim of this study, therefore, was to examine GPR40 expression and its clinical implications in humans. Methods: GPR40 mRNA expression in the human pancreas, pancreatic islets and islet cell tumours was analysed using TaqMan PCR. Results: GPR40 mRNA was detected in all human pancreases collected intraoperatively. It was enriched approximately 20-fold in isolated islets freshly prepared from the pancreases of the same individuals. The estimated mRNA copy number for the GPR40 gene in pancreatic islets was comparable to those for genes encoding sulfonylurea receptor 1, glucagon-like peptide 1 receptor and somatostatin receptors, all of which are known to be expressed abundantly in the human pancreatic islet. A large amount of GPR40 mRNA was detected in insulinoma tissues, whereas mRNA expression was undetectable in glucagonoma or gastrinoma. The GPR40 mRNA level in the pancreas correlated with the insulinogenic index, which reflects beta cell function (r=0.82, p=0.044), but not with glucose levels during the OGTT, the insulin area under the OGTT curve or the index for the homeostasis model assessment of insulin resistance (HOMA-IR). Conclusions/interpretation The present study provides evidence for GPR40 gene expression in pancreatic beta cells and implicates GPR40 in insulin secretion in humans.

Aims/hypothesis The aim of this study was to generate induced pluripotent stem (iPS) cells from patients with mitochondrial DNA (mtDNA) mutation. Methods Skin biopsies were obtained from two diabetic patients with mtDNA A3243G mutation. The fibroblasts thus obtained were infected with retroviruses encoding OCT4 (also known as POU5F1), SOX2, c-MYC (also known as MYC) and KLF4. The stem cell characteristics were investigated and the mtDNA mutation frequencies evaluated by Invader assay.Results From the two diabetic patients we isolated four and ten putative mitochondrial disease-specific iPS (Mt-iPS) clones, respectively. Mt-iPS cells were cytogenetically normal and positive for alkaline phosphatase activity, with the pluripotent stem cell markers being detectable by immunocytochemistry. The cytosine guanine dinucleotide islands in the promoter regions of OCT4 and NANOG were highly unmethylated, indicating epigenetic reprogramming to pluripotency. Mt-iPS clones were able to differentiate into derivatives of all three germ layers in vitro and in vivo. The Mt-iPS cells exhibited a bimodal degree of mutation heteroplasmy. The mutation frequencies decreased to an undetectable level in six of 14 clones, while the others showed several-fold increases in mutation frequencies (51-87%) compared with those in the original fibroblasts (18-24%). During serial cell culture passage and after differentiation, no recurrence of the mutation or no significant changes in the levels of heteroplasmy were seen. Conclusions/interpretation iPS cells were successfully generated from patients with the mtDNA A3243G mutation. Mutation-rich, stable Mt-iPS cells may be a suitable source of cells for human mitochondrial disease modelling in vitro. Mutation-free iPS cells could provide an unlimited, diseasefree supply of cells for autologous transplantation therapy.

We developed and analyzed two types of transgenic mice: rat insulin II promoter-ghrelin transgenic (RIP-G Tg) and rat glucagon promoter-ghrelin transgenic mice (RGP-G Tg). The pancreatic tissue ghrelin concentration measured by C-terminal radioimmunoassay (RIA) and plasma desacyl ghrelin concentration of RIP-G Tg were about 1000 and 3.4 times higher than those of nontransgenic littermates, respectively. The pancreatic tissue n-octanoylated ghrelin concentration measured by N-terminal RIA and plasma n-octanoylated ghrelin concentration of RIP-G Tg were not distinguishable from those of nontransgenic littermates. RIP-G Tg showed suppression of glucose-stimulated insulin secretion. Arginine-stimulated insulin secretion, pancreatic insulin mRNA and peptide levels, ␤ cell mass, islet architecture, and GLUT2 and PDX-1 immunoreactivity in RIP-G Tg pancreas were not significantly different from those of nontransgenic littermates. Islet batch incubation study did not show suppression of insulin secretion of RIP-G Tg in vitro. The insulin tolerance test showed lower tendency of blood glucose levels in RIP-G Tg. Taking lower tendency of triglyceride level of RIP-G Tg into consideration, these results may indicate that the suppression of insulin secretion is likely due to the effect of desacyl ghrelin on insulin sensitivity. RGP-G Tg, in which the pancreatic tissue ghrelin concentration measured by C-RIA was about 50 times higher than that of nontransgenic littermates, showed no significant changes in insulin secretion, glucose metabolism, islet mass, and islet architecture. The present study raises the possibility that desacyl ghrelin may have influence on glucose metabolism.Ghrelin is a 28-amino acid peptide with unique modification of acylation, which is essential for its biological action (1). Ghrelin was originally identified in rat stomach as an endogenous ligand for an orphan receptor, which has been so far called growth hormone secretagogue receptor (GHS-R) 1 (1). Ghrelin expression is detected in the stomach, intestine, hypothalamus, pituitary gland, kidney, placenta, and testis (2-6). Ghrelin is involved in a wide variety of the functions, including the regulation of growth hormone release, food intake, gastric acid secretion, gastric motility, blood pressure, and cardiac output (7-19).Recently Date et al. (20) reported that ghrelin is present in ␣ cells of normal human and rat pancreatic islets. Volante et al. (20,31,32). Salehi et al. have reported ghrelin has both inhibitory and stimulatory effects depending on its concentration (33). Therefore, there is still a lot of controversy about the localization of ghrelin in the pancreas and the effects of ghrelin on the insulin secretion. As for the effects of desacyl ghrelin on insulin secretion, Broglio et al. (34) have reported that acute desacyl ghrelin administration has no effect on insulin secretion in human but that it counteracts the inhibitory effect of n-octanoylated ghrelin on insulin secretion when co-administrated with n-octanoylated ghrelin (35).Here...

Abstract. An 82-year-old woman with type 2 diabetes had been treated with recombinant human insulin for 16 years. She developed large swellings in both sides of her lower abdomen. The masses were soft, painless, and located around her insulin injection sites. Based on the history and clinical features, a diagnosis of insulin-induced lipohypertrophy was made. Total resection revealed that the lesions were composed entirely of fatty tissue. Microscopic examination showed nests of mature adipocytes expanding toward the dermal reticular layer. The hypertrophic adipocytes were twice as large as those from normal subcutaneous areas and contained numerous small lipid droplets. Electron microscopic analysis also revealed a minor population of small adipocytes, suggesting active differentiation or proliferation. Thus, the possible in vivo effects of insulin on adipocytes were clearly observed in this case of insulin-induced lipohypertrophy. To our knowledge, this is the first report of insulin-induced lipohypertrophy with detailed histological examinations.

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