Junji Fujikura

An angiotensin II AT1 receptor antagonist, telmisartan augments glucose uptake and GLUT4 protein expression in 3T3-L1 adipocytes.

Evidence has accumulated that some of the angiotensin II AT1 receptor antagonists have insulin‐sensitizing property. We thus examined the effect of telmisartan on insulin action using 3T3‐L1 adipocytes. With standard differentiation inducers, a higher dose of telmisartan effectively facilitated differentiation of 3T3‐L1 preadipocytes. Treatment of both differentiating adipocytes and fully differentiated adipocytes with telmisartan caused a dose‐dependent increase in mRNA levels for PPARγ target genes such as aP2 and adiponectin. By contrast, telmisartan attenuated 11β‐hydroxysteroid dehydrogenase type 1 mRNA level in differentiated adipocytes. Of note, we demonstrated for the first time that telmisartan augmented GLUT4 protein expression and 2‐deoxy glucose uptake both in basal and insulin‐stimulated state of adipocytes, which may contribute, at least partly, to its insulin‐sensitizing ability.

Adipose Tissue–Specific Regulation of Angiotensinogen in Obese Humans and Mice: Impact of Nutritional Status and Adipocyte Hypertrophy

BACKGROUND The adipose tissue renin-angiotensin system (RAS) has been implicated in the pathophysiology of obesity and dysfunction of adipose tissue. However, neither regulation of angiotensinogen (AGT) expression in adipose tissue nor secretion of adipose tissue-derived AGT has been fully elucidated in humans. METHODS Human subcutaneous abdominal adipose tissue (SAT) biopsies were performed for 46 subjects with a wide range of body mass index (BMI). Considering the mRNA level of AGT and indices of body fat mass, the amount of adipose tissue-derived AGT secretion (A-AGT-S) was estimated. Using a mouse model of obesity and weight reduction, plasma AGT levels were measured with a newly developed enzyme-linked immunosorbent assay (ELISA), and the contribution of A-AGT-S to plasma AGT levels was assessed. RESULTS A-AGT-S was substantially increased in obese humans and the value was correlated with the plasma AGT level in mice. A-AGT-S and plasma AGT were higher in obese mice, whereas lower in mice with weight reduction. However, the AGT mRNA levels in the liver, kidney, and aorta were not altered in the mouse models. In both humans and mice, the AGT mRNA levels in mature adipocytes (MAs) were comparable to those in stromal-vascular cells. Coulter Multisizer analyses revealed that AGT mRNA levels in the MAs were inversely correlated with the average size of mature adipocytes. CONCLUSIONS This study demonstrates that adipose tissue-derived AGT is substantially augmented in obese humans, which may contribute considerably to elevated levels of circulating AGT. Adipose tissue-specific regulation of AGT provides a novel insight into the clinical implications of adipose tissue RAS in human obesity.

Expression of the gene for a membrane-bound fatty acid receptor in the pancreas and islet cell tumours in humans : evidence for GPR40 expression in pancreatic beta cells and implications for insulin secretion

Aims/hypothesisG 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: GPR40mRNA expression in the human pancreas, pancreatic islets and islet cell tumours was analysed using TaqMan PCR.Results: GPR40mRNA 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/interpretationThe present study provides evidence for GPR40 gene expression in pancreatic beta cells and implicates GPR40 in insulin secretion in humans.

Genetic and Pharmacological Inhibition of Rho-associated Kinase II Enhances Adipogenesis

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. Furthermore, Y-27632 restored inhibition of adipogenesis by lysophosphatidic acid, which activates Rho. Regarding actin cytoskeleton, Y-27632 disrupted stress fibers in 3T3-L1 preadipocytes. Next, we analyzed adipogenesis of mouse embryonic fibroblasts (MEFs) derived from ROCK-I and ROCK-II knock-out mice, respectively. Adipogenesis of ROCK-II (-/-) MEFs was markedly enhanced compared with wild-type MEFs while that of ROCK-I (-/-) MEFs was not. In contrast to pharmacological approaches, no obvious alteration was found in actin cytoskeleton of ROCK-II (-/-) MEFs compared with wild-type MEFs. In 3T3-L1 cells, knockdown of ROCK-II by RNA interference enhanced the expression of adipogenic transcription factors while that of ROCK-I did not. Moreover, Y-27632 inhibited IRS-1 serine phosphorylation and enhanced Akt phosphorylation in 3T3-L1 preadipocytes. Similarly, Akt phosphorylation in ROCK-II (-/-) MEFs was augmented compared with wild-type MEFs. In conclusion, inhibition of ROCK-II, not ROCK-I, enhances adipogenesis accompanied by the up-regulation of adipogenic transcription factors. Augmentation of insulin signaling may contribute to the enhancement of adipogenesis.

Induced pluripotent stem cells generated from diabetic patients with mitochondrial DNA A3243G mutation

Aims/hypothesisThe aim of this study was to generate induced pluripotent stem (iPS) cells from patients with mitochondrial DNA (mtDNA) mutation.MethodsSkin 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.ResultsFrom 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/interpretationiPS 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, disease-free supply of cells for autologous transplantation therapy.

Therapeutic Impact of Leptin on Diabetes, Diabetic Complications, and Longevity in Insulin-Deficient Diabetic Mice

OBJECTIVE The aim of the current study was to evaluate the long-term effects of leptin on glucose metabolism, diabetes complications, and life span in an insulin-dependent diabetes model, the Akita mouse. RESEARCH DESIGN AND METHODS We cross-mated Akita mice with leptin-expressing transgenic (LepTg) mice to produce Akita mice with physiological hyperleptinemia (LepTg:Akita). Metabolic parameters were monitored for 10 months. Pair-fed studies and glucose and insulin tolerance tests were performed. The pancreata and kidneys were analyzed histologically. The plasma levels and pancreatic contents of insulin and glucagon, the plasma levels of lipids and a marker of oxidative stress, and urinary albumin excretion were measured. Survival rates were calculated. RESULTS Akita mice began to exhibit severe hyperglycemia and hyperphagia as early as weaning. LepTg:Akita mice exhibited normoglycemia after an extended fast even at 10 months of age. The 6-h fasting blood glucose levels in LepTg:Akita mice remained about half the level of Akita mice throughout the study. Food intake in LepTg:Akita mice was suppressed to a level comparable to that in WT mice, but pair feeding did not affect blood glucose levels in Akita mice. LepTg:Akita mice maintained insulin hypersensitivity and displayed better glucose tolerance than did Akita mice throughout the follow-up. LepTg:Akita mice had normal levels of plasma glucagon, a marker of oxidative stress, and urinary albumin excretion rates. All of the LepTg:Akita mice survived for >12 months, the median mortality time of Akita mice. CONCLUSIONS These results indicate that leptin is therapeutically useful in the long-term treatment of insulin-deficient diabetes.

Augmentation of 11β-hydroxysteroid dehydrogenase type 1 in LPS-activated J774.1 macrophages – Role of 11β-HSD1 in pro-inflammatory properties in macrophages

Macrophage infiltration in obese adipose tissue provokes local inflammation and insulin resistance. Evidence has accumulated that activation of 11β‐HSD1 in adipocytes is critically involved in dysfunction of adipose tissue. However, the potential role of 11β‐HSD1 in macrophages still remains unclear. We here demonstrate that a murine macrophage cell line, J774.1 cells expressed 11β‐HSD1 mRNA and reductase activity, both of which were augmented by lipopolysaccharide (LPS)‐induced cell activation. Three kinds of pharmacological inhibition of 11β‐HSD1 in LPS‐treated macrophages significantly suppressed the expression and secretion of interleukin 1β, tumor necrosis factor α or monocyte chemoattractant protein 1, thereby highlighting a novel role of 11β‐HSD1 in pro‐inflammatory properties of activated macrophages.

Leptin Activates Hepatic 5′-AMP-activated Protein Kinase through Sympathetic Nervous System and α1-Adrenergic Receptor A POTENTIAL MECHANISM FOR IMPROVEMENT OF FATTY LIVER IN LIPODYSTROPHY BY LEPTIN

Background: AMPK activation promotes glucose and lipid metabolism. Results: Hepatic AMPK activities were decreased in fatty liver from lipodystrophic mice, and leptin activated the hepatic AMPK via the α-adrenergic effect. Conclusion: Leptin improved the fatty liver possibly by activating hepatic AMPK through the central and sympathetic nervous systems. Significance: Hepatic AMPK plays significant roles in the pathophysiology of lipodystrophy and metabolic action of leptin. Leptin is an adipocyte-derived hormone that regulates energy homeostasis. Leptin treatment strikingly ameliorates metabolic disorders of lipodystrophy, which exhibits ectopic fat accumulation and severe insulin-resistant diabetes due to a paucity of adipose tissue. Although leptin is shown to activate 5′-AMP-activated protein kinase (AMPK) in the skeletal muscle, the effect of leptin in the liver is still unclear. We investigated the effect of leptin on hepatic AMPK and its pathophysiological relevance in A-ZIP/F-1 mice, a model of generalized lipodystrophy. Here, we demonstrated that leptin activates hepatic AMPK through the central nervous system and α-adrenergic sympathetic nerves. AMPK activities were decreased in the fatty liver of A-ZIP/F-1 mice, and leptin administration increased AMPK activities in the liver as well as in skeletal muscle with significant reduction in triglyceride content. Activation of hepatic AMPK with A769662 also led to a decrease in hepatic triglyceride content and blood glucose levels in A-ZIP/F-1 mice. These results indicate that the down-regulation of hepatic AMPK activities plays a pathophysiological role in the metabolic disturbances of lipodystrophy, and the hepatic AMPK activation is involved in the therapeutic effects of leptin.

Glucocorticoid reamplification within cells intensifies NF-κB and MAPK signaling and reinforces inflammation in activated preadipocytes

Increased expression and activity of the intracellular glucocorticoid-reactivating enzyme 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) contribute to dysfunction of adipose tissue. Although the pathophysiological role of 11 beta-HSD1 in mature adipocytes has long been investigated, its potential role in preadipocytes still remains obscure. The present study demonstrates that the expression of 11 beta-HSD1 in preadipocyte-rich stromal vascular fraction (SVF) cells in fat depots from ob/ob and diet-induced obese mice was markedly elevated compared with lean control. In 3T3-L1 preadipocytes, the level of mRNA and reductase activity of 11 beta-HSD1 was augmented by TNF-alpha, IL-1 beta, and LPS, with a concomitant increase in inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), or IL-6 secretion. Pharmacological inhibition of 11 beta-HSD1 and RNA interference against 11 beta-HSD1 reduced the mRNA and protein levels of iNOS, MCP-1, and IL-6. In contrast, overexpression of 11 beta-HSD1 further augmented TNF-alpha-induced iNOS, IL-6, and MCP-1 expression. Moreover, 11 beta-HSD1 inhibitors attenuated TNF-alpha-induced phosphorylation of NF-kappaB p65 and p38-, JNK-, and ERK1/2-MAPK. Collectively, the present study provides novel evidence that inflammatory stimuli-induced 11 beta-HSD1 in activated preadipocytes intensifies NF-kappaB and MAPK signaling pathways and results in further induction of proinflammatory molecules. Not limited to 3T3-L1 preadipocytes, we also demonstrated that the notion was reproducible in the primary SVF cells from obese mice. These findings highlight an unexpected, proinflammatory role of reamplified glucocorticoids within preadipocytes in obese adipose tissue.

GPR119 expression in normal human tissues and islet cell tumors: evidence for its islet-gastrointestinal distribution, expression in pancreatic beta and alpha cells, and involvement in islet function

OBJECTIVE GPR119 is reportedly involved in regulating glucose metabolism and food intake in rodents, but little is known about its expression and functional significance in humans. To begin to assess the potential clinical importance of GPR119, the distribution of GPR119 gene expression in humans was examined. MATERIALS/METHODS Expression of GPR119 mRNA in fresh samples of normal human pancreas (n=19) and pancreatic islets (n=3) and in insulinomas (n=2) and glucagonomas (n=2), all collected at surgery, was compared with the mRNA expression of various receptors highly expressed and operative in human pancreatic islets. RESULTS GPR119 mRNA was most abundant in the pancreas, followed by the duodenum, stomach, jejunum, ileum and colon. Pancreatic levels of GPR119 mRNA were similar to those of GPR40 mRNA and were higher than those of GLP1R and SUR1 mRNA, which are strongly expressed in human pancreatic islets. Moreover, levels of GPR119 mRNA in pancreatic islets were more than 10 times higher than in adjacent pancreatic tissue, as were levels of GPR40 mRNA. GPR119 mRNA was also abundant in two cases of insulinoma and two cases of glucagonoma, but was undetectable in a pancreatic acinar cell tumor. Similar results were obtained with mouse pancreatic islets, MIN6 insulinoma cells and alpha-TC glucagonoma cells. CONCLUSIONS The results provide evidence of an islet-gastrointestinal distribution of GPR119, its expression in pancreatic beta and alpha cells, and its possible involvement in islet function. They also provide the basis for a better understanding of the potential clinical importance of GPR119.