海老原 健

Leptin enhances insulin sensitivity in addition to reducing food intake and body weight. Recently, amylin, a pancreatic β-cell-derived hormone, was shown to restore a weight-reducing effect of leptin in leptin-resistant diet-induced obesity. However, whether amylin improves the effect of leptin on insulin sensitivity in diet-induced obesity is unclear. Diet-induced obese (DIO) mice were infused with either saline (S), leptin (L; 500 μg·kg⁻¹·day⁻¹), amylin (A; 100 μg·kg⁻¹·day⁻¹), or leptin plus amylin (L/A) for 14 days using osmotic minipumps. Food intake, body weight, metabolic parameters, tissue triglyceride content, and AMP-activated protein kinase (AMPK) activity were examined. Pair-feeding and weight-matched calorie restriction experiments were performed to assess the influence of food intake and body weight reduction. Continuous L/A coadministration significantly reduced food intake, increased energy expenditure, and reduced body weight, whereas administration of L or A alone had no effects. L/A coadministration did not affect blood glucose levels during ad libitum feeding but decreased plasma insulin levels significantly (by 48%), suggesting the enhancement of insulin sensitivity. Insulin tolerance test actually showed the increased effect of insulin in L/A-treated mice. In addition, L/A coadministration significantly decreased tissue triglyceride content and increased AMPKα2 activity in skeletal muscle (by 67%). L/A coadministration enhanced insulin sensitivity more than pair-feeding and weight-matched calorie restriction. In conclusion, this study demonstrates the beneficial effect of L/A coadministration on glucose and lipid metabolism in DIO mice, indicating the possible clinical usefulness of L/A coadministration as a new antidiabetic treatment in obesity-associated diabetes.

We herein report a case of premature atherosclerosis in a patient with familial partial lipodystrophy (FPL), diabetes mellitus, hypertension and hypertriglyceridemia. Sequencing of the candidate genes LMNA, PPARG and CAV1 associated with FPL revealed no genetic abnormalities, which indicated the activity of a novel gene in this patient. The patient's son showed milder fat loss and similar fat distribution compared to the proband; however, the son showed no signs of any atherosclerotic disease. Although a cluster of atherogenic risk factors is likely to be the primary causes of atherosclerosis in our patient, other factors, including an unknown gene associated with FPL, the severity of fat loss and gender, might affect the development of atherosclerosis.

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.

Recent epidemiological studies indicate that obesity increases the incidence of depression. We examined the implication of leptin for obesity-associated depression. Leptin induced antidepressive behavior in normal mice in a forced swimming test (FST), and leptin-overexpressing transgenic mice with hyperleptinemia exhibited more antidepressive behavior in the FST than nontransgenic mice. In contrast, leptin-deficient ob/ob mice showed more severe depressive behavior in the FST than normal mice, and leptin administration substantially ameliorated this depressive behavior. Diet-induced obese (DIO) mice fed a high-fat diet showed more depressive behavior in the FST and in a sucrose preference test compared with mice fed a control diet (CD). In DIO mice, leptin induced neither antidepressive action nor increment of the number of c-Fos immunoreactive cells in the hippocampus. Diet substitution from high-fat diet to CD in DIO mice ameliorated the depressive behavior and restored leptin-induced antidepressive action. Brain-derived neurotrophic factor concentrations in the hippocampus were significantly lower in DIO mice than in CD mice. Leptin administration significantly increased hippocampal brain-derived neurotrophic factor concentrations in CD mice but not in DIO mice. The antidepressant activity of leptin in CD mice was significantly attenuated by treatment with K252a. These findings demonstrated that leptin induces an antidepressive state, and DIO mice, which exhibit severe depressive behavior, did not respond to leptin in both the FST and the biochemical changes in the hippocampus. Thus, depression associated with obesity is due, at least in part, to impaired leptin activity in the hippocampus.

Adipose tissue expresses all components of the renin-angiotensin system including angiotensinogen (AGT). Recent studies have highlighted a potential role of AGT in adipose tissue function and homeostasis. However, some controversies surround the regulatory mechanisms of AGT in obese adipose tissue. In this context, we here demonstrated that the AGT messenger RNA (mRNA) level in human subcutaneous adipose tissue was significantly reduced in obese subjects as compared with nonobese subjects. Adipose tissue AGT mRNA level in obese mice was also lower as compared with their lean littermates; however, the hepatic AGT mRNA level remained unchanged. When 3T3-L1 adipocytes were cultured for a long period, the adipocytes became hypertrophic with a marked increase in the production of reactive oxygen species. Expression and secretion of AGT continued to decrease during the course of adipocyte hypertrophy. Treatment of the 3T3-L1 and primary adipocytes with reactive oxygen species (hydrogen peroxide) or tumor necrosis factor alpha caused a significant decrease in the expression and secretion of AGT. On the other hand, treatment with the antioxidant N-acetyl cysteine suppressed the decrease in the expression and secretion of AGT in the hypertrophied 3T3-L1 adipocytes. Finally, treatment of obese db/db mice with N-acetyl cysteine augmented the expression of AGT in the adipose tissue, but not in the liver. The present study demonstrates for the first time that oxidative stress dysregulates AGT in obese adipose tissue, providing a novel insight into the adipose tissue-specific interaction between the regulation of AGT and oxidative stress in the pathophysiology of obesity.

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.

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.

This report describes a 46-year-old Japanese diabetic woman with an unusual type of familial partial lipodystrophy. She has marked loss of subcutaneous fat in her lower limbs and buttocks, with sparing of the face, neck, upper limbs, and trunk. This distribution of fat atrophy appears to be rare in comparison with previous reports. Sequencing of candidate genes LMNA, PPARG, AKT2, caveolin-1, as well as the PPARG4 promoter gene, which are known to be associated with familial partial lipodystrophy, revealed no genetic abnormalities, suggesting that this case may involve a novel gene. Pioglitazone was markedly effective in glycemic control in this case. Her diabetes remained uncontrolled despite a total daily dose of insulin of 30 U and combined treatment with 10 mg of glibenclamide and 0.6 mg of voglibose. We therefore attempted combined treatment with 30 mg of pioglitazone and 30 U/d insulin injection. The hemoglobin A(1c) level was reduced from 11.2% to 6.1% after 6 months of treatment and has since remained stable. Her body weight increased from 62.0 to 71.0 kg after 12 months of treatment, suggesting that weight gain may result from synergism between thiazolidinediones and insulin-promoting adipogenesis. Pioglitazone increased the fat mass in the upper limbs and trunk, while inducing less increase in the lower limbs, where fat atrophy exists in this patient. Pioglitazone may thus have improved the glycemic control in this case through adipocyte differentiation from progenitor cells mainly in the upper limbs and trunk.

Since the 1980s, a number of bioactive molecules, now known as cardiovascular hormones, have been isolated from the heart and blood vessels, particularly from the subset of vascular endothelial cells. The natriuretic peptide family is the prototype of the cardiovascular hormones. Over the following decade, a variety of hormones and cytokines, now known as adipokines or adipocytokines, have also been isolated from adipose tissue. Leptin is the only adipokine demonstrated to cause an obese phenotype in both animals and humans upon deletion. Thus, the past two decades have seen the identification of two important classes of bioactive molecules secreted by newly recognized endocrine cells, both of which differentiate from mesenchymal stem cells. To assess the physiological and clinical implications of these novel hormones, we have investigated their functions using animal models. We have also developed and analyzed mice overexpressing transgenic forms of these proteins and knockout mice deficient in these and related genes. Here, we demonstrate the current state of the translational research of these novel hormones, the natriuretic peptide family and leptin, and discuss how lessons learned from excellent animal models and rare human diseases can provide a better understanding of common human diseases.

The adipocyte-derived hormone, leptin controls feeding behavior, augments fatty acid beta-oxidation in the skeletal muscle, attenuates insulin secretion but enhances whole body insulin sensitivity and glucose disposal, thereby serving as a promising therapeutic candidate for the treatment of insulin resistance and dyslipidemia. Along with other researchers, we demonstrated the clinical efficacy and safety of leptin in the treatment of diabetes and dyslipidemia for patients with generalized lipodystrophy. However, the clinical application of leptin has been hampered by the notion that leptin does not fully exert its metabolic effects in human obesity and diet-induced obese rodents. We found that the activity of skeletal muscle AMP-activated protein kinase (AMPK) parallels hypothalamic leptin sensitivity and metabolic phenotype in transgenic mice overexpressing leptin. Our data indicate that the activation of skeletal muscle AMPK is mediated by the hypothalamic melanocortin pathway. In fact, intracerebroventricular administration of melanocortin agonist, MT-II in mice robustly overcomes high-fat diet-induced leptin resistance and ameliorates fuel dyshomeostasis and hyperphagia, with a concomitant recovery of AMPK activity in skeletal muscle. Conversely, AMPK/ACC phosphorylation by leptin was abrogated by the co-administration of melanocortin antagonist, SHU9119 and in the KKA(y) mice, which centrally express endogenous melanocortin antagonist. Importantly, high-fat diet-induced attenuation of AMPK/ACC phosphorylation in leptin-overexpressing transgenic mice was not reversed by central leptin per se, but was markedly recovered by MT-II. Our data provide evidence for the critical role of the central melanocortin system in leptin-skeletal muscle AMPK axis, and highlight the system as a therapeutic target for leptin insuffciency in obese humans.

OBJECTIVE: Dysregulation of tissue-specific intracellular glucocorticoid reactivation is implicated in obesity and related metabolic diseases in humans. The ratio of end products of glucocorticoid metabolism in fresh urine sample, tetrahydrocortisol (THF) + allo-tetrahydrocortisol (allo-THF) vs. tetrahydrocortisone (THE), i.e., the urinary ratio is regarded as an index of the systemic balance underlying intracellular glucocorticoid metabolism, where the enzymes, 11β-hydroxysteroid dehydrogenase type 1 and type 2 as well as 5α- and 5β-reductase are involved in a tissue-specific manner. METHODS: To explore the clinical implications of the urinary ratio in obesity and related metabolic diseases, the urinary ratio was determined by gas chromatography and mass spectrometry. RESULTS: The urinary ratio was shown to be constant and reproducible in the same individuals. The ratio was found to inversely correlate with BMI (P < 0.01), waist circumference (P < 0.01), and liver transaminase (P < 0.05) in a large cohort of ∼200 Japanese subjects. This finding suggests that the systemic balance underlying intracellular glucocorticoid reactivation was suppressed in obesity and liver dysfunction. Consistent with this notion, the ratio was decreased in patients with non-alcoholic steatohepatitis (P < 0.01). The urinary ratio was not altered in patients with type 2 diabetes on a 2-month mild calorie restriction. In contrast, the ratio was significantly reduced in patients who responded to the anti-diabetic pioglitazone (P < 0.01). CONCLUSION: The present study provides novel evidence that the urinary ratio reflects the facet of adipose tissue and liver function in humans, thereby offering a unique opportunity to evaluate obesity-related diseases.

Induced 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 fibronectin- coated dishes with adipogenic differentiation medium, human iPS cells exhibited lipid accumulation and transcription of adipogenesis-related molecules such as C/EBPalpha, PPARgamma2, leptin and aP2. These results demonstrate that human iPS cells have an adipogenic potential comparable to human ES cells.

Urinary neutrophil gelatinase-associated lipocalin (Ngal or lipocalin 2) is a very early and sensitive biomarker of kidney injury. Here we determined the origin and time course of Ngal appearance in several experimental and clinically relevant renal diseases. Urinary Ngal levels were found to be markedly increased in lipoatrophic- and streptozotocin-induced mouse models of diabetic nephropathy. In the latter mice, the angiotensin receptor blocker candesartan dramatically decreased urinary Ngal excretion. The reabsorption of Ngal by the proximal tubule was severely reduced in streptozotocin-induced diabetic mice, but upregulation of its mRNA and protein in the kidney was negligible, compared to those of control mice, suggesting that increased urinary Ngal was mainly due to impaired renal reabsorption. In the mouse model of unilateral ureteral obstruction, Ngal protein synthesis was dramatically increased in the dilated thick ascending limb of Henle and N was found in the urine present in the swollen pelvis of the ligated kidney. Five patients with nephrotic syndrome or interstitial nephritis had markedly elevated urinary Ngal levels at presentation, but these decreased in response to treatment. Our study shows that the urinary Ngal level may be useful for monitoring the status and treatment of diverse renal diseases reflecting defects in glomerular filtration barrier, proximal tubule reabsorption, and distal nephrons.

A variety of metabolic/molecular changes in obese adipose tissue considerably contribute to the pathophysiology of life style-related diseases. Fat cell-derived hormone leptin controls appetite and energy homeostasis, thereby enhancing whole body insulin sensitivity. However, clinical application of leptin for the treatment of obesity/metabolic syndrome has been hampered by the fact that leptin does not fully exert its beneficial metabolic impact on prevalent forms of obesity. In an attempt to elucidate underlying mechanism of leptin resistance in obesity, we found that the activity of skeletal muscle AMP-activated protein kinase (AMPK) tightly parallels hypothalamic leptin sensitivity and metabolic phenotype in transgenic mice overexpressing leptin. Actually, intracerebroventricular administration of melanocortin agonist MT-II robustly overcomes high fat diet-induced leptin resistance and ameliorates fuel dyshomeostasis and hyperphagia in mice, with a concomitant recovery of AMPK activity in skeletal muscle, thereby highlighting the system as a therapeutic target for leptin resistance. In this context, type 4 melanocortin receptor is a promising drug target for the treatment of obesity/metabolic syndrome.

Orexins and melanin-concentrating hormone (MCH) as orexigenic neuropeptides are present in the lateral hypothalamus, and their receptors are distributed in the cerebral cortex and hippocampus. In the present study, the regulatory effects of orexin-A, orexin-B and MCH on neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) expressions were examined in primary cortical neuron cultures using quantitative real-time PCR. Both orexin-A and orexin-B on 6-day exposure significantly increased the NT-3 mRNA at concentrations of 0.01, 0.1 and 1microM. Orexin-A and B at 1microM led to an increase of twofold or more over the control. However, no such NT-s mRNA increase occurred with exposure to MCH at the same concentrations as orexins. The mRNA expression of BDNF was significantly increased only by orexin-B at 1microM. These findings suggest that orexins, but not MCH, may be an inducer of NT-3 in the cerebral cortex.

Orexin is one of the orexigenic neuropeptides in the hypothalamus. Orexin neurons in the lateral hypothalamus (LH) project into the cerebral cortex and hippocampus in which the receptors are distributed in high concentrations. Therefore, to elucidate the actions of orexin in the cerebral cortex, we examined its effects on the mRNA expressions of N-methyl-d-aspartate (NMDA) receptor subunits (NR1, NR2A, NR2B) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits (GluR1, GluR2) following 6-day application of orexin-A or orexin-B to rat primary cortical neuron cultures. The mRNAs of NR1 and NR2A subunits were significantly decreased by orexin-A and orexin-B at concentrations over 0.1 microM and 0.01 microM, respectively. The mRNA expression of NR2B subunit was also significantly decreased by orexin-A and orexin-B only at the concentration of 1 microM. Moreover, orexin-A and orexin-B at concentrations over 0.01 microM significantly decreased the mRNA expressions of AMPA receptor subunits, GluR1 and GluR2. The present study demonstrated that orexins significantly suppressed RNA expressions of NMDA and AMPA receptor subunits in cortical neuron cultures, suggesting that orexin may regulate the higher functions of the cerebral cortex as well as be involved in energy regulation in the hypothalamus.

A single bout of exercise increases the rate of muscle glucose transport (GT) by both insulin-independent and insulin-dependent mechanisms. The purpose of this study was to determine whether high-fat diet (HFD) feeding interferes with the metabolic activation induced by moderate-intensity endurance exercise. Rats were fed an HFD or control diet (CD) for 4 weeks and then exercised on a treadmill for 1 hour (19 m/min, 15% incline). Insulin-independent GT was markedly higher in soleus muscle dissected immediately after exercise than in muscle dissected from sedentary rats in both dietary groups, but insulin-independent GT was 25% lower in HFD-fed than in CD-fed rats. Insulin-dependent GT in the presence of submaximally effective concentration of insulin (0.9 nmol/L) was also higher in both dietary groups in muscle dissected 2 hours after exercise, but was 25% lower in HFD-fed than in CD-fed rats. Exercise-induced activation of 5'adenosine monophosphate-activated protein kinase, a signaling intermediary leading to insulin-independent GT and regulating insulin sensitivity, was correspondingly blunted in the HFD group. High-fat diet did not affect glucose transporter 4 content or insulin-stimulated Akt phosphorylation. Our findings provide evidence that an HFD impairs the effects of short-term endurance exercise on glucose metabolism and that exercise does not fully compensate for HFD-induced insulin resistance in skeletal muscle. Although the underlying mechanism is unclear, reduced 5'adenosine monophosphate-activated protein kinase activation during exercise may play a role.

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.

Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development.

Little is known about the role of the central melanocortin system in the control of fuel metabolism in peripheral tissues. Skeletal muscle AMP-activated protein kinase (AMPK) is activated by leptin and serves as a master regulator of fatty acid beta-oxidation. To elucidate an unidentified role of the central melanocortin system in muscle AMPK regulation, we treated conscious, unrestrained mice intracerebroventricularly with the melanocortin agonist MT-II or the antagonist SHU9119. MT-II augmented phosphorylation of AMPK and its target acetyl-CoA carboxylase (ACC) independent of caloric intake. Conversely, AMPK/ACC phosphorylation by leptin was abrogated by the coadministration of SHU9119 or in KKA(y) mice, which centrally express endogenous melanocortin antagonist. Importantly, high-fat-diet-induced attenuation of AMPK/ACC phosphorylation in leptin-overexpressing transgenic mice was not reversed by central leptin but was markedly restored by MT-II. Our data provide evidence for the critical role of the central melanocortin system in the leptin-skeletal muscle AMPK axis and highlight the system as a therapeutic target in leptin resistance.

A clinically employed antihyperlipidemic drug, bezafibrate, has been characterized as a PPAR(alpha, -gamma, and -delta) pan-agonist in vitro. Recent extended trials have highlighted its antidiabetic properties in humans. However, the underlying molecular mechanism is not fully elucidated. The present study was designed to explore potential regulatory mechanisms of intracellular glucocorticoid reactivating enzyme, 11beta-HSD1 and anti-diabetic hormone, adiponectin by bezafibrate in murine adipose tissue, and cultured adipocytes. Treatment of db/db mice with bezafibrate significantly ameliorated hyperglycemia and insulin resistance, accompanied by a marked reduction of triglyceride and nonesterified fatty acids. Despite equipotent in lipid-lowering effects, another fibrate, fenofibrate, did not show such beneficial effects on glycemic control. Treatment of bezafibrate caused a marked decrease in the mRNA level of 11beta-HSD1 preferentially in adipose tissue of db/db mice (-47%, P<0.05), concomitant with a significant increase in plasma adiponectin level (+37%, P<0.01). Notably, treatment of bezafibrate caused a marked decrease in the mRNA level (-34%, P<0.01) and enzyme activity (-32%, P<0.01) of 11beta-HSD1, whereas the treatment substantially augmented the expression (+71%, P<0.01) and secretion (+27%, P<0.01) of adiponectin in 3T3-L1 adipocytes. Knockdown of 11beta-HSD1 by siRNA confirmed that 11beta-HSD1 acts as a distinct oxoreductase in adipocytes and validated the enzyme activity assays in the present study. Effects of bezafibrate on regulation of 11beta-HSD1 and adiponectin in murine adipocytes were comparable with those in thiazolidinediones. This is the first demonstration that bezafibrate directly regulates 11beta-HSD1 and adiponectin in murine adipocytes, both of which may contribute to metabolically-beneficial effects by bezafibrate.

5'-AMP-activated protein kinase (AMPK) has been implicated in glycogen metabolism in skeletal muscle. However, the physiological relevance of increased AMPK activity during exercise has not been fully clarified. This study was performed to determine the direct effects of acute AMPK activation on muscle glycogen regulation. For this purpose, we used an isolated rat muscle preparation and pharmacologically activated AMPK with 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR). Tetanic contraction in vitro markedly activated the alpha(1)- and alpha(2)-isoforms of AMPK, with a corresponding increase in the rate of 3-O-methylglucose uptake. Incubation with AICAR elicited similar enhancement of AMPK activity and 3-O-methylglucose uptake in rat epitrochlearis muscle. In contrast, whereas contraction stimulated glycogen synthase (GS), AICAR treatment decreased GS activity. Insulin-stimulated GS activity also decreased after AICAR treatment. Whereas contraction activated glycogen phosphorylase (GP), AICAR did not alter GP activity. The muscle glycogen content decreased in response to contraction but was unchanged by AICAR. Lactate release was markedly increased when muscles were stimulated with AICAR in buffer containing glucose, indicating that the glucose taken up into the muscle was catabolized via glycolysis. Our results suggest that AMPK does not mediate contraction-stimulated glycogen synthesis or glycogenolysis in skeletal muscle and also that acute AMPK activation leads to an increased glycolytic flux by antagonizing contraction-stimulated glycogen synthesis.

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

BACKGROUND: Lack of leptin is implicated in insulin resistance and other metabolic abnormalities in generalized lipodystrophy; however, the efficacy, safety, and underlying mechanisms of leptin-replacement therapy in patients with generalized lipodystrophy remain unclear. METHODS: Seven Japanese patients with generalized lipodystrophy, two acquired and five congenital type, were treated with the physiological replacement dose of recombinant leptin during an initial 4-month hospitalization followed by outpatient follow-up for up to 36 months. RESULTS: The leptin-replacement therapy with the twice-daily injection dramatically improved fasting glucose (mean +/- SE, 172 +/- 20 to 120 +/- 12 mg/dl, P < 0.05) and triglyceride levels (mean +/- SE, 700 +/- 272 to 260 +/- 98 mg/dl, P < 0.05) within 1 wk. The leptin-replacement therapy reduced insulin resistance evaluated by euglycemic clamp method and augmented insulin secretion at glucose tolerance test with different responses between acquired and congenital types. Improvement of the fatty liver was also observed. The efficacy and safety of the once-daily injection were comparable to those of the twice-daily injection. The leptin-replacement therapy ameliorated macro- and microalbuminuria and showed no deterioration of neuropathy and retinopathy of these patients. The leptin-replacement therapy is beneficial to diabetic complications and lipodystrophic ones. Two patients developed antileptin antibodies but not neutralizing antibodies. The therapy was well tolerated, and its effects were maintained for up to 36 months without any notable adverse effects such as hypoglycemia, high blood pressure, or reduction of bone mineral density. CONCLUSIONS: The present study demonstrates the efficacy and safety of the long-term leptin-replacement therapy and possible mechanisms of leptin actions in patients with generalized lipodystrophy.

Insulinoma is the most common cause of fasting hypoglycemia resulting from autonomous insulin hypersecretion. A 59-year-old woman who had previously had an insulinoma and had undergone a partial pancreatectomy was admitted to our hospital because of recurrence of hypoglycemia after 27 years. She had two unusual endocrinological features: 1) the serum insulin response to intravenous secretin injection was not impaired, and 2) the serum C-peptide levels and ratios of serum C-peptide to insulin were relatively low. Two pancreatic tumors were readily detectable by computed tomography (CT) and magnetic resonance imaging (MRI). The selective arterial calcium injection (SACI) test showed a hyperinsulinemic response by calcium administration to the gastroduodenal artery. A partial pancreatectomy was done and her hypoglycemia disappeared. Histology revealed that the tumors were composed of monotonous, small round cells that were positive for both insulin and cathepsin B. As previous in vitro studies have shown that C-peptide can be metabolized within human insulinoma cells by proteolytic cleavage by cathepsin B, our patient's low serum C-peptide levels might have been caused by degradation of C-peptide by cathepsin B. According to the data from the literature, the molar ratio of serum C-peptide to insulin is generally decreased in patients with insulinoma than normal subjects. This case highlights the need for careful interpretation of C-peptide levels and the intravenous secretin injection test in the diagnosis of insulinoma.

Skeletal muscle expresses two catalytic subunits, alpha1 and alpha2, of the 5'-AMP-activated protein kinase (AMPK), which has been implicated in contraction-stimulated glucose transport and fatty acid oxidation. Muscle contraction activates the alpha2-containing AMPK complex (AMPKalpha2), but this activation may occur with or without activation of the alpha1-containing AMPK complex (AMPKalpha1), suggesting that AMPKalpha2 is the major isoform responsible for contraction-induced metabolic events in skeletal muscle. We report for the first time that AMPKalpha1, but not AMPKalpha2, can be activated in contracting skeletal muscle. Rat epitrochlearis muscles were isolated and incubated in Krebs-Ringer bicarbonate buffer containing pyruvate. In muscles stimulated to contract at a frequency of 1 and 2 Hz during the last 2 min of incubation, AMPKalpha1 activity increased twofold and AMPKalpha2 activity remained unchanged. Muscle stimulation did not change the muscle AMP concentration or the AMP-to-ATP ratio. AMPK activation was associated with increased phosphorylation of Thr(172) of the alpha-subunit, the primary activation site. Muscle stimulation increased the phosphorylation of acetyl-CoA carboxylase (ACC), a downstream target of AMPK, and the rate of 3-O-methyl-d-glucose transport. In contrast, increasing the frequency (>or=5 Hz) or duration (>or=5 min) of contraction activated AMPKalpha1 and AMPKalpha2 and increased AMP concentration and the AMP/ATP ratio. These results suggest that 1) AMPKalpha1 is the predominant isoform activated by AMP-independent phosphorylation in low-intensity contracting muscle, 2) AMPKalpha2 is activated by an AMP-dependent mechanism in high-intensity contracting muscle, and 3) activation of each isoform enhances glucose transport and ACC phosphorylation in skeletal muscle.

5'Adenosine monophosphate-activated protein kinase (AMPK) has been implicated in exercise-induced stimulation of glucose metabolism in skeletal muscle. Although skeletal muscle expresses both the alpha1 and alpha2 isoforms of AMPK, the alpha2 isoform is activated predominantly in response to moderate-intensity endurance exercise in human and animal muscles. The purpose of this study was to determine whether activation of alpha2 AMPK plays a role in increasing the rate of glucose transport, promoting glucose transporter 4 (GLUT4) expression, and enhancing insulin sensitivity in skeletal muscle. To selectively activate the alpha2 isoform, we used 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR), which is metabolized in muscle cells and preferentially stimulates the alpha2 isoform. Subcutaneous administration of 250 mg/kg AICAR activated the alpha2 isoform for 90 minutes, but not the alpha1 isoform in hind limb muscles of the C57/B6J mouse. The maximal activation of the alpha2 isoform was observed 30 to 60 minutes after administration of AICAR and was similar to the activation induced by a 30-minute swim in a current pool. The increase in alpha2 activity paralleled the phosphorylation of Thr(172), the essential residue for full kinase activation, and the activity of acetyl-coenzyme A carboxylase beta, a known substrate of AMPK in skeletal muscle. Subcutaneous injection of AICAR rapidly increased, by 30%, the rate of 2-deoxyglucose (2DG) transport into soleus muscle; 2DG transport increased within 30 minutes and remained elevated for 4 hours after administration of AICAR. Repeated intraperitoneal injection of AICAR, 3 times a day for 4 to 7 days, increased soleus GLUT4 protein by 30% concomitant with a significant 20% increase in insulin-stimulated 2DG transport. These data suggest that moderate endurance exercise promotes glucose transport, GLUT4 expression, and insulin sensitivity in skeletal muscle at least partially via activation of the alpha2 isoform of AMPK.

To assess gene expression of a membrane-bound G-protein-coupled fatty acid receptor, GPR40, in the human pancreas and islet cell tumors obtained at surgery were analyzed. The mRNA level of the GPR40 gene in isolated pancreatic islets was approximately 20-fold higher than that in the pancreas, and the level was comparable to or rather higher than that of the sulfonylurea receptor 1 gene, which is known to be expressed abundantly in human pancreatic beta cells. A large amount of GPR40 mRNA was detected in tissue extracts from two cases of insulinoma, whereas the expression was undetectable in glucagonoma or gastrinoma. The present study demonstrates that GPR40 mRNA is expressed predominantly in pancreatic islets in humans and that GPR40 mRNA is expressed solely in human insulinoma among islet cell tumors. These results indicate that GPR40 is probably expressed in pancreatic beta cells in the human pancreas.

Steatosis is one of the most common liver diseases and is associated with the metabolic syndrome. A line of evidence suggests that peroxisome proliferator-activated receptor (PPAR) alpha and PPARgamma are involved in its pathogenesis. Hepatic overexpression of PPARgamma1 in mice provokes steatosis, whereas liver-specific PPARgamma disruption ameliorates steatosis in ob/ob mice, suggesting that hepatic PPARgamma functions as an aggravator of steatosis. In contrast, PPARalpha-null mice are susceptible to steatosis because of reduced hepatic fatty acid oxidation. PPARgamma with mutations in its C-terminal ligand-binding domain (L468A/E471A mutant PPARgamma1) have been reported as a constitutive repressor of both PPARalpha and PPARgamma activities in vitro. To elucidate the effect of co-suppression of PPARalpha and PPARgamma on steatosis, we generated mutant PPARgamma transgenic mice (Liver mt PPARgamma Tg) under the control of liver-specific human serum amyloid P component promoter. In the liver of transgenic mice, PPARalpha and PPARgamma agonist-induced augmentation of the expression of downstream target genes of PPARalpha and PPARgamma, respectively, was significantly attenuated, suggesting PPARalpha and PPARgamma co-suppression in vivo. Suppression of PPARalpha and PPARgamma target genes was also observed in the fasted and high-fat-fed conditions. Liver mt PPARgamma Tg were susceptible to fasting-induced steatosis while being protected against high-fat diet-induced steatosis. The opposite hepatic outcomes in Liver mt PPARgamma Tg as a result of fasting and high-fat feeding may indicate distinct roles of PPARalpha and PPARgamma in 2 different types of nutritionally provoked steatosis.

In 3T3-L1 preadipocytes, hormonal induction causes adipose conversion and facilitates the expression of insulin-sensitive glucose transporter, GLUT4. Evidence has accumulated that, in 3T3-L1 preadipocytes, the formation of GLUT4 storage vesicle and its translocation to plasma membrane precede both lipid accumulation and expression of GLUT4 and C/EBPalpha, a key transcription factor for adipose differentiation. On the other hand, 3T3-C2 fibroblastic cells, a subline of 3T3-L1, follow adipogenic process till mitotic clonal expansion stage (2 days after hormonal induction), but do not proceed to terminal differentiation stage (8 days after the induction), resulting in a lack of adipose conversion and GLUT4 expression. Here we show that, when myc-tagged GLUT4 was retrovirally expressed in 3T3-C2 cells, insulin-stimulated GLUT4 translocation did occur on day 2 after the induction. On day 8 after the induction, however, neither GLUT4 translocation nor the expression of C/EBPalpha was observed. We also created 3T3-C2 cells stably expressing both myc-tagged GLUT4 and C/EBPalpha, demonstrating that co-expressed cells showed insulin-stimulated GLUT4 translocation on day 8 after the induction, as well as adipose conversion coupling with PPARgamma expression. Our results provide evidence that C/EBPalpha has the potential to maintain the ability of insulin-stimulated GLUT4 translocation in C/EBPalpha-deficient 3T3-C2 fibroblastic cells.

Leptin augments glucose and lipid metabolism independent of its effect on satiety. Administration of leptin in rodents increases skeletal muscle beta-oxidation by activating AMP-activated protein kinase (AMPK). We previously reported that, as hyperleptinemic as obese human subjects, transgenic skinny mice overexpressing leptin in liver (LepTg) exhibit enhanced insulin sensitivity and lipid clearance. To assess skeletal muscle AMPK activity in leptin-sensitive and -insensitive states, we examined phosphorylation of AMPK and its target, acetyl CoA carboxylase (ACC), in muscles from LepTg under dietary modification. Here we show that phosphorylation of AMPK and ACC are chronically augmented in LepTg soleus muscle, with a concomitant increase in the AMP-to-ATP ratio and a significant decrease in tissue triglyceride content. Despite preexisting hyperleptinemia, high-fat diet (HFD)-fed LepTg develop obesity, insulin-resistance, and hyperlipidemia. In parallel, elevated soleus AMPK and ACC phosphorylation in regular diet-fed LepTg is attenuated, and tissue triglyceride content is increased in those given HFD. Of note, substitution of HFD with regular diet causes a robust recovery of soleus AMPK and ACC phosphorylation in LepTg, with a higher rate of body weight reduction and a regain of insulin sensitivity. In conclusion, soleus AMPK and ACC phosphorylation in LepTg changes in parallel with its insulin sensitivity under dietary modification, suggesting a close association between skeletal muscle AMPK activity and sensitivity to leptin.

Diabetic nephropathy is the leading cause of end-stage renal disease, for which effective therapy to prevent the progression at advanced stages remains to be established. There is also a long debate whether diabetic glomerular injury is reversible or not. Lipoatrophic diabetes, a syndrome caused by paucity of adipose tissue, is characterized by severe insulin resistance, dyslipidemia, and fatty liver. Here, we show that a genetic model of lipoatrophic diabetes (A-ZIP/F-1 mice) manifests a typical renal injury observed in human diabetic nephropathy that is associated with glomerular hypertrophy, diffuse and pronounced mesangial widening, accumulation of extracellular matrix proteins, podocyte damage, and overt proteinuria. By crossing A-ZIP/F-1 mice with transgenic mice overexpressing an adipocyte-derived hormone leptin, we also reveal that leptin completely prevents the development of hyperglycemia and nephropathy in A-ZIP/F-1 mice. Furthermore, continuous leptin administration to A-ZIP/F-1 mice by minipump beginning at 40 weeks of age significantly alleviates the glomerular injury and proteinuria. These findings demonstrate the therapeutic usefulness of leptin at least for a certain type of diabetic nephropathy. The model presented here will serve as a novel tool to analyze the molecular mechanism underlying not only the progression but also the regression of diabetic nephropathy.

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 PPARgamma target genes such as aP2 and adiponectin. By contrast, telmisartan attenuated 11beta-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.

Recent studies have suggested that 5'AMP-activated protein kinase (AMPK) is activated in response to metabolic stresses, such as contraction, hypoxia, and the inhibition of oxidative phosphorylation, which leads to insulin-independent glucose transport in skeletal muscle. In the present study, we hypothesized that acute oxidative stress increases the rate of glucose transport via an AMPK-mediated mechanism. When rat epitrochlearis muscles were isolated and incubated in vitro in Krebs buffer containing the oxidative agent H(2)O(2), AMPKalpha1 activity increased in a time- and dose-dependent manner, whereas AMPKalpha2 activity remained unchanged. The activation of AMPKalpha1 was associated with phosphorylation of AMPK Thr(172), suggesting that an upstream kinase is involved in the activation process. H(2)O(2)-induced AMPKalpha1 activation was blocked in the presence of the antioxidant N-acetyl-l-cysteine (NAC), and H(2)O(2) significantly increased the ratio of oxidized glutathione to glutathione (GSSG/GSH) concentrations, a sensitive marker of oxidative stress. H(2)O(2) did not cause an increase in the conventional parameters of AMPK activation, such as AMP and AMP/ATP. H(2)O(2) increased 3-O-methyl-d-glucose transport, and this increase was partially, but significantly, blocked in the presence of NAC. Results were similar when the muscles were incubated in a superoxide-generating system using hypoxanthine and xanthine oxidase. Taken together, our data suggest that acute oxidative stress activates AMPKalpha1 in skeletal muscle via an AMP-independent mechanism and leads to an increase in the rate of glucose transport, at least in part, via an AMPKalpha1-mediated mechanism.

Congenital generalized lipodystrophy (CGL), Berardinelli-Seip syndrome, is a rare metabolic disorder characterized by a near total lack of adipose tissue from birth or early infancy. Recently, seipin, encoding a 398-amino acid protein of unknown function, and AGPAT2, encoding 1-acyl-sn-glycerol-3-phosphate acyltransferase 2, were identified as causative genes for CGL. Seipin mutations were found in patients from families originating from Europe and the Middle East. AGPAT2 mutations were found predominantly in African ancestry. However, no information is available on these genes in the pathogenesis of CGL in Asian ancestry. We examined the sequences of the entire coding region of seipin and AGPAT2 in four Japanese CGL patients from independent families. Their average body fat content was 4.7 +/- 0.5%, and the plasma leptin level was 1.15 +/- 0.14 ng/ml. We identified a novel nonsense mutation of seipin at codon 275 (R275X). Of four CGL patients, three were homozygous for R275X. No seipin mutation was found in any exon in one patient. We did not find any AGPAT2 mutations in our Japanese patients, suggesting that AGPAT2 is a minor causative gene, if any, for CGL in Japanese. This is the first report on gene and phenotype analysis of CGL in Japanese.

Ghrelin is an acylated peptide, whose lipid modification is essential for its biological activities. Previous studies demonstrated that it strongly stimulates GH release and has a potent orexigenic action. Meanwhile, there is enough evidence showing that feeding states influence plasma ghrelin levels. Fasting stimulates ghrelin secretion, and feeding reduces plasma ghrelin levels. In this study we examined the regulation of plasma ghrelin by fasting in genetically obese animals considering its molecular forms. Plasma levels of active form of ghrelin as well as those of total ghrelin were reduced in ob/ob and db/db mice compared with those in their control mice. Zucker fatty (fa/fa) rats also showed lower plasma ghrelin levels by fasting than the control rats. Insulin-induced hypoglycemia, however, stimulated ghrelin secretion in the fasted fatty rats. Moreover, glucose injection was revealed to reduce plasma ghrelin levels in rats. The effect of the severity of obesity on secretory regulation of ghrelin was also studied. Older fatty rats showed low plasma ghrelin levels even after 48-h fasting. These data suggest that the short-term secretory regulation of total ghrelin and the active form of ghrelin is delayed in obese animals and that blood glucose levels may be involved in the delayed regulation.