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While hypothalamic leptin resistance can occur prior to establishment of obesity, clarification is needed as to whether the impaired response to leptin in the reward-related nuclei occurs independently of obesity. To answer this question, we attempted to dissociate the normally coexisting leptin resistance from obesity. We investigated phenotypes of leptin-overexpressing transgenic mice fed for 1 week with 60 % high-fat diet (HFD) (LepTg-HFD1W mice). After 1 week, we observed that LepTg-HFD1W mice weighed as same as wild type (WT) mice fed standard chow diet (CD) for 1 week (WT-CD1W mice). However, compared to WT-CD1W mice, LepTg-HFD1W mice exhibited attenuated leptin-induced anorexia, decreased leptin-induced c-fos immunostaining in nucleus accumbens (NAc), one of important site of reward system, decreased leptin-stimulated pSTAT3 immunostaining in hypothalamus. Furthermore, neither sucrose nor lipid preference was suppressed by leptin in LepTg-HFD1W mice. On the contrary, leptin significantly suppressed both preferences in WT mice fed HFD (WT-HFD1 W mice). These results indicate that leptin responsiveness decreases in NAc independently of obesity. Additionally, in this situation, suppressive effect of leptin on the hedonic feeding results in impaired regulation. Such findings suggest the impaired leptin responsiveness in NAc partially contributes to dysregulated hedonic feeding behavior independently of obesity.

Background: Cerebral amyloid angiopathy is a cerebrovascular disease directly implicated in Alzheimer’s disease pathogenesis through amyloid-β deposition. Growing evidence has shown a pivotal role of chronic neuroinflammation both in cerebral amyloid angiopathy and Alzheimer’s disease. Objective: The aim of this study was to investigate whether circulating levels of the complement 3, a crucial component of the innate immune system, are increased in patients with cerebral amyloid angiopathy. Methods: Serum complement 3 levels were retrospectively measured by a sandwich enzyme-linked immunosorbent assay in a single-center cohort of patients with mild cognitive impairment. The diagnosis of cerebral amyloid angiopathy was based on the modified Boston criteria. Logistic regression analysis was performed to identify the predictive factors for cerebral amyloid angiopathy. Results: We analyzed 55 mild cognitive impairment patients (mean age [standard deviation]: 76.3 [6.8] years; 33 [60% ] men). Complement 3 levels were significantly increased in cerebral amyloid angiopathy patients (n = 16) compared with those without cerebral amyloid angiopathy (n = 39) (median [interquartile range]: 0.43 [0.34–0.65] versus 0.35 [0.25–0.45], respectively; p = 0.040). Univariate and multivariate logistic regression analysis revealed that increased complement 3 levels were significantly associated with cerebral amyloid angiopathy. After selection of the best predictive model using stepwise selection, complement 3 was preserved as a significant independent predictive factor for cerebral amyloid angiopathy (odds ratio per 0.1 unit/mL increase [95% confidence interval]: 1.407 [1.042–1.899]; p = 0.026). Conclusion: Complement activation may play a pivotal role in cerebral amyloid angiopathy. Complement 3 may be a novel diagnostic biomarker for cerebral amyloid angiopathy.

Introduction Although the risk of dementia among patients with type 2 diabetes mellitus (T2DM) is double that of those without T2DM, the mechanism remains to be elucidated and the glycemic goal to prevent progression of cognitive impairment is unclear. Results from cross-sectional studies suggest that glucose fluctuations are associated with impairment of cognitive function among T2DM patients. Therefore, the aim of the longitudinal study described here is to evaluate the relationships between glucose fluctuation indexes assessed by continuous glucose monitoring (CGM) and cognitive function among elderly patients with T2DM. Methods This will be a prospective, single-center, 2-year longitudinal study in which a total of 100 elderly patients with T2DM showing mild cognitive impairment (MCI) will be enrolled. Glucose fluctuations, assessed using the FreeStyle Libre Pro continuous glucose monitoring system (Abbott Laboratories), and results of cognitive tests, namely the Montreal Cognitive Assessment (MoCA) and Alzheimer’s Disease Assessment Scale (ADAS), will be evaluated at baseline, 1-year visit and 2-year visit. The primary endpoint is the relationships between indexes of glucose fluctuation and change in MoCA and ADAS scores. Secondary endpoints are the relationships between the indexes of glucose fluctuation or cognitive scores and the following: indexes representing intracranial lesions obtained by magnetic resonance imaging and angiography of the head; Geriatric Depression Scale score; Apathy Scale score; carotid intima-media thickness assessed by echography; inflammatory markers; fasting glucose; glycated hemoglobin; blood pressure; and the development of cardiovascular and renal events. Planned Outcomes The current study is scheduled for completion in June 2022. The results could lead to the elucidation of novel glycemic goals to prevent the progression of cognitive impairment and/or of relationships between glucose fluctuations and cognitive function among T2DM patients. The findings of the study will be reported in publications and conference presentations. Trial Registration University Hospital Medical Information Network Clinical Trial Registry (UMIN000038546).

Background: Patients with familial hypercholesterolemia are known to have an extremely high risk of coronary artery disease owing to high levels of low-density lipoprotein-cholesterol since birth. In addition, aortic stenosis among patients with familial hypercholesterolemia has also been reported besides coronary disease. The aim of this study was to characterize the histopathological differences in excised aortic valves for aortic stenosis between patients with familial hypercholesterolemia and non-familial hypercholesterolemia. Subjects and Methods: Six familial patients (3 homozygous, 3 heterozygous familial hypercholesterolemia patients), and 18 non-familial hypercholesterolemia patients underwent pathological and immunohistochemical examinations for aortic valves macroscopically and microscopically at aortic valve replacement surgery for stenosis. Results: Our study revealed that calcification of aortic valves among homozygous hypercholesterolemia showed a much milder degree than those of non-familial patients. Moreover, the age at surgery for stenosis in the case of homozygotes was significantly less than that of heterozygous hypercholesterolemia and nonfamilial hypercholesterolemia patients. In addition, CD68-positive macrophages infiltrated the aorta side (fibrosa) in all familial hypercholesterolemia patients. However, the macrophage accumulation in the aortic valves of non-familial hypercholesterolemia patients was recognized in the middle layer (spongiosa) near calcification and left ventricular side (ventricularis) of the aortic valves. Conclusion: Lipid is one of the important factors for aortic valve fibrosis and stenosis in hypercholesterolemia. This study suggested that the non-familial atherosclerotic aortic stenosis in the elderly is qualitatively different from aortic valves in familial hypercholesterolemia, primarily owing to calcification resulting from age and long-term degeneration and inflammatory responses.

Background: Patients with type 2 diabetes mellitus (T2DM) are high-risk subjects who more frequently have micro- and macrovascular diseases including coronary artery disease (CAD). Since impaired glycemic homeostasis directly influences the formation and propagation of atherosclerotic plaques, optimal management of glycemic status is required for the prevention of diabetic atherosclerosis. Continuous glucose monitoring (CGM) provides not only average glucose level but also the degree of glucose fluctuation and hypoglycemia. Given the association of glycemic variability with diabetic macrovascular diseases, CGM-based glycemic management could favorably modulate glycemic fluctuation, thereby potentially modifying atheroma burden in T2DM subjects. To test this hypothesis, the Observation of Coronary Atheroma Progression under Continuous Glucose Monitoring Guidance in Patients with Type 2 Diabetes Mellitus (OPTIMAL) study has been designed (Japan Registry of Clinical Trials: jRCT1052180152, University Hospital Medical Information Network Clinical Trial Registry UMIN000036721). Methods: The OPTIMAL is a single-center, randomized trial to evaluate the efficacy of CGM-based glycemic control on atheroma progression in T2DM patients with CAD by using serial intravascular ultrasound (IVUS) and near-infrared spectroscopy (NIRS) imaging. A total of 90 eligible subjects will be randomized 1:1 into two groups to receive either CGM-based glycemic control or HbA1c-baded glycemic management. Coronary angiography and NIRS/IVUS imaging is repeated at the end of the assigned treatment period. Results: The primary endpoint is the normalized absolute change in total atheroma volume (TAV) from baseline to 12 months. The secondary endpoints include (I) the absolute change in percent atheroma volume, (II) the percent change in lipid core burden index, (III) the change in coefficient variance measured by CGM, (IV) the change in atherogenic markers (high-density lipoprotein functionality, proprotein convertase subxilisin/kexin type 9 and fatty-acid binding proteins), and (V) the frequency of hypoglycemia. Safety will also be evaluated. Conclusions: The collaboration of CGM use with serial NIRS/IVUS imaging will enable to compare atheroma progression rate under CGM-based glycemic management and HbA1c-based approach.

Intermittent-scanning continuous glucose monitoring (isCGM) is widely used in type 1 diabetes (T1D) patients; however, the education required to prevent hypoglycemia by using isCGM is not established. This study examines the combined effect of isCGM device usage and the education to reduce the time in hypoglycemia in comparison to conventional self-monitoring of blood glucose (SMBG).The Effect of Intermittent-Scanning Continuous Glucose Monitoring to Glycemic Control Including Hypoglycemia and Quality of Life of Patients with Type 1 Diabetes Mellitus Study (ISCHIA Study), a randomized, crossover trial, enrolls 104 T1D patients (age, 20-74 years) with T1D. Participants are randomized to use isCGM combined with structured education (Intervention period) or SMBG (Control period) for 84 days, followed by the other for a further 84 days. During the Intervention period, participants have access to the sensor glucose levels and trend arrow of the device. During the Control period, participants conduct SMBG at least three times a day, and retrospective CGM is used to record the blinded sensor glucose levels. The primary endpoint is the decrease of time in hypoglycemia (70 mg/dL) per day (hour/day) during the Intervention period compared with the Control period. The secondary endpoints include other indices of glycemic control, glycoalbumin, accuracy of isCGM, diabetes-related quality of life (QOL), adherence, and cost-effectiveness. The study protocol has received Certified Review Board (CRB) approval from National Hospital Organization Osaka National Hospital (N2018002, Feb 14, 2019). This study is carried out in accordance with the Declaration of Helsinki and the Clinical Trials Act. The findings will be published in peer-reviewed journals.The ISCHIA study will contribute to the standardization of patient education regarding the prevention of hypoglycemia by using isCGM.

Context: In the National Cerebral and Cardiovascular Center (NCVC) Biobank, buffy coats have been collected from patients and stored with cryoprotective agents as a possible source for viable blood...

Angiopoietin-like protein (ANGPTL)8 is a liver- and adipocyte-derived protein that controls plasma triglyceride (TG) levels. Most animal studies have used mouse models. Here, we generated an Angptl8 KO rat model using a clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system to clarify the roles of ANGPTL8 in glucose and lipid metabolism. Compared with WT rats, Angptl8 KO rats had lower body weight and fat content, associated with impaired lipogenesis in adipocytes; no differences existed between the groups in food intake or rectal temperature. Plasma TG levels in both the fasted and refed states were significantly lower in KO than in WT rats, and an oral fat tolerance test showed decreased plasma TG excursion in Angptl8 KO rats. Higher levels of lipase activity in the heart and greater expression of genes related to β-oxidation in heart and skeletal muscle were observed in Angptl8 KO rats. However, there were no significant differences between KO and WT rats in glucose metabolism or the histology of pancreatic β-cells on both standard and high-fat diets. In conclusion, we demonstrated that Angptl8 KO in rats resulted in lower body weight and plasma TG levels without affecting glucose metabolism. ANGPTL8 might be an important therapeutic target for obesity and dyslipidemia.

We previously established human induced pluripotent stem (iPS) cells in two diabetic patients from different families with the mitochondrial A3243G mutation and isolated isogenic iPS cell clones with either undetectable or high levels of the mutation in both patients. In the present study, we analyzed the mitochondrial functions of two mutation-undetectable and two mutation-high clones in each patient through four methods to assess complex I activity, mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production. In the first patient, complex I activity, mitochondrial respiration, and mitochondrial ATP production were decreased in the mutation-high clones compared with the mutation-undetectable clones, and mitochondrial membrane potential was decreased in a mutation-high clone compared with a mutation-undetectable clone. In the second patient, complex I activity was decreased in one mutation-high clone compared with the other clones. The other parameters showed no differences in any clones. In addition, the complex I activity and mitochondrial respiration of the mutation-undetectable clones from both patients were located in the range of those of iPS cells from healthy subjects. The present study suggests that the mitochondrial function of the mutation-undetectable iPS cell clones obtained from two patients with the A3243G mutation is comparable to the control iPS cells.

To elucidate the clinical implication of ghrelin, we have been trying to generate variable models of transgenic (Tg) mice overexpressing ghrelin. We generated Tg mice overexpressing des-acyl ghrelin in a wide variety of tissues under the control of β-actin promoter. While plasma des-acyl ghrelin level in the Tg mice was 44-fold greater than that of control mice, there was no differences in the plasma ghrelin level between des-acyl ghrelin Tg and the control mice. The des-acyl ghrelin Tg mice exhibited the lower body weight and the shorter body length due to modulation of GH-IGF-1 axis. We tried to generate Tg mice expressing a ghrelin analog, which possessed ghrelin-like activity (Trp3-ghrelin Tg mice). The plasma Trp3-ghrelin concentration in Trp3-ghrelin Tg mice was approximately 85-fold higher than plasma ghrelin (acylated ghrelin) concentration seen in the control mice. Because Trp3-ghrelin is approximately 24-fold less potent than ghrelin, the plasma Trp3-ghrelin concentration in Trp3-ghrelin Tg mice was calculated to have approximately 3.5-fold biological activity greater than that of ghrelin (acylated ghrelin) in the control mice. Trp3-ghrelin Tg mice did not show any phenotypes except for reduced insulin sensitivity in 1-year old. After the identification of ghrelin O-acyltransferase (GOAT), we generated doubly Tg mice overexpressing both mouse des-acyl ghrelin and mouse GOAT in the liver by cross-mating the two kinds of Tg mice. The plasma ghrelin concentration of doubly Tg mice was approximately 2-fold higher than that of the control mice. No apparent phenotypic changes in body weight and food intake were observed in doubly Tg mice. Further studies are ongoing in our laboratory to generate Tg mice with the increased plasma ghrelin level to a greater extent. The better understanding of physiological and pathophysiological significance of ghrelin from experiments using an excellent animal model may provide a new therapeutic approach for human diseases.

Leptin may reduce pancreatic lipid deposition, which increases with progression of obesity and can impair β-cell function. The insulinotropic effect of glucagon-like peptide-1 (GLP-1) and the efficacy of GLP-1 receptor agonist are reduced associated with impaired β-cell function. In this study, we examined whether leptin could restore the efficacy of exenatide, a GLP-1 receptor agonist, in type 2 diabetes with increased adiposity. We chronically administered leptin (500 μg·kg−1·day−1) and/or exenatide (20 μg·kg−1·day−1) for 2 wk in a mouse model of type 2 diabetes with increased adiposity induced by streptozotocin and high-fat diet (STZ/HFD mice). The STZ/HFD mice exhibited hyperglycemia, overweight, increased pancreatic triglyceride level, and reduced glucose-stimulated insulin secretion (GSIS); moreover, the insulinotropic effect of exenatide was reduced. However, leptin significantly reduced pancreatic triglyceride level, and adding leptin to exenatide (LEP/EX) remarkably enhanced GSIS. These results suggested that the leptin treatment restored the insulinotropic effect of exenatide in the mice. In addition, LEP/EX reduced food intake, body weight, and triglyceride levels in the skeletal muscle and liver, and corrected hyperglycemia to a greater extent than either monotherapy. The pair-feeding experiment indicated that the marked reduction of pancreatic triglyceride level and enhancement of GSIS by LEP/EX occurred via mechanisms other than calorie restriction. These results suggest that leptin treatment may restore the insulinotropic effect of exenatide associated with the reduction of pancreatic lipid deposition in type 2 diabetes with increased adiposity. Combination therapy with leptin and exenatide could be an effective treatment for patients with type 2 diabetes with increased adiposity.

Human induced pluripotent stem (iPS) and embryonic stem (ES) cells can differentiate into a variety of cell types. We reported on adipogenic potential of human iPS and ES cells in vitro. In the present study, we investigate the survival and maintenance of adipocytes differentiated in vitro from human iPS and ES cells after transplantation. Following adipogenic induction in vitro, the differentiated cells exhibited functional properties of adipocytes such as lipid storage, lipolysis, and insulin responsiveness. Subsequently, Matrigel containing the differentiated human iPS and ES cells was transplanted into the subcutaneous tissue of nude mice. After 1-4 weeks, the cells with adipocyte-like features were observed in transplanted Matrigel by histological analysis. The human origin of the cells, their lipid accumulation, and gene expression of adipocyte markers in transplanted cells were then confirmed, suggesting the presence of adipocytes in transplanted Matrigel. When the relative areas of these cells were calculated by dividing the adipocyte areas by the total Matrigel areas, we found that they peaked at 2 weeks after transplantation, and that the adipocytes persisted at 4 weeks. The present study demonstrates that human iPS and ES cells can differentiate into adipocytes with functional properties and that adipocytes derived from human iPS and ES cells can survive and maintain the differentiated properties of adipocytes for at least 4 weeks after transplantation. Adipocytes derived from human iPS and ES cells thus have the potential to open new avenues for stem cell-based research into metabolic diseases and future therapeutic applications.

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.

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.

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.

Objective Congenital generalized lipodystrophy (CGL) is an autosomal recessive disorder characterized by marked scarcity of adipose tissue, extreme insulin resistance, hypertriglyceridemia, hepatic steatosis and early-onset diabetes. Mutation of the BSCL2 / SEIPIN gene causes the most severe form of CGL. The aim of this study was to generate induced pluripotent stem (iPS) cells from patients with CGL harboring BSCL2 / SEIPIN mutations. Methods Skin biopsies were obtained from two Japanese patients with CGL harboring different nonsense mutations (E189X and R275X) in BSCL2 / SEIPIN . The fibroblasts thus obtained were infected with retroviruses encoding OCT4, SOX2, c-MYC, and KLF4. The generated iPS cells were evaluated for pluripotency by examining the expression of pluripotency markers (alkaline phosphatase, SSEA-4, TRA-1-60, and NANOG) and their ability to differentiate to three germ layers in vitro by forming embryoid bodies, and to form teratomas in vivo. Adipogenic capacity of differentiated BSCL2-iPS cells was determined by oil red O and adipose differentiation-related protein (ADRP) staining. Rescue experiments were also performed using stable expression of wild-type BSCL2. A coimmunoprecipitation assay was conducted to investigate the interaction of SEIPIN with ADRP. Results iPS cells were generated from fibroblasts of the two patients with CGL. Each of the patient-derived iPS (BSCL2-iPS) clones showed all of the hallmarks of pluripotency and could differentiate into derivatives of all three germ layers in vitro by forming embryoid bodies, and form teratomas after injection into mouse testes. BSCL2-iPS cells maintained the mutations in BSCL2 and lacked intact BSCL2. Upon adipogenic differentiation, BSCL2-iPS cells exhibited marked reduction of lipid droplet formation concomitant with diffuse cytoplasmic distribution of ADRP, compared with iPS cells from healthy individuals. Forced expression of BSCL2 not only rescued the lipid accumulation defects, but also restored cytoplasmic punctate localization of ADRP in BSCL2-iPS cells. Coimmunoprecipitation indicated SEIPIN interacted with ADRP. Conclusion BSCL2-iPS cells that recapitulate the lipodystrophic phenotypes in vitro could provide valuable models with which to study the physiology of lipid accumulation and the pathology of human lipodystrophy. We found that BSCL2 defines the localization of ADRP, which has a role in lipid accumulation and adipogenic differentiation.

Like many other human cancers, the development of malignant mesothelioma is closely associated with a chronic inflamma-tory condition. Both macrophages and mesothelial cells play crucial roles in the inflammatory response caused by asbestos exposure. Here, we show that adipocytes can also contribute to asbestos-induced inflammation through dysregulated adi-pocytokine production. 3T3-L1 preadipocytes were differenti-ated into mature adipocytes prior to use. These cells took up asbestos fibers (chrysotile, crocidolite and amosite) but were more resistant to asbestos-induced injury than macrophages and mesothelial cells. Expression microarray analysis fol-lowed by reverse transcription–PCR revealed that adipocytes respond directly to asbestos exposure with an increased pro-duction of proinflammatory adipocytokines [e.g. monocyte chemoattractant protein-1 (MCP-1)], whereas the production of anti-inflammatory adipocytokines (e.g. adiponectin) is sup-pressed. This was confirmed in epididymal fat pad of mice after intraperitoneal injection of asbestos fibers. Such dysregu-lated adipocytokine production favors the establishment of a proinflammatory environment. Furthermore, MCP-1 margin-ally promoted the growth of MeT-5A mesothelial cells and significantly enhanced the wound healing of Y-MESO-8A and Y-MESO-8D human mesothelioma cells. Our results suggest that increased levels of adipocytokines, such as MCP-1, can potentially contribute to the promotion of mesothelial carcino-genesis through the enhanced recruitment of inflammatory cells as well as a direct growth and migration stimulatory effect on mesothelial and mesothelioma cells. Taken together, our find-ings support a potential cancer-promoting role of adipocytes in asbestos-induced mesothelial carcinogenesis.Introduction

中規模養豚場で1990年9～11月に分娩した母豚のうち93例について, 分娩開始時刻 (第一子) を記録し, 産子のたびに, その時刻や体重を記録した。供試母豚は次子娩出までの間隔が30分以上となったものを対象とし, その時点で, 陣痛促進剤としてオキシトシン25単位 (Oxy 25), カルベトシン (Car; 長時間作用型オキシトン類似体) 0.4mg, 0.2mg, 0.1mgのいずれかを筋注し, 以下の結果が得られた.1. 分娩間隔が30分以上の発生率は非常に高く, 約50%に及び, Car群全て1回注射により, 全例が30分以内に分娩を再開したのに対して, Oxy群では, 10例中4例 (40%) において, 2回注射が必要であった。2. Oxy群では投与前の子豚1頭当たりの分娩所要時間が平均30.7分, 投与後22.8分となり, 投与を100とした場合, 74となり約25%短縮されたが, 投与前後間での有意差は認められなかった. これに対して, Car群では0.4mg群において48.8分が19.7分に, 0.2mg群では34.8分が9.8分に, また0.1mg群でも34.0分が11.2分に短縮され, 同様に投与前を100とした場合には, 40, 28, 33となり, Car全群がOxy群よりも有意に短縮した。しかもOxy群では認められなかった投与前後間の有意な短縮がCar群全てにおいて認められた。3. 母豚当たりの死産の発生率は, Oxy群60.0%と最も多く, 次にCar 0.4mg群, Car 0.2mg群, 無処置群, Car 0.1mg群の順であった。子豚当たりの死産の発生率は, 母豚のそれと同様な傾向であった。4. 分娩後7～21日にかけての子豚の1日の増体量では, Car群の方がOxy群よりも増体が良い傾向であった。5. 分娩後7日目と21日目の子豚の育成率では, 無処置群が92.7%と最も高く, 次にCar 0.2mg群, Car 0.1mg群, Oxy群, Car 0.4mg群の順であった。

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.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.The urinary ratio was shown to be constant and reproducible in the same individuals. The ratio was found to inversely correlate with BMI (P0.01), waist circumference (P0.01), and liver transaminase (P0.05) 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 (P0.01). 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 (P0.01).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.

Increased activity of intracellular glucocorticoid reactivating enzyme, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in obese adipose tissue contributes to adipose dysfunction. As recent studies have highlighted a potential role of preadipocytes in adipose dysfunction, we tested the hypothesis that a variety of metabolic stress mediated by ceramide or AMP-activated protein kinase (AMPK) would regulate 11beta-HSD1 in preadipocytes. The present study is the first to show that 1) expression of 11beta-HSD1 in 3T3-L1 preadipocytes was robustly induced when cells were treated with cell-permeable ceramide analogue C(2) ceramide, bacterial sphingomyelinase, and sphingosine 1-phosphate, 2) 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-induced activation of AMPK augmented the expression and enzyme activity of 11beta-HSD1, and 3) these results were reproduced in human preadipocytes. We demonstrate for the first time that C(2) ceramide and AICAR markedly induced the expression of CCAAT/enhancer-binding protein (C/EBP) beta and its binding to 11beta-HSD1 promoter. Transient knockdown of C/EBPbeta protein by small interfering RNA markedly attenuated the expression of 11beta-HSD1 induced by C(2) ceramide or AICAR. The present study provides novel evidence that ceramide- and AMPK-mediated signaling pathways augment the expression and activity of 11beta-HSD1 in preadipocytes by way of C/EBPbeta, thereby highlighting a novel, metabolic stress-related regulation of 11beta-HSD1 in a cell-specific manner.

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.

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.

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.GPR40 mRNA expression in the human pancreas, pancreatic islets and islet cell tumours was analysed using TaqMan PCR.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).The present study provides evidence for GPR40 gene expression in pancreatic beta cells and implicates GPR40 in insulin secretion in humans.

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, beta 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.

Fatigue tests on the three strand roller chains having various fitting conditions between middle link plates and pins were made. Results are as follows:1. The fatigue strength of treble strand M-type roller chains (standard) having clearance between middle link plates and pins is twice as that of single strand chains.2. The fatigue strength of treble strand P-type roller chains having interference between middle link plates and pins is 2.4 times as large as that of single strand chains.3. The endurance ratio of multiple strand roller chains is smaller than that of single strand roller chains.4. The fatigue strength reduction factor due to increase of strands is nearly independent to the number of cycles to estimate the fatigue strength.5. The fatigue strength reduction factor takes the smallest value at P-type. M-type is the next. The value is the largest at L-type.6. The load distribution for the each strand seems to be nearly uniform at P-type, considering from the form of destruction. In the other types, it seems to be not uniform.

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.

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.

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.