Insulinoma induces a hyperinsulinemia-mediated decrease of GLUT2 and GLP1 receptor in normal pancreatic ß-cells.

There have been several clinical reports of transient postoperative hyperglycemia in patients with insulinoma, but the effect of insulinoma on normal ß-cells has not been investigated. We examined the glucose transporter 2 (GLUT2) and glucagon-like peptide 1 receptor (GLP1R) expression in normal pancreatic ß-cells of five patients with insulinoma and five patients with normal glucose tolerance (NGT) as controls. The positive rate of GLUT2-or GLP1R-positive islets in the nontumor area was calculated by the ratio with the analyzed islets. For functional in vitro analyses, q-PCR and Western blotting were performed after insulin loading on MIN6 cells. The expression rates of both GLUT2 and GLP1R were significantly lower in nontumor area islets of insulinoma patients than in patients with NGT (GLUT2: 31.6 ± 15.3% vs 95.9 ± 6.7%, p < 0.01, GLP1R: 66.8 ± 15.0% vs 96.7 ± 5.0%, p < 0.01). Exposure of MIN6 cells to high concentrations of insulin resulted in a significant decrease in GLUT2 protein for 12 h and GLP1R protein for 24 h (GLUT2; 1.00 ± 0.079 vs 0.81 ± 0.04. p = 0.02, GLP1R; 1.00 ± 0.10 vs 0.50 ± 0.24, p = 0.03) but not in those mRNAs. Our findings show that insulinoma is associated with the downregulation of GLUT2 and GLP1R expression in nontumor area islets. These phenomena may be caused by high levels of insulin.

In tube immunocytochemistry for fluorescence-activated cell sorting that prevents RNA degradation in sorted cells.

Fluorescence-activated cell sorting (FACS) is a powerful tool for analyzing stem cells. When using fixed cells, however, it is sometimes difficult to analyze RNA extracted from sorted cells due to RNA degradation. We established a protocol for immunocytochemistry before FACS to prevent RNA degradation. Cells were fixed with a methanol-based fixative (UM-Fix), then subjected to immunocytochemistry. The addition of RNase inhibitor and dithiothreitol (DTT) to some buffers used for immunocytochemistry increased RNA integrity after cell recovery. We found increased copy numbers of mRNA in recovered cells using quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. When RNase inhibitor and DTT were added, amplification of mRNA using T7 promoter was possible with RNA extracted from recovered cells after FACS. Our protocol ensures high quality RNA in cells recovered by FACS; therefore, gene expression analysis with a smaller number of cells is possible using pre-amplification of mRNAs. Our protocol for immunocytochemistry also might be applicable to RNA recovery after immunostaining.

Predominance of ß-cell neogenesis rather than replication in humans with an impaired glucose tolerance and newly diagnosed diabetes.

CONTEXT: A decrease in pancreatic ß-cell mass is involved in the development of type 2 diabetes. OBJECTIVE: The purpose of this study was to evaluate the ß-cell mass and the incidence of ß-cell neogenesis, replication, and apoptosis at both the prediabetic and diabetic stages. METHODS: We conducted a cross-sectional study of pancreatic tissues obtained from 42 patients undergoing a pancreatectomy who were classified into 4 groups: normal glucose tolerance (n = 11), impaired glucose tolerance (n = 11), newly diagnosed diabetes (n = 10), and long-standing type 2 diabetes (n = 10). RESULTS: The relative ß-cell area decreased and the ß-cell apoptosis increased during the development of diabetes. The number of single and clustered ß-cells, some of which coexpressed nestin, increased in the patients with impaired glucose tolerance and newly diagnosed diabetes. The prevalence of cells positive for both insulin and glucagon or somatostatin also increased in these patients compared with those with normal glucose tolerance. These double-positive cells were mainly localized in single and clustered ß-cells, rather than large islets, and were also positive for Pdx1 or Ngn3. The percentage of insulin-positive cells embedded within ducts increased in the impaired glucose tolerance group. There were no significant differences in the incidence of cells positive for both insulin and Ki67 among the groups. CONCLUSIONS: These results suggest that ß-cell neogenesis, rather than replication, predominates during impaired glucose tolerance and newly diagnosed diabetes in humans and may serve as a compensatory mechanism for the decreased ß-cell mass.

Transforming growth factor ß1 T868C gene polymorphism is associated with cerebral infarction in Japanese patients with type 2 diabetes.

It is likely that the C allele of the polymorphism at position 29 of the translated sequence of transforming growth factor (TGF)-ß1 gene, which codes a pleiotropic cytokine expressed in a variety of cells, is a susceptibility allele for cerebral infarction in Japanese type 2 diabetic patients.

N-linked glycosylation of mouse adiponectin.

Adiponectin is an insulin-sensitizing adipokine with antidiabetic, anti-atherogenic, anti-inflammatory, and cardioprotective properties. Previously, some types of posttranslational modification on adiponectin have been reported. In this study, we demonstrate that mouse adiponectin protein migrated as 2 bands on SDS-PAGE gel. Slower migrating band of adiponectin was reduced by PNGase treatment. PNGase is known as N-glycosidase, and is able to change the mobility of N-glycosylated protein on SDS-PAGE gel. This result indicates the possibility that slower band shifted and overlapped with faster band by cleavage of N-glycan. To further clarify the N-glycosylation of adiponectin, we investigated the effect of N-glycosylation inhibitor tunicamycin on 3T3-L1 adipocytes. Tunicamycin significantly reduced the ratio of slower band to faster band in culture medium from 3T3-L1 adipocytes. This result also indicates the possibility that slower band of adiponectin is N-glycosylated. Lastly, to identify glycosylated asparagine residues, we established 3T3-L1 cell lines stably expressing wild type and mutant adiponectin in N-glycosylation sites. Wild-type adiponectin protein migrated as double bands, and mutant adiponectin in either asparagine at position 53 or threonine at 55 lacked slower band. These results suggest that a part of mouse adiponectin is modified by N-linked glycosylation at asparagine 53.

Aldose reductase C-106T gene polymorphism is associated with diabetic retinopathy in Japanese patients with type 2 diabetes.

It is likely that the C allele of the polymorphism at position -106 in the promoter of aldose reductase gene, which codes a rate-limiting enzyme of the polyol pathway, is a susceptibility allele for diabetic retinopathy in Japanese type 2 diabetic patients.

Measurement of adiponectin production from differentiated metabolic stem cells.

To treat metabolic syndrome, fat tissue dysfunction should be corrected rather than controlling conventional risk factors such as hypertension, dyslipidemia, and diabetes mellitus. For this purpose, accumulating evidence suggests increasing plasma adiponectin levels can be a key treatment strategy, especially in setting of food or drug selection. Here we report that adipocyte precursors obtained from several sites of fat tissue, which we call Metabolic Stem Cells (MSC), could be used as a novel screening system to identify adiponectin enhancing drugs or food for individual patients. MSC were prepared from fat tissues collected from 29 patients. They were differentiated in cultures into mature adipocytes. The time course of adiponectin production was independent of the number of mature adipocytes and gradually decreased at 48 h after differentiation. Pioglitazone, a full PPARgamma agonist, stabilized adiponectin production at days 8-16 after differentiation, whereas telmisartan, a partial PPARgamma agonist, showed variable response. Dividing the adiponectin secretion of day 12 by that of day 10 provided an estimate of adiponectin-producing activity irrespective of the number of MSC-derived adipocytes in culture. Using this score of adiponectin-production activity, we successfully assessed 16 agents in a 96-well plate. The effect of each agent on adiponectin production showed a similar pattern, independent of the site of isolated adipose tissue. Our results show that MSC can be used as a tool for selecting drugs that enhance adiponectin-production activity.

Insulin-mediated regulation of decidual protein induced by progesterone (DEPP) in adipose tissue and liver.

We analyzed the profile of the genes expressed in human adipose tissue and identified the fat-derived molecules, adiponectin and aquaporin 7, which modulate glucose and lipid metabolism. The same Bodymap analysis revealed abundant expression of the decidual protein induced by progesterone (DEPP) in the white adipose tissue. Northern blot analysis confirmed that human DEPP mRNA was highly expressed in white adipose tissue. Mouse DEPP mRNA was detected in heart, lung, skeletal muscle, and white adipose tissue under feeding state. In contrast, under fasting state, mouse DEPP mRNA was enhanced in lung, skeletal muscle, and white adipose tissue and it appeared also in the liver and kidney, suggesting up regulation of DEPP by fasting. Because fasting-induced DEPP expression was observed in insulin-sensitive organs, we investigated the regulation of DEPP in white adipose tissue and liver. During adipogenesis of mouse 3T3-L1 cells, DEPP mRNA increased in a differentiation-dependent manner similar to adiponectin and aquaporin 7. Treatment of cultured 3T3-L1 mature adipocytes, rat H4IIE, and human HepG2 hepatoma cells with insulin significantly decreased DEPP mRNA levels in dose- and time-dependent manners. IN VIVO experiments showed significant decrease of hepatic and adipose DEPP mRNA levels in refed mice, compared to fasted animals, and also showed significant increase in DEPP mRNA in streptozotocin-induced insulin-deficient diabetic mice. These results indicate that DEPP is a novel insulin-regulatory molecule expressed abundantly in insulin-sensitive tissues including white adipose tissue and liver.

Adiponectin plays a protective role in caerulein-induced acute pancreatitis in mice fed a high-fat diet.

BACKGROUND: Obesity is a risk factor for acute pancreatitis (AP), but the molecular mechanism remains unclear. Adiponectin, an adipose tissue-derived secretory factor, has anti-inflammatory properties in addition to various biological functions, and its plasma concentrations are reduced in obese subjects. However, the role of adiponectin in AP has not been investigated. AIM: To determine the effects of adiponectin on AP. METHODS: We investigated the effects of adiponectin on experimental AP by using adiponectin-knockout (APN-KO) mice and adenovirus-mediated adiponectin over-expression. AP was induced by 10 hourly intraperitoneal injections of low-dose caerulein (10 microg/kg) after 2 week feeding of normal chow or a high-fat diet (HFD) in wild-type (WT) and APN-KO mice. We evaluated the severity of AP biochemically and morphologically. RESULTS: Low-dose caerulein treatment did not induce pancreatic damage in either WT or APN-KO mice under normal chow feeding. APN-KO mice, but not WT mice, fed a HFD and then treated with caerulein developed pancreatic damage and inflammation, accompanied by increased macrophage/neutrophil infiltration and upregulation of pro-inflammatory mediators such as tumour necrosis factor alpha in the pancreas. Adenovirus-mediated over-expression of adiponectin attenuated the severity of HFD/caerulein-induced AP in APN-KO mice. CONCLUSIONS: Adiponectin plays a protective role in caerulein-induced AP in HFD-fed mice.

Continuous stimulation of human glucagon-like peptide-1 (7-36) amide in a mouse model (NOD) delays onset of autoimmune type 1 diabetes.

AIMS/HYPOTHESIS: We examined the effect of glucagon-like peptide-1 (GLP-1) on the development of diabetes and islet morphology in NOD mice by administering GLP-1 to prediabetic mice. METHODS: Eight-week-old female NOD mice were infused subcutaneously with human GLP-1 via a mini-osmotic pump for 4 or 8 weeks. In mice treated with GLP-1 for 4 weeks, blood glucose levels and body weight were measured. An intraperitoneal glucose tolerance test (IPGTT) and evaluation of insulitis score were also performed. Beta cell area, proliferation, apoptosis, neogenesis from ducts and subcellular localisation of forkhead box O1 (FOXO1) were examined by histomorphometrical, BrdU-labelling, TUNEL, insulin/cytokeratin and FOXO1/insulin double-immunostaining methods, respectively. RESULTS: Mice treated with human GLP-1 for 4 weeks had lower blood glucose levels until 2 weeks after completion of treatment, showing improved IPGTT data and insulitis score. This effect continued even after cessation of the treatment. In addition to the increase of beta cell neogenesis, BrdU labelling index was elevated (0.24 vs 0.13%, p < 0.001), while apoptosis was suppressed by 54.2% (p < 0.001) in beta cells. Beta cell area was increased in parallel with the translocation of FOXO1 from the nucleus to the cytoplasm. The onset of diabetes was delayed in mice treated with GLP-1 for 4 weeks, while mice treated with GLP-1 for 8 weeks did not develop diabetes by age 21 weeks compared with a 60% diabetes incidence in control mice at this age. CONCLUSIONS/INTERPRETATION: Continuous infusion of human GLP-1 to prediabetic NOD mice not only induces beta cell proliferation and neogenesis, but also suppresses beta cell apoptosis and delays the onset of type 1 diabetes.

Macrophages and dendritic cells infiltrating islets with or without beta cells produce tumour necrosis factor-alpha in patients with recent-onset type 1 diabetes.

AIMS/HYPOTHESIS: Type 1A diabetes results from autoimmune destruction of pancreatic beta cells. We examined the involvement of TNF-alpha and IL-1beta, as well as of T cells, macrophages and dendritic cells, in the destruction of beta cells in patients with recent-onset type 1 diabetes. MATERIALS AND METHODS: We obtained pancreatic biopsy specimens from six patients with recent-onset type 1 diabetes and analysed these by immunohistochemistry. RESULTS: T cell infiltration was less common in islets without beta cells (12.5 [0-33.3]%) than in those with beta cells (46.0 [17.4-83.3]%), while macrophages and dendritic cells showed a similar extent of infiltration into islets both with or without beta cells. TNF-alpha was detected in 25.0 (4.3-46.9)% of macrophages and 11.8 (0-40.0)% of dendritic cells infiltrating the islets in samples from each patient, but not at all in T cells. IL-1beta was detected in 1.8 (0-11.3)% of T cells infiltrating the islets with beta cells, while it was found in 19.2 (0-35.3)% of macrophages or 10.7 (0-31.3)% of dendritic cells infiltrating the islets in samples from each patient (all values median [range]). CONCLUSIONS/INTERPRETATION: Macrophages and dendritic cells infiltrate the islets and produce inflammatory cytokines (TNF-alpha and IL-1beta) during the development of type 1A diabetes.

Blockade of Angiotensin II type-1 receptor reduces oxidative stress in adipose tissue and ameliorates adipocytokine dysregulation.

Dysregulated production of adipocytokines may be involved in the development of atherosclerotic cardiovascular disease in metabolic syndrome and chronic kidney disease (CKD) associated with metabolic syndrome. The aim of this study was to determine the effects of treatment with angiotensin II (Ang II) type-1 receptor blocker (ARB) on the regulation of adipocytokines. Olmesartan, an ARB, significantly blunted the age- and body weight-associated falls in plasma adiponectin both in genetically and diet-induced obese mice, without affecting body weight, but had no effect on plasma adiponectin levels in lean mice. Olmesartan also ameliorated dysregulation of adipocytokines in obesity, such as tumor necrosis factor-alpha, plasminogen activator inhibitor-1, monocyte chemotactic protein-1, and serum amyloid A3. Olmesartan significantly reduced reactive oxygen species originating from accumulated fat and attenuated the expression of nicotinamide adenine dinucleotide phospho hydrogenase oxidase subunits in adipose tissue. In cultured adipocytes, olmesartan acted as an antioxidant and improved adipocytokine dysregulation. Our results indicate that blockade of Ang II receptor ameliorates adipocytokine dysregulation and that such action is mediated, at least in part, by targeting oxidative stress in obese adipose tissue. Ang II signaling and subsequent oxidative stress in adipose tissue may be potential targets for the prevention of atherosclerotic cardiovascular disease in metabolic syndrome and also in metabolic syndrome-based CKD.

Pancreatic beta and alpha cells are both decreased in patients with fulminant type 1 diabetes: a morphometrical assessment.

AIMS/HYPOTHESIS: We have previously reported that fulminant type 1 diabetes is characterised by an absence of diabetes-related antibodies and a remarkably abrupt onset. However, little is known about the mechanism of beta cell destruction in this diabetes subtype, and to obtain insights into the aetiology of the disease, we investigated residual endocrine cells and the expression of Fas and Fas ligand in fulminant type 1 diabetes. METHODS: Residual beta and alpha cells were morphologically assessed in pancreatic tissue obtained by biopsy from five patients with recent-onset fulminant type 1 diabetes and five patients with recent-onset typical autoimmune type 1 diabetes. In addition, the expression of Fas and Fas ligand was evaluated by immunohistochemistry. RESULTS: In fulminant type 1 diabetes, beta and alpha cell areas were decreased significantly, compared with autoimmune type 1 diabetes and control subjects. In contrast, the alpha cell area was not decreased significantly in autoimmune type 1 diabetes, compared with that in control subjects. No Fas expression in islets and Fas ligand expression in CD3(+) cells in the exocrine pancreas were found in the fulminant type 1 diabetic patients who underwent this evaluation. CONCLUSIONS/INTERPRETATION: Our study showed that beta and alpha cells are damaged in fulminant type 1 diabetes. In addition to the lack of Fas and Fas ligand expression, the results suggest that the mechanism of beta cell destruction in fulminant type 1 diabetes is different from that in autoimmune type 1 diabetes.

Production of adiponectin, an anti-inflammatory protein, in mesenteric adipose tissue in Crohn's disease.

BACKGROUND AND AIMS: A characteristic feature of Crohn's disease (CD) is mesenteric adipose tissue hypertrophy. Mesenteric adipocytes or specific proteins secreted by them may play a role in the pathogenesis of CD. We recently identified adiponectin as an adipocyte specific protein with anti-inflammatory properties. Here we report on expression of adiponectin in mesenteric adipose tissue of CD patients. METHODS AND RESULTS: Mesenteric adipose tissue specimens were obtained from patients with CD (n = 22), ulcerative colitis (UC) (n = 8) and, for controls, colon carcinoma patients (n = 28) who underwent intestinal resection. Adiponectin concentrations were determined by enzyme linked immunosorbent assay, and adiponectin mRNA levels were determined by real time quantitative reverse transcription-polymerase chain reaction. Tissue concentrations and release of adiponectin were significantly increased in hypertrophied mesenteric adipose tissue of CD patients compared with normal mesenteric adipose tissue of CD patients (p = 0.002, p = 0.040, respectively), UC patients (p = 0.002, p = 0.003), and controls (p<0.0001, p<0.0001). Adiponectin mRNA levels were significantly higher in hypertrophied mesenteric adipose tissue of CD patients than in paired normal mesenteric adipose tissue from the same subjects (p = 0.024). Adiponectin concentrations in hypertrophied mesenteric adipose tissue of CD patients with an internal fistula were significantly lower than those of CD patients without an internal fistula (p = 0.003). CONCLUSIONS: Our results suggest that adipocytes in hypertrophied mesenteric adipose tissue produce and secrete significant amounts of adiponectin, which could be involved in the regulation of intestinal inflammation associated with CD.

Plasma adiponectin levels in women with anorexia nervosa.

Adiponectin is a plasma protein exclusively secreted by adipose tissue, which plays a role in modulating lipid and glucose metabolism. The plasma adiponectin concentration shows an inverse correlation with the body mass index in normal and obese individuals, but it has not been investigated in subjects with an extremely low body weight and undernutrition such as anorexia nervosa patients. We investigated plasma adiponectin levels in 21 females with anorexia nervosa. Nineteen healthy females served as the lean control group. The subjects with anorexia nervosa had a significantly lower weight and showed a tendency towards higher adiponectin levels than the control group. No correlation between adiponectin and BMI was found in patients with anorexia nervosa, while a linear negative correlation was seen in lean controls. The patient who showed the lowest adiponectin level reached a life-threatening state and required intravenous feeding in hospital. In association with improved nutrition and weight gain, the adiponectin level increased gradually until the body mass index was about 16 and then decreased subsequently as would be expected in lean normal subjects. These observations suggest that adipose tissue secretes less adiponectin and the adiponectin levels do not show an inverse correlation simply with body mass index in some subjects with severe undernutrition.

Effects of soy protein diet on the expression of adipose genes and plasma adiponectin.

Many studies have reported the cholesterol-lowering, anti-lipogenic, anti-obesity and anti-hypertensive effects of soy protein. Adipose tissue-specific plasma protein, adiponectin, has anti-atherogenic and anti-insulin-resistance properties. Here, we investigated the effects of soy protein diet on body fat composition, plasma glucose, lipid and adiponectin levels and expression of genes involved in glucose and fatty acid metabolism in obese KK-A y mice. Body weights and adipose tissue weights of mesenteric, epididymal, and brown fat were lower in mice on calorie-restricted diet containing soy protein isolate. Plasma cholesterol, triglyceride, free fatty acid, and glucose levels were also decreased by this diet. Body fat content and plasma glucose levels in mice on a soy protein isolate diet were still lower than those treated with an isocaloric casein-protein-diet. Among the genes related to glucose and fatty acid metabolism, adiponectin mRNA levels in adipose tissue and adiponectin plasma concentrations were elevated in mice on a calorie-restricted diet, although there were no significant differences between soy protein and casein protein groups. Our results indicate that that soy protein diet decreased body fat content and plasma glucose levels more effectively than isocaloric casein-protein diet in obese mice.

Enhancement of the aquaporin adipose gene expression by a peroxisome proliferator-activated receptor gamma.

The current study demonstrates that aquaporin adipose (AQPap), an adipose-specific glycerol channel (Kishida, K., Kuriyama, H., Funahashi, T., Shimomura, I., Kihara, S., Ouchi, N., Nishida, M., Nishizawa, H., Matsuda, M., Takahashi, M., Hotta, K., Nakamura, T., Yamashita, S., Tochino, Y., and Matsuzawa, Y. (2000) J. Biol. Chem. 275, 20896-20902), is a target gene of peroxisome proliferator-activated receptor (PPAR) gamma. The AQPap mRNA amounts increased following the induction of PPARgamma in the differentiation of 3T3-L1 adipocytes. The AQPap mRNA in the adipose tissue increased when mice were treated with pioglitazone (PGZ), a synthetic PPARgamma ligand, and decreased in PPARgamma(+/-) heterozygous knockout mice. In 3T3-L1 adipocytes, PGZ augmented the AQPap mRNA expression and its promoter activity. Serial deletion of the promoter revealed the putative peroxisome proliferator-activated receptor response element (PPRE) at -93/-77. In 3T3-L1 preadipocytes, the expression of PPARgamma by transfection and PGZ activated the luciferase activity of the promoter containing the PPRE, whereas the PPRE-deleted mutant was not affected. The gel mobility shift assay showed the direct binding of PPARgamma-retinoid X receptor alpha complex to the PPRE. DeltaPPARgamma, which we generated as the dominant negative PPARgamma lacking the activation function-2 domain, suppressed the promoter activity in 3T3-L1 cells, dose-dependently. We conclude that AQPap is a novel adipose-specific target gene of PPARgamma through the binding of PPARgamma-retinoid X receptor complex to the PPRE region in its promoter.