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Tetsuhiro Kakimoto, Hirotaka Kimata, Satoshi Iwasaki, Atsushi Fukunari, and Hiroyuki Utsumi

Type 2 diabetes is characterized by impaired insulin secretion from pancreatic β-cells. Quantification of the islet area in addition to the insulin-positive area is important for detailed understanding of pancreatic islet histopathology. Here we show computerized automatic recognition of the islets of Langerhans as a novel high-throughput method to quantify islet histopathology. We utilized state-of-the-art tissue pattern recognition software to enable automatic recognition of islets, eliminating the need to laboriously trace islet borders by hand. After training by a histologist, the software successfully recognized even irregularly shaped islets with depleted insulin immunostaining, which were quite difficult to automatically recognize. The results from automated image analysis were highly correlated with those from manual image analysis. To establish whether this automated, rapid, and objective determination of islet area will facilitate studies of islet histopathology, we showed the beneficial effect of chronic exendin-4, a glucagon-like peptide-1 analog, treatment on islet histopathology in Zucker diabetic fatty (ZDF) rats. Automated image analysis provided qualitative and quantitative evidence that exendin-4 treatment ameliorated the loss of pancreatic insulin content and gave rise to islet hypertrophy. We also showed that glucagon-positive α-cell area was decreased significantly in ZDF rat islets with disorganized structure. This study is the first to demonstrate the utility of automatic quantification of digital images to study pancreatic islet histopathology. The proposed method will facilitate evaluations in preclinical drug efficacy studies as well as elucidation of the pathophysiology of diabetes.

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Ruben Rodriguez, Jacqueline N Minas, Jose Pablo Vazquez-Medina, Daisuke Nakano, David G Parkes, Akira Nishiyama, and Rudy M Ortiz

Obesity is associated with the inappropriate activation of the renin-angiotensin system (RAS), which increases arterial pressure, impairs insulin secretion and decreases peripheral tissue insulin sensitivity. RAS blockade reverses these detriments; however, it is not clear whether the disease state of the organism and treatment duration determine the beneficial effects of RAS inhibition on insulin secretion and insulin sensitivity. Therefore, the objective of this study was to compare the benefits of acute vs chronic angiotensin receptor type 1 (AT1) blockade started after the onset of obesity, hyperglycemia and hypertension on pancreatic function and peripheral insulin resistance. We assessed adipocyte morphology, glucose intolerance, pancreatic redox balance and insulin secretion after 2 and 11 weeks of AT1 blockade in the following groups of rats: (1) untreated Long-Evans Tokushima Otsuka (lean control; n = 10), (2) untreated Otsuka Long-Evans Tokushima Fatty (OLETF; n = 12) and (3) OLETF + ARB (ARB; 10 mg olmesartan/kg/day by oral gavage; n = 12). Regardless of treatment duration, AT1 blockade decreased systolic blood pressure and fasting plasma triglycerides, whereas chronic AT1 blockade decreased fasting plasma glucose, glucose intolerance and the relative abundance of large adipocytes by 22, 36 and 70%, respectively. AT1 blockade, however, did not improve pancreatic oxidative stress or reverse impaired insulin secretion. Collectively, these data show that AT1 blockade after the onset of obesity, hyperglycemia and hypertension improves peripheral tissue insulin sensitivity, but cannot completely reverse the metabolic derangement characterized by impaired insulin secretion once it has been compromised.

Free access

DR Brigstock

The CCN family comprises cysteine-rich 61 (CYR61/CCN1), connective tIssue growth factor (CTGF/CCN2), nephroblastoma overexpressed (NOV/CCN3), and Wnt-induced secreted proteins-1 (WISP-1/CCN4), -2 (WISP-2/CCN5) and -3 (WISP-3/CCN6). These proteins stimulate mitosis, adhesion, apoptosis, extracellular matrix production, growth arrest and migration of multiple cell types. Many of these activities probably occur through the ability of CCN proteins to bind and activate cell surface integrins. Accumulating evidence supports a role for these factors in endocrine pathways and endocrine-related processes. To illustrate the broad role played by the CCN family in basic and clinical endocrinology, this Article highlights the relationship between CCN proteins and hormone action, skeletal growth, placental angiogenesis, IGF-binding proteins and diabetes-induced fibrosis.

Free access

Zhenping Liu, Per Bendix Jeppesen, Søren Gregersen, Lotte Bach Larsen, and Kjeld Hermansen

Chronic hyperglycemia and hyperlipidemia cause deleterious effects on β-cell function. Interestingly, increased circulating amino acid (AA) levels are also a characteristic of the prediabetic and diabetic state. The chronic effects of AAs on β-cell function remain to be determined. Isolated mouse islets and INS-1E cells were incubated with or without excess leucine. After 72 h, leucine increased basal insulin secretion and impaired glucose-stimulated insulin secretion in both mouse islets and INS-1E cells, corroborating the existence of aminoacidotoxicity-induced β-cell dysfunction. This took place concomitantly with alterations in proteins and genes involved in insulin granule transport, trafficking (e.g. collapsin response mediator protein 2 and GTP-binding nuclear protein Ran), insulin signal transduction (proteasome subunit α type 6), and the oxidative phosphorylation pathway (cytochrome c oxidase). Leucine downregulated insulin 1 gene expression but upregulated pancreas duodenum homeobox 1 and insulin 2 mRNA expressions. Importantly, cholesterol (CH) accumulated in INS-1E cells concomitantly with upregulation of enzymes involved in CH biosynthesis (e.g. 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate (diphospho) decarboxylase, and squalene epoxidase) and LDL receptor, whereas triglyceride content was decreased. Our findings indicate that chronic exposure to elevated levels of leucine may have detrimental effects on both β-cell function and insulin sensitivity. Aminoacidotoxicity may play a pathogenic role in the development of type 2 diabetes.

Free access

K Takeda, K Toda, T Saibara, M Nakagawa, K Saika, T Onishi, T Sugiura, and Y Shizuta

Aromatase (CYP19) is a cytochrome P450 enzyme that catalyzes the formation of aromatic C18 estrogens from C19 androgens. It is expressed in various tissues and contributes to sex-specific differences in cellular metabolism. We have generated aromatase-knockout (ArKO) mice in order to study the role of estrogen in the regulation of glucose metabolism. The mean body weights of male ArKO (-/-) mice (n=7) and wild-type littermates (+/+) (n=7) at 10 and 12 weeks of age were 26.7+/-1.9 g vs 26.1+/-0.8 g and 28.8+/-1.4 g vs 26.9+/-1.0 g respectively. The body weights of the ArKO and wild-type mice diverged between 10 and 12 weeks of age with the ArKO males weighing significantly more than their wild-type littermates (P<0.05). The ArKO males showed significantly higher blood glucose levels during an intraperitoneal glucose tolerance test compared with wild-type littermates beginning at 18 weeks of age. By 24 weeks of age, they had higher fasting blood glucose levels compared with wild-type littermates (133.8+/-22.8 mg/dl vs 87.8+/-20.3 mg/dl respectively; P<0.01). An intraperitoneal injection of insulin (0.75 mU insulin/g) caused a continuous decline in blood glucose levels in wild-type mice whereas ArKO males at 18 weeks and older exhibited a rebound increase in glucose levels 30 min after insulin injection. Thus, ArKO male mice appear to develop glucose intolerance and insulin resistance in an age-dependent manner. There was no difference in fasting serum triglyceride and total cholesterol levels between ArKO male mice and wild-type littermates at 13 and 25 weeks of age. However, serum triglyceride and cholesterol levels were significantly elevated following a meal in ArKO mice at 36 weeks of age. Serum testosterone levels in ArKO male mice were continuously higher compared with wild-type littermates. Treatment of ArKO males with 17beta-estradiol improved the glucose response as measured by intraperitoneal glucose and insulin tolerance tests. Treatment with fibrates and thiazolidinediones also led to an improvement in insulin resistance and reduced androgen levels. As complete aromatase deficiency in man is associated with insulin resistance, obesity and hyperlipidemia, the ArKO mouse may be a useful animal model for examining the role of estrogens in the control of glucose and lipid homeostasis.

Free access

F Sentinelli, E Filippi, M G Cavallo, S Romeo, M Fanelli, and M G Baroni

The insulin receptor substrate-1 (IRS-1) plays a central role in insulin sensitivity, and association studies have shown that the IRS-1 G972R variant is a risk factor for insulin resistance. However, how this mutation may lead to impaired insulin sensitivity is still to be determined. Our study aimed to evaluate, after transfection of the IRS-1 G972R variant in 3T3L1 adipocytes, the effect of this mutation on insulin signaling and on cell differentiation. The 3T3L1 cells were transfected with pcDNA3 expression vector containing either the human wild-type IRS-1 or the G972R variant. After induction of differentiation, the 3T3L1 transfected with wild-type IRS-1 differentiated in 6–8 days, while the cells transfected with G972R variant did not differentiate. To determine whether the defect in IRS-1 was responsible for this, we analyzed the expression of several genes involved in the insulin signaling pathway. Results showed that PPARγ expression was significantly reduced in cells transfected with the mutated IRS-1, together with a significant decrease in binding of phosphatidylinositol-3 kinase (PI 3-kinase) to IRS-1 G972R and in PI 3-kinase activity. In addition, we observed that the interaction between the insulin receptor (IR) and the IRS-1 G972R protein was increased and that the autophosphorylation of the IR was significantly inhibited in 3T3L1-G972R cells compared with 3T3L1-WT. Treatment of the 3T3L1-G972R cells with pioglitazone (PIO), a PPARγ agonist, restored differentiation with higher level of PPARγ expression and restoration of PI 3-kinase binding to IRS-1 G972R and PI 3-kinase activity. IR autophosphorylation was also increased. Withdrawal of PIO in fully differentiated 3T3L1-G972R cells determined the reappearance of the insulin signaling defect. Finally, we observed higher levels of IRS-2 expression, suggesting that IRS-2 may play a more important role in adipocyte insulin signaling. In conclusion, IRS-1 G972R variant impairs insulin signaling, and treatment with PPARγ agonist restores the normal phenotype of 3T3L1 cells.

Free access

Sanhua Leng, Wenshuo Zhang, Yanbin Zheng, Ziva Liberman, Christopher J Rhodes, Hagit Eldar-Finkelman, and Xiao Jian Sun

High glucose (HG) has been shown to induce insulin resistance in both type 1 and type 2 diabetes. However, the molecular mechanism behind this phenomenon is unknown. Insulin receptor substrate (IRS) proteins are the key signaling molecules that mediate insulin's intracellular actions. Genetic and biological studies have shown that reductions in IRS1 and/or IRS2 protein levels are associated with insulin resistance. In this study we have shown that proteasome degradation of IRS1, but not of IRS2, is involved in HG-induced insulin resistance in Chinese hamster ovary (CHO) cells as well as in primary hepatocytes. To further investigate the molecular mechanism by which HG induces insulin resistance, we examined various molecular candidates with respect to their involvement in the reduction in IRS1 protein levels. In contrast to the insulin-induced degradation of IRS1, HG-induced degradation of IRS1 did not require IR signaling or phosphatidylinositol 3-kinase/Akt activity. We have identified glycogen synthase kinase 3β (GSK3β or GSK3B as listed in the MGI Database) as a kinase required for HG-induced serine332 phosphorylation, ubiquitination, and degradation of IRS1. Overexpression of IRS1 with mutation of serine332 to alanine partially prevents HG-induced IRS1 degradation. Furthermore, overexpression of constitutively active GSK3β was sufficient to induce IRS1 degradation. Our data reveal the molecular mechanism of HG-induced insulin resistance, and support the notion that activation of GSK3β contributes to the induction of insulin resistance via phosphorylation of IRS1, triggering the ubiquitination and degradation of IRS1.

Free access

Michael P Walker, Richard P DiAugustine, Ernest Zeringue, Maureen K Bunger, Martina Schmitt, Trevor K Archer, and R Gregg Richards

Estrogens are potent mitogens for some target organs, such as the uterus, and cancers that develop in this organ might be linked to the proliferative action of these hormones. However, the mechanism by which estrogens influence the cell cycle machinery is not known. We found that a null mutation for the insulin receptor substrate (IRS)-1, a docking protein that is important for IGF1 signaling, compromised hormone-induced mitosis in the uterine epithelium; BrdU incorporation was not affected. This selective effect on mitosis was associated with a reduction in uterine cyclin B-associated kinase activity; cyclin A-associated kinase activity was not changed. The null mutation also reduced the extent of hormone-induced phosphorylation of endogenous uterine histone H1, as determined with phospho-specific antiserum. Uterine epithelial cyclin dependent kinase (cdk)1 was induced in response to hormone, but the level of the kinase protein, as determined by immunoblotting, was noticeably less in the irs1 null mutant than that in the wild-type (WT) mouse, especially around the time of peak mitosis (24 h). Since IRS-1 binds/activates phosphatidylinositol 3-kinase (PI3K), the absence of this docking protein could impair signaling of a known pathway downstream of AKT that stimulates translation of cell cycle components. Indeed, we found that phosphorylation of uterine AKT (Ser473) in irs1 null mutants was less than that in WTs following treatment. Based on earlier studies, it is also possible that an IGF1/IRS-1/PI3K/AKT pathway regulates posttranslational changes in cdk1. This model may provide insights as to how a growth factor pathway can mediate hormone action on cell proliferation.

Free access

Stuart A Morgan, Zaki K Hassan-Smith, Craig L Doig, Mark Sherlock, Paul M Stewart, and Gareth G Lavery

The adverse metabolic effects of prescribed and endogenous glucocorticoid excess, ‘Cushing’s syndrome’, create a significant health burden. While skeletal muscle atrophy and resultant myopathy is a clinical feature, the molecular mechanisms underpinning these changes are not fully defined. We have characterized the impact of glucocorticoids upon key metabolic pathways and processes regulating muscle size and mass including: protein synthesis, protein degradation, and myoblast proliferation in both murine C2C12 and human primary myotube cultures. Furthermore, we have investigated the role of pre-receptor modulation of glucocorticoid availability by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in these processes. Corticosterone (CORT) decreased myotube area, decreased protein synthesis, and increased protein degradation in murine myotubes. This was supported by decreased mRNA expression of insulin-like growth factor (IGF1), decreased activating phosphorylation of mammalian target of rapamycin (mTOR), decreased phosphorylation of 4E binding protein 1 (4E-BP1), and increased mRNA expression of key atrophy markers including: atrogin-1, forkhead box O3a (FOXO3a), myostatin (MSTN), and muscle-ring finger protein-1 (MuRF1). These findings were endorsed in human primary myotubes, where cortisol also decreased protein synthesis and increased protein degradation. The effects of 11-dehydrocorticosterone (11DHC) (in murine myotubes) and cortisone (in human myotubes) on protein metabolism were indistinguishable from that of CORT/cortisol treatments. Selective 11β-HSD1 inhibition blocked the decrease in protein synthesis, increase in protein degradation, and reduction in myotube area induced by 11DHC/cortisone. Furthermore, CORT/cortisol, but not 11DHC/cortisone, decreased murine and human myoblast proliferative capacity. Glucocorticoids are potent regulators of skeletal muscle protein homeostasis and myoblast proliferation. Our data underscores the potential use of selective 11β-HSD1 inhibitors to ameliorate muscle-wasting effects associated with glucocorticoid excess.

Free access

R Hnasko, M McFarland, and N Ben-Jonathan

Plasmalemma vesicle protein-1 (PV-1) is an integral membrane protein associated with endothelial cell caveolae and fenestrae. Since endocrine glands are enriched with fenestrated endothelium, we examined the distribution of PV-1 mRNA and protein in endocrine glands and determined its cellular localization. A single transcript was detected by RT-PCR in all endocrine glands examined. A synthetic peptide was used to generate antibodies for Western blotting and immunohistochemistry (IHC). Western blotting of membrane fractions from lung, pituitary, adrenal, testis and PV-1-transfected Cos-1 cells revealed a major 65 kDa protein. This protein binds to heparin with high affinity. Using IHC, PV-1 was localized to both endothelial cells of the adrenal zona reticularis and chromaffin cells of the medulla. In the pancreas, PV-1 expression was restricted to a few cells in the islets of Langerhans that partially overlap with somatostatin-positive delta-cells. In both neonatal and adult pituitaries, strong PV-1 immunoreactivity was detected in neural lobe pituicytes in a pattern similar to that of glial fibrillary acidic protein (GFAP). PV-1 and GFAP expression was seen in the adult, but not neonatal, intermediate lobe. Endothelial cells throughout the neonatal anterior lobe were PV-1 positive, but PV-1 in the adult was restricted to some endothelial and endocrine cells localized near the margins of lobe. In the adult testis, strong PV-1 expression was seen in germ cells within the seminiferous tubules that varied with the stage of spermatogenesis. In contrast, PV-1 in the neonatal testis was localized to the interstitial cells but not seminiferous tubules. In the ovary, PV-1 was expressed in stromal endothelial cells as well as the thecal layer of developing follicles. Over half the corpus luteal cells were positive for PV-1. Our data have shown that PV-1 is not restricted to endothelial cells but is localized in many types of endocrine and non-endocrine cells. Furthermore, PV-1 expression in the pituitary and testis is developmentally regulated.