Cell death is increased in the anterior pituitary of poorly controlled diabetic rats, but anti-apoptotic mechanisms are also activated. We hypothesized that specific cell types are selectively protected against diabetes-induced cell death. To determine when anti-apoptotic mechanisms are activated, streptozotocin-induced diabetic rats were killed after 1, 4, 6 and 8 weeks of evolution. Anterior pituitaries were processed for western blot analysis to determine changes in the intrinsic cell death pathway and upstream kinases involved in cell protection mechanisms. An increase in cell death was detected by ELISA at 4 weeks of diabetes. TUNEL labelling demonstrated that this corresponded to death of primarily lactotrophs, a few somatotrophs, and no thyrotrophs, corticotrophs or gonadotrophs. Levels of phosphorylated (p) Akt were increased at 1 week of diabetes, while pERK1/2 levels increased at 4 weeks and pJNK at 6 weeks. Activation of caspase 3 decreased and anti-apoptotic members of the Bcl-2 protein family increased as early as 1 week after diabetes onset. These changes were coincident with increased IGF-I receptor levels. Levels of X-linked inhibitor of apoptosis protein (XIAP) increased significantly after 6 weeks of diabetes, as did activation of nuclear factor (NF)κB. Double immunohistochemistry indicated that XIAP was expressed in less than 1% of lactotrophs and gonadotrophs, approximately 50% of somatotrophs and more than 90% of corticotrophs and thyrotrophs. These results suggest that some cell survival mechanisms are rapidly activated in the anterior pituitary, even before increased cell death can be detected, while others are more delayed. Furthermore, both pituitary cell death and expression of protective mechanisms such as XIAP are cell-type specific.
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- Abstract: Diabetes x
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- Abstract: BetaCells x
- Abstract: Pancreas x
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- Abstract: Hypoglycemia x
- Abstract: Insulinoma x
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- Abstract: Type 1 x
- Abstract: Type 2 x
Ana I Arroba, Alfonso M Lechuga-Sancho, Laura M Frago, Jesús Argente and Julie A Chowen
Hui-Fang Wang, Qing-Qing Yu, Rui-Fang Zheng and Ming Xu
Cardiovascular complications of type 2 diabetes mellitus (T2DM) are associated with vascular remodeling in the arteries. Perivascular sympathetic neurons release an abundance of trophic factors to regulate vascular function via a paracrine signaling. Netrin-1, a diffusible protein that can be secreted outside the cell, is one of common signals of ‘conversation’ between nerve and vessel. The present study investigated whether netrin-1 is a novel modulator of sympathetic neurons paracrine signaling and played a critical role in vascular adventitial remodeling under T2DM. Vascular adventitial remodeling was observed in adventitial fibroblasts (AFs) responding to netrin-1 deficiency in the supernatant from primary rat superior cervical ganglia (SCG) neurons, shown as AFs proliferation, migration, and collagen deposition. Conditioned medium from the high glucose (HG)-treated SCG neurons contributed to AFs remodeling, which was effectively alleviated by exogenous netrin-1 supplementation. Further, it was found that uncoordinated-5-B (Unc5b) was mainly expressed in AFs among netrin-1 specific receptors. Treatment of netrin-1 inhibited H2O2 production derived from NADPH oxidase 4 (NOX4) through the UNC5b/CAMP/PKA signal pathway in AFs remodeling. In vivo, aorta adventitial remodeling was accompanied with the downregulation of netrin-1 in the perivascular sympathetic nerve in T2DM rats. Such abnormalities were restored by netrin-1 intervention, which was associated with the inhibition of NOX4 expression in the aorta adventitia. In conclusion, netrin-1 is a novel modulator of sympathetic neurons paracrine signaling to maintain AFs function. Vascular adventitial remodeling was aggravated by sympathetic neurons paracrine signaling under hyperglycemia, which was ameliorated by netrin-1 treatment through the UNC5b/CAMP/PKA/NOX4 pathway.
Mathieu Lafontaine-Lacasse, Geneviève Doré and Frédéric Picard
The activity and levels of SIRT1, which promotes cell survival in several models, are linked to glucose concentrations and cellular energy metabolism. The present study aimed to determine whether impaired Sirt1 activity is involved in the induction of apoptosis by the nutrient-sensing hexosamine biosynthesis pathway (HBP). Pancreatic Nit-1, Rin-m5F, and Min6 β-cells were acutely treated at different doses and times with glucosamine, which enters and stimulates the HBP. Sirt1 levels were genetically modulated by retroviral infection. Expression levels, cellular localization, and activity of apoptosis-related markers were determined by qPCR, immunoblotting, and co-immunoprecipitation. Glucosamine treatment dose- and time dependently induced cell apoptosis in all cell lines studied. HBP stimulation time dependently modified SIRT1 protein levels, notably in the cytoplasm. This was concomitant with increased E2F1 binding to the c-myc promoter. In both NIT-1 and min6 β-cells, genetic knockdown of Sirt1 expression resulted in higher susceptibility to HBP-stimulated apoptosis, whereas overexpression of Sirt1 had the opposite impact. These findings indicate that reduction of SIRT1 levels by hexosamines contributes to β-cell apoptosis. Methods to increase SIRT1 levels or activity could thus prevent the decrease in β-cell mass, notably that observed in type 2 diabetes.
A Amrani, M Jafarian-Tehrani, P Mormède, S Durant, J-M Pleau, F Haour, M Dardenne and F Homo-Delarche
Cytokines, particularly interleukin 1 (IL-1) and tumor necrosis factor, are known to induce hypoglycemia in normal rodents or different experimental models of type II diabetes. We investigated, at the pre-diabetic stage, the effect of short-term administration of murine recombinant interleukin-1α (mrIL-1α) on the levels of glucose, insulin and corticosterone in the non-obese diabetic (NOD) mouse, a spontaneous model of type I diabetes. Two-month-old, pre-diabetic NOD mice of both sexes were insensitive to mrIL-1α (12·5 and 50 μg/kg) 2 h after administration, the time at which the maximal decrease (around 50%) was observed in the C57BL/6 mouse strain. Kinetic studies however showed that mrIL-1α lowered glycemia in both sexes of NOD mice, but the effect was limited and delayed. In the NOD and C57BL/6 strains, mrIL-1α had no influence on insulin levels in females, but significantly increased them in males (P<0·0001). Castration of NOD males abrogated the stimulatory effect of mrIL-1α on insulin secretion. Corticosterone secretion was stimulated by mrIL-1α in both sexes of NOD and C57BL/6 mice, and this effect was faster and greater in NOD females than in C57BL/6 females. The incomplete hypoglycemic response to mrIL-1α in females may be attributed to the anti-insulin effect of glucocorticoids, an effect which can be demonstrated when mrIL-1α is administered to adrenalectomized animals or when mrIL-1α is administered together with the glucocorticoid antagonist RU38486. In NOD males, in contrast, glucocorticoids did not play a major role in the limited hypoglycemic response to mrIL-1α, since RU38486 and adrenalectomy were not able to unmask a hypoglycemic effect. Moreover, NOD mice of both sexes were less sensitive than C57BL/6 mice to the hypoglycemic effect of insulin (2·5 U/kg), which suggests some degree of insulin-resistance in NOD mice. With regard to the effect of IL-1 on NOD mouse glycemia, therefore, these results suggest that glucocorticoids and/or androgens, according to the animal's sex, may induce a state of insulin-resistance.
Journal of Endocrinology (1996) 148, 139–148
Anna Milanesi, Jang-Won Lee, Qijin Xu, Laura Perin and John S Yu
Promising results of pancreatic islet transplantation to treat type 1 diabetes mellitus, combined with severe shortage of donor pancreata, have spurred efforts to generate pancreatic islet-like cells and insulin-producing β-cells from various progenitor populations. In this study, we show for the first time that multipotent nestin-positive stem cells selected from rat bone marrow can be differentiated into pancreatic ductal and insulin-producing β-cells in vitro. We report an effective multistep protocol in a serum-free system, which could efficiently induce β-cell differentiation from multipotent nestin-positive bone marrow stem cells. To enhance the induction and differentiation toward pancreatic lineage we used trichostatin A, an important regulator of chromatin remodeling, and 5-aza 2′ deoxycytidine, an inhibitor of DNA methylase. All-trans retinoic acid was then utilized to promote pancreatic differentiation. We sequentially induced important transcription factor genes, such as Pdx1, Ngn3, and Pax6, following the in vivo development timeline of the pancreas in rats. Furthermore, in the final stage with the presence of nicotinamide, the induced cells expressed islet and ductal specific markers. The differentiated cells not only expressed insulin and glucose transporter 2, but also displayed a glucose-responsive secretion of the hormone. Our results delineate a new model system to study islet neogenesis and possible pharmaceutical targets. Nestin-positive bone marrow stem cells may be therapeutically relevant for β-cell replacement in type 1 diabetes.
Paul W Caton, Nanda K Nayuni, Julius Kieswich, Noorafza Q Khan, Muhammed M Yaqoob and Roger Corder
Abnormal elevation of hepatic gluconeogenesis is central to the onset of hyperglycaemia in patients with type 2 diabetes mellitus (T2DM). Metformin corrects hyperglycaemia through inhibition of gluconeogenesis, but its mechanism of action is yet to be fully described. SIRT1 and GCN5 (listed as KAT2A in the MGI Database) have recently been identified as regulators of gluconeogenic gene expression through modulation of levels and activity of the coactivators cAMP-response element binding protein-regulated transcription coactivator 2 (TORC2 or CRTC2 as listed in the MGI Database) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α or PPARGC1A as listed in the MGI Database). We report that in db/db mice, metformin (250 mg/kg per day; 7 days) increases hepatic levels of GCN5 protein and mRNA compared with the untreated db/db mice, as well as increases levels of SIRT1 protein and activity relative to controls and untreated db/db mice. These changes were associated with reduced TORC2 protein level and decreased gene expression and activation of the PGC1α gene target phosphoenolpyruvate carboxykinase, and lower plasma glucose and insulin. Inhibition of SIRT1 partially blocked the effects of metformin on gluconeogenesis. SIRT1 was increased through an AMP-activated protein kinase-mediated increase in gene expression of nicotinamide phosphoribosyltransferase, the rate-limiting enzyme of the salvage pathway for NAD+. Moreover, levels of GCN5 were dramatically reduced in db/db mice compared with the controls. This indicates that loss of GCN5-mediated inhibition of gluconeogenesis appears to constitute a major mechanism for the onset of abnormally elevated hepatic glucose production in db/db mice. In conclusion, induction of GCN5 and SIRT1 potentially represents a critical mechanism of action of metformin. In addition, these data identify induction of hepatic GCN5 as a potential therapeutic strategy for treatment of T2DM.
Galya Vassileva, Weiwen Hu, Lizbeth Hoos, Glen Tetzloff, Shijun Yang, Li Liu, Ling Kang, Harry R Davis, Joseph A Hedrick, Hong Lan, Timothy Kowalski and Eric L Gustafson
G-protein-coupled bile acid receptor 1 (GPBAR1/TGR5/M-Bar/GPR131) is a cell surface receptor involved in the regulation of bile acid metabolism. We have previously shown that Gpbar1-null mice are resistant to cholesterol gallstone disease when fed a lithogenic diet. Other published studies have suggested that Gpbar1 is involved in both energy homeostasis and glucose homeostasis. Here, we examine the functional role of Gpbar1 in diet-induced obese mice. We found that body weight, food intake, and fasted blood glucose levels were similar between Gpbar1-null mice and their wild-type (WT) littermates when fed a chow or high-fat diet (HFD) for 2 months. However, insulin tolerance tests revealed improved insulin sensitivity in male Gpbar1 −/− mice fed chow, but impaired insulin sensitivity when fed a HFD. In contrast, female Gpbar1 −/− mice exhibited improved insulin sensitivity when fed a HFD compared with their WT littermates. Female Gpbar1 −/− mice had significantly lower plasma cholesterol and triglyceride levels than their WT littermates on both diets. Male Gpbar1 −/− mice on HFD displayed increased hepatic steatosis when compared with Gpbar1 + / + males and Gpbar1 −/− females on HFD. These results suggest a gender-dependent regulation of Gpbar1 function in metabolic disease.
Guillermo Vazquez-Anaya, Bridget Martinez, José G Soñanez-Organis, Daisuke Nakano, Akira Nishiyama and Rudy M Ortiz
Both hypothyroidism and hyperthyroidism are associated with glucose intolerance, calling into question the contribution of thyroid hormones (TH) on glucose regulation. TH analogues and derivatives may be effective treatment options for glucose intolerance and insulin resistance (IR), but their potential glucoregulatory effects during conditions of impaired metabolism are not well described. To assess the effects of thyroxine (T4) on glucose intolerance in a model of insulin resistance, an oral glucose tolerance test (oGTT) was performed on three groups of rats (n = 8): (1) lean, Long Evans Tokushima Otsuka (LETO), (2) obese, Otsuka Long Evans Tokushima Fatty (OLETF) and (3) OLETF + T4 (8.0 µg/100 g BM/day × 5 weeks). T4 attenuated glucose intolerance by 15% and decreased IR index (IRI) by 34% in T4-treated OLETF compared to untreated OLETF despite a 31% decrease in muscle Glut4 mRNA expression. T4 increased the mRNA expressions of muscle monocarboxylate transporter 10 (Mct10), deiodinase type 2 (Di2), sirtuin 1 (Sirt1) and uncoupling protein 2 (Ucp2) by 1.8-, 2.2-, 2.7- and 1.4-fold, respectively, compared to OLETF. Activation of AMP-activated protein kinase (AMPK) and insulin receptor were not significantly altered suggesting that the improvements in glucose intolerance and IR were independent of enhanced insulin-mediated signaling. The results suggest that T4 treatment increased the influx of T4 in skeletal muscle and, with an increase of DI2, increased the availability of the biologically active T3 to upregulate key factors such SIRT1 and UCP2 involved in cellular metabolism and glucose homeostasis.
JJ Smink, JA Koedam, JG Koster and SC van Buul-Offers
High (pharmacological) doses of glucocorticoids inhibit the proliferation of growth plate chondrocytes, which leads to one of the side-effects of these steroids, namely suppression of longitudinal growth. Growth inhibition by glucocorticoids is thought to be mediated in part by impaired action of components of the IGF axis, which are important for chondrocyte regulation and hence for longitudinal growth. The aim of the present study was to determine whether glucocorticoid-induced growth retardation involves changes in IGF axis components. Chondrocytes were isolated from epiphyseal growth plates of neonatal piglets and treated with pharmacological doses of dexamethasone (DXM) for 24 h to study glucocorticoid-induced growth retardation. Under IGF-I-supplemented (10 nM) culture conditions, IGF-binding proteins (IGFBPs)-2, -4 and -5 were secreted by the growth plate chondrocytes and IGFBP-2 protein and mRNA levels were decreased by the DXM treatment, whereas IGFBP-4 and -5 were not affected. Proliferation of the chondrocytes, as measured by [(3)H]thymidine incorporation, was 3.5-fold higher in serum-supplemented medium in contrast to IGF-I-supplemented (10 nM) medium. In the presence of serum, DNA synthesis was significantly inhibited by 50-63% when treated with 100 nM DXM, which was prevented by the glucocorticoid-receptor antagonist Org34116. mRNA levels of IGF axis components were determined using Northern blot analysis. IGFBP-2 to -6 were expressed in the chondrocytes, IGFBP-1 was absent and both IGF-I and IGF-II, and the type I and type II IGF receptors were expressed. Treatment with DXM (100 nM) resulted in a 2-fold increase in mRNA levels of both IGFBP-5 and the type I IGF receptor, whereas IGFBP-2 mRNA levels decreased by 55%, in concert with the decrease in protein level observed under IGF-I-supplemented culture conditions. The changes in mRNA levels due to the DXM treatment were prevented by the glucocorticoid receptor antagonist. Our data show that exposure to pharmacological doses of DXM results in inhibition of proliferation and changes in components of the IGF axis, IGFBP-2 and -5 and the type I IGF receptor, suggesting a role for these components in glucocorticoid-induced growth retardation at the local level of the growth plate.
E. M. W. Maunder, A. V. Pillay and A. D. Care
An i.v. injection of calcitriol (1,25-(OH)2D3) had no effect within 2·5 h on plasma concentrations of calbindin-D9k (vitamin D-induced calcium-binding protein; CaBP) in hypocalcaemic pigs with inherited vitamin D-dependent rickets type I or in their normocalcaemic siblings or half-siblings. Three days later the plasma concentration of CaBP had doubled in the hypocalcaemic pigs, but was unaltered in the normocalcaemic siblings and half-siblings. Following daily i.v. injections of 1,25-(OH)2D3 for a further 5 days (days 4–8) plasma concentrations of CaBP increased in both the hypocalcaemic (days 4–8) and normocalcaemic (day 8) pigs, the effect being more rapid and greater in the hypocalcaemic 1,25-(OH)2D3-deficient animals. An i.v. injection of 1,25-(OH)2D3 to pure Yucatan pigs also had no effect on plasma concentrations of CaBP within 1·5 h, but in the following 1 h there was some indication of an increase in plasma CaBP levels.
In contrast to the normal pigs, insulin-induced hypoglycaemia did not lead to a peak in plasma CaBP concentrations in the hypocalcaemic pigs. There was also no change in the plasma concentrations of 1,25-(OH)2D3 associated with the peak in plasma CaBP following insulin-induced hypoglycaemia in normocalcaemic pigs. These results suggest that changes in plasma concentrations of 1,25-(OH)2D3 are not directly involved in mediating the increase in plasma CaBP which follows hypoglycaemia induced by insulin in normal pigs, although 1,25-(OH)2D3 probably plays a permissive role.
J. Endocr. (1987) 115, 129–134