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Free access

A Alidibbiat, C E Marriott, K T Scougall, S C Campbell, G C Huang, W M Macfarlane and J A M Shaw

Generation of new β-cells from the adult pancreas or the embryonic stem cells is being pursued by research groups worldwide. Success will be dependent on confirmation of true β-cell phenotype evidenced by capacity to process and store proinsulin. The aim of these studies was to robustly determine endocrine characteristics of the AR42J rat pancreatic acinar cell line before and after in vitro transdifferentiation. β-cell phenotypic marker expression was characterised by RT-PCR, immunostaining, western blotting, ELISA and in human preproinsulin transgene over-expression studies in wild-type AR42J cells and after culture on Matrigel basement membrane matrix with and without growth/differentiation factor supplementation. Pancreatic duodenal homeobox 1 (PDX1), forkhead box transcription factor a2 (Foxa2), glucokinase, pancreatic polypeptide and low-level insulin gene transcription in wild-type AR42J cells were confirmed by RT-PCR. Culture on Matrigel-coated plates and supplementation of medium with glucagon-like peptide 1 induced expression of the β-cell Glut 2 with maintained expression of insulin and PDX1. Increased biosynthesis and secretion of proinsulin were confirmed by immunocytochemical staining and sensitive ELISA. Absence of the regulated secretory pathway was demonstrated by undetectable prohormone convertase expression. In addition, inability to process and store endogenous proinsulin or human proinsulin translated from a constitutively over-expressed preproinsulin transgene was confirmed. The importance of robust phenotypic characterisation at the protein level in attempted β-cell transdifferentiation studies has been confirmed. Rodent and human sensitive/specific differential proinsulin/insulin ELISA in combination with human preproinsulin over-expression enables detailed elucidatation of core endocrine functions of proinsulin processing and storage in putative new β-cells.

Free access

Andreas Börjesson and Carina Carlsson

In order to elucidate a possible relationship between β-cell function and conversion of proinsulin to insulin, isolated rat pancreatic islets were maintained in tissue culture for 1 week at various glucose concentrations (5.6–56 mM). Studies were also conducted on islets cultured for 48 h with interleukin-1β (IL-1β). By pulse-chase labelling and immunoprecipitation, the relative contents of newly synthesized proinsulin and insulin were determined. ELISA was used to analyse insulin and proinsulin content in medium and within islets. Using real-time PCR, the mRNA levels of proinsulin converting enzymes (PC1 and PC2) were studied. Islets cultured at 56 mM glucose had an increased proportion of newly synthesized proinsulin when compared with islets cultured at 5.6 mM glucose after a 90-min chase periods, however, no difference was observed after culture at 11 and 28 mM glucose. ELISA measurements revealed that culture at increased glucose concentrations as well as islet exposure to IL-1β increased proinsulin accumulation in the culture media. The mRNA expression of PC1 was increased after culture at 11 and 28 mM glucose. Treatment for 48 h with IL-1β increased the proportion of proinsulin both at 45 and 90 min when compared with control islets. These islets also displayed a decreased mRNA level of PC1 as well as PC2. Calculations of the half-time for proinsulin demonstrated a significant prolongation after treatment with IL-1β. We conclude that a sustained functional stimulation by glucose of islets is coupled to a decreased conversion of proinsulin which is also true for islets treated with IL-1β. This may contribute to the elevated levels of proinsulin found both at the onset of type 1 diabetes as well as in type 2 diabetes.

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.

Open access

K E Lines, P J Newey, C J Yates, M Stevenson, R Dyar, G V Walls, M R Bowl and R V Thakker

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by the combined occurrence of parathyroid, pituitary and pancreatic islet tumours, and is due to mutations of the MEN1 gene, which encodes the tumour suppressor protein menin. Menin has multiple roles in genome stability, transcription, cell division and proliferation, but its mechanistic roles in tumourigenesis remain to be fully elucidated. miRNAs are non-coding single-stranded RNAs that post-transcriptionally regulate gene expression and have been associated with tumour development, although the contribution of miRNAs to MEN1-associated tumourigenesis and their relationship with menin expression are not fully understood. Alterations in miRNA expression, including downregulation of three putative ‘tumour suppressor’ miRNAs, miR-15a, miR-16-1 and let-7a, have been reported in several tumour types including non-MEN1 pituitary adenomas. We have therefore investigated the expression of miR-15a, miR-16-1 and let-7a in pituitary tumours that developed after 12 months of age in female mice with heterozygous knockout of the Men1 gene (Men1 +/ mice). The miRNAs miR-15a, miR-16-1 and let-7a were significantly downregulated in pituitary tumours (by 2.3-fold, P < 0.05; 2.1-fold P < 0.01 and 1.6-fold P < 0.05, respectively) of Men1 +/ mice, compared to normal WT pituitaries. miR-15a and miR-16-1 expression inversely correlated with expression of cyclin D1, a known pro-tumourigenic target of these miRNAs, and knockdown of menin in a human cancer cell line (HeLa), and AtT20 mouse pituitary cell line resulted in significantly decreased expression of miR-15a (P < 0.05), indicating that the decrease in miR-15a may be a direct result of lost menin expression.

Restricted access

Ziping Jiang, Junduo Wu, Fuzhe Ma, Jun Jiang, Linlin Xu, Lei Du, Wenlin Huang, Zhaohui Wang, Ye Jia, Laijin Lu and Hao Wu

Over a half of the diabetic individuals develop macrovascular complications that cause high mortality. Oxidative stress (OS) promotes endothelial dysfunction (ED) which is a critical early step toward diabetic macrovascular complications. Nuclear factor erythroid 2-related factor 2 (NRF2) is a master regulator of cellular antioxidant defense system and combats diabetes-induced OS. Previously, we found that impaired NRF2 antioxidant signaling contributed to diabetes-induced endothelial OS and dysfunction in mice. The present study has investigated the effect of microRNA-200a (miR-200a) on NRF2 signaling and diabetic ED. In aortic endothelial cells (ECs) isolated from C57BL/6 wild-type (WT) mice, high glucose (HG) reduced miR-200a levels and increased the expression of kelch-like ECH-associated protein 1 (Keap1) – a target of miR-200a and a negative regulator of NRF2. This led to the inactivation of NRF2 signaling and exacerbation of OS and inflammation. miR-200a mimic (miR-200a-M) or inhibitor modulated KEAP1/NRF2 antioxidant signaling and manipulated OS and inflammation under HG conditions. These effects were completely abolished by knockdown of Keap1, indicating that Keap1 mRNA is a major target of miR-200a. Moreover, the protective effect of miR-200a-M was completely abrogated in aortic ECs isolated from C57BL/6 Nrf2 knockout (KO) mice, demonstrating that NRF2 is required for miR-200a’s actions. In vivo, miR-200a-M inhibited aortic Keap1 expression, activated NRF2 signaling, and attenuated hyperglycemia-induced OS, inflammation and ED in the WT, but not Nrf2 KO, mice. Therefore, the present study has uncovered miR-200a/KEAP1/NRF2 signaling that controls aortic endothelial antioxidant capacity, which protects against diabetic ED.

Free access

Ghania Ramdani, Nadine Schall, Hema Kalyanaraman, Nisreen Wahwah, Sahar Moheize, Jenna J Lee, Robert L Sah, Alexander Pfeifer, Darren E Casteel and Renate B Pilz

NO/cGMP signaling is important for bone remodeling in response to mechanical and hormonal stimuli, but the downstream mediator(s) regulating skeletal homeostasis are incompletely defined. We generated transgenic mice expressing a partly-activated, mutant cGMP-dependent protein kinase type 2 (PKG2R242Q) under control of the osteoblast-specific Col1a1 promoter to characterize the role of PKG2 in post-natal bone formation. Primary osteoblasts from these mice showed a two- to three-fold increase in basal and total PKG2 activity; they proliferated faster and were resistant to apoptosis compared to cells from WT mice. Male Col1a1-Prkg2 R242Q transgenic mice had increased osteoblast numbers, bone formation rates and Wnt/β-catenin-related gene expression in bone and a higher trabecular bone mass compared to their WT littermates. Streptozotocin-induced type 1 diabetes suppressed bone formation and caused rapid bone loss in WT mice, but male transgenic mice were protected from these effects. Surprisingly, we found no significant difference in bone micro-architecture or Wnt/β-catenin-related gene expression between female WT and transgenic mice; female mice of both genotypes showed higher systemic and osteoblastic NO/cGMP generation compared to their male counterparts, and a higher level of endogenous PKG2 activity may be responsible for masking effects of the PKG2R242Q transgene in females. Our data support sexual dimorphism in Wnt/β-catenin signaling and PKG2 regulation of this crucial pathway in bone homeostasis. This work establishes PKG2 as a key regulator of osteoblast proliferation and post-natal bone formation.

Free access

R Wang, N Yashpal, F Bacchus and J Li

Hepatocyte growth factor (HGF) has been suggested to be a potent regulator of β-cell function and proliferation. The purpose of this study was to investigate whether HGF could regulate the proliferation and differentiation of islet-derived epithelial monolayers into insulin-producing cells. We have generated islet-derived epithelial monolayers that are enriched with cells expressing c-Kit, a tyrosine kinase receptor and putative marker, from isolated postnatal rat islets. Monolayers were cultured on type I collagen gel and treated in defined differentiation medium with or without HGF (50 ng/ml) for 7 days. Subsequently, the expression of transcription factors and pancreatic endocrine cell markers as well as c-Kit expression were compared between the HGF (HGF+), no HGF treatment (HGF) and monolayers without differentiation medium (control) groups, using immunocytochemical and RT-PCR approaches. We observed that the number of c-Kit-, glucose transport type 2 (Glut2)- and the transcription factor pancreatic duodenal homeobox-1 (PDX-1)-expressing cells were significantly increased in the HGF+ group. The expression of insulin at the mRNA and protein level was also increased in this treatment group with a 1.7-fold increase in basal insulin release and a 2.3-fold increase in insulin content in comparison with the HGF group. A high proliferative capacity was also found in the HGF+ group. Co-localization of insulin and PDX-1 or Glut2 was revealed frequently in cells treated with HGF+ with occasional co-staining of c-Kit and insulin observed. This study showed that HGF can activate the proliferation and differentiation of islet-derived epithelial monolayer into insulin-producing cells. However, no formation of islet-like clusters was observed. Taken together, this study implies that HGF mediates differentiation of immature cell types into insulin-expressing cells; however, HGF supplementation alone is insuffcient in restoring full β-cell function.

Free access

Noriko Tagawa, Ryosuke Yuda, Sayaka Kubota, Midori Wakabayashi, Yuko Yamaguchi, Daisuke Kiyonaga, Natsuko Mori, Erika Minamitani, Hiroaki Masuzaki and Yoshiharu Kobayashi

17β-Estradiol (E2) serves as an anti-obesity steroid; however, the mechanism underlying this effect has not been fully clarified. The effect of E2 on adipocytes opposes that of glucocorticoids, which potentiate adipogenesis and anabolic lipid metabolism. The key to the intracellular activation of glucocorticoid in adipocytes is 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which catalyses the production of active glucocorticoids (cortisol in humans and corticosterone in rodents) from inactive 11-keto steroids (cortisone in humans and 11-dehydrocorticosterone in rodents). Using differentiated 3T3-L1 adipocytes, we showed that E2 inhibited 11β-HSD1 activity. Estrogen receptor (ER) antagonists, ICI-182 780 and tamoxifen, failed to reverse this inhibition. A significant inhibitory effect of E2 on 11β-HSD1 activity was observed within 5–10 min. Furthermore, acetylation or α-epimerization of 17-hydroxy group of E2 attenuated the inhibitory effect on 11β-HSD1. These results indicate that the inhibition of 11β-HSD1 by E2 depends on neither an ER-dependent route, transcriptional pathway nor non-specific fashion. Hexose-6-phosphate dehydrogenase, which provides the cofactor NADPH for full activation of 11β-HSD1, was unaffected by E2. A kinetic study revealed that E2 acted as a non-competitive inhibitor of 11β-HSD1. The inhibitory effect of E2 on 11β-HSD1 was reproduced in adipocytes isolated from rat mesenteric fat depots. This is the first demonstration that E2 inhibits 11β-HSD1, thereby providing a novel insight into the anti-obesity mechanism of estrogen.

Free access

Qingling Huang, Elena Timofeeva and Denis Richard

The present study was conducted to investigate the long-term effects of subchronic elevation of central leptin levels on the expression of corticotropin-releasing factor (CRF) and its types 1 and 2 receptors in the brain of rats subjected to treadmill running-induced stress. PBS or recombinant murine leptin was infused continuously for a period of 5 days into the third ventricle of rats with the aid of osmotic minipumps at a delivery rate of 2 μg/day. On the fifth day of infusion, rats were killed under resting conditions or after a session of treadmill running, which is known to induce a stress response in rats. Leptin treatment significantly decreased food intake, body weight, white adipose tissue weight, glucose and insulin plasma contents, and blunted the treadmill running-induced elevation in plasma levels of corticosterone. Leptin infusion prevented stress-induced de novo synthesis of CRF in the paraventricular hypothalamic nucleus (PVN), which was measured using the intronic probe for CRF heteronuclear RNA. The induction of the type 1 CRF receptor (CRF1R) in the PVN and supraoptic nucleus in running rats was also significantly blunted by leptin. In contrast, leptin treatment strongly increased the expression of type 2 CRF receptor (CRF2R) in the ventromedial hypothalamic nucleus (VMH). The present results suggest that subchronic elevation of central levels of leptin blunts treadmill running-induced activation of the hypothalamic–pituitary–adrenal axis through the inhibition of activation of the CRFergic PVN neurons, and potentially enhances the anorectic CRF effects via the stimulation of expression of CRF2R in the VMH.

Free access

J Claustre, S Brechet, P Plaisancie, JA Chayvialle and JC Cuber

Postprandial release of peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) from L cells results from both nutrient transit in the ileal lumen and neural drive of endocrine cells. The adrenosympathetic system and its effectors have been shown to induce secretion of L cells in vivo or in vitro. Because these transmitters act through three receptors, beta, alpha1, alpha2, coupled to different intracellular pathways, we evaluated the responses of L cells to specific agonists, using the model of isolated vascularly perfused rat ileum. General stimulation of adrenergic receptors with epinephrine (10(-7) M) induced significant GLP-1 and PYY secretions (94+/-38 and 257+/-59 fmol/8 min respectively) which were abolished upon propranolol (10(-7) M) pretreatment and strongly decreased upon infusion with 10(-8) M prazosin. Blockade of alpha2-receptors with idazoxan (10(-8) M) did not alter epinephrine-induced peptide secretion. The beta-adrenergic agonist isoproterenol (10(-6) M) infused for 30 min induced a transient release of GLP-1 and PYY (integrated release over the 8 min of the peak secretion: 38+/-16 and 214+/-69 fmol for GLP-1 and PYY respectively, P<0.05). Because terbutaline but not dobutamine or BRL 37,344 (10(-5) M) induced significant GLP-1 and PYY secretions (135+/-30 and 305+/-39 fmol/8 min respectively), isoproterenol-induced secretions are suggested to result mainly from stimulation of the beta2-isoreceptor type. In contrast, the alpha1-agonist phenylephrine (10(-7) M) did not stimulate peptide release. When co-infused with 10(-6) M or 10(-7) M isoproterenol, 10(-7) M phenylephrine raised GLP-1 release to 174+/-53 and 108+/-28 fmol/8 min respectively (vs 38+/-16 and 35+/-10 fmol/8 min for isoproterenol alone, P<0.05) whereas PYY secretion was not significantly increased. Clonidine (10(-7) M), an alpha2-agonist, induced a moderate and delayed increase of GLP-1 and PYY but abolished the isoproterenol-induced peptide secretion. Our results showed that general stimulation of adrenergic receptors stimulates the secretory activity of ileal endocrine L cells. The net peptide secretion results from the activation of the beta2-isoreceptor type. Additionally, GLP-1 and PYY secretions are positively modulated by alpha1-receptor stimulation and inhibited by alpha2-receptor activation upon beta-receptor occupation.