ProSAAS is a neuroendocrine peptide precursor that potently inhibits prohormone convertase 1 in vitro. To explore the function of proSAAS and its derived peptides, transgenic mice were created which express proSAAS using the beta-actin promoter. The body weight of transgenic mice was normal until approximately 10-12 weeks, and then increased 30-50% over wild-type littermates. Adult transgenic mice had a fat mass approximately twice that of wild-type mice, and fasting blood glucose levels were slightly elevated. In the pituitary, the levels of several fully processed peptides in transgenic mice were not reduced compared with wild-type mice, indicating that the proSAAS transgene did not affect prohormone convertase 1 activity in this tissue. Because the inhibitory potency of proSAAS-derived peptides towards prohormone convertase 1 is much greater in the absence of carboxypeptidase E activity, the proSAAS transgene was also expressed in carboxypeptidase E-deficient Cpe (fat/fat) mice. Although the transgenic mice were born in the expected frequency, 21 of 22 proSAAS transgenic Cpe (fat/fat) mice died between 11 and 26 weeks of age, presumably due to greatly elevated blood glucose. The levels of several pituitary peptides were significantly reduced in the proSAAS transgenic Cpe (fat/fat) mice relative to non-transgenic Cpe (fat/fat) mice, suggesting that the transgene inhibited prohormone convertase 1 in these mice. Taken together, these results are consistent with a role for proSAAS-derived peptides as neuropeptides that influence body weight independently of their function as inhibitors of prohormone convertase 1.
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- Abstract: Diabetes x
- Abstract: Islets x
- Abstract: Insulin x
- Abstract: BetaCells x
- Abstract: Pancreas x
- Abstract: Obesity x
- Abstract: Glucose x
- Abstract: Hyperglycemia x
- Abstract: Hypoglycemia x
- Abstract: Insulinoma x
- Abstract: Glucagon x
- Abstract: IGF* x
- Abstract: Type 1 x
S Wei, Y Feng, FY Che, H Pan, N Mzhavia, LA Devi, AA McKinzie, N Levin, WG Richards and LD Fricker
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.
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.
V Csernus, AV Schally and K Groot
Antagonistic analogs of GHRH inhibit growth of various human cancers both in vivo and in vitro. To elucidate the mechanism of direct action of the antagonistic analogs of GHRH on tumor cells, cultured human cancer cells were exposed to GHRH, vasoactive intestinal peptide (VIP), secretin, glucagon, neuropeptide-Y (NPY), pituitary adenylate cyclase-activating peptide (PACAP), and VIP analogs in a superfusion system, and changes in cAMP and IGF-II release from the cells were measured. Various human cancer cell lines, such as mammary (MDAMB-468 and ZR-75-1), prostatic (PC-3), pancreatic (SW-1990 and Capan-2), ovarian (OV-1063), and colorectal (LoVo) responded to pulsatile stimuli with GHRH (0.5-20 nM), VIP (0.02-10 nM), and PACAP-38 (0.05-5 nM) with a rapid, transient increase in cAMP release from the cells. The VIP antagonist, PG-97-269, and the adenylate cyclase inhibitor, MDL-12330A, but not SQ-22536 or pertussis toxin, blocked the cAMP responses to these peptides. Stimulation of the cells with 100 nM secretin, glucagon or NPY did not alter the cAMP release. Our results suggest that GHRH receptors different from the type expressed in the pituitary are involved in mediating these effects. As cAMP is a potent second messenger controlling a wide variety of intracellular functions, including those required for cell growth, our results indicate that GHRH might have a direct stimulatory effect on growth of human cancers. Blockade of the autocrine/paracrine action of GHRH with its antagonistic analogs may provide a new approach to tumor control.
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.
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.
Qiongge Zhang, Chaoqun Wang, Yehua Tang, Qiangqiang Zhu, Yongcheng Li, Haiyan Chen, Yi Bao, Song Xue, Liangliang Sun, Wei Tang, Xiangfang Chen, Yongquan Shi, Lefeng Qu, Bin Lu and Jiaoyang Zheng
Hyperglycemia plays a major role in the development of diabetic macrovascular complications, including atherosclerosis and restenosis, which are responsible for the most of disability and mortality in diabetic patients. Osteopontin (OPN) is an important factor involved in atherogenesis, and hyperglycemia enhances the transcriptional activity of FoxO1 which is closely association with insulin resistance and diabetes. Here, we showed that plasma OPN levels were significantly elevated in type 2 diabetic patients and positively correlated with glycated albumin (GA). The more atherosclerotic lesions were observed in the aorta of diabetic ApoE−/− mice analyzed by Sudan IV staining. High glucose increased both the mRNA and protein expression levels of OPN and inhibited the phosphorylation of FoxO1 in RAW 264.7 cells. Overexpression of WT or constitutively active mutant FoxO1 promoted the expression levels of OPN, while the dominant-negative mutant FoxO1 decreased slightly the expression of OPN. Conversely, knockdown of FoxO1 reduced the expression of OPN. Luciferase reporter assay revealed that high glucose and overexpression of FoxO1 enhanced the activities of the OPN promoter region nt −1918 ~ −713. Furthermore, the interactions between FoxO1 and the OPN promoter were confirmed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). Our results suggest that high glucose upregulates OPN expression via FoxO1 activation, which would play a critical role in the development of diabetic atherogenesis.
RN Kulkarni, ZL Wang, RM Wang, DM Smith, MA Ghatei and SR Bloom
We have studied the effects of first and second generation sulphonylureas on the release of insulin and neuropeptide tyrosine (NPY) from hamster insulinoma tumour (HIT T15) cells and isolated rat islets. In the presence of 5.5 mmol/l glucose all sulphonylureas stimulated insulin release from the HIT cells (P<0.01 ANOVA, n> or =4) but only glibenclamide (GLIB, 10 micromol/l) stimulated the release of NPY (mean+/-s.e.m. control 11.1+/-1.3 vs GLIB 28.4+/-4.1 fmol/h per 10(6) cells, P<0001, n=16). In isolated perifused rat islets both glibenclamide (10 micromol/l) (control 3.5+/-0.3 vs GLIB 6. 3+/-0.2 fmol/min per islet, P<0.01, n=6) and tolbutamide (50 micromol/l) (control 4.7+/-0.1 vs TOLB 6.7+/-0.3 fmol/min per islet, P<0.01, n=6) enhanced glucose (8 mmol/l)-stimulated insulin release. However, only glibenclamide stimulated the release of NPY from the islets (control 3.4+/-0.8 vs GLIB 24.5+/-5 attomol/min per islet, P<0.01, n=6). Similar results were obtained in islets isolated from dexamethasonetreated rats. Glibenclamide treatment of HIT cells showed a prompt insulin release (10 min) while NPY secretion was slower (60 min), suggesting that internalization of the sulphonylurea is required to stimulate NPY release. Glibenclamide, the most common oral therapeutic agent in type 2 diabetes mellitus, is associated with release of the autocrine insulin secretion inhibitor, NPY.
TY Tai, JY Lu, CL Chen, MY Lai, PJ Chen, JH Kao, CZ Lee, HS Lee, LM Chuang and YM Jeng
This study aimed at elucidating the effects of interferon (IFN)-alpha on glucose metabolism in patients with chronic hepatitis B and C infections. Twenty-eight biopsy-proven patients with chronic hepatitis B (ten cases) and hepatitis C (18 cases) were given IFN-alpha for a total of 24 weeks. The patients received a 75 g oral glucose tolerance test (OGTT), glucagon stimulation test, tests for type 1 diabetes-related autoantibodies and an insulin suppression test before and after IFN-alpha therapy. Ten of the 28 patients responded to IFN-alpha therapy. Steady-state plasma glucose of the insulin suppression test decreased significantly in responders (13.32+/-1.48 (S.E.M.) vs 11.33+/-1.19 mmol/l, P=0.0501) but not in non-responders (12.29+/-1.24 vs 11.11+/-0.99 mmol/l, P=0.2110) immediately after completion of IFN-alpha treatment. In the oral glucose tolerance test, no significant difference was observed in plasma glucose in either responders (10.17+/-0.23 vs 10.03+/-0.22 mmol/l) or non-responders (10.11+/-0.22 vs 9.97+/-0.21 mmol/l) 3 Months after completion of IFN-alpha treatment. However, significant differences were noted in C-peptide in both responders (2.90+/-0.13 vs 2.20+/-0.09 nmol/l, P=0.0040) and non-responders (2.45+/-0.11 vs 2.22+/-0.08 nmol/l, P=0.0287) before vs after treatment. The changes of C-peptide in an OGTT between responders and non-responders were also significantly different (P=0.0028), with responders reporting a greater reduction in C-peptide. No case developed autoantibodies during the treatment. In patients who were successfully treated with IFN-alpha, insulin sensitivity improved and their plasma glucose stayed at the same level without secreting as much insulin from islet beta-cells.
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.