In this study, an insulinoma-associated antigen-1 (INSM1)-binding site in the proximal promoter sequence of the insulin gene was identified. The co-transfection of INSM1 with rat insulin I/II promoter-driven reporter genes exhibited a 40–50% inhibitory effect on the reporter activity. Mutational experiments were performed by introducing a substitution, GG to AT, into the INSM1 core binding site of the rat insulin I/II promoters. The mutated insulin promoter exhibited a three- to 20-fold increase in the promoter activity over the wild-type promoter in several insulinoma cell lines. Moreover, INSM1 overexpression exhibited no inhibitory effect on the mutated insulin promoter. Chromatin immunoprecipitation assays using βTC-1, mouse fetal pancreas, and Ad-INSM1-transduced human islets demonstrated that INSM1 occupies the endogenous insulin promoter sequence containing the INSM1-binding site in vivo. The binding of the INSM1 to the insulin promoter could suppress ∼50% of insulin message in human islets. The mechanism for transcriptional repression of the insulin gene by INSM1 is mediated through the recruitment of cyclin D1 and histone deacetylase-3 to the insulin promoter. Anti-INSM1 or anti-cyclin D1 morpholino treatment of fetal mouse pancreas enhances the insulin promoter activity. These data strongly support the view that INSM1 is a new zinc-finger transcription factor that modulates insulin gene transcription during early pancreas development.
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- Abstract: Diabetes x
- Abstract: Islets x
- Abstract: Insulin x
- Abstract: BetaCells x
- Abstract: Pancreas x
- Abstract: Obesity x
- Abstract: Hyperglycemia x
- Abstract: Hypoglycemia x
- Abstract: Insulinoma x
- Abstract: IGF* x
- Abstract: Type 1 x
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Hong-Wei Wang, Michelle Muguira, Wei-Dong Liu, Tao Zhang, Chiachen Chen, Rebecca Aucoin, Mary B Breslin, and Michael S Lan
Gemma Llauradó, Victòria Ceperuelo-Mallafré, Carme Vilardell, Rafael Simó, Pilar Gil, Albert Cano, Joan Vendrell, and José-Miguel González-Clemente
The aim of this study was to investigate the relationship between advanced glycation end products (AGEs) and arterial stiffness (AS) in subjects with type 1 diabetes without clinical cardiovascular events. A set of 68 patients with type 1 diabetes and 68 age- and sex-matched healthy subjects were evaluated. AGEs were assessed using serum concentrations of N-carboxy-methyl-lysine (CML) and using skin autofluorescence. AS was assessed by aortic pulse wave velocity (aPWV), using applanation tonometry. Patients with type 1 diabetes had higher serum concentrations of CML (1.18 vs 0.96 μg/ml; P=0.008) and higher levels of skin autofluorescence (2.10 vs 1.70; P<0.001) compared with controls. These differences remained significant after adjustment for classical cardiovascular risk factors. Skin autofluorescence was positively associated with aPWV in type 1 diabetes (r=0.370; P=0.003). No association was found between CML and aPWV. Skin autofluorescence was independently and significantly associated with aPWV in subjects with type 1 diabetes (β=0.380; P<0.001) after adjustment for classical cardiovascular risk factors. Additional adjustments for HbA1c, disease duration, and low-grade inflammation did not change these results. In conclusion, skin accumulation of autofluorescent AGEs is associated with AS in subjects with type 1 diabetes and no previous cardiovascular events. These findings indicate that determination of tissue AGE accumulation may be a useful marker for AS in type 1 diabetes.
A Shirakami, T Toyonaga, K Tsuruzoe, T Shirotani, K Matsumoto, K Yoshizato, J Kawashima, Y Hirashima, N Miyamura, CR Kahn, and E Araki
Insulin receptor substrate 1 (IRS-1) gene polymorphisms have been identified in type 2 diabetic patients; however, it is unclear how such polymorphisms contribute to the development of diabetes. Here we introduced obesity in heterozygous IRS-1 knockout (IRS-1(+/-)) mice by gold-thioglucose (GTG) injection and studied the impact of reduced IRS-1 expression on obesity-linked insulin resistance. GTG injection resulted in approximately 30% weight gain in IRS-1(+/-) and wild type (WT) mice, compared with saline-injected controls. There was no difference in insulin sensitivity between lean IRS-1(+/-) and lean WT. Elevated fasting insulin levels but no change in fasting glucose were noted in obese IRS-1(+/-) and WT compared with the respective lean controls. Importantly, fasting insulin in obese IRS-1(+/-) was 1.5-fold higher (P<0.05) than in obese WT, and an insulin tolerance test showed a profound insulin resistance in obese IRS-1(+/-) compared with obese WT. The islets of obese IRS-1(+/-) were 1.4-fold larger than those of obese WT. The expression of insulin receptor and IRS-1 and IRS-2 was decreased in obese IRS-1(+/-), which could in part explain the profound insulin resistance in these mice. Our results suggest that IRS-1 is the suspected gene for type 2 diabetes and its polymorphisms could worsen insulin resistance in the presence of other additional factors, such as obesity.
L Monetini, F Barone, L Stefanini, A Petrone, T Walk, G Jung, R Thorpe, P Pozzilli, and MG Cavallo
Enhanced cellular immune response to bovine beta-casein has been reported in patients with type 1 diabetes. In this study we aimed to establish beta-casein-specific T cell lines from newly diagnosed type 1 diabetic patients and to characterise these cell lines in terms of phenotype and epitope specificity. Furthermore, since sequence homologies exist between beta-casein and putative beta-cell autoantigens, reactivity to the latter was also investigated. T cell lines were generated from the peripheral blood of nine recent onset type 1 diabetic patients with different HLA-DQ and -DR genotypes, after stimulation with antigen pulsed autologous irradiated antigen presenting cells (APCs) and recombinant human interleukin-2 (rhIL-2). T cell line reactivity was evaluated in response to bovine beta-casein, to 18 overlapping peptides encompassing the whole sequence of beta-casein and to beta-cell antigens, including the human insulinoma cell line, CM, and a peptide from the beta-cell glucose transporter, GLUT-2. T cell lines specific to beta-casein could not be isolated from HLA-matched and -unmatched control subjects. beta-Casein T cell lines reacted to different sequences of the protein, however a higher frequency of T cell reactivity was observed towards the C-terminal portion (peptides B05-14, and B05-17 in 5/9 and 4/9 T cell lines respectively). Furthermore, we found that 1 out of 9 beta-casein-specific T cell lines reacted also to the homologous peptide from GLUT-2, and that 3 out of 4 of tested cell lines reacted also to extracts of the human insulinoma cell line, CM. We conclude that T cell lines specific to bovine beta-casein can be isolated from the peripheral blood of patients with type 1 diabetes; these cell lines react with multiple and different sequences of the protein particularly towards the C-terminal portion. In addition, reactivity of beta-casein T cell lines to human insulinoma extracts and GLUT-2 peptide was detected, suggesting that the potential cross-reactivity with beta-cell antigens deserves further investigation.
Xue Jiang, Jia Xiao, Mulan He, Ani Ma, and Anderson O L Wong
Type II suppressor of cytokine signaling (SOCS) serve as feedback repressors for cytokines and are known to inhibit growth hormone (GH) actions. However, direct evidence for SOCS modulation of GH-induced insulin-like growth factor 1 (Igf1) expression is lacking, and the post-receptor signaling for SOCS expression at the hepatic level is still unclear. To shed light on the comparative aspects of SOCS in GH functions, grass carp was used as a model to study the role of type II SOCS in GH-induced Igf1 expression. Structural identity of type II SOCS, Socs1–3 and cytokine-inducible SH2-containing protein (Cish), was established in grass carp by 5’/3’-RACE, and their expression at both transcript and protein levels were confirmed in the liver by RT-PCR and LC/MS/MS respectively. In carp hepatocytes, GH treatment induced rapid phosphorylation of JAK2, STATs, MAPK, PI3K, and protein kinase B (Akt) with parallel rises in socs1–3 and cish mRNA levels, and these stimulatory effects on type II SOCS were shown to occur before the gradual loss of igf1 gene expression caused by prolonged exposure of GH. Furthermore, GH-induced type II SOCS gene expression could be negated by inhibiting JAK2, STATs, MEK1/2, P38 MAPK, PI3K, and/or Akt respectively. In CHO cells transfected with carp GH receptor, over-expression of these newly cloned type II SOCS not only suppressed JAK2/STAT5 signaling with GH treatment but also inhibited GH-induced grass carp Igf1 promoter activity. These results, taken together, suggest that type II SOCS could be induced by GH in the carp liver via JAK2/STATs, MAPK, and PI3K/Akt cascades and serve as feedback repressors for GH signaling and induction of igf1 gene expression.
Sachiko Kitanaka, Utako Sato, and Takashi Igarashi
Mutations in hepatocyte nuclear factor-1β (HNF-1β) lead to type 5 maturity-onset diabetes of the young (MODY5). Moreover, mutations in the HNF-1β gene might cause multiorgan abnormalities including renal diseases, genital malformations, and abnormal liver function. The objective of this study was to investigate the molecular mechanism of diabetes mellitus, intrauterine growth retardation, and cholestasis observed in MODY5 patients. We analyzed the transactivity of wild-type and three mutant HNF-1β on native human insulin, IGF-I, and multidrug resistance protein 2 (MRP2) promoters in combination with HNF-1α, using a reporter-assay system in transiently transfected mammalian cells. In the human insulin gene promoter, we found that the cooperation of HNF-1α and HNF-1β is prominent. Absence of this cooperation was observed in all of the HNF-1β mutants. In the human IGF-I and MRP2 promoters, we found that the HNF-1β His153Asn (H153N) mutant had a mutant-specific repressive effect on both HNF-1α and wild-type HNF-1β transactivity. Absence of the cooperation of HNF-1β mutants with HNF-1α in the human insulin gene promoter might be one cause of defective insulin secretion. The H153N mutant-specific repression of HNF-1α and HNF-1β transactivity in human IGF-I and MRP2 promoters might explain the case-specific clinical features of growth retardation and cholestasis observed only in early infancy. We found differential property of HNF-1α/HNF-1β activity and the effect of HNF-1β mutants by the promoters. We consider that analyses of HNF-1β mutants on the intended human native promoters in combination with HNF-1α may be useful in investigating the molecular mechanisms of the various features in MODY5.
ST Dheen, K Rajkumar, and LJ Murphy
Transgenic mice which overexpress insulin-like growth factor binding protein-1 (IGFPB-1) demonstrate fasting hyperglycemia, hyperinsulinemia and glucose intolerance in adult life. Here we have examined the ontogeny of pancreatic endocrine dysfunction and investigated islet cell proliferation and apoptosis in this mouse model. In addition we have examined pancreatic insulin content in transgenic mice derived from blastocyst transfer into non-transgenic mice. Transgenic mice were normoglycemic at birth but had markedly elevated plasma insulin levels, 56.2 +/- 4.5 versus 25.4 +/- 1.5 pmol/l, p < 0.001, and pancreatic insulin concentration, 60.5 +/- 2.5 versus 49.0 +/- 2.6 ng/mg of tissue, P < 0.01, compared with wild-type mice. Transgenic mice derived from blastocyst transfer to wild-type foster mothers had an elevated pancreatic insulin content similar to that seen in pups from transgenic mice. There was an age-related decline in pancreatic insulin content and plasma insulin levels and an increase in fasting blood glucose concentrations, such that adult transgenic mice had significantly less pancreatic insulin than wild-type mice. Pancreatic islet number and the size of mature islets were increased in transgenic animals at birth compared with wild-type mice. Both islet cell proliferation, measured by 5-bromo-2'-deoxyuridine labeling, and apoptosis, assessed by the in situ terminal deoxynucleotidyl transferase and nick translation assay, were increased in islets of newborn transgenic mice compared with wild-type mice. In adult mice both islet cell proliferation and apoptosis were low and similar in transgenic and wild-type mice. Islets remained significantly larger and more numerous in adult transgenic mice despite a reduction in pancreatic insulin content. These data suggest that overexpression of IGFBP-1, either directly or indirectly via local or systemic mechanisms, has a positive trophic effect on islet development.
D. Janjic and M. Asfari
To investigate further the role of cytokines in the pathogenesis of type I insulin-dependent diabetes mellitus, the effects of interleukin-1β (IL-1), tumour necrosis factor-α (TNF) and γ-interferon (IFN) were tested on rat insulinoma INS-1 cells. Whereas TNF and IFN had, respectively, a minor or no effect on insulin production, IL-1 caused a time- and dose-dependent decrease in insulin release and lowered the insulin content as well as the preproinsulin mRNA content of INS-1 cells. Both IL-1 and TNF exerted a cytostatic effect, estimated by a decrease in [3H]thymidine incorporation, while only IL-1 decreased cell viability as measured by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test.
The glutathione content of INS-1 cells was shown to be modulated by the presence of 2-mercaptoethanol in the culture medium, but was not affected by IL-1 or TNF.
In conclusion, INS-1 cell culture is considered to be a useful model for studying the effect of cytokines on insulin-producing cells. The differentiated features of these cells will permit several questions to be addressed regarding the mechanism of action of IL-1 and eventually other cytokines, both at the level of gene expression and of intracellular signalling.
Journal of Endocrinology (1992) 132, 67–76
PM Jehle, DR Jehle, S Mohan, and BO Bohm
Osteopenia has been ascribed to diabetics without residual insulin secretion and high insulin requirement. However, it is not known if this is partially due to disturbances in the IGF system, which is a key regulator of bone cell function. To address this question, we performed a cross-sectional study measuring serum levels of IGF-I, IGF-binding protein-1 (IGFBP-1), IGFBP-3, IGFBP-4 and IGFBP-5 by specific immunoassays in 52 adults with Type 1 (n=27) and Type 2 (n=25) diabetes mellitus and 100 age- and sex-matched healthy blood donors. In the diabetic patients, we further determined serum levels of proinsulin, intact parathyroid hormone (PTH), 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and several biochemical bone markers, including osteocalcin (OSC), bone alkaline phosphatase (B-ALP), carboxy-terminal propeptide of type I procollagen (PICP), and type I collagen cross-linked carboxy-terminal telopeptide (ICTP). Urinary albumin excretion was ascertained as a marker of diabetic nephropathy. Bone mineral density (BMD) of hip and lumbar spine was determined by dual-energy X-ray absorptiometry. Data are presented as means+/-s.e.m. Differences between the experimental groups were determined by performing a one-way analysis of variance (ANOVA), followed by Newman-Keuls test. Correlations between variables were assessed using univariate linear regression analysis and partial correlation analysis. Type 1 diabetics showed significantly lower IGF-I (119+/-8 ng/ml) and IGFBP-3 (2590+/-104 ng/ml) but higher IGFBP-1 levels (38+/-10 ng/ml) compared with Type 2 patients (170+/-13, 2910+/-118, 11+/-3 respectively; P<0.05) or healthy controls (169+/-5, 4620+/-192, 3.5+/-0.4 respectively; P<0.01). IGFBP-5 levels were markedly lower in both diabetic groups (Type 1, 228+/-9; Type 2, 242+/-11 ng/ml) than in controls (460+/-7 ng/ml,P<0. 01), whereas IGFBP-4 levels were similar in diabetics and controls. IGF-I correlated positively with IGFBP-3 and IGFBP-5 and negatively with IGFBP-1 and IGFBP-4 in all subjects. Type 1 patients showed a lower BMD of hip (83+/-2 %, Z-score) and lumbar spine (93+/-2 %) than Type 2 diabetics (93+/-5 %, 101+/-5 % respectively), reaching significance in the female subgroups (P<0.05). In Type 1 patients, BMD of hip correlated negatively with IGFBP-1 (r=-0.34, P<0.05) and IGFBP-4 (r=-0.3, P<0.05) but positively with IGFBP-5 (r=0.37, P<0. 05), which was independent of age, diabetes duration, height, weight and body mass index, as assessed by partial correlation analysis. Furthermore, biochemical markers indicating bone loss (ICTP) and increased bone turnover (PTH, OSC) correlated positively with IGFBP-1 and IGFBP-4 but negatively with IGF-I, IGFBP-3 and IGFBP-5, while the opposite was observed with bone formation markers (PICP, B-ALP) and vitamin D3 metabolites. In 20 Type 2 patients in whom immunoreactive proinsulin could be detected, significant positive correlations were found between proinsulin and BMD of hip (r=0.63, P<0.005), IGF-I (r=0.59, P<0.01) as well as IGFBP-3 (r=0.49, P<0.05). Type 1 and Type 2 patients with macroalbuminuria showed a lower BMD of hip, lower IGFBP-5 but higher IGFBP-4 levels, suggesting that diabetic nephropathy may contribute to bone loss by a disturbed IGF system. In conclusion, the findings of this study support the hypothesis that the imbalance between individual IGF system components and the lack of endogenous proinsulin may contribute to the lower BMD in Type 1 diabetics.
Alexander Hennebry, Jenny Oldham, Tea Shavlakadze, Miranda D Grounds, Philip Sheard, Marta L Fiorotto, Shelley Falconer, Heather K Smith, Carole Berry, Ferenc Jeanplong, Jeremy Bracegirdle, Kenneth Matthews, Gina Nicholas, Mônica Senna-Salerno, Trevor Watson, and Christopher D McMahon
Insulin-like growth factors (IGFs) and myostatin have opposing roles in regulating the growth and size of skeletal muscle, with IGF1 stimulating, and myostatin inhibiting, growth. However, it remains unclear whether these proteins have mutually dependent, or independent, roles. To clarify this issue, we crossed myostatin null (Mstn −/−) mice with mice overexpressing Igf1 in skeletal muscle (Igf1 +) to generate six genotypes of male mice; wild type (Mstn +/+), Mstn +/−, Mstn −/−, Mstn +/+ :Igf1 +, Mstn +/− :Igf1 + and Mstn −/− :Igf1 +. Overexpression of Igf1 increased the mass of mixed fibre type muscles (e.g. Quadriceps femoris) by 19% over Mstn +/+, 33% over Mstn +/− and 49% over Mstn −/− (P < 0.001). By contrast, the mass of the gonadal fat pad was correspondingly reduced with the removal of Mstn and addition of Igf1. Myostatin regulated the number, while IGF1 regulated the size of myofibres, and the deletion of Mstn and Igf1 + independently increased the proportion of fast type IIB myosin heavy chain isoforms in T. anterior (up to 10% each, P < 0.001). The abundance of AKT and rpS6 was increased in muscles of Mstn −/− mice, while phosphorylation of AKTS473 was increased in Igf1 + mice (Mstn +/+ :Igf1 +, Mstn +/− :Igf1 + and Mstn −/− :Igf1 +). Our results demonstrate that a greater than additive effect is observed on the growth of skeletal muscle and in the reduction of body fat when myostatin is absent and IGF1 is in excess. Finally, we show that myostatin and IGF1 regulate skeletal muscle size, myofibre type and gonadal fat through distinct mechanisms that involve increasing the total abundance and phosphorylation status of AKT and rpS6.