The ubiquitously expressed G protein α-subunit Gsα mediates the intracellular cAMP response to glucagon-like peptide 1 (GLP1) and other incretin hormones in pancreatic islet cells. We have shown previously that mice with β-cell-specific Gsα deficiency (βGsKO) develop severe early-onset insulin-deficient diabetes with a severe defect in β-cell proliferation. We have now generated mice with Gsα deficiency throughout the whole pancreas by mating Gsα-floxed mice with Pdx1-cre transgenic mice (PGsKO). PGsKO mice also developed severe insulin-deficient diabetes at a young age, confirming the important role of Gsα signaling in β-cell growth and function. Unlike in βGsKO mice, islets in PGsKO mice had a relatively greater proportion of α-cells, which were spread throughout the interior of the islet. Similar findings were observed in mice with pancreatic islet cell-specific Gsα deficiency using a neurogenin 3 promoter-cre recombinase transgenic mouse line. Studies in the α-cell line αTC1 confirmed that reduced cAMP signaling increased cell proliferation while increasing cAMP produced the opposite effect. Therefore, it appears that Gsα/cAMP signaling has opposite effects on pancreatic α- and β-cell proliferation, and that impaired GLP1 action in α- and β-cells via Gsα signaling may be an important contributor to the reciprocal effects on insulin and glucagon observed in type 2 diabetics. In addition, PGsKO mice show morphological changes in exocrine pancreas and evidence for malnutrition and dehydration, indicating an important role for Gsα in the exocrine pancreas as well.
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- Abstract: Diabetes x
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- Abstract: Glucagon x
- Abstract: IGF* x
- Abstract: Type 1 x
- Abstract: Type 2 x
Tao Xie, Min Chen and Lee S Weinstein
RH McCusker and J Novakofski
Zinc (Zn(2+)), a multifunctional micronutrient, was recently shown to lower the affinity of cell-associated insulin-like growth factor (IGF) binding protein (IGFBP)-3 and IGFBP-5 for both IGF-I and IGF-II, but to increase the affinity of the cell surface type 1 IGF receptor (IGF-1R) for the same two ligands. However, there is a need for data concerning the effects of Zn(2+) on soluble IGFBPs and the type 2 IGF receptor (IGF-2R). In the current work, we demonstrate that Zn(2+) affects the affinity of IGFBP-5 secreted by myoblasts but not IGFBP-4. Zn(2+), at physiological levels, depressed binding of both IGF-I and IGF-II to IGFBP-5, affecting (125)I-IGF-I more than (125)I-IGF-II. Both (125)I-IGF-I and (125)I-IGF-II bound to high and low affinity sites on IGFBP-5. Zn(2+) converted the high affinity binding sites of IGFBP-5 into low affinity binding sites. An IGF-I analog, (125)I-R(3)-IGF-I, did not bind to the soluble murine IGFBP-5. Zn(2+) also decreased the affinity of the IGF-2R on L6 myoblasts. In contrast, Zn(2+) increased IGF-I, IGF-II and R(3)-IGF-I binding to the IGF-1R by increasing ligand binding affinity on both P(2)A(2a)-LISN and L6 myoblasts. Soluble IGFBP-5 and IGFBP-4 depressed the binding of (125)I-IGF-I and (125)I-IGF-II to the IGF-1R, but did not affect binding of (125)I-R(3)-IGF-I. By depressing the association of the IGFs with soluble IGFBP-5, Zn(2+) partitioned (125)I-IGF-I and (125)I-IGF-II from soluble IGFBP-5 onto cell surface IGF-1Rs. This effect is not seen when soluble L6-derived IGFBP-4 is present in extracellular fluids. We introduce a novel mechanism by which the trace micronutrient Zn(2+) may alter IGF distribution, i.e. Zn(2+) acts to increase IGF-1R binding at the expense of IGF binding to soluble IGFBP-5 and the IGF-2R.
E A Parker, A Hegde, M Buckley, K M Barnes, J Baron and O Nilsson
Previous studies of the GH–IGF system gene expression in growth plate using immunohistochemistry and in situ hybridization have yielded conflicting results. We therefore studied the spatial and temporal patterns of mRNA expression of the GH–IGF system in the rat proximal tibial growth plate quantitatively. Growth plates were microdissected into individual zones. RNA was extracted, reverse transcribed and analyzed by real-time PCR. In 1-week-old animals, IGF-I mRNA expression was minimal in growth plate compared with perichondrium, metaphyseal bone, muscle, and liver (70-, 130-, 215-, and 400-fold less). In contrast, IGF-II mRNA was expressed at higher levels than in bone and liver (65- and 2-fold). IGF-II expression was higher in the proliferative and resting zones compared with the hypertrophic zone (P < 0.001). GH receptor and type 1 and 2 IGF receptors were expressed throughout the growth plate. Expression of IGF-binding proteins (IGFBPs)-1 through -6 mRNA was low throughout the growth plate compared with perichondrium and bone. With increasing age (3-, 6-, 9-, and 12-week castrated rats), IGF-I mRNA levels increased in the proliferative zone (PZ) but remained at least tenfold lower than levels in perichondrium and bone. IGF-II mRNA decreased dramatically in PZ (780-fold; P < 0.001) whereas, type 2 IGF receptor and IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4 increased significantly with age in growth plate and/or surrounding perichondrium and bone. These data suggest that IGF-I protein in the growth plate is not produced primarily by the chondrocytes themselves. Instead, it derives from surrounding perichondrium and bone. In addition, the decrease in growth velocity that occurs with age may be caused, in part, by decreasing expression of IGF-II and increasing expression of type 2 IGF receptor and multiple IGFBPs.
Thangiah Geetha, Paul Langlais, Michael Caruso and Zhengping Yi
Skeletal muscle insulin resistance is an early abnormality in individuals with metabolic syndrome and type 2 diabetes (T2D). Insulin receptor substrate-1 (IRS1) plays a key role in insulin signaling, the function of which is regulated by both phosphorylation and dephosphorylation of tyrosine and serine/threonine residues. Numerous studies have focused on kinases in IRS1 phosphorylation and insulin resistance; however, the mechanism for serine/threonine phosphatase action in insulin signaling is largely unknown. Recently, we identified protein phosphatase 1 (PP1) regulatory subunit 12A (PPP1R12A) as a novel endogenous insulin-stimulated interaction partner of IRS1 in L6 myotubes. The current study was undertaken to better understand PPP1R12A's role in insulin signaling. Insulin stimulation promoted an interaction between the IRS1/p85 complex and PPP1R12A; however, p85 and PPP1R12A did not interact independent of IRS1. Moreover, kinase inhibition experiments indicated that insulin-induced interaction between IRS1 and PPP1R12A was reduced by treatment with inhibitors of phosphatidylinositide 3 kinase, PDK1, Akt, and mTOR/raptor but not MAPK. Furthermore, a novel insulin-stimulated IRS1 interaction partner, PP1 catalytic subunit (PP1cδ), was identified, and its interaction with IRS1 was also disrupted by inhibitors of Akt and mTOR/raptor. These results indicate that PPP1R12A and PP1cδ are new members of the insulin-stimulated IRS1 signaling complex, and the interaction of PPP1R12A and PP1cδ with IRS1 is dependent on Akt and mTOR/raptor activation. These findings provide evidence for the involvement of a particular PP1 complex, PPP1R12A/PP1cδ, in insulin signaling and may lead to a better understanding of dysregulated IRS1 phosphorylation in insulin resistance and T2D.
T Matsumoto, S E Gargosky, Y Oh and R G Rosenfeld
The aim of this study was to assess the regulation of insulin-like growth factor-binding proteins (IGFBPs) by IGFs in primary cultures of rat articular chondrocytes (RAC). Employing Western ligand blotting, immunoprecipitation and Northern blot analysis, RAC were found to secrete IGFBP-5 (29 kDa) and IGFBP-4 (24 kDa) as the predominant IGFBPs, as well as IGFBP-2 (32–30 kDa) and IGFBP-3 (43–39 kDa) as the minor species. Treatment of cells with IGF-I and IGF-II resulted in a dose-dependent increase of IGFBP-5 and a small increase in IGFBP-4 in conditioned media (CM). Des(1–3) IGF-I and [Gln6, Ala7,Tyr18, Leu19] IGF-II ([QAYL] IGF-II), which bind to the type 1 IGF receptor but not to IGFBPs, also induced IGFBP-5 peptide, although the increase was less than with IGF-I or IGF-II treatment of RAC. [Leu27] IGF-II, which does not bind to the type 1 IGF receptor but binds to IGFBPs, resulted in little induction of IGFBP-5, while [QAYL-Leu27] IGF-II, which has reduced affinity for both the type 1 IGF receptor and IGFBPs, did not increase IGFBP-5. These data suggest that the increase in IGFBP-5 in CM is modulated by both the type 1 IGF receptor and the interaction between IGFs and IGFBPs. Northern blotting analysis showed that IGF-I, IGF-II and des(1–3) IGF-I treatment of RAC increased steady state levels of IGFBP-5 mRNA, suggesting that the IGF-mediated increase in IGFBP-5 is transcriptionally modulated. Interestingly, the increase in IGFBP-5 peptide levels and mRNA were not parallel, suggesting the possibility of post-translational modifications of IGFBP-5, such as those seen with IGFBP-5 protease. IGFBP-5 protease activity was detectable in untreated CM, whereas treatment with IGF-I and IGF-II partially protected IGFBP-5 from proteolysis. In summary, treatment of RAC with IGF-I and IGF-II results in dose-dependent increases in both IGFBP-5 peptide in the CM and mRNA levels. These changes are mediated by interactions via the type 1 IGF receptor as well as IGFBPs, both transcriptionally and post-translationally.
Journal of Endocrinology (1996) 148, 355–369
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.
Gordon Moody, Pedro J Beltran, Petia Mitchell, Elaina Cajulis, Young-Ah Chung, David Hwang, Richard Kendall, Robert Radinsky, Pinchas Cohen and Frank J Calzone
Ganitumab is a fully human MAB to the human type 1 IGF receptor (IGF1R). Binding assays showed that ganitumab recognized murine IGF1R with sub-nanomolar affinity (K D=0.22 nM) and inhibited the interaction of murine IGF1R with IGF1 and IGF2. Ganitumab inhibited IGF1-induced activation of IGF1R in murine lungs and CT26 murine colon carcinoma cells and tumors. Addition of ganitumab to 5-fluorouracil resulted in enhanced inhibition of tumor growth in the CT26 model. Pharmacological intervention with ganitumab in naïve nude mice resulted in a number of physiological changes described previously in animals with targeted deletions of Igf1 and Igf1r, including inhibition of weight gain, reduced glucose tolerance and significant increase in serum levels of GH, IGF1 and IGFBP3. Flow cytometric analysis identified GR1/CD11b-positive cells as the highest IGF1R-expressing cells in murine peripheral blood. Administration of ganitumab led to a dose-dependent, reversible decrease in the number of peripheral neutrophils with no effect on erythrocytes or platelets. These findings indicate that acute IGF availability for its receptor plays a critical role in physiological growth, glucose metabolism and neutrophil physiology and support the presence of a pituitary IGF1R-driven negative feedback loop that tightly regulates serum IGF1 levels through Gh signaling.
Hong Liu, Jian Guo, Lin Wang, Ning Chen, Andrew Karaplis, David Goltzman and Dengshun Miao
To assess the roles of 1,25-dihydroxyvitamin D (1,25(OH)2D) and parathyroid hormone (PTH) in hard tissue formation in oro-facial tissues, we examined the effect of either 1,25(OH)2D or PTH deficiency on dentin and dental alveolar bone formation and mineralization in the mandibles, and osteoblastic bone formation in long bones of 1α-hydroxylase knockout (1α(OH)ase−/−) mice. Compared with wild-type mice, the mineral density was decreased in the teeth and mandibles, and unmineralized dentin (predentin and biglycan immunopositive dentin) and unmineralized bone matrix in the dental alveolar bone were increased in 1α(OH)ase−/− mice. The dental volume, reparative dentin volume, and dentin sialoprotein immunopositive areas were reduced in 1α(OH)ase−/− mice. The cortical thickness, dental alveolar bone volume, and osteoblast number were all decreased significantly in the mandibles; in contrast, the osteoblast number and surface were increased in the trabecular bone of the tibiae in 1α(OH)ase−/− mice consistent with their secondary hyperparathyroidism. The expression of PTH receptor and IGF1 was reduced slightly in mandibles, but enhanced significantly in the long bones in the 1α(OH)ase−/− mice. To control for the role of secondary hyperparathyroidism, we also examined teeth and mandibles in 6-week-old PTH−/− mice. In these animals, dental and bone volumes in mandibles were not altered when compared with their wild-type littermates. These results suggest that 1,25(OH)2D3 plays an anabolic role in both dentin and dental alveolar bone as it does in long bones, whereas PTH acts predominantly in long bones rather than mandibular bone.
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.
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.