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M. Tepel, S. Bauer, S. Husseini, A. Raffelsiefer, and W. Zidek


Cytosolic free sodium concentrations ([Na+]i) in intact platelets from 32 type 2 (non-insulin-dependent) diabetic patients and from 27 age- and sex-matched non-diabetic control subjects were measured with the novel sodium-sensitive fluorescent dye sodium-binding-benzofuran-isophthalate. [Na+]i was significantly higher in platelets from type 2 diabetic patients compared with control subjects (40·6 ± 2·4 vs 32·0 ± 2·0 mmol/l, means ± s.e.m., P<0·03). Both systolic and diastolic blood pressure were significantly elevated in diabetic patients compared with control subjects. Analysis of diabetic patients showed a significant association between [Na+]i and diastolic blood pressure (P =0·026). Stimulation of Na/H exchange by thrombin increased [Na+]i in both groups. After inhibition of Na/K/ATPase by ouabain (1 mmol/l), [Na+]i was significantly increased both in diabetic patients and non-diabetic subjects in a similar way (by 40·2 ± 7·3 and 31·7 ± 5·3 mmol/l respectively). It is concluded that increased [Na+]i in cells from type 2 diabetic patients may be related to hypertension.

Journal of Endocrinology (1993) 138, 565–572

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J. M. H. M. Reul, F. R. van den Bosch, and E. R. de Kloet


The rat brain contains two receptor systems for corticosterone: the type-I corticosterone-preferring receptor and the classical type-II glucocorticoid receptor. The two receptor populations can be distinguished in binding studies with the 'pure' synthetic glucocorticoid 11β,17β-dihydroxy-6-methyl-17α (1-propynyl)-androsta-1,4,6-trione-3-one (RU 28362). In-vitro autoradiography and quantitative image analysis showed that the type-I receptor was localized almost exclusively in the hippocampus, whereas the type-II receptor extended throughout the brain, with the highest levels in the nucleus paraventricularis, nucleus supraopticus and in the thalamic, amygdaloid, hippocampal and septal regions. Unoccupied type-I and type-II receptor sites, as measured in vitro by cytosol binding of 3H-labelled steroids, displayed a large difference in the rate of appearance after adrenalectomy. The availability of type-I receptors exhibited a marked increase, reaching maximal levels within 4–7 h, and then remained constant until 2 weeks after adrenalectomy. The availability of type-II receptors did not change considerably during the first 24 h after adrenalectomy, but displayed a large increase in capacity during the subsequent 2 weeks. After adrenocortical activation as a consequence of exposure to a novel environment, plasma concentrations of corticosterone increased to reach a peak of 811 nmol/l after 30 min and attained the basal concentration (43 nmol/l) after 240 min. During this time, occupation of type-I receptors increased from 77·8% at 0 min to 97% at 30–60 min and then declined to 84·8% after 240 min. Occupation of the type-II receptors was 28·1% at 0 min, 74·5% after 30 min and 32·8% after 240 min. Injection of dexamethasone (25 μg/100 g body wt) at 08.00 h resulted in suppression of basal plasma concentrations of corticosterone and prevented the circadian-driven rise in circulating corticosterone. Occupation of type-I receptors did not change considerably as a result of injection of dexamethasone, but occupation of type-II receptors was markedly increased till 16.00 h compared with that after injection of vehicle.

It was concluded that the type-I and type-II receptors are not only localized differently in the rat brain, but also exhibit a striking difference in occupation after manipulation of the pituitary-adrenocortical system. The data further support the concept of a type-I receptor-mediated tonic activating influence and a type-II receptor-mediated feedback action of corticosterone on brain function.

J. Endocr. (1987) 115, 459–467

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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

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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.

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P. J. Miettinen, T. Otonkoski, and R. Voutilainen


To understand the development of the human pancreas better, we studied the expression and regulation of insulin, insulin-like growth factor-II (IGF-II) and transforming growth factor-α (TGF-α) genes in the human fetal pancreas and islet-like cell clusters (ICC) from the second trimester human fetuses. Northern blot analysis revealed an abundant expression of IGF-II, insulin and TGF-α mRNAs in the intact pancreas and the cultured ICCs. Furthermore, transcripts for insulin receptor, type-1 and -2 IGF receptors, and GH receptor could be amplified by polymerase chain reaction analysis from the pancreas and the ICCs. With in-situ hybridization, IGF-II mRNA was found in abundance in both the exocrine and endocrine pancreas, exceeding the amount of insulin mRNA. In ICCs, insulin mRNA-containing cells were present as small clusters in the periphery and in the centre of the clusters corresponding to the immunolocation of insulin. The ICCs also contained many epidermal growth factor-, insulin- and type-1 IGF receptor- and TGF-α-positive cells.

When the ICCs were cultured in the presence of various secretagogues, only dibutyryl cyclic AMP was found to up-regulate insulin mRNA (39%; P < 0·05). IGF-II mRNA was also under cyclic AMP-dependent regulation (threefold increase; P = 0·025). Furthermore, blocking the type-1 IGF receptor with a monoclonal receptor antibody drastically reduced insulin expression (87%; P = 0·005) and additionally down-regulated IGF-II mRNA (49%; P = 0·005). IGF-1, IGF-II, TGF-α or epidermal growth factor-receptor antibody had no significant effect on either insulin or IGF-II mRNA. Exogenous TGF-α inhibited the release of insulin by the ICCs. It was concluded that IGF-II and TGF-α may be involved in the regulation of islet growth and differentiation.

Journal of Endocrinology (1993) 138, 127–136

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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.

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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.

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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.

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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.

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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.