Biomedical subjects

D E Moller

Publications and source records attributed to D E Moller.

At least 55 records · Page 3Linked to original sources

PubMed1996

Insulin- and mitogen-activated protein kinase-mediated phosphorylation and activation of peroxisome proliferator-activated receptor gamma.

Peroxisome proliferator-activated receptor (PPAR) gamma plays an important role in adipocyte differentiation and the regulation of adipocyte gene expression. Insulin also serves to promote adipogenesis. We report that insulin and a PPARgamma ligand (thiazolidinedione (TZD)) stimulate in a synergistic manner the expression of an adipocyte-specific gene (aP2) in rat adipocytes and 3T3-L1 cells. Potential cross-talk between insulin signaling and PPARgamma was studied in Chinese hamster ovary cells expressing insulin receptors (CHO.T), PPARgamma, and reporter genes. Both TZD and insulin independently stimulated PPARgamma-mediated transactivation of aP2 promoter-luciferase reporter genes; both agents combined resulted in a synergistic effect. Co-transfection of CHO.T cells with dominant-negative mitogen-activated protein (MAP) kinase-kinase (MKK1) abrogated both insulin- and TZD-mediated activation of PPARgamma; transactivation was markedly increased in cells co-transfected with constitutively active MKK1. Both insulin and constitutively active MKK1 also stimulated 32P incorporation into PPARgamma in vivo. The conclusions are: 1) Insulin synergizes with a PPARgamma ligand and can activate the receptor in a ligand-independent fashion. 2) PPARgamma is phosphorylated in vivo by insulin stimulation or activation of the MAP kinase pathway. 3) MAP kinase is an important mediator of cross-talk between insulin signal transduction pathways and PPARgamma function.

3T3 Cells↗

PubMed1996

Regulation and interaction of pp90(rsk) isoforms with mitogen-activated protein kinases.

Each of the three known mammalian 90-kDa S6 kinase (pp90(rsk)) isoforms (RSK1, RSK2, and RSK3) was expressed in transfected cells and further characterized. The kinase activity (immunocomplex toward S6 peptide) of each isoform was activated by in vivo growth factor (epidermal growth factor (EGF)) stimulation; RSK1 was more responsive (10-15-fold) versus RSK2 and RSK3 (2-4-fold). Pretreatment with PD98059 (MEK1 inhibitor) partially (80%) blocked EGF-mediated ERK1 activation and had similar effects on EGF stimulation of each ribosomal S6 kinase (RSK). Cotransfection with dominant-negative MEK1 inhibited activation of each RSK; furthermore, the kinase activity of RSK1, RSK2, and RSK3 was markedly increased by cotransfection with constitutively active MEK1. A specific association between mitogen-activated protein kinases (MAPKs) (ERK1 and ERK2) and RSK isoforms was tested by MAPK immunoblotting after immunoprecipitation of RSKs. ERK1 and ERK2 were present in RSK3 (and to a lesser extent, RSK2) immunoprecipitates, but were absent in RSK1 immunoprecipitates. Both dephosphorylated (from quiescent cells) and phosphorylated (from stimulated cells) MAPKs were associated with RSK2 and RSK3. Deletion mutants of RSK3 were characterized: the C terminus (33 residues) was shown to be required for association with MAPKs. The kinase activity of RSK1 or RSK2 was enhanced by in vitro incubation with ERK1. In contrast, RSK3 activity was not affected by exposure to ERK1. Furthermore, MAPKs in RSK3 immunoprecipitates were phosphorylated by purified MEK1; however, RSK3 kinase activity was unaffected. We conclude that 1) the MEK1-MAPK signaling pathway is both necessary and sufficient for in vivo growth factor-mediated activation of all three RSK isoforms; 2) RSK isoforms differ with respect to growth factor responsiveness and their physical association with MAPK; and 3) formation of the MAPK.RSK complex is mediated by the RSK C terminus.

Animals↗

PubMed1996

Regulation of acetyl coenzyme-A carboxylase gene in a transgenic animal model.

The gene for acetyl-CoA carboxylase, the rate-limiting enzyme in the biosynthesis of long-chain fatty acids, contains two promoters; promoter I (PI) and promoter II (PII) which are responsible for generation of class I and class II ACC mRNAs, respectively. Class I ACC mRNAs are present in adipose tissue, but only a trace was found in the liver under normal physiological conditions. However, class I mRNAs were induced under stimulated lipogenic conditions. To investigate how PI is regulated in vivo, we generated transgenic mice containing a reporter gene under the control of PI. In transgenic mice, PI is generally inactive and a small amount of PI activity was found only in the adipose tissues of female animals. Stimulated lipogenic conditions activated PI about 17-fold over normal conditions and again only in white adipose tissues of female animals.

Acetyl-CoA Carboxylase↗

PubMed1996

Molecular cloning, expression and characterization of human peroxisome proliferator activated receptors gamma 1 and gamma 2.

We describe the molecular cloning and expression of cDNAs encoding human PPAR gamma 1 and PPAR gamma 2. Our sequences are distinct from the published sequence at 3 positions, resulting in nonconservative amino acid substitutions. In humans, PPAR gamma mRNA is expressed in spleen, bone marrow, liver, testis, skeletal muscle and brain, in addition to fat. Three thiazolidinediones were found to 1) displace a radiolabeled thiazolidinedione from both receptors with essentially the same IC50s and 2) to transactivate both PPAR gamma isoforms with similar EC50s in transient cotransfection assays utilizing the adipocyte-specific aP2 promoter. Saturating concentrations of these 3 thiazolidinediones altered the conformation of in vitro synthesized PPAR gamma protein producing a 27 kDa protease-resistant fragment. These results indicate that the antidiabetic effects of thiazolidinediones in humans are likely to be mediated via binding to and transactivation of PPAR gamma 1 and gamma 2.

Amino Acid Sequence↗

PubMed1996

Overexpression of hexokinase II in transgenic mice. Evidence that increased phosphorylation augments muscle glucose uptake.

Hexokinase II (HKII) is the predominant isozyme expressed in peripheral insulin-responsive tissues. To explore the role of HKII in muscle glucose metabolism, two lines of transgenic mice were generated where overexpression was restricted to striated muscle; HKII protein levels and activity were increased by 3-8-fold. Oral glucose tolerance, intravenous insulin tolerance, and insulin and lactate levels were unaffected in transgenic mice. There was a trend toward increased levels of muscle glycogen; however, glucose-6-phosphate levels were increased by 43% in transgenic skeletal muscle following in vivo glucose and insulin administration. Using 2-[3H]deoxyglucose as a tracer, in vitro basal and insulin-stimulated glucose uptake were determined in extensor digitorum longus, soleus, and epitrochlearis muscles. Maximal insulin-stimulated glucose uptake was increased by 17% (extensor digitorum longus), 34% (soleus), and 90% (epitrochlearis) in transgenic muscles; basal and submaximal glucose uptake was also modestly increased in soleus and epitrochlearis. These data suggest that increased muscle HKII (corresponding to the upper end of the physiologic range) may not be sufficient to augment net in vivo glucose homeostasis. However, glucose phosphorylation can represent a rate-limiting step for skeletal muscle glucose utilization since muscle glucose-6-phosphate levels are increased during in vivo hyperinsulinemia and hyperglycemia; furthermore, basal and insulin-mediated muscle glucose uptake can be increased by a selective increase in HKII expression.

Amino Acid Sequence↗

PubMed1996

Regulation of PPAR gamma gene expression by nutrition and obesity in rodents.

The orphan nuclear receptor, peroxisome proliferator-activated receptor (PPAR) gamma, is implicated in mediating expression of fat-specific genes and in activating the program of adipocyte differentiation. The potential for regulation of PPAR gamma gene expression in vivo is unknown. We cloned a partial mouse PPAR gamma cDNA and developed an RNase protection assay that permits simultaneous quantitation of mRNAs for both gamma l and gamma 2 isoforms encoded by the PPAR gamma gene. Probes for detection of adipocyte P2, the obese gene product, leptin, and 18S mRNAs were also employed. Both gamma l and gamma 2 mRNAs were abundantly expressed in adipose tissue. PPAR gamma 1 expression was also detected at lower levels in liver, spleen, and heart; whereas, gamma l and gamma 2 mRNA were expressed at low levels in skeletal muscle. Adipose tissue levels of gamma l and gamma 2 were not altered in two murine models of obesity (gold thioglucose and ob/ob), but were modestly increased in mice with toxigene-induced brown fat ablation uncoupling protein diphtheria toxin A mice. Fasting (12-48 h) was associated with an 80% fall in PPAR gamma 2 and a 50% fall in PPAR gamma mRNA levels in adipose tissue. Western blot analysis demonstrated a marked effect of fasting to reduce PPAR gamma protein levels in adipose tissue. Similar effects of fasting on PPAR gamma mRNAs were noted in all three models of obesity. Insulin-deficient (streptozotocin) diabetes suppressed adipose tissue gamma l and gamma 2 expression by 75% in normal mice with partial restoration during insulin treatment. Levels of adipose tissue PPAR gamma 2 mRNA were increased by 50% in normal mice exposed to a high fat diet. In obese uncoupling protein diphtheria toxin A mice, high fat feeding resulted in de novo induction of PPAR gamma 2 expression in liver. We conclude (a) PPAR gamma 2 mRNA expression is most abundant in adipocytes in normal mice, but lower level expression is seen in skeletal muscle; (b) expression of adipose tissue gamma1 or gamma2 mRNAs is increased in only one of the three models of obesity; (c) PPAR gamma 1 and gamma 2 expression is downregulated by fasting and insulin-deficient diabetes; and (d) exposure of mice to a high fat diet increases adipose tissue expression of PPAR gamma (in normal mice) and induces PPAR gamma 2 mRNA expression in liver (in obese mice). These findings demonstrate in vivo modulation of PPAR gamma mRNA levels over a fourfold range and provide an additional level of regulation for the control of adipocyte development and function.

Adipocytes↗

PubMed1996

Two naturally occurring mutant insulin receptors phosphorylate insulin receptor substrate-1 (IRS-1) but fail to mediate the biological effects of insulin. Evidence that IRS-1 phosphorylation is not sufficient for normal insulin action.

Two naturally occurring mutant insulin receptors, Arg-1174 --> Gln and Leu-1178 --> Pro, found in patients with dominantly inherited Type A insulin resistance, showed unusual signaling properties when stably expressed in Chinese hamster ovary (CHO) cells. Both mutant receptors were expressed on the cell surface and bound insulin normally, but showed markedly impaired autophosphorylation in response to insulin. In addition, the in vitro tyrosine kinase activity of both mutant receptors toward an artificial substrate was also severely impaired. Despite these defects of kinase activity, anti-phosphotyrosine immunoblotting of whole cell lysates and anti-phosphotyrosine immunoprecipitation of 32P-labeled cells showed insulin-stimulated tyrosine phosphorylation of a protein of approximately 185 kDa to an extent comparable to that seen in CHO cells expressing wild-type human insulin receptors. Anti-insulin receptor substrate-1 (IRS-1) immunoprecipitation followed by anti-phosphotyrosine immunoblotting confirmed that this tyrosine-phosphorylated protein was IRS-1. In contrast, CHO cells expressing an insulin receptor mutated at the ATP binding site (Lys-1030 --> Arg) showed no insulin-stimulated autophosphorylation or phosphorylation of IRS-1. Despite exhibiting apparently normal insulin stimulation of IRS-1 tyrosine-phosphorylation, cells expressing the Arg-1174 --> Gln or Pro-1178 --> Leu receptors showed marked impairment in insulin stimulation of glycogen synthesis, thymidine incorporation, and activation of MAP kinase. The inability of these mutant receptors to signal normally to metabolic and mitogenic responses suggests that insulin-stimulated tyrosine phosphorylation of IRS-1 alone is insufficient to fully mediate insulin action.

Adolescent↗

PubMed1996

Effects of exercise and insulin on mitogen-activated protein kinase signaling pathways in rat skeletal muscle.

Studies in mammalian cells have established the existence of at least three distinct mitogen-activated protein kinase (MAP kinase) signaling pathways that are activated by a variety of growth factors and/or environmental stressors. We determined whether physical exercise, a physiological stressor, and insulin, a metabolic stimulator and growth factor, activate the c-jun NH2-terminus kinase (JNK), the p38 kinase, and/or the extracellular regulatory kinases (ERK; p42MAPK and p44MAPK) signaling pathways in rat skeletal muscle. Animals were studied immediately after running on a motorized treadmill for 10-60 min (20 m/min, 10% grade) or 5-30 min after an intraperitoneal injection of insulin (20 U/rat). Exercise increased skeletal muscle JNK activity by two- to threefold throughout the time course studied, whereas insulin did not significantly increase JNK activity. The p38 activity was slightly stimulated by exercise and not by insulin. The ERK kinase pathway, as assessed by ribosomal S6 kinase-2 activity assays and phosphospecific p42MAPK/p4NAPK immunoblotting, was stimulated by both exercise and insulin. These data are the first demonstration of exercise stimulating multiple intracellular signaling pathways in skeletal muscle. Activation of these MAP kinase signaling pathways may mediate changes in skeletal muscle growth and metabolism that occur in response to exercise.

Animals↗

PubMed1996

Transgenic mice with muscle-specific insulin resistance develop increased adiposity, impaired glucose tolerance, and dyslipidemia.

Impaired skeletal muscle insulin receptor function is a feature of common forms of insulin resistance, including obesity and noninsulin-dependent diabetes mellitus. However, the extent to which this defect accounts for impaired muscle glucose disposal or altered in vivo glucose homeostasis remains to be established. We recently showed that transgenic mice that overexpress dominant-negative insulin receptors specifically in striated muscle have a severe defect in muscle insulin receptor-mediated signaling and modest hyperinsulinemia. Here we performed hindlimb perfusion studies to determine the impact of this defect on muscle glucose uptake and metabolism. Maximal rates of insulin-stimulated muscle 3-O-methylglucose transport were reduced by 32-40% in transgenic mice with proportional defects involving total hindlimb [14C]glucose uptake, lactate production, and muscle glycogen synthesis. To address the hypothesis that muscle insulin resistance could promote an increase in the accretion of body fat, carcass analysis was performed using two independent lines of transgenic mice. Although body weights were normal, transgenic mice had a 22-38% increase in body fat, with a reciprocal decrease (10-15%) in body protein. Mean gonadal fat pad weight was also increased in transgenic mice. Skeletal muscle histology and fiber type distribution were not affected. To determine whether muscle-specific insulin resistance was sufficient to cause impaired glucose tolerance, oral glucose tolerance tests were performed with 6-month-old transgenic and control mice. Fasting glucose levels were increased by 25%, and peak values were 22-40% higher in transgenic mice. Transgenic mice also had a 37% decrease in plasma lactate levels and modest increases in levels of plasma triglycerides and FFA (29% and 15%, respectively). We conclude that 1) severe defects in muscle insulin receptor function result in impaired insulin-stimulated glucose uptake and metabolism in this tissue; 2) muscle-specific insulin resistance can contribute to the development of obesity; and 3) a "pure" defect in insulin-mediated muscle glucose disposal is sufficient to result in impaired glucose tolerance and other features of the insulin resistance syndrome, including hyperinsulinemia and dyslipidemia.

Adipose Tissue↗

PubMed1996

Examination of the phosphoenolpyruvate carboxykinase gene promoter in patients with noninsulin-dependent diabetes mellitus.

Expression of phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme in gluconeogenesis, is under dominant negative regulation by insulin. In this study, we sought to test the hypothesis that mutations in the PEPCK gene promoter may impair the ability of insulin to suppress hepatic glucose production, thereby contributing to both the insulin resistance and increased rate of gluconeogenesis characteristic of NIDDM. The proximal PEPCK promoter region in 117 patients with noninsulin-dependent diabetes mellitus and 20 obese Pima Indians was amplified by PCR and analyzed with single strand conformation polymorphism techniques. In addition, limited direct DNA sequencing was performed on the insulin response sequence and flanking regions. No DNA sequence polymorphisms were found in any patient. This result suggests that mutations in cis-acting PEPCK gene regulatory elements do not constitute a common cause of noninsulin-dependent diabetes mellitus. The significance of genetic variation in promoter regions to human disease is discussed.

Adolescent↗

PubMed1996

Negative regulation of peroxisome proliferator-activated receptor-gamma gene expression contributes to the antiadipogenic effects of tumor necrosis factor-alpha.

Recent studies indicate that a peroxisome proliferator-activated receptor, PPAR gamma, functions as an important adipocyte determination factor. In contrast, tumor necrosis factor-alpha (TNF alpha) inhibits adipogenesis, causes dedifferentiation of mature adipocytes, and reduces the expression of several adipocyte-specific genes. Here, we report that treatment of 3T3-L1 adipocytes with TNF alpha resulted in a time- and concentration-dependent decrease in PPAR gamma mRNA expression to the level detected in preadipocytes. PPAR gamma mRNA levels were reduced by 95% with 3 nM TNF alpha treatment for 24 h. Half-maximal effects were seen after 3 h treatment with 3 nM TNF alpha or with 50 pM TNF alpha (24-h exposure). Parallel reductions in PPAR gamma protein levels were also observed after treatment of 3T3-L1 adipocytes with TNF alpha. Using a ribonuclease protection assay, both alternatively spliced PPAR gamma isoforms (gamma 1 and gamma 2) were shown to be negatively regulated by TNF alpha. The down-regulation of PPAR gamma by TNF-alpha preceded the diminution in expression of other adipocyte-specific genes including CCAAT/enhancer binding protein and adipocyte fatty acid-binding protein (aP2). The effect of TNF alpha was specific for the gamma-isoform of PPARs, since the expression of PPAR delta mRNA was not affected by treatment with TNF alpha. Low level constitutive expression of PPAR gamma in 3T3-L1 adipocytes (at levels approximately 2- to 3-fold higher than in preadipocytes) partially blocked the inhibitory effect of TNF alpha on aP2 and adipsin expression. These findings support the following conclusions: 1) PPAR gamma expression is necessary for the maintenance of the adipocyte phenotype. 2) PPAR gamma, but not PPAR delta, expression is sufficient to attenuate TNF alpha-mediated effects on adipocyte phenotype. 3) Reduced PPAR gamma gene expression is likely to represent an important component of the mechanism by which TNF alpha exerts its antiadipogenic effects.

3T3 Cells↗

PubMed1996

Candidate genes for insulin resistance.

Insulin resistance confers increased susceptibility to NIDDM, atherosclerotic cardiovascular disease, ovarian hyperandrogenism, and possibly hypertension. Insulin resistance is largely inherited, in rare cases as a monogenic disorder or more commonly as a complex trait. The search for insulin resistance genes relies mainly on two complementary approaches: 1) positional cloning using random DNA markers present throughout the genome; and 2) the analysis of specific candidate genes. This report briefly summarizes the candidate gene approach to insulin resistance. Progress related to the analysis of genes encoding molecules that participate in insulin action is reviewed. In addition, the spectrum of potential genetic defects that might contribute to insulin resistance, both at the level of the target cell and secondarily (e.g., obesity genes), is discussed.

Animals↗

PubMed1995

Insulin stimulation of mitogen-activated protein kinase, p90rsk, and p70 S6 kinase in skeletal muscle of normal and insulin-resistant mice. Implications for the regulation of glycogen synthase.

UNLABELLED: Skeletal muscles from mice stimulated with insulin in vivo were used to evaluate relationships between the insulin receptor tyrosine kinase, mitogen-activated protein (MAP) kinase, p90rsk, p70 S6 kinase (p70S6k), and glycogen synthase. Two models of insulin resistance were also evaluated: (a) transgenic mice with a severe insulin receptor defect and (b) gold thioglucose (GTG) mice (obesity with minimal insulin receptor dysfunction). In normal mice, insulin stimulated MAP kinase (6-fold), p90rsk (RSK2, 5-fold), p70S6k (10-fold), and glycogen synthase (30-50% increase in fractional velocity). In transgenic mice, stimulation of MAP kinase and RSK2 were not detectable, whereas activation of p70S6k and glycogen synthase were preserved. In GTG mice, activation of MAP kinase, RSK2, p70S6k, and glycogen synthase were impaired. Since p70S6k and glycogen synthase were correlated, rapamycin was used to block p70S6k, and glycogen synthase activation was unaffected in normal mice; however, it was partially impaired in transgenic mice. CONCLUSIONS: (a) stimulation of p70S6k and glycogen synthase are selectively preserved in muscles with a severe insulin receptor kinase defect, indicating signal amplification in pathways leading to these effects; (b) MAP kinase-RSK2 and p70S6k activation are impaired in obese mice, suggesting multiple loci for postreceptor insulin resistance; (c) glycogen synthase was dissociated from MAP kinase and RSK2, indicating that they are not required for this effect of insulin; and (d) p70S6k is not essential for glycogen synthase activation, but it may participate in redundant signaling pathways leading to this effect of insulin.

Animals↗

PubMed1995

Divergent functional roles for p90rsk kinase domains.

A unique and highly conserved structural feature of approximately 90-kDa ribosomal S6 kinase (p90rsk or RSK) is the presence of two non-identical kinase domains. To explore the mechanism of RSK activation, a cloned human RSK cDNA (RSK3) was used to generate and characterize several site-directed RSK mutants; K91A (N-Lys, NH2-terminal ATP-binding mutant), K444A (C-Lys, COOH-terminal ATP-binding mutant), N/C-Lys (double ATP-binding mutant) T570A (C-Thr, mutant of the putative MAPK phosphorylation site in subdomain VIII of the C-domain), S218A (N-Ser, mutant of the corresponding NH2-terminal residue). Epitope-tagged RSKs were expressed in transfected COS cells followed by immunoprecipitation with or without prior in vivo epidermal growth factor stimulation. Kinase activity (S6 peptide) of N/C-Lys and N-Lys was ablated (and partially impaired with N-Ser). In contrast, both C-Lys and C-Thr retained high levels of kinase activity and were capable of responding to stimulation. C-Lys also retained partial kinase activity toward other substrates (c-Fos, S40 ribosomes, protein phosphatase 1 G-subunit, histones, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide)) whereas N-Lys did not. The isolated NH2-and COOH-terminal domains were also expressed; the C-domain was inactive, whereas the N-domain retained partial activity. Relative to wild-type, both N-Lys and C-Lys (as well as N-Ser and C-Thr) underwent partial in vitro autophosphorylation that was further stimulated by EGF protein tyrosine phosphatase. We conclude that 1) the NH2-terminal RSK kinase domain mediates substrate phosphorylation; 2) both domains contribute to autophosphorylation; 3) the putative MAPK phosphorylation site is not required for growth factor-stimulated autophosphorylation or kinase activation.

Amino Acid Sequence↗

PubMed1995

Impaired insulin signaling in skeletal muscles from transgenic mice expressing kinase-deficient insulin receptors.

Transgenic mice which overexpress kinase-deficient human insulin receptors in muscle were used to study the relationship between insulin receptor tyrosine kinase and the in vivo activation of several downstream signaling pathways. Intravenous insulin stimulated insulin receptor tyrosine kinase activity by 7-fold in control muscle versus < or = 1.5-fold in muscle from transgenic mice. Similarly, insulin failed to stimulate tyrosyl phosphorylation of receptor beta-subunits or insulin receptor substrate 1 (IRS-1) in transgenic muscle. Insulin substantially stimulated IRS-1-associated phosphatidylinositol (PI) 3-kinase in control versus absent stimulation in transgenic muscles. In contrast, insulin-like growth factor 1 modestly stimulated PI 3-kinase in both control and transgenic muscle. The effects of insulin to stimulate p42 mitogen-activated protein kinase and c-fos mRNA expression were also markedly impaired in transgenic muscle. Specific immunoprecipitation of human receptors followed by measurement of residual insulin receptors suggested the presence of hybrid mouse-human heterodimers. In contrast, negligible hybrid formation involving insulin-like growth factor 1 receptors was evident. We conclude that (i) transgenic expression of kinase-defective insulin receptors exerts dominant-negative effects at the level of receptor auto-phosphorylation and kinase activation; (ii) insulin receptor tyrosine kinase activity is required for in vivo insulin-stimulated IRS-1 phosphorylation, IRS-1-associated PI 3-kinase activation, phosphorylation of mitogen-activated protein kinase, and c-fos gene induction in skeletal muscle; (iii) hybrid receptor formation is likely to contribute to the in vivo dominant-negative effects of kinase-defective receptor expression.

Animals↗

PubMed1995

Binding of human, porcine and bovine insulin to insulin receptors from human brain, muscle and adipocytes and to expressed recombinant alternatively spliced insulin receptor isoforms.

Previous studies have suggested that human and porcine insulin exert identical effects on blood glucose and counter-regulatory hormones but elicit different neurophysiological reactions. A major goal of the present study was to investigate whether this could be caused by different relative affinities of the insulins from different species to insulin receptors from the brain compared to other tissues. Insulin receptors isolated from human brain, muscle or adipocytes as well as from cultured cells over-expressing either of the human insulin receptor isoforms (exon 11- or exon 11 +) were immobilized to microwells coated with monoclonal anti-insulin receptor antibody. Subsequently the binding of human, porcine and bovine insulin was measured. While the receptors derived from the different tissues had different affinities for insulin, there were no tissue-specific differences in the relative binding of the insulins of the three species. The insulins of the three species were also not different with regard to their binding to the receptor isoforms. Finally, in human brain homogenates no differences in the degradation rates for human, porcine and bovine insulin were detected. Thus, our data do not support the hypothesis that different neurophysiological reactions during hypoglycaemia due to human or porcine insulin are caused by differences of the binding of the insulins to human brain insulin receptors or their degradation in the human brain.

Adipocytes↗

PubMed1995

Insulin-like growth factor I (rhIGF-I) as a therapeutic agent for hyperinsulinemic insulin-resistant diabetes mellitus.

Insulin resistance is one of the major underlying abnormalities in NIDDM, however, its pathophysiologic mechanisms are not well understood. Many clues about the mechanisms of insulin action have come from patients with the most severe forms of insulin resistance, including those with genetic abnormalities in the insulin signal transduction cascade. We used rhIGF-I as a probe to differentiate insulin and IGF-I action and to study the therapeutic potential of IGF in states of insulin resistance. To date, we have studied six subjects with varying phenotypes of severe insulin resistance but without mutations in the insulin receptor itself. All subjects underwent baseline physiologic monitoring to quantitate carbohydrate tolerance, insulin secretion, and insulin action prior to receiving rhIGF-I at 100 micrograms/kg body wt s.c. bid for 1 month with interval testing of glycemic control and insulin sensitivity. None of the six subjects noted significant side effects from the rhIGF-I. Four of the six subjects had overt diabetes during control testing; three of these subjects demonstrated normalization of fasting and postprandial blood glucose concentrations during rhIGF-I administration on no other therapy. In the fourth patient, insulin requirements and fasting hypertriglyceridemia decreased without improvement in glycemic control. The two subjects with normal glucose tolerance (two sisters with congenital lipodystrophy) maintained normal glucose tolerance at dramatically lower insulin levels and had a dramatic reduction in triglyceride levels. The efficacy of IGF-I continued to increase over the duration of the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Glucose↗

PubMed1995

RSK3 encodes a novel pp90rsk isoform with a unique N-terminal sequence: growth factor-stimulated kinase function and nuclear translocation.

A novel pp90rsk Ser/Thr kinase (referred to as RSK3) was cloned from a human cDNA library. The RSK3 cDNA encodes a predicted 733-amino-acid protein with a unique N-terminal region containing a putative nuclear localization signal. RSK3 mRNA was widely expressed (but was predominant in lung and skeletal muscle). By using fluorescence in situ hybridization, the human RSK3 gene was localized to band q27 of chromosome 6. Hemagglutinin epitope-tagged RSK3 was expressed in transiently transfected COS cells. Growth factors, serum, and phorbol ester stimulated autophosphorylation of recombinant RSK3 and its kinase activity toward several protein substrates known to be phosphorylated by RSKs. However, the relative substrate specificity of RSK3 differed from that reported for other isoforms. RSK3 also phosphorylated potential nuclear target proteins including c-Fos and histones. Furthermore, although RSK3 was inactivated by protein phosphatase 2A in vitro, the enzyme was not activated by ERK2/mitogen-activated protein (MAP) kinase. In contrast, the kinase activity of another epitope-tagged RSK isoform (RSK-1) was significantly increased by in vitro incubation with ERK2/MAP kinase. Finally, we used affinity-purified RSK3 antibodies to demonstrate by immunofluorescence that endogenous RSK3 undergoes serum-stimulated nuclear translocation in cultured HeLa cells. These results provide evidence that RSK3 is a third distinct isoform of pp90rsk which translocates to the cell nucleus, phosphorylates potential nuclear targets, and may have a unique upstream activator. RSK3 may therefore subserve a discrete physiologic role(s) that differs from those of the other two known mammalian RSK isoforms.

Amino Acid Sequence↗