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Biomedical subjects

D J Withers

Publications and source records attributed to D J Withers.

At least 19 recordsLinked to original sources
PubMed2006

Liver-specific deletion of insulin receptor substrate 2 does not impair hepatic glucose and lipid metabolism in mice.

AIMS/HYPOTHESIS: Hepatic insulin resistance is thought to be a critical component in the pathogenesis of type 2 diabetes but the role of intrinsic insulin signalling pathways in the regulation of hepatic metabolism remains controversial. Global gene targeting in mice and in vitro studies have suggested that IRS2 mediates the physiological effects of insulin in the liver. Reduced hepatic production of IRS2 is found in many cases of insulin resistance. To investigate the role of IRS2 in regulating liver function in vivo, we generated mice that specifically lack Irs2 in the liver (LivIrs2KO). MATERIALS AND METHODS: Hepatic insulin signalling events were examined in LivIrs2KO mice by western blotting. Glucose homeostasis and insulin sensitivity were assessed by glucose tolerance tests and hyperinsulinaemic-euglycaemic clamp studies. The effects of high-fat feeding upon glucose homeostasis were also determined. Liver function tests were performed and expression of key metabolic genes in the liver was determined by RT-PCR. RESULTS: Proximal insulin signalling events and forkhead box O1 and A2 function were normal in the liver of LivIrs2KO mice, which displayed minimal abnormalities in glucose and lipid homeostasis, hepatic gene expression and liver function. In addition, hepatic lipid homeostasis and the metabolic response to a high-fat diet did not differ between LivIrs2KO and control mice. CONCLUSIONS/INTERPRETATION: Our findings suggest that liver IRS2 signalling, surprisingly, is not required for the long-term maintenance of glucose and lipid homeostasis, and that extra-hepatic IRS2-dependent mechanisms are involved in the regulation of these processes.

Animal Feed↗
PubMed2002

Adrenomedullin: receptor and signal transduction.

Adrenomedullin is a vascular tissue peptide and a member of the calcitonin family of peptides, which includes calcitonin, calcitonin-gene-related peptide (CGRP) and amylin. Its many biological actions are mediated via CGRP type 1 (CGRP(1)) receptors and by specific adrenomedullin receptors. Although the pharmacology of these receptors is distinct, they are both represented in molecular terms by the type II family G-protein-coupled receptor, calcitonin-receptor-like receptor (CRLR). The specificity here is defined by co-expression of receptor-activity-modifying proteins (RAMPs). CGRP(1) receptors are represented by CRLR and RAMP1, and specific adrenomedullin receptors by CRLR and RAMP2 or 3. Here we discuss how CRLR/RAMP2 relates to adrenomedullin binding, pharmacology and pathophysiology, and how chemical cross-linking of receptor-ligand complexes in tissue relates to that in CRLR/RAMP2-expressing cells. CRLR, like other type II family G-protein-coupled receptors, signals via G(s) and adenylate cyclase activation. We demonstrated that adrenomedullin signalling in cell lines expressing specific adrenomedullin receptors followed this expected pattern.

Adrenomedullin↗
PubMed2001

Insulin receptor substrate proteins and neuroendocrine function.

A family of insulin receptor substrate (IRS) proteins mediates the pleiotropic effects of insulin and insulin-like growth factor 1 (IGF-1) on cellular function by recruiting several intracellular signalling networks. Conventional murine knockout strategies have started to reveal distinct physiological roles for the IRS proteins. Deletion of Irs1 produces a mild metabolic phenotype with compensated insulin resistance but also causes marked growth retardation. In contrast, mice lacking IRS-2 display nearly normal growth but develop diabetes owing to a combination of peripheral insulin resistance and beta-cell failure. As well as the classical metabolic events regulated by insulin signalling pathways, studies in lower organisms have implicated insulin/IGF-1 signalling pathways in the control of food intake and reproductive function. Our analysis of IRS-2 knock-out mice shows that female mice are infertile owing to defects in the hypothalamus, pituitary and gonad. IRS-2(-/-) mice have small, anovulatory ovaries with reduced numbers of follicles. Levels of the pituitary hormones luteinizing hormone and prolactin and gonadal steroids are low in these animals. Pituitaries of IRS-2(-/-) animals are decreased in size and contain reduced numbers of gonadotrophs. Additionally, IRS-2(-/-) females display increased food intake and develop obesity, despite elevated leptin levels, suggesting abnormalities in hypothalamic function. Coupled with recent observations that brain-specific deletion of the insulin receptor causes a similar phenotype, these findings implicate IRS signalling pathways in the neuroendocrine regulation of reproduction and energy homeostasis.

Animals↗
PubMed2000

Contrasting effects of IRS-1 versus IRS-2 gene disruption on carbohydrate and lipid metabolism in vivo.

To examine the impact of homozygous genetic disruption of insulin receptor substrate (IRS)-1 (IRS-1(-/-)) or IRS-2 (IRS-2(-/-)) on basal and insulin-stimulated carbohydrate and lipid metabolism in vivo, we infused 18-h fasted mice (wild-type (WT), IRS-1(-/-), and IRS-2(-/-)) with [3-(3)H]glucose and [(2)H(5)]glycerol and assessed rates of glucose and glycerol turnover under basal (0-90 min) and hyperinsulinemic-euglycemic clamp (90-210 min; 5 mm glucose, and 5 milliunits of insulin.kg(-)(1).min(-)(1)) conditions. Both IRS-1(-)(/-) and IRS-2(-)(/-) mice were insulin-resistant as reflected by markedly impaired insulin-stimulated whole-body glucose utilization compared with WT mice. Insulin resistance in the IRS-1(-)(/-) mice could be ascribed mainly to decreased insulin-stimulated peripheral glucose metabolism. In contrast, IRS-2(-)(/-) mice displayed multiple defects in insulin-mediated carbohydrate metabolism as reflected by (i) decreased peripheral glucose utilization, (ii) decreased suppression of endogenous glucose production, and (iii) decreased hepatic glycogen synthesis. Additionally, IRS-2(-)(/-) mice also showed marked insulin resistance in adipose tissue as reflected by reduced suppression of plasma free fatty acid concentrations and glycerol turnover during the hyperinsulinemic-euglycemic clamp. These data suggest important tissue-specific roles for IRS-1 and IRS-2 in mediating the effect of insulin on carbohydrate and lipid metabolism in vivo in mice. IRS-1 appears to have its major role in muscle, whereas IRS-2 appears to impact on liver, muscle, and adipose tissue.

Adipose Tissue↗
PubMed2000

IRS-2 pathways integrate female reproduction and energy homeostasis.

Severe dietary restriction, catabolic states and even short-term caloric deprivation impair fertility in mammals. Likewise, obesity is associated with infertile conditions such as polycystic ovary syndrome. The reproductive status of lower organisms such as Caenorhabditis elegans is also modulated by availability of nutrients. Thus, fertility requires the integration of reproductive and metabolic signals. Here we show that deletion of insulin receptor substrate-2 (IRS-2), a component of the insulin/insulin-like growth factor-1 signalling cascade, causes female infertility. Mice lacking IRS-2 have small, anovulatory ovaries with reduced numbers of follicles. Plasma concentrations of luteinizing hormone, prolactin and sex steroids are low in these animals. Pituitaries are decreased in size and contain reduced numbers of gonadotrophs. Females lacking IRS-2 have increased food intake and obesity, despite elevated levels of leptin. Our findings indicate that insulin, together with leptin and other neuropeptides, may modulate hypothalamic control of appetite and reproductive endocrinology. Coupled with findings on the role of insulin-signalling pathways in the regulation of fertility, metabolism and longevity in C. elegans and Drosophila, we have identified an evolutionarily conserved mechanism in mammals that regulates both reproduction and energy homeostasis.

Animals↗
PubMed1999

Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation.

Growth factor induced activation of phosphoinositide 3-kinase and protein kinase B (PKB) leads to increased activity of the mammalian target of rapamycin (mTOR). This subsequently leads to increased phosphorylation of eIF4E binding protein-1 (4EBP1) and activation of p70 ribosomal S6 protein kinase (p70(S6K)), both of which are important steps in the stimulation of protein translation. The stimulation of translation is attenuated in cells deprived of amino acids and this is associated with the attenuation of 4EBP1 phosphorylation and p70(S6K) activation. It has been suggested that PKB regulates mTOR function by phosphorylation although direct phosphorylation of mTOR by PKB has not been demonstrated previously. In the present work, we have found that PKB directly phosphorylates mTOR and, using phosphospecific antibodies, we have shown this phosphorylation occurs at Ser(2448). Insulin also induces phosphorylation on Ser(2448) and this effect is blocked by wortmannin but not rapamycin, consistent with the effect being mediated by PKB. Amino-acid starvation rapidly attenuated the reactivity of the Ser(2448) phosphospecific antibody with mTOR and this could not be restored by either insulin stimulation of cells or incubation with PKB in vitro. Our findings demonstrate that mTOR is a direct target for PKB and support the conclusion that regulation of phosphorylation of Ser(2448) is a point of convergence for the counteracting regulatory effects of growth factors and amino acid levels.

Amino Acids↗
PubMed1999

Insulin receptor substrate-2 is not necessary for insulin- and exercise-stimulated glucose transport in skeletal muscle.

Insulin receptor substrate-2-deficient (IRS2(-/-)) mice develop type 2 diabetes. The purpose of this study was to determine whether there is a defect in basal, insulin-, and exercise-stimulated glucose transport in the skeletal muscle of these animals. IRS2(-/-) and wild-type (WT) mice (male, 8-10 weeks) exercised on a treadmill for 1 h or remained sedentary. 2-Deoxyglucose (2DG) uptake was measured in isolated soleus muscles incubated in vitro in the presence or absence of insulin. Resting blood glucose concentration in IRS2(-/-) mice (10.3 mM) was higher than WT animals (4.1 mM), but there was a wide range among the IRS2(-/-) mice (3-25 mM). Therefore, IRS2(-/-) mice were divided into two subgroups based on blood glucose concentrations (IRS2(-/-)L < 7.2 mM, IRS2(-/-)H > 7.2 mM). Only IRS2(-/-)H had lower basal, exercise-, and submaximally insulin-stimulated 2DG uptake, while maximal insulin-stimulated 2DG uptake was similar among the three groups. The ED(50) for insulin to stimulate 2DG uptake above basal in IRS2(-/-)H was higher than WT and IRS2(-/-)L mice, suggesting insulin resistance in the skeletal muscle from the IRS2(-/-) mice with high blood glucose concentrations. Furthermore, resting blood glucose concentrations from all groups were negatively correlated to submaximally insulin-stimulated 2DG uptake (r(2) = 0.33, p < 0.01). Muscle GLUT4 content was significantly lower in IRS2(-/-)H mice compared with WT and IRS2(-/-)L mice. These results demonstrate that the IRS2 protein in muscle is not necessary for insulin- or exercise-stimulated glucose transport, suggesting that the onset of diabetes in the IRS2(-/-) mice is not due to a defect in skeletal muscle glucose transport; hyperglycemia may cause insulin resistance in the muscle of IRS2(-/-) mice.

Animals↗
PubMed1999

Irs-2 coordinates Igf-1 receptor-mediated beta-cell development and peripheral insulin signalling.

Insulin receptor substrates (Irs proteins) mediate the pleiotropic effects of insulin and Igf-1 (insulin-like growth factor-1), including regulation of glucose homeostasis and cell growth and survival. We intercrossed mice heterozygous for two null alleles (Irs1+/- and Irs2+/-) and investigated growth and glucose metabolism in mice with viable genotypes. Our experiments revealed that Irs-1 and Irs-2 are critical for embryonic and post-natal growth, with Irs-1 having the predominant role. By contrast, both Irs-1 and Irs-2 function in peripheral carbohydrate metabolism, but Irs-2 has the major role in beta-cell development and compensation for peripheral insulin resistance. To establish a role for the Igf-1 receptor in beta-cells, we intercrossed mice heterozygous for null alleles of Igf1r and Irs2. Our results reveal that Igf-1 receptors promote beta-cell development and survival through the Irs-2 signalling pathway. Thus, Irs-2 integrates the effects of insulin in peripheral target tissues with Igf-1 in pancreatic beta-cells to maintain glucose homeostasis.

Age Factors↗
PubMed1998

Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling.

Insulin plays a key role in regulating a wide range of cellular processes. However, until recently little was known about the signalling pathways that are involved in linking the insulin receptor with downstream responses. It is now apparent that the activation of class 1a phosphoinositide 3-kinase (PI 3-kinase) is necessary and in some cases sufficient to elicit many of insulin's effects on glucose and lipid metabolism. The lipid products of PI 3-kinase act as both membrane anchors and allosteric regulators, serving to localize and activate downstream enzymes and their protein substrates. One of the major ways these lipid products of PI 3-kinase act in insulin signalling is by binding to pleckstrin homology (PH) domains of phosphoinositide-dependent protein kinase (PDK) and protein kinase B (PKB) and in the process regulating the phosphorylation of PKB by PDK. Using mechanisms such as this, PI 3-kinase is able to act as a molecular switch to regulate the activity of serine/threonine-specific kinase cascades important in mediating insulin's effects on endpoint responses.

Animals↗
PubMed1998

Disruption of IRS-2 causes type 2 diabetes in mice.

Human type 2 diabetes is characterized by defects in both insulin action and insulin secretion. It has been difficult to identify a single molecular abnormality underlying these features. Insulin-receptor substrates (IRS proteins) may be involved in type 2 diabetes: they mediate pleiotropic signals initiated by receptors for insulin and other cytokines. Disruption of IRS-1 in mice retards growth, but diabetes does not develop because insulin secretion increases to compensate for the mild resistance to insulin. Here we show that disruption of IRS-2 impairs both peripheral insulin signalling and pancreatic beta-cell function. IRS-2-deficient mice show progressive deterioration of glucose homeostasis because of insulin resistance in the liver and skeletal muscle and a lack of beta-cell compensation for this insulin resistance. Our results indicate that dysfunction of IRS-2 may contribute to the pathophysiology of human type 2 diabetes.

Animals↗
PubMed1997

Expression, enzyme activity, and subcellular localization of mammalian target of rapamycin in insulin-responsive cells.

The role of the mammalian target of rapamycin (mTOR) was investigated in insulin responsive cell lines. mTOR was expressed at high levels in insulin responsive cell types and in 3T3-L1 cells mTOR expression levels increased dramatically as cells differentiated from fibroblasts into insulin responsive adipocytes. mTOR localized to membrane fractions in all cells tested and in 3T3-L1 adipocytes mTOR was specifically localized to microsomal membranes. Protein kinase activity directed towards mTOR was tightly associated with mTOR immunoprecipitates and this kinase activity was inhibited by FKBP12-rapamycin indicating it was due to an autokinase activity present in mTOR. The mTOR autokinase and the protein kinase activity of the p110 alpha isoform of PI 3-kinase shared several notable similarities; (a) both were maximally active in the presence of Mn2+ but also showed significant activity in the presence of Mg2+ (b) neither were inhibited by the presence of non-ionic detergent and (c) both were inhibited by wortmannin and LY294002, known inhibitors of the PI 3-kinase lipid kinase activity. These data taken together indicate the autokinase activity lay in the PI 3-kinase homology domain. In summary mTOR is a membrane anchored protein kinase that is active in conditions encountered in vivo and the fact it is highly expressed in insulin responsive cell types is consistent with a role in insulin signalling.

3T3 Cells↗
PubMed1997

Leptin rapidly suppresses insulin release from insulinoma cells, rat and human islets and, in vivo, in mice.

Obesity is associated with diabetes, and leptin is known to be elevated in obesity. To investigate whether leptin has a direct effect on insulin secretion, isolated rat and human islets and cultured insulinoma cells were studied. In all cases, mouse leptin inhibited insulin secretion at concentrations within the plasma range reported in humans. Insulin mRNA expression was also suppressed in the cultured cells and rat islets. The long form of the leptin receptor (OB-Rb) mRNA was present in the islets and insulinoma cell lines. To determine the significance of these findings in vivo, normal fed mice were injected with two doses of leptin. A significant decrease in plasma insulin and associated rise in glucose concentration were observed. Fasted normal and leptin receptor-deficient db/db mice showed no response to leptin. A dose of leptin, which mimicked that found in normal mice, was administered to leptin-deficient, hyperinsulinemic ob/ob mice. This caused a marked lowering of plasma insulin concentration and a doubling of plasma glucose. Thus, leptin has a powerful acute inhibitory effect on insulin secretion. These results suggest that the action of leptin may be one mechanism by which excess adipose tissue could acutely impair carbohydrate metabolism.

Animals↗
PubMed1997

Rapamycin dissociates p70(S6K) activation from DNA synthesis stimulated by bombesin and insulin in Swiss 3T3 cells.

Phosphorylation and subsequent activation of p70 S6 kinase (S6K) are events that are highly conserved in the cellular response to mitogens. The neuropeptide bombesin, which is a potent mitogen for Swiss 3T3 cells, stimulated a time- and dose-dependent activation of p70(S6K) as determined by gel mobility shift and immune complex kinase assays. This effect was inhibited by the immunosuppressant rapamycin at 10 nM, which also completely abolished bombesin-stimulated DNA synthesis at identical concentrations. In striking contrast, the combination of bombesin and insulin in synergy stimulated maximum DNA synthesis in these cells despite persistent inhibition of p70(S6K) by rapamycin throughout G1. These results indicate that activation of p70(S6K) is not required for the transition of quiescent cells to the S phase of the cell cycle. The inhibitory effects of rapamycin on bombesin-stimulated cell cycle progression did not involve accumulation of the cyclin-dependent kinase inhibitor p27(kip1) but a striking inhibition of the expression of cyclin D1. This effect was circumvented by bombesin with insulin, suggesting that a rapamycin-insensitive pathway stimulated by this combination leads to cyclin D1 expression. Thus, these findings, dissociating the mitogenic effects of bombesin in synergy with insulin from activation of p70(S6K), support the hypothesis that this kinase is a component of one of the parallel pathways that can lead to DNA synthesis rather than an obligatory point of convergence in mitogenic signaling.

3T3 Cells↗
PubMed1997

Signalling pathways involved in the mitogenic effects of cAMP.

1. Elevation of intracellular cyclic AMP (cAMP) is a potent mitogenic signal for a number of cell types, including Swiss 3T3 cells, thyroid epithelial cells and the somatotroph cells of the anterior pituitary. 2. Activation of the mitogen-activated protein kinase (MAPK) cascade has been shown to underlie the mitogenic effects of many growth factors. However, the precise relationship between the mitogenic effects of cAMP and the MAPK cascade is not fully defined. 3. In Swiss 3T3 cells, elevation of cAMP did not stimulate kinases at all three levels of the MAPK cascade. Additionally, blockade of the MAPK pathway failed to inhibit cAMP-stimulated DNA synthesis. 4. Mitogenic combinations of cAMP strongly stimulated the phosphorylation and activation of the serine/threonine kinase p70 S6 kinase, p70S6K, an effect that was inhibited by rapamycin. This agent markedly inhibited cAMP-stimulated DNA synthesis, suggesting a critical role for p70S6K in cAMP mitogenic signalling. 5. Thus, multiple parallel but distinct signalling pathways may be involved in the action of mitogens. This redundancy has important implications for the pathogenesis and treatment of conditions characterized by inappropriate activation of growth factor signalling pathways.

3T3 Cells↗
PubMed1997

Regulation of anterior pituitary galanin and vasoactive intestinal peptide by oestrogen and prolactin status.

The neuropeptides vasoactive intestinal peptide (VIP) and galanin are synthesized in the anterior pituitary, galanin in the lactotroph and VIP probably in another cell type, and both stimulate prolactin secretion. Oestrogen regulates anterior pituitary VIP and galanin, galanin expression reflecting physiological variation in oestrogen status, whilst VIP is induced by pharmacological concentrations of oestrogen. Implanting anterior pituitaries under the renal capsule to induce hyperprolactinaemia we studied the regulation of anterior pituitary VIP and galanin synthesis and storage by prolactin and its interaction with oestrogen status. Five groups of animals were studied: control, hypophysectomized implanted, implanted, hyperoestrogenized (oestradiol-17 beta; 250 micrograms/day) and hyperoestrogenized implanted. Spontaneously cycling animals were followed through two cycles prior to implanting and were maintained for at least 1 week and then killed once they were in dioestrus. Circulating prolactin levels were significantly elevated in implanted animals but not in hypophysectomized implanted animals compared with controls. There was a more marked increase in prolactin levels in hyperoestrogenized animals and hyperoestrogenized implanted animals, with no significant difference between these two groups. Native anterior pituitary galanin and VIP content was suppressed in implanted animals, and markedly increased in hyperoestrogenized animals. Pituitary implantation only marginally reduced the effect of hyperoestrogenization on galanin content but abolished the effect of hyperoestrogenization on VIP content. Implant peptide content was suppressed to less than 10% of native anterior pituitary content. Galanin was not detected in implants from hypophysectomized-implanted animals but implant VIP content was unaffected by hypophysectomy. VIP content was increased in implants from hyperoestrogenized implanted animals but implant galanin content was unaffected by hyperoestrogenization. Peptide mRNA levels changed in parallel with peptide content except that the implant galanin mRNA levels were increased by hyperoestrogenization. Thus it appears that prolactin negatively regulates anterior pituitary galanin and VIP gene expression and content, probably due to a direct effect on the anterior pituitary and by altered secretion of hypothalamic factors. Oestrogen is a potent stimulus to expression of both peptide genes. Its positive effect on anterior pituitary peptide gene expression and content is greatly diminished by the effect of implant-induced hyperprolactinaemia, suggesting that circulating prolactin levels may be controlled by a negative feedback effect of prolactin on galanin and VIP. A similar effect of hyperoestrogenization is observed in the implants, except that galanin content remains at a low level, suggesting that the combination of hyperoestrogenization and the absence of dopamine may lead to uncontrolled release of high levels of galanin.

Animals↗
PubMed1996

Reduced requirement of mitogen-activated protein kinase (MAPK) activity for entry into the S phase of the cell cycle in Swiss 3T3 fibroblasts stimulated by bombesin and insulin.

Bombesin induced a marked and persistent activation of the mitogen-activated protein kinase kinase-1 (MEK-1), p42(mapk) and p90(rsk) in Swiss 3T3 cells by a pathway that was independent of p74(raf-1) but dependent on the activity of protein kinase C. Pretreatment of the cells with a specific inhibitor of MEK-1, PD 098059, markedly reduced the early and abolished the sustained phase of bombesin-induced p42(mapk) activation. In addition, PD 098059 prevented bombesin-induced DNA synthesis and progression of the cells through the cell cycle, indicating that the mitogenic effect of bombesin is dependent on the activation of p42(mapk). However, in the presence of insulin, which neither stimulated p42(mapk) activation nor DNA synthesis on its own in Swiss 3T3 cells, bombesin potently stimulated DNA synthesis even at concentrations of PD 098059 (15 microM) that completely abolished the mitogenic effect of bombesin alone. Furthermore, Swiss 3T3 cells stably transfected with interfering mutants of MEK-1 showed a marked decrease in the mitogenic effect of bombesin. In contrast, the combination of bombesin and insulin strongly stimulated DNA synthesis in these cells to levels comparable with that obtained in the wild type cells. Thus, our data demonstrate that insulin dramatically reduced the requirement for the mitogen-activated protein kinase pathway for reinitiation of DNA synthesis in bombesin-treated Swiss 3T3 cells and consequently indicate that the contribution of the mitogen-activated protein kinase cascade to mitogenesis depends on the combination of extracellular signals that are used to stimulate these cells.

3T3 Cells↗
PubMed1996

Adrenomedullin stimulates DNA synthesis and cell proliferation via elevation of cAMP in Swiss 3T3 cells.

Our results demonstrate that the novel vasoactive regulatory peptide adrenomedullin is a potent mitogen for Swiss 3T3 cells. Acting via a specific adrenomedullin receptor, it stimulates a dose-dependent increase in DNA synthesis in synergy with insulin. Additionally, adrenomedullin stimulates further progression through the cell cycle resulting in cell proliferation, an effect that was further enhanced by the presence of insulin. Adrenomedullin rapidly induces accumulation of intracellular cAMP but does not stimulate an increase in intracellular Ca2+, activation of protein kinase C, or tyrosine phosphorylation of intracellular substrates. Adrenomedullin-stimulated mitogenesis is markedly enhanced in Swiss 3T3 cells stably transfected with a constitutively activated Gs alpha, which are highly sensitive to agents that elevate cAMP, and is inhibited by the PKA inhibitor H-89. Adrenomedullin is, thus, identified as a novel mitogenic regulatory peptide acting via cAMP.

3T3 Cells↗