Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha.

Phosphoinositide 3-kinases produce 3'-phosphorylated phosphoinositides that act as second messengers to recruit other signalling proteins to the membrane. Pi3ks are activated by many extracellular stimuli and have been implicated in a variety of cellular responses. The Pi3k gene family is complex and the physiological roles of different classes and isoforms are not clear. The gene Pik3r1 encodes three proteins (p85 alpha, p55 alpha and p50 alpha) that serve as regulatory subunits of class IA Pi3ks (ref. 2). Mice lacking only the p85 alpha isoform are viable but display hypoglycaemia and increased insulin sensitivity correlating with upregulation of the p55 alpha and p50 alpha variants. Here we report that loss of all protein products of Pik3r1 results in perinatal lethality. We observed, among other abnormalities, extensive hepatocyte necrosis and chylous ascites. We also noted enlarged skeletal muscle fibres, brown fat necrosis and calcification of cardiac tissue. In liver and muscle, loss of the major regulatory isoform caused a great decrease in expression and activity of class IA Pi3k catalytic subunits; nevertheless, homozygous mice still displayed hypoglycaemia, lower insulin levels and increased glucose tolerance. Our findings reveal that p55 alpha and/or p50 alpha are required for survival, but not for development of hypoglycaemia, in mice lacking p85 alpha.

beta-cell-specific deletion of the Igf1 receptor leads to hyperinsulinemia and glucose intolerance but does not alter beta-cell mass.

Regulation of glucose homeostasis by insulin depends on the maintenance of normal beta-cell mass and function. Insulin-like growth factor 1 (Igf1) has been implicated in islet development and differentiated function, but the factors controlling this process are poorly understood. Pancreatic islets produce Igf1 and Igf2, which bind to specific receptors on beta-cells. Igf1 has been shown to influence beta-cell apoptosis, and both Igf1 and Igf2 increase islet growth; Igf2 does so in a manner additive with fibroblast growth factor 2 (ref. 10). When mice deficient for the Igf1 receptor (Igf1r(+/-)) are bred with mice lacking insulin receptor substrate 2 (Irs2(-/-)), the resulting compound knockout mice show a reduction in mass of beta-cells similar to that observed in pancreas of Igf1r(-/-) mice (ref. 11), suggesting a role for Igf1r in growth of beta-cells. It is possible, however, that the effects in these mice occur secondary to changes in vascular endothelium or in the pancreatic ductal cells, or because of a decrease in the effects of other hormones implicated in islet growth. To directly define the role of Igf1, we have created a mouse with a beta-cell-specific knockout of Igf1r (betaIgf1r(-/-)). These mice show normal growth and development of beta-cells, but have reduced expression of Slc2a2 (also known as Glut2) and Gck (encoding glucokinase) in beta-cells, which results in defective glucose-stimulated insulin secretion and impaired glucose tolerance. Thus, Igf1r is not crucial for islet beta-cell development, but participates in control of differentiated function.

Dilated cardiomyopathy and atrioventricular conduction blocks induced by heart-specific inactivation of mitochondrial DNA gene expression.

Mutations of mitochondrial DNA (mtDNA) cause several well-recognized human genetic syndromes with deficient oxidative phosphorylation and may also have a role in ageing and acquired diseases of old age. We report here that hallmarks of mtDNA mutation disorders can be reproduced in the mouse using a conditional mutation strategy to manipulate the expression of the gene encoding mitochondrial transcription factor A (Tfam, previously named mtTFA), which regulates transcription and replication of mtDNA. Using a loxP-flanked Tfam allele (TfamloxP) in combination with a cre-recombinase transgene under control of the muscle creatinine kinase promoter, we have disrupted Tfam in heart and muscle. Mutant animals develop a mosaic cardiac-specific progressive respiratory chain deficiency, dilated cardiomyopathy, atrioventricular heart conduction blocks and die at 2-4 weeks of age. This animal model reproduces biochemical, morphological and physiological features of the dilated cardiomyopathy of Kearns-Sayre syndrome. Furthermore, our findings provide genetic evidence that the respiratory chain is critical for normal heart function.

Identification of the gene altered in Berardinelli-Seip congenital lipodystrophy on chromosome 11q13.

Congenital generalized lipodystrophy, or Berardinelli-Seip syndrome (BSCL), is a rare autosomal recessive disease characterized by a near-absence of adipose tissue from birth or early infancy and severe insulin resistance. Other clinical and biological features include acanthosis nigricans, hyperandrogenism, muscular hypertrophy, hepatomegaly, altered glucose tolerance or diabetes mellitus, and hypertriglyceridemia. A locus (BSCL1) has been mapped to 9q34 with evidence of heterogeneity. Here, we report a genome screen of nine BSCL families from two geographical clusters (in Lebanon and Norway). We identified a new disease locus, designated BSCL2, within the 2.5-Mb interval flanked by markers D11S4076 and D11S480 on chromosome 11q13. Analysis of 20 additional families of various ethnic origins led to the identification of 11 families in which the disease cosegregates with the 11q13 locus; the remaining families provide confirmation of linkage to 9q34. Sequence analysis of genes located in the 11q13 interval disclosed mutations in a gene homologous to the murine guanine nucleotide-binding protein (G protein), gamma3-linked gene (Gng3lg) in all BSCL2-linked families. BSCL2 is most highly expressed in brain and testis and encodes a protein (which we have called seipin) of unknown function. Most of the variants are null mutations and probably result in a severe disruption of the protein. These findings are of general importance for understanding the molecular mechanisms underlying regulation of body fat distribution and insulin resistance.

Targeted disruption of the glucose transporter 4 selectively in muscle causes insulin resistance and glucose intolerance.

The prevalence of type 2 diabetes mellitus is growing worldwide. By the year 2020, 250 million people will be afflicted. Most forms of type 2 diabetes are polygenic with complex inheritance patterns, and penetrance is strongly influenced by environmental factors. The specific genes involved are not yet known, but impaired glucose uptake in skeletal muscle is an early, genetically determined defect that is present in non-diabetic relatives of diabetic subjects. The rate-limiting step in muscle glucose use is the transmembrane transport of glucose mediated by glucose transporter (GLUT) 4 (ref. 4), which is expressed mainly in skeletal muscle, heart and adipose tissue. GLUT4 mediates glucose transport stimulated by insulin and contraction/exercise. The importance of GLUT4 and glucose uptake in muscle, however, was challenged by two recent observations. Whereas heterozygous GLUT4 knockout mice show moderate glucose intolerance, homozygous whole-body GLUT4 knockout (GLUT4-null) mice have only mild perturbations in glucose homeostasis and have growth retardation, depletion of fat stores, cardiac hypertrophy and failure, and a shortened life span. Moreover, muscle-specific inactivation of the insulin receptor results in minimal, if any, change in glucose tolerance. To determine the importance of glucose uptake into muscle for glucose homeostasis, we disrupted GLUT4 selectively in mouse muscles. A profound reduction in basal glucose transport and near-absence of stimulation by insulin or contraction resulted. These mice showed severe insulin resistance and glucose intolerance from an early age. Thus, GLUT4-mediated glucose transport in muscle is essential to the maintenance of normal glucose homeostasis.

3-Hydroxyisobutyrate, A Strong Marker of Insulin Resistance in Type 2 Diabetes and Obesity That Modulates White and Brown Adipocyte Metabolism.

Circulating branched-chain amino acids (BCAAs) associate with insulin resistance and type 2 diabetes. 3-Hydroxyisobutyrate (3-HIB) is a catabolic intermediate of the BCAA valine. In this study, we show that in a cohort of 4,942 men and women, circulating 3-HIB is elevated according to levels of hyperglycemia and established type 2 diabetes. In complementary cohorts with measures of insulin resistance, we found positive correlates for circulating 3-HIB concentrations with HOMA2 of insulin resistance, as well as a transient increase in 3-HIB followed by a marked decrease after bariatric surgery and weight loss. During differentiation, both white and brown adipocytes upregulate BCAA utilization and release increasing amounts of 3-HIB. Knockdown of the 3-HIB-forming enzyme 3-hydroxyisobutyryl-CoA hydrolase decreases release of 3-HIB and lipid accumulation in both cell types. Conversely, addition of 3-HIB to white and brown adipocyte cultures increases fatty acid uptake and modulated insulin-stimulated glucose uptake in a time-dependent manner. Finally, 3-HIB treatment decreases mitochondrial oxygen consumption and generation of reactive oxygen species in white adipocytes, while increasing these measures in brown adipocytes. Our data establish 3-HIB as a novel adipocyte-derived regulator of adipocyte subtype-specific functions strongly linked to obesity, insulin resistance, and type 2 diabetes.

Role of atypical protein kinase C in activation of sterol regulatory element binding protein-1c and nuclear factor kappa B (NFkappaB) in liver of rodents used as a model of diabetes, and relationships to hyperlipidaemia and insulin resistance.

AIMS/HYPOTHESIS: Previous findings in rodents used as a model of diabetes suggest that insulin activation of atypical protein kinase C (aPKC) is impaired in muscle, but, unexpectedly, conserved in liver, despite impaired hepatic protein kinase B (PKB/Akt) activation. Moreover, aPKC at least partly regulates two major transactivators: (1) hepatic sterol receptor binding protein-1c (SREBP-1c), which controls lipid synthesis; and (2) nuclear factor kappa B (NFkappaB), which promotes inflammation and systemic insulin resistance. METHODS: In Goto-Kakizaki rats used as a model of type 2 diabetes, we examined: (1) whether differences in hepatic aPKC and PKB activation reflect differences in activation of IRS-1- and IRS-2-dependent phosphatidylinositol 3-kinase (PI3K); (2) whether hepatic SREBP-1c and NFkappaB are excessively activated by aPKC; and (3) metabolic consequences of excessive activation of hepatic aPKC, SREBP-1c and NFkappaB. RESULTS: In liver, as well as in muscle, IRS-2/PI3K activation by insulin was intact, whereas IRS-1/PI3K activation by insulin was impaired. Moreover, hepatic IRS-2 is known to control hepatic aPKC during insulin activation. Against this background, selective inhibition of hepatic aPKC by adenoviral-mediated expression of mRNA encoding kinase-inactive aPKC or short hairpin RNA targeting Irs2 mRNA and partially depleting hepatic IRS-2 diminished hepatic SREBP-1c production and NFkappaB activities, concomitantly improving serum lipids and insulin signalling in muscle and liver. Similar improvements in SREBP-1c, NFkappaB and insulin signalling were seen in ob/ob mice following inhibition of hepatic aPKC. CONCLUSIONS/INTERPRETATION: In diabetic rodent liver, diminished PKB activation may largely reflect impaired IRS-1/PI3K activation, while conserved aPKC activation reflects retained IRS-2/PI3K activity. Hepatic aPKC may also contribute importantly to excessive SREPB-1c and NFkappaB activities. Excessive hepatic aPKC-dependent activation of SREBP-1c and NFkappaB may contribute importantly to hyperlipidaemia and systemic insulin resistance.

Membrane receptors for hormones and neurotransmitters.

Receptors for peptide hormones and neurotransmitters are integral components of the plasma membrane of cells which serve to couple the external milieu to the intracellular regulators of metabolism. These macromolecules are usually high molecular weight glycoproteins, and in many cases appear to have more than one subunit capable of binding the hormone. The interaction of the hormone or neurotransmitter with its receptor is rapid, reversible, and of high affinity and specificity. Many receptors exhibit cooperative properties in hormone binding or biological function. The concentration of receptors on the membrane is a function of continued synthesis and degradation, and may be altered by a variety of factors including the hormone itself. The fluid mosaic nature of the membrane may allow hormone receptors and effectors to exist in free floating states. Further investigations of the hormone-receptor interaction will no doubt yield new insights into both the mechanism of hormone action and membrane structure and function.

Abnormality of insulin binding and receptor phosphorylation in an insulin-resistant melanoma cell line.

The insulin receptor possesses an insulin-stimulated tyrosine-kinase activity; however, the significance of receptor phosphorylation in terms of the binding and signaling function of the receptor is unclear. To help clarify this problem, we have studied insulin binding and receptor phosphorylation in a Cloudman S91 melanoma cell line and two of its variants: the wild type (1A) in which insulin inhibits cell growth, an insulin-resistant variant (111) in which insulin neither stimulates or inhibits growth, and a variant (46) in which insulin stimulates cell growth. 125I-insulin binding to intact cells was similar for the wild-type 1A and insulin-stimulated variant 46. The insulin-resistant variant 111, in contrast, showed approximately 30% decrease in insulin binding. This was due to a decrease of receptor affinity with no major difference in receptor number. When the melanoma cells were solubilized in 1% Triton X-100 and the insulin receptor was partially purified by chromatography on wheat germ agglutinin-agarose, a similar pattern of binding was observed. Phosphorylation was studied by incubation of the partially purified receptor with insulin and [gamma-32P]ATP, and the receptor was identified by immunoprecipitation and NaDodSO4 PAGE. Insulin stimulated phosphorylation of the 95,000-mol-wt beta-subunit of the receptor in all three cells types with similar kinetics. The amount of 32P incorporated into the beta-subunit in the insulin-resistant cell line 111 was approximately 50% of that observed with the two other cell lines. This difference was reflected throughout the entire dose-response curve (10(-9) M to 10(-6) M). Qualitatively similar results were obtained when phosphorylation was studied in the intact cell. Peptide mapping of the beta-subunit using tryptic digestion and reverse-phase high-performance liquid chromatography column separation indicated three sites of phosphorylation in receptor from the wild type and variant 46, but only two major sites of phosphorylation of variant 111. These data suggest that the insulin-resistant variant melanoma 111 possesses a specific defect in the insulin receptor which alters both its binding and autophosphorylation properties, and also suggests a possible role of receptor phosphorylation in both the binding and the signaling function of the insulin receptor.

Two steps of insulin receptor internalization depend on different domains of the beta-subunit.

The internalization of signaling receptors such as the insulin receptor is a complex, multi-step process. The aim of the present work was to determine the various steps in internalization of the insulin receptor and to establish which receptor domains are implicated in each of these by the use of receptors possessing in vitro mutations. We find that kinase activation and autophosphorylation of all three regulatory tyrosines 1146, 1150, and 1151, but not tyrosines 1316 and 1322 in the COOH-terminal domain, are required for the ligand-specific stage of the internalization process; i.e., the surface redistribution of the receptor from microvilli where initial binding occurs to the nonvillous domain of the cell. Early intracellular steps in insulin signal transduction involving the activation of phosphatidylinositol 3'-kinase are not required for this redistribution. The second step of internalization consists in the anchoring of the receptors in clathrin-coated pits. In contrast to the first ligand specific step, this step is common to many receptors including those for transport proteins and occurs in the absence of kinase activation and receptor autophosphorylation, but requires a juxta-membrane cytoplasmic segment of the beta-subunit of the receptor including a NPXY sequence. Thus, there are two independent mechanisms controlling insulin receptor internalization which depend on different domains of the beta-subunit.

Rad: a member of the Ras family overexpressed in muscle of type II diabetic humans.

To identify the gene or genes associated with insulin resistance in Type II (non-insulin-dependent) diabetes mellitus, subtraction libraries were prepared from skeletal muscle of normal and diabetic humans and screened with subtracted probes. Only one clone out of 4000 was selectively overexpressed in Type II diabetic muscle as compared to muscle of non-diabetic or Type I diabetic individuals. This clone encoded a new 29-kilodalton member of the Ras-guanosine triphosphatase superfamily and was termed Rad (Ras associated with diabetes). Messenger ribonucleic acid of Rad was expressed primarily in skeletal and cardiac muscle and was increased an average of 8.6-fold in the muscle of Type II diabetics as compared to normal individuals.

Insulin stimulates the phosphorylation of the 95,000-dalton subunit of its own receptor.

Cultured human lymphocytes and rat hepatoma cells were labeled with [32P]orthophosphate and the insulin receptor subunits identified by immunoprecipitation and sodium dodecyl sulfate-gel electrophoreses. In both cell types the 95,000-dalton (beta) subunit of the insulin receptor was selectively phosphorylated. Phosphorylation was specifically stimulated by insulin in a dose-dependent fashion after 1 and 15 minutes of hormone treatment, whereas human growth hormone was without effect. This phosphorylation may be a very early event in insulin action.

Radioimmunoassay of the insulin receptor: a new probe of receptor structure and function.

A sensitive and specific radioimmunoassay for the insulin receptor has been developed employing receptor autoantibodies from the serum of a patient with insulin-resistant diabetes. The assay detects insulin binding sites at concentrations as low as 0.1 nanomolar; distinguishes between receptors originating from human placental membranes, human lymphoblastoid cells, and mouse liver membranes; and measures the receptor independently of its binding function. Down-regulation, or loss of binding after exposure to insulin, is associated with loss of immunoreactive receptor.

Antibodies that impair insulin receptor binding in an unusual diabetic syndrome with severe insulin resistance.

Six patients with a unique form of diabetes associated with extreme insulin resistance have markedly reduced insulin binding to specific receptors on their circulating monocytes. When normal insulin receptors were exposed to serum or immunoglobulin fractions from three of these patients in vitro the specific binding defect was reproduced.

Role of brain insulin receptor in control of body weight and reproduction.

Insulin receptors (IRs) and insulin signaling proteins are widely distributed throughout the central nervous system (CNS). To study the physiological role of insulin signaling in the brain, we created mice with a neuron-specific disruption of the IR gene (NIRKO mice). Inactivation of the IR had no impact on brain development or neuronal survival. However, female NIRKO mice showed increased food intake, and both male and female mice developed diet-sensitive obesity with increases in body fat and plasma leptin levels, mild insulin resistance, elevated plasma insulin levels, and hypertriglyceridemia. NIRKO mice also exhibited impaired spermatogenesis and ovarian follicle maturation because of hypothalamic dysregulation of luteinizing hormone. Thus, IR signaling in the CNS plays an important role in regulation of energy disposal, fuel metabolism, and reproduction.

Visualization of viral clearance in the living animal.

The early events in viral dissemination via the bloodstream were identified by monitoring the fate of 123I-radiolabeled reovirus after it was injected intravenously in rats. Continuous scintillation camera imaging showed that reovirus serotypes 1 and 3 were cleared from the circulation in less than 10 minutes by specific and distinct target organs. Reovirus serotype 1 accumulated predominantly in the lungs and the liver, whereas serotype 3 accumulated in the liver and the spleen with very little virus uptake by the lungs. Incubation of reovirus serotype 1 with a monoclonal antibody directed against the viral hemagglutinin before injection totally inhibited the clearance of the virus by the lungs. Similar results were obtained when viruses biolabeled with 35S were used. These results demonstrate that viruses can be rapidly transported through the bloodstream to specific target organs and that the localization of the viruses depends on the interaction between specific viral surface components and the target organ.

Male Brown Fat-Specific Double Knockout of IGFIR/IR: Atrophy, Mitochondrial Fission Failure, Impaired Thermogenesis, and Obesity.

It is unknown how the lack of insulin receptor (IR)/insulinlike growth factor I receptor (IGFIR) in a tissue-specific manner affects brown fat development and mitochondrial integrity and function, as well as its effect on the redistribution of the adipose organ and the metabolic status. To address this important issue, we developed IR/IGFIR double-knockout (DKO) in a brown adipose tissue-specific manner. Lack of those receptors caused severe brown fat atrophy, enhanced beige cell clusters in inguinal fat; loss of mitochondrial mass; mitochondrial damage related to cristae disruption; and the loss of proteins involved in autophagosome formation, mitophagy, mitochondrial quality control, and dynamics and thermogenesis. More important, DKO mice showed an impaired thermogenesis upon cold exposure, based on a failure in the mitochondrial fission mechanisms and a much lower uncoupling protein 1 transcription rate and content. As a result, DKO mice under normal conditions showed an obesity susceptibility, revealed by increased body fat mass and insulin resistance. Upon consumption of a high-fat diet, DKO mice displayed frank obesity, as shown by increased body weight, increased adiposity, insulin resistance, hyperinsulinemia, and hypertriglyceridemia, all consistent with a metabolic syndrome. Collectively, our data suggest a cause-and-effect relationship between failure in brown fat thermogenesis and increased adiposity and obesity.

Nonsuppressible insulin-like activity of human serum. A potent inhibitor of insulin degradation.

Nonsuppressible insulin-like activity soluble in acid ethanol (NSILA-s) is a well-characterized peptide derived from human serum which has previously been shown to have insulin-like bioactivity and react with both insulin and NSILA-s receptor sites in liver plasma membranes. In the present study we find that NSILA-s is also a potent competitive inhibitor of the insulin-degrading system of the liver plasma membrane. The most purified NSILA-s preparation tested was 20-fold more potent than insulin itself, and significant inhibition of insulin degradation occurred at concentrations of NSILA-s similar to those found in plasma.

Epidermal growth factor receptor defects in leprechaunism. A multiple growth factor-resistant syndrome.

Leprechaunism is a rare genetic disorder characterized by severe growth retardation and insulin resistance. Maximal epidermal growth factor (EGF) binding was reduced in fibroblasts from three unrelated patients with leprechaunism (Ark-1, Can-1, and Minn-1) compared with control (0.8-2.2%/mg protein vs. 5.5%/mg protein). This was due to a decrease in receptor affinity in Ark-1 and Can-1 and a decrease in receptor number in Minn-1. In all cell lines, EGF-stimulated receptor autophosphorylation was also decreased to 18-60% of control, whereas EGF internalization and degradation was normal. Sphingosine (40 microM), a protein kinase C inhibitor, increased EGF receptor affinity twofold in control cells and six- to nine-fold in cells of leprechaunism. However, sphingosine did not enhance EGF-stimulated receptor autophosphorylation in either the controls or the patients' cells. By contrast, only one of the three cell lines of patients with the type A syndrome demonstrated a decrease in EGF binding and all demonstrated normal or near normal EGF-stimulated receptor autophosphorylation. These data indicate that in patients with leprechaunism, there are functional abnormalities of the EGF receptor, as well as of the insulin receptor, that may contribute to the severity of the syndrome. These data also suggest a role for the insulin receptor in maintaining normal EGF receptor function in these cells.