INSULIN RESISTANCE AND PATHOGENESIS OF POSTMENOPAUSAL OSTEOPOROSIS.

Osteoporosis (OP) is a disease predisposing postmenopausal women to fractures, and often accompanied by insulin resistance (IR) and metabolic syndrome (MetS). Previous studies provided contradictory results concerning prevalence of MetS in postmenopausal OP. To better understand the pathogenesis of IR, we reviewed cellular and molecular aspects and systematically reviewed studies providing homeostasis model assessment (HOMA) index. Bone is an active endocrine organ maintaining its integrity by orchestrated balance between bone formation and resorption. Both osteoblasts and osteoclasts contain receptors for insulin and insulin-like growth factor-1 (IGF-1) operating in skeletal development and in the adult life. Defects in this system generate systemic IR and bone-specific IR, which in turn regulates glucose homeostasis and energy metabolism through osteocalcin. Examination of genetic syndromes of extreme IR revealed intriguing features namely high bone mineral density (BMD) or accelerated growth. Studies of moderate forms of IR in postmenopausal women reveal positive correlations between HOMA index and BMD while correlations with osteocalcin were rather negative. The relation with obesity remains complex involving regulatory factors such as leptin and adiponectin to which the contribution of potential genetic factors and in particular, the correlation with the degree of obesity or body composition should be added.

HOW GENETICISTS CONTRIBUTE TO UNDERSTANDING OF COVID-19 DISEASE PATHOGENICITY.

Human populations are faced to the COVID-19 pandemic due to the emerging SARS-CoV-2 coronavirus originating from Wuhan (China) and with dramatic Public Health consequences. Despite periods of panic, the scientific community demonstrated an incredible innovation potential and energy ending up in one year with new vaccines to be used in population. Researchers are interrogating on how individual genetic differences contribute to the diversity of clinical manifestations or ethnic and geographic disparities of COVID-19. While efforts were spent to understand mechanistically the infectious potential of the virus, recent progresses in molecular genetics and bioinformatics allowed the characterization of viral sequence and construction of phylogeographical maps of viral dispersion worldwide. These data will help understanding epidemiological disparities among continents and ethnic populations. Much effort was also spent in analyzing host genetics by studying individual genes involved in innate and immune responses or explaining pathogenesis of comorbidities that complicate the fate of elderly patients. Several international consortia launched already Genome wide Association Studies (GWAS) and whole genome sequencing strategies to identify genetic markers with immediate application in patients at risk of respiratory failure. These new genetic data are important not only for understanding susceptibility factors for COVID-19 but they also contain an important message of hope for mankind warranting our survival and health.

PORTABILITY OF GWAS RESULTS BETWEEN ETHNIC POPULATIONS: GENETIC MARKERS FOR POLYCYSTIC OVARY SYNDROME (PCOS) IN MEDITERRANEAN AREA.

Genome Wide Association Studies (GWAS) are excellent opportunities to define culprit genes in complex disorders such as the polycystic ovary syndrome (PCOS). PCOS is a prevalent disorder characterized by anovulation, hyperandrogenism and polycystic ovaries, which benefitted from several GWASs in Asians and Europeans revealing more than 20 potential culprit genes near associated single nucleotide variations (SNV). Translation of these findings into the clinical practice raises difficulties since positive hits are surrogate SNVs linked with causative mutations by linkage disequilibrium (LD). Studies in Mediterranean populations (e.g. Southern Europe and North Africa) raise supplementary problems because of a different LD-pattern, which may disrupt the link with causative mutations. Our experience in MEDIGENE program between Tunisia and France enforces the necessity of genetic anthropology studies before translating GWAS data. Tunisians are a heterogeneous population with ancestral Berbers, European, Arab and Sub-Saharan African components while South Europeans display a high level of genetic diversity, partially explained by gene flow from North Africa. Human diversity studies require sampling from Middle East and North Africa (MENA) region that will help to understand genetic factors in complex diseases.

BRANCHED CHAIN AMINO ACIDS AT THE EDGE BETWEEN MENDELIAN AND COMPLEX DISORDERS.

Branched chained amino acids (BCAA) are essential components of the human diet and important nutrient signals, which regain particular interest in recent years with the avenue of metabolomics studies suggesting their potential role as biomarkers. There is now compelling evidence for predictive role of BCAA in progression of diabetes, but causality relationship is still debated concerning insulin resistance and genetic versus non-genetic pathogenesis. Mendelian randomization studies in large cohorts of diabetes indicated pathogenic role of PPM1K (protein phosphatase Mg2+/Mn2+ dependent 1K) on Chr 4q22.1 gene, encoding for a phosphatase that activates BCKDH (branched chain keto acid dehydrogenase) complex. Recent studies indicated that insulin rapidly and dose-dependently regulates gene expression of the same complex, but the relationship with systemic insulin resistance and glucose levels is complex. Rare genetic syndromes due to Mendelian mutations in key genes in BCAA catabolism may be good models to understand potential role of gene of BCAA catabolism. However, in studying complex disorders geneticists are faced to complete new aspects of metabolic regulation complicating understanding genetics of obesity, diabetes or metabolic syndrome. A review of genetic syndromes of BCAA metabolism suggests that insulin resistance is not present, except rare cases of methylmalonic aciduria due to MUT (methylmalonyl-coA mutase) gene on Chr 6p12.3. Another aspect that complicates understanding is the new role of central nervous system (CNS) in insulin resistance. For a long time the hypothalamic hunger/satiety neuronal system was considered a key site of nutrient regulation. Genes may also affect the brain rewarding system (BRS) that would regulate food intake by modulating the motivation to obtain food and considering hedonic properties. Nutrigenomic and nutrigenetic investigations taking into account concurrently BCAA intake, metabolic regulation and gene variation have large perspectives to merge genetic and nutritional understanding in complex disorders.

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.

Impact of FTO genotypes on BMI and weight in polycystic ovary syndrome: a systematic review and meta-analysis.

AIMS/HYPOTHESIS: FTO gene single nucleotide polymorphisms (SNPs) have been shown to be associated with obesity-related traits and type 2 diabetes. Several small studies have suggested a greater than expected effect of the FTO rs9939609 SNP on weight in polycystic ovary syndrome (PCOS). We therefore aimed to examine the impact of FTO genotype on BMI and weight in PCOS. METHODS: A systematic search of medical databases (PubMed, EMBASE and Cochrane CENTRAL) was conducted up to the end of April 2011. Seven studies describing eight distinct PCOS cohorts were retrieved; seven were genotyped for SNP rs9939609 and one for SNP rs1421085. The per allele effect on BMI and body weight increase was calculated and subjected to meta-analysis. RESULTS: A total of 2,548 women with PCOS were included in the study; 762 were TT homozygotes, 1,253 had an AT/CT genotype, and 533 were AA/CC homozygotes. Each additional copy of the effect allele (A/C) increased the BMI by a mean of 0.19 z score units (95% CI 0.13, 0.24; p = 2.26 × 10(-11)) and body weight by a mean of 0.20 z score units (95% CI 0.14, 0.26; p = 1.02 × 10(-10)). This translated into an approximately 3.3 kg/m(2) increase in BMI and an approximately 9.6 kg gain in body weight between TT and AA/CC homozygotes. The association between FTO genotypes and BMI was stronger in the cohorts with PCOS than in the general female populations from large genome-wide association studies. Deviation from an additive genetic model was observed in heavier populations. CONCLUSIONS/INTERPRETATION: The effect of FTO SNPs on obesity-related traits in PCOS seems to be more than two times greater than the effect found in large population-based studies. This suggests an interaction between FTO and the metabolic context or polygenic background of PCOS.

Increased copeptin levels in metabolic syndrome from a Romanian population.

Rationale: Arginine vasopressin (AVP) is secreted under conditions of water deprivation. Since AVP has a low half-life in the plasma, the C-terminal fragment of AVP-precursor (copeptin) was used to estimate the AVP levels. High copeptin levels increase the risk for the development of diabetes mellitus. Aim: This study was aimed to measure copeptin levels in the metabolic syndrome (MetS) in Romanians using a competitive enzyme immunoassay. Methods and results: Patients prone to present MetS (n = 63) were compared to controls (n = 42). In the MetS group, the syndrome was confirmed in 93.6%. Affected patients displayed 85.7% obesity and insulin resistance (HOMAIR of 4.9 ± 0.4 versus 1.1 ± 0.8 in controls). Low HDL-cholesterol was less represented (47.5%). Copeptin levels were 0.6 ± 0.0 in MetS versus 0.42 ± 0.0 ng/ mL in controls (P < 0.004). Higher copeptin (0.79 to 1.83 ng/ mL) was associated with MetS, P < 0.0018, OR 20, 95%CI [3.03 - 131.7]. In ANOVA, high copeptin was equally explained by MetS or obesity (P < 0.05,α = 3.8). The best correlation was found with high triglyceride levels (P < 0.013,α = 6.3) while the correlation with HOMAIR remained not significant. Discussion: These data indicated a concordant correlation between increased copeptin and MetS or its components. In the light of epidemiological data, indicating that more than 50% of the European population has a lower daily water intake and a fraction of 25% displaying high copeptin, our data further sustained that copeptin may be a good biomarker for MetS and/ or obesity, which should be further investigated with other members of the osmoregulation pathway at both pathogenesis and genetic levels.

Effects of heparin on insulin binding and biological activity.

The effect of heparin, a polyanionic glycosaminoglycan known to alter the function of many proteins, on insulin binding and bioactivity was studied. Cultured human lymphocytes (IM-9) were incubated with varying concentrations of heparin, then extensively washed, and 125I-labeled insulin binding was measured. Heparin at concentrations used clinically for anticoagulation (1-50 U/ml) inhibited binding in a dose-dependent manner; 50% inhibition of binding occurred with 5-10 U/ml. Scatchard analysis indicated that the decrease in binding was due to a decrease in both the affinity and the apparent number of available insulin receptors. The effect occurred within 10 min at 22 degrees C and persisted even after the cells were extensively washed. Inhibition of insulin binding also occurred when cells were preincubated with heparinized plasma or heparinized serum but not when cells were incubated with normal serum or plasma from blood anticoagulated with EDTA. By contrast, other polyanions and polycations, e.g., poly-L-glutamic acid, poly-L-lysine, succinylated poly-L-lysine, and histone, did not inhibit binding. Heparin also inhibited insulin binding in Epstein-Barr (EB) virus-transformed lymphocytes but had no effect on insulin binding to isolated adipocytes, human erythrocytes, or intact hepatoma cells. When isolated adipocytes were incubated with heparin, there was a dose-dependent inhibition of insulin-stimulated glucose oxidation and, to a lesser extent, of basal glucose oxidation. Although heparin has no effect on insulin binding to intact hepatoma cells, heparin inhibited both insulin binding and insulin-stimulated autophosphorylation in receptors solubilized from these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of binding and phosphorylation defects of erythrocyte insulin receptors in the type A syndrome of insulin resistance.

The type A syndrome of insulin resistance and acanthosis nigricans is characterized by severe insulin resistance due to a cellular defect in insulin action. To better understand the molecular nature of this defect, we have investigated insulin binding to circulating monocytes, erythrocytes, and the Triton X-100-solubilized erythrocyte receptor, and insulin-stimulated receptor autophosphorylation using cells and receptor from three type A patients. Insulin binding in both circulating cells and the soluble extract of erythrocytes indicated a heterogeneity of defects. Patients A1 and A2 both presented a major decrease in tracer insulin binding to intact cells and soluble insulin receptor. Determination of stoichiometric binding parameters using a cooperative model indicated that in patient A1 this was due to a reduction in the number of receptors, whereas in patient A2 the affinity constant for binding was decreased. Patient A3 presented near-normal insulin binding to erythrocytes and normal binding in intact monocytes, solubilized erythrocyte receptors, and cultured fibroblasts. Affinity labeling of erythrocyte receptor from this patient revealed a normal alpha-subunit and also a normal relative distribution of the higher-molecular-weight, nonreduced oligomeric forms of the receptor. Receptor autophosphorylation was measured using the solubilized insulin receptor from erythrocytes. The maximal stimulated phosphorylation was reduced by 79%, 76%, and 52% in patients A1, A2, and A3, respectively, relative to the simultaneous control. In all three patients, the autophosphorylation was stimulated only 1.0-3.5 times the basal level compared with controls, in which the stimulation was 5.7-fold +/- 1.2 (mean +/- 1 SD, P less than 0.005). In addition, in patients A1 and A2 a decrease in basal phosphorylation was observed and in patient A2 there was a rightward shift of the dose-response curve for insulin stimulation. These data and the correlation of coupling of receptor phosphorylation with the fractional occupancy of the receptor measured in the same extract suggest that these patients exhibit three types of defects. In patient A1, there is a loss in receptor number manifested by a parallel decrease in insulin binding and receptor phosphorylation. In patient A2, there is an additional decrease in the affinity constant leading to a decrease in both binding and receptor phosphorylation with an almost linear coupling between receptor occupancy and receptor phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

Prevalence of mutations in the insulin receptor gene in subjects with features of the type A syndrome of insulin resistance.

Mutations of the insulin receptor gene are a cause of the type A syndrome of extreme insulin resistance. This study assessed the prevalence of such mutations in women with clinical features of the type A syndrome including ovarian hyperandrogenism, moderate-to-severe degrees of insulin resistance, and acanthosis nigricans. We studied 22 unrelated women with insulin resistance (fasting insulin > 300 pM [50 microU/ml] and/or peak during an oral glucose tolerance test (OGTT) > 1,800 pM [300 microU/ml]), acanthosis nigricans, and the polycystic ovary syndrome (hyperandrogenemia, oligoamenorrhea, and hirsutism). Two insulin-resistant probands with congenital generalized lipodystrophy and one male proband with severe insulin resistance also were included in the study. Southern blotting experiments were performed to exclude gross gene deletions, insertions, or rearrangements. Exons 2-22 of the insulin receptor gene were polymerase chain reaction (PCR) amplified from genomic DNA and screened for nucleotide variation using single-strand conformation polymorphism (SSCP). No nucleotide variation between study subjects was detected in exons 4-6, 10-12, 15, 16, 18, 19, or 21. Sequencing of amplified DNA revealed that SSCP variants in exons 2, 3, 8, 9, and 17 corresponded to known silent polymorphisms within the coding region. Variants in exons 2, 9, 13, and 14 were caused by novel silent polymorphisms; variants in exons 7 and 22 were caused by nucleotide substitutions in flanking introns. One proband was found to have a heterozygous point mutation in exon 20 (CGG-->CAG, Arg1174-->Gln) that involves the intracellular receptor beta-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of allelic variants Gly972Arg of IRS-1 and Gly1057Asp of IRS-2 in moderate-to-severe insulin resistance of women with polycystic ovary syndrome.

To assess the role of insulin receptor, insulin receptor substrate (IRS)-1, and IRS-2 genes in insulin resistance, we explored the genomic DNA in women with polycystic ovary syndrome (PCOS) and a variable degree (mean +/- SE) of insulin resistance (homeostasis model assessment index for insulin resistance [HOMA(IR)] 3.2 +/- 0.6, n = 53; control subjects 1.56 +/- 0.34, n = 102) using direct sequencing. Whereas no novel mutations were found in these genes, gene-dosage effects were found on fasting insulin for the Gly972Arg IRS-1 variant and on 2-h plasma glucose for the Gly1057Asp IRS-2 variant. The Gly972Arg IRS-1 variant was more prevalent in insulin-resistant patients compared with non-insulin-resistant individuals or control subjects (39.3 vs. 4.0 and 16.6%, P < 0.0031, respectively). A multivariate model that included BMI as a variable revealed significant effects of the Gly1057Asp IRS-2 variant on insulin resistance (P < 0.016, odds ratio [OR] 7.2, 95% CI 1.29-43.3). HOMA(IR) was higher in carriers of both IRS variants than in those with IRS-2 mutations only or those with wild-type variants (6.2 +/- 2.3, 2.8 +/- 0.5, and 1.8 +/- 0.2, respectively; P < 0.01), and it was significantly associated with this genotype (P < 0.0085, OR 1.7, 95% CI 1.09-2.99). We conclude that polymorphic alleles of both IRS-1 and IRS-2, alone or in combination, may have a functional impact on the insulin-resistant component of PCOS.

Genotype-phenotype relationships in Berardinelli-Seip congenital lipodystrophy.

Generalised lipodystrophy of the Berardinelli-Seip type (BSCL) is a rare autosomal recessive human disorder with severe adverse metabolic consequences. A gene on chromosome 9 (BSCL1) has recently been identified, predominantly in African-American families. More recently, mutations in a previously undescribed gene of unknown function (BSCL2) on chromosome 11, termed seipin, have been found to be responsible for this disorder in a number of European and Middle Eastern families. We have studied the genotype/phenotype relationships in 70 affected subjects from 44 apparently unrelated pedigrees of diverse ethnic origin. In all subjects, hepatic dysfunction, hyperlipidaemia, diabetes mellitus, and hypertrophic cardiomyopathy were significant contributors to morbidity with no clear differences in their prevalence between subjects with BSCL1 or BSCL2 and those with evidence against cosegregation with either chromosome 9 or 11 (designated BSCLX). BSCL2 appears to be a more severe disorder than BSCL1 with a higher incidence of premature death and a lower prevalence of partial and/or delayed onset of lipodystrophy. Notably, subjects with BSCL2 had a significantly higher prevalence of intellectual impairment than those with BSCL1 or BSCLX (p<0.0001, OR 17.0, CI 3.6 to 79.0). The higher prevalence of intellectual impairment and the increased risk of premature death in BSCL2 compared to BSCL1 emphasise the importance of molecular diagnosis of this syndrome and have clear implications for genetic counselling.

The human erythrocyte insulin-like growth factor I receptor: characterization and demonstration of ligand-stimulated autophosphorylation.

To characterize the insulin-like growth factor I (IGF-I) receptor on human erythrocytes, cells were purified from peripheral blood by Ficoll-Hypaque gradient centrifugation and incubated with [125I]IGF-I. Specific binding was maximal at pH 8.0 after 24 h at 4 C and increased linearly with cell number to 3.9 +/- 0.2% (+/- SEM) for 3.0 X 10(9) cells/ml. The Scatchard plot of the binding data was linear, with 7 fmol [125I]IGF-I bound/10(9) cells and an affinity constant (K) of 1.8 X 10(9) M-1. Unlabeled IGF-I inhibited tracer binding half-maximally at 6 ng/ml. Multiplication-stimulating activity (or rat IGF-II) was 40% as potent (ED50, 15 ng/ml), whereas insulin and proinsulin were 30- to 500-fold less potent. A monoclonal antibody to the IGF-I receptor (alpha IR-3) inhibited IGF-I binding by 50% at a 1:1000 dilution and by 80% at a 1:250 dilution. Insulin binding was unaffected by the same dilutions. IGF-I receptor phosphorylation was studied in erythrocyte ghosts prepared by hypotonic lysis and solubilized in 1% Triton. The extract was preincubated with and without 100 ng/ml IGF-I or porcine insulin and incubated with [gamma-32P]ATP in the presence of Mn2+, and the receptor was identified by immunoprecipitation with alpha IR-3 antibody and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IGF-I stimulated 4-fold the incorporation of 32P into a protein of 95,000 mol wt, which was immunoprecipitated by alpha IR-3; insulin produced a 2-fold stimulation of this protein. This protein corresponds to the beta-subunit of the IGF-I receptor. These data demonstrate that human erythrocytes have specific receptors for IGF-I, and that this IGF-I receptor, like the insulin receptor, undergoes ligand-stimulated autophosphorylation. Thus, analysis of erythrocyte IGF-I binding and receptor phosphorylation may be useful tools for the study of patients with a variety of growth disorders.

Defects in insulin binding and autophosphorylation of erythrocyte insulin receptors in patients with syndromes of severe insulin resistance and their parents.

Genetic forms of severe insulin resistance are often characterized by alterations in binding and/or kinase properties of the insulin receptor. To evaluate whether alterations in insulin receptor kinase of erythrocytes can be used as genetic markers, we studied patients with two apparently inherited conditions of severe insulin resistance (leprechaunism and the type A syndrome of insulin resistance) and their families. In the two propositi, [125I]insulin binding to intact erythrocytes was decreased by 64% and 45%, respectively. This was primarily due to a decrease in receptor number and was found in intact cells and solubilization of the receptors. Similar insulin binding defects were found on monocytes. Insulin-stimulated tyrosine kinase activity of the solubilized receptor from erythrocytes was also decreased and to a similar extent as binding. Parents of neither patient had clinical manifestations of leprechaunism or the type A syndrome. Furthermore, no alterations in insulin receptor binding or kinase activity were found in erythrocytes from the mothers. Insulin binding in the father of the type A patient was also normal, whereas the father of the leprechaun had decreased receptor affinity. Receptors extracted from the both fathers' cells had a 40-60% decrease in maximal insulin-stimulated phosphorylation and significant rightward shifts of the insulin dose-response curves (ED50, 141 and 42 ng/mL, respectively; control ED50, 16 ng/mL). The finding of biochemical defects in insulin receptor kinase activity in clinically unaffected parents of patients suggests that these alterations may be useful genetic markers and more sensitive than insulin binding studies for studying pattern of inheritance of these diseases.

Distribution and characterization of insulin and insulin-like growth factor I receptors in normal human ovary.

Insulin-resistant hyperinsulinemic states are now widely known to be associated with ovarian hyperandrogenism, and this is thought to be due to an action of insulin on the ovary. However, the identity of the receptor that is responsible for insulin action in these patients, whose insulin receptors on classical target tissues are severely impaired, is unclear. We now report the presence of insulin receptors in stromal and follicular compartments as well as in granulosa cells obtained from normal ovaries. After 15-h incubations at 4 C with [125I]insulin and tissue fragments, specific insulin binding was 6-19% and 7-13%/mg protein (n = 8) to stroma and theca, respectively. Granulosa cells obtained in the course of in vitro fertilization were separated from red cells on a Percoll gradient; specific insulin binding ranged from 9-15%/10(6) cells. Insulin binding was characterized by sensitive insulin competition (half-maximal, 10 ng/ml), appropriately shifted proinsulin competition (20 times to the right), and complete inhibition by specific anti-insulin receptor antibodies (B-2). An antibody to the insulin-like growth factor I (IGF-I) receptor (alpha IR-3) that inhibits IGF-I binding to IGF-I receptors in other cell systems had no effect on insulin binding. Further proof that this binding is to classic insulin receptors was obtained from measurement of insulin-stimulated receptor autophosphorylation. When insulin receptors from stroma were extracted with Triton X-100 and incubated with [gamma-32P]ATP and Mn, insulin increased the incorporation of 32P into the beta-subunit of the receptor 5-fold. In parallel studies with [125I-]IGF-I and specific blocking antibodies to its receptor, no detectable IGF-I binding to stroma or follicles was found. We conclude that specific high affinity insulin receptors possessing tyrosine kinase activity are widely distributed in normal human ovary. IGF-I receptors in normal ovary are either absent or present at very low density. Binding of insulin to its own receptor (as opposed to IGF-I receptors) appears to be the most likely first step in the stimulation of ovarian steroidogenesis by insulin in normal ovaries and possibly in insulin-resistant states as well.

Tyrosine-kinase defect of the insulin receptor in cultured fibroblasts from patients with lipoatropic diabetes.

Postbinding defects in insulin action were described previously in cultured fibroblasts from six patients with lipoatropic diabetes. To define the contribution of the insulin receptor tyrosine kinase in these defects, we studied autophosphorylation and kinase activity of lectin purified receptors from these six patients and six normal cell lines. The patients' insulin receptors, prepared by precipitation with polyethylene glycol, had normal insulin binding characteristics and autophosphorylation properties, but a 56% decrease in the tyrosine kinase activity toward an exogenous substrate. To identify more subtle qualitative defects in autophosphorylation, insulin receptors were sequentially immunoprecipitated and analyzed for their phosphoaminoacid content. The phosphorylated receptors precipitated with an antiphosphotyrosine antibody contained labeled phosphotyrosine, whereas those in the supernatant, when further precipitated with an antireceptor antibody, contained only phosphoserine. Under these conditions, the insulin-stimulated autophosphorylation of tyrosine was significantly decreased by 54% in the patient receptors compared to normal subjects' receptors. In addition, insulin-like growth factor-I stimulation of autophosphorylation of its receptor was reduced by 59% in the patients' cells compared to those from normal subjects. We conclude that fibroblasts from patients with lipoatropic diabetes have defects in the tyrosine kinase activity of their insulin and their insulin-like growth factor-I receptors that might give rise to the in vitro hormone resistance and be related to the in vivo hormone resistance that occurs in these patients.

Insulin-mediated pseudoacromegaly: clinical and biochemical characterization of a syndrome of selective insulin resistance.

We have performed clinical, physiological, in vitro biochemical and genetic studies of a patient with severe insulin resistance associated with the phenotype of "pseudoacromegaly," defined as the presence of acromegaloid features in the absence of elevated levels of GH or insulin-like growth factor-I (IGF-I). Despite marked hyperinsulinemia, insulin and IGF-I binding to circulating blood cells and cultured skin fibroblasts was normal. Insulin and IGF-I-stimulated autophosphorylation of their respective receptors in cultured skin fibroblasts was also normal. However, neither insulin nor IGF-I were able to stimulate 2-deoxy D-glucose uptake by cultured skin fibroblasts. In contrast, the ability of insulin and IGF-I (or IGF-II) to stimulate amino acid uptake and thymidine incorporation into DNA was not impaired. This unique discordant signaling defect through both insulin and IGF-I receptors appeared not to be the consequence of altered expression or primary structure of the insulin receptor or the GLUT-4 glucose transporter, as assessed by several genetic and biochemical techniques. GLUT-4 expression in muscle was normal on Western blots, and SSCP screening of all 11 exons of the gene for nucleotide variation revealed no variations from normal. DNA sequencing and SSCP screening of exons 2-22 of the insulin receptor gene revealed only one variation predicted to alter the amino acid sequence (Val985-->Met). No functional differences between Met985 and wild-type human insulin receptors were evident in studies performed with Chinese hamster ovary cell transfectants that overexpress either receptor. This data combined with our previously published epidemiological data concerning the frequency of the Met985 allele, indicate that this variant insulin receptor is not responsible for the insulin resistant glucose uptake or the clinical syndrome of pseudoacromegaly. We conclude that: 1) The molecular lesion responsible for the selective biochemical defect in this individual appears to involve a signaling intermediate required for insulin and IGF-I regulation of glucose transport, and/or an effector mechanism operative in this process. 2) Cells derived from this patient may be a valuable tool in the search for such molecular mechanisms. 3) The Met985 allele is a relatively common variant which has no demonstrable adverse consequences for insulin receptor function. 4) Pseudoacromegaly can be viewed as the expected result of hyperinsulinemia driving the unopposed mitogenic and anabolic actions of insulin.

Studies of the variability of the genes encoding the insulin-like growth factor I receptor and its ligand in relation to type 2 diabetes mellitus.

Insulin-like growth factor I (IGF-I) is an important regulator of many aspects of growth, differentiation, and development, and as low birth weight has been associated with impaired glucose tolerance and overt type 2 diabetes in adult life, we considered the genes encoding the IGF-I and the IGF-I receptor (IGF-IR) as candidates for low birth weight, insulin resistance, and type 2 diabetes. Here we report the mutational analysis of the coding regions of the IGF-I and IGF-IR performed on genomic DNA from probands of 82 Danish type 2 diabetic families. No mutations predicting changes in the amino acid sequences of the IGF-I or IGF-IR genes were detected, but several silent and intronic polymorphisms were found. The impact of the most prevalent polymorphism, GAG1013GAA of the IGF-IR, was evaluated in a population-based sample of 349 young healthy subjects, where the variant had an allele frequency of 0.44 (95% confidence interval, 0.40-0.48). No significant relationships between this variant and birth weight, birth length, or insulin sensitivity index were detected. In addition, we did not observe any significant differences in allelic frequencies of the codon 1013 variant between 395 type 2 diabetic patients (allele frequency, 0.52; 95% confidence interval, 0.49-0.55) and 238 matched glucose-tolerant control subjects (allelic frequency, 0.47; 95% confidence interval, 0.43-0.50). In conclusion, variability in the coding regions of IGF-I and the IGF-IR does not associate with reduced birth weight, insulin sensitivity index, or type 2 diabetes in the Danish population.

Identification and sequence analysis of arginine vasopressin mRNA in normal and Brattleboro rat aortic tissue.

Arginine vasopressin (AVP), a hormone of the hypothalamic-pituitary axis, was also localized in peripheral tissues. To explore AVP precursor gene expression at the vascular level, we have investigated gene transcripts by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing in aortic tissue of normal rat and in the particular genetic condition of the homozygous (di/di) Brattleboro rat strain suffering from diabetes insipidus. In these rats, a gene deletion induces an unprocessed AVP precursor in the hypothalamus with undetectable immunoreactive AVP, in contrast to the detection of immunoreactive material at the vascular level. In normal rats, using primers complementary to exon 1 and 3 of the AVP neurophysin precursor gene, RT-PCR and sequencing revealed transcripts of the expected size from aorta, mesenteric artery and hypothalamus with normal, authentic sequences. Removal of aortic endothelium severely reduced the amounts of transcripts, suggesting their main endothelial origin. In Brattleboro rats, transcripts of similar size were obtained from aorta and hypothalamus and sequencing revealed the homozygous deletion (deltaG316) in both tissues, identical to that found in genomic DNA (deltaG1864). While sequence data from normal rats provide the first direct evidence for the presence of AVP precursor transcripts in rat aortic tissue, identification of the deleted sequence of transcripts in Brattleboro rat aorta suggests that tissue-specific mechanisms are operating for the expression of vasopressin neurophysin precursor in peripheral vascular tissue compared with the hypothalamus.