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Biomedical subjects

S O'Rahilly

Publications and source records attributed to S O'Rahilly.

At least 109 records · Page 6Linked to original sources
PubMed1997

Two naturally occurring insulin receptor tyrosine kinase domain mutants provide evidence that phosphoinositide 3-kinase activation alone is not sufficient for the mediation of insulin's metabolic and mitogenic effects.

We have recently reported (1) that two naturally occurring mutants of the insulin receptor tyrosine kinase domain, Arg-1174 --> Gln and Pro-1178 --> Leu (Gln-1174 and Leu1178, respectively), both found in patients with inherited severe insulin resistance, markedly impaired receptor tyrosine autophosphorylation, with both mutant receptors being unable to mediate the stimulation of glycogen synthesis or mitogenesis by insulin when expressed in Chinese hamster ovary cells. However, these mutations did not fully prevent IRS-1 phosphorylation in response to insulin in these cells, suggesting that IRS-1 alone may not be sufficient to mediate insulin's metabolic and mitogenic effects. In the present study, we have demonstrated that these mutations also impair the ability of the insulin receptor to activate the transcription factor Elk-1 and promote GLUT4 translocation to the plasma membrane. Although at low concentrations of insulin, the mutant receptors were impaired in their ability to stimulate the tyrosine phosphorylation of IRS-1, at higher insulin concentrations we confirmed that the cells expressing the mutant receptors showed significantly increased tyrosine phosphorylation of IRS-1 compared with parental nontransfected cells. In addition, at comparable insulin concentrations, the association of the p85alpha subunit of phosphoinositide 3-kinase (PI3-kinase) with IRS-1 and the enzymatic activity of IRS-1-associated PI3-kinase were significantly enhanced in cells expressing the mutant receptors. In contrast, no significant stimulation of the tyrosine phosphorylation of Shc, GTP loading of Ras, or mitogen-activated protein kinase phosphorylation was seen in cell lines expressing these mutant receptors. Thus, no activation of any measurable mitogenic or metabolic response was detectable, despite significant insulin-induced phosphorylation of IRS-1 and its association with PI3-kinase in cells stably expressing the mutant insulin receptors. These findings suggest that PI3-kinase activation alone may be insufficient to mediate a wide range of the metabolic and mitogenic effects of insulin. Additionally, the data provide support for the notion that insulin activation of Ras is more closely linked with Shc, and not IRS-1, phosphorylation.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Differential signaling to glycogen synthesis by the intracellular domain of the insulin versus the insulin-like growth factor-1 receptor. Evidence from studies of TrkC-chimeras.

Insulin and insulin-like growth factor-1 (IGF-1) have similar cell-surface receptors yet subserve different physiological functions. To examine whether these differences relate to intrinsic signaling properties of the intracellular domains of their respective receptors, chimeric receptors were constructed using the extracellular domain of the neurotrophin-3 (NT-3) receptor, TrkC, and the intracellular domain of either the insulin receptor or the IGF-1 receptor. TrkC-IR (TIR) and TrkC-IGF-1R (TIGR) were stably expressed in 3T3-L1 cells. While TIR and TIGR cell lines expressing similar numbers of chimeric receptors showed a similar dose-response relationship in NT-3 stimulated DNA synthesis, NT-3 stimulated glycogen synthesis was greater in TIR than in TIGR cells (maximum TIGR response was 35% of maximum TIR response). Additionally, the concentration of NT-3 at which significant stimulation of glycogen synthesis was seen was 0.1 ng/ml in TIR and 1 ng/ml in TIGR cells. Basal levels of thymidine incorporation but not glycogen synthesis were consistently higher in TIR than in TIGR expressing cells. No detectable basal autophosphorylation of chimeric receptors was seen in any of the cell lines. However, exposure of cell lines to the phosphatase inhibitor bisperoxovanadate resulted in greater basal autophosphorylation of the TIR and endogenous murine IR than the TIGR and endogenous murine IGF-1R. Thus, in this system, the intracellular domain of the IR appears to couple more effectively to glycogen synthesis than that of the IGF-1R, whereas the intracellular domains of both receptors have a similar capacity to stimulate DNA synthesis.

3T3 Cells↗
PubMed1997

Congenital leptin deficiency is associated with severe early-onset obesity in humans.

The extreme obesity of the obese (ob/ob) mouse is attributable to mutations in the gene encoding leptin, an adipocyte-specific secreted protein which has profound effects on appetite and energy expenditure. We know of no equivalent evidence regarding leptin's role in the control of fat mass in humans. We have examined two severely obese children who are members of the same highly consanguineous pedigree. Their serum leptin levels were very low despite their markedly elevated fat mass and, in both, a homozygous frame-shift mutation involving the deletion of a single guanine nucleotide in codon 133 of the gene for leptin was found. The severe obesity found in these congenitally leptin-deficient subjects provides the first genetic evidence that leptin is an important regulator of energy balance in humans.

Adult↗
PubMed1997

Science, medicine, and the future. Non-insulin dependent diabetes mellitus: the gathering storm.

A massive increase in the global prevalence of non-insulin dependent diabetes is likely to occur as "Westernisation" of dietary habits and patterns of physical activity becomes more widespread. Advances in molecular and cellular science may provide some useful insights and therapeutic tools to assist in the fight against the severe consequences of this epidemic. These will include the better identification of specific aetiological subtypes of the disease; the identification of new drugs through the better understanding of the biology of insulin secretion and action; and the targeting of therapies to specific subtypes of the disease. In addition, knowledge of the precise mode of action and antidiabetes drugs may facilitate the design of more effective non-pharmacological manipulations; the genetic identification of "high risk" asymptomatic people may allow us to target screening and preventive strategies more effectively; and investigations into the mechanisms that underlie the link between low birth weight and later diabetes should provide new routes towards treatment and prevention. Barriers to the implementation of the global measures required to stem the predicted flood of non-insulin dependent diabetes may prove insuperable, but if we are to have any success, then close collaboration between clinicians, epidemiologists, public health physicians, and laboratory scientists will be essential.

B-Lymphocytes↗
PubMed1997

Homozygosity for a common polymorphism in the islet-specific promoter of the glucokinase gene is associated with a reduced early insulin response to oral glucose in pregnant women.

A commonly occurring sequence variant in the islet-specific promoter of the glucokinase gene (-30 G to A) has been variably reported to be associated with reduced insulin secretory responses to oral glucose. The effect of this promoter variant may be subtle and only become apparent under conditions of beta-cell 'stress'. As late pregnancy is a time of increased insulin secretory demand, we have examined whether this common genetic variant was associated with impairment of insulin secretory responses to oral glucose in 92 women in the third trimester of pregnancy. The three women who were homozygous for the variant sequence had a markedly diminished 30' insulin incremental response to oral glucose (10.4, 11.4, and 17.2 pmol insulin mmol-1 glucose, respectively) compared to either heterozygous (49.3 (37.6-64.6 pmol insulin mmol-1 glucose)) (p < 0.002) or homozygous wild-type (51.4 (40.9-64.7 pmol insulin mmol-1 glucose)) (p < 0.002) Mann-Whitney U test) women. In a subset of 35 British Caucasian women with gestational diabetes, no mutations resulting in a change of amino acid sequence were detected by molecular scanning of all exons of the glucokinase gene. In summary, in a cohort of 35 British Caucasian women with gestational diabetes neither missense nor nonsense glucokinase mutations were found. However, in women in the third trimester of pregnancy, homozygosity for a common polymorphic variant in the islet-specific promoter of the glucokinase gene was associated with a highly significant reduction of early insulin secretory responsiveness to oral glucose. Under the conditions of increased secretory demand represented by late pregnancy, a promoter variant in the glucokinase gene may influence the early insulin secretory response to oral glucose.

Base Sequence↗
PubMed1997

Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene.

Human obesity has an inherited component, but in contrast to rodent obesity, precise genetic defects have yet to be defined. A mutation of carboxypeptidase E (CPE), an enzyme active in the processing and sorting of prohormones, causes obesity in the fat/fat mouse. We have previously described a women with extreme childhood obesity (Fig. 1), abnormal glucose homeostasis, hypogonadotrophic hypogonadism, hypocortisolism and elevated plasma proinsulin and pro-opiomelanocortin (POMC) concentrations but a very low insulin level, suggestive of a defective prohormone processing by the endopeptidase, prohormone convertase 1 (PC1; ref. 4). We now report this proband to be a compound heterozygote for mutations in PC1. Gly-->Arg483 prevents processing of proPC1 and leads to its retention in the endoplasmic reticulum (ER). A-->C+4 of the intro-5 donor splice site causes skipping of exon 5 leading to loss of 26 residues, a frameshift and creation of a premature stop codon within the catalytic domain. PC1 acts proximally to CPE in the pathway of post-translational processing of prohormones and neuropeptides. In view of the similarity between the proband and the fat/fat mouse phenotype, we infer that molecular defects in prohormone conversion may represent a generic mechanism for obesity, common to humans and rodents.

Amino Acid Sequence↗
PubMed1997

Regulation of adipose cell number in man.

1. Adipose tissue mass is dependent on both the average volume and the number of its constituent adipocytes. Significant alteration in body mass involves alteration in both adipocyte volume and number. 2. Increases in adipocyte number occur via replication and differentiation of preadipocytes, a process which occurs throughout life. Decreases in adipocyte number occur via preadipocyte and adipocyte apoptosis, and possibly adipocyte dedifferentiation. 3. Overall regulation of adipose mass involves endocrine, paracrine and possibly autocrine systems. Hypothalamic centres appear to control appetite, metabolic rate and activity levels in a co-ordinated manner. Within the hypothalamus, known weight regulatory molecules include glucagon-like peptide-1, neuropeptide Y and leptin. Leptin is a major afferent signal from adipose tissue to the hypothalamus, providing information on overall adipose tissue mass. However, the precise means by which the hypothalamus signals to adipose tissue is less well understood. 4. In adipose tissue, known molecular regulators of adipose cell number include insulin, ligands for the peroxisome proliferator activated receptor-gamma, retinoids, corticosteroids and tumour necrosis factor-alpha. The net effect of these and other regulators is to effect a concerted alteration in adipocyte volume and number. This review largely focuses on the control of fat cell acquisition and loss and the influence of these processes on adipose tissue mass and regional distribution.

Adipocytes↗
PubMed1997

Expression of the putative inhibitor of the insulin receptor tyrosine kinase PC-1 in dermal fibroblasts from patients with syndromes of severe insulin resistance.

OBJECTIVE: To date, mutations in the insulin receptor gene are the only clearly defined cause of extreme insulin resistance in man. Recently, however, some patients with severe insulin resistance have been reported to have marked over-expression of the transmembrane glycoprotein PC-1 in their cultured fibroblasts. This protein appears to act as an endogenous inhibitor of the insulin receptor tyrosine kinase which suggests that primary over-expression of PC-1 may play an aetiological role in some forms of insulin resistance. One sub-type of extreme insulin resistance in which the insulin receptor gene has been reported to be normal is pseudo-acromegalic insulin resistance. The main aim of this study was to determine whether overexpression of PC-1 might contribute to the severe insulin resistance exhibited by some patients with pseudo-acromegaly. DESIGN AND PATIENTS PC-1 phosphodiesterase activity and PC-1 protein and mRNA content were measured in cultured dermal fibroblast from three severely insulin resistant pseudo-acromegalic patients. These were compared with fibroblasts from normoinsulinaemic normoglycaemic controls and from subjects with known genetic defects in the insulin receptor or IRS-1. RESULTS: In the fibroblasts from pseudo-acromegalic insulin resistant subjects PC-1 activity and PC-1 protein and mRNA levels were indistinguishable from the normoinsulinaemic controls. Consistent with this observation, insulin receptor tyrosine kinase activity was similar in extracts from fibroblasts of pseudo-acromegalic subjects and normal controls. Surprisingly, subjects with insulin receptor or IRS-1 mutations had a profound reduction in PC-1 activity (p < or = 0.005), protein (p < or = 0.05) and mRNA levels (P < or = 0.005). CONCLUSIONS: The results indicate that PC-1 over-expression does not appear to contribute to the insulin resistant state of pseudo-acromegalic patients. The finding of normal insulin receptor tyrosine kinase activity in these subjects suggests that the site of defective insulin signalling is likely to be distal to the receptor. The unexpected finding that PC-1 activity, protein and mRNA were all dramatically reduced in patients with lesions early in the insulin signalling cascade provides further evidence for a link, albeit as yet poorly understood, between cellular insulin action and the expression of PC-1.

Cells, Cultured↗
PubMed1997

Dunnigan-Kobberling syndrome: an autosomal dominant form of partial lipodystrophy.

Dunnigan-Kobberling syndrome is a form of partial lipodystrophy characterized by sparing of the face. Despite descriptions of six families since 1974, details of total body adipose tissue distribution and studies of carbohydrate and fat metabolism are lacking. The mode of inheritance also remains unclear, with most authors favouring an X-linked dominant transmission lethal in the hemizygous male. We examined 23 members of a family, of whom at least eight had partial lipodystrophy. Auxological evaluation and cross-sectional imaging showed absence of subcutaneous fat, presence of adipose tissue inside the body cavities, and skeletal muscle hypertrophy. Biochemical evaluation identified insulin resistance but revealed inadequate suppression of non-esterified fatty acids. In this family, male-to-male transmission supports an autosomal dominant mode of inheritance for Dunnigan-Kobberling syndrome.

Acanthosis Nigricans↗
PubMed1997

Effect of metformin on glucose disposal and hyperinsulinaemia in a 14-year-old boy with acanthosis nigricans.

We report the treatment of a 14-year-old Indian boy with acanthosis nigricans and hyperinsulinaemia with metformin in an attempt to improve his skin lesions. Oral metformin was used for 6 months with assessment of insulin status during an intravenous glucose tolerance test and hyperinsulinaemic-euglycaemic clamping before and after treatment. The first-phase insulin response reduced from 19,593 to 5,410 pmol/l/min (normal 1,900-13,400), and the second-phase insulin response improved from 59,120 to 34,020 pmol/l/min (normal 2,900-18,100). During hyperinsulinaemic-euglycaemic clamping hepatic glucose production was normally suppressed prior to therapy, but peripheral glucose remained abnormally low, 152 and 138% of basal (expected 199%). The acanthosis nigricans remained unaltered but over this period puberty progressed and his body mass index increased. We conclude that, in this patient, metformin had a minimal effect on the hyperinsulinism and none on the acanthosis nigricans in the relatively short term, but further studies in more patients over longer time intervals are warranted.

Acanthosis Nigricans↗
PubMed1997

Tumor necrosis factor-alpha induces apoptosis of human adipose cells.

Tumor necrosis factor-alpha (TNF-alpha) production by adipocytes is elevated in obesity, as shown by increased adipose tissue TNF-alpha mRNA and protein levels and by increased circulating concentrations of the cytokine. Furthermore, TNF-alpha has distinct effects on adipose tissue including induction of insulin resistance, induction of leptin production, stimulation of lipolysis, suppression of lipogenesis, induction of adipocyte dedifferentiation, and impairment of preadipocyte differentiation in vitro. Taken together, these effects all tend to decrease adipocyte volume and number and suggest a role for TNF-alpha in limiting increase in fat mass. The aim of the present study was to determine if TNF-alpha could induce apoptosis in human adipose cells, hence delineating another mechanism by which the cytokine could act to limit the development of, or extent of, obesity. Cultured human preadipocytes and mature adipocytes in explant cultures were exposed in vitro to human TNF-alpha at varying concentrations for up to 24 h. Apoptosis was assessed using morphological (histology, nuclear morphology following acridine orange staining, electron microscopy) and biochemical (demonstration of internucleosomal DNA cleavage by gel electrophoresis and of annexin V staining using immunocytochemistry) criteria. In control cultures, apoptotic indexes were between 0 and 2.3% in all experiments. In the experimental systems, TNF-alpha induced apoptosis in both preadipocytes and adipocytes, with indexes between 5 and 25%. Therefore, TNF-alpha induces apoptosis of human preadipocytes and adipocytes in vitro. In view of the major metabolic role of TNF-alpha in human adipose tissue, and the knowledge that adipose tissue is dynamic (with cell acquisition via preadipocyte replication/differentiation and cell loss via apoptosis), these findings describe a further mechanism whereby adipose tissue mass may be modified by TNF-alpha.

Acridine Orange↗
PubMed1997

Depot- and sex-specific differences in human leptin mRNA expression: implications for the control of regional fat distribution.

Obese subjects with excess intra-abdominal fat deposition suffer greater adverse metabolic consequences than do similarly overweight subjects with a predominantly subcutaneous distribution of adiposity. Little is known about the factors regulating the regional distribution of body fat. Leptin is a recently characterized protein secreted by adipocytes that appears to provide a long-term hormonal feedback signal regulating fat mass. No systematic evaluation of site-related differences in human adipocyte leptin expression has been reported to date. Levels of leptin mRNA were examined by quantitative reverse transcription-polymerase chain reaction in adipocytes isolated from omental and subcutaneous adipose depots of nonobese and mildly obese individuals undergoing elective surgery. In all individuals studied (n = 24), leptin mRNA levels were higher in subcutaneous than in omental adipocytes (P < 0.0001). In contrast, there were no consistent site-specific differences in the expression of glycerol-3-phosphate dehydrogenase mRNA. The subcutaneous-to-omental ratio of leptin mRNA expression was markedly higher in women (5.5 +/- 1.1-fold) than in men (1.9 +/- 0.2-fold) (P < 0.02). A significant relationship between BMI and leptin mRNA expression was demonstrable in the subcutaneous adipocytes of women (P < 0.006). Thus, leptin mRNA appears to be expressed predominantly by subcutaneous adipocytes, particularly in women. These findings suggest a possible role for leptin in the control of adipose tissue distribution and mass.

Abdomen↗
PubMed1996

Functional activation of mutant human insulin receptor by monoclonal antibody.

BACKGROUND: A mutant insulin receptor, Ser323Leu, has been reported in two severely insulin-resistant patients with Rabson-Mendenhall syndrome. In both cases, extreme hyperglycaemia could not be controlled by conventional antidiabetic therapy. The SER323Leu mutant insulin receptor is inserted normally in the plasma membrane but has very low binding affinity for insulin. A monoclonal antibody directed against the extracellular domain of the insulin receptor (83.14) can mimic the natural ligand as far as the first step after ligand binding--autophosphorylation of the intracellular domain of the receptor. We have investigated whether antibody binding can imitate autophosphorylation of the Ser323Leu mutant receptor and lead to metabolic events within the cell. METHODS: The effects of insulin and the insulin-receptor monoclonal antibody on receptor autophosphorylation and glycogen synthesis were compared in Chinese hamster ovary cells expressing the wild-type human insulin receptor, mock-transfected cells, cells expressing an insulin-receptor mutant without autophosphorylation capacity, and cells expressing the Ser323Leu mutant receptor. FINDINGS: Cells expressing the SER323Leu mutant receptor had very low specific insulin binding and, unlike cells expressing wild-type insulin receptors, did not show autophosphorylation or stimulation of glycogen synthesis in response to insulin. However, exposure of cells expressing the Ser323Leu mutant receptor to monoclonal antibody 83.14 resulted in autophosphorylation and stimulation of glycogen synthesis similar to that seen in cells expressing wild-type insulin receptors. INTERPRETATION: Although insulin does not bind to cells expressing the Ser323Leu mutation, insulin signalling can be mimicked by exposure of the cells to an antibody to the extracellular domain of the insulin receptor. Activation by monoclonal antibodies of mutant transmembrane receptors that show normal cell-surface expression but defective ligand binding may provide an approach to the therapy of some subtypes of inherited hormone resistance for which little effective treatment is available.

Adult↗
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

Mutant insulin receptors in syndromes of insulin resistance.

To date, mutations of the insulin receptor remain the only well-established causes of severe insulin resistance. There is a broad correlation between the extent of impairment of signal transduction seen when the mutant receptors are expressed in vitro with the severity of the clinical phenotype. Thus leprechaunism, Rabson-Mendenhall syndrome and Type A insulin resistance appear to represent points on a continuum of severity of receptor dysfunction, rather than completely distinct syndromes. In other syndromes of insulin resistance, insulin receptor abnormalities remain the exception. However, functional studies of expressed naturally occurring insulin receptor mutations have acted as experiments of nature and greatly aided attempts to dissect the structure-function relationships of the receptor. The next few years will no doubt begin to reveal the contributions made by defects in the post-receptor signalling cascade to the syndromes of insulin resistance in man.

Humans↗