Insulin resistance in skeletal muscle and adipose tissue in polycystic ovary syndrome: are the molecular mechanisms distinct from type 2 diabetes?

The association of polycystic ovary syndrome (PCOS) with insulin resistance was recognized almost three decades ago. Despite the pivotal role of insulin resistance in the pathogenesis of PCOS, the precise cellular and molecular mechanisms of impaired insulin action remain elusive. This review has two aims: 1) to review the mechanisms of insulin resistance, specifically impaired insulin-stimulated glucose transport, in skeletal muscle and adipose tissue in PCOS, and 2) to assess whether mechanisms of insulin resistance in PCOS are distinct from those in type 2 diabetes. As in type 2 diabetes, studies in skeletal muscle in PCOS support the existence of intrinsic defects in insulin signalling but also underscore the importance of in vivo environmental factors for the development of insulin resistance. In PCOS and type 2 diabetes, similar insulin signalling defects in muscle have been described i.e. impaired signalling via IRS-1 and up-regulation of ERK signalling. Similar defects in insulin signalling have also been described in adipose tissue in PCOS and type 2 diabetes, but data are limited. As for type 2 diabetes, PCOS is characterized by chronic inflammation, mitochondrial dysfunction and cellular stress. Androgen excess, a key feature of PCOS, has a genetic component: the relationship of hyperandrogenemia to the development of insulin resistance requires further study. In conclusion, although similar insulin signalling defects have been identified in muscle and adipose tissue in PCOS and type 2 diabetes, these defects probably reflect a common final pathway resulting from genetic and environmental influences on insulin action that are unique to each disorder.

Effects of androgens on insulin action in women: is androgen excess a component of female metabolic syndrome?

Hyperinsulinemia as a consequence of insulin resistance causes hyperandrogenemia in women. The objective was to review evidence for the converse situation, i.e. whether androgens adversely influence insulin action. Androgen excess could potentially contribute to the pathogenesis of insulin resistance in women with polycystic ovary syndrome (PCOS), metabolic syndrome/type 2 diabetes, and in obese peripubertal girls. An Entrez-PubMed search was conducted to identify studies addressing the relationship of androgens with metabolic syndrome/type 2 diabetes in women. Studies reporting outcomes of androgen administration, interventions to reduce androgen effects in hyperandrogenemic women, and basic studies investigating androgen effects on insulin target tissues were reviewed. Multiple studies showed associations between serum testosterone and insulin resistance or metabolic syndrome/type 2 diabetes risk in women, but their cross-sectional nature did not allow conclusions about causality. Androgen administration to healthy women was associated with development of insulin resistance. Intervention studies in women with hyperandrogenism were limited by small subject numbers and use of indirect methods for assessing insulin sensitivity. However, in three of the seven studies using euglycemic hyperinsulinemic clamps, reduction of androgen levels or blockade of androgen action improved insulin sensitivity. Testosterone administration to female rats caused skeletal muscle insulin resistance. Testosterone induced insulin resistance in adipocytes of women in vitro. In conclusion, the metabolic consequences of androgen excess in women have been under-researched. Studies of long-term interventions that lower androgen levels or block androgen effects in young women with hyperandrogenism are needed to determine whether these might protect against metabolic syndrome/type 2 diabetes in later life.

Effects of spironolactone on glucose transport and interleukin-6 secretion in adipose cells of women.

Adipose tissue inflammation and insulin resistance are central to the pathogenesis of the metabolic syndrome. Spironolactone, an antagonist of mineralocorticoid receptor, glucocorticoid receptor and androgen receptor, and agonist of progesterone receptor, has anti-inflammatory activity. Blockade of the renin-angiotensin-aldosterone system has been shown to improve glucose metabolism. We have investigated whether spironolactone has direct effects on glucose uptake and interleukin-6 secretion in human adipocytes. Spironolactone, but not its active metabolite canrenoic acid, significantly increased basal and insulin-stimulated glucose uptake in cultured IN VITRO-differentiated adipocytes of women, without affecting insulin sensitivity. The effect was not due to changes in abundance of glucose transporters 1 or 4 or in degree of cell differentiation. Spironolactone, but not canrenoic acid, significantly reduced basal interleukin-6 secretion by cultured stromal-vascular cells. These effects of spironolactone were not mediated by ligand-dependent antagonism of the mineralocorticoid, glucocorticoid, or androgen receptors. Spironolactone may have a novel role in increasing glucose uptake into adipose cells and attenuating adipose tissue inflammation, with implications for management of metabolic syndrome.

Chronic testosterone treatment induces selective insulin resistance in subcutaneous adipocytes of women.

Adipose tissue plays a central role in determining whole body insulin sensitivity. Several aspects of adipose cell function are regulated by androgens. Given that high androgen levels and insulin resistance are linked in women, we proposed that androgens may influence insulin-mediated glucose metabolism in adipose cells. Preadipocytes harvested from s.c. adipose tissue of healthy women aged 37 +/- 5 years were differentiated in vitro, then treated with testosterone (T) and/or androgen receptor (AR) antagonists (cyproterone acetate, flutamide) for 48 h. Maximal insulin-stimulated glucose uptake (insulin 10 nM) and increment following insulin stimulation were significantly impaired in cells treated with T 10 and 100 nmol/l. This defect was abolished by cyproterone acetate and partially reversed by flutamide. The effect of T could not be accounted for by altered differentiation status of the adipocytes. In the glucose metabolic pathway of insulin signaling, treatment of cells with T 10 nmol/l did not alter insulin-stimulated phosphorylation of insulin receptor substrate-1 or Akt, but insulin-stimulated phosphorylation of protein kinase C (PKC) zeta was impaired. Insulin signaling via the mitogenic/gene regulatory pathway, as assessed by extracellular signal-regulated kinase phosphorylation, was unchanged. We conclude that (1) T, or an androgenic metabolite of T, induces insulin resistance in adipocytes of women, selective for metabolic signaling pathways; (2) this defect is via AR; and (3) the defect in signaling is independent of phosphatidylinositol 3-kinase activation and involves impaired phosphorylation of PKCzeta. These findings are relevant to understanding the pathogenesis of insulin resistance in hyperandrogenic women.

The effect of obesity on the ratio of type 3 17beta-hydroxysteroid dehydrogenase mRNA to cytochrome P450 aromatase mRNA in subcutaneous abdominal and intra-abdominal adipose tissue of women.

OBJECTIVES: To investigate (1) whether type 3 17beta-hydroxysteroid dehydrogenase (17beta-HSD), the enzyme which catalyzes the conversion of androstenedione to testosterone in the testis, is co-expressed with P450aromatase in the preadipocytes of women, and (2) whether the relative expression of type 3 17beta-HSD and aromatase varies in subcutaneous abdominal vs intra-abdominal adipose tissue of women. SUBJECTS: Subcutaneous abdominal and intra-abdominal adipose tissue was obtained from women undergoing elective abdominal surgery (age 22-78 y, body mass index (BMI) 22.4-52.9 kg/m(2)). MEASUREMENTS: Expression of type 3 17beta-HSD in adipose cell fractions was determined using RT-PCR. Preadipocyte steroidogenesis was investigated in primary cultures using androstenedione as substrate. Messenger RNA levels for type 3 17beta-HSD and aromatase were measured in adipose tissue from the subcutaneous abdominal and intra-abdominal depots using a quantitative multiplex competitive RT-PCR assay. RESULTS: Type 3 17beta-HSD is co-expressed with aromatase in the abdominal preadipocytes of women. Cultured preadipocytes from both subcutaneous abdominal (n=5) and intra-abdominal (n=5) sites converted androstenedione to testosterone, and there was minimal conversion of androstenedione to estrone. Consistent with this, the levels of type 3 17beta-HSD mRNA were significantly higher than aromatase mRNA at both sites (P<0.05; n=8 subcutaneous abdominal, n=12 intra-abdominal adipose tissue). The ratio of levels of 17beta-HSD mRNA to aromatase mRNA in intra-abdominal adipose tissue was positively correlated with BMI (n=11, r=0.61, P<0.05) and waist circumference (n=10, r=0.65, P<0.05). The converse was found in subcutaneous abdominal adipose tissue. CONCLUSION: The intra-abdominal adipose tissue of women may be substantially androgenic, increasingly so with increasing obesity, particularly central obesity. While androgen production by this adipose tissue deposit may not contribute to circulating testosterone levels due to hepatic clearance, it may have hitherto unrecognised local effects in the intra-abdominal adipose tissue and also on the liver via the hepatic portal system. These studies suggest a mechanism linking central obesity with insulin resistance and dyslipidaemia.

Insulin resistance in polycystic ovary syndrome: progress and paradoxes.

Over the past 20 years, it has been clearly documented that 1) polycystic ovary syndrome (PCOS) has major metabolic sequelae related to insulin resistance and 2) insulin resistance plays an important role in the pathogenesis of the reproductive abnormalities of the disorder. Women with PCOS are at significantly increased risk of developing type 2 diabetes mellitus (DM). Studies in isolated adipocytes and in cultured skin fibroblasts from PCOS women have demonstrated intrinsic postbinding defects in insulin-mediated glucose metabolism. In fibroblasts, the mitogenic pathway of insulin action is intact, consistent with a selective defect in insulin signaling. While PCOS skeletal muscle is resistant to insulin in vivo, cultured muscle cells have normal insulin sensitivity, consistent with a major role of extrinsic factors in producing insulin resistance in this tissue. Excessive serine phosphorylation of the insulin receptor or downstream signaling proteins may be involved in the pathogenesis of insulin resistance in PCOS. The putative serine kinase is extrinsic to the insulin receptor but its identity is unknown. The explanations for tissue-specific and signaling pathway-specific differences in insulin action in PCOS are unknown but may involve differential roles of insulin receptor substrate (IRS)-1 and IRS-2 in insulin signal transduction.

Insulin/IGF-1 and TNF-alpha stimulate phosphorylation of IRS-1 at inhibitory Ser307 via distinct pathways.

Serine/threonine phosphorylation of IRS-1 might inhibit insulin signaling, but the relevant phosphorylation sites are difficult to identify in cultured cells and to validate in isolated tissues. Recently, we discovered that recombinant NH2-terminal Jun kinase phosphorylates IRS-1 at Ser307, which inhibits insulin-stimulated tyrosine phosphorylation of IRS-1. To monitor phosphorylation of Ser307 in various cell and tissue backgrounds, we prepared a phosphospecific polyclonal antibody designated alphapSer307. This antibody revealed that TNF-alpha, IGF-1, or insulin stimulated phosphorylation of IRS-1 at Ser307 in 3T3-L1 preadipocytes and adipocytes. Insulin injected into mice or rats also stimulated phosphorylation of Ser307 on IRS-1 immunoprecipitated from muscle; moreover, Ser307 was phosphorylated in human muscle during the hyperinsulinemic euglycemic clamp. Experiments in 3T3-L1 preadipocytes and adipocytes revealed that insulin-stimulated phosphorylation of Ser307 was inhibited by LY294002 or wortmannin, whereas TNF-alpha-stimulated phosphorylation was inhibited by PD98059. Thus, distinct kinase pathways might converge at Ser307 to mediate feedback or heterologous inhibition of IRS-1 signaling to counterregulate the insulin response.

Expression of types 1, 2, and 3 17 beta-hydroxysteroid dehydrogenase in subcutaneous abdominal and intra-abdominal adipose tissue of women.

Human adipose tissue is known to have 17 beta-oxidoreductase activity, interconverting estrone (E1) and estradiol (E2), as well as androstenedione (A) and testosterone (T). We examined both the subcutaneous abdominal and intra-abdominal (visceral) adipose tissue of women for expression of types 1, 2, and 3 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) using ribonuclease (RNase) protection assay and RT-PCR/Southern blotting. Type 1 17 beta-HSD, which encodes the enzyme responsible for the conversion of E1 to E2 in the placenta and ovary, was expressed in the subcutaneous abdominal and intra-abdominal adipose tissue of women, but the messenger RNA transcripts were predominantly incompletely spliced and therefore unlikely to encode an active protein. A pseudogene for type 1 17 beta-HSD was also expressed in these tissues, but messenger RNA transcripts were again unspliced. Type 2 17 beta-HSD, which encodes an enzyme that can catalyze the conversion of T to A and E2 to E1, was expressed in both the subcutaneous abdominal and intra-abdominal adipose tissue of women. Type 3 17 beta-HSD was also expressed in adipose tissue from both sites studied. Type 3 17 beta-HSD encodes the enzyme that catalyzes the conversion of A to T in the testis and also converts E1 to E2. Together with aromatase, which is known to be expressed in adipose tissue, the expression of types 2 and 3 17 beta-HSD indicates that sex steroid production in the adipose tissue of women is a complex process. The association of visceral obesity with the development of insulin resistance and dyslipidaemia raises the question of the role of steroid production in adipose tissue in the pathogenesis of these disorders.

Regulation of the insulin-like growth factors and their binding proteins by glucocorticoid and growth hormone in nonislet cell tumor hypoglycemia.

Hypoglycemia in patients with nonislet cell tumors is often secondary to overexpression of tumor insulin-like growth factor (IGF) II. In these patients the formation of serum complexes between IGFs, IGF binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS) is impaired. An 87-yr-old woman with nonislet cell tumor hypoglycemia resulting from a localized fibrous tumor of the pleura was treated for 97 days with graded doses of prednisolone (30, 10, and 5 mg/day) followed by GH (1, 4, 8, 4, and 2 U/day). Both prednisolone and GH alleviated the hypoglycemia, concomitantly with increases in IGF-I, IGFBP-3, and ALS levels. Pretreatment serum IGFBP-2 and IGFBP-6 levels were greatly elevated, but as glucose normalized with treatment, only IGFBP-2 decreased, showing an inverse correlation with glucose (r = 0.716). IGFBP-1 gave a variable pattern not clearly related to blood glucose. Both treatments caused a redistribution of serum IGFBP-3 from binary- to ternary-complexed forms. However, only prednisolone improved the ability of IGFBP-3 to bind ALS in vitro. Prednisolone also suppressed IGF-II, the effect being confined to pro-IGF-II forms. Compared with normal IGF-II, pro-IGF-II inhibited ALS binding to IGFBP-3 in vitro. Although prednisolone and GH reverse hypoglycemia by different mechanisms, with only prednisolone suppressing tumor IGF-II secretion, both increase the formation of ternary IGF-IGFBP-3 complexes. We conclude that the failure of serum IGFBP-3 and tumor IGF-II to complex with ALS is a primary cause of hypoglycemia in nonislet cell tumor hypoglycemia.

Serological studies of Australian and Papua New Guinean cattle and Australian sheep for the presence of antibodies against bluetongue group viruses.

Following isolation of a virus (CSIRO19) from insects in Australia and its identification as bluetongue virus serotype 20 (BTV20), a nationwide survey of antibodies in cattle and sheep sera was undertaken. Initial studies using the serum neutralization (SN) test showed that the distribution of BTV20 antibodies in cattle was confined to the northern part of Australia. Group-reactive antibody tests (agar gel diffusion precipitin, AGDP, and complement-fixation, CF) showed group-reactive cattle sera south of the BTV20 zone (northern Australia), and southwards from Queensland to New South Wales. Very few group-reactive sheep sera (45 out of 16213) were found and these were of doubtful epidemiological significance. Some of these BTV group-reactive, BTV20-negative, sera were tested in SN tests against BTV1 to 17 and Ibaraki (IBA) virus. The results indicated that BTV1, or a closely related orbivirus, was active in cattle in Queensland, northern Western Australia, and New South Wales, and that antibody to BTV15 was present in some of the cattle sera in northern Western Australia and the Northern Territory. Antibody to IBA virus was present in some cattle sera in Queensland, northern Western Australia and New South Wales. SN antibody titres greater than or equal to 60 were also found to a number of other BTV serotypes in cattle sera in northern Western Australia and Queensland (principally, BTV2 and BTV7). Low level reactions were commonly observed against these and a number of other BTV serotypes, often in the same serum samples. Further, 22% of the group-reactive cattle sera did not react with any of the viruses in the SN tests. Such results were difficult to interpret in terms of known Australian BTV or BTV-related isolates.

Colisepticaemia of lambs.

In 1979 and 1980, 23 and 29 cases of ovine colisepticaemia from 16 and 18 properties respectively were diagnosed. All cases but one were caused by Escherichia coli 078: NM (Non Motile). Mortality ranged from 1% to 5% with an age distribution of 3 to 12 weeks. Most cases occurred in October. Thirty-three of the isolates were tested against a wide range of antibiotics and all had similar sensitivity patterns. E. coli 078 was detected less frequently in rectal cultures than in bile, small intestinal or nasal cultures from colisepticaemic lambs. In addition, E. coli 078 was recovered more frequently from nasal than from rectal cultures of lambs dying from causes other than colisepticaemia in colisepticaemic flocks. These findings suggest that perhaps the nasal route may be the more important portal of infection in ovine colisepticaemia caused by E. Coli 078 organisms.

Serological responses of sheep and cattle exposed to natural Toxoplasma infection.

During a four and a half year period a group of cattle was grazed continuously with a group of sheep and observed for Toxoplasma antibodies. Initially, there were 25 cattle and 31 sheep, but these were reduced to 19 and 18, respectively, by the end of the observation. Only transient antibody responses were noted in 2 cattle, whereas persistent titres developed in 12 sheep. Interpreted in the light of available information, these results were taken to indicate that cattle do not readily acquire persistent T. gondii infections.

Immunoglobulin classes of antibodies produced against Toxoplasma gondii by ovine fetuses.

Sera from aborted or newborn lambs with congenital toxoplasmosis were fractionated on Sephadex G-200. Sera and fractions were tested for Toxoplasma antibodies using the indirect fluorescent antibody test. Most of the antibody was found to reside in the IgG fraction.