Metabolic dyslipidemia is characterized by high circulating triglyceride (TG) and low HDL cholesterol levels and is frequently accompanied by hepatic steatosis. Increased hepatic lipogenesis contributes to both of these problems. Because insulin fails to suppress gluconeogenesis but continues to stimulate lipogenesis in both obese and lipodystrophic insulin-resistant mice, it has been proposed that a selective postreceptor defect in hepatic insulin action is central to the pathogenesis of fatty liver and hypertriglyceridemia in these mice. Here we show that humans with generalized insulin resistance caused by either mutations in the insulin receptor gene or inhibitory antibodies specific for the insulin receptor uniformly exhibited low serum TG and normal HDL cholesterol levels. This was due at least in part to surprisingly low rates of de novo lipogenesis and was associated with low liver fat content and the production of TG-depleted VLDL cholesterol particles. In contrast, humans with a selective postreceptor defect in AKT2 manifest increased lipogenesis, elevated liver fat content, TG-enriched VLDL, hypertriglyceridemia, and low HDL cholesterol levels. People with lipodystrophy, a disorder characterized by particularly severe insulin resistance and dyslipidemia, demonstrated similar abnormalities. Collectively these data from humans with molecularly characterized forms of insulin resistance suggest that partial postreceptor hepatic insulin resistance is a key element in the development of metabolic dyslipidemia and hepatic steatosis.

In many chronic inflammatory disorders, glucocorticoid (GC) insensitivity is a challenging clinical problem associated with life-threatening disease progression. The molecular basis of GC insensitivity, however, is unknown. Alternative splicing of the GC receptor (R) pre–messenger RNA generates a second GCR, termed GCR-β, which does not bind GCs but antagonizes the transactivating activity of the classic GCR, termed GCR-α. In the current study, we demonstrate that GC-insensitive asthma is associated with a significantly higher number of GCR-β–immunoreactive cells in peripheral blood than GC-sensitive asthmatics or normal controls. Furthermore, we show that patients with GC-insensitive asthma have cytokine-induced abnormalities in the DNA binding capability of the GCR. These abnormalities can be reproduced by transfection of cell lines with the GCR-β gene resulting in significant reduction of their GCR-α DNA binding capacity. We conclude that increased expression of GCR-β is cytokine inducible and may account for GC insensitivity in this common inflammatory condition.

Neutrophils are markedly less sensitive to glucocorticoids than T cells, making it difficult to control inflammation in neutrophil-mediated diseases. Development of new antiinflammatory strategies for such diseases would be aided by an understanding of mechanisms underlying differential steroid responsiveness. Two protein isoforms of the human glucocorticoid receptor (GR) exist, GRα and GRβ, which arise from alternative splicing of the GR pre-mRNA primary transcripts. GRβ does not bind glucocorticoids and is an inhibitor of GRα activity. Relative amounts of these two GRs can therefore determine the level of glucocorticoid sensitivity. In this study, human neutrophils and peripheral blood mononuclear cells (PBMCs) were studied to determine the relative amounts of each GR isoform.The mean fluorescence intensity (MFI) using immunofluorescence analysis for GRα was 475 ± 62 and 985 ± 107 for PBMCs and neutrophils, respectively. For GRβ, the MFI was 350 ± 60 and 1,389 ± 143 for PBMCs and neutrophils, respectively (P < 0.05). After interleukin (IL)-8 stimulation of neutrophils, there was a statistically significant increase in intensity of GRβ staining to 2,497 ± 140 (P < 0.05). No change in GRα expression was observed. This inversion of the GRα/GRβ ratio in human neutrophils compared with PBMCs was confirmed by quantitative Western analysis. Increased GRβ mRNA expression in neutrophils at baseline, and after IL-8 exposure, was observed using RNA dot blot analysis. Increased levels of GRα/GRβ heterodimers were found in neutrophils as compared with PBMCs using coimmunoprecipitation/Western analysis. Transfection of mouse neutrophils, which do not contain GRβ, resulted in a significant reduction in the rate of cell death when treated with dexamethasone.We conclude that high constitutive expression of GRβ by human neutrophils may provide a mechanism by which these cells escape glucocorticoid-induced cell death. Moreover, upregulation of this GR by proinflammatory cytokines such as IL-8 further enhances their survival in the presence of glucocorticoids during inflammation.

The parental origin of the missing short arm of the X chromosome has an impact on overweight, kidney, eye, and lipids, which suggests a potential effect of an as-yet-undetermined X chromosome gene imprinting.

Glucocorticoid resistance is a rare, familial, or sporadic condition characterized by partial end-organ insensitivity to glucocorticoids. The clinical spectrum of the condition ranges from completely asymptomatic to severe hyperandrogenism, fatigue, and/or mineralocorticoid excess. The molecular basis of glucocorticoid resistance in several families and sporadic cases has been ascribed to mutations in the human glucocorticoid receptor-alpha (hGRalpha) gene, which impair the ability of the receptor to transduce the glucocorticoid signal. We systematically investigated the molecular mechanisms through which natural, ligand-binding domain hGRalpha mutants, including hGRalphaI559N, hGRalphaV571A, hGRalphaD641V, hGRalphaV729I, and hGRalphaI747M, produce a defective signal and determined whether their differential effects on hGRalpha function might account for the type of genetic transmission of the disorder and the variable clinical phenotype of the affected subjects. Our findings suggest that all five mutant receptors studied have ligand-binding domains with decreased intrinsic transcriptional activity. Unlike hGRalphaI559N and I747M previously shown to exert a dominant negative effect upon the transcriptional activity of hGRalpha, hGRalphaV571A, D641V, and V729I do not have such an effect. All five mutants studied demonstrate varying degrees of decreased affinity for the ligand in a standard dexamethasone binding assay, but preserve their ability to bind DNA. The nondominant negative mutants, hGRalphaV571A, D641V, and V729I, show delayed translocation into the nucleus after exposure to ligand. Finally, hGRalphaI559N, V571A, D641V, and V729I display an abnormal interaction with the glucocorticoid receptor-interacting protein-1 coactivator in vitro, as this was previously shown also for hGRalphaI747M. We conclude that each of the above hGRalpha mutations imparts different functional defects upon the glucocorticoid signal transduction pathway, which explains the autosomal recessive or dominant transmission of the disorder, but might only explain in part its variable clinical phenotype.

Mitochondrial dysfunction is associated with insulin resistance and type 2 diabetes. It has thus been suggested that primary and/or genetic abnormalities in mitochondrial function may lead to accumulation of toxic lipid species in muscle and elsewhere, impairing insulin action on glucose metabolism. Alternatively, however, defects in insulin signaling may be primary events that result in mitochondrial dysfunction, or there may be a bidirectional relationship between these phenomena. To investigate this, we examined mitochondrial function in patients with genetic defects in insulin receptor (INSR) signaling. We found that phosphocreatine recovery after exercise, a measure of skeletal muscle mitochondrial function in vivo, was significantly slowed in patients with INSR mutations compared with that in healthy age-, fitness-, and BMI-matched controls. These findings suggest that defective insulin signaling may promote mitochondrial dysfunction. Furthermore, consistent with previous studies of mouse models of mitochondrial dysfunction, basal and sleeping metabolic rates were both significantly increased in genetically insulin-resistant patients, perhaps because mitochondrial dysfunction necessitates increased nutrient oxidation in order to maintain cellular energy levels.

OBJECTIVE:To elucidate the hormonal regulation of interleukin-6 (IL-6) production by human adipose tissue and its relation to leptin. DESIGN: In vitro study. Human adipocytes were incubated with dexamethasone (with or without RU486), norepinephrine and epinephrine (with or without propranolol), or insulin. MEASUREMENTS: IL-6 and leptin secretion by human adipocytes. RESULTS: A gradual increase in IL-6 secretion by adipocytes during differentiation was observed. A positive correlation was found between basal IL-6 release and both glycerol 3-phosphate dehydrogenase activity -a marker of adipocyte differentiation -and leptin release. Dexamethasone decreased IL-6 secretion and increased leptin secretion in a dose-dependent manner. Both catecholamines increased IL-6 and leptin secretion. The effects of dexamethasone and catecholamines on IL-6 and leptin were abrogated by RU486 and propranolol, respectively. Incubation with insulin resulted in a dose-dependent stimulation of IL-6 and leptin secretion. CONCLUSION: IL-6 is produced by human adipocytes and is a potential marker of adipocyte differentiation. Furthermore it is a hormonally regulated cytokine, suppressed by glucocorticoids, and stimulated by catecholamines and insulin in physiological concentrations.

The human glucocorticoid receptor (hGR) beta, a splicing variant of the classic receptor hGRalpha, functions as a dominant-negative inhibitor of hGRalpha. We explored the mechanism(s) underlying this effect of hGRbeta by evaluating the interactions of this isoform with known steroid receptor coactivators. We found that hGRbeta suppressed the transcriptional activity of both activation function (AF)-1 and AF-2 of hGRalpha, indicating that hGRbeta may exert its dominant-negative effect by affecting the function of coactivators that are attracted to these transactivation domains. hGRbeta bound to one of the p160 coactivators, the glucocorticoid receptor-interacting protein 1 (GRIP1) via its preserved AF-1 but not via its defective AF-2 in vitro. In a chromatin immunoprecipitation assay, hGRbeta prevented coprecipitation of GRIP1 with hGRalpha tethered to glucocorticoid response elements of the endogenous tyrosine aminotransferase promoter, whereas deletion of the AF-1 of hGRbeta abolished this effect. In further experiments, overexpression of GRIP1 attenuated the suppressive effect of hGRbeta on hGRalpha-mediated transactivation of the mouse mammary tumor virus promoter. Competition for binding to glucocorticoid response elements or heterodimerization with hGRalpha via the D loop dimerization interface occurred, but they were not necessary for the suppressive effect of hGRbeta on the transcriptional activity of hGRalpha. Our findings suggest that hGRbeta suppresses the transcriptional activity of hGRalpha by competing with hGRalpha for binding to GRIP1, and possibly other p160 coactivators, through its preserved AF-1. These findings suggest that participation of hGRbeta in the formation of a coactivator complex renders this complex ineffective.