Acute Hypercortisolemia Exerts Depot-Specific Effects on Abdominal and Femoral Adipose Tissue Function.

CONTEXT: Glucocorticoids have pleiotropic metabolic functions, and acute glucocorticoid excess affects fatty acid metabolism, increasing systemic lipolysis. Whether glucocorticoids exert adipose tissue depot-specific effects remains unclear. OBJECTIVE: To provide an in vivo assessment of femoral and abdominal adipose tissue responses to acute glucocorticoid administration. DESIGN AND OUTCOME MEASURES: Nine healthy male volunteers were studied on two occasions, after a hydrocortisone infusion (0.2 mg/kg/min for 14 hours) and a saline infusion, respectively, given in randomized double-blind order. The subjects were studied in the fasting state and after a 75-g glucose drink with an in vivo assessment of femoral adipose tissue blood flow (ATBF) using radioactive xenon washout and of lipolysis and glucose uptake using the arteriovenous difference technique. In a separate study (same infusion design), eight additional healthy male subjects underwent assessment of fasting abdominal ATBF and lipolysis only. Lipolysis was assessed as the net release of nonesterified fatty acids (NEFAs) from femoral and abdominal subcutaneous adipose tissue. RESULTS: Acute hypercortisolemia significantly increased basal and postprandial ATBF in femoral adipose tissue, but the femoral net NEFA release did not change. In abdominal adipose tissue, hypercortisolemia induced substantial increases in basal ATBF and NEFA release. CONCLUSIONS: Acute hypercortisolemia induces differential lipolysis and ATBF responses in abdominal and femoral adipose tissue, suggesting depot-specific glucocorticoid effects. Abdominal, but not femoral, adipose tissue contributes to the hypercortisolemia-induced systemic NEFA increase, with likely contributions from other adipose tissue sources and intravascular triglyceride hydrolysis.

SFRP2 Is Associated with Increased Adiposity and VEGF Expression.

AIMS: The aim of this study was to assess depot-specific expression and secretion of secreted frizzled-related protein 2 (sFRP2) by adipose tissue and its effect on adipocyte biology. We measured serum sFRP2 concentrations in 106 patients in vivo to explore its relationship to fat mass, glycaemia and insulin resistance. METHODS: Expression of sFRP2 in mouse and human tissues was assessed using polymerase chain reaction and Western blot. Western blot confirmed secretion of sFRP2 by adipose tissue into cell culture medium. Effects of recombinant sFRP2 on lipogenesis and preadipocyte proliferation were measured. Preadipocyte expression of the angiogenic genes vascular endothelial growth factor (VEGF) and nuclear factor of activated T-cells 3 (NFATC3) was measured after recombinant sFRP2 exposure. Complementary clinical studies correlating human serum sFRP2 with age, gender, adiposity and insulin secretion were also performed. RESULTS: sFRP2 messenger RNA (mRNA) was expressed in mouse and human adipose tissue. In humans, sFRP2 mRNA expression was 4.2-fold higher in omental than subcutaneous adipose. Omental adipose tissue secreted 63% more sFRP2 protein than subcutaneous. Treatment with recombinant sFRP2 did not impact on lipogenesis or preadipocyte proliferation but was associated with increased VEGF mRNA expression. In human subjects, circulating insulin levels positively correlated with serum sFRP2, and levels were higher in patients with abnormal glucose tolerance (34.2ng/ml) compared to controls (29.5ng/ml). A positive correlation between sFRP2 and BMI was also observed. CONCLUSIONS: Circulating sFRP2 is associated with adipose tissue mass and has a potential role to drive adipose angiogenesis through enhanced VEGF expression.

5α-Reductase Type 2 Regulates Glucocorticoid Action and Metabolic Phenotype in Human Hepatocytes.

Glucocorticoids and androgens have both been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); androgen deficiency in males, androgen excess in females, and glucocorticoid excess in both sexes are associated with NAFLD. Glucocorticoid and androgen action are regulated at a prereceptor level by the enzyme 5α-reductase type 2 (SRD5A2), which inactivates glucocorticoids to their dihydrometabolites and converts T to DHT. We have therefore explored the role of androgens and glucocorticoids and their metabolism by SRD5A2 upon lipid homeostasis in human hepatocytes. In both primary human hepatocytes and human hepatoma cell lines, glucocorticoids decreased de novo lipogenesis in a dose-dependent manner. Whereas androgen treatment (T and DHT) increased lipogenesis in cell lines and in primary cultures of human hepatocytes from female donors, it was without effect in primary hepatocyte cultures from men. SRD5A2 overexpression reduced the effects of cortisol to suppress lipogenesis and this effect was lost following transfection with an inactive mutant construct. Conversely, pharmacological inhibition using the 5α-reductase inhibitors finasteride and dutasteride augmented cortisol action. We have demonstrated that manipulation of SRD5A2 activity can regulate lipogenesis in human hepatocytes in vitro. This may have significant clinical implications for those patients prescribed 5α-reductase inhibitors, in particular augmenting the actions of glucocorticoids to modulate hepatic lipid flux.

11ß-HSD1 is the major regulator of the tissue-specific effects of circulating glucocorticoid excess.

The adverse metabolic effects of prescribed and endogenous glucocorticoid (GC) excess, Cushing syndrome, create a significant health burden. We found that tissue regeneration of GCs by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), rather than circulating delivery, is critical to developing the phenotype of GC excess; 11ß-HSD1 KO mice with circulating GC excess are protected from the glucose intolerance, hyperinsulinemia, hepatic steatosis, adiposity, hypertension, myopathy, and dermal atrophy of Cushing syndrome. Whereas liver-specific 11ß-HSD1 KO mice developed a full Cushingoid phenotype, adipose-specific 11ß-HSD1 KO mice were protected from hepatic steatosis and circulating fatty acid excess. These data challenge our current view of GC action, demonstrating 11ß-HSD1, particularly in adipose tissue, is key to the development of the adverse metabolic profile associated with circulating GC excess, offering 11ß-HSD1 inhibition as a previously unidentified approach to treat Cushing syndrome.

Cortisol biosynthesis in the human ocular surface innate immune response.

Innate immune responses have a critical role in regulating sight-threatening ocular surface (OcS) inflammation. While glucocorticoids (GCs) are frequently used to limit tissue damage, the role of intracrine GC (cortisol) bioavailability via 11-beta-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) in OcS defense, remains unresolved. We found that primary human corneal epithelial cells (PHCEC), fibroblasts (PHKF) and allogeneic macrophages (M1, GM-CSF; M2, M-CSF) were capable of generating cortisol (M1>PHKF>M2>PHCEC) but in corneal cells, this was independent of Toll-like receptor (TLR) activation. While PolyI∶C induced maximal cytokine and chemokine production from both PHCEC (IFNγ, CCL2, CCL3, and (CCL4), IL6, CXCL10, CCL5, TNFα) and PHKF (CCL2, IL-6, CXCL10, CCL5), only PHKF cytokines were inhibited by GCs. Both Poly I∶C and LPS challenged-corneal cells induced M1 chemotaxis (greatest LPS-PHKF (250%), but down-regulated M1 11ß-HSD1 activity (30 and 40% respectively). These data were supported by clinical studies demonstrating reduced human tear film cortisol∶cortisone ratios (a biomarker of local 11ß-HSD1 activity) in pseudomonas keratitis (1∶2.9) versus healthy controls (1∶1.3; p<0.05). This contrasted with putative TLR3-mediated OcS disease (Stevens-Johnson Syndrome, Mucous membrane pemphigoid) where an increase in cortisol∶cortisone ratio was observed (113.8∶1; p<0.05). In summary, cortisol biosynthesis in human corneal cells is independent of TLR activation and is likely to afford immunoprotection under physiological conditions. Contribution to ocular mucosal innate responses is dependent on the aetiology of immunological challenge.

Dehydroepiandrosterone exerts antiglucocorticoid action on human preadipocyte proliferation, differentiation, and glucose uptake.

Glucocorticoids increase adipocyte proliferation and differentiation, a process underpinned by the local reactivation of inactive cortisone to active cortisol within adipocytes catalyzed by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1). The adrenal sex steroid precursor dehydroepiandrosterone (DHEA) has been shown to inhibit 11ß-HSD1 in murine adipocytes; however, rodent adrenals do not produce DHEA physiologically. Here, we aimed to determine the effects and underlying mechanisms of the potential antiglucocorticoid action of DHEA and its sulfate ester DHEAS in human preadipocytes. Utilizing a human subcutaneous preadipocyte cell line, Chub-S7, we examined the metabolism and effects of DHEA in human adipocytes, including adipocyte proliferation, differentiation, 11ß-HSD1 expression, and activity and glucose uptake. DHEA, but not DHEAS, significantly inhibited preadipocyte proliferation via cell cycle arrest in the G1 phase independent of sex steroid and glucocorticoid receptor activation. 11ß-HSD1 oxoreductase activity in differentiated adipocytes was inhibited by DHEA. DHEA coincubated with cortisone significantly inhibited preadipocyte differentiation, which was assessed by the expression of markers of early (LPL) and terminal (G3PDH) adipocyte differentiation. Coincubation with cortisol, negating the requirement for 11ß-HSD1 oxoreductase activity, diminished the inhibitory effect of DHEA. Further consistent with glucocorticoid-opposing effects of DHEA, insulin-independent glucose uptake was significantly enhanced by DHEA treatment. DHEA increases basal glucose uptake and inhibits human preadipocyte proliferation and differentiation, thereby exerting an antiglucocorticoid action. DHEA inhibition of the amplification of glucocorticoid action mediated by 11ß-HSD1 contributes to the inhibitory effect of DHEA on human preadipocyte differentiation.

Regulation of lipid metabolism by glucocorticoids and 11ß-HSD1 in skeletal muscle.

The prevalences of insulin resistance and type 2 diabetes mellitus are rising dramatically, and, as a consequence, there is an urgent need to understand the pathogenesis underpinning these conditions to develop new and more efficacious treatments. We have tested the hypothesis that glucocorticoid (GC)-mediated changes in insulin sensitivity may be associated with changes in lipid flux. Furthermore, prereceptor modulation of GC availability by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) may represent a critical regulatory step. Dexamethasone (DEX) decreased lipogenesis in both murine C2C12 and human LHC-NM2 myotubes. Inactivating p-Ser-79/218 of acetyl-CoA carboxylase 1/2 and activating p-Thr-172 of AMP-activated protein kinase were both increased after DEX treatment in C2C12 myotubes. In contrast, DEX increased ß-oxidation. Selective 11ß-HSD1 inhibition blocked the 11-dehydrocorticosterone (11DHC)-mediated decrease in lipogenic gene expression and increase in lipolytic gene expression. Lipogenic gene expression was decreased, whereas lipolytic and ß-oxidative gene expression increased in corticosterone (CORT)- and 11DHC-treated wild-type mice and CORT (but not 11DHC)-treated 11ß-HSD1(-/-) mice. Furthermore, CORT- and 11DHC-treated wild-type mice and CORT (but not 11DHC)-treated 11ß-HSD1(-/-) mice had increased p-Ser-79/218 acetyl-CoA carboxylase 1/2, p-Thr-172 AMP-activated protein kinase and intramyocellular diacylglyceride content. In summary, we have shown that GCs have potent actions on intramyocellular lipid homeostasis by decreasing lipid storage, increasing lipid mobilization and utilization, and increasing diacylglyceride content. It is plausible that dysregulated intramyocellular lipid metabolism may underpin GC-induced insulin resistance of skeletal muscle.

Expression of 11ß-hydroxysteroid dehydrogenase enzymes in human osteosarcoma: potential role in pathogenesis and as targets for treatments.

Osteosarcoma (OS) is a primary malignant tumour of bone occurring predominantly in children and young adults. Despite chemotherapy, relapse is common and mortality remains high. Non-transformed osteoblasts are highly sensitive to glucocorticoids, which reduce proliferation and induce apoptosis. Previously, we observed that OS cells, but not normal osteoblasts, express 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2). This enzyme inactivates cortisol (active) to cortisone (inactive) and expression of 11ß-HSD2 renders OS cells resistant to glucocorticoids. By contrast, the related enzyme 11ß-HSD1 converts cortisone to cortisol and reduces OS cell proliferation in vitro. Some synthetic glucocorticoids (e.g. dehydrodexamethasone (DHD), inactive counterpart of dexamethasone (DEX)) have been reported to be activated by 11ß-HSD2. We therefore investigated expression and enzymatic activity of 11ß-HSD isozymes in human OS tissue, determined whether 11ß-HSD expression has prognostic value in the response to therapy, and evaluated the potential use of synthetic glucocorticoids to selectively target OS cells. OS samples expressed both 11ß-HSD1 and 11ß-HSD2. 11ß-HSD1 expression in pretreatment biopsy specimens positively correlated with primary tumour size. Expression and activity of 11ß-HSD1 in post-treatment biopsies were unrelated to the degree of tumour necrosis following chemotherapy. However, high 11ß-HSD2 expression in post-treatment biopsies correlated with a poor response to therapy. OS cells that expressed 11ß-HSD2 inactivated endogenous glucocorticoids; but these cells were also able to generate DEX from DHD. These results suggest that OS treatment response is related to 11ß-HSD2 enzyme expression. Furthermore, OS cells expressing this enzyme could be targeted by treatment with synthetic glucocorticoids that are selectively reactivated by the enzyme.

A switch in hepatic cortisol metabolism across the spectrum of non alcoholic fatty liver disease.

CONTEXT: Non alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. NAFLD represents a spectrum of liver disease ranging from reversible hepatic steatosis, to non alcoholic steato-hepatitis (NASH) and cirrhosis. The potential role of glucocorticoids (GC) in the pathogenesis of NAFLD is highlighted in patients with GC excess, Cushing's syndrome, who develop central adiposity, insulin resistance and in 20% of cases, NAFLD. Although in most cases of NAFLD, circulating cortisol levels are normal, hepatic cortisol availability is controlled by enzymes that regenerate cortisol (F) from inactive cortisone (E) (11ß-hydroxysteroid dehydrogenase type 1, 11ß-HSD1), or inactivate cortisol through A-ring metabolism (5α- and 5ß-reductase, 5αR and 5ßR). OBJECTIVE AND METHODS: In vitro studies defined 11ß-HSD1 expression in normal and NASH liver samples. We then characterised hepatic cortisol metabolism in 16 patients with histologically proven NAFLD compared to 32 obese controls using gas chromatographic analysis of 24 hour urine collection and plasma cortisol generation profile following oral cortisone. RESULTS: In patients with steatosis 5αR activity was increased, with a decrease in hepatic 11ß-HSD1 activity. Total cortisol metabolites were increased in this group consistent with increased GC production rate. In contrast, in patients with NASH, 11ß-HSD1 activity was increased both in comparison to patients with steatosis, and controls. Endorsing these findings, 11ß-HSD1 mRNA and immunostaining was markedly increased in NASH patients in peri septal hepatocytes and within CD68 positive macrophages within inflamed cirrhotic septa. CONCLUSION: Patients with hepatic steatosis have increased clearance and decreased hepatic regeneration of cortisol and we propose that this may represent a protective mechanism to decrease local GC availability to preserve hepatic metabolic phenotype. With progression to NASH, increased 11ß-HSD1 activity and consequent cortisol regeneration may serve to limit hepatic inflammation.

Regulation of lipogenesis by glucocorticoids and insulin in human adipose tissue.

Patients with glucocorticoid (GC) excess, Cushing's syndrome, develop a classic phenotype characterized by central obesity and insulin resistance. GCs are known to increase the release of fatty acids from adipose, by stimulating lipolysis, however, the impact of GCs on the processes that regulate lipid accumulation has not been explored. Intracellular levels of active GC are dependent upon the activity of 11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) and we have hypothesized that 11ß-HSD1 activity can regulate lipid homeostasis in human adipose tissue (Chub-S7 cell line and primary cultures of human subcutaneous (sc) and omental (om) adipocytes. Across adipocyte differentiation, lipogenesis increased whilst ß-oxidation decreased. GC treatment decreased lipogenesis but did not alter rates of ß-oxidation in Chub-S7 cells, whilst insulin increased lipogenesis in all adipocyte cell models. Low dose Dexamethasone pre-treatment (5 nM) of Chub-S7 cells augmented the ability of insulin to stimulate lipogenesis and there was no evidence of adipose tissue insulin resistance in primary sc cells. Both cortisol and cortisone decreased lipogenesis; selective 11ß-HSD1 inhibition completely abolished cortisone-mediated repression of lipogenesis. GCs have potent actions upon lipid homeostasis and these effects are dependent upon interactions with insulin. These in vitro data suggest that manipulation of GC availability through selective 11ß-HSD1 inhibition modifies lipid homeostasis in human adipocytes.

Cortisone-reductase deficiency associated with heterozygous mutations in 11beta-hydroxysteroid dehydrogenase type 1.

In peripheral target tissues, levels of active glucocorticoid hormones are controlled by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), a dimeric enzyme that catalyzes the reduction of cortisone to cortisol within the endoplasmic reticulum. Loss of this activity results in a disorder termed cortisone reductase deficiency (CRD), typified by increased cortisol clearance and androgen excess. To date, only mutations in H6PD, which encodes an enzyme supplying cofactor for the reaction, have been identified as the cause of disease. Here we examined the HSD11B1 gene in two cases presenting with biochemical features indicative of a milder form of CRD in whom the H6PD gene was normal. Novel heterozygous mutations (R137C or K187N) were found in the coding sequence of HSD11B1. The R137C mutation disrupts salt bridges at the subunit interface of the 11ß-HSD1 dimer, whereas K187N affects a key active site residue. On expression of the mutants in bacterial and mammalian cells, activity was either abolished (K187N) or greatly reduced (R137C). Expression of either mutant in a bacterial system greatly reduced the yield of soluble protein, suggesting that both mutations interfere with subunit folding or dimer assembly. Simultaneous expression of mutant and WT 11ß-HSD1 in bacterial or mammalian cells, to simulate the heterozygous condition, indicated a marked suppressive effect of the mutants on both the yield and activity of 11ß-HSD1 dimers. Thus, these heterozygous mutations in the HSD11B1 gene have a dominant negative effect on the formation of functional dimers and explain the genetic cause of CRD in these patients.

The role of 11beta-hydroxysteroid dehydrogenase 1 in adipogenesis in thyroid-associated ophthalmopathy.

CONTEXT: Thyroid-associated ophthalmopathy (TAO) is a sight-threatening autoimmune disease in which de novo adipogenesis has been identified as a fundamental pathogenic mechanism. 11beta-Hydroxysteroid dehydrogenase 1 (11beta-HSD1) increases cortisol bioavailability and is pivotal in mediating glucocorticoid responses in adipose tissue and inflammation. OBJECTIVE: In this study we characterize 11beta-HSD1 as a determinant of the adipogenic and inflammatory pathways in TAO orbital fat (OF) compared with normal OF. PATIENTS AND METHODS: OF was harvested from 46 TAO and 44 control patients undergoing orbital surgery. Samples were examined by a combination of immunohistochemistry, real-time RT-PCR, primary cell culture, specific enzyme assays, colorimetric proliferation assays, and bead-based ELISA. RESULTS: Glucocorticoid (glucocorticoid receptor-alpha,11beta-HSD1, hexose-6-phosphate dehydrogenase) and inflammatory cytokines (IL-1beta, IL-1 receptor, IL-6, TNF-alpha, TNF-alpha inductible protein, TGF-beta2) target genes together with markers of late adipocyte differentiation (fatty-acid-binding-protein-4, glycerol-6-phosphate-dehydrogenase) were highly expressed in TAO whole OF (P < 0.05) compared with controls. Primary cultures of TAO OF stromal cells demonstrated greater 11beta-HSD1 oxoreductase activity (P < 0.05), which was regulated by cytokines, most notably TNF-alpha (P < 0.01), compared with controls. Activity increased across differentiation, and this was most marked in TAO cells (P < 0.01). Similarly, stromal cell proliferation was limited by incubation with cortisol in TAO cells only. Furthermore, cortisone decreased IL-6 (P < 0.005), IL-8 (P < 0.05), and macrophage chemoattractant protein-1 (P < 0.05) production by cultured TAO cells only, an effect that was abrogated by inhibition of 11beta-HSD1. CONCLUSIONS: Induction of 11beta-HSD1 activity and expression by inflammatory cytokines (TNF-alpha, IL-6) may enhance orbital adipocyte differentiation (adipogenesis) and limit proliferation in TAO. 11beta-HSD1 may also have a role in regulating the local orbital inflammatory response.

11beta-hydroxysteroid dehydrogenase type 1 regulates glucocorticoid-induced insulin resistance in skeletal muscle.

OBJECTIVE: Glucocorticoid excess is characterized by increased adiposity, skeletal myopathy, and insulin resistance, but the precise molecular mechanisms are unknown. Within skeletal muscle, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts cortisone (11-dehydrocorticosterone in rodents) to active cortisol (corticosterone in rodents). We aimed to determine the mechanisms underpinning glucocorticoid-induced insulin resistance in skeletal muscle and indentify how 11beta-HSD1 inhibitors improve insulin sensitivity. RESEARCH DESIGN AND METHODS: Rodent and human cell cultures, whole-tissue explants, and animal models were used to determine the impact of glucocorticoids and selective 11beta-HSD1 inhibition upon insulin signaling and action. RESULTS: Dexamethasone decreased insulin-stimulated glucose uptake, decreased IRS1 mRNA and protein expression, and increased inactivating pSer(307) insulin receptor substrate (IRS)-1. 11beta-HSD1 activity and expression were observed in human and rodent myotubes and muscle explants. Activity was predominantly oxo-reductase, generating active glucocorticoid. A1 (selective 11beta-HSD1 inhibitor) abolished enzyme activity and blocked the increase in pSer(307) IRS1 and reduction in total IRS1 protein after treatment with 11DHC but not corticosterone. In C57Bl6/J mice, the selective 11beta-HSD1 inhibitor, A2, decreased fasting blood glucose levels and improved insulin sensitivity. In KK mice treated with A2, skeletal muscle pSer(307) IRS1 decreased and pThr(308) Akt/PKB increased. In addition, A2 decreased both lipogenic and lipolytic gene expression. CONCLUSIONS: Prereceptor facilitation of glucocorticoid action via 11beta-HSD1 increases pSer(307) IRS1 and may be crucial in mediating insulin resistance in skeletal muscle. Selective 11beta-HSD1 inhibition decreases pSer(307) IRS1, increases pThr(308) Akt/PKB, and decreases lipogenic and lipolytic gene expression that may represent an important mechanism underpinning their insulin-sensitizing action.

Adipose cell-adrenal interactions: current knowledge and future perspectives.

The central role of adipose tissue in the development of cardiovascular and metabolic pathology has been highlighted by the discovery of mediators (adipokines) secreted by adipose tissue and their involvement in the regulation of various biological processes. In light of recent experimental data, cross-talk between adipose tissue and the adrenal gland, particularly via the mineralocorticoid aldosterone, has been proposed. Aldosterone can induce adipogenesis, and human white adipose tissue is reported to release as-yet-uncharacterized factors that stimulate adrenocortical steroidogenesis and aldosterone production. These data could provide new insights into the pathophysiology of obesity-related disorders, including hypertension and aldosterone excess, with further studies necessary for confirming and better defining such adipose-adrenal interactions.

Lack of hexose-6-phosphate dehydrogenase impairs lipid mobilization from mouse adipose tissue.

In adipose tissue, glucocorticoids regulate lipogenesis and lipolysis. Hexose-6-phosphate dehydrogenase (H6PDH) is an enzyme located in the endoplasmic reticulum that provides a cofactor for the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), regulating the set point of its activity and allowing for tissue-specific activation of glucocorticoids. The aim of this study was to examine the adipose tissue biology of the H6PDH null (H6PDH/KO) mouse. Real-time PCR analysis confirmed similar mRNA levels of 11beta-HSD1 and glucocorticoid receptor-alpha in wild-type (WT) and H6PDH/KO mice in liver and gonadal fat depots. Microsomal 11beta-HSD1 protein levels shown by Western blot analysis corresponded well with mRNA expression in gonadal fat of WT and H6PDH/KO mice. Despite this, the enzyme directionality in these tissues changed from predominately oxoreductase in WT to exclusively dehydrogenase activity in the H6PDH/KO mice. In the fed state, H6PDH/KO mice had reduced adipose tissue mass, but histological examination revealed no difference in average adipocyte size between genotypes. mRNA expression levels of the key lipogenic enzymes, acetyl CoA carboxylase, adiponutrin, and stearoyl-coenzyme A desaturase-2, were decreased in H6PDH/KO mice, indicative of impaired lipogenesis. In addition, lipolysis rates were also impaired in the H6PDH/KO as determined by lack of mobilization of fat and no change in serum free fatty acid concentrations upon fasting. In conclusion, in the absence of H6PDH, the set point of 11beta-HSD1 enzyme activity is switched from predominantly oxoreductase to dehydrogenase activity in adipose tissue; as a consequence, this leads to impairment of fat storage and mobilization.

Glucocorticoid modulation of insulin signaling in human subcutaneous adipose tissue.

CONTEXT: Glucocorticoid (GC) excess is characterized by central obesity, insulin resistance, and in some cases, type 2 diabetes. However, the impact of GC upon insulin signaling in human adipose tissue has not been fully explored. OBJECTIVE: We have examined the effect of GC upon insulin signaling in both human sc primary preadipocyte cultures and a novel human immortalized sc adipocyte cell line (Chub-S7) and contrasted this with observations in primary cultures of human skeletal muscle. DESIGN AND SETTING: This is an in vitro study characterizing the impact of GC upon insulin signaling in human tissues. PATIENTS: Biopsy specimens were from healthy volunteers who gave their full and informed written consent. INTERVENTIONS: Combinations of treatments, including GC, RU38486, and wortmannin, were used. MAIN OUTCOME MEASURES: Insulin signaling cascade gene and protein expression and insulin-stimulated glucose uptake were determined. RESULTS: In human adipocytes, pretreatment with GC induced a dose-dependent [1.0 (control); 1.2 +/- 0.1 (50 nm); 2.2 +/- 0.2 (250 nm), P < 0.01 vs. control; 3.4 +/- 0.2 (1000 nm), P < 0.001 vs. control] and time-dependent [1.0 (1 h); 3.2 +/- 2.0 (6 h); 9.1 +/- 5.9 (24 h), P < 0.05 vs. 1 h; 4.5 +/- 2.2 (48 h)] increase in insulin-stimulated protein kinase B/akt phosphorylation. In addition, whereas insulin receptor substrate (IRS)-1 protein expression did not change, IRS-1 tyrosine phosphorylation increased. Furthermore, GC induced IRS-2 mRNA expression (2.8-fold; P < 0.05) and increased insulin-stimulated glucose uptake [1.0 (control) 1.8 +/- 0.1 (insulin) vs. 2.8 +/- 0.2 (insulin + GC); P < 0.05]. In contrast, in primary cultures of human muscle, GC decreased insulin-stimulated glucose uptake [1.0 (control) 1.9 +/- 0.2 (insulin) vs. GC 1.3 +/- 0.1 (insulin + GC); P < 0.05]. CONCLUSIONS: We have demonstrated tissue-specific regulation of insulin signaling by GC. Within sc adipose tissue, GCs augment insulin signaling, yet in muscle GCs cause insulin resistance. We propose that enhanced insulin action in adipose tissue increases adipocyte differentiation, thereby contributing to GC-induced obesity.

Characterisation of 11beta-hydroxysteroid dehydrogenase 1 in human orbital adipose tissue: a comparison with subcutaneous and omental fat.

Glucocorticoids (GCs) have a profound effect on adipose biology increasing tissue mass causing central obesity. The pre-receptor regulation of GCs by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) that activates cortisol from cortisone has been postulated as a fundamental mechanism underlying the metabolic syndrome mediating adipocyte hyperplasia and hypertrophy in the omental (OM) depot. Orbital adipose tissue (OF) is the site of intense inflammation and tissue remodelling in several orbital inflammatory disease states. In this study, we describe features of the GC metabolic pathways in normal human OF depot and compare it with subcutaneous (SC) and OM depots. Using an automated histological characterisation technique, OF adipocytes were found to be significantly smaller (parameters: area, maximum diameter and perimeter) than OM and SC adipocytes (P<0 x 001). Although immunohistochemical analyses demonstrated resident CD68+ cells in all three whole tissue adipose depots, OF CD68 mRNA and protein expression exceeded that of OM and SC (mRNA, P<0 x 05; protein, P<0 x 001). In addition, there was higher expression of glucocorticoid receptor (GR)alpha mRNA in the OF whole tissue depot (P<0 x 05). Conversely, 11beta-HSD1 mRNA together with the markers of late adipocyte differentiation (FABP4 and G3PDH) were significantly lower in OF. Primary cultures of OF preadipocytes demonstrated predominant 11beta-HSD1 oxo-reductase activity with minimal dehydrogenase activity. Orbital adipocytes are smaller, less differentiated, and express low levels of 11beta-HSD1 but abundant GRalpha compared with SC and OM. OF harbours a large CD68+ population. These characteristics define an orbital microenvironment that has the potential to respond to sight-threatening orbital inflammatory disease.

Depot-specific prostaglandin synthesis in human adipose tissue: a novel possible mechanism of adipogenesis.

Despite the magnitude of the obesity epidemic, the mechanisms that contribute to increases in fat mass and to differences in fat depots are still poorly understood. Prostanoids have been proposed as potent adipogenic hormones, e.g. metabolites of prostaglandin J2 (PGJ2) bind and activate PPARgamma. We hypothesize that an altered expression of enzymes in PGJ2 synthesis may represent a novel pathogenic mechanism in human obesity. We characterized adipose depot-specific expression of enzymes in PGJ2 synthesis, prostaglandin transporter and PPARgamma isoforms. Paired omental and subcutaneous adipose tissue samples were obtained from 26 women undergoing elective abdominal surgery and gene expression examined in whole tissue and cultured preadipocytes using an Affymetrix cDNA microarray technique and validated with quantitative real-time PCR. All enzymes involved in prostaglandin synthesis were expressed in both adipose tissues. Expression of prostaglandin synthase-1 (PGHS1), prostaglandin D synthase (PTGDS), human prostaglandin transporter (hPGT) and PPARgamma2 was higher in OM adipose tissue compared to SC, whereas 17beta-hydroxysteroid dehydrogenase 5 (AKR1C3) showed predominance in SC adipose tissue. In SC adipose tissue, PGHS1 mRNA expression increased with BMI. The differential, depot-specific expression of key enzymes involved in transport, synthesis and metabolism of prostaglandins may have an important impact upon fat cell biology and may help to explain some of the observed depot-specific differences. In addition, the positive correlation between PGHS1 and BMI offers the novel hypothesis that the regulation of PG synthesis may have a role in determining fat distribution in human obesity.

Androgen receptor-mediated regulation of the alpha-subunit of the epithelial sodium channel in human kidney.

Rodents studies suggest that androgens are involved in sex-specific differences in blood pressure. In humans, there is no difference in blood pressure between boys and girls, but after puberty, blood pressure increases more in men than in women. We investigated androgen-dependent regulation of the alpha-subunit of the epithelial sodium channel (alphaEnaC) in human kidney and in the human renal cell line immortalized human renal proximal tubular cell line (HKC-8). We used microarray technique to analyze androgen-dependent gene regulation and performed quantitative RT-PCR for verification. Promoter constructs for human alphaENaC were used in transfection studies to analyze the regulation by testosterone. We investigated the in vivo effect of testosterone on alphaENaC in a rat model and used the mouse collecting duct cell line M-1 for transepithelial electrophysiological measurements. The androgen receptor (AR) was expressed in male kidney and HKC-8 cells. AlphaENaC mRNA expression increased 2- to 3-fold after treatment with testosterone in HKC-8 cells. The induction by testosterone was completely blocked by adding the AR antagonist flutamide. Analysis of the alphaENaC promoter sequence identified a putative AR response element (ARE) located 140 nucleotides upstream from the transcription start site. HKC-8 cell transfection studies showed that testosterone directly upregulated gene expression via this ARE. In vivo, testosterone treatment of orchiectomized rats resulted in an increased renal alphaENaC mRNA expression. In testosterone-treated mouse M-1 cells, amiloride caused a significant stronger decrease in short circuit current than in control cells. These data show that alphaENaC expression is directly regulated by androgens in vitro and in vivo and highlight a potential mechanism explaining the reported gender differences in blood pressure.

Hexose-6-phosphate dehydrogenase confers oxo-reductase activity upon 11 beta-hydroxysteroid dehydrogenase type 1.

Two isozymes of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) interconvert active cortisol and inactive cortisone. 11 beta-HSD2 (renal) acts only as a dehydrogenase, converting cortisol to cortisone. 11 beta-HSD1 (liver) is a bi-directional enzyme in cell homogenates, whereas in intact cells it typically displays oxo-reductase activity, generating cortisol from cortisone. We recently established that cortisone reductase deficiency is a digenic disease requiring mutations in both the gene encoding 11 beta-HSD1 and in the gene for a novel enzyme located within the lumen of the endoplasmic reticulum (ER), hexose-6-phosphate dehydrogenase (H6PDH). This latter enzyme generates NADPH, the co-factor required for oxo-reductase activity. Therefore, we hypothesized that H6PDH expression may be an important determinant of 11 beta-HSD1 oxo-reductase activity. Transient transfection of chinese hamster ovary (CHO) cells with 11 beta-HSD1 resulted in the appearance of both oxo-reductase and dehydrogenase activities in intact cells. Co-transfection of 11 beta-HSD1 with H6PDH increased oxo-reductase activity whilst virtually eliminating dehydrogenase activity. In contrast, H6PDH had no effect on reaction direction of 11 beta-HSD2, nor did the cytosolic enzyme, glucose-6-phosphate dehydrogenase (G6PD) affect 11 beta-HSD1 oxo-reductase activity. Conversely in HEK 293 cells stably transfected with 11 beta-HSD1 cDNA, transfection of an H6PDH siRNA reduced 11 beta-HSD1 oxo-reductase activity whilst simultaneously increasing 11 beta-HSD1 dehydrogenase activity. In human omental preadipocytes obtained from 15 females of variable body mass index (BMI), H6PDH mRNA levels positively correlated with 11 beta-HSD1 oxo-reductase activity, independent of 11 beta-HSD1 mRNA levels. H6PDH expression increased 5.3-fold across adipocyte differentiation (P < 0.05) and was associated with a switch from 11 beta-HSD1 dehydrogenase to oxo-reductase activity. In conclusion, H6PDH is a crucial determinant of 11 beta-HSD1 oxo-reductase activity in intact cells. Through its interaction with 11 beta-HSD1, H6PDH may represent a novel target in the pathogenesis and treatment of obesity.