Increased adipose expression of the uncoupling protein-3 gene by thiazolidinediones in Wistar fatty rats and in cultured adipocytes.

Uncoupling protein (UCP) 3 and UCP2, mitochondrial carrier proteins dissipating electrochemical gradient across the mitochondrial inner membrane, have been implicated in the regulation of energy metabolism. The UCP3 gene is expressed abundantly in the skeletal muscle, while the UCP2 gene is detected in the white adipose tissue (WAT) with diffuse localization throughout the body. Uncoupling of electron transport and ATP synthesis has been reported to increase glucose uptake, suggesting that UCP may be involved in glucose metabolism. Thiazolidinediones (TZDs), which are insulin-sensitizing agents for NIDDM, have been reported to increase energy expenditure. To elucidate the pathophysiologic significance of UCP3 and UCP2 in the effect of TZDs on glucose metabolism and energy expenditure, we examined their basal mRNA levels in the WAT, brown adipose tissue (BAT), and skeletal muscle from Wistar fatty rats, a rat model of NIDDM and obesity with leptin receptor defect, and investigated expression of the genes encoding UCP3 and UCP2 in Wistar fatty rats and in Wistar lean rats with 2-week oral administration of 3 mg x kg(-1) x day(-1) pioglitazone, a TZD derivative. Basal UCP3 mRNA levels were significantly lower (38 +/- 8, 45 +/- 13, and 76 +/- 6%) in the retroperitoneal WAT, BAT, and skeletal muscle from Wistar fatty rats than in those from Wistar lean rats, while basal UCP2 mRNA levels were significantly higher by 2.1-, 1.8-, and 2.5-fold in the subcutaneous WAT, retroperitoneal WAT, and BAT from Wistar fatty rats, respectively, than in those from Wistar lean rats. In pioglitazone-treated Wistar fatty rats, UCP3 mRNA levels were significantly increased by 2.1-, 2.0-, and 1.6-fold in the epididymal WAT, retroperitoneal WAT, and BAT, respectively, as compared with those in nontreated fatty rats. In pioglitazone-treated lean rats, UCP3 mRNA levels were significantly increased by 1.3-fold in the BAT as compared with those in nontreated lean rats. No significant change of UCP2 mRNA levels was observed in pioglitazone-treated fatty and lean rats. In addition, to examine the direct effect of TZDs on adipocytes, we examined the regulation of UCP3 and UCP2 gene expression using the primary culture of rat mature adipocytes from Sprague-Dawley rats. In rat cultured mature adipocytes, UCP3 mRNA levels were increased in a dose-responsive manner by 10(-5) to 10(-4) mol/l pioglitazone, while there was no significant change of UCP2 mRNA levels. These results clearly demonstrate that UCP3 gene expression is upregulated by TZDs in the WAT and BAT in Wistar fatty rats, an obese model with leptin receptor defect, and that adipose UCP3 gene expression is increased in response to TZDs in vitro. The present study suggests the involvement of UCP3 in the effects of TZDs on energy and glucose metabolism.

Glucocorticoid regulation of leptin synthesis and secretion in humans: elevated plasma leptin levels in Cushing's syndrome.

Leptin, the obese (ob) gene product, is an adipocyte-derived satiety factor that is involved in the regulation of food ingestion and body weight. To investigate glucocorticoid regulation of leptin synthesis and secretion in humans, we measured plasma leptin levels in patients with Cushing's syndrome with adrenal or pituitary adenoma and in patients with iatrogenic Cushing's syndrome. Plasma leptin levels in patients with Cushing's syndrome were significantly elevated compared to those in nonobese healthy subjects and obese subjects without any metabolic or endocrine diseases at a given percentage of body fat by analysis of covariance. In patients with adrenal or pituitary adenoma, after the tumor resection, plasma leptin levels were reduced, with a concurrent decrease in plasma cortisol levels. With no significant changes in body weight, plasma leptin levels were also elevated significantly in lean healthy volunteers 24 h after the administration of 1 mg dexamethasone. Dexamethasone potently induced ob gene expression and leptin secretion in the organ culture of human adipose tissue. The data demonstrate that glucocorticoids act, at least in part, directly on the adipose tissue and increase leptin synthesis and secretion in humans.

Tumor necrosis factor receptors in the pituitary cells.

To clarify the site and mode of action of tumor necrosis factor (TNF) in the pituitary, we studied the effects, binding sites of TNF and its receptor mRNA in the two types of mouse pituitary-derived cell lines, AtT-20, ACTH-producing cells and TtT/GF, folliculo-stellate (FS)-like cells. First, we examined the expression of TNF receptor mRNA in these cells. Using Northern blot analyses with radiolabeled cDNA to murine TNF receptor p60 and p80 mRNAs as probes, we identified both types of mRNA in the poly(A)-containing RNA prepared from AtT-20 cells and p60 TNF receptor mRNA from TtT/GF. The identified mRNA were compatible in size with those detected in the immune-competent cells. Next, we studied the TNF-binding sites on these cells. Scatchard plot analysis of the significant binding of [125I]TNF revealed a single type of binding site with a Kd (dissociation constant) of 210 pM and 131 binding sites/cell on AtT-20. Similarly on TtT/GF, [125I]TNF showed 353 binding sites/cell with a Kd of 900 pM. [125I]TNF binding on both types of cells competed with TNF and lymphotoxin (TNF beta) in an equimolar fashion. Third, TNF stimulates ACTH synthesis in AtT-20 cells, while TNF increases immunoreactive interleukin (IL)-6 release from TtT/GF cells. These findings demonstrate that AtT-20 and TtT/GF cells are equipped with fully functional TNF receptor system, and suggest that ligand of the receptor, TNF alpha and/or TNF beta, can modulate ACTH synthesis and release as a direct hormonal effector on corticotrophs or indirect modulator through another paracrine mediator, such as IL-6 from FS cells.

Development of radioimmunoassay for human leptin.

Using recombinant human leptin, we have produced an antiserum for human leptin and developed a radioimmunoassay (RIA) specific and sensitive for human leptin. We detected leptin-like immunoreactivity (-LI) in culture media of adipose tissue from subcutaneous abdominal fat in human. The plasma leptin-LI concentration in nonobese subjects (17.6

Effect of IL-1 receptor antagonist and antiserum to TNF-alpha on LPS-induced plasma ACTH and corticosterone rise in rats.

Using an antiserum against tumor necrosis factor (TNF)-alpha and an interleukin (IL-1) receptor antagonist, we studied putative roles of these cytokines in mediating the endotoxin-induced elevation of plasma adrenocorticotropic hormone (ACTH) and corticosterone levels in freely moving rats. Intravenous administration of Escherichia coli lipopolysaccharide (LPS) increased plasma ACTH and corticosterone levels in a dose-dependent manner. The plasma corticosterone reached to its highest level among a series of experiments after the administration of even the smallest dose (0.03 microgram/kg) tested. Plasma ACTH and corticosterone levels in these rats were completely inhibited by the intravenous administration of anti-murine TNF-alpha-rabbit antiserum (anti-TNFAS) after the administration of LPS but not by the intravenous administration of IL-1 receptor antagonist (IL-1RA). On the other hand, both recombinant human IL-1RA and anti-TNFAS significantly inhibited plasma ACTH increase stimulated with 10 micrograms/kg LPS. These findings indicate that 1) when the plasma corticosterone increase induced by intravenous LPS remains below its maximum, the effect is exclusively mediated by TNF-alpha, and 2) when a larger amount of LPS is administered, both IL-1 beta and TNF-alpha participate at least in part in the hypothalamic-pituitary-adrenal axis activation.