Allan Green

Tumor Necrosis Factor α Stimulates Lipolysis in Adipocytes by Decreasing Gi Protein Concentrations

Prolonged treatment (12–24 h) of adipocytes with tumor necrosis factor α (TNFα) stimulates lipolysis. We have investigated the hypothesis that TNFα stimulates lipolysis by blocking the action of endogenous adenosine. Adipocytes were incubated for 48 h with TNFα, and lipolysis was measured in the absence or presence of adenosine deaminase. Without adenosine deaminase, the rate of glycerol release was 2–3-fold higher in the TNFα-treated cells, but with adenosine deaminase lipolysis increased in the controls to approximately that in the TNFα-treated cells. This suggests that TNFα blocks adenosine release or prevents its antilipolytic effect. Both N6 -phenylisopropyl adenosine and nicotinic acid were less potent and efficacious inhibitors of lipolysis in treated cells. A decrease in the concentration of α-subunits of all three Gi subtypes was detected by Western blotting without a change in Gsproteins or β-subunits. Gi2α was about 50% of control, whereas Gi1α and Gi3α were about 20 and 40% of control values, respectively. The time course of Gidown-regulation correlated with the stimulation of lipolysis. Furthermore, down-regulation of Gi by an alternative approach (prolonged incubation withN6 -phenylisopropyl adenosine) stimulated lipolysis. These findings indicate that TNFα stimulates lipolysis by blunting endogenous inhibition of lipolysis. The mechanism appears to be a Gi protein down-regulation.

Troglitazone inhibits progesterone production in porcine granulosa cells

Troglitazone (a thiazolidinedione that improves insulin resistance) lowers elevated androgen concentrations in women with polycystic ovarian syndrome. In this study, we assessed the direct effects of troglitazone on steroidogenesis in porcine granulosa cells. Troglitazone inhibited progesterone production in a doseand time-dependent manner (earliest effects at 4 h, maximum at 24 h) without affecting cell viability. Progesterone production was also inhibited by troglitazone in the presence of 25-hydroxycholesterol, indicating that the drug does not affect intracellular cholesterol transport. Troglitazone also inhibited FSHand forskolin-stimulated progesterone secretion. The reduced progesterone production was accompanied by marked elevations of pregnenolone concentrations, suggesting inhibition of 3b-hydroxysteroid dehydrogenase (3b-HSD). The activity of 3b-HSD in troglitazone-treated granulosa cells was decreased by more than 60%, compared with controls after 24 h. Troglitazone did not affect aromatase activity in porcine granulosa cells. In summary, troglitazone has direct effects on porcine granulosa cell steroidogenesis. The drug specifically inhibits 3b-HSD activity, resulting in impaired progesterone production. The clinical relevance of this direct in vitro effect on steroidogenesis needs further investigation. (Endocrinology 139: 4962–4966, 1998) T represent a new class of drugs that have been demonstrated to significantly improve the treatment of type 2 diabetes mellitus (1, 2). Thiazolidinediones act primarily at adipose and muscle tissue, where they increase insulin sensitivity at the postreceptor level (3). This improvement in insulin resistance is the basis for their use in diabetes. Thiazolidinediones are high-affinity ligands for the peroxisome-proliferator activated receptor g (4). Thiazolidinediones bind to and activate peroxisome-proliferator activated receptor g that forms a heterodimer with retinoic acid receptor and binds to an orphan nuclear receptor-binding motif [direct repeat one (DR-1)] in gene promoters (5). By acting at the transcriptional level, thiazolidinediones selectively increase the expression of genes that regulate glucose homeostasis (6), and they decrease the expression of genes that oppose insulin action (7). These agents also stimulate adipocyte differentiation from preadipocyte fibroblasts (8) and counteract negative effects of some cytokines on glucose and lipid metabolism (9). Because thiazolidinediones improve peripheral insulin resistance and decrease hyperinsulinemia, they may also be used for the treatment of several disorders other than type 2 diabetes. One such disease is polycystic ovarian disease (PCO), where insulin resistance in peripheral tissues and resulting hyperinsulinemia are associated with increased androgen concentrations and oligomenorrhea (10). The thiazolidinedione troglitazone, when used for the treatment of PCO, improved insulin resistance and lowered androgen concentrations (11, 12). This beneficial effect of troglitazone was attributed to its lowering of serum insulin concentrations that are proposed to hyperstimulate the ovary (11, 12). Another possibility is that troglitazone affects ovarian function by direct effects on steroidogenesis. In this study, we used porcine granulosa cells in primary culture to investigate the in vitro effects of troglitazone on progesterone production. We found that troglitazone inhibits progesterone production in granulosa cells by inhibiting the activity of 3bhydroxysteroid dehydrogenase (3b-HSD). Materials and Methods

Evidence for Impaired Coupling of Receptors to Gi Protein in Adipocytes From Streptozocin-Induced Diabetic Rats

Adenosine and prostaglandins of the E series inhibit lipolysis in adipocytes by binding to cell surface receptors. This inhibition is mediated via Gi. It has been reported that Gi is almost absent in livers from diabetic rats. Therefore, we have evaluated the sensitivity of adipocytes from diabetic rats to the adenosine analogue N6-phenylisopropyl adenosine (PIA) and to prostaglandin E1 (PGE1). Diabetes was induced with streptozocin (65 mg/kg i.v.), and after 7 days, adipocytes were isolated. Lipolysis (measured in the presence of adenosine deaminase) was inhibited by PIA and PGE, in both control and diabetic cells. However, the dose-response curves were markedly shifted to the right in the cells from diabetic rats. The IC50 for PIA was 0.30 ± 0.02 nM in controls and 0.83 ± 0.08 in diabetic rats (P < 0.001), and the IC50 for PGE, was 3.16 ± 0.18 nM in controls and 5.26 ± 0.57 nM in diabetic rats (P < 0.02). These findings indicate decreased sensitivity to both adenosine and PGE1. Adipocyte membranes were isolated from control and diabetic rats. Adenosine receptors (measured by binding of 125I-labeled hydroxy-PIA) were not altered in cells from diabetic rats. However, the ability of Gpp(NH)p (a nonhydrolyzable GTP analogue) to inhibit adenosine-receptor binding was markedly decreased in membranes from diabetic rats, suggesting a change at the level of G1. The α-subunits of G11, G12, G13, and Gs were quantitated on Western blots with a series of recently characterized anti-peptide antisera. This revealed that the amounts of each of these G proteins were normal in membranes from the diabetic rats. These findings suggest that there is a functional but not quantitative abnormality in G1 in adipocytes from diabetic rats that results in abnormal coupling of both A1-adenosine receptors and prostaglandin receptors and would explain the decreased sensitivity to PIA and PGE1.

Gi down-regulation and heterologous desensitization in adipocytes after treatment with the α2-agonist UK 14304

Prolonged treatment of rat adipocytes with the A1-adenosine receptor agonist [-]N(6)-phenylisopropyl adenosine (PIA) or prostaglandin E1 down-regulates Gi and induces heterologous desensitization. alpha 2-Adrenergic receptors also inhibit adenylyl cyclase through Gi, but whether alpha 2-receptors are present on rat adipocytes has been controversial. We have investigated the effects of the highly specific alpha 2-adrenergic agonist UK 14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) on rat adipocytes. In previous studies on young rats, we were unable to demonstrate an effect of the alpha 2-agonist. We now report that, in cells isolated from older, more obese rats (300-400 g), UK 14304 inhibited lipolysis (measured as the rate of glycerol release) by approximately 40% (EC50 approximately 40 nM). To determine whether UK 14304 would induce heterologous desensitization, we incubated adipocytes with or without 1 microM UK 14304 for 4 days in primary culture. The cells were then washed, and the rate of lipolysis was determined during a 30-min incubation in the presence of various concentrations of PIA. The concentration-response curve for PIA-induced inhibition of lipolysis was shifted to the right, with the EC50 for UK 14304-treated cells about 2-fold higher than in the control cells. This finding demonstrates that the alpha 2-agonist can desensitize the response to PIA and indicates heterologous desensitization. To investigate the mechanism of this phenomenon, we isolated crude membrane fractions from the cells and analyzed them on Western blots using antibodies against Gi alpha 1, 2 and 3. In cells treated with UK 14304 for 4 days, Gi1 alpha and Gi2 alpha were down-regulated to about 15% of the control level, and Gi3 alpha was decreased to 30% of control. We conclude that prolonged treatment of adipocytes with the alpha 2-agonist induces heterologous desensitization of lipolysis and causes down-regulation of Gi. The findings suggest that G-protein down-regulation is a mechanism for heterologous desensitization.

High affinity antibody from hen's eggs directed against the human insulin receptor and the human IGF-I receptor☆

Large quantities of high affinity antibodies directed against the human insulin receptor and the human insulin-like growth factor-I (IGF-I) receptor were obtained from hens eggs. Hens were immunized with human placental membranes and human liver membranes by intramuscular injections. Specific antibodies to the receptors were demonstrated in serum and egg yolks at 5 weeks and these antibodies presisted for at least 6 months. Antibodies from egg yolks were purified by the polyethylene glycol precipitation technique of Polson et al. The eggs provided the equivalent of about 450 ml of immunized serum per month per bird. The purified antibodies were approximately equally reactive with receptors for insulin or IGF-I. Antibodies immunoprecipitated affinity-labeled receptors, inhibited binding of each ligand, and were capable of stimulating 2-deoxyglucose uptake in rat adipocytes and thymidine incorporation in cultured fibroblasts. The presence of antibodies directed against the IGF-I receptor in those hens immunized with human liver membranes was unexpected, since liver membranes possess little or no IGF-I binding activity. We conclude that antibodies against human antigens may be relatively easily obtained by immunization of hens and purification of those antibodies from eggs.

Characterization of human adipocyte adenosine receptors

125I-Hydroxyphenylisopropyl adenosine (125I-HPIA) was used to characterize adenosine receptors in human adipocyte plasma membranes. Steady state binding was achieved after 6 h at 37 degrees. Scatchard plots were linear, with a KD of approx. 2.5 nM, and Bmax of 360-1800 fmol/mg protein. (-)N6-phenylisopropyl adenosine (PIA) was a more potent inhibitor of binding than N-ethyl carboxamido adenosine, and (+)PIA was more than 10-fold less potent than (-)PIA, consistent with A1 adenosine receptor binding. Theophylline was a potent inhibitor of binding (IC50 approx. 10 microM). Photoaffinity cross-linking studies demonstrated that the receptor is a single subunit, Mr approx. 43 kDa. The findings demonstrate that the human adipocyte adenosine receptor is similar to the A1 adenosine receptor of rat adipocytes, although its molecular weight is higher, and its affinity for HPIA is lower than that of the rat.

Photochemical cross-linking of 125I-hydroxyphenylisopropyl adenosine to the A1 adenosine receptor of rat adipocytes

Rat adipocyte plasma membranes were incubated with the A1 adenosine receptor agonist, 125I‐hydroxyphenylisopropyl adenosine (1 nM) and then treated with the photoactive cross‐linking agent, ANB‐NOS. The membranes were solubilized and analyzed by SDS‐PAGE and autoradiography. A single protein, M r approx. 38 000, was specifically labeled. Reduction with 2‐mercaptoethanol did not affect the apparent M r of the labeled protein. Labeling was inhibited by the adenosine receptor agonists, HPIA, PIA and NECA, and by the antagonist, theophylline, but was unaffected by inosine. We conclude that the A1 adenosine receptor is a single protein of M r approx. 38 000.