Quantification of the alpha and beta subunits of the transducing elements (Gs and Gi) of adenylate cyclase in adipocyte membranes from lean and obese (ob/ob) mice.

The abundance of the alpha and beta subunits of the GTP-binding proteins (G-proteins) that transduce hormonal messages to adenylate cyclase was assessed in adipocyte membranes from lean (+/+) and obese (ob/ob) mice, using ADP-ribosylation with bacterial toxin and immunodetection. Both methods revealed two Gs alpha species (48 and 42 kDa) in the membranes. Compared with those of lean mice, the membranes from obese mice contained substantially less of the 48 kDa species of Gs alpha, as assessed by both methods. ADP-ribosylation by pertussis toxin showed that only half as much ADP-ribose was incorporated into Gi alpha in the membranes from obese as compared with lean mice. Immunodetection revealed two separate Gi alpha peptides (39 and 40 kDa) and showed that the 40 kDa species was less abundant in the membranes from obese mice, whereas the amount of the 39 kDa species was similar in membranes from both lean and obese animals. Based on ADP-ribosylation assays, in membranes from lean mice the ratio Gs alpha/Gi alpha was 1:16, whereas in the membranes from obese mice it was 1:10. Similar amounts of immunodetectable beta peptide were found in both types of membranes. On the basis of the currently accepted dissociation model of adenylate cyclase activation, the decrease in the abundance of the Gi alpha subunit in adipocyte membranes from obese mice could account for the abnormal kinetics of the enzyme in these membranes.     

Concurrent up-regulation of guanine-nucleotide-binding proteins Gi1 alpha, Gi2 alpha and Gi3 alpha in adipocytes of hypothyroid rats.

Rat white adipocytes express three distinct 'Gi-like' guanine-nucleotide-binding proteins (G-proteins) [Mitchell, Griffiths, Saggerson, Houslay, Knowler & Milligan (1989) Biochem. J. 262, 403-408]. We have previously noted elevated levels of Gi in membranes of adipocytes from hypothyroid rats [Milligan, Spiegel, Unson & Saggerson (1987) Biochem. J. 247, 223-227]. Using a series of anti-peptide antisera able to discriminate between the individual gene products we have examined levels of each Gi-like G-protein in adipocyte membranes of hypothyroid rats compared with euthyroid controls. We demonstrate that up-regulation of Gi in adipocytes of hypothyroid rats is not restricted to a single subtype of Gi but that each of Gi1 alpha, Gi2 alpha and Gi3 alpha is present at markedly higher levels compared with euthyroid animals. In contrast, levels of both the 45 and 42 kDa forms of Gs alpha were not altered substantially in the hypothyroid state.     

Presence of a functional inhibitory GTP-binding regulatory component, Gi, linked to adenylate cyclase in adipocytes of ob/ob mice

It has been reported recently (Begin-Heick, N. (1985) J. Biol. Chem. 260, 6187-6193) that adipocytes from the obese mouse strain (ob/ob), unlike normal mice (+/+), lack functional Gi, a GTP-regulated protein complex that mediates inhibition of adenylate cyclase. In contrast, we have found functional Gi linked to inhibition of adenylate cyclase in adipocyte membranes from both ob/ob and +/+ mice. This conclusion is based on observation of: 1) GTP-dependent inhibition of adenylate cyclase by antilipolytic agents, such as prostaglandin E2, nicotinic acid, and the adenosine receptor agonist, phenylisopropyladenosine (PIA); 2) classical biphasic GTP kinetics, with stimulation by low and inhibition by high concentrations of GTP; and 3) elimination of cyclase inhibition by antilipolytic agents upon treatment of ob/ob adipocytes with pertussis toxin. Upon treatment with pertussis toxin and [32P] NAD, purified adipocyte membranes from ob/ob mice incorporated twice as much radioactivity per unit membrane protein than those from +/+ mice in the 40,000-42,000 region. The inhibitory actions of PIA on adenylate cyclase were blocked by the adenosine receptor antagonists, theophylline and isobutylmethylxanthine. However, in contrast to other known inhibitory adenosine receptors, relatively high (100 nM) PIA concentrations were required for half-maximal inhibition of adenylate cyclases from both +/+ and ob/ob adipocytes. The adipocyte adenylate cyclase from both mouse strains were approximately equally susceptible to inhibition by nicotinic acid and prostaglandin E2. However, the ob/ob cyclase was inhibited by 47% with PIA, whereas the enzyme from the +/+ mouse was inhibited by only 27% (p less than 0.01). This greater inhibition by adenosine may contribute to abnormal fat metabolism in adipocytes from ob/ob mice.     

Cross-regulation between G-protein-mediated pathways. Activation of the inhibitory pathway of adenylylcylclase increases the expression of beta 2-adrenergic receptors.

Cross-regulation from the stimulatory (Gs alpha)-mediated) to the inhibitory (Gi alpha-mediated) pathways controlling adenylylcyclase has been described (Hadcock, J. R., Ros, M., Watkins, D. C., and Malbon, C. C. (1990) J. Biol. Chem. 265, 14784-14790). The extent to which cross-regulation occurs from inhibitory to stimulatory pathways for adenylylcyclase was explored. Persistent activation of the inhibitory pathway of adenylylcyclase by the A1-adenosine receptor agonist (-)-N6 (R-phenylisopropyl) adenosine (PIA) in hamster smooth muscle DDT1 MF-2 cells enhanced the stimulatory pathway of adenylylcyclase and its activation by the beta 2-adrenergic receptor agonist isoproterenol. PIA treatment (48 h) of cells increased isoproterenol-stimulated adenylylcyclase by 2-fold. In addition, the ED50 for stimulation of adenylylcyclase by isoproterenol decreased 50-fold to approximately 1 nM. Persistent activation of cells with PIA increased beta 2-adrenergic receptor number in a time- and dose-dependent manner. The steady-state levels of beta 2-adrenergic receptors (radioligand binding and immunoblotting) and receptor mRNA levels increased by more than 70%, while the half-life of the receptor (24 h) was unaltered. Both A1-adenosine receptor binding and Gi alpha 2 levels declined by half in cells persistently activated with PIA. Although Gi alpha 2 mRNA levels and the relative rate of synthesis of Gi alpha 2 protein upon persistent activation of the inhibitory pathway were found to increase, a decrease in the half-life of Gi alpha 2 from approximately 75 h in naive cells to approximately 40 in cells provides the basis for the decline in Gi alpha 2 levels. The steady-state level of mRNA and half-life of Gs alpha protein were unaltered in persistently activated cells. Thus, activation of the inhibitory pathway of adenylylcyclase cross-regulates the stimulatory, hormone-sensitive adenylylcyclase system by: (i) up-regulating beta 2-adrenergic receptors and enhancing the activation of the stimulatory adenylylcyclase pathway and (ii) down-regulating elements of the inhibitory adenylylcyclase pathway (Gi alpha 2 and A1-adenosine receptor binding).     

Gi down-regulation as a mechanism for heterologous desensitization in adipocytes.

Prolonged incubation of rat adipocytes with (-)N6-phenylisopropyl adenosine (PIA) (an A1 adenosine receptor agonist) leads to down-regulation of each of the three subtypes of Gi (Green, A., Johnson, J. L., and Milligan, G. (1990) J. Biol. Chem. 265, 5206-5210). To determine whether other inhibitors of adenylylcyclase would have similar actions, we incubated adipocytes in primary culture with PIA, prostaglandin E1 (PGE1), or nicotinic acid. After various times cells were homogenized, and crude membrane fractions were analyzed on Western blots using antipeptide antisera to alpha- and beta-subunits of G-proteins (SG1 (which binds to alpha i1 and alpha i2), I3B (which binds to alpha i3), BN2 (binds to beta-subunits) and CS1 (recognizes forms of alpha s)). PIA and PGE1 caused approximately 90% down-regulation of alpha i1 and alpha i3, and about 50% loss of alpha i2 and beta-subunits. In contrast, nicotinic acid at concentrations up to 1 mM had no effect on levels of any of these Gi subtypes. None of the compounds altered levels of either a 43- or 47-kDa form of alpha s. PIA caused about a 50% decrease in binding of [3H]DPCPX (an A1 adenosine receptor antagonist), indicating adenosine receptor down-regulation; however, neither PGE1 nor nicotinic acid treatment altered [3H]DPCPX binding. None of the treatments affected the activity of adenylylcyclase when measured in the presence of 100 microM forskolin and 10 mM Mn2+, indicating that the catalytic subunit of adenylylcyclase is not altered. To determine whether Gi down-regulation results in heterologous desensitization, we incubated adipocytes with maximally effective concentrations of PIA (300 nM), PGE1 (3 microM), or nicotinic acid (1 mM) for 4 days. The cells were then washed and incubated for an additional 30 min with various concentrations of these compounds to determine their ability to inhibit lipolysis. PIA caused a (marked) decrease in the sensitivity of the cells to both PIA and PGE1, thus indicating heterologous desensitization. Similarly, PGE1 decreased the sensitivity of the cells to both PGE1 and PIA, again demonstrating heterologous desensitization. In contrast, prolonged incubation with nicotinic acid decreased the sensitivity of the cells to nicotinic acid but had no effect on the sensitivity of the cells to PIA. Adenylylcyclase in membranes from PGE1-treated cells showed decreased sensitivity to inhibition by PIA. In contrast, adenylylcyclase showed normal sensitivity to PIA in membranes from nicotinic acid-treated cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chromosomal assignment of the murine Gi alpha and Gs alpha genes. Implications for the obese mouse

The G protein family of transmembrane signaling molecules includes Gs and Gi, the stimulatory and inhibitory regulators of adenylate cyclase. These and other characterized G proteins are comprised of beta, gamma, and alpha chains, the latter being the most variable among the proteins and thus serving to distinguish them. Previous results (Begin-Heick, N. (1985) J. Biol. Chem. 260, 6187-6193) suggested that the autosomal recessive mouse mutation obese (ob), which results in an abnormal response of adipose tissue to lipolytic hormones, is due to a defect in the gene coding for the alpha chain of Gi. In order to test this hypothesis we used a cloned cDNA probe representing murine Gi alpha mRNA in conjunction with a panel of Chinese hamster-mouse somatic cell hybrids segregating mouse chromosomes to map the Gi alpha gene in the mouse. In addition, we used a cDNA probe representing the murine Gs alpha gene to a specific mouse chromosome. Our results indicate that the Gi alpha locus maps to mouse chromosome 9, while Gs alpha is localized to region 2E1-2H3 of mouse chromosome 2. Localization of the Gi alpha gene to chromosome 9 excludes this gene as a site of the ob mutation, since the ob locus maps to chromosome 6. Furthermore, our findings indicate that certain members of the murine G protein alpha gene family have dispersed to different chromosomes since diverging from a common ancestral gene.     

Influence of genetic obesity in mice on the lipolytic response of isolated adipocytes to isoproterenol and ACTH-(1-24).

The lipolytic response of isolated adipocytes from genetic obese (C57/BL/64 ob/ob) and lean (C57BL/6J +/?) mice to ACTH-(1-24), isoproterenol and glucagon has been studied. The mean cell idameter of adipocytes form ob/ob mice was approximately twice that of lean controls. The adipocytes from obese mice contained on the average approximately six times the amount of triacylglycerol present in the smaller lean mouse adipocyte. Lipolysis was calculated both on a per cell basis (10(5) cells) and per mu mole of triacylglycerol and when expressed on a cell number basis, the larger adipocytes from obese mice showed an ACTH-(1-24) stimulated glycerol release which was quantitatively similar to that of smaller adipocytes from lean mice. When expressed per mu mole of triacylglycerol, the smaller cells from lean animals appeared to be dramatically more responsive to either isoproterenol or ACTH-(1-24). On either basis, ACTH-(1-24) stimulated glycerol release from obese mouse cells was greater than the isoproterenol response. The obese mouse of adipocyte showed selective loss of response to isoproterenol compared to its lean control.     

G-protein mRNA levels during adipocyte differentiation

G-protein-mediated transmembrane signaling in 3T3-L1 cells is modulated by differentiation. The regulation of G-protein expression in differentiating 3T3-L1 cells was probed at the level of mRNA by DNA-excess solution hybridization. Pertussis toxin-catalyzed ADP-ribosylation of G-protein alpha-subunits increased as fibroblasts differentiate to adipocytes. Steady-state levels of mRNA for Gi alpha 2 and Go alpha, in contrast, declined sharply. Immunoblotting with antipeptide antibodies specific for Gi alpha 2, too, revealed a decline in the steady-state expression of this pertussis toxin substrate. ADP-ribosylation of Gs alpha by cholera toxin was less in the adipocyte than fibroblast. Analysis by immunoblotting revealed only a modest decline in Gs alpha. Analysis of mRNA levels also demonstrated a decline for Gs alpha. mRNA levels for the G beta-subunits rose initially (25%) on day 1, declined from day 1 to day 3, and remained 25% lower in adipocytes than in fibroblasts. In 3T3-L1 adipocytes the molar amounts of subunit mRNAs were: 60.6 (Gs alpha); 2.1 (Gi alpha 2); and 1.5 (Go alpha) amol/microgram total cellular RNA. In rat fat cells these mRNA levels were 19.4 (Gs alpha); 7.0 (Gi alpha 2); and 2.3 (Go alpha). These data demonstrate that for Gi alpha 2 and Go alpha alike mRNA and protein expression decrease, not increase, in differentiation. A substrate for pertussis toxin other than Gi alpha 2 and Go alpha appears to be responsible for the increase in toxin-catalyzed labeling that accompanies differentiation of 3T3-L1 cells.     

Regulation of G-proteins in differentiation. Altered ratio of alpha- to beta-subunits in 3T3-L1 cells

The complexion of the adenylate cyclase system and in particular, the regulation of G-proteins was examined in 3T3-L1 cells during differentiation from a fibroblast-like to an adipocyte-like phenotype. Gs alpha (the identified regulatory component of hormone-sensitive adenylate cyclase that mediates stimulation), measured by cholera toxin-catalyzed ADP-ribosylation, increased by approximately 6-fold from day 0 to day 8. Gs alpha, measured by functional reconstitution, increased in specific activity by approximately 3-fold from day 0 to day 8. Both Gi alpha (the G-protein with alpha-subunit Mr 40,000-41,000 whose function is in part the mediation of inhibition of adenylate cyclase) and Go alpha (the highly abundant G-protein first isolated from bovine brain whose effector system remains to be established) measured by pertussis toxin-catalyzed ADP-ribosylation increased by approximately 4-fold over this same period. 3T3-L1 cells possess beta-subunits of G-proteins displaying Mr = 36,000 (beta 36) and Mr = 35,000 (beta 35). The increase in the beta 35 as well as beta 36 subunits was approximately 2-fold. Using quantitative immunoblotting techniques and specific antisera, the total amount of beta-subunits was determined to be 150 as compared to 70 pmol/mg of membrane protein, while the amount of Go alpha was 40 and 10 pmol/mg of membrane protein in adipocytes and fibroblasts, respectively. Since Go alpha is the most abundant G-protein alpha-subunit observed to date in both phenotypes, the overall ratio of beta- to alpha-subunits of G-proteins appears to decrease from approximately 4.7 in fibroblasts to 2.5 in adipocytes. These data suggest that in differentiation not only is the complexion of G-proteins altered but more importantly, the relative amounts of alpha- to beta-subunits are regulated.     

Increased lipolysis in transgenic animals overexpressing the epithelial fatty acid binding protein in adipose cells

Fatty acid binding proteins (FABPs) are low-molecular-mass, soluble, intracellular lipid carriers. Previous studies on adipocytes from adipocyte fatty acid binding protein (A-FABP)-deficient mice have revealed that both basal and isoproterenol-stimulated lipolysis were markedly reduced (Coe et al. 1999. J. Lipid Res. 40: 967-972). Herein, we report the construction of transgenic mice overexpressing the FABP5 gene encoding the epithelial fatty acid binding protein (E-FABP) in adipocytes, thereby allowing evaluation of the effects on lipolysis of increased FABP levels and of type specificity. In adipocytes from FABP5 transgenic mice, the total FABP protein level in the adipocyte was increased to 150% as compared to the wild type due to a 10-fold increase in the level of E-FABP and an unanticipated 2-fold down-regulation of the A-FABP. There were no significant differences in body weight, serum FFA, or fat pad mass between wild-type and FABP5 transgenic mice. Importantly, both basal and hormone-stimulated lipolysis increased in adipocytes from the FABP5 transgenic animals. The molecular composition of the fatty acid pool from either the intracellular compartment or that effluxed from the adipocyte was unaltered. These results demonstrate that there is a positive relationship between lipolysis and the total level of FABP but not between lipolysis and a specific FABP type.     

Effect of aging and cellularity on lipolysis in isolated mouse fat cells

The effects of age and cellularity on lipolysis have been investigated in isolated epididymal fat cells from both Swiss albino mice and Sprague-Dawley rats. No significant lipolytic response to glucagon could be demonstrated with adipocytes from either young or old mice, while glycerol output was increased by this hormone with fat cells from young rats. Larger adipocytes from older mice showed significantly greater isoproterenol-stimulated lipolysis than those from younger animals if the glycerol output was expressed on a per cell basis. However, the lipolytic response per cell appeared to be equivalent in young and old rat adipocytes with either isoproterenol or ACTH-(1-24). In a complete aging study, relationships between body weight, epididymal fat pad weight and cellularity were examined covering the life span of the mouse. ACTH-(1-24)- and dibutyryl cyclic AMP-stimulated lipolysis increased with age and cell size but fell at senescence when adipocyte size diminished. Although an effect of aging per se cannot be ruled out with the experimental techniques used in the present study, a dominant influence of adipocyte size on the lipolytic process was demonstrated.     

Catestatin (chromogranin A(352-372)) and novel effects on mobilization of fat from adipose tissue through regulation of adrenergic and leptin signaling

Chromogranin A knock-out (Chga-KO) mice display increased adiposity despite high levels of circulating catecholamines and leptin. Consistent with diet-induced obese mice, desensitization of leptin receptors caused by hyperleptinemia is believed to contribute to the obese phenotype of these KO mice. In contrast, obesity in ob/ob mice is caused by leptin deficiency. To characterize the metabolic phenotype, Chga-KO mice were treated with the CHGA-derived peptide catestatin (CST) that is deficient in these mice. CST treatment reduced fat depot size and increased lipolysis and fatty acid oxidation. In liver, CST enhanced oxidation of fatty acids as well as their assimilation into lipids, effects that are attributable to the up-regulation of genes promoting fatty acid oxidation (Cpt1α, Pparα, Acox, and Ucp2) and incorporation into lipids (Gpat and CD36). CST did not affect basal or isoproterenol-stimulated cAMP production in adipocytes but inhibited phospholipase C activation by the α-adrenergic receptor (AR) agonist phenylephrine, suggesting inhibition of α-AR signaling by CST. Indeed, CST mimicked the lipolytic effect of the α-AR blocker phentolamine on adipocytes. Moreover, CST reversed the hyperleptinemia of Chga-KO mice and improved leptin signaling as determined by phosphorylation of AMPK and Stat3. CST also improved peripheral leptin sensitivity in diet-induced obese mice. In ob/ob mice, CST enhanced leptin-induced signaling in adipose tissue. In conclusion, our results implicate CST in a novel pathway that promotes lipolysis and fatty acid oxidation by blocking α-AR signaling as well as by enhancing leptin receptor signaling.     

Cross-regulation between G-protein-mediated pathways. Stimulation of adenylyl cyclase increases expression of the inhibitory G-protein, Gi alpha 2

The hormone-sensitive adenylyl cyclase system is under dual control, receiving both stimulatory and inhibitory inputs. Guanine nucleotide-binding regulatory proteins (G-proteins) transduce signals from cell surface receptors to effectors such as adenylyl cyclase. Hormonal stimulation is propagated via Gs, inhibition by Gi. Persistent (24-h) activation of the stimulatory pathway of adenylyl cyclase by the diterpene forskolin or the beta-adrenergic agonist isoproterenol in S49 mouse lymphoma cells enhanced the effects of somatostatin mediated via the inhibitory pathway of adenylyl cyclase. Stimulating cells with forskolin or isoproterenol for 24 h resulted in a 3-fold increase in the steady-state levels of Gi alpha 2 and a 25% decline in Gs alpha, as quantified by immunoblotting. Within 12 h of stimulation of adenylyl cyclase, Gi alpha 2 mRNA levels increased 4-fold, measured by DNA-excess solution hybridization. Gs alpha mRNA levels, in contrast, increased initially (25%), but then declined to 75% of control. In S49 variants that lack functional protein kinase A (kin-), stimulation by isoproterenol failed to alter Gi alpha 2 expression at either the protein or the mRNA levels. A 3-fold increase in relative synthesis rate and no change in the half-life (approximately 80 h) of Gi alpha 2 was observed in response to forskolin stimulation. Although Gs alpha synthesis increased (70%) modestly in response to forskolin stimulation, the half-life of Gs alpha actually decreased from 55 h in naive cells to 34 h in treated cells. Thus, the two G-protein-mediated pathways controlling adenylyl cyclase display "cross-regulation." Persistent activation of the stimulatory pathway increases Gi alpha 2 mRNA and expression. Transiently elevated Gs alpha mRNA levels are counterbalanced by a reduction in the half-life of the protein.     

Effects of zinc supplementation on the plasma glucose level and insulin activity in genetically obese (ob/ob) mice

The effects of zinc supplementation (20 mM ZnCl₂ from the drinking water for eight weeks) on plasma glucose and insulin levels, as well as its in vitro effect on lipogenesis and lipolysis in adipocytes were studied in genetically obese (ob/ob) mice and their lean controls (+/?). Zinc supplementation reduced the fasting plasma glucose levels in both obese and lean mice by 21 and 25%, respectively (p < 0.05). Fasting plasma insulin levels were significantly decreased by 42% in obese mice after zinc treatment. In obese mice, zinc supplementation also attenuated the glycemic response by 34% after the glucose load. The insulin-like effect of zinc on lipogenesis in adipocytes was significantly increased by 80% in lean mice. However, the increment of 74% on lipogenesis in obese mice was observed only when the zinc plus insulin treatment was given. This study reveals that zinc supplementation alleviated the hyperglycemia of ob/ob mice, which may be related to its effect on the enhancement of insulin activity.     

G-protein subunit mRNA levels in rat heart, liver, and adipose tissues: analysis by DNA-excess solution hybridization

The steady-state levels of mRNAs for the G-proteins Gi alpha 2, Go alpha, and the G beta-subunits common to each were established in rat adipose, heart and liver. Uniformly-radiolabeled, single-stranded antisense probes were constructed from cDNAs or assembled from oligonucleotides. Direct comparison of the steady-state levels of the G-protein mRNAs was performed under identical assay conditions, and on a molar basis. In adipose, liver and heart, Gs alpha mRNA was more abundant than mRNA for Go alpha, Gi alpha, and G beta. In adipose tissue, mRNA levels were as follows: 19.4, 7.6, 7.0, and 2.3 amol mRNA per micrograms total cellular RNA for Gs alpha, G beta, Gi alpha 2, and Go alpha, respectively. In heart Gs alpha mRNA was less abundant than in adipose, but the relative trend among the G-protein subunits was the same. In liver, G beta mRNA was more abundant than either Go alpha or Gi alpha 2. Go alpha mRNA levels ranged from 1.2 to 2.3 amol/micrograms total RNA in liver and adipose, respectively. The present work demonstrates the many advantages of this strategy when applied to the study of a family of homologous, low-abundance proteins and establishes for the first time the molar levels of Gi alpha 2, Gs alpha, Go alpha, and G beta-subunit mRNAs in several mammalian tissues.     

Determination of G-protein levels, ADP-ribosylation by cholera and pertussis toxins and the regulation of adenylyl cyclase activity in liver plasma membranes from lean and genetically diabetic (db/db) mice

Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of Gi alpha-2, Gi alpha-3 and G-protein beta-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of Gs alpha-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of Gs alpha-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 microM), GTP (100 microM), p[NH]ppG (100 microM), NaF (10 mM) and glucagon (10 microM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 microM) was lower by about 22%. Lower concentrations (EC50-60 nM) of p[NH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC50-158 nM). As well as causing activation, p[NH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p[NH]ppG through a process suggested to be mediated by the abolition of functional Gi activity. The lower levels of expression of G-protein beta-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p[NH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals.     

Effects of obesity on the relationship of leptin mRNA expression and adipocyte size in anatomically distinct fat depots in mice

In support of leptin's physiological role as humoral signal of fat mass, we have shown that adipocyte volume is a predominant determinant of leptin mRNA levels in anatomically distinct fat depots in lean young mice in the postabsorptive state. In this report, we investigated how obesity may affect the relationship between leptin mRNA levels and adipocyte volume in anatomically distinct fat depots in mice with genetic (Lep(ob)/Lep(ob) and A(y)/+), diet-induced, and aging-related obesity. In all of the obese mice examined, tissue leptin mRNA levels relative to the average adipocyte volume were lower in the perigonadal and/or retroperitoneal than in the inguinal fat depots and were lower than those of the lean young mice in the perigonadal fat depot. A close, positive correlation between leptin mRNA level and adipocyte volume was present from small to hypertrophic adipocytes within each perigonadal and inguinal fat pad in the obese mice, but the slopes of the regression lines relating leptin mRNA level to adipocyte volume were significantly lower in the perigonadal than in the inguinal fat pads of the same mice. These results suggest that obesity per se is associated with a decreased leptin gene expression per unit of fat mass in mice and that the positive correlation between leptin mRNA level and adipocyte volume is an intrinsic property of adipocytes that is not disrupted by adipocyte hypertrophy in obese mice.     

Tumor necrosis factor alpha stimulates lipolysis in adipocytes by decreasing Gi protein concentrations

Prolonged treatment (12-24 h) of adipocytes with tumor necrosis factor alpha (TNFalpha) stimulates lipolysis. We have investigated the hypothesis that TNFalpha stimulates lipolysis by blocking the action of endogenous adenosine. Adipocytes were incubated for 48 h with TNFalpha, 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 TNFalpha-treated cells, but with adenosine deaminase lipolysis increased in the controls to approximately that in the TNFalpha-treated cells. This suggests that TNFalpha 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 alpha-subunits of all three Gi subtypes was detected by Western blotting without a change in Gs proteins or beta-subunits. Gi2alpha was about 50% of control, whereas Gi1alpha and Gi3alpha were about 20 and 40% of control values, respectively. The time course of Gi down-regulation correlated with the stimulation of lipolysis. Furthermore, down-regulation of Gi by an alternative approach (prolonged incubation with N6-phenylisopropyl adenosine) stimulated lipolysis. These findings indicate that TNFalpha stimulates lipolysis by blunting endogenous inhibition of lipolysis. The mechanism appears to be a Gi protein down-regulation.     

Evidence for altered expression of the GTP-dependent regulatory proteins, Gs and Gi, in adipocytes from aged rats.

The alpha-subunits of Gi and Gs were quantified in adipocyte membranes from young (2-month) and older (18-month) rats by pertussis-toxin and cholera-toxin labelling respectively. Aging was associated with a 3-fold increase in Gi alpha-subunit, but only a 2-fold increase in one of the two Gs alpha-subunit species labelled. The findings may explain the altered sensitivity of adipocytes from aged rats to lipolytic and anti-lipolytic stimuli.     

Differences in lipogenesis and lipolysis in obese and non-obese adult human adipocytes

It has been proposed that differences in adipocyte function and/or metabolism between obese and lean individuals may manifest themselves in functional adipose tissue abnormalities that lead to metabolic disorders in obesity. We studied lipogenesis and lipolysis of omental adipocytes from obese (OB) and non-obese (NOB) humans. The specific activity of the lipogenic marker enzyme G3PDH was 50% lower in total adipocytes of OB compared to that of NOB subjects. Omental adipocytes from OB subjects also had lower basal lipolytic levels, and a lower lipolytic response to beta-adrenergic stimulus. Cholesterol depletion of adipocyte plasma membrane using methyl b-cyclodextrin caused a lipolytic effect on adipocytes of both groups together, but when obese and lean subjects were analyzed separately, the response was significant only in the obese. We present evidence of a different lipogenic and lipolytic profile in obese individuals' omental adipocytes, and propose a relevant role of plasma membrane cholesterol, where the impact of its removal in OB and NOB adipocyte lipolysis differs.