guanosine-5--o-(3-thiotriphosphate) and Obesity

The endocannabinoid system plays a crucial role in the pathophysiology of obesity. However, the clinical use of cannabinoid antagonists has been recently stopped because of its central side-effects. The aim of this study was to compare the effects of a chronic treatment with the CB(1) cannabinoid antagonist rimonabant or the CB(1) inverse agonist taranabant in diet-induced obese female rats to clarify the biological consequences of CB(1) blockade at central and peripheral levels. As expected, chronic treatment with rimonabant and taranabant reduced body weight and fat content. Interestingly, a decrease in the number of CB(1) receptors and its functional activity was observed in all the brain areas investigated after chronic taranabant treatment in both lean and obese rats. In contrast, chronic treatment with rimonabant did not modify the density of CB(1) cannabinoid receptor binding, and decreased its functional activity to a lower degree than taranabant. Six weeks after rimonabant and taranabant withdrawal, CB(1) receptor density and activity recovered to basal levels. These results reveal differential adaptive changes in CB(1) cannabinoid receptors after chronic treatment with rimonabant and taranabant that could be related to the central side-effects reported with the use of these cannabinoid antagonists.

Topics: Amides; Analysis of Variance; Animals; Autoradiography; Benzoxazines; Body Weight; Brain; Cyclohexanols; Diet Fads; Disease Models, Animal; Eating; Female; Guanosine 5'-O-(3-Thiotriphosphate); International Cooperation; Morpholines; Naphthalenes; Obesity; Piperidines; Protein Binding; Pyrazoles; Pyridines; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Rimonabant; Sulfur Isotopes; Time Factors; Tomography Scanners, X-Ray Computed; Tritium; Whole Body Imaging

Reactive oxygen species play a key role in pathophysiology of cardiovascular diseases by modulating G-protein-coupled receptor signaling. We have shown that treatment of animal models of diabetes and aging with tempol decreases oxidative stress and restores renal dopamine D1 receptor (D1R) function. In present study, we determined whether oxidation of D1R and upregulation of mitogen-activated protein kinases (MAPK) were responsible for decreased D1R signaling in obese animals. Male lean and obese Zucker rats were supplemented with antioxidants tempol or lipoic acid for 2 weeks. Compared to lean, obese animals were hyperglycemic and hyperinsulinemic with increased oxidative stress, D1R oxidation and decreased glutathione levels. These animals had decreased renal D1R affinity and basal coupling to G-proteins. SKF-38393, a D1R agonist failed to stimulate G-proteins and adenylyl cyclase. Obese animals showed marked increase in renal MAPK activities. Treatment of obese rats with tempol or lipoic acid decreased blood glucose, reduced oxidative stress, and restored the basal D1R G-protein coupling. Antioxidants also normalized MAPK activities and restored D1R affinity and SKF-38393 induced D1R G-protein coupling and adenylyl cyclase stimulation. These studies show that D1R oxidation and MAPK upregulation contribute to D1R dysfunction in obese animals. Consequently, antioxidants while reducing the oxidative stress normalize the MAPK activities and restore D1R signaling.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Animals; Antioxidants; Blood Glucose; Cyclic AMP; Cyclic N-Oxides; Dopamine Agonists; GTP-Binding Protein alpha Subunits, Gs; Guanosine 5'-O-(3-Thiotriphosphate); Immunoprecipitation; Kidney; Male; Mitogen-Activated Protein Kinases; Obesity; Oxidative Stress; Rats; Rats, Zucker; Receptors, Dopamine D1; Spin Labels; Thioctic Acid; Up-Regulation

The dopamine D1 receptors (D1R), expressed in renal proximal tubules, participate in the regulation of sodium transport. A defect in the coupling of the D1R to its G protein/effector complex in renal tubules has been reported in various conditions associated with oxidative stress. Because G protein-coupled receptor kinases (GRKs) are known to play an important role in D1R desensitization, we tested the hypothesis that increased oxidative stress in obese Zucker rats may cause GRK2 upregulation and, subsequently, D1R dysfunction. Lean and obese rats were given normal diet or diet supplemented with antioxidant lipoic acid for 2 wk. Compared with lean rats, obese rats exhibited oxidative stress, D1R were uncoupled from G(q/11)alpha at basal level, and SKF-38393 failed to elicit D1R-G protein coupling, stimulate phospholipase C (PLC), and inhibit Na-K-ATPase activity. These animals showed increased basal protein kinase C (PKC) activity and membranous translocation of GRK2 and increased GKR2-G(q/11)alpha interaction and D1R serine phosphorylation. Enzymatic dephosphorylation of D1R restored SKF-38393-induced adenylyl cyclase stimulation but not PLC activation. Treatment of obese rats with lipoic acid restored D1R-G protein coupling and SKF-38393-induced PLC stimulation and Na-K-ATPase inhibition. Lipoic acid treatment also normalized PKC activity, GRK2 sequestration, and GKR2-G(q/11)alpha interaction. In conclusion, these data show that oxidative stress increases PKC activity causing GRK2 membranous translocation. GRK2 interacts with G(q/11)alpha and acts, at least in part, as a regulator of G protein signaling leading to the D1R-G(q/11)alpha uncoupling, causing inability of SKF-38393 to stimulate PLC and inhibit Na/K-ATPase. Lipoic acid, while reducing oxidative stress, normalized PKC activity and restored D1R-G(q/11)alpha-PLC signaling and the ability of SKF-38393 to inhibit Na-K-ATPase activity.

Topics: Alkaline Phosphatase; Animals; Antioxidants; beta-Adrenergic Receptor Kinases; Blotting, Western; Diet; Dopamine Agonists; Dopamine Antagonists; Enzyme Inhibitors; G-Protein-Coupled Receptor Kinase 2; GTP-Binding Protein alpha Subunits, Gq-G11; Guanosine 5'-O-(3-Thiotriphosphate); Kidney Tubules, Proximal; Male; Obesity; Oxidative Stress; Phosphatidylinositol 4,5-Diphosphate; Protein Kinase C; Rats; Rats, Zucker; Receptors, Dopamine D1; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Thioctic Acid; Type C Phospholipases

alpha-Melanocyte stimulating hormone (MSH) has generally been assumed to be the endogenous ligand acting at the melanocortin-4 receptor (MC4-R), activation of which in the hypothalamus leads to reduced feeding. However, beta-MSH is also capable of activating MC4-R and inhibiting feeding. Here, we investigated the possibility that beta-MSH acts as an endogenous MC4-R agonist and that this melanocortin peptide plays a role in the regulation of feeding and energy balance. We found that beta-MSH had significantly higher affinities than alpha-MSH at both human MC4-R transfected into CHO cells (K(i): beta-MSH, 11.4+/-0.4 nmol/l versus alpha-MSH, 324+/-16 nmol/l, P<0.001) and MC4-R in rat hypothalamic homogenates (K(i): beta-MSH, 5.0+/-0.4 nmol/l versus alpha-MSH, 22.5+/-2.3 nmol/l, P<0.001). Incubation of brain slices with 5 microM beta-MSH significantly increased [35S]GTPgammaS binding by 140-160% (P<0.001), indicating activation of G-protein-coupled receptors (GPCRs), in the hypothalamic ventromedial (VMH), dorsomedial (DMH), arcuate (ARC) and paraventricular (PVN) nuclei. These sites match the distribution of beta-MSH immunoreactive fibres and also the distribution of MC4-R binding sites which we and others previously reported. Food-restriction significantly increased beta-MSH levels in the VMH, DMH and ARC (all P<0.05) above freely-fed controls, whilst alpha-MSH concentrations were unchanged. We propose that increased beta-MSH concentrations reflect blockade of the peptide's release in these sites, consistent with the increased hunger and the known up-regulation of MC4-R in the same nuclei. Thus, we conclude that (1). beta-MSH has higher affinity at MC4-R than alpha-MSH; (2). beta-MSH activates GPCR in these sites, which are rich in MC4-R; and (3). beta-MSH is present in hypothalamic nuclei that regulate feeding and its concentrations alter with nutritional state. We suggest that beta-MSH rather than alpha-MSH is the key ligand at the MC4-R populations that regulate feeding, and that inhibition of tonic release of beta-MSH is one mechanism contributing to hunger in under-feeding.

Topics: Animals; beta-MSH; CHO Cells; Cricetinae; Energy Metabolism; Food Deprivation; Guanosine 5'-O-(3-Thiotriphosphate); Homeostasis; Humans; Hypothalamus; Insulin; Ligands; Male; Obesity; Rats; Rats, Wistar; Receptor, Melanocortin, Type 4

In fa/fa Zucker rats, leptin receptor deficiency is responsible for both a deficit of energy expenditure and hyperphagia which lead to massive obesity and insulin resistance in adulthood. This obesity is also characterised by alterations of the beta-adrenergic signaling pathway.. To determine whether alterations in beta-adrenergic pathway could occur at the onset of obesity when fa/fa rats are not yet hyperinsulinemic.. Fourteen-day-old suckling fa/fa and Fa/fa littermates (from heterozygous lean (Fa/fa) female and homozygous obese (fa/fa) male mating).. Membranes were prepared from isolated adipocytes after collagenase treatment of inguinal adipose tissue. The response of adenylyl-cyclase activity to stimulation by isoprenaline, GTPgamma-S or forskolin was studied. Bmax and Kd of (beta1+beta2) and of beta3 adrenoceptors were measured using 3H-CGP saturation binding experiments. mRNA concentration of beta1- and beta3-AR was determined by semi-quantitative RT-PCR. G(s)alpha protein was quantified by Western blotting and Gi protein by ADP-ribosylation.. Despite an almost normal body weight, inguinal fat pad weight was increased two-fold by the expression of fa mutation. This increase was entirely accounted for by fat cell hypertrophy (x2.5 in volume). In fa/fa compared to Fa/fa pups, response of adenylyl cyclase to isoprenaline was decreased two-fold but responses to GTPgammaS or forskolin were unchanged. Density of (beta1+beta2) and beta3-AR was not affected by the fa/fa genotype, as well as G(s)alpha and Gi concentration.. Response of inguinal fat cells to catecholamines was decreased without any quantitative modifications of the different elements of the adenylyl cyclase cascade. This suggests an alteration in the coupling between beta-AR and G proteins. Due to the important increase in fat cell volume we hypothesize that changes in the physical properties of plasma membranes and/or changes in cytoskeleton-extracellular-matrix interactions could disturb the beta-adrenergic pathway responsiveness. In addition to the excess of lipid storage, which occurs very early at the onset of obesity, the impairment of the responsiveness to catecholamines reported in this study might worsen the obesity syndrome.

Topics: Adenylyl Cyclases; Adipocytes; Animals; Animals, Newborn; Blotting, Western; Catecholamines; Colforsin; Female; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gs; Guanosine 5'-O-(3-Thiotriphosphate); Isoproterenol; Male; Obesity; Rats; Rats, Zucker; Receptors, Adrenergic, beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction

It is reported that dopamine promotes renal sodium excretion via activation of D1-like dopamine receptors located on the proximal tubules. In spontaneously hypertensive rats the natriuretic and diuretic response to exogenously administered and endogenously produced dopamine is reduced, which results from a diminished dopamine-induced inhibition of the enzyme, Na+,K+-ATPase. The present study was designed to examine dopamine-receptor mediated inhibition of Na+,K+-ATPase and its associated signal transduction pathway in the proximal tubules of Zucker obese and lean control rats. The obese animals were hypertensive, hyperinsulinaemic and hyperglycaemic compared with the lean rats. While dopamine caused inhibition of Na+,K+-ATPase activity in lean rats, this effect was significantly attenuated in the obese animals. There was significant reduction in D1-like receptor numbers in the basolateral membranes of obese rats compared with lean rats with no change in the affinity to the ligand [3H]SCH 23390 between the two groups of rats. Dopamine failed to stimulate G proteins as measured by [35S]GTPgammaS binding in the obese rats. Also, dopamine was unable to cause phospholipase-C activation in obese rats, but it did activate phospholipase-C in lean rats. These results show that reduction in D1-like receptor numbers and a defect in receptor-G protein coupling may account for the inability of dopamine to activate the D1-like receptor-coupled signal transduction pathway and cause inhibition of Na+,K+-ATPase in the obese hypertensive rats.

Topics: Animals; Benzazepines; Dopamine Antagonists; Guanosine 5'-O-(3-Thiotriphosphate); Hypertension; Inositol 1,4,5-Trisphosphate; Kidney; Male; Obesity; Rats; Rats, Zucker; Receptors, Dopamine D1; Reference Values; Signal Transduction; Sodium-Potassium-Exchanging ATPase

Some of the pathophysiological consequences of obesity include insulin resistance, increased renal sodium reabsorption, and the development of hypertension. Dopamine promotes renal sodium excretion via activation of D(1)-like receptors present on the proximal tubules. Reduced dopamine-induced natriuresis and a defect in D(1)-like receptor function have been reported in the proximal tubules of hypertensive animals. The present study investigated D(1)-like dopamine receptors and associated G proteins as the initial signaling components in the proximal tubular basolateral membranes of obese Zucker and control lean Zucker rats. We found that the obese rats were hyperinsulinemic, hyperglycemic, and hypertensive compared with the lean rats. Dopamine produced concentration-dependent inhibition of Na,K-ATPase activity in the proximal tubules of lean rats, whereas the inhibitory effect of dopamine was reduced in obese rats. The D(1)-like receptors measured by [(3)H]SCH 23390 binding revealed an approximately 45% decrease in B(max) without a change in K(d) in the basolateral membranes of obese rats compared with lean rats. Although we found an increase in G(q)/11alpha and no change in G(s)alpha in the basolateral membranes of obese rats, dopamine and SKF 38393 failed to stimulate G proteins as measured by [(35)S]GTPgammaS binding in obese rats, suggesting a receptor-G protein coupling defect. We conclude that decrease in D(1)-like dopamine receptor binding sites and diminished activation of G proteins, resulting perhaps from defective coupling, led to the reduced inhibition by dopamine of Na,K-ATPase activity in the proximal tubules of obese Zucker rats. Such a defect in renal dopamine receptor function may contribute to sodium retention and development of hypertension in obese rats.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Animals; Benzazepines; Dopamine; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Kidney Tubules, Proximal; Male; Obesity; Rats; Rats, Zucker; Receptors, Dopamine D1; Sodium-Potassium-Exchanging ATPase

This study was undertaken to determine whether receptor and non-receptor components of the adenylate cyclase (AC) cascade were altered in brown adipose tissue (BAT) of 14-day-old pre-obese (fa/fa) rats, before endocrine status is strongly modified by fa gene expression. Activity of the AC catalytic subunit did not differ between the two genotypes. In fa/fa rats compared with control Fa/fa rats, there was a 50% decrease in the activity of alpha Gs (stimulated by NaF or guanosine 5'-[gamma-thio]triphosphate) but no change in protein content (Western blotting). alpha Gi function, assessed by the inhibitory action of low concentrations of guanosine 5'-[beta gamma-imido]triphosphate upon 10(-4) M forskolin-stimulated AC activity, was equally low in both genotypes. Analysis of dose-response curves for different beta-agonists revealed that (i) both the basal and the maximally stimulated activity of AC were 2-fold lower in fa/fa rats than in Fa/fa rats; (ii) BRL37344 and CGP12177 (beta 3 agonists) were less potent in fa/fa than in Fa/fa rats (Kact. multiplied by 2); (iii) noradrenaline and isoprenaline (Iso), at the low-affinity site (beta 3-AR), were less potent in fa/fa than in Fa/fa pups (Kact. increased by 30 and 20% respectively). At the high-affinity site (mainly beta 1) these two agonists were more potent in fa/fa than in Fa/fa rats (Kact. decreased by 40 and 80% respectively). In good agreement with the latter result, the beta 1-adrenergic receptor (beta 1-AR)-selective antagonist CGP20712A had more effect on the Iso-stimulated AC activity in pre-obese than in lean pups (2-fold decreased in IC50). Binding experiments with [3H]CGP12177 show that in BAT of suckling rats, beta 3-ARs represent 80% of the total beta-ARs. Bmax values for the two sites were not affected by the genotype, although the beta 3-AR mRNA concentration in BAT (quantitative reverse-transcriptase PCR) was 3-fold lower in fa/fa rats than in Fa/fa pups. In conclusion, these results provide evidence for alterations in beta 1- and beta 3-AR signalling in BAT of 14-day-old suckling pre-obese Zucker rats with a decreased activity of alpha Gs. The impaired AC responsiveness to catecholamines might be a primary contributor to the development of this genetic obesity.

Topics: Adenylyl Cyclases; Adipose Tissue, Brown; Adrenergic beta-Antagonists; Animals; Animals, Suckling; Base Sequence; Female; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanylyl Imidodiphosphate; Imidazoles; Isoproterenol; Male; Molecular Sequence Data; Norepinephrine; Obesity; Rats; Rats, Zucker; Receptors, Adrenergic, beta; RNA, Messenger

A novel pathway for physiological "cross-talk" between the insulin receptor and the regulatory Gi-protein has been demonstrated. We tested the hypothesis that a coupling defect between Gi and the insulin receptor is present in the liver of obese patients with and without type II diabetes. Insulin 1 x 10(-9) M (approximately ED50) and 1 x 10(-7) M (Max) inhibited pertussis toxin-catalyzed ADP ribosylation of Gi in human liver plasma membranes from lean and obese nondiabetic patients. However, 1 x 10(-7) M insulin was without effect in membranes from patients with type II diabetes. This coupling defect was not intrinsic to Gi, since Mg2+ and GTP gamma S inhibited pertussis toxin-catalyzed ADP ribosylation in both diabetic and nondiabetic patients. Binding of insulin of the alpha-subunit and activation of the tyrosine kinase intrinsic to the beta-subunit of the insulin receptor are not responsible for the coupling defect. 125I insulin binding is the same in obese patients with or without diabetes. Tyrosine kinase of the insulin receptor is decreased in diabetes. However, a monoclonal antibody to the insulin receptor (MA-20) at equimolar concentrations with insulin equally inhibits pertussis toxin-catalyzed ADP ribosylation of Gi without activating tyrosine kinase or insulin receptor autophosphorylation. Immunodetection of G-proteins suggested that Gi3 alpha was normal in diabetes and Gi1-2 alpha was decreased by 40% in the diabetic group as compared to the obese nondiabetic group but was normal when compared to the lean non diabetic group. We conclude that the novel pathway of insulin signaling involving the regulatory Gi proteins via biochemical mechanisms not directly involving the tyrosine kinase of the insulin receptor is altered in obese type II diabetes and offers a new target for the search of the mechanism(s) of insulin resistance.

Topics: Adenosine Diphosphate Ribose; Antibodies, Monoclonal; Cholera Toxin; Cytosol; Diabetes Mellitus; Diabetes Mellitus, Type 2; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Insulin; Insulin Resistance; Liver; Membrane Proteins; Obesity; Pertussis Toxin; Poly(ADP-ribose) Polymerases; Receptor Protein-Tyrosine Kinases; Receptor, Insulin; Signal Transduction; Virulence Factors, Bordetella

Attenuated maximal activations by forskolin, Mn+. NaF or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) were noted for adenylate cyclase activity in adipocytes from obese (fa/fa) Zucker rats compared with their lean (Fa/Fa) littermates. GTP[S] achieved half-maximal activation of adenylate cyclase at some 10-fold lower concentrations in membranes from lean animals compared with those from obese. Levels of the 42 and 45 kDa forms of Gs were some 40-50% lower in membranes from obese animals, and levels of Gi-1 and Gi-3 were some 62-65% lower. No differences in levels of Gi-2 alpha-subunits or G-protein beta-subunits were observed. Gi function, as assessed by inhibiting forskolin-stimulated adenylate cyclase, achieved by prostaglandin E1, nicotinate and phenylisopropyladenosine, was similar in membranes from both lean and obese animals. Levels of beta-adrenoceptors were some 50% lower in membranes from obese animals. It is suggested that the attenuated activation of adenylate cyclase by stimulatory ligands in membranes from obese animals may be caused by decreases in both Gs and receptors, and that this may contribute to the attenuated lipolytic response seen in adipocytes from such animals.

Topics: Adenylyl Cyclases; Adipose Tissue; Animals; Cell Membrane; Cells, Cultured; Glucagon; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Isoproterenol; Kinetics; Macromolecular Substances; Male; Obesity; Rats; Rats, Zucker; Secretin; Thyrotropin

Adenylate cyclase activity was determined in membranes of liver, muscle, white adipose tissue, and brown adipose tissue (BAT) of lean (Fa/) and obese (fa/fa) Zucker rats. Responses were monitored following beta-adrenergic receptor stimulation and addition of GTP, GTP gamma S, or forskolin. beta-Adrenergic responses in liver, white adipose tissue, and BAT were lower in obese than in lean animals. No such difference was observed in muscle membranes. Production of cAMP after addition of guanine nucleotides was lower in liver and white adipose tissue membranes from obese rats compared with their lean littermates. Synthesis of cAMP in muscle membranes of obese animals after addition of GTP was either not different, or slightly higher, than that observed in muscle membranes from lean animals. Furthermore, production of cAMP after forskolin addition to muscle membranes of obese rats was significantly higher than that observed from lean rats under the same conditions. Interestingly, BAT membranes of obese rats were significantly more sensitive to guanine nucleotide activation than those of lean animals. The results confirm recent findings indicating inferior function of G proteins in liver plasma membranes of obese Zucker rats, and extend this observation to adipose tissue. The present results further suggest that the "nonreceptor" components (e.g., G proteins) responsible for the activation of adenylate cyclase in BAT membranes of obese rats are more responsive to stimulation than those of lean animals. Such sensitivity may be related to and perhaps compensate for the reduced thermogenic activity in the obese Zucker rat during the development of obesity.

Topics: Adenylyl Cyclases; Adipose Tissue; Adipose Tissue, Brown; Animals; Body Weight; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Female; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Isoproterenol; Liver; Muscles; Norepinephrine; Obesity; Organ Size; Rats; Rats, Zucker; Receptors, Adrenergic, beta