guanylyl-imidodiphosphate and Obesity

Studies were designed to find the molecular basis for previous observations that lipolysis is less active and A1 adenosine receptor signaling is more active in adipocytes from obese than from lean Zucker rats. With quantitative immunoblot procedures for detection, Gi alpha 1 and Gs alpha 45 levels were found anomalously low in obese compared with lean membranes (50 and 30%, respectively), but other G alpha subunit levels were normal. However, the sensitivity of the receptor-Gi protein to GTP was about 5- to 10-fold higher in obese compared with lean membranes when assessed from 1) the ability of GTP to inhibit forskolin-stimulated adenylyl cyclase in the presence of an adenosine receptor agonist and 2) the ability of a nonhydrolyzable guanine nucleotide analogue to alter A1 adenosine receptor agonist binding. Alkaline phosphatase treatment of isolated adipocyte membranes from obese but not lean animals decreased guanine nucleotide sensitivity of agonist binding. Surprisingly, solubilized adipocyte A1 adenosine receptors from all animals exhibited the same high sensitivity to guanine nucleotides as that of intact obese membranes, and this high sensitivity could be decreased 20-fold by treatment with alkaline phosphatase. These data suggest that protein phosphorylation may regulate coupling of the A1 adenosine receptor in rat adipocyte membranes.

Topics: Adenosine; Adenylyl Cyclases; Adipocytes; Animals; Cell Membrane; Colforsin; Cyclic AMP; Guanine Nucleotides; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Iodine Radioisotopes; Iodobenzenes; Obesity; Phosphorylation; Proteins; Purinergic P1 Receptor Agonists; Rats; Rats, Sprague-Dawley; Rats, Zucker; Receptors, Purinergic P1

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

Lipolysis in rat adipocytes is controlled by the hormonally mediated stimulation and inhibition of adenylate cyclase. This dual regulation involves stimulatory (Gs) and inhibitory (Gi) GTP-binding proteins which control cAMP production in a GTP dependent manner. Adenosine, acting via the A1 receptor-Gi complex provides tonic regulation of adenylate cyclase and lipolysis in rat adipocytes. Adipocytes prepared from young obese Zucker (fa/fa) rats exhibit less stimulation (or greater inhibition) in response to adenosine deaminase, alone or in combination with lipolytic hormones, as compared with their lean littermates. Adenylate cyclase, measured in membranes prepared from obese adipocytes, showed decreased sensitivity to activation by low concentrations of GTP and was not inhibited by higher concentrations of the guanine nucleotide which, in lean control rats results in a biphasic activity curve. Adenosine A1 receptor binding, measured in these same membranes, demonstrated an increased sensitivity to activation by the GTP analogue, guanylyl imidodiphosphate. The presence of the analogue results in the dissociation of the receptor-Gi complex and conversion to the low affinity form in a greater proportion of receptors in the obese membranes. These results are consistent with an increased sensitivity to adenosine mediated inhibition of adenylate cyclase and lipolysis in the fat cells of the young obese (fa/fa) Zucker rat.

Topics: Adenosine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Adipose Tissue; Animals; Awards and Prizes; Cell Membrane; Cyclic AMP; Dietetics; Female; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Isoproterenol; Lipolysis; Obesity; Phenylisopropyladenosine; Rats; Rats, Zucker; Receptors, Purinergic; Societies, Medical; United States

Hepatocyte membranes from both lean and obese Zucker rats exhibited adenylate cyclase activity that could be stimulated by glucagon, forskolin, NaF and elevated concentrations of p[NH]ppG. In membranes from lean animals, functional Gi was detected by the ability of low concentrations of p[NH]ppG to inhibit forskolin-activated adenylate cyclase. This activity was abolished by treatment of hepatocytes with either pertussis toxin or the phorbol ester TPA, prior to making membranes for assay of adenylate cyclase activity. In hepatocyte membranes from obese animals no functional Gi activity was detected. Quantitative immunoblotting, using an antibody able to detect the alpha subunit of Gi, showed that hepatocyte plasma membranes from both lean and obese Zucker rats had similar amounts of Gi-alpha subunit. This was 6.2 pmol/mg plasma membrane for lean and 6.5 pmol/mg plasma membrane for obese animals. Using thiol pre-activated pertussis toxin and [32P]-NAD+, similar degrees of labelling of the 40 kDa alpha subunit of Gi were found using plasma membranes of both lean and obese Zucker rats. We suggest that liver plasma membranes from obese Zucker rats express an inactive Gi alpha subunit. Thus lesions in liver Gi functioning are seen in insulin-resistant obese rats and in alloxan- and streptozotocin-induced diabetic rats which also show resistance as regards the acute actions of insulin. Liver plasma membranes of obese animals also showed an impairment in the coupling of glucagon receptors to Gs-controlled adenylate cyclase, with the Kd values for activation by glucagon being 17.3 and 126 nM for lean and obese animals respectively. Membranes from obese animals also showed a reduced ability for high concentration of p[NH]ppG to activate adenylate cyclase. The use of [32P]-NAD+ and thiol-preactivated cholera toxin to label the 43 kDa and 52 kDa forms of the alpha-subunit of Gs showed that a reduced labelling occurred using liver plasma membranes from obese animals. It is suggested that abnormalities in the levels of expression of primarily the 52 kDa form of alpha-Gs may give rise to the abnormal coupling between glucagon receptors and adenylate cyclase in liver membranes from obese (fa/fa) Zucker rats.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Blotting, Western; Cell Membrane; Cholera Toxin; Colforsin; Diabetes Mellitus, Experimental; Glucagon; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Insulin Resistance; Liver; Male; NAD; Obesity; Pertussis Toxin; Phosphorus Radioisotopes; Rats; Rats, Inbred Strains; Rats, Zucker; Receptors, Gastrointestinal Hormone; Receptors, Glucagon; Virulence Factors, Bordetella

Adenylate cyclase activity and its modulation by guanine nucleotides and isoproterenol were assessed in adipocyte membranes of mice with mutations causing different genetic obesity syndromes. The object was to determine whether the defect in inhibitory modulation observed in the obese (ob/ob) mouse was also present in the diabetes (db/db) mouse. The data show that adipocyte adenylate cyclase in both the ob/ob and the db/db mouse is resistant to activation by isoproterenol. The response to guanosine triphosphate (GTP) differed between the two mutants, such that an inhibitory phase was visible in the db/db but not in the ob/ob membranes. Moreover, pertussis toxin attenuated the inhibitory effect of GTP and significantly stimulated cyclase activity in the db/db but not in the ob/ob membranes. The data show that the two mutations affect the expression of adenylate cyclase activity via different mechanisms.

Topics: Adenylyl Cyclases; Adipose Tissue; Animals; Cell Membrane; Diabetes Mellitus, Experimental; Enzyme Activation; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Isoproterenol; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mutation; Obesity

The activation of brown adipose tissue adenylate cyclase by catecholamines was studied in genetically obese (ob/ob) and lean mice. In obese mice, the maximum activation of the enzyme by several beta-adrenergic agonists was only two-thirds that in lean mice and, as an activator, noradrenaline was only one-eighth as potent. The adenylate cyclase was also less responsive to guanine nucleotides. In these respects, the defect in catecholamine-stimulated adenylate cyclase was similar in both white and brown adipose tissue of the obese mouse. The enzyme in brown adipose tissue differed from that in white adipose tissue in its sensitivity to other beta-adrenergic agonists and in its requirement for Mg2+. It is suggested that this abnormal catecholamine-activated adenylate cyclase in brown adipose tissue may be relate to the thermoregulatory defect of the obese mouse and hence may contribute to the obesity syndrome.

Topics: Adenylyl Cyclases; Adipose Tissue, Brown; Adrenergic beta-Agonists; Animals; Catecholamines; Enzyme Activation; Guanylyl Imidodiphosphate; Male; Mice; Mice, Inbred C57BL; Mice, Obese; Obesity

Experimental obesity syndromes are characterized by considerable fat accretion which may be the result of hyperinsulinemia. Adipose tissue accretion may be the result both of the excessive synthesis of triglycerides and a defect in their mobilization from adipose tissue stores. The adenylate cyclase system which appears to be the site of the defect in mobilization is studies in more detail.

Topics: Adenylyl Cyclases; Adipose Tissue; Animals; Cell Membrane; Disease Models, Animal; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Lipolysis; Liver; Mice; Mice, Obese; Obesity; Rats; Rats, Inbred Strains; Rats, Zucker