Treatment of streptozotocin-diabetic rats with metformin restores the ability of insulin to inhibit adenylate cyclase activity and demonstrates that insulin does not exert this action through the inhibitory guanine nucleotide regulatory protein Gi

Insulin caused the inhibition of glucagon-stimulated adenylate cyclase activity in liver plasma membranes, but failed to inhibit this activity in liver membranes from rats made diabetic by treatment with either alloxan or streptozotocin. Treatment of streptozotocin-diabetic rats with insulin, to normalize their blood glucose concentrations, restored this action of insulin. Rats treated with the biguanide drug metformin exhibited a decreased content of the inhibitory guanine nucleotide regulatory protein Gi in liver plasma membranes assessed both structurally, by using a specific polyclonal antibody (AS7), and functionally. Treatment of normal rats with metformin did not alter insulin's ability to inhibit adenylate cyclase in liver plasma membranes; however, metformin treatment of streptozotocin-diabetic rats completely restored this inhibitory action of insulin. Liver plasma membranes from streptozotocin-diabetic animals which either had or had not been treated with metformin had contents of Gi which were less than 10% of those seen in control animals. We conclude that: (i) insulin does not inhibit adenylate cyclase activity through the inhibitory guanine nucleotide regulatory protein Gi; (ii) streptozotocin- and alloxan-induced diabetes elicit a selective insulin-resistant state; and (iii) metformin can exert a post-receptor effect, at the level of the liver plasma membrane, which restores the ability of insulin to inhibit adenylate cyclase.

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Evidence that a beta-adrenergic receptor-associated guanine nucleotide regulatory protein conveys guanosine 5'-O-(3-thiotriphosphate)- dependent adenylate cyclase activity.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)68903-9 ◽

1981 ◽

Vol 256 (16) ◽

pp. 8718-8723

Author(s):

J.M. Stadel ◽

R.G. Shorr ◽

L.E. Limbird ◽

R.J. Lefkowitz

Keyword(s):

Adenylate Cyclase ◽

Adrenergic Receptor ◽

Regulatory Protein ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Guanine Nucleotide ◽

Beta Adrenergic Receptor ◽

Beta Adrenergic ◽

Guanine Nucleotide Regulatory Protein

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Challenge of hepatocytes by glucagon triggers a rapid modulation of adenylate cyclase activity in isolated membranes

Biochemical Journal ◽

10.1042/bj2140093 ◽

1983 ◽

Vol 214 (1) ◽

pp. 93-98 ◽

Cited By ~ 46

Author(s):

C M Heyworth ◽

M D Houslay

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Regulatory Protein ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Maximum Effect ◽

Guanine Nucleotide ◽

Rapid Modulation ◽

Guanine Nucleotide Regulatory Protein ◽

Dose Dependent

Membrane fractions obtained from hepatocytes treated with glucagon exhibited a decreased glucagon (with or without GTP)-stimulated adenylate cyclase activity. A maximum effect was seen in around 5 min. No change in the rate of cyclic AMP production was observed for the basal, NaF-, p[NH]ppG (guanosine 5′-[beta, gamma-imido]-triphosphate)- and GTP-stimulated states of the enzyme. The lag observed in the p[NH]ppG-stimulated adenylate cyclase activity of native membranes was abolished when membranes from glucagon-pretreated cells were used. When Mn2+ replaced Mg2+ in the assays, the magnitude of the apparent desensitization was decreased. Mn2+ abolished the lag of onset of p[NH]ppG-stimulated activity in native membranes. The desensitization process was dose-dependent on glucagon, which exhibited a Ka of 4 X 10(-10) M. Depletion of intracellular ATP did not affect this process. It is suggested that this desensitization occurs at the level of the guanine nucleotide-regulatory protein.

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Differential effects of fluoride on adenylate cyclase activity and guanine nucleotide regulation of agonist high-affinity receptor binding

Biochemical Journal ◽

10.1042/bj2540015 ◽

1988 ◽

Vol 254 (1) ◽

pp. 15-20 ◽

Cited By ~ 19

Author(s):

J M Stadel ◽

S T Crooke

Keyword(s):

Adenylate Cyclase ◽

Plasma Membranes ◽

Regulatory Protein ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Adenylate Cyclase System ◽

Guanine Nucleotide ◽

Beta Adrenergic ◽

High Affinity ◽

Platelet Membranes

Fluoride ion, presumably an Al3+-F- complex, has been proposed to activate the guanine nucleotide regulatory protein (G-protein) of the visual system, transducin, by associating with GDP at the nucleotide-binding site and thus mimicking the effects of non-hydrolysable GTP analogues [Bigay, Deterre, Pfister & Chabre (1985) FEBS Lett. 191, 181-85]. We have examined this proposed model by using the adenylate cyclase complexes of frog erythrocytes, S49 lymphoma cells and human platelets. Preincubation of plasma membranes from frog erythrocytes and S49 cells with 20 mM-fluoride for 20 min at 30 degrees C strongly stimulated adenylate cyclase activity. In contrast, the preactivated membranes were still able to bind beta-adrenergic agonist with high affinity, as determined by radioligand-binding techniques. Moreover, high-affinity agonist binding in fluoride-treated membranes was fully sensitive to guanine nucleotide, which decreased beta-adrenergic-receptor affinity for agonist. Very similar results were obtained for [3H]prostaglandin E1 binding to S49 membranes pretreated with fluoride. Incubation of human platelet membranes with increasing concentrations of fluoride (1-50 mM) resulted in biphasic regulation of adenylate cyclase activity, with inhibition observed at concentrations greater than 10 mM. Preincubation of platelet membranes with 20 mM-fluoride did not affect agonist high-affinity binding to alpha 2-adrenergic receptors, nor receptor regulation by guanine nucleotide. These results suggest that the model developed from the study of transducin may not be generally applicable to the G-proteins of the adenylate cyclase system.

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Islet-activating protein blocks glucagon desensitization in intact hepatocytes

Biochemical Journal ◽

10.1042/bj2220189 ◽

1984 ◽

Vol 222 (1) ◽

pp. 189-194 ◽

Cited By ~ 36

Author(s):

C M Heyworth ◽

E Hanski ◽

M D Houslay

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Plasma Membranes ◽

Regulatory Protein ◽

Inhibitory Effect ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Inhibitory Input ◽

Pronounced Increase ◽

Guanine Nucleotide Regulatory Protein

Treatment of intact hepatocytes with islet-activating protein, from Bordatella pertussis, led to a pronounced increase in the ability of glucagon to raise intracellular cyclic AMP concentrations. Islet-activating protein, however, caused no apparent increase in the intracellular concentration of cyclic AMP under basal conditions. These effects were attributed to an enhanced ability of adenylate cyclase, in membranes from hepatocytes treated with islet-activating protein, to be stimulated by glucagon. When forskolin was used to amplify the basal adenylate cyclase activity, elevated GTP concentrations were shown to inhibit adenylate cyclase activity in membranes from control hepatocytes. This inhibitory effect of GTP was abolished if the hepatocytes had been pre-treated with islet activating protein. In isolated liver plasma membranes, islet-activating protein caused the NAD-dependent ribosylation of a Mr-40000 protein, the putative inhibitory guanine nucleotide regulatory protein, Ni. This effect was inhibited if guanosine 5′-[beta‐thio]diphosphate rather than GTP was present in the ribosylation incubations. The ability of glucagon to uncouple or desensitize the activity of adenylate cyclase in intact hepatocytes was also blocked by pre-treating hepatocytes with islet-activating protein. Islet-activating protein thus heightens the response of hepatocytes to the stimulatory hormone glucagon. It achieves this by both inhibiting the expression of desensitization and also removing a residual inhibitory input expressed in the presence of glucagon.

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Decrease in adenylate cyclase activity in dog livers during endotoxic shock

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1983.245.5.r737 ◽

1983 ◽

Vol 245 (5) ◽

pp. R737-R742

Author(s):

S. Ghosh ◽

M. S. Liu

Keyword(s):

Adenylate Cyclase ◽

Plasma Membranes ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Guanine Nucleotide ◽

Endotoxin Administration ◽

Liver Plasma Membranes ◽

Dog Liver

The effects of endotoxin administration on adenylate cyclase in dog liver plasma membranes were studied. The basal, fluoride-, guanine nucleotide-, isoproterenol-, and glucagon-stimulated adenylate cyclase activities were decreased by 61, 62, 69, 83, and 63%, respectively, 2 h after in vivo administration of endotoxin. Endotoxin (100 micrograms/ml) in vitro decreased the guanine nucleotide-, isoproterenol-, and glucagon-stimulated adenylate cyclase activities by 24, 25, and 21%, respectively. These data demonstrate that endotoxin administered in vivo or in vitro had an inhibitory effect on the adenylate cyclase enzyme system in dog liver plasma membranes. Because of the involvement of the adenylate cyclase-adenosine 3',5'-cyclic monophosphate (cAMP) system in the regulation of hepatic carbohydrate metabolism, the finding that endotoxin administration decreased adenylate cyclase activity in the liver should contribute to the understanding of the pathophysiology of altered hepatic glucose homeostasis during shock.

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Phorbol esters inhibit adenylate cyclase activity in cultured collecting tubular cells

AJP Cell Physiology ◽

10.1152/ajpcell.1988.254.1.c183 ◽

1988 ◽

Vol 254 (1) ◽

pp. C183-C191 ◽

Cited By ~ 28

Author(s):

B. S. Dixon ◽

R. Breckon ◽

C. Burke ◽

R. J. Anderson

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Adenylate Cyclase ◽

Regulatory Protein ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Guanine Nucleotide ◽

Tubular Cells ◽

Kinase C ◽

Guanine Nucleotide Regulatory Protein

Activators of protein kinase C, a calcium- and phospholipid-dependent protein kinase, inhibit vasopressin-stimulated water flow in toad bladder. To determine the biochemical mechanisms of this inhibition, we examined the effects of activators of protein kinase C on arginine vasopressin (AVP)-stimulated adenylate cyclase activity in cultured rabbit cortical collecting tubular cells. The phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA), the diacylglycerol, 1-oleyl-2-acetyl glycerol (OAG), and the diacylglycerol kinase inhibitor, R59022, all rapidly activate protein kinase C in collecting tubular cells. Pretreatment with PMA produces a delayed inhibition (greater than or equal to 4 h) of AVP-stimulated adenylate cyclase activity. The 4-h time lag suggests that the effects of protein kinase C are mediated indirectly, possibly as a consequence of stimulating cell proliferation. PMA does not inhibit cholera toxin- or forskolin-stimulated adenylate cyclase activity, suggesting an effect on the vasopressin receptor or coupling of the receptor to the stimulatory guanine nucleotide regulatory protein. Neither prostaglandins nor the inhibitory guanine nucleotide regulatory protein appear to mediate this effect. In contrast, treatment with either OAG or R59022 produces a rapid inhibition of both AVP- and forskolin-stimulated adenylate cyclase activity suggesting a prominent distal site of action, presumably at the catalytic subunit of adenylate cyclase. The results demonstrate that different activators of protein kinase C inhibit AVP-stimulated adenylate cyclase activity by distinctly different mechanisms possibly by altering the substrate specificity or activating multiple forms of the kinase. These results have important implications when using different activators to study the biological effects of protein kinase C.

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Lysophosphatidylcholines can modulate the activity of the glucagon-stimulated adenylate cyclase from rat liver plasma membranes

Biochemical Journal ◽

10.1042/bj1780217 ◽

1979 ◽

Vol 178 (1) ◽

pp. 217-221 ◽

Cited By ~ 27

Author(s):

M D Houslay ◽

R W Palmer

Keyword(s):

Adenylate Cyclase ◽

Rat Liver ◽

Plasma Membranes ◽

Hormone Receptors ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Liver Plasma Membranes ◽

Rat Liver Plasma Membranes ◽

Increased Sensitivity

1. Synthetic lysophosphatidylcholines inhibit the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes at concentrations two to five times lower than those needed to inhibit the fluoride-stimulated activity. 2. Specific 125I-labelled glucagon binding to hormone receptors is inhibited at concentrations similar to those inhibiting the fluoride-stimulated activity. 3. At concentrations of lysophosphatidylcholines immediately below those causing inhibition, an activation of adenylate cyclase activity or hormone binding was observed. 4 These effects are essentially reversible. 5. We conclude that the increased sensitivity of glucagon-stimulated adenylate cyclase to inhibition may be due to the lysophosphatidylcholines interfering with the physical coupling between the hormone receptor and catalytic unit of adenylate cyclase. 6. We suggest that, in vivo, it is possible that lysophosphatidylcholines may modulate the activity of adenylate cyclase only when it is in the hormone-stimulated state.

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Multiple defects occur in the guanine nucleotide regulatory protein system in liver plasma membranes of obese (fa/fa) but not lean (Fa/Fa) zucker rats: Loss of functional G1 and abnormal Gs function

Cellular Signalling ◽

10.1016/0898-6568(89)90016-8 ◽

1989 ◽

Vol 1 (1) ◽

pp. 9-22 ◽

Cited By ~ 24

Author(s):

Miles D. Houslay ◽

Debra J. Gawler ◽

Graeme Milligan ◽

Andrew Wilson

Keyword(s):

Plasma Membranes ◽

Regulatory Protein ◽

Zucker Rats ◽

Guanine Nucleotide ◽

Multiple Defects ◽

Protein System ◽

Liver Plasma Membranes ◽

Guanine Nucleotide Regulatory Protein

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Elevated membrane cholesterol concentrations inhibit glucagon-stimulated adenylate cyclase

Biochemical Journal ◽

10.1042/bj2100437 ◽

1983 ◽

Vol 210 (2) ◽

pp. 437-449 ◽

Cited By ~ 61

Author(s):

A D Whetton ◽

L M Gordon ◽

M D Houslay

Keyword(s):

Fatty Acid ◽

Adenylate Cyclase ◽

Spin Probe ◽

Plasma Membranes ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Cholesterol Concentration ◽

Lipid Phase ◽

Lipid Order ◽

Liver Plasma Membranes

A method was devised which increases the cholesterol concentration of rat liver plasma membranes by exchange from cholesterol-rich liposomes at low temperature (4 degrees C). When the cholesterol concentration of liver plasma membranes is increased, there is an increase in lipid order as detected by a decrease in mobility of an incorporated fatty acid spin probe. This is accompanied by an inhibition of adenylate cyclase activity. The various ligand-stimulated adenylate cyclase activities exhibit different sensitivities to inhibition by cholesterol, with inhibition of glucagon-stimulated greater than fluoride-stimulated greater than basal activity. The bilayer-fluidizing agent benzyl alcohol is able to reverse the inhibitory effect of cholesterol on adenylate cyclase activity in full. The thermostability of fluoride-stimulated cyclase is increased in the cholesterol-rich membranes. Elevated cholesterol concentrations abolish the lipid-phase separation occurring at 28 degrees C in native membranes as detected by an incorporated fatty acid spin probe. This causes Arrhenius plots of glucagon-stimulated adenylate cyclase activity to become linear, rather than exhibiting a break at 28 degrees C. It is suggested that the cholesterol contents of both halves of the bilayer are increased by the method used and that inhibition of adenylate cyclase ensues, owing to the increase in lipid order and promotion of protein-protein and specific cholesterol-phospholipid interactions.

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