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The pyruvate dehydrogenase multienzyme complex catalyses the oxidative decarboxylation of pyruvate, which is an important regulatory step in oxidative metabolism. Phosphorylation of the E1 (pyruvate decarboxylase) subunit on one of three specific serine residues results in loss of enzyme activity. Four dedicated PDHK (pyruvate dehydrogenase kinase) isoenzymes have been identified, each of which display a distinct tissue-specific expression profile, and have differential regulatory properties. Thus PDHK play a key role in controlling the balance between glucose and lipid oxidation according to substrate supply. Increasing glucose oxidation by inhibiting PDHK may be an effective mechanism to increase glucose utilization; additionally, increasing pyruvate oxidation may further contribute to lowering of glucose level by decreasing the supply of gluconeogenic substrates. A number of PDHK inhibitors are now available to enable this mechanism to be evaluated as a therapy for diabetes. The isoenzyme selectivity profile of AZD7545 and related compounds will be described and evidence for their non-ATP-competitive mode of action presented. These compounds increase PDH activity in vivo, and when dosed chronically, improve glycaemic control in Zucker rats. Furthermore, glucose lowering has been demonstrated in the hyperglycaemic Zucker diabetic fatty rat. This result supports the hypothesis that inhibition of PDHK may be an effective therapy for Type II diabetes.

The PDH (pyruvate dehydrogenase) multi-enzyme complex catalyses a key regulatory step in oxidative glycolysis. Phosphorylation of the E1 subunit of the complex on serine residues results in the inactivation of enzyme activity. A family of four dedicated PDH kinase isoenzymes exists, each of which displays a distinct tissue-specific expression profile. AZD7545 is one of a series of PDH kinase inhibitors developed for the treatment of type 2 diabetes. The isoenzyme-selectivity profile of AZD7545 and related compounds is described and the consequences for their in vivo mode of action are discussed.

PDH (pyruvate dehydrogenase) is a key enzyme controlling the rate of glucose oxidation, and the availability of gluconeogenic precursors. Activation of PDH in skeletal muscle and liver may increase glucose uptake and reduce glucose production. This study describes the properties of AZD7545, a novel, small-molecule inhibitor of PDHK (PDH kinase). In the presence of PDHK2, AZD7545 increased PDH activity with an EC50 value of 5.2 nM. In rat hepatocytes, the rate of pyruvate oxidation was stimulated 2-fold (EC50 105 nM). A single dose of AZD7545 to Wistar rats increased the proportion of liver PDH in its active, dephosphorylated form in a dose-related manner from 24.7 to 70.3% at 30 mg/kg; and in skeletal muscle from 21.1 to 53.3%. A single dose of 10 mg/kg also significantly elevated muscle PDH activity in obese Zucker (fa/fa) rats. Obese, insulin-resistant, Zucker rats show elevated postprandial glucose levels compared with their lean counterparts (8.7 versus 6.1 mM at 12 weeks old). AZD7545 (10 mg/kg) twice daily for 7 days markedly improved the 24-h glucose profile, by eliminating the postprandial elevation in blood glucose. These results suggest that PDHK inhibitors may be beneficial agents for improving glucose control in the treatment of type 2 diabetes.

The molecular mechanisms which control the terminal differentiation of mature fat cells remain largely unknown. Various studies have shown that the signalling molecule cyclic AMP (CAMP) can modulate the growth of cells. It has been reported that i n vivo sympathetic innervation exerts an inhibitory influence on the proliferation of rat preadipocytes [l] and that i n v i t r o CAMP promotes the differentiation of primary cultures of rat adipocytes [2]. However studies using preadipocyte cell lines have produced conflicting results showing that CAMP either has no effect upon [3], promotes [4] or inhibits [5] differentiation. In order to investigate this apparent anomaly we have carried out a thorough study of the effect of CAMP on the differentiation of 3T3-F442A preadipocytes. 3T3-F442A fibroblasts were grown to confluence in Dulbecco’s modified Eagle‘s medium (DMEM) containing 10% calf serum. Confluent cultures were induced to differentiate by replacing the growth medium with either DMEM containing 10% foetal calf serum (FCS) and 5 pg/ml insulin or with a defined differentiation medium (DDM) containing growth hormone ( 2 nM), insulin (1.8 pM) , T, (0.1 ng/ml) , EGF (50 ng/ml) and other factors as previously described [6] along with the drugs being tested. After three days the drugs were removed and after a further three days lipid filling of the cells was assessed by staining with Oil Red 0 and the activity of a-glycerophosphate dehydrogenase (GPDH), a marker for adipocyte differentiation, was measured [6]. Initial studies demonstrated that, during differentiation of cells with FCS/insulin, raising intracellular CAMP levels with forskolin ( 5 0 pM)’ cholera toxin (10 ng/ml) or CPT-CAMP (0.25 mM) severely attenuated lipid filling and GPDH activity (~90% decrease relative to control cells). Dideoxyforskolin (50 pM) , an inactive analogue, had no effect on the extent of differentiation. In contrast, inclusion of the phosphodiesterase inhibitor isobutylmethyl xanthine (IBMX, 500 pM) in the FCS/insulin medium promoted differentiation (88% increase in GPDH activity). Similar effects were observed upon the differentiation of cells in DDM. Thus, forskolin (50 pM) , cholera toxin (10 ng/ml) and CPT-CAMP (0.25 mM) decreased the GPDH activity of differentiated cells by ~ 7 0 % and IBMX increased GPDH activity by 21%. These results suggest that a small increase in intracellular CAMP levels, as occurs in the presence of IBMX, promotes differentiation while the larger increases in CAMP levels achieved with the forskolin, cholera toxin and CPT-CAMP treatments inhibit the differentiation process. To confirm that CAMP exerts differential effects upon differentiation, cells were incubated with FCS/insulin medium in the 0.0 L I . . I I . . .d . . .,,.,.1 . ..,,,,.I , ,,I... d , ,.I... * . . .,,d

Adenosine is a paracrine/autocrine antilipolytic agent that binds to A, receptors of adipocyte membranes, thereby activating GTP-binding proteins, G,, which inhibit adenylate cyclase activity and hence lipolysis. The mechanism whereby GH facilitates lipolysis is not fully explained, however it is possible that GH exerts its effects by modulating the acute regulation of this process by other hormones rather than having a direct effect on lipolysis itself. Initially studies focused on modulation of response of adipocytes to catecholamines, but very recent studies [ I , 21 suggest that the G,-mediated antilipolytic system is a major target of GH action. We have investigated this further by (a) treating sheep with recombinant bovine growth hormone, (Monsanto) 10 mg per day for 7 days in vivo and (b) by maintaining sheep adipose tissue explants in culture with GH (metabolic activities and other processes are particularly well maintained in sheep adipose tissue during culture [3]. For culture, adipose tissue explants were preincubated for 24h with no hormones and then GH (100ng/ml) was added for the next 24h. In both cases, response to adenosine in vitro was determined using N6-phenylisopropyladenosine (PIA) as described previously [2] (in essence lipolysis was activated with 100 nM isoprenaline and inhibited with various concentrations Treatment of sheep in vivo with GH decreased maximum inhibition of isoproterenol-stimulated lipolysis by 100 nM PIA from 68.8 f 11.4 to 27.8 f 9.2% (P

T h e detailed pathways which link occupancy of the insulin receptor with actions on target cells remain t o be defined. The insulin receptor expresses a tyrosyl kinase activity for which considerable evidence has accrued indicating that it plays a fundamental role in transducing at least certain o f insulin's actions. Nevertheless, the mechanism whcrcby tyrosyl phosphorylation o f proteins might lead to cellular responses is still as much of an enigma a s the physiological role o f the few substrates that have been identified 11.21. T h e functioning o f ii wide variety of cell surface receptors for hormones and neurotransmitters is transduced by their interaction with specific guanine nucleotide regulatory proteins (G-proteins) I3,4]. Not only that, but considerable evidence suggests that there is substantial 'cross-talk' bctwecn the machinery o f cellular signalling systems [ 51. This can lead to heterologous desensitization or t o supersensitization depending upon the signalling system and cell type. Here we discuss evidence which supports the contention we made earlier 16, 71, that insulin can interact with the G-protein system. This shows that ( i ) insulin, acting through its receptor can interact with both individual G-proteins and specific G-protein-controlled systems, ( i i ) that a specific G-protein may be involved in transducing certain of insulin's actions and ( i i i ) that in diabetes and in insulin-resistant states one sees the functional inactivation o f the inhibitory Gprotein G, in both liver 18.91 and white adipose tissue. MARY-ANNE McGREGOR. ELAINE KILGOUR.