The Glucose/Fatty Acid Cycle 1963–2003
AZD7545, a novel inhibitor of pyruvate dehydrogenase kinase 2 (PDHK2), activates pyruvate dehydrogenase in vivo and improves blood glucose control in obese (fa/fa) Zucker rats R.M. Mayers1 , R.J. Butlin, E. Kilgour, B. Leighton, D. Martin, J. Myatt, J.P. Orme and B.R. Holloway AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, U.K.
Abstract 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.
Introduction PDH (pyruvate dehydrogenase) plays a pivotal role in controlling the balance between glucose and fatty acid oxidation, and its activation state is tightly controlled by the balance between specific PDHK (PDH kinase) and PDP (PDH phosphatase) activities. In type 2 diabetes, glucose oxidation is inappropriately low, possibly the result of the effect of elevated non-esterified (‘free’) fatty acids on PDH. Fatty acid oxidation results in elevated acetyl-CoA/CoA, ATP/ADP and NADH/NAD+ ratios, which increase PDHK activity. In addition, fatty acids exert a more long-term effect on glucose oxidation by increasing the expression of the high-specific-activity isoform PDHK4, which may occur at least in part via activation of peroxisome-proliferatoractivated receptor α [1]. Diabetes is also associated with elevated hepatic glucose production, so activation of PDH may have the potential to decrease blood glucose, not only by increasing glucose oxidation, but also reducing the supply of the gluconeogenic substrates lactate and alanine. DCA (dichloroacetate) is a non-specific inhibitor of PDHK which has been shown to increase both muscle and liver PDH activity in a number of rat models of diabetes: streptozotocin- [2] and alloxan-induced [3] insulin-
deficient states and models of type 2 diabetes such as the dexamethasone-induced [4] and the ZDF (Zucker diabetic fatty) rat [5]. In these models, with the exception of alloxandiabetes, DCA causes a decrease in blood glucose. Clinically, DCA has shown some effect in hyperglycaemic patients [6]; consistent with the hypothesis that PDH activation will decrease the availability of gluconeogenic substrates, glucose lowering was associated with a marked decrease in plasma lactate and alanine. In the normal fed rat or non-diabetic human, PDH activation by DCA does not affect glucose levels, but it does lower glucose in fasted animals [7]. DCA is unsuitable as a therapeutic agent because of low potency, metabolism and toxicity [8]. The search for novel, small-molecule inhibitors of PDHK offering improved potency and specificity has been ongoing for some years. Halogenated acetophenones were described in 1995 [9]; these were of relatively low potency (>1 µM) and do not seem to have been developed further. Several other structural types have been identified by high-throughput screening techniques; these and their proposed mechanisms of action are reviewed in [10].
Activation of PDH in vitro Key words: AZD7545, diabetes, glucose oxidation, pyruvate dehydrogenase (PDH), pyruvate dehydrogenase kinase (PDHK), Zucker. Abbreviations used: PDH, pyruvate dehydrogenase; PDHK, pyruvate dehydrogenase kinase; PDP, pyruvate dehydrogenase phosphatase; DCA, dichloroacetate; ZDF, Zucker diabetic fatty. 1 To whom correspondence should be addressed (e-mail
[email protected]).
Compound screening at AstraZeneca has identified a novel series of anilide tertiary carbinols, including AZD7545, which are potent and specific inhibitors of PDHK2. AstraZeneca utilized a functional enzyme assay where native porcine PDH C 2003
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complex was preincubated with recombinant PDHK and ATP. The degree of phosphorylation and hence inactivation of the complex was assessed by subsequent measurement of PDH activity following addition of substrates. This method is essentially similar to that described by Espinal and co-workers [9], who used purified bovine kinase, and by Jackson et al. [11], who utilized the residual kinase present in the porcine PDH preparation. Using recombinant human PDHK2, AZD7545 increased PDH activity in a concentration-dependent manner with an EC50 of 5.2 nM (95% confidence limits, 3.2–8.5 nM). In the absence of ATP in the preincubation step, no increased PDH activity was observed, leading us to conclude that PDH activation is kinase-dependent. DCA shows very weak activity (EC50 113 µM); a similar result was obtained by Espinal et al. (EC50 100–150 µM) [9] and even weaker inhibition (EC50 >3000 µM) was seen in the uncertain isoform constitution of the Jackson protocol [11]. Demonstration of PDH activation in intact cells is more technically challenging due to low expression of the enzyme in cultured cells, and requires a sensitive assay. This was resolved by Aicher et al. [12], who monitored the conversion of [14 C]lactate to 14 CO2 in human skin fibroblasts. Primary rat hepatocytes, following overnight culture, are more relevant to target tissue and more amenable to rapid throughput. Cells preincubated with PDHK inhibitor showed a doubling of the rate of conversion of [14 C]pyruvate to 14 CO2 from 2.7 to 4.8 nmol/min per 106 cells. DCA has a similar effect at a high concentration (1 mM), but results with DCA have been more variable, probably due to hepatic metabolism of this compound. The EC50 for AZD7545 in this cell-based assay is 105 nM (95% confidence limits, 69– 160 nM).
PDH activation in vivo PDH activity was assessed ex vivo in tissue extracts via the linked spectrophotometric enzyme assay described by Coore et al. [13]. Activity was assessed before and after complete dephosphorylation by purified pig heart phosphatase. A single acute dose of AZD7545 to fed Wistar rats increased the percentage of active PDH in the liver in a dose-dependent manner from a basal (vehicle-dosed) level of 24.7 ± 6.2% to 70.3 ± 2.6% at the highest dose evaluated (30 mg/kg), where it is evident that maximal activation was not attained (Figure 1). PDH in gastrocnemius muscle is increased at similar doses of compound, but again full activation is not achieved at the doses tested (from 21.1 ± 1.9 to 53.3 ± 4.0%). Aicher et al. [12,14] demonstrated in vivo activity in rats which had undergone a period of fasting; PDH activation was determined either indirectly via plasma lactate [12] or by measuring PDH activity ex vivo [14]. The increases in PDH in liver and tibialis anterior muscle elicited by representative compounds were relatively modest following doses of 20 µmol/kg (approx. 8 mg/kg), but results are not expressed as a proportion of PDH in the active state and hence cannot be compared with results from our own study. C 2003
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Figure 1 Effect of AZD7545 on PDH activity Male Wistar rats (200–240 g) were dosed orally at 08:00 h with AZD7545 in suspension in 0.5% (w/w) methocel/0.1% polysorbate 80. After 2 h animals were anaesthetized with sodium pentabarbitone (60 mg/kg, intraperitoneally) and tissues excised, freeze-clamped and stored in liquid nitrogen prior to assay. Tissue extracts were prepared and PDH activity determined by the method of Coore et al. [13]. Total PDH activity (PDHt) in the extract was determined by assay following dephosphorylation by porcine heart PDP in the presence of 20 mM MgCl2 /0.8 mM CaCl2 . PDH activity is given as the proportion in the active (dephosphorylated) form in extracts from (䊏) liver and (䊐) gastrocnemius muscle. Results are compared with tissues from control, vehicle-treated animals using Student’s t test, where ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001.
Effect of AZD7545 in Zucker (fa/fa) rats The obese ( fa/fa) Zucker rat is a frequently used model of the insulin-resistant or prediabetic state. It exhibits impaired glucose tolerance, hyperphagia, hyperinsulinaemia and hyperlipidaemia. While not overtly hyperglycaemic, the fa/fa rat exhibits an abnormal glucose profile following feeding compared with its lean counterpart (4 h into the dark feeding phase, blood glucose levels are 8.7 mM, compared with 6.1 mM in lean animals). This is associated with a small but consistent and significantly elevated glycated haemoglobin level (3.49 versus 3.26%). At the age used in our study (12 weeks), PDH activity in the fa/fa rat was elevated compared with that in lean Zucker or Wistar rats. We have measured no difference in expression levels of PDHK2 or PDHK4 between obese and lean Zucker rats [15]. As in Wistar rats, PDH in fed fa/fa rats can be further activated by PDHK inhibitors (for example 10 mg/kg AZD7545 increases muscle PDH from 61.0 to 97.0% active, and liver PDH from 33.5 to 72.8%). Obese Zucker rats were treated with the PDHK inhibitor AZD7545 orally for 7 days, and at the end of this period the glucose profile was monitored for 24 h (Figure 2). In control, vehicle-treated rats, blood glucose rose to a maximum of 9.45 ± 1.11 mM, whereas in rats treated with AZD7545 once daily at 08:00 h, the concentration was 6.55 ± 0.58 mM at the
The Glucose/Fatty Acid Cycle 1963–2003
Figure 2 Effect of AZD7545 on 24 h glucose profile Obese male (fa/fa) Zucker rats, housed in a 06:00 h on/18:00 h off light cycle, were dosed for 7 days with either 10 mg/kg AZD7545, given orally, at 08:00 h (䊊) or 08:00 and 18:00 h (䉱) or with vehicle (䊏). On day 8, glucose was measured using a hand-held glucose monitor (GlucotrendTM ).
same time. A similar obliteration of the postprandial glucose elevation was seen after administration twice daily. This is the first report of the testing of a novel PDHK inhibitor in the obese Zucker rat and provides clear evidence that a PDHK inhibitor can improve the control of blood glucose levels in an animal model with impaired glucose homoeostasis. This is in contrast to the statement by Aicher et al. [14] that small-molecule inhibitors were ineffective in animal models of diabetes (ob/ob mice and ZDF rats). Clearly
our data would suggest that an inhibitor of PDHK will be an effective novel therapy for type 2 diabetes.
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