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Targeted Upregulation of Pyruvate Dehydrogenase Kinase (PDK)

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Targeted Upregulation of Pyruvate Dehydrogenase Kinase (PDK) Targeted Upregulation of Pyruvate Dehydrogenase Kinase (PDK)-4 in Slow-Twitch Skeletal Muscle Underlies the Stable Modification of the Regulatory Characteristics of PDK Induced by High-Fat Feeding Mark J. Holness, Alexandra Kraus, Robert A. Harris, and Mary C. Sugden In using Western blot analysis with antibodies raised expression may underlie the stable modification of the against recombinant pyruvate dehydrogenase kinase regulatory characteristics of PDK observed in slow- (PDK) isoforms PDK2 and PDK4, this study demon- twitch muscle in response to high-fat feeding. Diabetes strates selective PDK isoform switching in specific 49:775–781, 2000 skeletal muscle types in response to high-fat feeding that is associated with altered regulation of PDK activ- ity by pyruvate. The administration of a diet high in sat- urated fats led to stable (~2-fold) increases in PDK egulation of the activity of the pyruvate dehy- activities in both a typical slow-twitch (soleus [SOL]) drogenase complex (PDC) is an important com- muscle and a typical fast-twitch (anterior tibialis [AT]) ponent of the regulation of glucose homeostasis. muscle. Western blot analysis revealed that high-fat Activation of PDC promotes glucose disposal, feeding significantly increased (~2-fold; P < 0.001) R whereas suppression of PDC activity is crucial to glucose PDK4 protein expression in SOL, with a modest conservation in starvation when 3 carbon compounds (1.3-fold) increase in PDK2 protein expression. The (including pyruvate) are required for gluconeogenesis to relative increase in PDK4 protein expression in SOL was maintain glycemia. PDC is rendered inactive by phospho- associated with a 7.6-fold increase in the pyruvate con- ␣ centration that was required to elicit a 50% active rylation of the -subunit of its pyruvate dehydrogenase com- pyruvate dehydrogenase complex, which indicates a ponent by pyruvate dehydrogenase kinase (PDK) (1,2). PDK marked decrease in the sensitivity of PDK to inhibi- activity is itself regulated. Short-term mechanisms of PDK tion by pyruvate. In AT muscle, high-fat feeding elicited regulation include suppression of activity by pyruvate gen- comparable (1.5- to 1.7-fold) increases (P < 0.05) in erated by glycolysis or from circulating lactate and activation PDK4 and PDK2 protein expression. Loss of sensitivity resulting from high mitochondrial acetyl-CoA/CoA and of PDK to inhibition by pyruvate was less marked. The NADH/NAD+ concentration ratios that are generated by data suggest that a positive correlation exists between increased rates of fatty acid (FA) ␤-oxidation. In addition, increases in PDK4 expression and the propensity with PDK activity can be enhanced by a longer-term mechanism which muscles use lipid-derived fuels as respiratory that is independent of the acute effects of small molecular substrates rather than with the degree of insulin resis- tance induced in skeletal muscles by high-fat feeding. In weight effectors (3). A total of 4 PDK isoforms have been conclusion, high-fat feeding leads to selective upregu- identified in mammalian tissues (4,5). Studies with the indi- lation of PDK4 expression in slow-twitch muscle in vidual recombinant PDK isoforms expressed in Escherichia response to high-fat feeding in vivo, which is associated coli have demonstrated important differences in their specific with a pronounced loss of sensitivity of PDK activity to activities and in their regulatory characteristics (4). In stud- acute inhibition by pyruvate. Thus, increased PDK4 ies in the rat in vivo, stable effects of starvation to enhance PDK activity have been observed in association with the From the Department of Diabetes and Metabolic Medicine (M.J.H., A.K., upregulation of the protein expression of at least 2 of these M.C.S.), Division of General and Developmental Medicine, St. Bartholomew’s 4 PDK isoforms. PDK4 is upregulated in response to starva- and the Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, University of London, London, U.K.; and the Department tion in the heart (6). PDK4 is a high–specific activity PDK iso- of Biochemistry and Molecular Biology (R.A.H.), Indiana University School form whose activity is relatively insensitive to suppression of Medicine, Indianapolis, Indiana. by dichloroacetate (4), which is a highly specific synthetic Address correspondence and reprint requests to M.C. Sugden, MA, DPhil, DSc, Department of Diabetes and Metabolic Medicine, Medical Sci- inhibitor of PDK that is believed to mimic the effect of the ences Bldg., Queen Mary and Westfield College, Mile End Rd., London E1 physiological inhibitor pyruvate. This response to starva- 4NS, U.K. E-mail: [email protected]. tion appears to be specific for PDK4 with no change in pro- Received for publication 11 May 1999 and accepted in revised form 21 January 2000. tein expression of PDK2 (a lower–specific activity pyruvate- [3H]2DG, 2-deoxy-D-[1-3H]glucose; AT, anterior tibialis; ECL, enhanced sensitive isoform) (6). By contrast, PDK2 protein expres- chemiluminescence; FA, fatty acid; GUI, glucose utilization index; PDC, sion is upregulated by starvation both in the liver (7) and in pyruvate dehydrogenase complex; PDHa, active pyruvate dehydrogenase the kidneys (8). This suggests that the responses of individ- complex; PDK, pyruvate dehydrogenase kinase; PPAR-␣, peroxisome pro- ␣ ual PDK isoforms to insulin deficiency are dictated by tissue liferator-activated receptor- ; Rd, glucose disappearance rate; SOL, soleus; TBS, Tris-buffered saline; TBST, Tris-buffered saline with Tween. type and presumably function. DIABETES, VOL. 49, MAY 2000 775 PDK ISOFORM EXPRESSION The skeletal muscle mass is quantitatively of major impor- products, Ely, Cambridgeshire, U.K.). Other chemicals and biochemicals were tance for glucose disposal (9). In nondiabetic Pima Indians (a purchased from Bio-Rad (Hemel Hempstead, Hertfordshire, U.K.), Boehringer population with a high prevalence of type 2 diabetes associ- Mannheim (Lewes, East Sussex, U.K.), or Sigma (Poole, Dorset, U.K.). Female Wistar rats were purchased from Charles River (Margate, Kent, U.K.). Diets ated with obesity), the expression of PDK2 and PDK4 mRNA and individual components of diets were purchased from Special Diet Services in muscle biopsies correlates positively with fasting plasma (Witham, Essex, U.K.). insulin levels (an indicator of insulin resistance) and nega- Rats and diets. Adult female Wistar rats (initial weights 230–250 g) with free tively with insulin-mediated glucose uptake (10). This study access to food and water were housed in individual cages in a temperature- suggested that insufficient downregulation of PDK by insulin (21 ± 2°C) and light-controlled room (a 12-h light–dark cycle). Age-matched rats were randomly allocated to 2 groups. The first group was maintained on in the skeletal muscles of insulin-resistant individuals may be standard high-carbohydrate/low-fat rodent laboratory diet (72% digestible a cause of increased PDK expression, thus leading to carbohydrate, 20% protein, 8% lipid by energy), whereas the second group was impaired glucose oxidation followed by increased FA oxida- maintained on a semisynthetic low-carbohydrate high–saturated fat diet (33% tion (10). Recent findings have also implicated a direct role carbohydrate, 20% protein, and 47% lipid by energy) (17) for 28 days. In all of the experimental protocols, the 2 individual muscles were sampled from the for FA acting via the peroxisome proliferator-activated recep- same animal and were analyzed in parallel. tor-␣ (PPAR-␣) in signaling increased PDK4 expression in Euglycemic-hyperinsulinemic clamps. For the euglycemic-hyperinsulinemic skeletal muscle (11). However, the skeletal muscle mass dis- clamp studies, each rat was fitted with 2 chronic indwelling cannulas. One can- plays considerable heterogeneity regarding glucose utilization nula was placed in the right jugular vein, and the other cannula was placed in (both in the basal [postabsorptive] state and during insulin the left jugular vein (for infusion and sampling, respectively) under Hypnorm (Janssen Pharmaceuticals, Oxford, U.K.) (fentanyl citrate [0.315 mg/ml]/ stimulation) and the capacity for FA utilization. Under rest- fluanisone [10 mg/ml], 1 ml/kg body wt via intraperitoneal injection) and ing conditions, the slow-twitch muscles (e.g., soleus [SOL] Diazepam (Phoenix Pharmaceuticals, Gloucester, U.K.) (5 mg/ml, 1 ml/kg body muscle) display higher rates of glucose utilization (12,13) wt via intraperitoneal injection) anesthesia at 5–7 days before study (18). On and greater insulin sensitivity (13) than the fast-twitch mus- the day of the experiment, food was withdrawn at the end of the dark (feed- cles (e.g., anterior tibialis [AT] muscle). Furthermore, slow- ing) phase, and the rats were studied in the postabsorptive state at 1400 (i.e., 6 h after food withdrawal). Euglycemic-hyperinsulinemic clamps were per- twitch muscles (which contain higher proportions of oxida- formed in conscious unstressed freely moving rats (18). In brief, a primed con- tive fibers [14]) avidly oxidize FA, and glucose utilization is tinuous intravenous infusion of insulin was administered at a fixed rate markedly reduced in these metabolically active muscles to a (4.2 mU · kg body wt–1 · min–1) for 2.5 h. This insulin dose was selected on the greater degree than the reduction in whole-body glucose dis- basis of previous studies demonstrating that it produces plasma insulin con- centrations comparable to those observed after ingesting a carbohydrate-rich posal after high-fat feeding (15). In contrast, fast-twitch mus- meal (16). A variable rate of glucose infusion was initiated at 1 min after the cles containing mainly glycolytic fibers are little affected by start of insulin infusion. Blood was sampled from the right jugular vein at 5- high-fat feeding (15). PDK2 and PDK4 mRNA expression has to 10-min intervals. A steady state was reached after 60–90 min. Coefficients been demonstrated in rat gastrocnemius muscle (4); in the rat, of variation of blood glucose concentrations during the hyperinsulinemic this muscle displays characteristics of fast-twitch muscle clamp were <12% in all studies.
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