Sepsis induces profound alterations in carbohydrate metabolism which results from the development and clinical complications of this disease. In particular, hyperlactatemia is a major clinical discriminator for organ dysfunction in septic patients. The hyperlactatemia arises from an overproduction of lactate in peripheral tissues which is in excess of the clearing ability of the liver. In skeletal muscle, the increased lactate production results from an accelerated glucose uptake and decreased rate of glucose oxidation. Despite the importance of the mitochondrial oxidative pathway in maintaining lactate homeostasis, the mechanisms underlying the inhibition of glucose oxidation in sepsis have not been elucidated. At the enzyme level, the pyruvate dehydrogenase (PDH) complex is the primary determinant of mitochondrial glucose oxidation in animals and man. The PDH complex is inactivated by a PDH kinase and activated by PDH phosphatases. Our previous studies have provided evidence that sepsis causes a decreased proportion of skeletal muscle PDH complex in the active, unphosphorylated form (PDHa) without altering the total amount of the PDH complex. A decreased PDHa would be expected to limit glucose oxidation and increase the diversion of pyruvate away from oxidation to lactate and alanine formation in muscle. Part of the rationale for implicating a role for the PDH complex in sepsis is the observation that activation of the PDH complex with dichloroacetate normalizes plasma and skeletal muscle lactate concentrations and glucose turnover rates in septic rats to values observed in control rats. The inhibition of the PDH complex in sepsis is mediated by an increased PDH kinase activity. PDH kinase activity may be enhanced by increasing the activity of the PDH kinase intrinsic to the PDH complex and/or another distinct protein, termed kinase activator protein (KAP). There are two potential mechanisms leading to a stimulation of PDH kinase or KAP activity. First, the amount of PDH kinase or KAP protein may be increased. Second, specific activity of the PDH kinase or KAP protein, of which activation through covalent modification is the most obvious example, is stimulated in sepsis. The overall goal of the studies described herein is to provide fundamental information regarding the mechanisms by which sepsis alters the activity of the PDH complex.
The specific aims of the proposal are: l) to investigate whether the increased PDH kinase activity in sepsis is mediated by an increased KAP activity, an increased PDH kinase activity intrinsic to the complex, or a combination of both; (2) to investigate the mechanism(s) responsible for the stimulation of KAP or PDH kinase activity by establishing whether the effect of sepsis results from increased expression or stable activation of these proteins; (3) to investigate whether the sepsis-induced inhibition of the PDH complex activity is mediated by cytokines, particularly tumor necrosis factor or interleukin- l. The studies will involve a number of methodologies including isolation and purification of proteins, peptide microsequencing, cloning of KAP and beta-subunit of the PDH kinase, and immunoblot analysis of KAP and PDH kinase protein. Elucidating the regulation of the PDH kinase and KAP will contribute to a better understanding of the pathologic alterations in glucose metabolism in sepsis.
