OBJECTIVE-Pyruvate dehydrogenase complex (PDC) serves as the metabolic switch between glucose and fatty acid utilization. PDC activity is inhibited by PDC kinase (PDK). PDC shares the same substrate, i.e., pyruvate, as glyceroneogenesis, a pathway controlling fatty acid release from white adipose tissue (WAT). Thiazolidinediones activate glyceroneogenesis. We studied the regulation by rosiglitazone of PDK2 and PDK4 isoforms and tested the hypothesis that glyceroneogenesis could be controlled by PDK. RESEARCH DESIGN AND METHODS-Rosiglitazone was administered toZucker fa/fa rats, and then PDK4 and PDK2 mRNAs were examined in subcutaneous, periepididymal, and retroperitoneal WAT, liver, and muscle by real-time RT-PCR. Cultured WAT explants from humans and rats and 3T3-F442A adipocytes were rosiglitazone-treated before analyses of PDK2 and PDK4 mRNA and protein. Small interfering RNA (siRNA) was transfected by electroporation. Glyceroneogenesis was determined using [1-14 C]pyruvate incorporation into lipids.RESULTS-Rosiglitazone increased PDK4 mRNA in all WAT depots but not in liver and muscle. PDK2 transcript was not affected. This isoform selectivity was also found in ex vivotreated explants. In 3T3-F442A adipocytes, Pdk4 expression was strongly and selectively induced by rosiglitazone in a direct and transcriptional manner, with a concentration required for halfmaximal effect at 1 nmol/l. The use of dichloroacetic acid or leelamine, two PDK inhibitors, or a specific PDK4 siRNA demonstrated that PDK4 participated in glyceroneogenesis, therefore altering nonesterified fatty acid release in both basal and rosiglitazone-activated conditions.CONCLUSIONS-These data show that PDK4 upregulation in adipocytes participates in the hypolipidemic effect of thiazolidinediones through modulation of glyceroneogenesis. Diabetes

Aims/hypothesis Regulation of glyceroneogenesis and its key enzyme cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) plays a major role in the control of fatty acid release from adipose tissue. Here we investigate the effect of rosiglitazone on the expression of genes involved in fatty acid metabolism and the resulting metabolic consequences.

Hyperinsulinism and hyperammonemia syndrome has been reported as a cause of moderately severe hyperinsulinism with diffuse involvement of the pancreas. The disorder is caused by gain of function mutations in the GLUD1 gene, resulting in a decreased inhibitory effect of guanosine triphosphate on the glutamate dehydrogenase (GDH) enzyme. Twelve unrelated patients (six males, six females) with hyperinsulinism and hyperammonemia syndrome have been investigated. The phenotypes were clinically heterogeneous, with neonatal and infancy-onset hypoglycemia and variable responsiveness to medical (diazoxide) and dietary (leucine-restricted diet) treatment. Hyperammonemia (90 -200 mol/L, normal Ͻ50 mol/L) was constant and not influenced by oral protein, by protein-and leucinerestricted diet, or by sodium benzoate or N-carbamylglutamate administration. The patients had mean basal GDH activity (18.3 Ϯ 0.9 nmol/min/mg protein) not different from controls (17.9 Ϯ 1.8 nmol/min/mg protein) in cultured lymphoblasts. The sensitivity of GDH activity to inhibition by guanosine triphosphate was reduced in all patient lymphoblast cultures (IC 50 , or concentrations required for 50% inhibition of GDH activity, ranging from 140 to 580 nM, compared with control IC 50 value of 83 Ϯ 1.0 nmol/L). The allosteric effect of ADP was within the normal range. The activating effect of leucine on GDH activity varied among the patients, with a significant decrease of sensitivity that was correlated with the negative clinical response to a leucinerestricted diet in plasma glucose levels in four patients. Molecular studies were performed in 11 patients. Heterozygous mutations were localized in the antenna region (four patients in exon 11, two patients in exon 12) as well as in the guanosine triphosphate binding site (two patients in exon 6, two patients in exon 7) of the GLUD1 gene. No mutation has been found in one patient after sequencing the exons 5-13 of the gene. Hyperinsulinism is a common cause of recurrent hypoglycemia in early infancy. It is caused by a permanent increase in insulin secretion (1, 2). Insulin secretion depends on the ATP/ ADP ratio in B cells, which causes their depolarization, enhances Ca 2ϩ influx and the exocytosis of insulin. Glucose and leucine regulate insulin secretion by regulating glutaminolysis by glucose (3) and by the direct action of glucose and leucine on GDH. GDH is a mitochondrial enzyme that oxidizes glutamate to ␣-ketoglutarate using NAD and/or NADP as cofactor (4 -8).A new syndrome associating hyperinsulinism with hyperammonemia, HHS, was recently reported to be a cause of diffuse and moderate hyperinsulinism (9 -11). It results in excessive GDH (EC 1.4.1.3) activity resulting from a change in its regulation through decreased sensitivity to inhibition by GTP. It has been suggested that the elevated oxidation of glutamate to ␣-ketoglutarate stimulates insulin secretion by the pancreatic B cell by increasing the ATP/ADP ratio, although this has yet to be tested experimentally.

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We have identified and sequenced a cDNA that encodes an apparent human orthologue of a yeast protein-X component (ScPDX1) of pyruvate dehydrogenase multienzyme complexes. The new human cDNA that has been referred to as "HsPDX1" cDNA was cloned by use of the "database cloning" strategy and had a 1,506-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 20% identical with that encoded by the yeast PDX1 gene and 40% identical with that encoded by the lipoate acetyltransferase component of the pyruvate dehydrogenase and included a lipoyl-bearing domain that is conserved in some dehydrogenase enzyme complexes. Northern blot analysis demonstrated that the major HsPDX1 mRNA was 2.5 kb in length and was expressed mainly in human skeletal and cardiac muscles but was also present, at low levels, in other tissues. FISH analysis performed with a P1-derived artificial chromosome (PAC)-containing HsPDX1 gene sublocalized the gene to 11p1.3. Molecular investigation of PDX1 deficiency in four patients with neonatal lactic acidemias revealed mutations 78del85 and 965del59 in a homozygous state, and one other patient had no PDX1 mRNA expression.

We report studies of four patients with pyruvate dehydrogenase complex (PDH) deficiency caused by mutations in the E1 alpha subunit. Two unrelated male patients presented with Leigh syndrome and a R263G missense mutation in exon 8. This mutation has previously been described in males with the same phenotype. The two other patients had different novel mutations: (1) an 8-bp deletion at the C-terminus (exon 11) was found in one allele of a young girl suffering from microcephaly and (2) a C88S missense mutation (exon 3) in a boy who only presented with motor neuropathy. These mutations were not found in the mothers of any of the four cases. Immunoblot analysis revealed decreased immunoreactivity for the E1 alpha and E1 beta subunits in three out of the four patients. These findings confirm that: (1) PDH deficiencies are genetically heterogeneous, (2) the R263G mutation is more frequent in male cases than are other mutations and this amino acid is a hot spot for gene mutations, (3) the last eight amino acids may be important for the conformation of the tetrameric E1-PDH enzyme, and (4) the amino acids at positions 88, 263 and 382-387 are essential for the linking of the alpha subunit with the beta subunit and for the activity of the holoenzyme.

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Platelet‐activating factor (Paf‐acether, 1‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphorylcholine) induced full aggregation and a limited release reaction of human platelets in plasma or in blood. Cyclo‐oxygenase inhibition with aspirin only reduced aggregation when induced by threshold amounts of Paf‐acether, whereas higher concentrations surmounted inhibition whether tested in citrated or in heparinized platelet‐rich plasma or blood. Aspirin‐induced inhibition of platelet secretion by Paf‐acether was insurmountable and independent of the anti‐coagulant used. Paf‐acether and adrenaline acted synergistically in inducing aggregation in citrate and in heparin. Aspirin in vitro or after oral ingestion at doses that suppressed aggregation induced by arachidonic acid alone, failed to reduce significantly the synergized aggregation induced by Paf‐acether alone or combined with adrenaline. Twenty‐four hours after the oral ingestion of aspirin, when aggregation by arachidonic acid remained blocked, a slight inhibitory activity on the effect of Paf‐acether noted 4 h after aspirin, had ceased. This was probably accounted for by the synthesis of thromboxane A2 by newly formed platelets, since the in vitro addition of aspirin, or of the thromboxane/endoperoxide receptor inhibitor 13‐azaprostanoic acid caused the 24 h platelets to behave in a manner similar to platelets collected 4 h after aspirin. The α2‐adrenoceptor inhibitor, yohimbine, blocked the direct effect of adrenaline as well as its synergism with Paf‐acether. Since the synergistic effect of Paf‐acether and adrenaline was maintained when thrombin‐degranulated platelets were used, and aspirin remained ineffective against it, it is clear that the augmented platelet responsiveness is not accounted for by the platelet release reaction. Paf‐acether and adrenaline act synergistically and stimulate platelets by cyclo‐oxygenase‐independent mechanisms, which may be relevant in human physiopathological conditions.