Regulation of the activity of the PDC (pyruvate dehydrogenase complex) is achieved in large part by phosphorylation of its dehydrogenase component by PDHKs (pyruvate dehydrogenase kinases) and dephosphorylation by PDPs (pyruvate dehydrogenase phosphatases) [ 1 , 2 ]. [Proc Natl Acad Sci U S A. 1969] Review The 2-oxo acid dehydrogenase complexes: recent advances. [J Biol Chem. 1996]. Biochem J. Biochem J. Mice with a deletion in the gene for CCAAT/enhancer-binding protein beta have an attenuated response to cAMP and impaired carbohydrate metabolism. [Biochem J. 1974]. Biochem J. [Biochem J. 1986]. Metabolism. Gene.

Harris* 2

RESULTS. DISCUSSION. References. Abstract The PDC (pyruvate dehydrogenase complex) is strongly inhibited by phosphorylation during starvation to conserve substrates for gluconeogenesis. The role of PDHK4 (pyruvate dehydrogenase kinase isoenzyme 4) in regulation of PDC by this mechanism was investigated with PDHK4&x02212;/&x02212; mice (homozygous PDHK4 knockout mice). Starvation lowers blood glucose more in mice lacking PDHK4 than in wild-type mice. The activity state of PDC (percentage dephosphorylated and active) is greater in kidney, gastrocnemius muscle, diaphragm and heart but not in the liver of starved PDHK4&x02212;/&x02212; mice.

Intermediates of the gluconeogenic pathway are lower in concentration in the liver of starved PDHK4&x02212;/&x02212; mice, consistent with a lower rate of gluconeogenesis due to a substrate supply limitation. The concentration of gluconeogenic substrates is lower in the blood of starved PDHK4&x02212;/&x02212; mice, consistent with reduced formation in peripheral tissues. Isolated diaphragms from starved PDHK4&x02212;/&x02212; mice accumulate less lactate and pyruvate because of a faster rate of pyruvate oxidation and a reduced rate of glycolysis.

BCAAs (branched chain amino acids) are higher in the blood in starved PDHK4&x02212;/&x02212; mice, consistent with lower blood alanine levels and the importance of BCAAs as a source of amino groups for alanine formation. Non-esterified fatty acids are also elevated more in the blood of starved PDHK4&x02212;/&x02212;mice, consistent with lower rates of fatty acid oxidation due to increased rates of glucose and pyruvate oxidation due to greater PDC activity.

Abbreviations: BCAA, branched chain amino acid; DCA, dichloroacetate; DTT, dithiothreitol; PEG, poly(ethylene glycol); PDC, pyruvate dehydrogenase complex; PDHK2, pyruvate dehydrogenase kinase isoenzyme 2; PDHK4&x02212;/&x02212; mice, homozygous PDHK4 knockout mice; PDP, pyruvate dehydrogenase phosphatase; Tos-Phe-CH2Cl, tosylphenylalanylchloromethane

Regulation of the activity of the PDC (pyruvate dehydrogenase complex) is achieved in large part by phosphorylation of its dehydrogenase component by PDHKs (pyruvate dehydrogenase kinases) and dephosphorylation by PDPs (pyruvate dehydrogenase phosphatases) [ 1 , 2 ]. In the well-fed state, PDC is relatively dephosphorylated and therefore active to promote oxidation of three-carbon compounds.

Two of them, PDHK2 (PDHK isoenzyme 2) and PDHK4, increase in response to starvation and diabetes in a tissue-specific manner in the rat [ 9 , 11 , 12 ]. PDHK2 increases significantly in liver and kidney and only slightly or not at all in other tissues. PDHK4 increases greatly in heart, skeletal muscle and kidney, but only slightly in the liver. These observations suggest that control of the amount of PDHK4, and to a lesser extent the amount of PDHK2, is important in long-term regulation of PDC activity and therefore glucose homoeostasis.

Findings of the present study with mice lacking PDHK4 indicate that up-regulation of PDHK4 in peripheral tissues is indeed important for glucose homoeostasis during starvation.

RESULTS.

Genotyping of mice was performed by PCR (40 cycles at 95&x000a0;&x000b0;C for 30&x000a0;s, 57&x000a0;&x000b0;C for 30&x000a0;s and 65&x000a0;&x000b0;C for 4&x000a0;min). Primer 1 (5&x02032;-gactttcataacacccagtctcc-3&x02032;) and primer 2 (5&x02032;-cgcttttctggattcatcgactgtggc-3&x02032;) were used to amplify a 1.4&x000a0;kb fragment from the targeted allele.

Glucose and lactate were measured in tail blood by an Accu-Chek advantage glucometer (Roche, Indianapolis, IN, U.S.A.) and a Lactate Prolactate test meter (Arkray, Shiga, Japan) respectively. Acetoacetate and &x003b2;-hydroxybutyrate [ 14 ], pyruvate [ 15 ] and BCAAs (branched chain amino acids) [ 16 ] were assayed by enzymatic methods with deproteinized serum.

. For the determination of actual PDC activity, pulverized tissue was homogenized in 5 vol. (w/v) of extraction buffer [ 19 ] containing 30&x000a0;mM Hepes/KOH (pH&x000a0;7.5), 0.5&x000a0;mM thiamin pyrophosphate, 3% (v/v) Triton X-100, 5&x000a0;mM EDTA, 2% (v/v) bovine serum, 5&x000a0;mM DTT (dithiothreitol), 10&x000a0;&x003bc;M Tos-Phe-CH2Cl (tosylphenylalanylchloromethane; &x02018;TPCK&x02019;), 10&x000a0;&x003bc;g/ml trypsin inhibitor, 1&x000a0;&x003bc;M leupeptin, 2&x000a0;mM DCA (dichloroacetate) and 50&x000a0;mM KF.

The supernatant obtained by centrifugation at 10000&x000a0;g for 10&x000a0;min at 4&x000a0;&x000b0;C was made 9% (w/v) in PEG [poly(ethylene glycol)] 6000 to precipitate PDC. Pellets produced by centrifugation at 12000&x000a0;g for 10&x000a0;min were suspended in a suspension buffer containing 30&x000a0;mM Hepes/KOH (pH&x000a0;7.5), 1% Triton X-100, 0.2&x000a0;mM EDTA, 2% bovine serum, 1&x000a0;&x003bc;M leupeptin and 5&x000a0;mM DTT.

For the determination of total PDC activity, tissues were homogenized in 5 vol. of the extraction buffer described above but without DCA and KF. The supernatant (200&x000a0;&x003bc;l) obtained by centrifugation of the homogenate at 10000&x000a0;g for 10&x000a0;min at 4&x000a0;&x000b0;C was added to 100&x000a0;&x003bc;l of an activation buffer (suspension buffer containing 25&x000a0;mM MgCl2, 1.5&x000a0;mM CaCl2 and 1&x000a0;&x003bc;g of recombinant PDP1 protein).

INTRODUCTION. EXPERIMENTAL. RESULTS. DISCUSSION. References. RESULTS Generation of PDHK4&x02212;/&x02212; mice

Blood glucose levels of PDHK4&x02212;/&x02212; mice were 30% lower than that of wild-type mice after starvation for 24&x000a0;h ( Figure 2 ). A rebound back to a higher level of blood glucose occurred between 24 and 48&x000a0;h of starvation in wild-type mice but not in PDHK4&x02212;/&x02212; mice ( Figure 2 ).

However, more glycogen was lost from the livers of PDHK4&x02212;/&x02212; mice during 24&x000a0;h of starvation, and analogous to what was observed for blood glucose, less rebound in the amount of liver glycogen occurred between 24 and 48&x000a0;h of starvation in mice lacking PDHK4 ( Figure 2 ). Skeletal-muscle glycogen levels of PDHK4&x02212;/&x02212; mice and wild-type mice were also the same in the fed state (20.3&x000b1;1.6 versus 20.6&x000b1;1.5&x000a0;&x003bc;mol of glucose/g wet weight; means&x000b1;S.E.M.; n=6 in each group).

Since PDHK4 expression is relatively low in tissues of fed rats [ 11 ], lack of PDHK4 was expected to have little or no effect on actual PDC activities (measured as extracted from the tissue without activation by dephosphorylation) and PDC activity states (percentage of total PDC that is active) in tissues of mice in the fed state. Indeed, this was found to be the case for kidney and the gastrocnemius muscle ( Table 5 ).

Since it is well established in studies with rats that starvation decreases the activity state of PDC and increases PDHK4 expression [ 11 ], we expected that the activity states of PDC would be maintained at higher levels during starvation in tissues of mice lacking PDHK4 than in the tissues of wild-type mice. To our surprise, the activity state of PDC was still markedly reduced by starvation in all tissues of PDHK4&x02212;/&x02212; mice examined ( Table 5 ).

Tissue-specific expression of PDHK1 may contribute to the very low activity state induced in the heart by starvation. Nevertheless, a significantly higher PDC activity state was observed in the heart, muscle, kidney and diaphragm of PDHK4&x02212;/&x02212; mice ( Table 5 ), consistent with a role for PDHK4 in inactivation of PDC in these tissues.

The amount of PDHK4 protein in gastrocnemius muscle of wild-type mice was very low in the fed state, but markedly increased by starvation ( Figures 4 A and &x200B;and4B). 4 B). The amounts of PDHK2 protein were similar in gastrocnemius muscle in the two groups of mice in the fed state ( Figure 4 C). Starvation had no effect in wild-type mice but, surprisingly, caused a decrease in PDHK2 protein in PDHK4&x02212;/&x02212; mice.

PDHK4 protein in heart of wild-type mice was very low in the fed state, but markedly increased by starvation ( Figures 5 A and &x200B;and5B). 5 B). PDHK2 protein in heart was greater in PDHK4&x02212;/&x02212; mice, perhaps partially compensating for the absence of PDHK4 ( Figure 5 C). Starvation induced an increase in PDHK2 protein of wild-type mice, but had no effect on the already elevated amount of PDHK2 protein in the PDHK4&x02212;/&x02212; mice.

The response of the kidney was similar to that of the heart ( Figure 6 ). PDHK4 protein was markedly increased in response to starvation in the wild-type mice ( Figures 6 A and &x200B;and6B). 6 B). PDHK2 protein was greater in the heart of fed PDHK4&x02212;/&x02212; mice, but in contrast with the heart, starvation caused an additional increment in PDHK2 protein in the PDHK4&x02212;/&x02212; mice as well as the PDHK4+/+ mice ( Figure 6 C).

The response of the liver to starvation was qualitatively the same as the other tissues ( Figure 7 ). Starvation also increased PDHK4 protein in the liver of wild-type mice ( Figures 7 A and &x200B;and7B). 7 B). PDHK2 protein was similar in amount in the livers of fed PDHK4+/+ mice and PDHK4&x02212;/&x02212; mice ( Figure 7 C). Starvation increased the amounts of PDHK2 protein in both groups of mice.

The amount of PDHK4 protein in the diaphragm of fed wild-type mice was low but detectable by Western-blot analysis ( Figure 8 A).

This is consistent with the low level of PDHK4 expression in tissues in the fed state and, therefore, less effect on the activity state of PDC. After overnight fasting, blood glucose levels are significantly lower in PDHK4&x02212;/&x02212; mice than in wild-type mice, suggesting that the increase in PDHK4 protein is important for maintaining euglycaemia during fasting.

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Activity recording is turned off

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Genes & Expression.

Gene. Protein.

Mouse Genome.