Partially active in the fasted state in DKO mice, glucose should contribute more carbon to the synthesis of ketone bodies in these mice. This was examined by measuring the incorporation of carbon from glucose into ketone bodies in wild-type and DKO mice. As anticipated, greater hydroxybutyrate enrichment with 
two carbons was found in the plasma of the DKO mice, which, combined with the greater concentration of ketone bodies in the DKO mice, established that more ketone bodies were produced from glucose in the DKO mice than in the wild-type mice. This finding is consistent with greater flux through the PDH complex with subsequent conversion of acetyl-CoA into ketone bodies. However, the relative contribution of glucose carbon to the formation of ketone bodies was minuscule relative to other carbon sources, which presumably were almost entirely fatty acids. Since serum levels of NEFAs were similar between DKO and wild-type mice, greater availability of fatty acids for oxidation does not explain the increase in ketone bodies. Fasting induces ketoacidosis and hypothermia PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19847069 in the DKO mice Debio1347 web PDHK4-KO mice tolerate fasting without evidence of metabolic decompensation. Since preliminary studies suggested that DKO mice are more sensitive to fasting, the metabolic effects of fasting for various periods 
of time were determined with wild-type, single-KO and DKO mice. Relative to wild-type mice, a modest, but Biochem J. Author manuscript; available in PMC 2015 February 10. Jeoung et al. Page 10 significant, increase in -hydroxybutyrate occurred after 12 h, but not after 24 and 36 h, of fasting in PDHK2-KO mice. In PDHK4-KO mice, acetoacetate was significantly increased after 24 and 36 h of fasting and -hydroxybutyrate after 36 h of fasting. In the DKO mice, fasting induced much higher levels of both ketone bodies throughout the study than observed in the other genotypes. Fasting for 36 h induced nearly a 5-fold increase in acetoacetate in DKO mice MedChemExpress HC-067047 compared with wild-type mice and a 2.5-fold increase compared with PDHK4 KO. In addition, the concentration of hydroxybutyrate was elevated approximately 4-fold in the DKO mice compared with wildtype mice and 2-fold compared with PDHK4-KO mice. Because ketosis can induce metabolic acidosis, the blood pH of the DKO mice was determined. Fasting for 4 h significantly lowered blood pH in the DKO mice compared with wild-type mice. After 24 h of fasting, blood pH of the DKO mice reached dangerously low levels owing to severe ketoacidosis. Unlike the response of the DKO mice, 36 h of fasting did not lower blood pH of PDHK2-KO and PDHK4-KO mice. As expected, with the presence of acidosis, the concentration of bicarbonate was dramatically reduced in the DKO mice compared with wild- type mice . Furthermore, pCO2 was significantly reduced in DKO mice. In addition to suffering from ketoacidosis, the DKO mice experienced hypothermia after 36 h of fasting, leading ultimately to their death. Expression of PDHK4 does not compensate for a lack of PDHK2 in PDHK2-KO mice and vice versa PDHK2 and PDHK4 were measured by Western blot analysis to assess whether altered expression of these proteins compensates for the lack of PDHK2 and PDHK4 in the corresponding KO mice. Protein levels of PDHK2 were not changed in the tissues of the PDHK4-KO mice compared with wild-type mice. Protein levels of PDHK4 were likewise similar in heart, liver and skeletal muscle of PDHK2-KO mice and wild-type mice. These findings suggest that, in the fast.Partially active in the fasted state in DKO mice, glucose should contribute more carbon to the synthesis of ketone bodies in these mice. This was examined by measuring the incorporation of carbon from glucose into ketone bodies in wild-type and DKO mice. As anticipated, greater hydroxybutyrate enrichment with two carbons was found in the plasma of the DKO mice, which, combined with the greater concentration of ketone bodies in the DKO mice, established that more ketone bodies were produced from glucose in the DKO mice than in the wild-type mice. This finding is consistent with greater flux through the PDH complex with subsequent conversion of acetyl-CoA into ketone bodies. However, the relative contribution of glucose carbon to the formation of ketone bodies was minuscule relative to other carbon sources, which presumably were almost entirely fatty acids. Since serum levels of NEFAs were similar between DKO and wild-type mice, greater availability of fatty acids for oxidation does not explain the increase in ketone bodies. Fasting induces ketoacidosis and hypothermia PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19847069 in the DKO mice PDHK4-KO mice tolerate fasting without evidence of metabolic decompensation. Since preliminary studies suggested that DKO mice are more sensitive to fasting, the metabolic effects of fasting for various periods of time were determined with wild-type, single-KO and DKO mice. Relative to wild-type mice, a modest, but Biochem J. Author manuscript; available in PMC 2015 February 10. Jeoung et al. Page 10 significant, increase in -hydroxybutyrate occurred after 12 h, but not after 24 and 36 h, of fasting in PDHK2-KO mice. In PDHK4-KO mice, acetoacetate was significantly increased after 24 and 36 h of fasting and -hydroxybutyrate after 36 h of fasting. In the DKO mice, fasting induced much higher levels of both ketone bodies throughout the study than observed in the other genotypes. Fasting for 36 h induced nearly a 5-fold increase in acetoacetate in DKO mice compared with wild-type mice and a 2.5-fold increase compared with PDHK4 KO. In addition, the concentration of hydroxybutyrate was elevated approximately 4-fold in the DKO mice compared with wildtype mice and 2-fold compared with PDHK4-KO mice. Because ketosis can induce metabolic acidosis, the blood pH of the DKO mice was determined. Fasting for 4 h significantly lowered blood pH in the DKO mice compared with wild-type mice. After 24 h of fasting, blood pH of the DKO mice reached dangerously low levels owing to severe ketoacidosis. Unlike the response of the DKO mice, 36 h of fasting did not lower blood pH of PDHK2-KO and PDHK4-KO mice. As expected, with the presence of acidosis, the concentration of bicarbonate was dramatically reduced in the DKO mice compared with wild- type mice . Furthermore, pCO2 was significantly reduced in DKO mice. In addition to suffering from ketoacidosis, the DKO mice experienced hypothermia after 36 h of fasting, leading ultimately to their death. Expression of PDHK4 does not compensate for a lack of PDHK2 in PDHK2-KO mice and vice versa PDHK2 and PDHK4 were measured by Western blot analysis to assess whether altered expression of these proteins compensates for the lack of PDHK2 and PDHK4 in the corresponding KO mice. Protein levels of PDHK2 were not changed in the tissues of the PDHK4-KO mice compared with wild-type mice. Protein levels of PDHK4 were likewise similar in heart, liver and skeletal muscle of PDHK2-KO mice and wild-type mice. These findings suggest that, in the fast.