Discussion of STAR. J.O.W. was supported by the Swedish

Discussion of STAR. J.O.W. was supported by the Swedish Society for Medical Research. P.M. was supported by the CIHR. S.E.C. and B.R.G. were supported by the NHGRI modENCODE Project, contract U54-HG006994 under DOE contract #DE-AC02-05CH11231. E.C.L.’s group was supported by the Burroughs Wellcome Fund and NIH R01-GM083300 and R01-NS083833. The PDH complex plays a pivotal role in controlling the concentrations of GFT505 chemical information glucose in the fed and fasted state. In the well-fed state, the PDH complex is highly active, promoting glucose oxidation by generating acetyl-CoA, which can be oxidized by the citric acid cycle or used for fatty acid and cholesterol synthesis. In the fasted state, the PDH complex is inactivated by phosphorylation by PDHKs to conserve three carbon compounds for the production of glucose. The four PDHK isoenzymes responsible for phosphorylating the PDH complex are expressed in a tissue-specific manner. Among the four, PDHK2 and PDHK4 are most abundantly expressed in the heart, skeletal muscle and liver of fasted mice. Of these two, PDHK2 is of interest because PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19847069 of its greater sensitivity to activation by acetyl-CoA and NADH and inhibition by pyruvate. However, PDHK4 has received greater attention because its expression is increased in many tissues by fasting and diabetes and transcription of its gene is regulated by insulin, glucocorticoids, thyroid hormone and fatty acids. Inactivation of the PDH complex by phosphorylation helps to maintain euglycaemia during fasting, but contributes to hyperglycaemia in Type 2 diabetics. The increase in PDHK activity in diabetes raises the question of whether the PDHKs should be considered therapeutic targets for the treatment of diabetes. Support for this possibility has been provided by the finding that mice lacking PDHK4 are AZ-6102 web euglycaemic in the fasted state and are more glucose tolerant than wild-type mice fed on a high-fat diet. In the present study, PDHK2-KO mice were produced to determine the importance of this isoform in glucose homoeostasis. In contrast with PDHK4-KO mice, blood glucose levels were not lowered in the fasting state and glucose tolerance was not improved in mice lacking PDHK2. This raised the question of whether the presence of PDHK4 compensates for the lack of PDHK2 and vice versa. To answer this question, PDHK2/PDHK4-DKO mice were produced and characterized. In contrast with the relatively mild phenotypes of the single-KO mice, the DKO mice are unable to tolerate fasting for extended periods of time. The findings show that survival during fasting depends upon inactivation of the PDH complex by PDHK2 and/or PDHK4. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Materials and Methods Generation of the PDHK2/PDHK4-DKO mice The procedures used to generate Pdk4-/- C57BL/6J black mice and Pdk2-/- C57BL/6J black mice have been described previously. PDHK2-KO mice were crossed with PDHK4-KO mice to produce PDHK2/PDHK4-DKO mice. Age-matched wild-type mice were produced from the C57BL/6J black mice that were used to stabilize the genetic backgrounds of the PDHK2-KO and PDHK4-KO mice. Biochem J. Author manuscript; available in PMC 2015 February 10. Jeoung et al. Page 3 Animal protocols Mice were housed in an AALAC approved pathogen-free barrier facility and ad libitum fed on a standard rodent chow diet. Studies were conducted with the approval of the Institutional Animal Care and Use Committee of Indiana University School of Medicine. Blood was collected.Discussion of STAR. J.O.W. was supported by the Swedish Society for Medical Research. P.M. was supported by the CIHR. S.E.C. and B.R.G. were supported by the NHGRI modENCODE Project, contract U54-HG006994 under DOE contract #DE-AC02-05CH11231. E.C.L.’s group was supported by the Burroughs Wellcome Fund and NIH R01-GM083300 and R01-NS083833. The PDH complex plays a pivotal role in controlling the concentrations of glucose in the fed and fasted state. In the well-fed state, the PDH complex is highly active, promoting glucose oxidation by generating acetyl-CoA, which can be oxidized by the citric acid cycle or used for fatty acid and cholesterol synthesis. In the fasted state, the PDH complex is inactivated by phosphorylation by PDHKs to conserve three carbon compounds for the production of glucose. The four PDHK isoenzymes responsible for phosphorylating the PDH complex are expressed in a tissue-specific manner. Among the four, PDHK2 and PDHK4 are most abundantly expressed in the heart, skeletal muscle and liver of fasted mice. Of these two, PDHK2 is of interest because PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19847069 of its greater sensitivity to activation by acetyl-CoA and NADH and inhibition by pyruvate. However, PDHK4 has received greater attention because its expression is increased in many tissues by fasting and diabetes and transcription of its gene is regulated by insulin, glucocorticoids, thyroid hormone and fatty acids. Inactivation of the PDH complex by phosphorylation helps to maintain euglycaemia during fasting, but contributes to hyperglycaemia in Type 2 diabetics. The increase in PDHK activity in diabetes raises the question of whether the PDHKs should be considered therapeutic targets for the treatment of diabetes. Support for this possibility has been provided by the finding that mice lacking PDHK4 are euglycaemic in the fasted state and are more glucose tolerant than wild-type mice fed on a high-fat diet. In the present study, PDHK2-KO mice were produced to determine the importance of this isoform in glucose homoeostasis. In contrast with PDHK4-KO mice, blood glucose levels were not lowered in the fasting state and glucose tolerance was not improved in mice lacking PDHK2. This raised the question of whether the presence of PDHK4 compensates for the lack of PDHK2 and vice versa. To answer this question, PDHK2/PDHK4-DKO mice were produced and characterized. In contrast with the relatively mild phenotypes of the single-KO mice, the DKO mice are unable to tolerate fasting for extended periods of time. The findings show that survival during fasting depends upon inactivation of the PDH complex by PDHK2 and/or PDHK4. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Materials and Methods Generation of the PDHK2/PDHK4-DKO mice The procedures used to generate Pdk4-/- C57BL/6J black mice and Pdk2-/- C57BL/6J black mice have been described previously. PDHK2-KO mice were crossed with PDHK4-KO mice to produce PDHK2/PDHK4-DKO mice. Age-matched wild-type mice were produced from the C57BL/6J black mice that were used to stabilize the genetic backgrounds of the PDHK2-KO and PDHK4-KO mice. Biochem J. Author manuscript; available in PMC 2015 February 10. Jeoung et al. Page 3 Animal protocols Mice were housed in an AALAC approved pathogen-free barrier facility and ad libitum fed on a standard rodent chow diet. Studies were conducted with the approval of the Institutional Animal Care and Use Committee of Indiana University School of Medicine. Blood was collected.