Requirement of hepatic pyruvate carboxylase during fasting, high fat, and ketogenic diet

Abstract

Pyruvate has two major fates upon entry into mitochondria, the oxidative decarboxylation to acetyl-CoA via the pyruvate decarboxylase complex or the biotin-dependent carboxylation to oxaloacetate via pyruvate carboxylase (Pcx). Here, we have generated mice with a liver-specific KO of pyruvate carboxylase (PcxL−/−) to understand the role of Pcx in hepatic mitochondrial metabolism under disparate physiological states. PcxL−/− mice exhibited a deficit in hepatic gluconeogenesis and enhanced ketogenesis as expected but were able to maintain systemic euglycemia following a 24 h fast. Feeding a high-fat diet to PcxL−/− mice resulted in animals that were resistant to glucose intolerance without affecting body weight. However, we found that PcxL−/− mice fed a ketogenic diet for 1 week became severely hypoglycemic, demonstrating a requirement for hepatic Pcx for long-term glycemia under carbohydrate-limited diets. Additionally, we determined that loss of Pcx was associated with an induction in the abundance of lysine-acetylated proteins in PcxL−/− mice regardless of physiologic state. Furthermore, liver acetyl-proteomics revealed a biased induction in mitochondrial lysine-acetylated proteins. These data show that Pcx is important for maintaining the proper balance of pyruvate metabolism between oxidative and anaplerotic pathways. Pyruvate has two major fates upon entry into mitochondria, the oxidative decarboxylation to acetyl-CoA via the pyruvate decarboxylase complex or the biotin-dependent carboxylation to oxaloacetate via pyruvate carboxylase (Pcx). Here, we have generated mice with a liver-specific KO of pyruvate carboxylase (PcxL−/−) to understand the role of Pcx in hepatic mitochondrial metabolism under disparate physiological states. PcxL−/− mice exhibited a deficit in hepatic gluconeogenesis and enhanced ketogenesis as expected but were able to maintain systemic euglycemia following a 24 h fast. Feeding a high-fat diet to PcxL−/− mice resulted in animals that were resistant to glucose intolerance without affecting body weight. However, we found that PcxL−/− mice fed a ketogenic diet for 1 week became severely hypoglycemic, demonstrating a requirement for hepatic Pcx for long-term glycemia under carbohydrate-limited diets. Additionally, we determined that loss of Pcx was associated with an induction in the abundance of lysine-acetylated proteins in PcxL−/− mice regardless of physiologic state. Furthermore, liver acetyl-proteomics revealed a biased induction in mitochondrial lysine-acetylated proteins. These data show that Pcx is important for maintaining the proper balance of pyruvate metabolism between oxidative and anaplerotic pathways. The liver can exhibit dramatic metabolic shifts depending on nutritional and/or dietary state. This metabolic flexibility is most aptly demonstrated by the shift between ad libitum feeding and fasting where the liver becomes a net consumer or producer of blood glucose, respectively (1Rui L. Energy metabolism in the liver.Compr. Physiol. 2014; 4: 177-197Crossref PubMed Scopus (1243) Google Scholar). Conversely, the liver is a net producer and then consumer of fatty acids between the carbohydrate replete and fasted states. This shift in macronutrient metabolism is accomplished by shifts in tricarboxylic acid (TCA) cycle flux whereby carbon is partitioned into either the reductive or oxidative branches of the TCA cycle to facilitate gluconeogenesis or fatty acid synthesis. While the fate of pyruvate is unique among the fed and fasted states, pyruvate carboxylase is central to both by generating oxaloacetate (OAA) from pyruvate (2Utter M.F. Keech D.B. Formation of oxaloacetate from pyruvate and carbon dioxide.J. Biol. Chem. 1960; 235: PC17-PC18Abstract Full Text PDF PubMed Google Scholar). Pyruvate entry into mitochondria is accompanied by the concomitant partitioning of pyruvate into two major fates, the oxidative decarboxylation to acetyl-CoA and CO2 via the pyruvate decarboxylase complex or its biotin-dependent carboxylation to OAA via pyruvate carboxylase (3Jitrapakdee S. St Maurice M. Rayment I. Cleland W.W. Wallace J.C. Attwood P.V. Structure, mechanism and regulation of pyruvate carboxylase.Biochem. J. 2008; 413: 369-387Crossref PubMed Scopus (306) Google Scholar). Pyruvate carboxylase contributes to both gluconeogenesis and fatty acid synthesis. Pyruvate carboxylase generates OAA as a mitochondrial-derived gluconeogenic substrate. Alternatively, pyruvate carboxylase generates OAA for the balanced synthesis of citrate by citrate synthase and is therefore highly expressed in lipogenic and steroidogenic tissues. Lipogenesis is a highly cataplerotic process and pyruvate carboxylase is a major mechanism by which the TCA cycle is replenished (4Owen O.E. Kalhan S.C. Hanson R.W. The key role of anaplerosis and cataplerosis for citric acid cycle function.J. Biol. Chem. 2002; 277: 30409-30412Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar). Fatty acid oxidation is required for gluconeogenesis in part by generating ample mitochondrial acetyl-CoA to allosterically activate pyruvate carboxylase in addition to its roles in generating NADH, FADH2, and ATP. The activation of pyruvate carboxylase by acetyl-CoA derived from fatty acids has been proposed to play a major regulatory role in potentiating inappropriate gluconeogenesis in insulin resistance (5Perry R.J. Camporez J.P. Kursawe R. Titchenell P.M. Zhang D. Perry C.J. et al.Hepatic acetyl CoA links adipose tissue inflammation to hepatic insulin resistance and type 2 diabetes.Cell. 2015; 160: 745-758Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). Interfering with this activation could be a beneficial strategy in the treatment of type II diabetes (6Kumashiro N. Beddow S.A. Vatner D.F. Majumdar S.K. Cantley J.L. Guebre-Egziabher F. et al.Targeting pyruvate carboxylase reduces gluconeogenesis and adiposity and improves insulin resistance.Diabetes. 2013; 62: 2183-2194Crossref PubMed Scopus (93) Google Scholar, 7Lao-On U. Attwood P.V. Jitrapakdee S. Roles of pyruvate carboxylase in human diseases: from diabetes to cancers and infection.J. Mol. Med. (Berl). 2018; 96: 237-247Crossref PubMed Scopus (56) Google Scholar). Here, we generated liver-specific pyruvate carboxylase KO mice in order to determine the requirements of hepatic pyruvate carboxylase in the liver and systemic metabolism following disparate nutritional and physiological states. We found that the liver-specific loss of pyruvate carboxylase was surprisingly well tolerated during a 24 h fast and even improved glucose tolerance during high fat feeding. However, a carbohydrate-limited ketogenic diet resulted in rapid metabolic decompensation. These data show the requirement of pyruvate carboxylase mediated anaplerosis in the liver under disparate metabolic conditions. Upon fasting, pyruvate carboxylase is activated by acetyl-CoA derived from fatty acid ß-oxidation to generate OAA from pyruvate for hepatic gluconeogenesis. To better understand the requirements for Pcx in hepatic metabolism, we generated a hepatocyte-specific deletion of pcx in mice. Pcx transgenic mice from the European Mutant Mouse Archive (C57BL/6NTac-Pcx Tm1a) were obtained and bred to Flpe germline deleter mice (Jax #5705) to generate mice with Pcx that contain a floxed exon 10. These mice were then bred to Albumin-Cre mice to generate a hepatocyte-specific deletion of pyruvate carboxylase (PcxL−/−) and littermate controls (Pcxf/f). The loss of Pcx in the liver of PcxL−/− mice was validated at the mRNA and protein level (Fig. 1, A and B). The loss of Pcx did not alter the body weight of male or female mice under fed or fasted conditions (Fig. 1C). The hepatic-specific loss of Pcx resulted in a statistically significant suppression of both fed and fasted blood glucose of PcxL−/− mice (Fig. 1D). However, this small suppression in blood glucose is not likely physiologically significant. The loss of Pcx did result in an increase in circulating lactate, consistent with human inborn errors of pyruvate carboxylase deficiency (OMIM # 266150) (8Haworth J.C. Robinson B.H. Perry T.L. Lactic acidosis due to pyruvate carboxylase deficiency.J. Inherit. Metab. Dis. 1981; 4: 57-58Crossref PubMed Scopus (34) Google Scholar) (Fig. 1E). PcxL−/− mice exhibited normal circulating nonesterified fatty acid (NEFA) and triglycerides (TGs) (Fig. The body was in fed PcxL−/− a on fatty acid oxidation as an (Fig. liver was in PcxL−/− mice following a 24 h fast consistent with an on fatty acid (Fig. the loss of hepatic pyruvate carboxylase resulted in mice that a 24 h fast with surprisingly physiologically normal that PcxL−/− mice were able to maintain circulating fasting glucose we the requirement for pyruvate carboxylase in hepatic gluconeogenesis We from and PcxL−/− mice. We then the with or and the of glucose from (Fig. The loss of pyruvate carboxylase the of to gluconeogenesis as the of glucose in was (Fig. and glucose from (Fig. or (Fig. was consistent with pyruvate carboxylase loss of S. M. et anaplerosis by TCA cycle and metabolism in Metab. Full Text Full Text PDF PubMed Scopus Google Scholar). show that pyruvate carboxylase is required for gluconeogenic can for the loss of hepatic gluconeogenesis during a 24 h fast M. flux of gluconeogenesis in hepatic PubMed Scopus Google Scholar). These data show that a in gluconeogenesis from mitochondria, mice a systemic to fasting We to to understand the of hepatic pyruvate carboxylase on the liver following a 24 h fast The most of in PcxL−/− liver were cycle and were in PcxL−/− liver as well as (Fig. with the increase in cycle a of was (Fig. J.L. L. and Scholar). of acids were in PcxL−/− liver (Fig. hepatic gluconeogenesis is to in insulin resistance and type II To understand the role of pyruvate carboxylase and hepatic gluconeogenesis in insulin we male and female and PcxL−/− mice on a high-fat diet for We did not in body weight between and PcxL−/− mice and we did not in adipose adipose or liver and but not male PcxL−/− mice exhibited a significant increase in weight (Fig. PcxL−/− mice exhibited a significant suppression in blood glucose and were not (Fig. to fed PcxL−/− were in male PcxL−/− mice (Fig. mice fed a exhibited in circulating (Fig. male and female PcxL−/− mice resulted in a suppression in blood glucose and increase in blood and and were in male and female PcxL−/− mice without affecting or and tolerance revealed an in blood glucose tolerance with glucose and insulin in male PcxL−/− mice (Fig. and insulin was improved (Fig. tolerance revealed an in blood glucose in male PcxL−/− mice (Fig. and PcxL−/− mice did not exhibit in glucose or insulin tolerance (Fig. These data show that hepatic pyruvate carboxylase hepatic ketogenesis and improves glucose intolerance in a pyruvate carboxylase KO mice from high-fat glucose fasted blood glucose, lactate, and of male and PcxL−/− mice fed a high fat diet for fasted blood and of male and PcxL−/− mice fed a high fat diet for glucose tolerance of male and PcxL−/− mice fed a high fat diet for under the of glucose tolerance of male and PcxL−/− mice fed a high fat diet for fasted insulin and glucose of male and PcxL−/− mice fed a high fat diet for insulin tolerance of male and PcxL−/− mice fed a high fat diet for of insulin tolerance of male and PcxL−/− mice fed a high fat diet for expressed as nonesterified fatty PcxL−/− mice tolerated a 24 h fast and feeding with normal and improved glucose we the of male and female PcxL−/− mice to maintain glucose under a carbohydrate ketogenic the PcxL−/− and mice body and a suppression of blood glucose and blood consistent with However, of ketogenic diet male and female PcxL−/− mice body weight and exhibited metabolic with in blood glucose and and While mice exhibited a statistically significant physiologically suppression in blood glucose, of a ketogenic diet resulted in a physiologically significant circulating blood Furthermore, ketogenic diet and in male PcxL−/− mice to littermate controls and We circulating of and in PcxL−/− female mice following to ketogenic the of PcxL−/− female mice to (Fig. of and liver were not in male and female but the of PcxL−/− male mice were with and These data the requirement of hepatic pyruvate carboxylase for during carbohydrate-limited conditions. order to the of PcxL−/− mice to fasting, we on liver from 24 h fasted and PcxL−/− mice 1 and of were in PcxL−/− mice with and by at (Fig. of did not the However, were of the was in PcxL−/− mice and in PcxL−/− liver in the and ketogenic states. While has been in hepatic insulin and fatty acid its role and requirement in glucose and has been of for a role of as an of insulin in J. PubMed Scopus Google Scholar, R. D. et deletion of the normal hepatic insulin and glucose PubMed Scopus Google Scholar). A of was and The and were in the fasted and ketogenic (Fig. the and were in fasted PcxL−/− liver (Fig. While was in PcxL−/− liver in the fasted and were in the and ketogenic (Fig. to the has been in the mitochondrial C.J. N. The a mitochondrial Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). These data that PcxL−/− liver is from of 24 h fasted male and PcxL−/− liver that were by of fatty acid acid protein in a The metabolic we in the liver of PcxL−/− mice to protein in PcxL−/− revealed an increase in the abundance of lysine-acetylated proteins to in both the fed and fasting (Fig. fed PcxL−/− liver exhibited abundance of lysine-acetylated proteins even 24 h fasted PcxL−/− mice fed a exhibited a increase in the abundance of lysine-acetylated proteins to the increase by feeding (Fig. However, ketogenic PcxL−/− and mice exhibited a of lysine-acetylated that the increase in is associated with To a of the proteins with an increase in abundance of lysine-acetylated we and the abundance of proteins via We found that the abundance of lysine-acetylated proteins in mitochondria was in the of PcxL−/− consistent with mitochondrial metabolism in mice (Fig. the is that mitochondrial protein is the in PcxL−/− liver with in of mitochondria (Fig. and To the of proteins was by the loss of we on mitochondrial proteins in the of and PcxL−/− of the protein we were even were the most highly lysine-acetylated proteins. These data that the loss of hepatic pyruvate carboxylase is associated with a dramatic increase in the abundance of lysine-acetylated proteins in mitochondria in the regulatory from acetyl-proteomics of 24 h fasted male and PcxL−/− liver in a derived from and via two major fates the mitochondrial To to the oxidative TCA is by the pyruvate complex generating acetyl-CoA and this pyruvate can mitochondria and to The major fate for pyruvate is the biotin-dependent carboxylation to OAA via pyruvate Pyruvate carboxylase is important for hepatic TCA cycle in both the fed and fasted by an anaplerotic for gluconeogenesis and fatty acid Here, we have pyruvate carboxylase in the liver to better understand the of pyruvate mediated anaplerosis to liver and systemic We have that the liver pyruvate carboxylase for but its loss is well tolerated following a 24 h fast with a metabolic the suppression in hepatic gluconeogenesis PcxL−/− mice from glucose However, feeding of carbohydrate to mice in rapid metabolic decompensation. The of pyruvate is into the mitochondrial via the Pyruvate in the mitochondrial J.C. et mitochondrial pyruvate required for pyruvate in and PubMed Scopus Google Scholar, S. S. J.L. N. et and of the mitochondrial pyruvate PubMed Scopus Google Scholar). The loss of either of the of the or is not to mitochondrial pyruvate metabolism as pyruvate can be generated by as or by anaplerosis L. for the mitochondrial pyruvate in revealed by a Biol. PubMed Scopus Google Scholar, et of mitochondrial pyruvate 2 in the liver to in gluconeogenesis and via Metab. 2015; Full Text Full Text PDF PubMed Scopus Google Scholar, L. S.C. et al.Hepatic mitochondrial pyruvate 1 is required for regulation of gluconeogenesis and glucose Metab. 2015; Full Text Full Text PDF PubMed Scopus Google Scholar). is to for the loss of pyruvate we found that pyruvate carboxylase was required for gluconeogenesis and as could not gluconeogenesis in the of pyruvate consistent with the of S. M. et anaplerosis by TCA cycle and metabolism in Metab. Full Text Full Text PDF PubMed Scopus Google Scholar). The for a KO of pyruvate carboxylase be required for of anaplerosis is not Fatty acid oxidation and pyruvate carboxylase fasting or fatty acids in mitochondria to with and to facilitate gluconeogenesis and the carbon for This the liver to blood glucose and for highly oxidative during The loss of pyruvate carboxylase was associated with likely on with disparate inborn errors in fatty acid oxidation exhibit following a fast. Fatty acids to a net increase in the carbon of glucose during gluconeogenesis in of the important roles of hepatic fatty acid oxidation is to gluconeogenesis. this in part by acetyl-CoA to allosterically activate pyruvate we a loss of of liver with a of hepatic fatty acid oxidation and that exhibited a in hepatic gluconeogenesis but exhibited glycemia following a 24 h fast J. J. S. fatty acid oxidation systemic during Full Text Full Text PDF PubMed Scopus Google Scholar). to PcxL−/− animals with a in hepatic fatty acid oxidation exhibited a rapid metabolic following a ketogenic diet at a J. J. S. fatty acid oxidation systemic during Full Text Full Text PDF PubMed Scopus Google Scholar). The between the of mice with liver-specific in pyruvate carboxylase and fatty acid oxidation The liver-specific loss of in a as PcxL−/− mice fasting euglycemia with ketogenesis M. et of blood glucose in the of hepatic glucose during fasting in induction of and gluconeogenesis by PubMed Scopus Google Scholar, N. I. S. et deletion of liver type of Full Text Full Text PDF PubMed Scopus Google Scholar). This that hepatic glucose from as or not required to maintain fasting glycemia and that a to fasting This is likely mediated by the of metabolic of PcxL−/− mice demonstrated a of a mitochondrial the induction of as and These were in the fed but upon metabolic fasting or high fat These a mechanism for and to metabolic PcxL−/− mice as well as mice with a in hepatic fatty acid oxidation resistant to glucose intolerance J. J. L. S. et of hepatic mitochondrial fatty acid oxidation resistance to and glucose Full Text Full Text PDF PubMed Scopus Google Scholar). resistance in two in the exhibit an to gluconeogenesis during carbohydrate replete conditions. insulin is a of fatty acid synthesis. These at and have been insulin resistance J.L. insulin a Metab. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). The mechanism of this insulin has been that the liver insulin and therefore and that gluconeogenesis is mediated by activation of pyruvate carboxylase by acetyl-CoA derived from fatty acid oxidation (5Perry R.J. Camporez J.P. Kursawe R. Titchenell P.M. Zhang D. Perry C.J. et al.Hepatic acetyl CoA links adipose tissue inflammation to hepatic insulin resistance and type 2 diabetes.Cell. 2015; 160: 745-758Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). insulin resistance in the adipose and the to and fatty acid to the liver play a role in gluconeogenesis. from PcxL−/− mice exhibit a dramatic increase in the abundance of lysine-acetylated proteins. 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