Tracking the carbons supplying gluconeogenesis

As the burden of type 2 diabetes mellitus (T2DM) grows in the 21st century, the need to understand glucose metabolism heightens. Increased gluconeogenesis is a major contributor to the hyperglycemia seen in T2DM. Isotope tracer experiments in humans and animals over several decades have offered insights into gluconeogenesis under euglycemic and diabetic conditions. This review focuses on the current understanding of carbon flux in gluconeogenesis, including substrate contribution of various gluconeogenic precursors to glucose production. Alterations of gluconeogenic metabolites and fluxes in T2DM are discussed. We also highlight ongoing knowledge gaps in the literature that require further investigation. A comprehensive analysis of gluconeogenesis may enable a better understanding of T2DM pathophysiology and identification of novel targets for treating hyperglycemia. As the burden of type 2 diabetes mellitus (T2DM) grows in the 21st century, the need to understand glucose metabolism heightens. Increased gluconeogenesis is a major contributor to the hyperglycemia seen in T2DM. Isotope tracer experiments in humans and animals over several decades have offered insights into gluconeogenesis under euglycemic and diabetic conditions. This review focuses on the current understanding of carbon flux in gluconeogenesis, including substrate contribution of various gluconeogenic precursors to glucose production. Alterations of gluconeogenic metabolites and fluxes in T2DM are discussed. We also highlight ongoing knowledge gaps in the literature that require further investigation. A comprehensive analysis of gluconeogenesis may enable a better understanding of T2DM pathophysiology and identification of novel targets for treating hyperglycemia. Glucose serves as a fuel source for many tissues and is the primary source of energy for neurons, renal medullary cells, and red blood cells (1Rizza R.A. Pathogenesis of fasting and postprandial hyperglycemia in type 2 diabetes: implications for therapy.Diabetes. 2010; 59 (20705776): 2697-270710.2337/db10-1032Crossref PubMed Scopus (231) Google Scholar). Circulating blood glucose levels are maintained in a narrow range (3.9–7.1 mmol/liter), and the liver plays a critical role in maintaining glucose homeostasis (2Moore M.C. Coate K.C. Winnick J.J. An Z. Cherrington A.D. Regulation of hepatic glucose uptake and storage in vivo.Adv. Nutr. 2012; 3 (22585902): 286-29410.3945/an.112.002089Crossref PubMed Scopus (218) Google Scholar). The liver stores glucose in the form of glycogen and releases glucose into circulation by either glycogenolysis or gluconeogenesis. In the fed state, hepatic glucose production is suppressed by insulin secretion, and the glucose ingested is stored in part as glycogen. During a short-term fast, the liver maintains euglycemia through glycogenolysis. During longer periods of fasting, as glycogen stores are depleted, the liver relies on gluconeogenesis to maintain euglycemia (3Ekberg K. Landau B.R. Wajngot A. Chandramouli V. Efendic S. Brunengraber H. Wahren J. Contributions by kidney and liver to glucose production in the postabsorptive state and after 60 h of fasting.Diabetes. 1999; 48 (10334304): 292-29810.2337/diabetes.48.2.292Crossref PubMed Scopus (197) Google Scholar). Gluconeogenesis is an intricate process that requires several enzymatic steps (Fig. 1), which are under the regulation of hormones, nutrient intake, stress conditions, and substrate concentrations. Occurring in hepatocytes and renal cortical cells, gluconeogenesis functions as a biosynthetic pathway responsible for countering the glycolytic breakdown of glucose. T2DM, a chronic medical condition characterized by hyperglycemia, has reached pandemic proportions affecting over 400 million adults globally (4Ogurtsova K. da Rocha Fernandes J.D. Huang Y. Linnenkamp U. Guariguata L. Cho N.H. Cavan D. Shaw J.E. Makaroff L.E. IDF Diabetes Atlas: global estimates for the prevalence of diabetes for 2015 and 2040.Diabetes Res. Clin. Pract. 2017; 128 (28437734): 40-5010.1016/j.diabres.2017.03.024Abstract Full Text Full Text PDF PubMed Scopus (2243) Google Scholar). A major pathophysiological tenet of T2DM is increased hepatic gluconeogenesis with rates elevated up to 40% (5Gastaldelli A. Baldi S. Pettiti M. Toschi E. Camastra S. Natali A. Landau B.R. Ferrannini E. Influence of obesity and type 2 diabetes on gluconeogenesis and glucose output in humans: a quantitative study.Diabetes. 2000; 49 (10923639): 1367-137310.2337/diabetes.49.8.1367Crossref PubMed Scopus (255) Google Scholar). In T2DM, gluconeogenesis remains a significant contributor to hepatic glucose production both under fasting conditions and after meal intake (6Petersen K.F. Price T. Cline G.W. Rothman D.L. Shulman G.I. Contribution of net hepatic glycogenolysis to glucose production during the early postprandial period.Am. J. Physiol. 1996; 270 (8772491): E186-E19110.1152/ajpendo.1996.270.1.E186PubMed Google Scholar). Hyperinsulinemia during a hyperinsulinemic-euglycemic clamp, where exogenous insulin is infused as supraphysiologic amounts with concurrent infusion of glucose to maintain a certain blood glucose level, completely suppressed glycogenolysis but only reduced gluconeogenesis by about 20% (7Gastaldelli A. Toschi E. Pettiti M. Frascerra S. Quinones-Galvan A. Sironi A.M. Natali A. Ferrannini E. Effect of physiological hyperinsulinemia on gluconeogenesis in nondiabetic subjects and in type 2 diabetic patients.Diabetes. 2001; 50 (11473042): 1807-181210.2337/diabetes.50.8.1807Crossref PubMed Scopus (118) Google Scholar). Longstanding hyperglycemia is associated with both macrovascular complications, such as heart attacks and stroke, and microvascular complications affecting retinal, renal, and nerve tissues (8Cade W.T. Diabetes-related microvascular and macrovascular diseases in the physical therapy setting.Phys. Ther. 2008; 88 (18801863): 1322-133510.2522/ptj.20080008Crossref PubMed Scopus (571) Google Scholar), which help drive the costs of diabetes care to over $322 billion annually in the United States alone (9Fang M. Trends in the prevalence of diabetes among U.S. adults: 1999–2016.Am. J. Prev. Med. 2018; 55 (30126668): 497-50510.1016/j.amepre.2018.05.018Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The rise in obesity has led to increased prevalence of T2DM and nonalcoholic fatty liver disease (NAFLD). More than 1 in 3 adult Americans have obesity (10Flegal K.M. Kruszon-Moran D. Carroll M.D. Fryar C.D. Ogden C.L. Trends in obesity among adults in the United States, 2005 to 2014.JAMA. 2016; 315 (27272580): 2284-229110.1001/jama.2016.6458Crossref PubMed Scopus (2035) Google Scholar), whereas 1 in 4 have NAFLD (11Andronescu C.I. Purcarea M.R. Babes P.A. Nonalcoholic fatty liver disease: epidemiology, pathogenesis and therapeutic implications.J. Med. Life. 2018; 11 (29696060): 20-23PubMed Google Scholar) and nearly 1 in 10 have T2DM (12Bullard K.M. Cowie C.C. Lessem S.E. Saydah S.H. Menke A. Geiss L.S. Orchard T.J. Rolka D.B. Imperatore G. Prevalence of diagnosed diabetes in adults by diabetes type—United States, 2016.MMWR Morb. Mortal. Wkly. Rep. 2018; 67 (29596402): 359-36110.15585/mmwr.mm6712a2Crossref PubMed Scopus (222) Google Scholar). Gluconeogenesis rates are elevated in patients with obesity even without overt diabetes (5Gastaldelli A. Baldi S. Pettiti M. Toschi E. Camastra S. Natali A. Landau B.R. Ferrannini E. Influence of obesity and type 2 diabetes on gluconeogenesis and glucose output in humans: a quantitative study.Diabetes. 2000; 49 (10923639): 1367-137310.2337/diabetes.49.8.1367Crossref PubMed Scopus (255) Google Scholar) as well in patients with NAFLD (13Fletcher J.A. Deja S. Satapati S. Fu X. Burgess S.C. Browning J.D. Impaired ketogenesis and increased acetyl-CoA oxidation promote hyperglycemia in human fatty liver.JCI Insight. 2019; 410.1172/jci.insight.127737Crossref Scopus (76) Google Scholar). Based on these epidemiologic data, most patients with obesity and NAFLD do not develop overt hyperglycemia, highlighting fundamental differences within these patient populations. Understanding gluconeogenesis across distinct but related metabolic conditions might lead to greater insights into underlying pathophysiology and more targeted therapies. Many studies support the notion that increased gluconeogenesis in T2DM stems from dysregulation of two key gluconeogenic enzymes: phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) (14Chakravarty K. Cassuto H. Reshef L. Hanson R.W. Factors that control the tissue-specific transcription of the gene for phosphoenolpyruvate carboxykinase-C.Crit. Rev. Biochem. Mol. Biol. 2005; 40 (15917397): 129-15410.1080/10409230590935479Crossref PubMed Scopus (176) Google Scholar, 15Barzilai N. Rossetti L. Role of glucokinase and glucose-6-phosphatase in the acute and chronic regulation of hepatic glucose fluxes by insulin.J. Biol. Chem. 1993; 268 (8227065): 25019-25025Abstract Full Text PDF PubMed Google Scholar). PEPCK converts oxaloacetate to phosphoenolpyruvate, allowing Krebs cycle intermediates to contribute to gluconeogenesis (16Petersen M.C. Vatner D.F. Shulman G.I. Regulation of hepatic glucose metabolism in health and disease.Nat. Rev. Endocrinol. 2017; 13 (28731034): 572-58710.1038/nrendo.2017.80Crossref PubMed Scopus (499) Google Scholar). G6Pase converts glucose 6-phosphate to glucose, the final step in gluconeogenesis, which allows glucose to exit the hepatocyte and enter circulation via the GLUT2 hepatocyte transporter (16Petersen M.C. Vatner D.F. Shulman G.I. Regulation of hepatic glucose metabolism in health and disease.Nat. Rev. Endocrinol. 2017; 13 (28731034): 572-58710.1038/nrendo.2017.80Crossref PubMed Scopus (499) Google Scholar, 17Fukumoto H. Seino S. Imura H. Seino Y. Eddy R.L. Fukushima Y. Byers M.G. Shows T.B. Bell G.I. Sequence, tissue distribution, and chromosomal localization of mRNA encoding a human glucose transporter-like protein.Proc. Natl. Acad. Sci. U. S. A. 1988; 85 (3399500): 5434-543810.1073/pnas.85.15.5434Crossref PubMed Scopus (368) Google Scholar). Many hormones regulate PEPCK expression, including glucagon, epinephrine, insulin, and glucocorticoids (14Chakravarty K. Cassuto H. Reshef L. Hanson R.W. Factors that control the tissue-specific transcription of the gene for phosphoenolpyruvate carboxykinase-C.Crit. Rev. Biochem. Mol. Biol. 2005; 40 (15917397): 129-15410.1080/10409230590935479Crossref PubMed Scopus (176) Google Scholar). Similarly, insulin, glucocorticoids, cAMP, and glucose all affect G6Pase expression (18van Schaftingen E. Gerin I. The glucose-6-phosphatase system.Biochem. J. 2002; 362 (11879177): 513-53210.1042/0264-6021:3620513Crossref PubMed Scopus (340) Google Scholar). Given the health burden of T2DM and the public health impact, there has been significant research on underlying disease processes leading to varied pharmacologic therapies for the disease. Despite 14 distinct T2DM medication classes currently approved, hyperglycemia remains a persistent challenge for patients, and physicians need to be mindful of avoiding hypoglycemia and minimizing side effects (19Miller B.R. Nguyen H. Hu C.J.-H. Lin C. Nguyen Q.T. New and emerging drugs and targets for type 2 diabetes: reviewing the evidence.Am. Health Drug Benefits. 2014; 7 (25558307): 452-463PubMed Google Scholar, 20Rines A.K. Sharabi K. Tavares C.D.J. Puigserver P. Targeting hepatic glucose metabolism in the treatment of type 2 diabetes.Nat. Rev. Drug Discov. 2016; 15 (27516169): 786-80410.1038/nrd.2016.151Crossref PubMed Scopus (196) Google Scholar). Thus, novel therapeutic approaches are warranted. To better target gluconeogenesis, a key question becomes the origin of the carbons that account for the increased glucose production in T2DM. Further, many medications for T2DM affect gluconeogenesis rates directly and indirectly (20Rines A.K. Sharabi K. Tavares C.D.J. Puigserver P. Targeting hepatic glucose metabolism in the treatment of type 2 diabetes.Nat. Rev. Drug Discov. 2016; 15 (27516169): 786-80410.1038/nrd.2016.151Crossref PubMed Scopus (196) Google Scholar), although their mechanisms of action could be better known if we had an accurate assessment of gluconeogenesis flux. This review discusses the current understanding of gluconeogenic flux based on isotope tracer data primarily from experiments in humans but also from selected animal and in vitro models to fill in where human data are lacking. We focus on how different gluconeogenic precursors contribute to the process, how these contributions may differ in T2DM, and new findings that may question each precursor's relative role in the process. We also discuss how these precursors' concentrations change in T2DM and how precursors themselves may regulate gluconeogenesis. Given the complexities behind biochemical processes, including gluconeogenesis, researchers have studied metabolites directly to gain insight. The term metabolites refers to all endogenous small molecules (<1,500 Da) involved in metabolic reactions, including substrates, intermediates, and products (21Goodacre R. Vaidyanathan S. Dunn W.B. Harrigan G.G. Kell D.B. Metabolomics by numbers: acquiring and understanding global metabolite data.Trends Biotechnol. 2004; 22 (15109811): 245-25210.1016/j.tibtech.2004.03.007Abstract Full Text Full Text PDF PubMed Scopus (999) Google Scholar). A metabolite's circulating concentration is based on its synthesis, dietary intake, and degradation as well as uptake and release from other body compartments, such as liver, muscle, and adipose tissue (22Umpleby A.M. Hormone measurement guidelines: tracing lipid metabolism: the value of stable isotopes.J. Endocrinol. 2015; 226 (26047888): G1-G1010.1530/JOE-14-0610Crossref PubMed Scopus (17) Google Scholar). Metabolites most directly reflect physiologic and pathologic conditions in an organism. The entire complement of metabolites in cells, tissues, or whole organisms makes up the metabolome, and metabolomics can measure these molecules with precision and accuracy. There are 6,500 and counting discrete metabolites in the human metabolome (23Wishart D.S. Jewison T. Guo A.C. Wilson M. Knox C. Liu Y. Djoumbou Y. Mandal R. Aziat F. Dong E. Bouatra S. Sinelnikov I. Arndt D. Xia J. Liu P. et al.HMDB 3.0–the Human Metabolome Database in 2013.Nucleic Acids Res. 2013; 41 (23161693): D801-D80710.1093/nar/gks1065Crossref PubMed Scopus (2280) Google Scholar, 24Zamboni N. Saghatelian A. Patti G.J. Defining the metabolome: size, flux, and regulation.Mol. Cell. 2015; 58 (26000853): 699-70610.1016/j.molcel.2015.04.021Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). 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Mass appeal: metabolite identification in mass spectrometry-focused untargeted.Metabolomics. 2013; 9: 44-6610.1007/s11306-012-0434-4Crossref Scopus (367) Google Scholar). Paired with isotope-labeled metabolites, targeted metabolomics can measure metabolic flux as heavy atoms from a labeled substrate are detected in downstream metabolic products across different time points. Several different methodologies can help with determining metabolic flux. NMR and MS are two commonly used analytical platforms for metabolite detection and quantification. NMR is a highly reproducible technique that can provide fractional abundance of an isotope at a specific atom position (27Leenders J. Frédérich M. de Tullio P. Nuclear magnetic resonance: a key metabolomics platform in the drug discovery process.Drug Discov. Today Technol. 2015; 13 (26190682): 39-4610.1016/j.ddtec.2015.06.005Crossref PubMed Scopus (24) Google Scholar). For example, a 12C-1H interaction gives a different peak than a 13C-1H interaction on an NMR spectrum. NMR yields significant structural information about a molecule as adjacent nuclei within that molecule interact via spin-spin coupling to produce distinct peaks. Disadvantages of NMR include low sensitivity, making measurement of metabolites with low concentrations difficult (28Shao Y. Le W. Recent advances and perspectives of metabolomics-based investigations in Parkinson's disease.Mol. Neurodegener. 2019; 14 (30634989): 310.1186/s13024-018-0304-2Crossref PubMed Scopus (102) Google Scholar). Because there is little sample preparation with NMR, there is no chromatographic separation of structurally similar compounds leading to overlapping resonances, which can make the charting of biochemical pathways difficult. MS is a highly sensitive technique that can detect metabolites even at low concentrations. MS involves fragmenting labeled or unlabeled compounds through ionization by electron impact ionization or chemical impact ionization (29Wolfe R.R. Chinkes D.L. Wolfe R.R. Isotope Tracers in Metabolic Research: Principles and Practice of Kinetic Analysis. 2nd Ed. Wiley-Liss, Hoboken, NJ2005Google Scholar). After going through the ionization source, fragmented ions pass through a mass analyzer with a specific mass/charge (m/z) ratio and retention time (29Wolfe R.R. Chinkes D.L. Wolfe R.R. Isotope Tracers in Metabolic Research: Principles and Practice of Kinetic Analysis. 2nd Ed. Wiley-Liss, Hoboken, NJ2005Google Scholar). MS can detect the subtle mass differences between isotopes. For example, 3-[13C]lactate (m + 1), which has a label only on lactate's third carbon, has an m/z ratio and retention time in the mass spectrometer different from those of the unlabeled lactate (m + 0). Chromatographic separation provides high resolution even between structurally similar molecules. Disadvantages of MS include the need for sample derivatization, which can lead to sample loss (30Dunn W.B. Broadhurst D. Begley P. Zelena E. Francis-McIntyre S. Anderson N. Brown M. Knowles J.D. Halsall A. Haselden J.N. Nicholls A.W. Wilson I.D. Kell D.B. Goodacre R. Human Serum Metabolome (HUSERMET) ConsortiumProcedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry.Nat. Protoc. 2011; 6 (21720319): 1060-108310.1038/nprot.2011.335Crossref PubMed Scopus (1720) Google Scholar). MS often cannot tell you specifically where in the molecule is the labeled atom (i.e. which carbon is labeled in an M + 1 lactate molecule). For a detailed description of the established methods of measuring gluconeogenesis and glycogenolysis using MS and NMR, please refer to a review by Chung et al. (31Chung S.T. Chacko S.K. Sunehag A.L. Haymond M.W. Measurements of gluconeogenesis and glycogenolysis: a methodological review.Diabetes. 2015; 64 (26604176): 3996-401010.2337/db15-0640Crossref PubMed Scopus (48) Google Scholar). Others have written on the practical applications related to in vivo research with metabolomics (32Kim I.-Y. Suh S.-H. Lee I.-K. Wolfe R.R. Applications of stable, nonradioactive isotope tracers in in vivo human metabolic research.Exp. Mol. Med. 2016; 48 (26795236): e20310.1038/emm.2015.97Crossref PubMed Scopus (73) Google Scholar, 33Chokkathukalam A. Jankevics A. Creek D.J. Achcar F. Barrett M.P. Breitling R. mzMatch-ISO: an R tool for the annotation and relative quantification of isotope-labelled mass spectrometry data.Bioinformatics. 2013; 29 (23162054): 281-28310.1093/bioinformatics/bts674Crossref PubMed Scopus (68) Google Scholar, 34Sas K.M. Karnovsky A. Michailidis G. Pennathur S. 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The two most relevant amino acids for gluconeogenesis are alanine and glutamine. Glutamine gluconeogenesis is predominantly in the kidney, whereas alanine gluconeogenesis is predominantly in the liver (36Stumvoll M. Perriello G. Meyer C. Gerich J. Role of glutamine in human carbohydrate metabolism in kidney and other tissues.Kidney Int. 1999; 55 (10027916): 778-79210.1046/j.1523-1755.1999.055003778.xAbstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Infusion of a carbon-labeled precursor of glucose is commonly used to study gluconeogenesis. Using isotope dilution techniques, the ratio of labeled glucose over the labeled precursor equates to the percentage contribution of the precursor to glucose production. Numerous studies assessing substrate contribution to gluconeogenesis in humans were done in the 1960s–1990s using advanced tools for the time. Results vary based on the isotope tracers used, test conditions, and methods of calculation. Although we cannot cover all tracer experiments conducted, we will highlight relevant studies (Table 1) to illustrate key concepts as well as point out inconsistencies in the literature that require reconciliation.Table 1Direct contribution of gluconeogenesis precursors and glycogen to hepatic glucose production after an overnight fast in humans as determined by isotope tracer experimentsHealthyT2DMLactate7–18% (40Consoli A. Nurjhan N. Reilly J.J. Bier D.M. Gerich J.E. Contribution of liver and skeletal muscle to alanine and lactate metabolism in humans.Am. J. Physiol. 1990; 259 (2240206): E677-E68410.1152/ajpendo.1990.259.5.E677PubMed Google Scholar, 41Jenssen T. Nurjhan N. Consoli A. Gerich J.E. Failure of substrate-induced gluconeogenesis to increase overall glucose appearance in normal humans: demonstration of hepatic autoregulation without a change in plasma glucose concentration.J. Clin. 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Bier D.M. Toft I. Jenssen T.G. Gerich J.E. Glutamine: a major gluconeogenic precursor and vehicle for interorgan carbon transport in man.J. Clin. Invest. 1995; 95 (7814625): 272-27710.1172/JCI117651Crossref PubMed Google Scholar)2-Fold increase (47Stumvoll M. Perriello G. Nurjhan N. Welle S. Gerich J. Bucci A. Jansson P.-A. Dailey G. Bier D. Jenssen T. Gerich J. Glutamine and alanine metabolism in NIDDM.Diabetes. 1996; 45 (8666134): 863-86810.2337/diabetes.45.7.863Crossref PubMed Scopus (51) Google Scholar)Glycerol3–7% (51Nurjhan N. Campbell P.J. Kennedy F.P. Miles J.M. Gerich J.E. Insulin dose-response characteristics for suppression of glycerol release and conversion to glucose in humans.Diabetes. 1986; 35 (3533681): 1326-133110.2337/diabetes.35.12.1326Crossref PubMed Google Scholar52Nurjhan N. Consoli A. Gerich J. Increased lipolysis and its consequences on gluconeogenesis in non-insulin-dependent diabetes mellitus.J. Clin. 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