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Tracking the carbons supplying gluconeogenesis

Open AccessPublished:August 13, 2020DOI:https://doi.org/10.1074/jbc.REV120.012758
      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 (
      • Rizza R.A.
      Pathogenesis of fasting and postprandial hyperglycemia in type 2 diabetes: implications for therapy.
      ). 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 (
      • Moore M.C.
      • Coate K.C.
      • Winnick J.J.
      • An Z.
      • Cherrington A.D.
      Regulation of hepatic glucose uptake and storage in vivo.
      ). 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 (
      • Ekberg 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.
      ).
      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.
      Figure thumbnail gr1
      Figure 1Glucose metabolism in the context of glycolysis and gluconeogenesis. α-KG, α-ketoglutarate; G6Pase, glucose-6-phosphatase; OAA, oxaloacetate; PEPCK, phosphoenolpyruvate carboxykinase.
      T2DM, a chronic medical condition characterized by hyperglycemia, has reached pandemic proportions affecting over 400 million adults globally (
      • Ogurtsova 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.
      ). A major pathophysiological tenet of T2DM is increased hepatic gluconeogenesis with rates elevated up to 40% (
      • Gastaldelli 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.
      ). In T2DM, gluconeogenesis remains a significant contributor to hepatic glucose production both under fasting conditions and after meal intake (
      • Petersen 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.
      ). 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% (
      • Gastaldelli 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.
      ). Longstanding hyperglycemia is associated with both macrovascular complications, such as heart attacks and stroke, and microvascular complications affecting retinal, renal, and nerve tissues (
      • Cade W.T.
      Diabetes-related microvascular and macrovascular diseases in the physical therapy setting.
      ), which help drive the costs of diabetes care to over $322 billion annually in the United States alone (
      • Fang M.
      Trends in the prevalence of diabetes among U.S. adults: 1999–2016.
      ).
      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 (
      • Flegal 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.
      ), whereas 1 in 4 have NAFLD (
      • Andronescu C.I.
      • Purcarea M.R.
      • Babes P.A.
      Nonalcoholic fatty liver disease: epidemiology, pathogenesis and therapeutic implications.
      ) and nearly 1 in 10 have T2DM (
      • Bullard 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.
      ). Gluconeogenesis rates are elevated in patients with obesity even without overt diabetes (
      • Gastaldelli 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.
      ) as well in patients with NAFLD (
      • Fletcher 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.
      ). 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) (
      • Chakravarty K.
      • Cassuto H.
      • Reshef L.
      • Hanson R.W.
      Factors that control the tissue-specific transcription of the gene for phosphoenolpyruvate carboxykinase-C.
      ,
      • Barzilai N.
      • Rossetti L.
      Role of glucokinase and glucose-6-phosphatase in the acute and chronic regulation of hepatic glucose fluxes by insulin.
      ). PEPCK converts oxaloacetate to phosphoenolpyruvate, allowing Krebs cycle intermediates to contribute to gluconeogenesis (
      • Petersen M.C.
      • Vatner D.F.
      • Shulman G.I.
      Regulation of hepatic glucose metabolism in health and disease.
      ). 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 (
      • Petersen M.C.
      • Vatner D.F.
      • Shulman G.I.
      Regulation of hepatic glucose metabolism in health and disease.
      ,
      • Fukumoto 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.
      ). Many hormones regulate PEPCK expression, including glucagon, epinephrine, insulin, and glucocorticoids (
      • Chakravarty K.
      • Cassuto H.
      • Reshef L.
      • Hanson R.W.
      Factors that control the tissue-specific transcription of the gene for phosphoenolpyruvate carboxykinase-C.
      ). Similarly, insulin, glucocorticoids, cAMP, and glucose all affect G6Pase expression (
      • van Schaftingen E.
      • Gerin I.
      The glucose-6-phosphatase system.
      ).
      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 (
      • Miller 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.
      ,
      • Rines A.K.
      • Sharabi K.
      • Tavares C.D.J.
      • Puigserver P.
      Targeting hepatic glucose metabolism in the treatment of type 2 diabetes.
      ). 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 (
      • Rines A.K.
      • Sharabi K.
      • Tavares C.D.J.
      • Puigserver P.
      Targeting hepatic glucose metabolism in the treatment of type 2 diabetes.
      ), 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.

      Metabolomics overview

      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 (
      • Goodacre R.
      • Vaidyanathan S.
      • Dunn W.B.
      • Harrigan G.G.
      • Kell D.B.
      Metabolomics by numbers: acquiring and understanding global metabolite data.
      ). 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 (
      • Umpleby A.M.
      Hormone measurement guidelines: tracing lipid metabolism: the value of stable isotopes.
      ). 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 (
      • Wishart 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.
      ,
      • Zamboni N.
      • Saghatelian A.
      • Patti G.J.
      Defining the metabolome: size, flux, and regulation.
      ).
      Nontargeted, or untargeted, metabolomics compares two different biological conditions, including different disease states, genetic alterations, or drug treatments, and identifies metabolite changes in response to a manipulation. This unbiased approach can generate novel hypotheses regarding metabolites and pathways. However, one needs to study metabolic flux (metabolite flow per time) to fully understand pathway activity (
      • Sauer U.
      Metabolic networks in motion: 13C-based flux analysis.
      ). To obtain a more thorough understanding of metabolite regulation and quantify fluxes under various conditions, one must introduce a labeled metabolite and “follow the label” (
      • Dunn W.B.
      • Erban A.
      • Weber R.J.M.
      • Creek D.J.
      • Brown M.
      • Breitling R.
      • Hankemeier T.
      • Goodacre R.
      • Neumann S.
      • Kopka J.
      • Viant M.R.
      Mass appeal: metabolite identification in mass spectrometry-focused untargeted.
      ). 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 (
      • Leenders J.
      • Frédérich M.
      • de Tullio P.
      Nuclear magnetic resonance: a key metabolomics platform in the drug discovery process.
      ). 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 (
      • Shao Y.
      • Le W.
      Recent advances and perspectives of metabolomics-based investigations in Parkinson's disease.
      ). 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 (
      • Wolfe R.R.
      • Chinkes D.L.
      • Wolfe R.R.
      ). After going through the ionization source, fragmented ions pass through a mass analyzer with a specific mass/charge (m/z) ratio and retention time (
      • Wolfe R.R.
      • Chinkes D.L.
      • Wolfe R.R.
      ). 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 (
      • Dunn 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) Consortium
      Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry.
      ). 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. (
      • Chung S.T.
      • Chacko S.K.
      • Sunehag A.L.
      • Haymond M.W.
      Measurements of gluconeogenesis and glycogenolysis: a methodological review.
      ). Others have written on the practical applications related to in vivo research with metabolomics (
      • Kim I.-Y.
      • Suh S.-H.
      • Lee I.-K.
      • Wolfe R.R.
      Applications of stable, nonradioactive isotope tracers in in vivo human metabolic research.
      ,
      • Chokkathukalam 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.
      ,
      • Sas K.M.
      • Karnovsky A.
      • Michailidis G.
      • Pennathur S.
      Metabolomics and diabetes: analytical and computational approaches.
      ,
      • Jang C.
      • Chen L.
      • Rabinowitz J.D.
      Metabolomics and isotope tracing.
      ).

      Carbon contribution to gluconeogenesis

      Given the powerful tools of NMR and MS within metabolomics, one can study how the liver makes glucose under fasting conditions. Glucose is a six-carbon molecule whose concentrations remain relatively constant in the fasted state in metabolically healthy individuals but can rise in subjects with T2DM (
      • Rizza R.A.
      Pathogenesis of fasting and postprandial hyperglycemia in type 2 diabetes: implications for therapy.
      ). Gluconeogenic precursors come from noncarbohydrate sources, including lactate, glycerol, and amino acids. 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 (
      • Stumvoll M.
      • Perriello G.
      • Meyer C.
      • Gerich J.
      Role of glutamine in human carbohydrate metabolism in kidney and other tissues.
      ).
      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 experiments
      HealthyT2DM
      Lactate7–18% (
      • Consoli 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.
      ,
      • Jenssen 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.
      )
      2-Fold increase (
      • Consoli A.
      • Nurjhan N.
      • Reilly J.J.
      • Bier D.M.
      • Gerich J.E.
      Mechanism of increased gluconeogenesis in noninsulin-dependent diabetes mellitus: role of alterations in systemic, hepatic, and muscle lactate and alanine metabolism.
      ,
      • De Meutter R.C.
      • Shreeve W.W.
      Conversion of dl-lactate-2-C14 or -3-C14 or pyruvate-2-C14 to blood glucose in humans: effects of diabetes, insulin, tolbutamide, and glucose load.
      )
      Alanine6–11% (
      • Consoli 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.
      ,
      • Jenssen 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.
      ,
      • Chochinov R.H.
      • Perlman K.
      • Moorhouse J.A.
      Circulating alanine production and disposal in healthy subjects.
      )
      1.5-Fold increase (
      • Perriello G.
      • Pampanelli S.
      • Del Sindaco P.
      • Lalli C.
      • Ciofetta M.
      • Volpi E.
      • Santeusanio F.
      • Brunetti P.
      • Bolli G.B.
      Evidence of increased systemic glucose production and gluconeogenesis in an early stage of NIDDM.
      ,
      • Stumvoll 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.
      ), 0.70-fold decrease (
      • Consoli A.
      • Nurjhan N.
      • Reilly J.J.
      • Bier D.M.
      • Gerich J.E.
      Mechanism of increased gluconeogenesis in noninsulin-dependent diabetes mellitus: role of alterations in systemic, hepatic, and muscle lactate and alanine metabolism.
      ), or no change (
      • Chochinov R.H.
      • Bowen H.F.
      • Moorhouse J.A.
      Circulating alanine disposal in diabetes mellitus.
      )
      Glutamine5–8% (
      • Hankard R.G.
      • Haymond M.W.
      • Darmaun D.
      Role of glutamine as a glucose precursor in fasting humans.
      ,
      • Nurjhan N.
      • Bucci A.
      • Perriello G.
      • Stumvoll M.
      • Dailey G.
      • Bier D.M.
      • Toft I.
      • Jenssen T.G.
      • Gerich J.E.
      Glutamine: a major gluconeogenic precursor and vehicle for interorgan carbon transport in man.
      )
      2-Fold increase (
      • Stumvoll 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.
      )
      Glycerol3–7% (
      • Nurjhan 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.
      • Nurjhan N.
      • Consoli A.
      • Gerich J.
      Increased lipolysis and its consequences on gluconeogenesis in non-insulin-dependent diabetes mellitus.
      ,
      • Puhakainen I.
      • Koivisto V.A.
      • Yki-Järvinen H.
      Lipolysis and gluconeogenesis from glycerol are increased in patients with noninsulin-dependent diabetes mellitus.
      • Baba H.
      • Zhang X.J.
      • Wolfe R.R.
      Glycerol gluconeogenesis in fasting humans.
      )
      1.5-Fold increase (
      • Nurjhan N.
      • Consoli A.
      • Gerich J.
      Increased lipolysis and its consequences on gluconeogenesis in non-insulin-dependent diabetes mellitus.
      ,
      • Puhakainen I.
      • Koivisto V.A.
      • Yki-Järvinen H.
      Lipolysis and gluconeogenesis from glycerol are increased in patients with noninsulin-dependent diabetes mellitus.
      )
      Glycogen40–70% (
      • Petersen 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.
      ,
      • Landau B.R.
      • Wahren J.
      • Chandramouli V.
      • Schumann W.C.
      • Ekberg K.
      • Kalhan S.C.
      Contributions of gluconeogenesis to glucose production in the fasted state.
      • Landau B.R.
      • Wahren J.
      • Chandramouli V.
      • Schumann W.C.
      • Ekberg K.
      • Kalhan S.C.
      Use of 2H2O for estimating rates of gluconeogenesis: application to the fasted state.
      ,
      • Kunert O.
      • Stingl H.
      • Rosian E.
      • Krssak M.
      • Bernroider E.
      • Seebacher W.
      • Zangger K.
      • Staehr P.
      • Chandramouli V.
      • Landau B.R.
      • Nowotny P.
      • Waldhausl W.
      • Haslinger E.
      • Roden M.
      Measurement of fractional whole-body gluconeogenesis in humans from blood samples using 2H nuclear magnetic resonance spectroscopy.
      • Tayek J.A.
      • Katz J.
      Glucose production, recycling, and gluconeogenesis in normals and diabetics: a mass isotopomer [U-13C]glucose study.
      )
      0.5-Fold decrease (
      • Magnusson I.
      • Rothman D.L.
      • Katz L.D.
      • Shulman R.G.
      • Shulman G.I.
      Increased rate of gluconeogenesis in type II diabetes mellitus: a 13C nuclear magnetic resonance study.
      )

      Lactate

      As shown in Fig. 2, the Cori cycle depicts shuttling of lactate from anaerobic glycolysis in skeletal muscle cells to the liver to feed gluconeogenesis (
      • Lehninger A.L.
      • Nelson D.L.
      • Cox M.M.
      ). Many consider lactate the predominant gluconeogenic precursor (
      • Kreisberg R.A.
      Glucose-lactate inter-relations in man.
      ,
      • Kaloyianni M.
      • Freedland R.A.
      Contribution of several amino acids and lactate to gluconeogenesis in hepatocytes isolated from rats fed various diets.
      ,
      • Consoli 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.
      ). Studies in healthy humans have shown that lactate contributes as little as 7% (
      • Jenssen 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.
      ) to as much as 18% (
      • Consoli 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.
      ) to plasma glucose after an overnight fast. Comparing subjects with T2DM and healthy controls, there was a 2-fold increase in lactate incorporation into glucose in T2DM (
      • Consoli A.
      • Nurjhan N.
      • Reilly J.J.
      • Bier D.M.
      • Gerich J.E.
      Mechanism of increased gluconeogenesis in noninsulin-dependent diabetes mellitus: role of alterations in systemic, hepatic, and muscle lactate and alanine metabolism.
      ,
      • De Meutter R.C.
      • Shreeve W.W.
      Conversion of dl-lactate-2-C14 or -3-C14 or pyruvate-2-C14 to blood glucose in humans: effects of diabetes, insulin, tolbutamide, and glucose load.
      ).
      Figure thumbnail gr2
      Figure 2Carbon flow in Cori cycle (A), glucose-alanine cycle (B), and glucose-glutamine cycle (C). Stoichiometry and cofactors for reactions were omitted for clarity. α-KG, α-ketoglutatarate; PEP, phosphoenolpyruvate; OAA, oxaloacetate.

      Alanine

      With muscle catabolism, alanine is released into circulation, undergoes deamination in the liver to become pyruvate, and later glucose as depicted in Fig. 2 (
      • Felig P.
      • Pozefsky T.
      • Marliss E.
      • Cahill G.F.
      Alanine: key role in gluconeogenesis.
      ). Studies have shown a 6–11% contribution of the amino acid to glucose production after an overnight fast in healthy humans (
      • Consoli 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.
      ,
      • Jenssen 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.
      ,
      • Chochinov R.H.
      • Perlman K.
      • Moorhouse J.A.
      Circulating alanine production and disposal in healthy subjects.
      ). The role of alanine's contribution to gluconeogenesis in T2DM remains less clear. Some have documented a 2-fold increase of gluconeogenesis from alanine in subjects with T2DM compared with healthy controls (
      • Perriello G.
      • Pampanelli S.
      • Del Sindaco P.
      • Lalli C.
      • Ciofetta M.
      • Volpi E.
      • Santeusanio F.
      • Brunetti P.
      • Bolli G.B.
      Evidence of increased systemic glucose production and gluconeogenesis in an early stage of NIDDM.
      ,
      • Stumvoll 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.
      ). However, Consoli et al (
      • Consoli A.
      • Nurjhan N.
      • Reilly J.J.
      • Bier D.M.
      • Gerich J.E.
      Mechanism of increased gluconeogenesis in noninsulin-dependent diabetes mellitus: role of alterations in systemic, hepatic, and muscle lactate and alanine metabolism.
      ). concluded that those with T2DM did not have an increase in alanine's contribution to glucose production compared with controls. In a separate study, Chochinov et al. (
      • Chochinov R.H.
      • Bowen H.F.
      • Moorhouse J.A.
      Circulating alanine disposal in diabetes mellitus.
      ) concluded that gluconeogenesis from alanine decreased from 11% in controls to just 3% in subjects with T2DM. These conflicting studies make it difficult to assess what role, if any, alanine has in T2DM hyperglycemia.

      Glutamine

      Glutamine contributes to gluconeogenesis by converting to glutamate, which gets deamidated to α-ketoglutarate (
      • Lehninger A.L.
      • Nelson D.L.
      • Cox M.M.
      ). Fig. 1 shows how α-ketoglutarate can then enter the Krebs cycle and ultimately feed gluconeogenesis. Glutamine contributed 5–8% to glucose production in healthy humans in prior studies (
      • Hankard R.G.
      • Haymond M.W.
      • Darmaun D.
      Role of glutamine as a glucose precursor in fasting humans.
      ,
      • Nurjhan N.
      • Bucci A.
      • Perriello G.
      • Stumvoll M.
      • Dailey G.
      • Bier D.M.
      • Toft I.
      • Jenssen T.G.
      • Gerich J.E.
      Glutamine: a major gluconeogenic precursor and vehicle for interorgan carbon transport in man.
      ). With T2DM, the conversion of glutamine to glucose nearly doubled (
      • Stumvoll 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.
      ).

      Glycerol

      Lipolysis of triglycerides in adipocytes releases glycerol into circulation, which can become glucose in the liver. The contribution of glycerol to glucose production in metabolically healthy humans ranged from 3 to 7% (
      • Nurjhan 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.
      ,
      • Nurjhan N.
      • Consoli A.
      • Gerich J.
      Increased lipolysis and its consequences on gluconeogenesis in non-insulin-dependent diabetes mellitus.
      ,
      • Puhakainen I.
      • Koivisto V.A.
      • Yki-Järvinen H.
      Lipolysis and gluconeogenesis from glycerol are increased in patients with noninsulin-dependent diabetes mellitus.
      ,
      • Baba H.
      • Zhang X.J.
      • Wolfe R.R.
      Glycerol gluconeogenesis in fasting humans.
      ). In T2DM, glycerol's contribution to glucose production increased to 6–10% which was significantly higher compared with healthy controls (
      • Nurjhan N.
      • Consoli A.
      • Gerich J.
      Increased lipolysis and its consequences on gluconeogenesis in non-insulin-dependent diabetes mellitus.
      ,
      • Puhakainen I.
      • Koivisto V.A.
      • Yki-Järvinen H.
      Lipolysis and gluconeogenesis from glycerol are increased in patients with noninsulin-dependent diabetes mellitus.
      ).

      Direct versus net carbon contribution

      Like many biologic processes, the paths of gluconeogenic precursors to glucose production exist in both directions, such that glucose itself can lead to the production of many gluconeogenic precursors. Many tracer studies on gluconeogenesis, including references in this review, primarily report direct carbon contribution of precursors to gluconeogenesis but not net carbon contribution. For example, as in Fig. 3, molecules M and N can contribute to each other's production via reversible reactions. For molecule M, 60% of its flux goes toward the molecule N, whereas 40% goes to the molecule O. For molecule N, only 40% of its flux goes to molecule M, whereas 60% goes to P. If one were to give an isotope-labeled tracer of molecule M, one would see a 60% direct contribution of M to N. However, a dual tracer study with M and N tracers would show smaller net efflux from M to N. In general, introducing a labeled tracer of a molecule can give an idea of where that molecule is going. However, it does not give information about where that specific molecule is coming from. This requires introduction of other labeled substrates to obtain an integrated flux network that can distinguish direct and net contributions.
      Figure thumbnail gr3
      Figure 3Relative fluxes (numbers) for molecules M and N. Given the net efflux from M to N, molecule Q must contribute to the production of molecule M so that molecule M can remain at steady-state concentrations.
      Glucose itself is major contributor to many gluconeogenic precursors. Perriello et al. (
      • Perriello G.
      • Jorde R.
      • Nurjhan N.
      • Stumvoll M.
      • Dailey G.
      • Jenssen T.
      • Bier D.M.
      • Gerich J.E.
      Estimation of glucose-alanine-lactate-glutamine cycles in postabsorptive humans: role of skeletal muscle.
      ) showed in metabolically healthy humans that circulating glucose provided 67, 41, and 13% of the carbons for plasma lactate, alanine, and glutamine, respectively. These contributions were via the Cori cycle, glucose-alanine cycle, and glucose-glutamine cycle (Fig. 2). No human data exist to show how much, if at all, glucose contributes to glycerol production. However, studies in metabolically healthy dogs showed that less than 2% of glycerol's carbons come from glucose (
      • Nurjhan N.
      • Kennedy F.
      • Consoli A.
      • Martin C.
      • Miles J.
      • Gerich J.
      Quantification of the glycolytic origin of plasma glycerol: implications for the use of the rate of appearance of plasma glycerol as an index of lipolysis in vivo.
      ).
      Given the reciprocal fluxes between glucose and its precursors, infusing only 13C tracers of gluconeogenic precursors may not be enough. As the direct and net contribution of a precursor may not be congruent, studies that also give a [13C6]glucose tracer are needed to determine the source of non-glucose-derived carbons that fuel gluconeogenesis.
      The difference between direct and net contribution may not merely be academic but also practical. A better understanding of gluconeogenesis determinants could lead to more rationally designed and targeted T2DM treatments. For example, one could find a pharmacologic means to stop hepatic conversion of a specific gluconeogenesis substrate to glucose by blocking either key enzymes or substrate-specific transporters in the liver. Alternatively, one could block reabsorption of specific gluconeogenesis substrates in the renal tubules, leading to increased urinary excretion analogous to currently approved sodium-glucose co-transporter-2 inhibitors, which block renal reabsorption of glucose (
      • Hsia D.S.
      • Grove O.
      • Cefalu W.T.
      An update on sodium-glucose co-transporter-2 inhibitors for the treatment of diabetes mellitus.
      ). Such theoretical agents would need to be tested in preclinical models and assessed for off-target metabolic effects.

      Fluxes between gluconeogenic precursors

      Not only can gluconeogenic precursors contribute to glucose production, they can also contribute to the production of other gluconeogenic precursors. Returning to our example, in Fig. 3, for concentrations of molecule M to remain constant, molecule Q must feed molecule M. Otherwise, molecule M concentrations would decrease, given its net effluxes toward molecules N and O.
      Lactate may be an important direct carbon contributor to gluconeogenesis, given its high turnover and intimate coupling with glucose via the Cori cycle. However, the Cori cycle is not a net producer of glucose and cannot sufficiently explain the hyperglycemia seen in T2DM (
      • Kreisberg R.A.
      • Pennington L.F.
      • Boshell B.R.
      Lactate turnover and gluconeogenesis in normal and obese humans: effect of starvation.
      ). Similarly, many argue that the glucose-alanine cycle does not provide net substrate for gluconeogenesis and that its main physiologic function is ammonia transport (
      • Felig P.
      • Pozefsky T.
      • Marliss E.
      • Cahill G.F.
      Alanine: key role in gluconeogenesis.
      ,
      • Chochinov R.H.
      • Perlman K.
      • Moorhouse J.A.
      Circulating alanine production and disposal in healthy subjects.
      ). Therefore, for increased gluconeogenesis to contribute to T2DM hyperglycemia, the carbons supplying gluconeogenesis must come from other substrates.
      Recently our group has shown in mice, using [13C3]lactate, [13C3]glycerol, and [13C3]glucose tracers administered over different experimental days, that lactate is mainly recycled during the fasting period such that lactate is the largest direct contributor to gluconeogenesis but provides minimal new glucose carbons (
      • Wang Y.
      • Kwon H.
      • Su X.
      • Wondisford F.E.
      Glycerol not lactate is the major net carbon source for gluconeogenesis in mice during both short and prolonged fasting.
      ). In the same study, glycerol contributed to the carbons of glucose both by direct conversion in the liver and by first converting to lactate, which then became glucose. Glycerol was the most significant source of new carbons for gluconeogenesis after a 12-h fast, contributing over 50% of the net carbons for gluconeogenesis. Lactate contributed a small proportion to glycerol molecules in mice, and this coincides with rat data using a single intraperitoneal injection of [13C3]lactate that labeled intramuscular glycerol (
      • Jin E.S.
      • Sherry A.D.
      • Malloy C.R.
      Lactate contributes to glyceroneogenesis and glyconeogenesis in skeletal muscle by reversal of pyruvate kinase.
      ). The enzymes glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase are needed to generate glycerol from the triose phosphate pool (
      • Possik E.
      • Madiraju S.R.M.
      • Prentki M.
      Glycerol-3-phosphate phosphatase/PGP: role in intermediary metabolism and target for cardiometabolic diseases.
      ). To date, no human experiments have assessed how glycerol and lactate may contribute to each other's production.
      Glycerol may have two different metabolic fates based on the initial site of metabolism, and several tissues have high expression of glycerol kinase, which converts glycerol to glycerol 3-phosphate (
      • Dipple K.M.
      • Zhang Y.H.
      • Huang B.L.
      • McCabe L.L.
      • Dallongeville J.
      • Inokuchi T.
      • Kimura M.
      • Marx H.J.
      • Roederer G.O.
      • Shih V.
      • Yamaguchi S.
      • Yoshida I.
      • McCabe E.R.
      Glycerol kinase deficiency: evidence for complexity in a single gene disorder.
      ). Glycerol 3-phosphate can then enter the triose phosphate pool as an intermediate for glycolysis or gluconeogenesis (Fig. 1). The liver and kidney have high expression of glycerol kinase so that glycerol can contribute directly to glucose production in these two gluconeogenic organs (
      • Uhlen M.
      • Fagerberg L.
      • Hallstrom B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • Sivertsson A.
      • Kampf C.
      • Sjostedt E.
      • Asplund A.
      • Olsson I.
      • Edlund K.
      • Lundberg E.
      • Navani S.
      • Szigyarto C.A.-K.
      • et al.
      Proteomics: tissue-based map of the human proteome.
      ). Peripheral tissues such as intestines, lymphatics, and spleen also express glycerol kinase but not gluconeogenic enzymes, allowing glycerol to become a source for lactate (
      • Uhlen M.
      • Fagerberg L.
      • Hallstrom B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • Sivertsson A.
      • Kampf C.
      • Sjostedt E.
      • Asplund A.
      • Olsson I.
      • Edlund K.
      • Lundberg E.
      • Navani S.
      • Szigyarto C.A.-K.
      • et al.
      Proteomics: tissue-based map of the human proteome.
      ). The fates of glycerol delivered directly to gluconeogenic and nongluconeogenic organs have not been directly tested in humans. This would require invasive cannulation of certain arteries and veins to isolate certain organs, adding significant risks for subjects.
      Infusing [13C3]glycerol into metabolically healthy humans that fasted for 60 h, Landau et al. (
      • Landau B.R.
      • Wahren J.
      • Previs S.F.
      • Ekberg K.
      • Chandramouli V.
      • Brunengraber H.
      Glycerol production and utilization in humans: sites and quantitation.
      ) estimated that the gastrointestinal, renal, and muscle tissues accounted for 63% of the glycerol utilization and that the remaining 37% must be metabolized by other tissues that express the enzyme glycerol kinase. Knowing which tissues in the body process this remaining glycerol can help us further understand glycerol's role in T2DM hyperglycemia. In T2DM, where there is increased lipolysis and circulating glycerol, it is unknown whether that additional circulating glycerol gets evenly distributed between gluconeogenic or nongluconeogenic organs or if one set of organs has an increased metabolism of the substrate.
      Alanine can also supplement the lactate pool via a pyruvate intermediate, and one study showed that alanine contributed 16% of the carbons to circulating lactate in healthy humans (
      • Chochinov R.H.
      • Perlman K.
      • Moorhouse J.A.
      Circulating alanine production and disposal in healthy subjects.
      ). In contrast, a study in healthy dogs showed that lactate contributed to 70% of the alanine pool (
      • Wolfe R.R.
      • Jahoor F.
      • Miyoshi H.
      Evaluation of the isotopic equilibration between lactate and pyruvate.
      ). There are no known human studies to assess how much glutamine contributes to lactate production or vice versa. However, in vitro work from human fibroblasts has depicted glutamine converting to lactate (
      • Zielke H.R.
      • Sumbilla C.M.
      • Sevdalian D.A.
      • Hawkins R.L.
      • Ozand P.T.
      Lactate: a major product of glutamine metabolism by human diploid fibroblasts.
      ).
      Glutamine and alanine can contribute to each other's production as glutamine-derived glutamate can interchange with alanine via the enzyme alanine aminotransferase (
      • Kovacevic Z.
      • McGivan J.D.
      Mitochondrial metabolism of glutamine and glutamate and its physiological significance.
      ). Using [14C5]glutamate and 3-[13C]alanine tracers at the same time, studies have shown glutamine to be more quantitatively important in delivering protein-derived carbons to glucose in both healthy human subjects (
      • Nurjhan N.
      • Bucci A.
      • Perriello G.
      • Stumvoll M.
      • Dailey G.
      • Bier D.M.
      • Toft I.
      • Jenssen T.G.
      • Gerich J.E.
      Glutamine: a major gluconeogenic precursor and vehicle for interorgan carbon transport in man.
      ) and those with T2DM (
      • Stumvoll 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.
      ).
      Finally, it is unknown whether amino acids and glycerol provide any carbon contribution to each other. Prior tracer studies gave labeled gluconeogenic substrates and assessed a limited number of downstream products. However, the metabolome is more interconnected, and gluconeogenic precursors can supply each other with carbons directly or through intermediate metabolites.
      Given the various fluxes between gluconeogenic precursors as shown in Fig. 4 in humans with and without T2DM, there is a need for comprehensive experiments spanning multiple tracers in the same human or animal subject to assess the carbon flow. Such experiments may yield information that may lead to certain precursors as the significant culprit carbon contributors. Blocking these substrates pharmacologically from becoming glucose or other gluconeogenic precursors could be a potential treatment strategy. Blocking one pathway may or may not be sufficient to lower hepatic glucose production, as pathways may be redundant or leaky such that overall glucose production may not be affected even if one precursor is prevented from becoming glucose. However, attempts at blocking such pathways can only be made with a sound understanding of carbon flux in the first place.
      Figure thumbnail gr4
      Figure 4Schematic of gluconeogenesis recognizing fluxes between glucose and its precursors along with fluxes between the precursors themselves.

      Changes to gluconeogenic precursor levels in T2DM

      Whereas it is critical to study metabolic fluxes of gluconeogenic precursors, it is also pertinent to look at circulating concentrations of precursors under healthy and T2DM conditions. For the liver to make an excess of glucose in T2DM, one might expect circulating precursor levels to change in T2DM compared with metabolically heathy controls. Precursor levels could be elevated to allow for increased hepatic substrate delivery, or precursor levels could decrease due to increased hepatic substrate utilization. Both increased substrate delivery and utilization could also occur without affecting overall circulating precursor levels. Accounting for both metabolite levels and fluxes may yield a more thorough understanding of gluconeogenic changes in T2DM.

      Lactate

      Plasma lactate levels are elevated in T2DM compared with metabolically healthy humans in some studies (
      • Consoli A.
      • Nurjhan N.
      • Capani F.
      • Gerich J.
      Predominant role of gluconeogenesis in increased hepatic glucose production in NIDDM.
      ,
      • Crawford S.O.
      • Hoogeveen R.C.
      • Brancati F.L.
      • Astor B.C.
      • Ballantyne C.M.
      • Schmidt M.I.
      • Young J.H.
      Association of blood lactate with type 2 diabetes: the Atherosclerosis Risk in Communities Carotid MRI Study.
      ) but not all (
      • Consoli A.
      • Nurjhan N.
      • Reilly J.J.
      • Bier D.M.
      • Gerich J.E.
      Mechanism of increased gluconeogenesis in noninsulin-dependent diabetes mellitus: role of alterations in systemic, hepatic, and muscle lactate and alanine metabolism.
      ). Lactate turnover, or the amount of lactate appearing in circulation at any given moment, is also increased in T2DM (
      • Consoli A.
      • Nurjhan N.
      • Reilly J.J.
      • Bier D.M.
      • Gerich J.E.
      Mechanism of increased gluconeogenesis in noninsulin-dependent diabetes mellitus: role of alterations in systemic, hepatic, and muscle lactate and alanine metabolism.
      ). Increased levels of lactate occur in obesity due to decreased blood flow in adipose tissue causing local hypoxia and increased lactate production (
      • Hosogai N.
      • Fukuhara A.
      • Oshima K.
      • Miyata Y.
      • Tanaka S.
      • Segawa K.
      • Furukawa S.
      • Tochino Y.
      • Komuro R.
      • Matsuda M.
      • Shimomura I.
      Adipose tissue hypoxia in obesity and its impact on adipocytokine dysregulation.
      ). Insulin resistance in skeletal muscles was associated with decreased oxidative capacity and greater lactate production (
      • Lowell B.B.
      • Shulman G.I.
      Mitochondrial dysfunction and type 2 diabetes.
      ,
      • Del Prato S.
      • Bonadonna R.C.
      • Bonora E.
      • Gulli G.
      • Solini A.
      • Shank M.
      • DeFronzo R.A.
      Characterization of cellular defects of insulin action in type 2 (non-insulin-dependent) diabetes mellitus.
      ,
      • Kelley D.E.
      • Slasky B.S.
      • Janosky J.
      Skeletal muscle density: effects of obesity and non-insulin-dependent diabetes mellitus.
      ). Despite these mechanisms, the carbon sources for the increased lactate level in T2DM would still be primarily glucose, so lactate alone could not account for hyperglycemia seen in T2DM. Of note, there are no storage reservoirs of lactate in the body compared with amino acids (skeletal muscle tissue) and glycerol (adipose tissue).

      Amino acids

      Circulating alanine levels were increased in T2DM in some studies (
      • Consoli A.
      • Nurjhan N.
      • Capani F.
      • Gerich J.
      Predominant role of gluconeogenesis in increased hepatic glucose production in NIDDM.
      ,
      • Chen S.
      • Akter S.
      • Kuwahara K.
      • Matsushita Y.
      • Nakagawa T.
      • Konishi M.
      • Honda T.
      • Yamamoto S.
      • Hayashi T.
      • Noda M.
      • Mizoue T.
      Serum amino acid profiles and risk of type 2 diabetes among Japanese adults in the Hitachi Health Study.
      ) but not others (
      • Consoli A.
      • Nurjhan N.
      • Reilly J.J.
      • Bier D.M.
      • Gerich J.E.
      Mechanism of increased gluconeogenesis in noninsulin-dependent diabetes mellitus: role of alterations in systemic, hepatic, and muscle lactate and alanine metabolism.
      ,
      • Stumvoll 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.
      ,
      • Chochinov R.H.
      • Bowen H.F.
      • Moorhouse J.A.
      Circulating alanine disposal in diabetes mellitus.
      ). Alanine turnover was also increased in T2DM in the studies that have measured it (
      • Consoli A.
      • Nurjhan N.
      • Reilly J.J.
      • Bier D.M.
      • Gerich J.E.
      Mechanism of increased gluconeogenesis in noninsulin-dependent diabetes mellitus: role of alterations in systemic, hepatic, and muscle lactate and alanine metabolism.
      ,
      • Stumvoll 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.
      ). Some studies have shown that increased glutamine levels were associated with a decreased risk of developing T2DM (
      • Chen S.
      • Akter S.
      • Kuwahara K.
      • Matsushita Y.
      • Nakagawa T.
      • Konishi M.
      • Honda T.
      • Yamamoto S.
      • Hayashi T.
      • Noda M.
      • Mizoue T.
      Serum amino acid profiles and risk of type 2 diabetes among Japanese adults in the Hitachi Health Study.
      ,
      • Stancakova A.
      • Civelek M.
      • Saleem N.K.
      • Soininen P.
      • Kangas A.J.
      • Cederberg H.
      • Paananen J.
      • Pihlajamaki J.
      • Bonnycastle L.L.
      • Morken M.A.
      • Boehnke M.
      • Pajukanta P.
      • Lusis A.J.
      • Collins F.S.
      • Kuusisto J.
      • et al.
      Hyperglycemia and a common variant of GCKR are associated with the levels of eight amino acids in 9,369 Finnish men.
      ,
      • Ferrannini E.
      • Natali A.
      • Camastra S.
      • Nannipieri M.
      • Mari A.
      • Adam K.-P.
      • Milburn M.V.
      • Kastenmüller G.
      • Adamski J.
      • Tuomi T.
      • Lyssenko V.
      • Groop L.
      • Gall W.E.
      Early metabolic markers of the development of dysglycemia and type 2 diabetes and their physiological significance.
      ), whereas others showed no association between glutamine levels and T2DM risk (
      • Wang T.J.
      • Larson M.G.
      • Vasan R.S.
      • Cheng S.
      • Rhee E.P.
      • McCabe E.
      • Lewis G.D.
      • Fox C.S.
      • Jacques P.F.
      • Fernandez C.
      • O'Donnell C.J.
      • Carr S.A.
      • Mootha V.K.
      • Florez J.C.
      • Souza A.
      • et al.
      Metabolite profiles and the risk of developing diabetes.
      ,
      • Floegel A.
      • Stefan N.
      • Yu Z.
      • Mühlenbruch K.
      • Drogan D.
      • Joost H.-G.
      • Fritsche A.
      • Häring H.-U.
      • Hrabě de Angelis M.
      • Peters A.
      • Roden M.
      • Prehn C.
      • Wang-Sattler R.
      • Illig T.
      • Schulze M.B.
      • et al.
      Identification of serum metabolites associated with risk of type 2 diabetes using a targeted metabolomic approach.
      ,
      • Tillin T.
      • Hughes A.D.
      • Wang Q.
      • Würtz P.
      • Ala-Korpela M.
      • Sattar N.
      • Forouhi N.G.
      • Godsland I.F.
      • Eastwood S.V.
      • McKeigue P.M.
      • Chaturvedi N.
      Diabetes risk and amino acid profiles: cross-sectional and prospective analyses of ethnicity, amino acids and diabetes in a South Asian and European cohort from the SABRE (Southall And Brent REvisited) Study.
      ). One study comparing glutamine turnover in T2DM and healthy controls showed no difference between the two cohorts (
      • Stumvoll 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.
      ).
      Insulin is an anabolic hormone that promotes protein synthesis and prevents its breakdown (
      • Adegoke O.A.J.
      • Chevalier S.
      • Morais J.A.
      • Gougeon R.
      • Kimball S.R.
      • Jefferson L.S.
      • Wing S.S.
      • Marliss E.B.
      Fed-state clamp stimulates cellular mechanisms of muscle protein anabolism and modulates glucose disposal in normal men.
      ). Chevalier et al. (
      • Chevalier S.
      • Burgess S.C.
      • Malloy C.R.
      • Gougeon R.
      • Marliss E.B.
      • Morais J.A.
      The greater contribution of gluconeogenesis to glucose production in obesity is related to increased whole-body protein catabolism.
      ) showed that increased protein catabolism in obese nondiabetic subjects correlated with gluconeogenesis derived from amino acids. However, in T2DM, a condition with insulin resistance and higher compensatory insulin levels, muscle turnover as assessed by leucine turnover was unchanged in two studies (
      • Sreekumar R.
      • Halvatsiotis P.
      • Schimke J.C.
      • Nair K.S.
      Insulin effect on leucine kinetics in type 2 diabetes mellitus.
      ,
      • Pereira S.
      • Marliss E.B.
      • Morais J.A.
      • Chevalier S.
      • Gougeon R.
      Insulin resistance of protein metabolism in type 2 diabetes.
      ). Under isoaminoacidemic, hyperinsulinemic, euglycemic clamp conditions where amino acid, insulin levels, and glucose levels are held at constant levels by exogenous infusions, protein anabolism was blunted in men with T2DM compared with healthy controls (
      • Pereira S.
      • Marliss E.B.
      • Morais J.A.
      • Chevalier S.
      • Gougeon R.
      Insulin resistance of protein metabolism in type 2 diabetes.
      ). This suggests a defect in protein synthesis under insulin-resistant conditions in men. In women, this effect was not seen, leading to the potential sex differences in protein metabolism under diabetic conditions. T2DM is associated with lower skeletal muscle mass (
      • Munhoz da Rocha Lemos Costa T.
      • Costa F.M.
      • Jonasson T.H.
      • Moreira C.A.
      • Boguszewski C.L.
      • Borba V.Z.C.
      Body composition and sarcopenia in patients with chronic obstructive pulmonary disease.
      ,
      • Mesinovic J.
      • Zengin A.
      • De Courten B.
      • Ebeling P.R.
      • Scott D.
      Sarcopenia and type 2 diabetes mellitus: a bidirectional relationship.
      ), and decreased muscle mass is correlated with poorer glycemic control (
      • Sugimoto K.
      • Tabara Y.
      • Ikegami H.
      • Takata Y.
      • Kamide K.
      • Ikezoe T.
      • Kiyoshige E.
      • Makutani Y.
      • Onuma H.
      • Gondo Y.
      • Ikebe K.
      • Ichihashi N.
      • Tsuboyama T.
      • Matsuda F.
      • Kohara K.
      • et al.
      Hyperglycemia in non-obese patients with type 2 diabetes is associated with low muscle mass: the multicenter study for clarifying evidence for sarcopenia in patients with diabetes mellitus.
      ). Given these findings of decreased muscle mass with variable amino acid flux in T2DM, it remains difficult to quantify how much amino acids supply gluconeogenic carbons under diabetic conditions.

      Glycerol

      Circulating glycerol concentrations and glycerol turnover were consistently higher in subjects with T2DM compared with healthy controls in three studies that assessed the parameters (
      • Nurjhan N.
      • Consoli A.
      • Gerich J.
      Increased lipolysis and its consequences on gluconeogenesis in non-insulin-dependent diabetes mellitus.
      ,
      • Puhakainen I.
      • Koivisto V.A.
      • Yki-Järvinen H.
      Lipolysis and gluconeogenesis from glycerol are increased in patients with noninsulin-dependent diabetes mellitus.
      ,
      • Mahendran Y.
      • Cederberg H.
      • Vangipurapu J.
      • Kangas A.J.
      • Soininen P.
      • Kuusisto J.
      • Uusitupa M.
      • Ala-Korpela M.
      • Laakso M.
      Glycerol and fatty acids in serum predict the development of hyperglycemia and type 2 diabetes in Finnish men.
      ). Insulin resistance leads to increased lipolysis, which allows for greater release of glycerol into circulation, and T2DM is routinely linked with increased fat mass (
      • Morigny P.
      • Houssier M.
      • Mouisel E.
      • Langin D.
      Adipocyte lipolysis and insulin resistance.
      ). This would allow glycerol to be a net carbon contributor to gluconeogenesis in T2DM.

      Insights from in vitro experiments

      The tracer studies described so far in this review have been in vivo experiments, which are ideally suited to study whole-body metabolism accounting for organ cross-talk via hormones and substrates circulating at physiologic concentrations. However, in vitro experiments have utility, including the ability to more closely control testing conditions. In vitro experiments can be done much more quickly and cheaply to generate hypotheses and assess feasibility prior to scaling up to animal and human studies. Further, in vitro experiments can discern differences in metabolism of metabolites across different tissues without having to invasively cannulate blood vessels.
      As an example, investigators can use hepatocytes given labeled precursors and glucose production assays to assess precursor utilization. Kaloyianni et al. (
      • Kaloyianni M.
      • Freedland R.A.
      Contribution of several amino acids and lactate to gluconeogenesis in hepatocytes isolated from rats fed various diets.
      ) used 14C-labeled precursors at physiologic concentrations in rat primary hepatocytes and showed lactate as the major precursor of glucose, accounting for 60% of the glucose formed. Glutamine and alanine each accounted for ∼10% of glucose production, whereas serine, glycine, and threonine accounted for less than 5% each. One notable omission in this model was glycerol.
      In contrast, our group showed, using mouse primary hepatocytes given 13C-labeled substrates at physiologic concentrations, that glycerol accounted for over 75% of the glucose carbons labeled (
      • Kalemba K.M.
      • Wang Y.
      • Xu H.
      • Chiles E.
      • McMillin S.M.
      • Kwon H.
      • Su X.
      • Wondisford F.E.
      Glycerol induces G6pc in primary mouse hepatocytes and is the preferred substrate for gluconeogenesis both in vitroin vivo.
      ). Specifically, labeled glycerol yielded enrichments of m + 3 and m + 6 glucose, signifying glycerol as a direct carbon contributor to glucose, whereas labeled pyruvate/lactate yielded a mixed distribution pattern (m + 1 through m + 6), suggesting carbon loss via tricarboxylic acid cycle intermediates. This is consistent with findings by Hui et al. (
      • Hui S.
      • Ghergurovich J.M.
      • Morscher R.J.
      • Jang C.
      • Teng X.
      • Lu W.
      • Esparza L.A.
      • Reya T.
      • Zhan L.
      • Yanxiang Guo J.
      • White E.
      • Rabinowitz J.D.
      Glucose feeds the TCA cycle via circulating lactate.
      ) that showed that circulating [13C3]lactate primarily labeled Krebs cycle intermediates in fasting mice in all tissues except brain.
      Studying fatty livers from rats given a high caloric diet and nonfatty livers from rats given a control diet, Maeda Junior et al. (
      • Maeda Junior A.S.
      • Constantin J.
      • Utsunomiya K.S.
      • Gilglioni E.H.
      • Gasparin F.R.S.
      • Carreño F.O.
      • de Moraes S.M.F.
      • Rocha M.
      • Natali M.R.M.
      • Ghizoni C.V.C.
      • Bracht A.
      • Ishii-Iwamoto E.L.
      • Constantin R.P.
      Cafeteria diet feeding in young rats leads to hepatic steatosis and increased gluconeogenesis under fatty acids and glucagon influence.
      ) studied glucose production rates by perfusing labeled gluconeogenic precursors. Glycerol infusion led to increased glucose production rates in fatty livers, whereas lactate and lactate plus pyruvate infusions decreased glucose production in fatty livers. The addition of glucagon or the long-chain fatty acid stearate increased glucose production from these substrates, although only in fatty livers. In a separate study using labeled glutamine and labeled alanine, the same research group showed decreased capacity to produce glucose from these two amino acids in fatty rat livers compared with nonfatty rat livers (
      • de Castro Ghizoni C.V.
      • Gasparin F.R.S.
      • Júnior A.S.M.
      • Carreño F.O.
      • Constantin R.P.
      • Bracht A.
      • Ishii Iwamoto E.L.
      • Constantin J.
      Catabolism of amino acids in livers from cafeteria-fed rats.
      ). With fatty liver disease, commonly seen in conjunction with T2DM, hepatocytes may have a shift in substrate utilization for gluconeogenesis. However, this requires further exploration in humans.
      One must consider how gluconeogenic precursors regulate enzymes relevant to gluconeogenesis. In vitro studies have shown that glycerol induces G6Pase expression in mouse primary hepatocytes (
      • Kalemba K.M.
      • Wang Y.
      • Xu H.
      • Chiles E.
      • McMillin S.M.
      • Kwon H.
      • Su X.
      • Wondisford F.E.
      Glycerol induces G6pc in primary mouse hepatocytes and is the preferred substrate for gluconeogenesis both in vitroin vivo.
      ) and rat hepatoma FAO cells (
      • Yoshida M.
      • Lee E.Y.
      • Kohno T.
      • Tanaka T.
      • Miyazaki M.
      • Miki T.
      Importance of hepatocyte nuclear factor 4α in glycerol-induced glucose-6-phosphatase expression in liver.
      ). Yoshida et al. (
      • Yoshida M.
      • Lee E.Y.
      • Kohno T.
      • Tanaka T.
      • Miyazaki M.
      • Miki T.
      Importance of hepatocyte nuclear factor 4α in glycerol-induced glucose-6-phosphatase expression in liver.
      ) showed that glycerol induced G6Pase expression in mouse hepatocytes via binding of the G6Pase promoter region in conjunction with hepatocyte nuclear factor 4α (HNF4α) binding to the promoter region. Glycerol reduced PEPCK expression in some in vitro studies (
      • Kalemba K.M.
      • Wang Y.
      • Xu H.
      • Chiles E.
      • McMillin S.M.
      • Kwon H.
      • Su X.
      • Wondisford F.E.
      Glycerol induces G6pc in primary mouse hepatocytes and is the preferred substrate for gluconeogenesis both in vitroin vivo.
      ) but not all (
      • Yoshida M.
      • Lee E.Y.
      • Kohno T.
      • Tanaka T.
      • Miyazaki M.
      • Miki T.
      Importance of hepatocyte nuclear factor 4α in glycerol-induced glucose-6-phosphatase expression in liver.
      ). In contrast, lactate and pyruvate did not affect G6Pase and PEPCK expression in mouse primary hepatocytes (
      • Kalemba K.M.
      • Wang Y.
      • Xu H.
      • Chiles E.
      • McMillin S.M.
      • Kwon H.
      • Su X.
      • Wondisford F.E.
      Glycerol induces G6pc in primary mouse hepatocytes and is the preferred substrate for gluconeogenesis both in vitroin vivo.
      ). It is unknown whether amino acids affect expression of these two key gluconeogenic enzymes.
      Whereas changes in mRNA expression of PEPCK and G6Pase did not correlate with changes in gluconeogenic flux in prior studies (
      • Burgess S.C.
      • He T.
      • Yan Z.
      • Lindner J.
      • Sherry A.D.
      • Malloy C.R.
      • Browning J.D.
      • Magnuson M.A.
      Cytosolic phosphoenolpyruvate carboxykinase does not solely control the rate of hepatic gluconeogenesis in the intact mouse liver.
      ,
      • Samuel V.T.
      • Beddow S.A.
      • Iwasaki T.
      • Zhang X.-M.
      • Chu X.
      • Still C.D.
      • Gerhard G.S.
      • Shulman G.I.
      Fasting hyperglycemia is not associated with increased expression of PEPCK or G6Pc in patients with type 2 diabetes.
      ), these changes might provide insight into the substrates used to maintain such fluxes. Glycerol has a much shorter pathway to generate glucose as it enters into the middle portion of the gluconeogenic pathway. Pyruvate, and lactate via pyruvate, enter gluconeogenesis after converting to oxaloacetate in mitochondria and require transport to the cytosol via the malate-aspartate shuttle (
      • Rui L.
      Energy metabolism in the liver.
      ). Glycerol can potentially shift hepatocyte glucose production away from pyruvate and lactate and toward glycerol gluconeogenesis by inducing G6Pase and repressing PEPCK. More investigation is needed to study how T2DM affects gluconeogenic enzymes and whether acute and chronic changes in precursor concentrations, as discussed above, affect gluconeogenic flux.

      Gluconeogenesis contribution to glycogen stores

      The liver produces glucose for release into systemic circulation as well as storing glucose in the form of glycogen. Hepatic glucose production is a combination of both glycogenolysis and gluconeogenesis. In humans, glycogen is the single greatest source of hepatic glucose production after an overnight fast. However, reports vary on the exact contribution of gluconeogenesis to hepatic glucose output, ranging from 30 to 60% after an overnight fast in healthy humans, depending on the method used (
      • Petersen 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.
      ,
      • Landau B.R.
      • Wahren J.
      • Chandramouli V.
      • Schumann W.C.
      • Ekberg K.
      • Kalhan S.C.
      Contributions of gluconeogenesis to glucose production in the fasted state.
      ,
      • Landau B.R.
      • Wahren J.
      • Chandramouli V.
      • Schumann W.C.
      • Ekberg K.
      • Kalhan S.C.
      Use of 2H2O for estimating rates of gluconeogenesis: application to the fasted state.
      ,
      • Kunert O.
      • Stingl H.
      • Rosian E.
      • Krssak M.
      • Bernroider E.
      • Seebacher W.
      • Zangger K.
      • Staehr P.
      • Chandramouli V.
      • Landau B.R.
      • Nowotny P.
      • Waldhausl W.
      • Haslinger E.
      • Roden M.
      Measurement of fractional whole-body gluconeogenesis in humans from blood samples using 2H nuclear magnetic resonance spectroscopy.
      ,
      • Tayek J.A.
      • Katz J.
      Glucose production, recycling, and gluconeogenesis in normals and diabetics: a mass isotopomer [U-13C]glucose study.
      ). In T2DM, glycogen stores and glycogenolysis rates are diminished, and gluconeogenesis accounts for a higher percentage of hepatic glucose output (
      • Magnusson I.
      • Rothman D.L.
      • Katz L.D.
      • Shulman R.G.
      • Shulman G.I.
      Increased rate of gluconeogenesis in type II diabetes mellitus: a 13C nuclear magnetic resonance study.
      ). Further, a portion of the glycogen pool undergoes simultaneous synthesis (glycogenesis) and breakdown (glycogenolysis) in a process called glycogen cycling, and a review by Landau discusses different methods to measure glycogen cycling (
      • Landau B.R.
      Methods for measuring glycogen cycling.
      ).
      Fig. 1 shows that hepatic glycogen is synthesized from glucose 6-phosphate (
      • Shulman G.I.
      • Landau B.R.
      Pathways of glycogen repletion.
      ), whose carbons come from an intact glucose molecule (
      • Radziuk J.
      Hepatic glycogen in humans. I. Direct formation after oral and intravenous glucose or after a 24-h fast.
      ) or gluconeogenic precursors (
      • Radziuk J.
      Hepatic glycogen in humans. II. Gluconeogenetic formation after oral and intravenous glucose.
      ). Thus, it is possible for a gluconeogenic precursor to get stored as glycogen and later be released as a glucose molecule. Hellerstein et al. (
      • Hellerstein M.K.
      • Neese R.A.
      • Linfoot P.
      • Christiansen M.
      • Turner S.
      • Letscher A.
      Hepatic gluconeogenic fluxes and glycogen turnover during fasting in humans: a stable isotope study.
      ) showed in healthy humans that two-thirds of the glucose produced from gluconeogenesis was released into circulation, whereas one-third remained in the liver for glycogen deposition and cycling after an overnight fast. Thus, current methods that assess precursor contribution to gluconeogenesis underestimate the exact quantity of contribution as a significant portion of the carbons are stored in glycogen for later release. Whether the partitioning of gluconeogenic products between hepatic glucose release and glycogen storage is altered in T2DM also remains unknown.

      Untargeted metabolomics studies

      Alongside targeted metabolomics studies with isotope tracers, many studies have used untargeted metabolomics to find plasma and urine biomarkers for T2DM, and several reviews expound on this topic (
      • Park S.
      • Sadanala K.C.
      • Kim E.K.
      A metabolomic approach to understanding the metabolic link between obesity and diabetes.
      ,
      • Guasch-Ferre M.
      • Hruby A.
      • Toledo E.
      • Clish C.B.
      • Martínez-González M.A.
      • Salas-Salvadó J.
      • Hu F.B.
      Metabolomics in prediabetes and diabetes: a systematic review and meta-analysis.
      ,
      • Del Coco L.
      • Vergara D.
      • De Matteis S.
      • Mensà E.
      • Sabbatinelli J.
      • Prattichizzo F.
      • Bonfigli A.R.
      • Storci G.
      • Bravaccini S.
      • Pirini F.
      • Ragusa A.
      • Casadei-Gardini A.
      • Bonafè M.
      • Maffia M.
      • Fanizzi F.P.
      • et al.
      NMR-based metabolomic approach tracks potential serum biomarkers of disease progression in patients with type 2 diabetes mellitus.
      ,
      • Tam Z.Y.
      • Ng S.P.
      • Tan L.Q.
      • Lin C.-H.
      • Rothenbacher D.
      • Klenk J.
      • Boehm B.O
      ActiFE Study Group
      Metabolite profiling in identifying metabolic biomarkers in older people with late-onset type 2 diabetes mellitus.
      ,
      • Urpi-Sarda M.
      • Almanza-Aguilera E.
      • Tulipani S.
      • Tinahones F.J.
      • Salas-Salvadó J.
      • Andres-Lacueva C.
      Metabolomics for biomarkers of type 2 diabetes mellitus: advances and nutritional intervention trends.
      ). Collectively, these studies show that a myriad of metabolites derived from amino acids, lipids, carbohydrates, and nucleotides are altered in T2DM (
      • Park S.
      • Sadanala K.C.
      • Kim E.K.
      A metabolomic approach to understanding the metabolic link between obesity and diabetes.
      ), and these metabolites vary across disease progression (
      • Del Coco L.
      • Vergara D.
      • De Matteis S.
      • Mensà E.
      • Sabbatinelli J.
      • Prattichizzo F.
      • Bonfigli A.R.
      • Storci G.
      • Bravaccini S.
      • Pirini F.
      • Ragusa A.
      • Casadei-Gardini A.
      • Bonafè M.
      • Maffia M.
      • Fanizzi F.P.
      • et al.
      NMR-based metabolomic approach tracks potential serum biomarkers of disease progression in patients with type 2 diabetes mellitus.
      ). Despite these broad changes, it is difficult to know what they mean in terms of underlying pathophysiologic mechanisms and whether they contribute to the insulin resistance and hyperglycemia in T2DM or are a byproduct of the underlying disease process. Specifically, we are not aware of any studies that correlate biomarkers directly with gluconeogenic flux as determined by isotope tracer infusion. Such studies could be enlightening as they could inform us of the determinants of hyperglycemia in an individual. Given the phenotypic heterogeneity of T2DM (
      • Ahlqvist E.
      • Storm P.
      • Käräjämäki A.
      • Martinell M.
      • Dorkhan M.
      • Carlsson A.
      • Vikman P.
      • Prasad R.B.
      • Aly D.M.
      • Almgren P.
      • Wessman Y.
      • Shaat N.
      • Spégel P.
      • Mulder H.
      • Lindholm E.
      • et al.
      Novel subgroups of adult-onset diabetes and their association with outcomes: a data-driven cluster analysis of six variables.
      ), having biomarkers that correlate with increased gluconeogenic flux could lead to more personalized treatments for patients with T2DM that directly target gluconeogenesis.

      Perspective and future directions

      There is no normal range for carbon contribution to gluconeogenesis. Current analytical techniques offer precise and reproducible measurements but not necessarily absolute measurements. Whereas some studies have been more comprehensive than others, all remain limited in scope, given the complexity of the subject matter. Investigations using multiple tracers in the same subject using the same analytical technique might bring us closer to reconciling direct and net carbon contribution to gluconeogenesis.
      Consensus is also needed among thought leaders in the field regarding optimal analytical techniques, sample preparation methods, and data calculations. Subject preparation prior to experimentation, including fasting duration, preceding meal intake, and medication management, need to be addressed to make studies comparable. If investigators conducted isotope tracer experiments in a more uniform fashion, results across different studies could become more comparable. Data from different experimenters could be then integrated into a flux network to better understand carbon flow in metabolism.
      In summary, experiments with isotope tracers have led to significant advances in the quantification of gluconeogenic flux. To fully understand hepatic glucose output, one needs an accurate assessment of the input from gluconeogenic precursors and glycogen, which requires ongoing investigation. Such results can shed further insight into human physiology as well as relevant clinical conditions, including T2DM, obesity, and fatty liver disease.

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