REVIEWS

Tracking the carbons supplying gluconeogenesis

Received for publication, January 22, 2020, and in revised form, August 12, 2020 Published, Papers in Press, August 13, 2020, DOI 10.1074/jbc.REV120.012758
Ankit M. Shah and Fredric E. Wondisford*
From the Department of Medicine, Robert Wood Johnson Medical School, Rutgers University, New Brunswick, New Jersey, USA
Edited by Qi-Qun Tang

As the burden of type 2 diabetes mellitus (T2DM) grows in completely suppressed glycogenolysis but only reduced gluco-
the 21st century, the need to understand glucose metabolism neogenesis by about 20% (7). Longstanding hyperglycemia is
heightens. Increased gluconeogenesis is a major contributor to associated with both macrovascular complications, such as
the hyperglycemia seen in T2DM. Isotope tracer experiments in heart attacks and stroke, and microvascular complications
humans and animals over several decades have offered insights affecting retinal, renal, and nerve tissues (8), which help drive
into gluconeogenesis under euglycemic and diabetic conditions. the costs of diabetes care to over $322 billion annually in the
This review focuses on the current understanding of carbon flux United States alone (9).
in gluconeogenesis, including substrate contribution of various
The rise in obesity has led to increased prevalence of T2DM
gluconeogenic precursors to glucose production. Alterations
and nonalcoholic fatty liver disease (NAFLD). More than 1 in 3
of gluconeogenic metabolites and fluxes in T2DM are discussed.
adult Americans have obesity (10), whereas 1 in 4 have NAFLD
We also highlight ongoing knowledge gaps in the literature that
require further investigation. A comprehensive analysis of (11) and nearly 1 in 10 have T2DM (12). Gluconeogenesis rates
gluconeogenesis may enable a better understanding of T2DM are elevated in patients with obesity even without overt diabe-
pathophysiology and identification of novel targets for treating tes (5) as well in patients with NAFLD (13). Based on these epi-
hyperglycemia. demiologic data, most patients with obesity and NAFLD do not
develop overt hyperglycemia, highlighting fundamental differ-
ences within these patient populations. Understanding gluco-
Glucose serves as a fuel source for many tissues and is the neogenesis across distinct but related metabolic conditions
primary source of energy for neurons, renal medullary cells, might lead to greater insights into underlying pathophysiology
and red blood cells (1). Circulating blood glucose levels are and more targeted therapies.
maintained in a narrow range (3.9–7.1 mmol/liter), and the Many studies support the notion that increased gluconeo-
liver plays a critical role in maintaining glucose homeostasis genesis in T2DM stems from dysregulation of two key gluconeo-
(2). The liver stores glucose in the form of glycogen and releases genic enzymes: phosphoenolpyruvate carboxykinase (PEPCK)
glucose into circulation by either glycogenolysis or gluconeo- and glucose-6-phosphatase (G6Pase) (14, 15). PEPCK converts
genesis. In the fed state, hepatic glucose production is sup- oxaloacetate to phosphoenolpyruvate, allowing Krebs cycle inter-
pressed by insulin secretion, and the glucose ingested is stored mediates to contribute to gluconeogenesis (16). G6Pase converts
in part as glycogen. glucose 6-phosphate to glucose, the final step in gluconeogenesis,
During a short-term fast, the liver maintains euglycemia which allows glucose to exit the hepatocyte and enter circulation
through glycogenolysis. During longer periods of fasting, as gly- via the GLUT2 hepatocyte transporter (16, 17). Many hormones
cogen stores are depleted, the liver relies on gluconeogenesis to regulate PEPCK expression, including glucagon, epinephrine, in-
maintain euglycemia (3). sulin, and glucocorticoids (14). Similarly, insulin, glucocorticoids,
Gluconeogenesis is an intricate process that requires several cAMP, and glucose all affect G6Pase expression (18).
enzymatic steps (Fig. 1), which are under the regulation of hor- Given the health burden of T2DM and the public health
mones, nutrient intake, stress conditions, and substrate con- impact, there has been significant research on underlying dis-
centrations. Occurring in hepatocytes and renal cortical cells, ease processes leading to varied pharmacologic therapies for
gluconeogenesis functions as a biosynthetic pathway responsi- the disease. Despite 14 distinct T2DM medication classes cur-
ble for countering the glycolytic breakdown of glucose. rently approved, hyperglycemia remains a persistent challenge
T2DM, a chronic medical condition characterized by hyper- for patients, and physicians need to be mindful of avoiding
glycemia, has reached pandemic proportions affecting over 400 hypoglycemia and minimizing side effects (19, 20). Thus, novel
million adults globally (4). A major pathophysiological tenet of therapeutic approaches are warranted. To better target gluco-
T2DM is increased hepatic gluconeogenesis with rates elevated neogenesis, a key question becomes the origin of the carbons
up to 40% (5). In T2DM, gluconeogenesis remains a significant that account for the increased glucose production in T2DM.
contributor to hepatic glucose production both under fasting Further, many medications for T2DM affect gluconeogenesis
conditions and after meal intake (6). Hyperinsulinemia during rates directly and indirectly (20), although their mechanisms of
a hyperinsulinemic-euglycemic clamp, where exogenous insu- action could be better known if we had an accurate assessment
lin is infused as supraphysiologic amounts with concurrent of gluconeogenesis flux.
infusion of glucose to maintain a certain blood glucose level, This review discusses the current understanding of gluco-
neogenic flux based on isotope tracer data primarily from
* For correspondence: Fredric E. Wondisford, few11@rwjms.rutgers.edu. experiments in humans but also from selected animal and in

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© 2020 Shah and Wondisford. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
JBC REVIEWS: Carbon flux in gluconeogenesis

Figure 1. Glucose metabolism in the context of glycolysis and gluconeogenesis. a-KG, a-ketoglutarate; G6Pase, glucose-6-phosphatase; OAA, oxaloacetate;
PEPCK, phosphoenolpyruvate carboxykinase.

vitro models to fill in where human data are lacking. We focus genetic alterations, or drug treatments, and identifies metabolite
on how different gluconeogenic precursors contribute to the changes in response to a manipulation. This unbiased approach
process, how these contributions may differ in T2DM, and new can generate novel hypotheses regarding metabolites and path-
findings that may question each precursor’s relative role in the ways. However, one needs to study metabolic flux (metabolite
process. We also discuss how these precursors’ concentrations flow per time) to fully understand pathway activity (25). To
change in T2DM and how precursors themselves may regulate obtain a more thorough understanding of metabolite regulation
gluconeogenesis. and quantify fluxes under various conditions, one must introduce
a labeled metabolite and “follow the label” (26). Paired with iso-
Metabolomics overview tope-labeled metabolites, targeted metabolomics can measure
metabolic flux as heavy atoms from a labeled substrate are
Given the complexities behind biochemical processes,
detected in downstream metabolic products across different time
including gluconeogenesis, researchers have studied metabo-
points.
lites directly to gain insight. The term metabolites refers to all Several different methodologies can help with determining
endogenous small molecules (,1,500 Da) involved in meta- metabolic flux. NMR and MS are two commonly used analyti-
bolic reactions, including substrates, intermediates, and prod- cal platforms for metabolite detection and quantification. NMR
ucts (21). A metabolite’s circulating concentration is based on is a highly reproducible technique that can provide fractional
its synthesis, dietary intake, and degradation as well as uptake abundance of an isotope at a specific atom position (27). For
and release from other body compartments, such as liver, mus- example, a 12C-1H interaction gives a different peak than a 13C-
cle, and adipose tissue (22). Metabolites most directly reflect 1
H interaction on an NMR spectrum. NMR yields significant
physiologic and pathologic conditions in an organism. The structural information about a molecule as adjacent nuclei
entire complement of metabolites in cells, tissues, or whole within that molecule interact via spin-spin coupling to produce
organisms makes up the metabolome, and metabolomics can distinct peaks. Disadvantages of NMR include low sensitivity,
measure these molecules with precision and accuracy. There making measurement of metabolites with low concentrations
are 6,500 and counting discrete metabolites in the human difficult (28). Because there is little sample preparation with
metabolome (23, 24). NMR, there is no chromatographic separation of structurally
Nontargeted, or untargeted, metabolomics compares two similar compounds leading to overlapping resonances, which
different biological conditions, including different disease states, can make the charting of biochemical pathways difficult.

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 JBC REVIEWS: Carbon flux in gluconeogenesis

MS is a highly sensitive technique that can detect metabolites Alanine

even at low concentrations. MS involves fragmenting labeled or With muscle catabolism, alanine is released into circulation,
unlabeled compounds through ionization by electron impact undergoes deamination in the liver to become pyruvate, and
ionization or chemical impact ionization (29). After going later glucose as depicted in Fig. 2 (44). Studies have shown a 6–
through the ionization source, fragmented ions pass through a 11% contribution of the amino acid to glucose production after
mass analyzer with a specific mass/charge (m/z) ratio and an overnight fast in healthy humans (40, 41, 45). The role of ala-
retention time (29). MS can detect the subtle mass differences
nine’s contribution to gluconeogenesis in T2DM remains less
between isotopes. For example, 3-[13C]lactate (m 1 1), which
clear. Some have documented a 2-fold increase of gluconeogen-
has a label only on lactate’s third carbon, has an m/z ratio and
esis from alanine in subjects with T2DM compared with
retention time in the mass spectrometer different from those
healthy controls (46, 47). However, Consoli et al (42). con-
of the unlabeled lactate (m 1 0). Chromatographic separation
cluded that those with T2DM did not have an increase in ala-
provides high resolution even between structurally similar mol-
ecules. Disadvantages of MS include the need for sample deri- nine’s contribution to glucose production compared with con-
vatization, which can lead to sample loss (30). MS often cannot trols. In a separate study, Chochinov et al. (48) concluded that
tell you specifically where in the molecule is the labeled atom gluconeogenesis from alanine decreased from 11% in controls
(i.e. which carbon is labeled in an M 1 1 lactate molecule). to just 3% in subjects with T2DM. These conflicting studies
For a detailed description of the established methods of make it difficult to assess what role, if any, alanine has in T2DM
measuring gluconeogenesis and glycogenolysis using MS and hyperglycemia.
NMR, please refer to a review by Chung et al. (31). Others have
written on the practical applications related to in vivo research Glutamine
with metabolomics (32–35). Glutamine contributes to gluconeogenesis by converting to
glutamate, which gets deamidated to a-ketoglutarate (37). Fig.
Carbon contribution to gluconeogenesis 1 shows how a-ketoglutarate can then enter the Krebs cycle
Given the powerful tools of NMR and MS within metabolo- and ultimately feed gluconeogenesis. Glutamine contributed
mics, one can study how the liver makes glucose under fasting 5–8% to glucose production in healthy humans in prior studies
conditions. Glucose is a six-carbon molecule whose concentra- (49, 50). With T2DM, the conversion of glutamine to glucose
tions remain relatively constant in the fasted state in metabol- nearly doubled (47).
ically healthy individuals but can rise in subjects with T2DM
(1). Gluconeogenic precursors come from noncarbohydrate Glycerol
sources, including lactate, glycerol, and amino acids. The two Lipolysis of triglycerides in adipocytes releases glycerol into
most relevant amino acids for gluconeogenesis are alanine and circulation, which can become glucose in the liver. The contri-
glutamine. Glutamine gluconeogenesis is predominantly in the bution of glycerol to glucose production in metabolically
kidney, whereas alanine gluconeogenesis is predominantly in healthy humans ranged from 3 to 7% (51–54). In T2DM, glycer-
the liver (36). ol’s contribution to glucose production increased to 6–10%
Infusion of a carbon-labeled precursor of glucose is com- which was significantly higher compared with healthy controls
monly used to study gluconeogenesis. Using isotope dilution (52, 53).
techniques, the ratio of labeled glucose over the labeled precur-
sor equates to the percentage contribution of the precursor to Direct versus net carbon contribution
glucose production. Numerous studies assessing substrate con-
tribution to gluconeogenesis in humans were done in the Like many biologic processes, the paths of gluconeogenic
1960s–1990s using advanced tools for the time. Results vary precursors to glucose production exist in both directions, such
based on the isotope tracers used, test conditions, and methods that glucose itself can lead to the production of many gluconeo-
of calculation. Although we cannot cover all tracer experiments genic precursors. Many tracer studies on gluconeogenesis,
conducted, we will highlight relevant studies (Table 1) to illus- including references in this review, primarily report direct car-
trate key concepts as well as point out inconsistencies in the lit- bon contribution of precursors to gluconeogenesis but not net
erature that require reconciliation. carbon contribution. For example, as in Fig. 3, molecules M
and N can contribute to each other’s production via reversible
Lactate reactions. For molecule M, 60% of its flux goes toward the mol-
As shown in Fig. 2, the Cori cycle depicts shuttling of lactate ecule N, whereas 40% goes to the molecule O. For molecule N,
from anaerobic glycolysis in skeletal muscle cells to the liver to only 40% of its flux goes to molecule M, whereas 60% goes to P.
feed gluconeogenesis (37). Many consider lactate the predomi- If one were to give an isotope-labeled tracer of molecule M, one
nant gluconeogenic precursor (38–40). Studies in healthy would see a 60% direct contribution of M to N. However, a dual
humans have shown that lactate contributes as little as 7% (41) tracer study with M and N tracers would show smaller net
to as much as 18% (40) to plasma glucose after an overnight efflux from M to N. In general, introducing a labeled tracer of a
fast. Comparing subjects with T2DM and healthy controls, molecule can give an idea of where that molecule is going.
there was a 2-fold increase in lactate incorporation into glucose However, it does not give information about where that specific
in T2DM (42, 43). molecule is coming from. This requires introduction of other

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Table 1
Direct contribution of gluconeogenesis precursors and glycogen to hepatic glucose production after an overnight fast in humans as deter-
mined by isotope tracer experiments
The T2DM column indicates relative changes to glucose contribution from precursor in the setting of T2DM as compared with metabolically healthy controls.
Healthy T2DM
Lactate 7–18% (40, 41) 2-Fold increase (42, 43)
Alanine 6–11% (40, 41, 45) 1.5-Fold increase (46, 47), 0.70-fold decrease (42), or no change (48)
Glutamine 5–8% (49, 50) 2-Fold increase (47)
Glycerol 3–7% (51–54) 1.5-Fold increase (52, 53)
Glycogen 40–70% (6, 97–100) 0.5-Fold decrease (101)

Figure 2. Carbon flow in Cori cycle (A), glucose-alanine cycle (B), and glucose-glutamine cycle (C). Stoichiometry and cofactors for reactions were omit-
ted for clarity. a-KG, a-ketoglutatarate; PEP, phosphoenolpyruvate; OAA, oxaloacetate.

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 (57). Such theoretical agents would
Figure 3. Relative fluxes (numbers) for molecules M and N. Given the net need to be tested in preclinical models and assessed for off-tar-
efflux from M to N, molecule Q must contribute to the production of mole- get metabolic effects.
cule M so that molecule M can remain at steady-state concentrations.

Fluxes between gluconeogenic precursors

labeled substrates to obtain an integrated flux network that can
distinguish direct and net contributions. Not only can gluconeogenic precursors contribute to glucose
Glucose itself is major contributor to many gluconeogenic production, they can also contribute to the production of other
precursors. Perriello et al. (55) showed in metabolically healthy gluconeogenic precursors. Returning to our example, in Fig. 3,
humans that circulating glucose provided 67, 41, and 13% of for concentrations of molecule M to remain constant, molecule
the carbons for plasma lactate, alanine, and glutamine, respec- Q must feed molecule M. Otherwise, molecule M concentra-
tively. These contributions were via the Cori cycle, glucose-ala- tions would decrease, given its net effluxes toward molecules N
nine cycle, and glucose-glutamine cycle (Fig. 2). No human and O.
data exist to show how much, if at all, glucose contributes to Lactate may be an important direct carbon contributor to
glycerol production. However, studies in metabolically healthy gluconeogenesis, given its high turnover and intimate coupling
dogs showed that less than 2% of glycerol’s carbons come from with glucose via the Cori cycle. However, the Cori cycle is not a
glucose (56). net producer of glucose and cannot sufficiently explain the
Given the reciprocal fluxes between glucose and its precur- hyperglycemia seen in T2DM (58). Similarly, many argue that
sors, infusing only 13C tracers of gluconeogenic precursors may the glucose-alanine cycle does not provide net substrate for glu-
not be enough. As the direct and net contribution of a precur- coneogenesis and that its main physiologic function is ammo-
sor may not be congruent, studies that also give a [13C6]glucose nia transport (44, 45). Therefore, for increased gluconeogenesis
tracer are needed to determine the source of non-glucose- to contribute to T2DM hyperglycemia, the carbons supplying
derived carbons that fuel gluconeogenesis. gluconeogenesis must come from other substrates.
The difference between direct and net contribution may not Recently our group has shown in mice, using [13C3]lactate,
merely be academic but also practical. A better understanding [ C3]glycerol, and [13C3]glucose tracers administered over dif-
13

of gluconeogenesis determinants could lead to more rationally ferent experimental days, that lactate is mainly recycled during
designed and targeted T2DM treatments. For example, one the fasting period such that lactate is the largest direct contrib-
could find a pharmacologic means to stop hepatic conversion utor to gluconeogenesis but provides minimal new glucose car-
of a specific gluconeogenesis substrate to glucose by blocking bons (59). In the same study, glycerol contributed to the

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 JBC REVIEWS: Carbon flux in gluconeogenesis

carbons of glucose both by direct conversion in the liver and by number of downstream products. However, the metabolome is
first converting to lactate, which then became glucose. Glycerol more interconnected, and gluconeogenic precursors can supply
was the most significant source of new carbons for gluconeo- each other with carbons directly or through intermediate
genesis after a 12-h fast, contributing over 50% of the net car- metabolites.
bons for gluconeogenesis. Lactate contributed a small propor- Given the various fluxes between gluconeogenic precursors
tion to glycerol molecules in mice, and this coincides with rat as shown in Fig. 4 in humans with and without T2DM, there is
data using a single intraperitoneal injection of [13C3]lactate that a need for comprehensive experiments spanning multiple trac-
labeled intramuscular glycerol (60). The enzymes glycerol-3- ers in the same human or animal subject to assess the carbon
phosphate dehydrogenase and glycerol-3-phosphate phospha- flow. Such experiments may yield information that may lead to
tase are needed to generate glycerol from the triose phosphate certain precursors as the significant culprit carbon contribu-
pool (61). To date, no human experiments have assessed how tors. Blocking these substrates pharmacologically from becom-
glycerol and lactate may contribute to each other’s production. ing glucose or other gluconeogenic precursors could be a
Glycerol may have two different metabolic fates based on the potential treatment strategy. Blocking one pathway may or may
initial site of metabolism, and several tissues have high expres- not be sufficient to lower hepatic glucose production, as path-
sion of glycerol kinase, which converts glycerol to glycerol 3- ways may be redundant or leaky such that overall glucose pro-
phosphate (62). Glycerol 3-phosphate can then enter the triose duction may not be affected even if one precursor is prevented
phosphate pool as an intermediate for glycolysis or gluconeo- from becoming glucose. However, attempts at blocking such
genesis (Fig. 1). The liver and kidney have high expression of pathways can only be made with a sound understanding of car-
glycerol kinase so that glycerol can contribute directly to glu- bon flux in the first place.
cose production in these two gluconeogenic organs (63). Pe-
ripheral tissues such as intestines, lymphatics, and spleen also Changes to gluconeogenic precursor levels in T2DM
express glycerol kinase but not gluconeogenic enzymes, allow- Whereas it is critical to study metabolic fluxes of gluconeo-
ing glycerol to become a source for lactate (63). The fates of genic precursors, it is also pertinent to look at circulating con-
glycerol delivered directly to gluconeogenic and nongluconeo- centrations of precursors under healthy and T2DM conditions.
genic organs have not been directly tested in humans. This For the liver to make an excess of glucose in T2DM, one might
would require invasive cannulation of certain arteries and veins expect circulating precursor levels to change in T2DM com-
to isolate certain organs, adding significant risks for subjects. pared with metabolically heathy controls. Precursor levels
Infusing [13C3]glycerol into metabolically healthy humans could be elevated to allow for increased hepatic substrate deliv-
that fasted for 60 h, Landau et al. (64) estimated that the gastro- ery, or precursor levels could decrease due to increased hepatic
intestinal, renal, and muscle tissues accounted for 63% of the substrate utilization. Both increased substrate delivery and uti-
glycerol utilization and that the remaining 37% must be metab- lization could also occur without affecting overall circulating
olized by other tissues that express the enzyme glycerol kinase. precursor levels. Accounting for both metabolite levels and
Knowing which tissues in the body process this remaining glyc- fluxes may yield a more thorough understanding of gluconeo-
erol can help us further understand glycerol’s role in T2DM genic changes in T2DM.
hyperglycemia. In T2DM, where there is increased lipolysis and
circulating glycerol, it is unknown whether that additional cir-
Lactate
culating glycerol gets evenly distributed between gluconeogenic
or nongluconeogenic organs or if one set of organs has an Plasma lactate levels are elevated in T2DM compared with
increased metabolism of the substrate. metabolically healthy humans in some studies (68, 69) but not
Alanine can also supplement the lactate pool via a pyruvate all (42). Lactate turnover, or the amount of lactate appearing in
intermediate, and one study showed that alanine contributed circulation at any given moment, is also increased in T2DM
16% of the carbons to circulating lactate in healthy humans (42). Increased levels of lactate occur in obesity due to de-
(45). In contrast, a study in healthy dogs showed that lactate creased blood flow in adipose tissue causing local hypoxia and
contributed to 70% of the alanine pool (65). There are no increased lactate production (70). Insulin resistance in skeletal
known human studies to assess how much glutamine contrib- muscles was associated with decreased oxidative capacity and
utes to lactate production or vice versa. However, in vitro work greater lactate production (71–73). Despite these mechanisms,
from human fibroblasts has depicted glutamine converting to the carbon sources for the increased lactate level in T2DM
lactate (66). would still be primarily glucose, so lactate alone could not
Glutamine and alanine can contribute to each other’s pro- account for hyperglycemia seen in T2DM. Of note, there are no
duction as glutamine-derived glutamate can interchange with storage reservoirs of lactate in the body compared with amino
alanine via the enzyme alanine aminotransferase (67). Using acids (skeletal muscle tissue) and glycerol (adipose tissue).
[14C5]glutamate and 3-[13C]alanine tracers at the same time,
studies have shown glutamine to be more quantitatively impor- Amino acids
tant in delivering protein-derived carbons to glucose in both Circulating alanine levels were increased in T2DM in some
healthy human subjects (50) and those with T2DM (47). studies (68, 74) but not others (42, 47, 48). Alanine turnover
Finally, it is unknown whether amino acids and glycerol pro- was also increased in T2DM in the studies that have measured
vide any carbon contribution to each other. Prior tracer studies it (42, 47). Some studies have shown that increased glutamine
gave labeled gluconeogenic substrates and assessed a limited levels were associated with a decreased risk of developing

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JBC REVIEWS: Carbon flux in gluconeogenesis

and substrates circulating at physiologic concentrations. How-

ever, 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 tis-
sues without having to invasively cannulate blood vessels.
As an example, investigators can use hepatocytes given la-
beled precursors and glucose production assays to assess pre-
cursor utilization. Kaloyianni et al. (39) used 14C-labeled precur-
sors at physiologic concentrations in rat primary hepatocytes
and showed lactate as the major precursor of glucose, account-
ing for 60% of the glucose formed. Glutamine and alanine each
accounted for ;10% of glucose production, whereas serine, gly-
Figure 4. Schematic of gluconeogenesis recognizing fluxes between glucose
and its precursors along with fluxes between the precursors themselves. cine, and threonine accounted for less than 5% each. One nota-
ble omission in this model was glycerol.
T2DM (74–76), whereas others showed no association between In contrast, our group showed, using mouse primary hepato-
glutamine levels and T2DM risk (77–79). One study comparing cytes given 13C-labeled substrates at physiologic concentra-
glutamine turnover in T2DM and healthy controls showed no tions, that glycerol accounted for over 75% of the glucose
difference between the two cohorts (47). carbons labeled (89). Specifically, labeled glycerol yielded en-
Insulin is an anabolic hormone that promotes protein syn- richments of m 1 3 and m 1 6 glucose, signifying glycerol as
thesis and prevents its breakdown (80). Chevalier et al. (81) a direct carbon contributor to glucose, whereas labeled pyru-
showed that increased protein catabolism in obese nondiabetic vate/lactate yielded a mixed distribution pattern (m 1 1
subjects correlated with gluconeogenesis derived from amino through m 1 6), suggesting carbon loss via tricarboxylic acid
acids. However, in T2DM, a condition with insulin resistance cycle intermediates. This is consistent with findings by Hui et
and higher compensatory insulin levels, muscle turnover as al. (90) that showed that circulating [13C3]lactate primarily
assessed by leucine turnover was unchanged in two studies (82, labeled Krebs cycle intermediates in fasting mice in all tissues
83). Under isoaminoacidemic, hyperinsulinemic, euglycemic except brain.
clamp conditions where amino acid, insulin levels, and glucose Studying fatty livers from rats given a high caloric diet and
levels are held at constant levels by exogenous infusions, pro- nonfatty livers from rats given a control diet, Maeda Junior et
tein anabolism was blunted in men with T2DM compared with al. (91) studied glucose production rates by perfusing labeled
healthy controls (83). This suggests a defect in protein synthesis gluconeogenic precursors. Glycerol infusion led to increased
under insulin-resistant conditions in men. In women, this effect glucose production rates in fatty livers, whereas lactate and lac-
was not seen, leading to the potential sex differences in protein tate plus pyruvate infusions decreased glucose production in
metabolism under diabetic conditions. T2DM is associated fatty livers. The addition of glucagon or the long-chain fatty
with lower skeletal muscle mass (84, 85), and decreased muscle acid stearate increased glucose production from these sub-
mass is correlated with poorer glycemic control (86). Given strates, although only in fatty livers. In a separate study using la-
these findings of decreased muscle mass with variable amino beled glutamine and labeled alanine, the same research group
acid flux in T2DM, it remains difficult to quantify how much showed decreased capacity to produce glucose from these two
amino acids supply gluconeogenic carbons under diabetic amino acids in fatty rat livers compared with nonfatty rat livers
conditions. (92). With fatty liver disease, commonly seen in conjunction
with T2DM, hepatocytes may have a shift in substrate utiliza-
Glycerol tion for gluconeogenesis. However, this requires further explo-
ration in humans.
Circulating glycerol concentrations and glycerol turnover One must consider how gluconeogenic precursors regulate
were consistently higher in subjects with T2DM compared enzymes relevant to gluconeogenesis. In vitro studies have
with healthy controls in three studies that assessed the parame- shown that glycerol induces G6Pase expression in mouse pri-
ters (52, 53, 87). Insulin resistance leads to increased lipolysis, mary hepatocytes (89) and rat hepatoma FAO cells (93). Yosh-
which allows for greater release of glycerol into circulation, and ida et al. (93) showed that glycerol induced G6Pase expression
T2DM is routinely linked with increased fat mass (88). This in mouse hepatocytes via binding of the G6Pase promoter
would allow glycerol to be a net carbon contributor to gluco- region in conjunction with hepatocyte nuclear factor 4a
neogenesis in T2DM. (HNF4a) binding to the promoter region. Glycerol reduced
PEPCK expression in some in vitro studies (89) but not all
Insights from in vitro experiments (93). In contrast, lactate and pyruvate did not affect G6Pase
The tracer studies described so far in this review have been and PEPCK expression in mouse primary hepatocytes (89).
in vivo experiments, which are ideally suited to study whole- It is unknown whether amino acids affect expression of
body metabolism accounting for organ cross-talk via hormones these two key gluconeogenic enzymes.

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 JBC REVIEWS: Carbon flux in gluconeogenesis

Whereas changes in mRNA expression of PEPCK and they mean in terms of underlying pathophysiologic mecha-
G6Pase did not correlate with changes in gluconeogenic flux in nisms and whether they contribute to the insulin resistance and
prior studies (94, 95), these changes might provide insight into hyperglycemia in T2DM or are a byproduct of the underlying
the substrates used to maintain such fluxes. Glycerol has a disease process. Specifically, we are not aware of any studies
much shorter pathway to generate glucose as it enters into the that correlate biomarkers directly with gluconeogenic flux as
middle portion of the gluconeogenic pathway. Pyruvate, and determined by isotope tracer infusion. Such studies could be
lactate via pyruvate, enter gluconeogenesis after converting to enlightening as they could inform us of the determinants of
oxaloacetate in mitochondria and require transport to the cyto- hyperglycemia in an individual. Given the phenotypic heter-
sol via the malate-aspartate shuttle (96). Glycerol can poten- ogeneity of T2DM (112), having biomarkers that correlate
tially shift hepatocyte glucose production away from pyruvate with increased gluconeogenic flux could lead to more per-
and lactate and toward glycerol gluconeogenesis by inducing sonalized treatments for patients with T2DM that directly
G6Pase and repressing PEPCK. More investigation is needed to target gluconeogenesis.
study how T2DM affects gluconeogenic enzymes and whether
acute and chronic changes in precursor concentrations, as dis- Perspective and future directions
cussed above, affect gluconeogenic flux.
There is no normal range for carbon contribution to glu-
Gluconeogenesis contribution to glycogen stores coneogenesis. Current analytical techniques offer precise
and reproducible measurements but not necessarily absolute
The liver produces glucose for release into systemic circula-
measurements. Whereas some studies have been more com-
tion as well as storing glucose in the form of glycogen. Hepatic
prehensive than others, all remain limited in scope, given the
glucose production is a combination of both glycogenolysis and
complexity of the subject matter. Investigations using multi-
gluconeogenesis. In humans, glycogen is the single greatest
ple tracers in the same subject using the same analytical
source of hepatic glucose production after an overnight fast.
technique might bring us closer to reconciling direct and net
However, reports vary on the exact contribution of gluconeo-
carbon contribution to gluconeogenesis.
genesis to hepatic glucose output, ranging from 30 to 60% after
Consensus is also needed among thought leaders in the field
an overnight fast in healthy humans, depending on the method
used (6, 97–100). In T2DM, glycogen stores and glycogenolysis regarding optimal analytical techniques, sample preparation
rates are diminished, and gluconeogenesis accounts for a higher methods, and data calculations. Subject preparation prior to
percentage of hepatic glucose output (101). Further, a portion experimentation, including fasting duration, preceding meal
of the glycogen pool undergoes simultaneous synthesis (glyco- intake, and medication management, need to be addressed to
genesis) and breakdown (glycogenolysis) in a process called gly- make studies comparable. If investigators conducted isotope
cogen cycling, and a review by Landau discusses different meth- tracer experiments in a more uniform fashion, results across
ods to measure glycogen cycling (102). different studies could become more comparable. Data from
Fig. 1 shows that hepatic glycogen is synthesized from glu- different experimenters could be then integrated into a flux
cose 6-phosphate (103), whose carbons come from an intact network to better understand carbon flow in metabolism.
glucose molecule (104) or gluconeogenic precursors (105). In summary, experiments with isotope tracers have led to
Thus, it is possible for a gluconeogenic precursor to get stored significant advances in the quantification of gluconeogenic
as glycogen and later be released as a glucose molecule. Heller- flux. To fully understand hepatic glucose output, one needs an
stein et al. (106) showed in healthy humans that two-thirds of accurate assessment of the input from gluconeogenic precur-
the glucose produced from gluconeogenesis was released into sors and glycogen, which requires ongoing investigation. Such
circulation, whereas one-third remained in the liver for glyco- results can shed further insight into human physiology as well
gen deposition and cycling after an overnight fast. Thus, cur- as relevant clinical conditions, including T2DM, obesity, and
rent methods that assess precursor contribution to gluconeo- fatty liver disease.
genesis underestimate the exact quantity of contribution as a
significant portion of the carbons are stored in glycogen for Funding and additional information—F. E. W. was supported by
later release. Whether the partitioning of gluconeogenic prod- NIDDK, National Institutes of Health, Grant R01DK063349. A. M.
ucts between hepatic glucose release and glycogen storage is S. was supported by NCATS, National Institutes of Health, Grant
altered in T2DM also remains unknown. KL2TR003018. The content is solely the responsibility of the
authors and does not necessarily represent the official views of the
Untargeted metabolomics studies National Institutes of Health.
Alongside targeted metabolomics studies with isotope trac-
ers, many studies have used untargeted metabolomics to find Conflict of interest—The authors declare that they have no conflicts
plasma and urine biomarkers for T2DM, and several reviews of interest with the contents of this article.
expound on this topic (107–111). Collectively, these studies
Abbreviations—The abbreviations used are: T2DM, type 2 diabetes
show that a myriad of metabolites derived from amino acids,
mellitus; NAFLD, nonalcoholic fatty liver disease; PEPCK, phos-
lipids, carbohydrates, and nucleotides are altered in T2DM
phoenolpyruvate carboxykinase; G6Pase, glucose-6-phosphatase.
(107), and these metabolites vary across disease progression
(109). Despite these broad changes, it is difficult to know what

J. Biol. Chem. (2020) 295(42) 14419–14429 14425

JBC REVIEWS: Carbon flux in gluconeogenesis

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