23. 9 Regulation of glucose metabolism from a liver-centric perspective - Biology

23. 9  Regulation of glucose metabolism from a liver-centric perspective - Biology

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Glucose homeostasis is tightly regulated to meet the energy requirements of the vital organs and maintain an individual’s health. The liver has a major role in the control of glucose homeostasis by controlling various pathways of glucose metabolism, including glycogenesis, glycogenolysis, glycolysis and gluconeogenesis. Both the acute and chronic regulation of the enzymes involved in the pathways are required for the proper functioning of these complex interwoven systems. Allosteric control by various metabolic intermediates, as well as post-translational modifications of these metabolic enzymes constitute the acute control of these pathways, and the controlled expression of the genes encoding these enzymes is critical in mediating the longer-term regulation of these metabolic pathways. Notably, several key transcription factors are shown to be involved in the control of glucose metabolism including glycolysis and gluconeogenesis in the liver. In this review, we would like to illustrate the current understanding of glucose metabolism, with an emphasis on the transcription factors and their regulators that are involved in the chronic control of glucose homeostasis.

Overview of glucose metabolism in the liver

Under feeding conditions, dietary carbohydrates are digested and processed by various glucosidases in the digestive tract, and the resultant monosaccharides, mainly hexose glucose, are transported into various tissues as a primary fuel for ATP generation.1 In most mammalian tissues, the catabolism of glucose into pyruvate, termed glycolysis, is preserved as a major pathway in eliciting ATP. In tissues with abundant mitochondria, cytosolic pyruvate is transported into the mitochondrial matrix, converted to acetyl-CoA by pyruvate dehydrogenase complex, and incorporated into the tricarboxylic acid cycle in conjunction with oxaloacetate. The cycle generates energy equivalent to ATP (that is, GTP) as well as both NADH and FADH2, which serve as important electron carriers for electron transport chain-oxidative phosphorylation, resulting in the generation of ATP.

In some cases, such as red blood cells lacking mitochondria or cells under ischemic conditions, pyruvate is converted into lactate in the cytosol to regenerate the NAD+ that is necessary for the continued generation of ATP by substrate-level phosphorylation via anaerobic glycolysis. Excessive carbohydrates in the liver are first converted into glycogen, a storage form of glucose in animals, by glycogenesis. In addition, in a carbohydrate-rich diet, the excessive carbohydrates are also converted into fatty acids via lipogenesis using the acetyl-CoA generated from glycolysis-driven pyruvate, which is incorporated into very low density lipoproteins for transport to white adipose tissue for the storage.2 The regulation of glycogen metabolism is examined in detail in this section, and the transcriptional control of glycolysis and lipogenesis is delineated in the following section.

Under fasting conditions, the liver has a major role in generating glucose as a fuel for other tissues, such as the brain, red blood cells and muscles. Initially, an increase in the pancreatic hormone glucagon initiates the cascade of kinase action (stated below in detail) that releases glucose from the stored glycogen via glycogenolysis.1 Normally, stored glycogen is critical for maintaining glucose homeostasis in mammals during an overnight fasting period. During a longer term fast or starvation, essentially all of the stored glycogen in the liver is depleted (after ~30 h of fasting), and de novo glucose synthesis or gluconeogenesis is responsible for the generation of glucose as a fuel for other tissues. Major non-carbohydrate precursors for gluconeogenesis are lactate, which is transported from peripheral tissues such as skeletal muscles or red blood cells, and glycerol, which is released from the adipose tissues via enhanced lipolysis during fasting. Most of the initial precursors for gluconeogenesis are generated in the mitochondria (except glycerol 3-phosphate via glycerol kinase activity), but the majority of the reaction occurs in the cytosolic part of the cell. The complex regulatory mechanism is delineated in detail in the following section, with an emphasis on the transcriptional control of key regulatory enzyme genes.

Regulation of glycogen metabolism in the liver

The accumulation of glycogen in the liver during feeding conditions provides a storage form of glucose that can be used in times of reduced food intake (Figure 1). Multiple layers of regulation are required for this process for both the activation of glycogen synthase, which is a key enzyme of glycogenesis (glycogen synthesis), and the inhibition of glycogen phosphorylase, which is a key enzyme of glycogenolysis (glycogen breakdown) in the liver. Glycogen synthase is a major enzyme that facilitates the elongation of glycogen chains by catalyzing the transfer of the glucose residue of UDP-glucose to the non-reducing end of a pre-existing glycogen branch to produce a new α1→4 glycosidic linkage. The regulation of glycogen synthase has been mostly studied using a muscle-specific isoform. In the muscle, glycogen synthase is inactivated via phosphorylation on multiple serine residues by various serine/threonine kinases such as casein kinase-1, protein kinase A (PKA), and glycogen synthase kinase-3 (GSK-3). Most notably, the phosphorylation of glycogen synthase by GSK-3 at serine residues 640, 644 and 648 has been linked to the most important inhibitory post-translational modification for its catalytic activity.

Regulation of hepatic glycogen metabolism. Under fasting conditions, glucagon and epinephrine induce cAMP-dependent signaling cascades, leading to the activation of glycogen phosphorylase and glycogenolysis while inhibiting glycogenesis. Conversely, feeding enhances insulin-mediated signaling in the liver, leading to the activation of both PP1 and Akt, thus promoting glycogen synthesis in response to increased glucose uptake in the liver. See the main text for more specific regulatory pathways. cAMP, cyclic AMP.

Under fasting conditions, dephosphorylated and active GSK-3 phosphorylate and inactivate glycogen synthase, leading to the inhibition of hepatic glycogen synthesis. On feeding, increased insulin signaling activates Akt in the cell, which in turn phosphorylates and inactivates GSK-3, thus resulting in the activation of glycogen synthase. In addition, increased concentrations of glucose 6-phosphate allosterically activate this enzyme, thus potentiating its catalytic activity under feeding conditions.3, 4 One recent publication argues against the role of GSK-3 in the regulation of the liver-specific isoform of glycogen synthase. In that study, Guinovart et al.5 mutated the corresponding serine residues that are shown to be regulated by GSK-3 in the liver-isoform of glycogen synthase. They found that the mutation of those residues did not affect the overall enzyme activity but that the mutation of serine 7 to alanine, a site that is recognized and regulated by PKA, led to the increased activity of this enzyme. Further study is necessary to determine whether these results can be verified in vivo using animal models such as liver-specific knock-in mice for S7A liver glycogen synthase. The protein phosphatase 1 (PP1) may be responsible for the dephosphorylation and activation of glycogen synthase. Accordingly, both glucose and insulin have been shown to activate PP1 activity, whereas glucagon and epinephrine have been linked to the inhibition of its activity.

Glycogen phosphorylase is a major enzyme involved in glycogenolysis (Figure 1). This enzyme catalyzes the reaction of the removal of a glucose residue from the non-reducing end of a glycogen chain, leading to the generation of glucose 1-phosphate.6 Glucose 1-phosphate can be converted into glucose 6-phosphate by phosphoglucomutase, and glucose 6-phosphate can be incorporated into glycolysis or further converted into glucose by glucose 6-phosphatase, depending on the energy status of the organism. Glycogen phosphorylase is active when it is phosphorylated at its serine 14 residue. The phosphorylation of glycogen phosphorylase requires a cascade mechanism of epinephrine and glucagon in the liver. On the activation of Gαs by the binding of hormones to cell surface G protein-coupled receptors (beta adrenergic receptor or glucagon receptor), the intracellular cyclic AMP (cAMP) levels increase via adenylate cyclase, leading to the activation of PKA. PKA is then responsible for the phosphorylation and activation of glycogen phosphorylase kinase, which in turn phosphorylates and activates glycogen phosphorylase to enhance glycogen breakdown. Under feeding conditions, this kinase cascade is inactive due to the lack of secretion of catabolic hormones. In addition, insulin promotes the activation of PP1, which dephosphorylates and inactivates glycogen phosphorylase. In essence, the anabolic hormone insulin promotes glycogenesis and inhibits glycogenolysis via the activation of PP1, leading to the dephosphorylation of glycogen phosphorylase (inactivation) and glycogen synthase (activation), and via the activation of Akt, leading to the phosphorylation of GSK-3 (inactivation) that is unable to phosphorylate and inactivate glycogen synthase.

Control of hepatic glycolysis

As stated above, glycolysis is critical to the catabolism of glucose in most cells to generate energy. The key rate-limiting enzymes for this pathway include glucokinase (GK, also termed hexokinase IV), which converts glucose into glucose 6-phosphate; phosphofructokinase-1 (PFK-1), which converts fructose 6-bisphosphate into fructose 1,6-bisphosphate; and liver-type pyruvate kinase (L-PK), which converts phosphoenolpyruvate (PEP) into pyruvate in the liver. These enzymes are tightly regulated by allosteric mediators that generally promote the catabolism of glucose in the cell.2, 7, 8, 9

GK is a high Km hexokinase that is present in the liver and the pancreatic beta cells, thus functioning as a glucose sensor for each cell type. Unlike the other hexokinase isotypes, GK activity is not allosterically inhibited by its catalytic product, glucose 6-phosphate in the cell, thus enabling the liver to continuously utilize glucose for glycolysis during conditions of increased glucose availability, such as during feeding conditions. GK is regulated via its interaction with glucokinase regulatory protein (GKRP). In the low intracellular glucose concentration during fasting, the binding of GK and GKRP is enhanced by fructose 6-phosphate, leading to the nuclear localization of this protein complex. Higher concentrations of glucose during feeding compete with fructose 6-phosphate to bind this complex, which promotes the cytosolic localization of GK that is released from GKRP, thus causing the increased production of glucose 6-phosphate in this setting.10

PFK-1 catalyzes the metabolically irreversible step that essentially commits glucose to glycolysis. This enzyme activity is allosterically inhibited by ATP and citrate, which generally indicate a sign of energy abundance. Reciprocally, it is allosterically activated by ADP or AMP, making it more efficient to bring about glycolysis to produce more ATP in the cell. In addition, PFK-1 activity is allosterically activated by fructose 2,6-bisphosphate (F26BP), a non-glycolytic metabolite that is critical for the regulation of glucose metabolism in the liver. F26BP is generated from fructose 6-phosphate by the kinase portion of a bifunctional enzyme that contains both a kinase domain (phosphofructokinase-2, PFK-2) and a phosphatase domain (fructose 2,6-bisphosphatase, F-2,6-Pase). PFK-2 is activated by the insulin-dependent dephosphorylation of a bifunctional enzyme that activates PFK-2 activity and simultaneously inhibits F-2,6-Pase activity to promote the increased F26BP concentration. Glucagon-mediated activation of PKA is shown to be responsible for the phosphorylation and inactivation of the kinase portion of this enzyme.7

Unlike its muscle counterpart, L-PK is also a critical regulatory step in the control of glycolysis in the liver. As in the case of other glycolytic enzymes, L-PK activity is regulated by both allosteric mediators and post-translational modifications. L-PK activity is allosterically activated by fructose 1,6-bisphosphate, an indicator for the active glycolysis. By contrast, its activity is allosterically inhibited by ATP, acetyl-CoA, and long-chain fatty acids, all of which signal an abundant energy supply. Additionally, the amino acid alanine inhibits its activity, as it can be readily converted to pyruvate by a transamination reaction. L-PK is inhibited by PKA following a glucagon-mediated increase in intracellular cAMP during fasting and is activated by insulin-mediated dephosphorylation under feeding conditions.7

In addition to the acute regulation of key regulatory enzymes, glycolysis is regulated by a transcriptional mechanism that is activated during feeding conditions. Two major transcription factors, sterol regulatory element binding protein 1c (SREBP-1c) and carbohydrate response element binding protein (ChREBP), are responsible for the transcriptional activation of not only glycolytic enzyme genes but also the genes involved in fatty acid biosynthesis (such as fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase 1 (SCD1)) and triacylglycerol formation (such as glycerol 3-phosphate acyltransferase (GPAT) and diacylglycerol acyltransferase 2 (DGAT2)), a process that is normally activated by a carbohydrate-rich diet (Figure 2).11 Because these processes are often coordinately regulated, that is activated during feeding and inhibited by fasting, they are sometimes collectively called lipogenesis.

Regulation of hepatic glycolysis. Under feeding conditions, increased glucose uptake in hepatocytes promotes glycolysis and lipogenesis to generate triglycerides as storage forms of fuel. This process is transcriptionally regulated by two major transcription factors in the liver, SREBP-1c and ChREBP-Mlx heterodimer, which mediate the insulin and glucose response, respectively. See the main text for more specific regulatory pathways.

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SREBPs are the major regulators of lipid metabolism in mammals. They are members of the basic helix-loop-helix leucine zipper (b/HLH/LZ) type transcription factor families comprising SREBP-1a, SREBP-1c, and SREBP-2. SREBP is translated as an endoplasmic reticulum (ER)-bound precursor form that contains the N-terminal transcription factor domain and the C-terminal regulatory domain linked with the central transmembrane domain.12 Within this family of transcription factors, SREBP-2 is linked to the control of cholesterol uptake or biosynthesis in the liver by the transcriptional activation of the genes involved in the pathway including low density lipoprotein receptor (LDLR), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1), and farnesyl diphosphate synthase (FDPS). SREBP-1c, however, activates the genes encoding the enzymes for lipogenesis (FAS, ACC, SCD1, and DGAT2) as well as GK, which is a first enzyme in the commitment step of glucose utilization in the liver. Indeed, liver-specific SREBP-1c knockout mice showed an impaired activation of lipogenic genes in a high carbohydrate diet, thus confirming the importance of this transcription factor in the regulation of hepatic glycolysis and fatty acid biosynthesis.13 SREBP-1a is not highly expressed in the liver but was shown to be involved in the formation of inflammasomes in response to lipopolysaccharide (LPS) treatment in macrophages by transcriptional activation of Nlrp1.14 The regulation of SREBP-2 and SREBP-1c are quite distinct in the liver. The expression of SREBP-2 is not controlled by sterols, but its proteolytic processing is tightly regulated by intracellular concentrations of cholesterol. It is normally bound in the ER via the interaction of SREBP-cleavage-activating protein (SCAP) and insulin-induced gene protein (INSIG) in the presence of high intracellular cholesterol levels, and the reduction in the cholesterol concentration releases the interaction of SCAP and SREBP-2/INSIG complex, resulting in the translocation of the latter complex into the Golgi apparatus and the liberation of the active SREBP-2 factor by sequential proteolytic cleavages.15 Unlike SREBP-2, SREBP-1c is mainly regulated at the transcription level by insulin. The exact transcription factor that mediates this insulin-dependent signal is not yet clear, although SREBP-1c itself might be involved in the process as part of an auto-regulatory loop. Interestingly, the oxysterol-sensing transcription factor liver X receptor (LXR) is shown to control the transcription of SREBP-1c, suggesting that SREBP-1c and SREBP-2 could be regulated differently in response to cellular cholesterol levels.16 Recent studies have revealed the involvement of various kinases in the control of SREBP-1c activity. In HepG2 cells, PKA was shown to reduce the DNA binding ability of SREBP-1a by the phosphorylation of serine 338 (equivalent of serine 265 for SREBP-1c).17 A report by Bengoechea and Ericsson suggested that GSK-3, a kinase known to reduce glycogen synthesis by targeting glycogen synthase, downregulates SREBP-1 activity via the phosphorylation of the C-terminal residue that promotes the ubiquitin ligase Fbw7-dependent degradation of SREBP-1 proteins.18 In addition, both AMP activated protein kinase (AMPK) and its related kinase salt-inducible kinase (SIK) 1 are involved in the down-regulation of its activity through inhibitory phosphorylation (serine 372 for AMPK, which blocks proteolysis and nuclear localization of SREBP-1c, and serine 329 for SIK1, which directly reduces its transcriptional activity).19, 20 These data suggest that the fine-tuning of SREBP-1c activity is critical to the maintenance of glucose and lipid homeostasis in the liver.

The other prominent transcription factor for controlling glycolysis and fatty acid biosynthesis in the liver is ChREBP. ChREBP was initially known as Williams-Beuren syndrome critical region 14 (WBSCR14) and was considered one of the potential genes that instigate Williams-Beuren syndrome. Later, by using a carbohydrate response element (ChoRE) from L-PK, ChREBP was isolated as a bona fide transcription factor for binding ChoRE of glycolytic promoters.21 Indeed, ChREBP is highly expressed in tissues that are active in lipogenesis such as the liver, brown adipocytes, white adipocytes, small intestine, and kidney. As in the case for SREBP, ChREBP belongs to the b/HLH/LZ transcription factor family and forms a heterodimer with another b/HLH/LZ transcription factor Max-like protein X (Mlx) on the glycolytic promoter.22 As in the case for the SREBP-1c, the expression of ChREBP is increased in the liver as a result of a high carbohydrate diet, and the effect was recapitulated in primary hepatocytes with high glucose treatment.

A recent report indeed suggested a role for LXR in the transcriptional activation of ChREBP in response to glucose, although the study needs to be further verified because the transcriptional response is shown not only by the treatment of D-glucose, a natural form of glucose present in animals, but also by the treatment of unnatural L-glucose, a form of glucose that is not known to activate lipogenesis in the liver.23 Moreover, studies performed in LXR knockout mice revealed no changes in ChREBP expression in the liver, arguing against the role of LXR in the control of ChREBP.24 Glucose is also shown to regulate ChREBP activity by controlling its nuclear localization. There are three prominent serine/threonine residues that are targeted by serine/threonine kinases. PKA is shown to phosphorylate serine 196, which is critical for cellular localization, and threonine 666, which is critical for its DNA binding ability, whereas AMPK phosphorylate serine 568 dictates its DNA binding ability. All three sites are phosphorylated under fasting conditions by these kinases and are dephosphorylated under feeding by xylulose 5-phosphate (X5P)-mediated activity of protein phosphatase 2A (PP2A).25, 26 However, the current model needs to be further verified due to the contrasting data that have been published regarding the role of these phosphorylations on ChREBP activity.

First, high glucose concentrations in primary hepatocytes do not result in decreased cAMP levels or PKA activity, suggesting that other signals might be necessary to mediate the high glucose-dependent nuclear translocation of ChREBP. In addition, a serine to alanine mutant of ChREBP still requires high glucose for its full activity, suggesting that additional actions are necessary to recapitulate the high glucose-mediated activation/nuclear localization of ChREBP in the liver.27, 28 The physiological role of ChREBP in liver glucose metabolism was verified by in vivo studies. ChREBP knockout mice were born in a Mendelian ratio and showed no developmental problems. The knockout animals showed reduced liver triacylglycerol levels together with a reduction in lipogenic gene expression, thus confirming the role of ChREBP in the control of hepatic glycolysis and fatty acid synthesis.29 Interestingly, the compensatory increase in glycogen was observed in the livers of these mice, suggesting that these mice adapted to store more glycogen as a storage form of fuel as opposed to triacylglycerol. In ob/ob mouse liver, increased accumulation of nuclear ChREBP was shown, suggesting that this phenomenon might be causal to the fatty liver phenotype in these mice. Indeed, knockdown of ChREBP in ob/ob mice reduced the rate of lipogenesis with decreased expression of most lipogenic genes.30 Furthermore, the depletion of hepatic ChREBP in ob/ob mice improved hyperglycemia, hyperlipidemia, and hyperinsulinemia, suggesting that regulation of ChREBP might be critical in the control of metabolic disorders in the presence of obesity and insulin resistance.

Control of hepatic gluconeogenesis

Prolonged fasting or starvation induces de novo glucose synthesis from non-carbohydrate precursors, termed hepatic gluconeogenesis. This process initiates from the conversion of pyruvate to oxaloacetate by pyruvate carboxylase (PC) in the mitochondria and eventually concludes in the conversion into glucose via several enzymatic processes in the cytosol.7, 8, 9 Among the substrates for gluconeogenesis are amino acids, which can be converted into either pyruvate or intermediates of the tricarboxylic acid cycle; lactate, which can be converted into pyruvate by lactate dehydrogenase; and glycerol (from increased lipolysis in the white adipocytes under fasting), which can be converted into dihydroxyacetone phosphate, a gluconeogenic intermediate (a two-step process that is catalyzed by glycerol kinase and glycerol 3-phosphate dehydrogenase). Key regulatory enzymes in that pathway, including glucose 6-phosphatase (G6Pase), fructose 1,6-bisphosphatase (Fbpase1), PC, and phosphoenolpyruvate carboxykinase (PEPCK), are activated under fasting conditions to enhance gluconeogenic flux in that setting.

Mitochondrial acetyl-CoA (derived from the increased fatty acid oxidation under fasting) functions as a key allosteric activator of PC, leading to the increased production of oxaloacetate for the gluconeogenesis. In addition, F26BP, which is a key allosteric regulator for glycolysis by activating PFK-1, was shown to inhibit gluconeogenesis via the allosteric inhibition of Fbpase1, which helps reciprocally control gluconeogenesis and glycolysis under different dietary statuses. Because Fbpase2 is activated but PFK-2 is inhibited under fasting, the lack of F26BP enables the activation of Fbpase1 and the increased production of fructose 6-phosphate in gluconeogenesis. The chronic activation of gluconeogenesis is ultimately achieved via transcriptional mechanisms. Major transcriptional factors that are shown to induce gluconeogenic genes include CREB, FoxO1, and several nuclear receptors (Figure 3).31

Regulation of hepatic gluconeogenesis. Under fasting conditions, hepatic gluconeogenesis is enhanced via a decreased concentration of insulin and an increased concentration of insulin counterregulatory hormones such as glucagon. CREB/CRTC2, FoxO1, and a family of nuclear receptors are critical in coordinating the fasting-mediated activation of gluconeogenesis in the liver. FoxO1, forkhead box O 1

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Under fasting conditions, glucagon and epinephrine can increase the cAMP concentration in the liver via the activation of adenylate cyclase, leading to the activation of PKA and the subsequent induction of CREB via its serine 133 phosphorylation. The phosphorylation event is crucial in the recruitment of histone acetyltransferases (HAT) CBP/p300, leading to the histone H3 and H4 acetylation and the transcriptional activation of target genes.32, 33 CREB-dependent transcription is further enhanced by association with additional transcriptional coactivators CREB regulated transcription coactivators (CRTCs), which are a target for CBP/p300-mediated acetylation, which in turn promotes a tighter association of CREB, CBP/p300, and CRTC on the promoter.34, 35, 36 The role of CREB in the control of hepatic gluconeogenesis has been confirmed by in vivo studies by utilizing albumin promoter-driven ACREB (CREB inhibitor) transgenic mice and siRNA-mediated CREB knockdown mouse models.37, 38 In both mouse models, the inactivation of CREB reduced blood glucose levels and reduced the expression of gluconeogenic genes in mice, showing that CREB is a bona fide physiological transcriptional regulator of hepatic gluconeogenesis in vivo. In contrast, the role for CBP in gluconeogenesis is still controversial. Disruption of CREB-CBP interaction does not appear to affect glucose homeostasis because mice exhibiting a stable expression of mutant CBP that was unable to bind CREB showed normal glycemia.36 Furthermore, mutant mice producing CH1 null products (ΔCH1-a domain that is critical for insulin-mediated depression of CBP activity) displayed normal fasting gluconeogenesis.39 Thus, further studies are required to describe the potential role of HATs in the transcriptional control of CREB activity in this setting.

The CRTC family of transcriptional coactivators consists of CRTC1, CRTC2 and CRTC3, which were isolated by the expression library screening as activaters of CREB-dependent transcription.34 CRTC activity is regulated by cellular localization, and the AMPK family of serine/threonine kinases, such as AMPK, SIK1 or SIK2, was shown to be involved in the inhibitory phosphorylation of this factor (serine 171 for CRTC2).40 In addition, the phosphorylation status of CRTC is regulated by a pair of serine/threonine phosphatases (PP2B or PP4) in response to cAMP signaling or calcium concentration in the cell.41, 42 CRTC activity is also further enhanced by O-GlcNAcylation on serine 171 and arginine methylation by protein arginine methyltransferase (PRMT) 6.43, 44 Among the family members, CRTC2 is the prominent isoform in the liver. Recent studies have delineated the role of CRTC2 in the regulation of hepatic gluconeogenesis in vivo. Knockdown of CRTC2 in mice by RNAi reduced blood glucose levels and led to a concomitant repression of gluconeogenic gene expression.36 In addition, CRTC2 knockout mice displayed lower plasma glucose levels and improved glucose tolerance, indeed showing that CRTC2 is crucial in controlling hepatic glucose metabolism in vivo.45 A recent study indicated that CRTC2 could also coactivate other bZIP transcription factors that are implicated in the regulation of glucose homeostasis.46, 47 Further study is required to delineate the potential contributions from other bZIP factors in the control of hepatic gluconeogenesis by using tissue-specific knockout mouse models.

The forkhead box O (FoxOs) belongs to a class of forkhead families of transcription, which recognize the AT-rich insulin response element on the promoter.48, 49 Of the four major isoforms in mammals (FoxO1, FoxO3, FoxO4, and FoxO6), FoxO1 is the predominant isoform in the liver. The activity of this protein is also regulated by phosphorylation-dependent subcellular localization, and three major serine and threonine residues (threonine 24, serine 253 and serine 316 for murine FoxO1) are targeted by the insulin/Akt pathway. Following phosphorylation, FoxO1 moves to the cytosol via an association with 14-3-3, where it is degraded by the ubiquitin/proteasome-dependent pathway.50, 51, 52 In addition to phosphorylation, FoxO1 was shown to be regulated by the HAT-dependent acetylation of specific lysine residues (lysine 242, 245 and 262 for murine FoxO1), which also inhibit its transcriptional activity.53 In the liver, FoxO1 regulates hepatic gluconeogenesis via the transcriptional regulation of key genes in the pathway such as PEPCK and G6Pase and is considered a major regulatory point for the insulin-mediated repression of hepatic gluconeogenesis.54 Indeed, mice with liver-specific knockout of FoxO1 showed lower plasma glucose levels that those associated with reduced hepatic glucose output, thus underscoring the physiological significance of this factor in the control of glucose homeostasis in vivo.54, 55 As in the case for CREB, FoxO1 requires transcriptional coactivators for optimal transcriptional activity.

Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), a known coactivator for nuclear receptors, functions as a key transcriptional coactivator for FoxO1 in hepatic gluconeogenesis.56 PGC-1α was originally isolated in brown adipocytes and was shown to control adaptive thermogenesis in response to cold shock in that setting.57 In the liver, the expression of PGC-1α is upregulated under fasting conditions via a CRTC2-CREB-dependent mechanism and is critical in maintaining prolonged gluconeogenesis under starvation by enhancing the transcriptional activity of FoxO1 as a coactivator.38, 57, 58 Indeed, the depletion of hepatic PGC-1α in mice results in lower fasting glucose levels with a concomitant reduction in hepatic gluconeogenesis, thus showing the physiological relevance of this coactivator in the control of glucose homeostasis.59, 60 As is the case for CRTC2, FoxO1 activity is enhanced by arginine methylation by PRMT. In this case, PRMT1 promotes the asymmetric dimethylation of arginine 248 and 250 in FoxO1, which blocks the binding of Akt and the subsequent Akt-mediated phosphorylation of the adjacent serine residue (serine 253), thus enhancing the nuclear localization of FoxO1.61 Consequently, the chromatin occupancy of FoxO1 onto the gluconeogenic promoter and gluconeogenesis itself are increased due to the PRMT1-dependent arginine methylation.62 Acute knockdown of hepatic PRMT1 in mice reduces FoxO1-mediated glucose production, confirming that PRMT1 is crucial in modulating FoxO1 activity and subsequent gluconeogenesis in the physiological context.

Nuclear receptors belong to the superfamily of transcription factors that possess two Cys2-His2 type zinc finger motifs as a DNA binding domain as well as both ligand-independent and ligand-dependent transactivation domains.63 The latter activation domain also contains a ligand-binding domain. Nuclear receptors can be classified into one of three subgroups based on their dimer-forming potential. Homodimeric nuclear receptors are also called cytosolic receptors because they reside in the cytosol and associate with molecular chaperones such as heat-shock proteins. On binding to the ligand, they form homodimers and translocate to the nucleus to bind a specific response element termed the hormone response element to elicit the ligand-dependent transcriptional response. Most of the steroid hormone receptors, such as the glucocorticoid receptor (GR), estrogen receptor (ER), and progesterone receptor (PR), belong to this subfamily. By contrast, heterodimeric nuclear receptors reside in the nucleus and are bound to their cognate binding sites together with the universal binding partner retinoid X receptor (RXR). In the absence of the ligands, these factors repress the transcription of target genes in association with transcriptional corepressors such as histone deacetylase or nuclear receptor corepressor (NCoR)/silencing mediator of retinoid and thyroid hormone receptors (SMRT). Ligand binding initiates the conformational changes of these heterodimeric nuclear receptors, which promotes the dissociation of corepressors and the association of coactivators such as CBP/p300, p160 steroid receptor coactivator family, and PGC-1α.

Examples of this class of nuclear receptors include members of peroxisome proliferator-activated receptors, LXRs, vitamin D receptors and thyroid hormone receptors. The final subclasses of nuclear receptors are types that function as monomers. They usually lack specific endogenous ligands and are often called orphan nuclear receptors. Some of them also lack DNA binding domain and thus function as transcriptional repressors of various transcription factors, including members of nuclear receptors. They are called atypical orphan nuclear receptors. Among the homodimeric nuclear receptors, the role of GR has been linked to the control of hepatic gluconeogenesis. GR is activated by cortisol, which is released from the adrenal cortex in response to chronic stresses such as prolonged fasting.64, 65 GR was shown to directly bind to the cognate binding sites found in the promoters of gluconeogenic genes such as PEPCK and G6Pase and to enhance transcription of these genes under fasting conditions. The same response elements were also shown to be recognized and regulated by hepatocyte nuclear factor 4 (HNF4), a member of heterodimeric nuclear receptors, which suggests that these nuclear receptors could coordinately function to control hepatic gluconeogenesis in response to fasting.58

In accordance with this idea, the activity of these nuclear receptors can be effectively integrated by the function of transcriptional co-activator PGC-1α. Recently, estrogen-related receptor gamma (ERRγ), a member of monomeric nuclear receptors, was shown to be involved in the regulation of hepatic gluconeogenesis.66, 67 In the liver, ERRγ expression is increased under fasting or by insulin resistance in a CRTC2-CREB-dependent manner. This factor regulates hepatic gluconeogenesis by binding to unique response elements that are distinct from the known nuclear receptor-binding sites in the promoters of PEPCK and G6Pase. Inhibition of ERRγ activity by injecting either RNAi or the inverse agonist GSK5182 effectively reduced hyperglycemia in diabetic mice, suggesting that the control of this factor might potentially be beneficial in the treatment of patients with metabolic diseases. As is the case for other nuclear receptors that control hepatic gluconeogenesis, ERRγ activity is further enhanced by interaction with the transcriptional coactivator PGC-1α, showing that this coactivator functions as a master regulator for the hepatic glucose metabolism.

Three members of atypical orphan nuclear receptors, the small heterodimer partner (SHP, also known as NR0B2); the dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X (DAX-1, also known as NR0B1); and the SHP-interacting leucine zipper protein (SMILE) are implicated in the transcriptional repression of hepatic gluconeogenesis.68, 69, 70 SHP is ubiquitously expressed in mammalian tissues, with the highest expression occurring in the liver. Interestingly, metformin directly activates the transcription of SHP via an AMPK-mediated pathway. SHP directly inhibits cAMP-dependent transcription by binding to CREB, resulting in the reduced association of CREB with CRTC2.71, 72 The adenovirus-mediated overexpression of SHP could effectively reduce blood glucose levels in diabetic mice, thus showing the importance of this pathway in the control of hepatic glucose metabolism.

These results provide a dual mechanism for a metformin-AMPK dependent pathway to inhibit hepatic gluconeogenesis at the transcriptional level; an acute regulation of CRTC2 phosphorylation to inhibit the CRTC2-CREB-dependent transcriptional circuit; and a longer-term regulation of gluconeogenic transcription by enhanced SHP expression. Both DAX-1 and SMILE were shown to repress hepatic gluconeogenesis by inhibiting HNF4-dependent transcriptional events.73, 74 SIK1, a member of the AMPK-related kinases, was shown to enhance DAX-1 expression in the liver, whereas Akt was shown to activate the transcription of SMILE to target the HNF4 pathway under feeding conditions. Interestingly, SMILE was shown to directly replace PGC-1α from HNF4 and the gluconeogenic promoters, suggesting that this factor could potentially function as a major transcriptional repressor of hepatic gluconeogenesis in response to insulin signaling. Further study is necessary to fully understand the relative contribution of these nuclear receptors in the control of glucose homeostasis in both physiological conditions and pathological settings.

Concluding remarks

In this review, we attempted to describe the current understanding of the regulation of glucose metabolism in the mammalian liver. Under feeding conditions, glucose, a major hexose monomer of dietary carbohydrate, is taken up in the liver and oxidized via glycolysis. The excess glucose that is not utilized as an immediate fuel for energy is stored initially as glycogen and is later converted into triacylglycerols via lipogenesis. Glycogenesis is activated via the insulin-Akt-mediated inactivation of GSK-3, leading to the activation of glycogen synthase and the increased glycogen stores in the liver. Insulin is also critical in the activation of PP1, which functions to dephosphorylate and activate glycogen synthase. In addition, PP1 inhibits glycogenolysis via the dephosphorylation/inactivation of glycogen phosphorylase. Glycolysis is controlled by the regulation of three rate-limiting enzymes: GK, PFK-1 and L-PK. The activities of these enzymes are acutely regulated by allosteric regulators such as ATP, AMP, and F26BP but are also controlled at the transcription level. Two prominent transcription factors are SREBP-1c and ChREBP, which regulate not only the aforementioned glycolytic enzyme genes but also the genes encoding enzymes for fatty acid biosynthesis and triacylglycerol synthesis (collectively termed as lipogenesis).

The importance of these transcription factors in the control of glycolysis and fatty acid biosynthesis has been verified by knockout mouse studies, as described in the main text. The liver also has a critical role in controlling glucose homeostasis under fasting conditions. Initially, insulin counterregulatory hormones such as glucagon and epinephrine are critical in activating the PKA-driven kinase cascades that promote glycogen phosphorylase and glycogenolysis in the liver, thus enabling this tissue to provide enough fuel for peripheral tissues such as the brain, red blood cells and muscles. Subsequently, these hormones together with adrenal cortisol are crucial in initiating the transcriptional activation of gluconeogenesis such as PC, PEPCK and G6Pase. The major transcription factors involved in the pathway include CREB, FoxO1 and members of nuclear receptors, with aid from transcriptional coactivators such as CRTC, PGC-1α and PRMTs. These adaptive responses are critical for maintaining glucose homeostasis in times of starvation in mammals. Further study is necessary by using liver-specific knockout mice for each regulator of hepatic glucose metabolism to provide better insights into the intricate control mechanisms of glucose homeostasis in mammals.


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This work is supported by the National Research Foundation of Korea (grant nos.: NRF-2012M3A9B6055345, NRF-2015R1A5A1009024 and NRF- 2015R1A2A1A01006687), funded by the Ministry of Science, ICT & Future Planning, Republic of Korea, a grant of the Korean Health technology R&D Project (grant no: HI13C1886), Ministry of Health & Welfare, Republic of Korea and a grant from Korea University, Seoul, Republic of Korea.


  1. Division of Life Sciences, College of Life Sciences & Biotechnology, Korea University, Seoul, 136-713, Korea

    Hye-Sook Han, Geon Kang, Jun Seok Kim, Byeong Hoon Choi & Seung-Hoi Koo

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Metabolic host response and therapeutic approaches to influenza infection

Based on available metabolomic studies, influenza infection affects a variety of cellular metabolic pathways to ensure an optimal environment for its replication and production of viral particles. Following infection, glucose uptake and aerobic glycolysis increase in infected cells continually, which results in higher glucose consumption. The pentose phosphate shunt, as another glucose-consuming pathway, is enhanced by influenza infection to help produce more nucleotides, especially ATP. Regarding lipid species, following infection, levels of triglycerides, phospholipids, and several lipid derivatives undergo perturbations, some of which are associated with inflammatory responses. Also, mitochondrial fatty acid β-oxidation decreases significantly simultaneously with an increase in biosynthesis of fatty acids and membrane lipids. Moreover, essential amino acids are demonstrated to decline in infected tissues due to the production of large amounts of viral and cellular proteins. Immune responses against influenza infection, on the other hand, could significantly affect metabolic pathways. Mainly, interferon (IFN) production following viral infection affects cell function via alteration in amino acid synthesis, membrane composition, and lipid metabolism. Understanding metabolic alterations required for influenza virus replication has revealed novel therapeutic methods based on targeted inhibition of these cellular metabolic pathways.


Glucagon plays a key role in glucose metabolism in vivo. Administration of exogenous glucagon increases glucose levels in fasted or fed animals (63, 96), and similar observations were made in humans (29, 42, 57). Consistent with its role as a counterregulatory hormone of insulin, glucagon raises plasma glucose levels in response to insulin-induced hypoglycemia (29). In fact, glucagon administration is used clinically to treat hypoglycemia in humans (14, 29, 35). Numerous ex vivo or in vitro studies have directly demonstrated that glucagon stimulates glucose output from intact perfused rat livers (7, 28, 43) resulting from increases in both glycogenolysis and gluconeogenesis. Similarly, glucagon also stimulates glucose output from primary hepatocytes in culture (60, 92, 93).

Several lines of evidence indicate that glucagon is a sensitive and timely regulator of glucose homeostasis in vivo. Small doses of glucagon are sufficient to induce significant glucose elevations (35, 57, 63). The effect of glucagon can occur within minutes and dissipate rapidly (27). Glucagon is secreted from islets in a pulsatile fashion (65), and such pulsatile deliveries of glucagon are more effective in inducing hepatic glucose output in vitro, ex vivo, and in vivo (49, 66,92).

Conversely, there is ample evidence demonstrating that inhibition of glucagon signaling in vivo leads to a reduction in plasma glucose, or hypoglycemia. It was shown that administration of polyclonal glucagon-neutralizing antibodies abolished the hyperglycemic response to exogenous glucagon in animals (83). A similar observation was made using a high-affinity monoclonal anti-glucagon antibody (11). Additionally, the monoclonal antibody reduced ambient blood glucose by neutralizing endogenous glucagon in normal or diabetic animals (9-11). In these experiments, the glucagon antibodies reduced free glucagon in circulation to undetectable levels (9-11).

As discussed previously, glucagon is processed from proglucagon in pancreatic α-cells by PC2 (32, 74, 76). In PC2-null (PC2 −/− ) mice, circulating glucagon was undetectable due to a severe defect in the processing of proglucagon (30). Interestingly, PC2 −/− mice had reduced fasting blood glucose as well as improved glucose tolerance. Moreover, PC2 −/− mice had significant α-cell hypertrophy, which was consistent with the compensatory response for the lack of functional glucagon. Whereas the correlation between the hypoglycemia phenotype and the lack of circulating glucagon in the PC2 −/− mice is consistent with a major role of glucagon in glycemic control, the proposal is complicated by the finding that the mice were also defective in processing proinsulin to insulin (30, 32). It was recently shown, however, that glucagon replacement via microosmotic pump corrected hypoglycemia and α-cell hypertrophy in the PC2 −/− mice, proving an unequivocal role of glucagon in glucose homeostasis in vivo (91).

A small acidic protein, 7B2, is exclusively localized to neuroendocrine tissues, and it binds to and activates PC2 (62). It was shown that 7B2-null mice displayed hypoglucagonemia as well as hypoglycemia (94). Finally, mice lacking the glucagon receptor gene (GCGR −/− ) exhibited a phenotype of decreased glycemia under both fed and fasting states compared with control mice. No overt hypoglycemia was observed inGCGR −/− mice under ambient conditions, and these mice also had improved glucose tolerance (67). Together, these results support an important role of glucagon in glycemic control in vivo.

Metabolic regulation in time and space

In contrast to the general view of metabolism as being homogeneous within and between cells, recent findings indicate striking spatial compartmentalization of metabolism at both the intercellular/tissue and subcellular levels. In addition, it is becoming clear that energy metabolism is dynamically regulated not only in space, but also in time, across many scales. Below, we highlight a few examples of such spatiotemporal compartmentalization of metabolism.

Regulation at the intercellular/tissue level

Metabolic activity is spatially regulated at the cellular and tissue level. In the brain, for example, glucose metabolism is compartmentalized between neurons and astrocytes. Neurons show a lower glycolytic activity than astrocytes because of the constant degradation of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatese-3 (PFKFB3), which produces a potent allosteric activator of a key glycolytic enzyme phosphofructokinase 1 (PFK-1) (Almeida et al., 2004 Herrero-Mendez et al., 2009). Such intercellular compartmentalization generates a gradient of the glycolytic end-product lactate from astrocytes to neurons (Mächler et al., 2016), which leads to lactate flow from astrocytes to neurons via facilitated transport. Neurons, in turn, oxidize lactate into CO2 through the tricarboxylic acid (TCA) cycle, facilitating generation of ATP via OXPHOS (Pellerin and Magistretti, 2012). This astrocyte-neuron lactate shuttle allows neurons to use glucose preferentially for the maintenance of cellular redox balance, rather than for energy production. Neurons preferentially metabolize glucose through the pentose phosphate pathway (PPP), which ensures production of the reducing agent NADPH (Herrero-Mendez et al., 2009). When neurons are forced to activate glycolysis at the expense of glucose flux via the PPP, the lack of reducing equivalents leads to neuronal apoptosis due to oxidative stress (Herrero-Mendez et al., 2009). A similar metabolic interaction has been observed in intestinal organoids in this case, the shuttling of lactate from Paneth cells to intestinal stem cells promotes mitochondrial respiration and ROS production to drive crypt formation (Rodríguez-Colman et al., 2017).

Another striking example of spatial intercellular metabolic differences is observed during the asymmetric cell division of T cells. Upon T-cell activation by antigen-producing cells, Myc, a regulator of glycolysis and glutaminolysis (Dang, 2017 Wang et al., 2011), is asymmetrically partitioned into daughter cells, leading to a metabolic asymmetry (Verbist et al., 2016). It has been suggested that this Myc asymmetry is maintained by the differential activation of mammalian target of rapamycin complex 1 (mTORC1) between daughter cells via the asymmetric distribution of amino acid transporters during cell division. Functionally, daughter cells with high Myc levels show elevated glycolysis and glutaminolysis compared with cells that have low Myc levels and are more prone to differentiate into actively proliferating effector T cells than into memory T cells (Verbist et al., 2016). This metabolic switch to a high glycolytic state also has important functional consequences (discussed below).

Regulation at the subcellular level

Cellular energy metabolism is also highly compartmentalized at the subcellular level. Although glycolytic reactions are mediated by soluble glycolytic enzymes, classical studies have suggested that these enzymes are not uniformly distributed throughout the cytoplasm, but rather are assembled into protein complexes named glycolytic metabolons, which facilitate the channeling of glycolytic intermediates (Clarke and Masters, 1975 Kurganov et al., 1985 Menard et al., 2014). Whereas the assembly of a glycolytic metabolon has not yet been confirmed in vivo, it has been demonstrated that glycolytic enzymes bind to cellular structures, including the cytoskeleton and intracellular vesicles. In a striking case, glycolytic enzymes were shown to be sequestered in an organelle, the glycosome, in Kinetoplastea, a large group of flagellated free-living and parasitic protozoans (Szöör et al., 2014). Such subcellular compartmentalization of enzymes enables local and efficient energy production at sites of the highest energy demand, as well as rapid adaptation of metabolism to environmental changes.

Subcellular compartmentalization of glycolysis is also prominent in neurons with arborization (branches). For example, the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has been found to be anchored to intracellular vesicles (Zala et al., 2013). The downstream glycolytic enzyme phosphoglycerate kinase (PGK) also localizes to vesicles, and this is essential for local ATP production and axonal transport of vesicles (Zala et al., 2013). In addition, glycolytic enzymes have been found to form clusters near presynaptic sites to facilitate synaptic vesicle cycling upon energy stress (Jang et al., 2016).

Further examples of subcellular compartmentalization of glycolysis are found during blood vessel sprouting, in which glycolytic enzymes and activity are enriched in lamellipodia at the leading edge of migrating endothelial tip cells. The subcellular localization of these enzymes to lamellipodia generates ‘ATP hotspots’ that are proposed to meet the high energy demands associated with actin remodeling and cell motility (De Bock et al., 2013). Dynamic remodeling of the actin cytoskeleton is also accompanied by mobilization of active aldolase A from F-actin, thereby coupling acceleration of glycolysis to actin remodeling (Hu et al., 2016).

Temporal regulation of metabolism across scales

Cellular energy metabolism can be compartmentalized not only in space but also in time, over various time scales. A well known example includes the coordination of energy metabolism with the cell cycle in order to meet specific bioenergetic demands of each phase of the cell cycle (Salazar-Roa and Malumbres, 2017). At the G1/S transition of the cell cycle, glycolysis is activated and mitochondria show hyperfused morphology, with greater ATP output than in any other cell cycle stages (Almeida et al., 2010 Bao et al., 2013 Mitra et al., 2009 Tudzarova et al., 2011). Mitochondrial respiration is also found to be enhanced at the G2/M transition (Wang et al., 2014). These changes in energy metabolism during the cell cycle are mediated by the cell cycle machinery [e.g. cyclin-dependent kinases (CDKs) and E3 ubiquitin ligases]. Importantly, however, the link between the cell cycle and energy metabolism is bidirectional, with metabolic state functioning as a checkpoint of the cell cycle (Jones et al., 2005 Salazar-Roa and Malumbres, 2017).

Periodic metabolic rhythms are also linked to circadian clock activity, across the kingdoms of life (Eckel-Mahan et al., 2012 Qian and Scheer, 2016). In addition, circannual rhythms in metabolism are observed in hibernators (Dark, 2005). These rhythmic activity profiles of metabolism allow the coordination of physiology with external environmental cycles.

Finally, cells also show ultradian (i.e. shorter than 24 h period) rhythms and dynamics in metabolic activity, such as glycolytic oscillations, which were first identified in budding yeast decades ago (Ghosh and Chance, 1964 Richard, 2003). More recently it has been found that, during continuous culture in glucose-limited conditions, budding yeast exhibits robust cycles (with a period of ∼4 h) of oxygen consumption, designated the yeast metabolic cycle (YMC) (Cai et al., 2011 Tu et al., 2005, 2007). Interestingly, the iterations of oxidative and reductive phases are tightly coordinated with gene expression and cell proliferation/division (Cai et al., 2011 Papagiannakis et al., 2017). The YMC provides a striking example of the potential benefit of temporally compartmentalizing metabolic processes in order to enable optimal coordination with cellular programs (Tu et al., 2005). It has been shown that restricting DNA replication to the reductive phase minimizes the risk of oxidative DNA damage (Chen et al., 2007). Ultradian rhythms of metabolism have also been found in higher eukaryotic cells and in multicellular contexts. For example, glycolytic oscillations have been found in pancreatic β cells, and the link between these oscillations and pulsatile insulin secretion is being investigated (Merrins et al., 2013).

A direct consequence of spatiotemporal compartmentalization of metabolic activity is also that metabolite levels dynamically change over time and space. In turn, cellular levels of selected intermediate metabolites, such as acetyl coenzyme A (acetyl-CoA), can have a direct impact on epigenetic modifications, establishing an intriguing link between metabolic state and, for example, gene expression and cell signaling (see below). As technologies such as metabolite sensors (Paige et al., 2012 San Martín et al., 2014) and mass spectrometry imaging methods (Passarelli et al., 2017) keep improving at a fast pace, we expect that more examples of spatiotemporal compartmentalization of metabolism will be discovered in the coming years. Clearly, a major task will be to mechanistically link such spatiotemporally regulated metabolism to distinct cellular programs and, therefore, to developmental and/or physiological outcomes.

Irreversible steps of gluconeogenesis

As previously said, gluconeogenesis is in essence glycolysis in reverse. And, of the ten reactions that constitute gluconeogenesis, seven are shared with glycolysis these reactions have a ΔG close to zero, therefore easily reversible. However, under intracellular conditions, the overall ΔG of glycolysis is about -63 kJ/mol (-15 kcal/mol) and of gluconeogenesis about -16 kJ/mol (-3.83 kcal/mol), namely, both the pathways are irreversible.
The irreversibility of the glycolytic pathway is due to three strongly exergonic reactions, that cannot be used in gluconeogenesis, and listed below.

  • The phosphorylation of glucose to glucose 6-phosphate, catalyzed by hexokinase (EC or glucokinase (EC
    ΔG = -33.4 kJ/mol (-8 kcal/mol)
    ΔG°’ = -16.7 kJ/mol (-4 kcal/mol)
  • The phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate, catalyzed by phosphofructokinase-1 or PFK-1 (EC
    ΔG = -22.2 kJ/mol (-5.3 kcal/mol)
    ΔG°’ = -14.2 kJ/mol (-3.4 kcal/mol)
  • The conversion of phosphoenolpyruvate or PEP to pyruvate, catalyzed by pyruvate kinase (EC
    ΔG = -16.7 kJ/mol (-4.0 kcal/mol)
    ΔG°’ = -31.4 kJ/mole (-7.5 kcal/mol)

In gluconeogenesis, these three steps are bypassed by enzymes that catalyze irreversible steps in the direction of glucose synthesis: this ensures the irreversibility of the metabolic pathway.
Below, such reactions are analyzed.

From pyruvate to phosphoenolpyruvate

The first step of gluconeogenesis that bypasses an irreversible step of glycolysis, namely the reaction catalyzed by pyruvate kinase, is the conversion of pyruvate to phosphoenolpyruvate.
Phosphoenolpyruvate is synthesized through two reactions catalyzed, in order, by the enzymes:

  • pyruvate carboxylase (EC
  • phosphoenolpyruvate carboxykinase or PEP carboxykinase (EC

Pyruvate → Oxaloacetate → Phosphoenolpyruvate

Pyruvate carboxylase catalyzes the carboxylation of pyruvate to oxaloacetate, with the consumption of one ATP. The enzyme requires the presence of magnesium or manganese ions

Pyruvate + HCO3 – + ATP → Oxaloacetate + ADP + Pi

The enzyme, discovered in 1960 by Merton Utter, is a mitochondrial protein composed of four identical subunits, each with catalytic activity. The subunits contain a biotin prosthetic group, covalently linked by amide bond to the ε-amino group of a lysine residue, that acts as a carrier of activated CO2 during the reaction. An allosteric binding site for acetyl-CoA is also present in each subunit.
It should be noted that the reaction catalyzed by pyruvate carboxylase, leading to the production of oxaloacetate, also provides intermediates for the citric acid cycle or Krebs cycle.
Phosphoenolpyruvate carboxykinase is present, approximately in the same amount, in mitochondria and cytosol of hepatocytes. The isoenzymes are encoded by separate nuclear genes.
The enzyme catalyzes the decarboxylation and phosphorylation of oxaloacetate to phosphoenolpyruvate, in a reaction in which GTP acts as a donor of high-energy phosphate. PEP carboxykinase requires the presence of both magnesium and manganese ions. The reaction is reversible under normal cellular conditions.

Oxaloacetate + GTP ⇄ PEP + CO2 + GDP

During this reaction, a CO2 molecule, the same molecule that is added to pyruvate in the reaction catalyzed by pyruvate carboxylase, is removed. Carboxylation-decarboxylation sequence is used to activate pyruvate, since decarboxylation of oxaloacetate facilitates, makes thermodynamically feasible, the formation of phosphoenolpyruvate.
More generally, carboxylation-decarboxylation sequence promotes reactions that would otherwise be strongly endergonic, and also occurs in the citric acid cycle, in the pentose phosphate pathway, also called the hexose monophosphate pathway, and in the synthesis of fatty acids.
The levels of PEP carboxykinase before birth are very low, while its activity increases several fold a few hours after delivery. This is the reason why gluconeogenesis is activated after birth.
The sum of the reactions catalyzed by pyruvate carboxylase and phosphoenolpyruvate carboxykinase is:

Pyruvate + ATP + GTP + HCO3 – → PEP + ADP + GDP + Pi + CO2

ΔG°’ of the reaction is equal to 0.9 kJ/mol (0.2 kcal/mol), while standard free energy change associated with the formation of pyruvate from phosphoenolpyruvate by reversal of the pyruvate kinase reaction is + 31.4 kJ/mol (7.5 kcal/mol).
Although the ΔG°’ of the two steps leading to the formation of PEP from pyruvate is slightly positive, the actual free-energy change (ΔG), calculated from intracellular concentrations of the intermediates, is very negative, -25 kJ/mol (-6 kcal/mol). This is due to the fast consumption of phosphoenolpyruvate in other reactions, that maintains its concentration at very low levels. Therefore, under cellular conditions, the synthesis of PEP from pyruvate is irreversible.
It is noteworthy that the metabolic pathway for the formation of phosphoenolpyruvate from pyruvate depends on the precursor: pyruvate or alanine, or lactate.

Phosphoenolpyruvate precursors: pyruvate or alanine

The bypass reactions described below predominate when alanine or pyruvate is the glucogenic precursor.
Pyruvate carboxylase is a mitochondrial enzyme, therefore pyruvate must be transported from the cytosol into the mitochondrial matrix. This is mediated by transporters located in the inner mitochondrial membrane, referred to as MPC1 and MPC2. These proteins, associating, form a hetero-oligomer that facilitates pyruvate transport.
Pyruvate can also be produced from alanine in the mitochondrial matrix by transamination, in the reaction catalyzed by alanine aminotransferase (EC

Conversion of Pyruvate and Alanine to Phosphoenolpyruvate

Since the enzymes involved in the later steps of gluconeogenesis, except glucose-6-phosphatase, are cytosolic, the oxaloacetate produced in the mitochondrial matrix is transported into the cytosol. However, there are no oxaloacetate transporters in the inner mitochondrial membrane. The transfer to the cytosol occurs as a result of its reduction to malate, that, on the contrary, can cross the inner mitochondrial membrane. The reaction is catalyzed by mitochondrial malate dehydrogenase (EC, an enzyme also involved in the citric acid cycle, where the reaction proceeds in the reverse direction. In the reaction NADH is oxidized to NAD + .

Oxaloacetate + NADH + H + ⇄ Malate + NAD +

Although ΔG°’ of the reaction is highly positive, under physiological conditions, ΔG is close to zero, and the reaction is easily reversible.
Malate crosses the inner mitochondrial membrane through a component of the malate-aspartate shuttle, the malate-α-ketoglutarate transporter. Once in the cytosol, the malate is re-oxidized to oxaloacetate in the reaction catalyzed by cytosolic malate dehydrogenase. In this reaction NAD + is reduced to NADH.

Malate + NAD + → Oxaloacetate + NADH + H +

Note: Malate-aspartate shuttle is the most active shuttle for the transport of NADH-reducing equivalents from the cytosol into the mitochondria. It is found in mitochondria of liver, kidney, and heart.
The reaction enables the transport into the cytosol of mitochondrial reducing equivalents in the form of NADH. This transfer is needed for gluconeogenesis to proceed, as in the cytosolic the NADH, oxidized in the reaction catalyzed by glyceraldehydes 3-phosphate dehydrogenase (EC, is present in very low concentration, with a [NADH]/[NAD + ] ratio equal to 8吆 -4 , about 100,000 times lower than that observed in the mitochondria.
Finally, the oxaloacetate is converted to phosphoenolpyruvate in the reaction catalyzed by PEP carboxykinase.

Phosphoenolpyruvate precursor: lactate

Lactate is one of the major gluconeogenic precursors. It is produced for example by:

  • red blood cells, that are completely dependent on anaerobic glycolysis for ATP production
  • skeletal muscle during intense exercise, that is, under low oxygen condition, when the rate of glycolysis exceeds the rate of the citric acid cycle and oxidative phosphorylation.

When lactate is the gluconeogenic precursor, PEP synthesis occurs through a different pathway than that previously seen. In the hepatocyte cytosol NAD + concentration is high and the lactate is oxidized to pyruvate in the reaction catalyzed by the liver isoenzyme of lactate dehydrogenase (EC In the reaction NAD + is reduced to NADH.

Lactate + NAD + → Pyruvate + NADH + H +

The production of cytosolic NADH makes unnecessary the export of reducing equivalents from the mitochondria.
Pyruvate enters the mitochondrial matrix to be converted to oxaloacetate in the reaction catalyzed by pyruvate carboxylase. In the mitochondria, oxaloacetate is converted to phosphoenolpyruvate in the reaction catalyzed by mitochondrial pyruvate carboxylase. Phosphoenolpyruvate exits the mitochondria through an anion transporter located in the inner mitochondrial membrane, and, once in the cytosol, continues in the gluconeogenesis pathway.
Note: The synthesis of glucose from lactate may be considered as the part of the Cori cycle that takes place in the liver.

From fructose 1,6-bisphosphate to fructose 6-phosphate

The second step of gluconeogenesis that bypasses an irreversible step of the glycolytic pathway, namely the reaction catalyzed by PFK-1, is the dephosphorylation of fructose 1,6-bisphosphate to fructose 6-phosphate.
This reaction, catalyzed by fructose 1,6-bisphosphatase or FBPasi-1 (EC, a Mg2 + dependent enzyme located in the cytosol, leads to the hydrolysis of the C-1 phosphate of fructose 1,6-bisphosphate, without production of ATP.

Fructose 1,6-bisphosphate + H2O → Fructose 6-phosphate + Pi

The ΔG°’ of the reaction is -16.3 kJ/mol (-3.9 kcal/mol), therefore an irreversible reaction.

From glucose 6-phosphate to glucose

The third step of gluconeogenesis that bypasses an irreversible step of the glycolytic pathway, namely the reaction catalyzed by hexokinase or glucokinase, is the dephosphorylation of glucose 6-phosphate to glucose.
This reaction is catalyzed by the catalytic subunit of glucose 6-phosphatase, a protein complex located in the membrane of the endoplasmic reticulum of hepatocytes, enterocytes and cells of the proximal tubule of the kidney. Glucose 6-phosphatase complex is composed of a glucose 6-phosphatase catalytic subunit and a glucose 6-phosphate transporter called glucose 6-phosphate translocase or T1.
Glucose 6-phosphatase catalytic subunit has the active site on the luminal side of the organelle. This means that the enzyme catalyzes the release of glucose not in the cytosol but in the lumen of the endoplasmic reticulum.
Glucose 6-phosphate, both resulting from gluconeogenesis, produced in the reaction catalyzed by glucose 6-phosphate isomerase or phosphoglucose isomerase (EC, and glycogenolysis, produced in the reaction catalyzed by phosphoglucomutase (EC, is located in the cytosol, and must enter the lumen of the endoplasmic reticulum to be dephosphorylated. Its transport is mediated by glucose-6-phosphate translocase.

The catalytic subunit of glucose 6-phosphatase, a Mg 2+ -dependent enzyme, catalyzes the last step of both gluconeogenesis and glycogenolysis. And, like the reaction catalyzed by fructose 1,6-bisphosphatase, this reaction leads to the hydrolysis of a phosphate ester.

Glucose 6-phosphate + H2O → Glucose + Pi

It should also be underlined that, due to orientation of the active site, the cell separates this enzymatic activity from the cytosol, thus avoiding that glycolysis, that occurs in the cytosol, is aborted by enzyme action on glucose 6-phosphate.
The ΔG°’ of the reaction is -13.8 kJ/mol (-3.3 kcal/mol), therefore it is an irreversible reaction. If instead the reaction were that catalyzed by hexokinase/glucokinase in reverse, it would require the transfer of a phosphate group from glucose 6-phosphate to ADP. Such a reaction would have a ΔG equal to +33.4 kJ/mol (+8 kcal/mol), and then strongly endergonic. Similar considerations can be made for the reaction catalyzed by FBPase-1.
Glucose and Pi group seem to be transported into the cytosol via different transporters, referred to as T2 and T3, the last one an anion transporter.
Finally, glucose leaves the hepatocyte via the membrane transporter GLUT2, enters the bloodstream and is transported to tissues that require it. Conversely, under physiological conditions, as previously said, glucose produced by the kidney is mainly used by the medulla of the kidney itself.

Gluconeogenesis: energetically expensive

Like glycolysis, much of the energy consumed is used in the irreversible steps of the process.
Six high-energy phosphate bonds are consumed: two from GTP and four from ATP. Furthermore, two molecules of NADH are required for the reduction of two molecules of 1,3-bisphosphoglycerate in the reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase. The oxidation of NADH causes the lack of production of 5 molecules of ATP that are synthesized when the electrons of the reduced coenzyme are used in oxidative phosphorylation.
Also these energetic considerations show that gluconeogenesis is not simply glycolysis in reverse, in which case it would require the consumption of two molecules of ATP, as shown by the overall glycolytic equation.

Glucose + 2 ADP + 2 Pi + 2 NAD + → 2 Pyruvate + 2 ATP + 2 NADH + 2 H + + 2 H2O

Below, the overall equation for gluconeogenesis:

2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + + 2 H + + 4 H2O → Glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD +

At least in the liver, ATP needed for gluconeogenesis derives mostly from the oxidation of fatty acids or of the carbon skeletons of the amino acids, depending on the available “fuel”.


The liver has a major role in the control of glucose homeostasis in the body.[1] The association between chronic liver disease (CLD) and diabetes mellitus (DM) is known since long. Such association may be due to a common mechanism that leads to both diseases such as non-alcoholic fatty liver disease (NAFLD), hemochromatosis, autoimmune liver diseases, and chronic hepatitis C.[2,3] A 10-year follow-up study of Veteran Affairs cohort revealed 2-fold increased risk of CLD in the subjects with type 2 DM (T2DM) compared to those without T2DM, after adjusting the confounding variables.[4] However, more commonly, CLD per se can lead to diabetes as known as hepatogenous diabetes (HD).[5] The term HD was first used by Megyesi et al.[6] in the 60's. This term did not get attention, as the entity was then poorly understood. Though, enough data now exist to support HD as a separate entity, it is still a neglected condition and surprisingly even American Diabetes Association does not recognize it. HD appears after the onset of liver disease in individuals without risk factors of T2DM such as high body mass index, hyperlipidemia, and previous or family history of DM.


Heterogeneity occurs in most glucose intolerance disorders, including diabetes mellitus syndromes.

Type 1 diabetes mellitus

Type 1 diabetes mellitus is characterized by absolute insulin deficiency. In type 1A, a cellular-mediated autoimmune destruction of beta cells of the pancreas occurs. The disease process is initiated by an environmental factor—that is, an infectious or noninfectious agent in genetically susceptible individuals.

Some genes in the histocompatibility leukocyte antigen (HLA) system are thought to be crucial. A stress-induced epinephrine release, which inhibits insulin release (with resultant hyperglycemia), sometimes occurs and may be followed by a transient asymptomatic period known as "the honeymoon." Lasting weeks to months, the honeymoon precedes the onset of overt, permanent diabetes.

Amylin, a beta-cell hormone that is normally cosecreted with insulin in response to meals, is also completely deficient in persons with type 1 diabetes mellitus. Amylin exhibits several glucoregulatory effects that complement those of insulin in postprandial glucose regulation. Idiopathic forms of type 1 diabetes also occur, without evidence of autoimmunity or HLA association this subset is termed type 1B diabetes.

The underlying pathophysiology of beta cell demise or dysfunction is currently more understood in type 1 diabetes than in type 2 diabetes. The rate of progression in type 1 diabetes is dependent on the age at first detection of antibody, number of antibodies, antibody specificity, and antibody titer. [1, 8] Three distinct stages of type 1 diabetes have been recognized. [1, 8] Both stages 1 and 2 are characterized by autoimmunity and a presymptomatic status although there is still normoglycemia in stage 1, dysglycemia (impaired fasting glucose [IFG] and/or impaired glucose tolerance [IGT]) is present in stage 2. Stage 3 is characterized by new-onset symptomatic hyperglycemia. [1, 8]

Type 2 diabetes mellitus

In a state of health, normoglycemia is maintained by fine hormonal regulation of peripheral glucose uptake and hepatic production. Type 2 diabetes mellitus results from a defect in insulin secretion and an impairment of insulin action in hepatic and peripheral tissues, especially muscle tissue and adipocytes. [9] A postreceptor defect is also present, causing resistance to the stimulatory effect of insulin on glucose use. As a result, a relative insulin deficiency develops, unlike the absolute deficiency found in patients with type 1 diabetes. The specific etiologic factors are not known, but genetic input is much stronger in type 2 diabetes than in the type 1 form. [10]

Impaired glucose tolerance (IGT) is a transitional state from normoglycemia to frank diabetes, but patients with impaired glucose tolerance exhibit considerable heterogeneity. Type 2 diabetes, or glucose intolerance, is part of a dysmetabolic syndrome (syndrome X) that includes insulin resistance, hyperinsulinemia, obesity, hypertension, and dyslipidemia. Current knowledge suggests that the development of glucose intolerance or diabetes is initiated by insulin resistance and worsened by the compensatory hyperinsulinemia. Insulin resistance is not only predictive for type 2 diabetes and associated with myriad metabolic derangements in fasting conditions, but nondiabetic insulin-resistant individuals are subjected to a similar adverse postprandial metabolic setting and cardiometabolic risk as those with type 2 diabetes. [11] In addition, the prevalence of hypertension rises with exacerbation of stages of impaired glucose metabolism however, only in the early stages of impaired insulin metabolism do hyperglycemia and hyperinsulinemia appear to be significant contributors to the presence of hypertension. [12]

The paths to beta-cell dysfunction or demise are less well defined in type 1 diabetes. The progression to type 2 diabetes is influenced by genetics and environmental or acquired factors, such as a sedentary lifestyle and dietary habits that promote obesity. Most patients with type 2 diabetes are obese, and obesity is associated with insulin resistance. Insulin resistance is not only predictive for type 2 diabetes and associated with myriad metabolic derangements in fasting conditions, but nondiabetic insulin-resistant individuals are subjected to a similar adverse postprandial metabolic setting and cardiometabolic risk as those with type 2 diabetes. [11] Central adiposity is more important than increased generalized fat distribution. In patients with frank diabetes, glucose toxicity and lipotoxicity may further impair insulin secretion by the beta cells. [13] [14] [15] [16] Moreover, "in obesity, inflammation, with increased accumulation and inflammatory polarization of immune cells, takes place in various tissues, including adipose tissue, skeletal muscle, liver, gut, pancreatic islet, and brain, and may contribute to obesity-linked metabolic dysfunctions, leading to insulin resistance and type 2 diabetes." [17]

Gestational diabetes mellitus

Gestational diabetes mellitus (GDM) was previously described as any degree of glucose intolerance in which onset or first recognition occurs during pregnancy. [5] The definition was limited by imprecision. Women diagnosed with diabetes in the first trimester are now classified as having type diabetes. GDM is diabetes diagnosed in the second or third trimester of pregnancy that is not clearly overt diabetes. Insulin requirements are increased during pregnancy because of the presence of insulin antagonists, such as human placental lactogen or chorionic somatomammotropin, and cortisol these promote lipolysis and decrease glucose use.

Another factor in increased insulin requirements during pregnancy is the production of insulinase by the placenta. Various genetic defects of the beta cell, insulin action, diseases of the exocrine pancreas, endocrinopathies, drugs, chemical agents, infections, immune disorders, and genetic syndromes can cause variable degrees of glucose intolerance, including diabetes.

To see complete information on Diabetes Mellitus and Pregnancy, please go to the main article by clicking here.

Other specific types of diabetes mellitus

These are specific types of diabetes due to other causes, which include monogenic diabetes syndromes, diseases of the exocrine pancreas, and drug- or chemical induced diabetes. Various genetic defects of the beta cell, insulin action, diseases of the exocrine pancreas, endocrinopathies, drugs, chemical agents, infections, immune disorders, and genetic syndromes can cause variable degrees of glucose intolerance, including diabetes.

Varying forms of glucose intolerance

Glucose intolerance may be present in many patients with cirrhosis due to decreased hepatic glucose uptake and glycogen synthesis. Other underlying mechanisms include hepatic and peripheral resistance to insulin and serum hormonal abnormalities. Abnormal glucose homeostasis may also occur in uremia, as a result of increased peripheral resistance to the action of insulin.

The gastrointestinal tract plays a significant role in glucose tolerance. [18] With food ingestion, incretin hormones glucagonlike peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are synthesized and secreted by specialized gut cells. Oral glucose administration results in a higher insulin secretory response than does intravenous glucose administration this difference is due in part to incretin hormones.

The significance of incretin hormones has been noted as a result of efforts to develop agents that may improve glycemic control in patients with type 2 diabetes through new mechanisms. [19] These strategies include inhibition of dipeptidyl peptidase IV (DPP-4), the major enzyme responsible for degrading incretin hormones in vivo, and the use of GLP-1 agonists. [20] Incretin hormones also significantly affect the differentiation, mitogenesis, and survival of beta cells.

Pathologic defects observed in type 2 diabetes mellitus and sometimes in impaired glucose tolerance include postprandial hyperglucagonemia, dysregulation of gastric emptying, and loss of incretin effect.

Postprandial hyperglycemia in diabetes and impaired glucose tolerance (IGT) is related to a lower rate of glucose disposal, whereas insulin secretion and action, as well as postprandial turnover, are essentially normal in individuals with isolated IGT. [21]

Metformin lowers glucose 6-phosphate in hepatocytes by activation of glycolysis downstream of glucose phosphorylation

The chronic effects of metformin on liver gluconeogenesis involve repression of the G6pc gene, which is regulated by the carbohydrate-response element-binding protein through raised cellular intermediates of glucose metabolism. In this study we determined the candidate mechanisms by which metformin lowers glucose 6-phosphate (G6P) in mouse and rat hepatocytes challenged with high glucose or gluconeogenic precursors. Cell metformin loads in the therapeutic range lowered cell G6P but not ATP and decreased G6pc mRNA at high glucose. The G6P lowering by metformin was mimicked by a complex 1 inhibitor (rotenone) and an uncoupler (dinitrophenol) and by overexpression of mGPDH, which lowers glycerol 3-phosphate and G6P and also mimics the G6pc repression by metformin. In contrast, direct allosteric activators of AMPK (A-769662, 991, and C-13) had opposite effects from metformin on glycolysis, gluconeogenesis, and cell G6P. The G6P lowering by metformin, which also occurs in hepatocytes from AMPK knockout mice, is best explained by allosteric regulation of phosphofructokinase-1 and/or fructose bisphosphatase-1, as supported by increased metabolism of [3- 3 H]glucose relative to [2- 3 H]glucose by an increase in the lactate m2/m1 isotopolog ratio from [1,2- 13 C2]glucose by lowering of glycerol 3-phosphate an allosteric inhibitor of phosphofructokinase-1 and by marked G6P elevation by selective inhibition of phosphofructokinase-1 but not by a more reduced cytoplasmic NADH/NAD redox state. We conclude that therapeutically relevant doses of metformin lower G6P in hepatocytes challenged with high glucose by stimulation of glycolysis by an AMP-activated protein kinase-independent mechanism through changes in allosteric effectors of phosphofructokinase-1 and fructose bisphosphatase-1, including AMP, Pi, and glycerol 3-phosphate.

Keywords: glucose 6-phosphate glycolysis hepatocyte liver metformin phosphofructokinase.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

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Science Translational Medicine

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02 September 2020

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By Magdalene K. Montgomery , Jacqueline Bayliss , Camille Devereux , Ayenachew Bezawork-Geleta , David Roberts , Cheng Huang , Ralf B. Schittenhelm , Andrew Ryan , Scott L. Townley , Luke A. Selth , Trevor J. Biden , Gregory R. Steinberg , Dorit Samocha-Bonet , Ruth C. R. Meex , Matthew J. Watt

Science Translational Medicine 02 Sep 2020

SMOC1 is a liver-secreted glucose-responsive protein that improves glycemic control through CREB-mediated suppression of hepatic glucose output.


Y. Wang and Q. Ge designed the research, analyzed data, and wrote the manuscript Y. Wang, W. Zhao, J. Shi, and J. Wang performed the research J. Hao, X. Pang, X. Huang, X. Chen, Y. Li, and R. Jin contributed reagents and technical support and all authors reviewed the manuscript.

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