Effects of type 2 diabetes and non-alcoholic fatty liver disease on hepatic glucose metabolism and de novo lipogenesis

The liver maintains blood glucose homeostasis by net production of glucose in the post-absorptive state and net uptake of glucose in the postprandial state in response to the rise in blood glucose in the portal vein which delivers the products of carbohydrate digestion from the gut. In the post-absorptive state the liver produces glucose by glycogen degradation and gluconeogenesis. The glucose 6-phosphate (G6P) generated in the cytoplasm from these pathways is transported into the endoplasmic reticulum on the G6P-transporter (encoded by SLC37A4) and hydrolysed within the lumen by glucose 6-phosphatase (G6pc) to glucose and inorganic phosphate (Pi)⁽ Reference van Schaftingen and Gerin ^{13 )}. After a meal when the glucose concentration in the portal vein rises, hepatic glucose metabolism undergoes a large metabolic transition from net production to net uptake. The liver extracts about one-third of the glucose load and of this about 60 % is converted to glycogen; the rest is metabolised by glycolysis and the pyruvate is either transported into mitochondria or exported as lactate⁽ Reference Cherrington ^{14 )}. This rapid metabolic transition is mediated by endocrine and neural signals, and by metabolite signals linked to the increase in glucose concentration in the portal vein⁽ Reference Cherrington ^{14 ,} Reference Agius ^{15 )}.

T2D is characterised by defects in secretion of insulin and glucagon and by elevated hepatic glucose production, lack of suppression of hepatic glucose production by insulin and lower hepatic glucose uptake⁽ Reference Rizza ^{1 )}. Poorly controlled T2D is associated with low flux through glucokinase⁽ Reference Rizza ^{1 )} and low glucokinase activity⁽ Reference Caro, Triester and Patel ^{16 )} and mRNA expression⁽ Reference Haeusler, Camastra and Astiarraga ^{17 )}. T2D is also associated with raised plasma TAG levels and a greater fractional contribution of hepatic de novo lipogenesis (DNL), representing the synthesis of fatty acids from acetyl units⁽ Reference Hellerstein, Schwarz and Neese ^{18 )}, to TAG secretion⁽ Reference Vedala, Wang and Neese ^{19 )}.

In NAFLD, glycerolipid accumulates in hepatocytes because of an imbalance between fatty acid uptake (from NEFA and dietary lipids) plus synthesis (via DNL) relative to fatty acid disposal by mitochondrial β-oxidation plus secretion as TAG in VLDL⁽ Reference Donnelly, Smith and Schwarzenberg ^{20 –} Reference Lambert, Ramos-Roman and Browning ^{22 )}. In the healthy fasted state, the major sources of substrate for hepatic TAG secretion are NEFA derived from adipose lipolysis with a smaller contribution of NEFA from dietary chylomicrons that escape uptake by extra hepatic tissues and a negligible contribution of DNL⁽ Reference Hellerstein, Schwarz and Neese ^{18 )}. NAFLD is associated with increased fatty acid supply by adipose tissue lipolysis⁽ Reference Lambert, Ramos-Roman and Browning ^{22 )} and an increased fractional contribution of DNL to TAG secretion⁽ Reference Donnelly, Smith and Schwarzenberg ^{20 –} Reference Lambert, Ramos-Roman and Browning ^{22 )} from <5 % in controls to 15–20 % in patients⁽ Reference Donnelly, Smith and Schwarzenberg ^{20 ,} Reference Lambert, Ramos-Roman and Browning ^{22 )}. Both the rate of VLDL–TAG secretion derived from DNL and the fractional contribution of DNL to VLDL secretion are elevated in NAFLD and correlate with liver fat content⁽ Reference Lambert, Ramos-Roman and Browning ^{22 )}. Other hepatic metabolic abnormalities in NAFLD include impaired suppression of hepatic glucose production by insulin⁽ Reference Seppälä-Lindroos, Vehkavaara and Häkkinen ^{23 )}; elevated gluconeogenesis and increased flux through tricarboxylic acid cycle oxidation and anaplerosis, without increased turnover of ketone bodies⁽ Reference Sunny, Parks and Browning ^{24 )}.

A key paradox is that despite a greater fractional contribution of DNL to TAG secretion in NAFLD, the net flux through DNL is very small (<5 %) relative to carbohydrate turnover⁽ Reference Hellerstein ^{25 )}. Accordingly DNL is not a quantitative route for disposal of carbohydrate. Similar considerations apply in relation to ethanol which increases fractional DNL to >30%; however, the major disposal product is acetate released into the circulation rather than DNL which represents <5 % of ethanol metabolism⁽ Reference Hellerstein ^{25 )}. However, DNL has a regulatory role through changes in cellular levels of malonyl-CoA (the product of acetyl-carboxylase) which inhibits mitochondrial fatty acid oxidation⁽ Reference Savage, Choi and Samuel ^{26 )}. This may explain the correlation between raised fractional DNL and raised TAG secretion rate⁽ Reference Diraison, Moulin and Beylot ^{21 )}. DNL may represent a spill over of acetyl-units (as citrate efflux from mitochondria) as suggested by the association between intrahepatic lipid and flux through tricarboxylic acid cycle oxidation plus anaplerosis (and increased gluconeogenic flux), in the absence of elevated ketone body production⁽ Reference Sunny, Parks and Browning ^{24 )}.

Effects of dietary carbohydrate on de novo lipogenesis and hepatic glucose production

The fractional contribution of hepatic DNL to hepatic TAG secretion is dependent on dietary carbohydrate intake⁽ Reference Parks ^{27 )}. Healthy subjects consuming diets with lower (25–50 %) or higher (25–50 %) carbohydrate content for 5 d showed a near-linear relation between fractional DNL and dietary carbohydrate⁽ Reference Schwarz, Neese and Turner ^{28 )}. Raised fractional DNL with high carbohydrate was also observed in isoenergetic conditions⁽ Reference Hudgins, Hellerstein and Seidman ^{29 )} and more so with dietary sugars than with starch⁽ Reference Hudgins, Seidman and Diakun ^{30 )}. Fractional DNL correlates with raised VLDL–TAG secretion rates and raised plasma TAG levels⁽ Reference Hudgins, Hellerstein and Seidman ^{31 )}. Fructose has a greater effect than glucose on DNL, plasma lipid levels and hepatic steatosis⁽ Reference Parks, Skokan and Timlin ^{32 –} Reference Sevastianova, Santos and Kotronen ^{34 )}. Fructose increases DNL more strongly and more rapidly (within hours) than glucose when administered acutely⁽ Reference Hellerstein, Schwarz and Neese ^{18 ,} Reference Hellerstein ^{25 )}. This rapid effect of fructose is attributed to its high flux of metabolism, whereas the effect of chronic consumption of glucose-containing high-carbohydrate diets is thought to be predominantly transcriptional. Despite the large fractional increase in DNL (from 1 to about 30 %) caused by either acute fructose infusion or chronic carbohydrate surplus⁽ Reference Schwarz, Neese and Turner ^{28 ,} Reference Lammert, Grunnet and Faber ^{35 )} the net daily flux through DNL in comparison with dietary carbohydrate load is small (⩽5 %) in both man and rodents⁽ Reference Hellerstein ^{25 ,} Reference Lammert, Grunnet and Faber ^{35 )} indicating that even in healthy subjects on high-carbohydrate diets the role of DNL is regulatory for fuel selection for oxidation⁽ Reference Savage, Choi and Samuel ^{26 ,} Reference Schwarz, Neese and Turner ^{28 )} and not as a quantitative route of disposal of dietary carbohydrate⁽ Reference Hellerstein ^{25 )}.

Another effect of high-carbohydrate diets in man is an increase in hepatic glucose production.  During chronic carbohydrate under-feeding or over-feeding (for 5 d) hepatic glucose production increased in parallel with DNL with increasing carbohydrate load despite the elevated plasma insulin⁽ Reference Schwarz, Neese and Turner ^{28 )}. Elevation in basal glucose production also occurred with a high-carbohydrate diet in isoenergetic conditions⁽ Reference Bisschop, de Metz and Ackermans ^{36 )}. The parallel elevation in glucose production and DNL with high-carbohydrate diets are similar to the metabolic characteristics of NAFLD⁽ Reference Sunny, Parks and Browning ^{24 )}.

Effects of dietary carbohydrate on hepatic gene expression: role of carbohydrate-response element-binding protein

In animal models high-carbohydrate diets induce liver enzymes of DNL⁽ Reference Uyeda and Repa ^{8 ,} Reference Towle ^{9 )}. Two transcription factors involved are sterol regulatory element binding protein 1c that is induced by insulin and targets predominantly enzymes of DNL and ChREBP that is induced by high glucose and targets enzymes of DNL but also several additional genes, including pyruvate kinase ⁽ Reference Yamashita, Takenoshita and Sakurai ^{7 –} Reference Towle ^{9 )}. Because ChREBP is induced by dietary carbohydrate, the generally held explanation for ChREBP function is that it represents a mechanism for efficient conversion of dietary carbohydrate to fat as a ‘thrifty gene’⁽ Reference Uyeda and Repa ^{8 –} Reference Oosterveer and Schoonjans ^{11 )}. The decrease in DNL⁽ Reference Iizuka, Bruick and Liang ^{37 –} Reference Dentin, Benhamed and Hainault ^{39 )} and in steatosis⁽ Reference Iizuka, Miller and Uyeda ^{38 ,} Reference Dentin, Benhamed and Hainault ^{39 )} in ChREBP knock-down models⁽ Reference Iizuka, Bruick and Liang ^{37 )} is consistent with such a hypothesis but it does not exclude the alternative explanation⁽ Reference Hellerstein ^{25 )} that the role of DNL is regulatory for fuel selection because it is not a quantitative route of carbohydrate disposal⁽ Reference Hellerstein ^{25 ,} Reference Schwarz, Neese and Turner ^{28 )}.

ChREBP binds to the promoter of its target genes in complex with Max-like protein X (Mlx), a member of the Myc/Max superfamily of transcription factors⁽ Reference Towle ^{9 ,} Reference Peterson and Ayer ^{40 )}. Mlx has other partners, including MondoA, the paralogue of ChREBP⁽ Reference Peterson and Ayer ^{40 )}. Gene microarrays in hepatocytes using a dominant-negative Mlx, identified several candidate target genes of Mlx, including not only enzymes of DNL and L-pyruvate kinase (PKLR), but also two enzymes of gluconeogenesis (G6pc and fructose bisphosphatase-1) which catalyse generation of Pi from PE and the inhibitory protein of glucokinase encoded by the Gckr gene⁽ Reference Ma, Robinson and Towle ^{41 )}. These target genes were also confirmed by chromatin immunoprecipitation-PCR⁽ Reference Arden, Petrie and Tudhope ^{42 ,} Reference Arden, Tudhope and Petrie ^{43 )} or chromatin immunoprecipitation-sequencing⁽ Reference Poungvarin, Chang and Imamura ^{44 )}. Glucokinase which is the major determinant of hepatic glucose disposal⁽ Reference Agius ^{15 )}, is not a target gene for ChREBP and it is repressed rather than induced by high glucose⁽ Reference Arden, Petrie and Tudhope ^{42 )}. The induction by high glucose via ChREBP-Mlx of the two major counter-regulators of glucokinase, the glucokinase inhibitor protein (Gckr) and G6pc which dephosphorylates the product of the glucokinase reaction, argues against a role for ChREBP-Mlx in net conversion of glucose to fat.

To evaluate the hypothesis that the function of ChREBP is to maintain homeostasis of PE and ATP in conditions of high-carbohydrate diets or compromised intracellular metabolite homeostasis, the following issues need to be considered: (1) To what extent are the hepatic concentrations of PE of glucose metabolism elevated postprandially? What is the function of these changes? (2) What are the consequences of disrupted regulation on cell Pi and ATP homeostasis? (3) What mechanisms exist to restrain the elevation in PE within physiological limits? (4) What is the effect of ChREBP deletion on hepatic PE and ATP homeostasis? (5) What are the signals for ChREBP activation?

Hepatic concentrations of phosphate esters in the fasted-to-fed transition

In the rodent model, the hepatic concentration of G6P, the first intermediate of glucose metabolism (Fig. 1) and also downstream PE intermediates of glycolysis and the pentose phosphate pathway are elevated about 2-fold in the fed state on a starch-containing diet compared with the fasted state⁽ Reference Casazza and Veech ^{45 )}. The increase in G6P is sustained during the absorptive state⁽ Reference Fernández-Novell, Roca and Bellido ^{46 )}. Larger elevations in PE, particularly of the pentose pathway intermediates, occur on a sucrose-containing diet⁽ Reference Casazza and Veech ^{45 )}. Changes in G6P are associated with parallel changes in fructose 6-phosphate (F6P) which is in equilibrium with G6P via phosphoglucoisomerase, and with elevation in fructose 2,6-bisphosphate (F2,6P₂) which is a ‘regulatory’ rather than ‘intermediate’ metabolite that is a potent activator of phosphofructokinase-1 and thereby glycolysis⁽ Reference Hue and Rider ^{47 )}. F2,6P₂ is generated from F6P by the bifunctional enzyme phosphofructokinase-2/fructose biphosphatase-2, which also degrades F2,6P₂ to F6P. The postprandial elevation in G6P has a major role in stimulating glycogen storage (Fig. 1) because G6P is an allosteric effector of glycogen phosphorylase and glycogen synthase⁽ Reference Agius ^{15 )}. The elevation in F2,6P₂ mediates stimulation of glycolysis by activation of phosphofructokinase-1, with consequent elevation in its product fructose 1,6-bisphosphate, which is an allosteric activator of pyruvate kinase which converts phosphoenolpyruvate,  the last PE intermediate of glycolysis to pyruvate. The greater elevation in PE caused by a sucrose-diet compared with a starch-diet⁽ Reference Casazza and Veech ^{45 )} can be explained by the different pathways of glucose (Fig. 1) and fructose (Fig. 2) metabolism in liver as discussed later.

Fig. 1. Glucose stimulates glucose disposal by allosteric mechanisms but it also causes feed-back regulation of gene expression. Elevated glucose concentration in the portal vein causes rapid dissociation of glucokinase (GK) from the Gckr regulatory protein (RP) resulting in a moderate increase in the cellular concentrations of glucose 6-P (G6P) and fructose 2,6-bisphosphate (F2,6P₂) which cause allosteric activation of enzymes promoting glycogen storage and glycolysis. Chronic elevation in G6P and F2,6P₂ causes activation of transcription factors which down regulate the GK gene (Gck) and upregulate the G6pc and Pklr genes. This gene regulation serves to maintain homeostasis of the cell phosphometabolite pool and results in restraint of glucose uptake and elevated glucose production.

Fig. 2. Fructose is transported and phosphorylated by a distinct mechanism from glucose. Low concentrations of fructose cause moderate elevation in fructose 1-phosphate (F1P), which dissociates glucokinase (GK) from its regulatory protein (RP) leading to stimulation of glucose metabolism (1). Millimolar concentrations of fructose cause marked elevation in F1P (2) because the activity of ketohexokinase (KHK) is higher than that of downstream enzymes. During reconversion of ADP to ATP by oxidative phosphorylation (OP; 3), the cell content of inorganic phosphate (Pi) becomes depleted (4) resulting in depletion of ATP (5) and degradation of AMP (6) to uric acid (7).

Carbohydrate-response element-binding protein and liver ATP homeostasis: evidence from carbohydrate-response element-binding protein-deletion models and human gene variants

If the function of ChREBP is to maintain hepatic ATP homeostasis by controlling gene expression of enzymes or regulatory proteins that influence PE concentrations (e.g. G6pc, fructose biphosphatase-1, Gckr, Pklr), then ChREBP deletion models would be expected to be associated with raised PE levels, compromised ATP homeostasis and elevated uric acid production. Consistent with this hypothesis, ChREBP deletion models have 2- to 3-fold higher levels of hepatic G6P, dihydroxyacetone-P, 3-phosphoglycerate and phoshoenolpyruvate on either chow or starch diets⁽ Reference Iizuka, Bruick and Liang ^{37 )} with somewhat greater differences (about 5-fold) in the hyperphagic ob/ob mouse⁽ Reference Iizuka, Miller and Uyeda ^{38 ,} Reference Dentin, Benhamed and Hainault ^{39 )}. A study by Burgess and colleagues⁽ Reference Burgess, Iizuka and Jeoung ^{69 )} that determined the hepatic phosphorylation potential, representing the ratio of free concentrations of [ATP]/[ADP]×[Pi] in ChREBP^−/− mice showed a 50 % lower phosphorylation potential associated with a 24 % decrease in total ATP and 4-fold elevation of free AMP. The ChREBP^−/− mice were viable on chow or starch diets but not on sucrose or fructose diets⁽ Reference Iizuka, Bruick and Liang ^{37 ,} Reference Burgess, Iizuka and Jeoung ^{69 )}. Two possible explanations for the mortality on fructose are either increased expression of fructose transporters and/or KHKc leading to greater F1P elevation and more severe ATP depletion; or a defect in ATP recovery. The first possibility seems unlikely because Khk mRNA levels were lower in the ChREBP^−/− model⁽ Reference Iizuka, Bruick and Liang ^{37 )}. In support of the second possibility, gene microarrays in cells with ChREBP knock-down identified dihydrofolate reductase which is involved in purine biosynthesis in the down-regulated genes⁽ Reference Tong, Zhao and Mancuso ^{70 )}, and genes involved in purine metabolism were also identified as ChREBP targets by chromatin immunoprecipitation-sequencing⁽ Reference Poungvarin, Chang and Imamura ^{44 )}. A role for ChREBP in ATP homeostasis is also indicated by raised plasma uric acid levels in rats with partial ChREBP knock-down on a high-fructose diet⁽ Reference Erion, Popov and Hsiao ^{71 )}.

Plasma levels of uric acid in man are elevated in association with insulin resistance⁽ Reference Vuorinen-Markkola and Yki-Järvinen ^{72 )} and NAFLD⁽ Reference Li, Xu and Yu ^{73 )} and increased hepatic production of uric acid is inferred in these conditions⁽ Reference Vuorinen-Markkola and Yki-Järvinen ^{72 )}. Genome-wide studies in man have identified twenty-eight loci in association with raised uric acid levels⁽ Reference Köttgen, Albrecht and Teumer ^{74 )}. They include variants for MLXIPL and two of the ChREBP target genes, PKLR and GCKR. The association of these genes with raised uric acid, a marker of ATP depletion⁽ Reference Petrie, Patman and Sinha ^{62 )} is consistent with a role for ChREBP, Pklr and GCKR in ATP homeostasis.

Mechanism for glucose-induced activation of carbohydrate-response element-binding protein: glucose 6-phosphate or multiple phosphate esters?

The mechanism by which high glucose activates ChREBP has not been resolved. ChREBP is transcribed as either a full-length ChREBP-α (864 residues), which is cytoplasmic at low glucose and activated by high glucose which causes translocation to the nucleus, recruitment to target genes and transcriptional activation, or as a shorter ChREBP-β isoform (lacking the first 117-residues at the N-terminus), that is constitutively active at low glucose⁽ Reference Herman, Peroni and Villoria ^{75 )}. Several aspects of the post-transcriptional regulation of ChREBP-α have been explored, including covalent modification by phosphorylation⁽ Reference Uyeda and Repa ^{8 ,} Reference Sakiyama, Wynn and Lee ^{76 –} Reference Tsatsos, Davies and O'Callaghan ^{78 )}, O-GlcNAc-modification⁽ Reference Guinez, Filhoulaud and Rayah-Benhamed ^{79 ,} Reference Sakiyama, Fujiwara and Noguchi ^{80 )} and acetylation⁽ Reference Bricambert, Miranda and Benhamed ^{81 )}; mutational studies for mapping of the N-terminal glucose sensory domain and 14-3-3 binding sites⁽ Reference Li, Chang and Imamura ^{82 –} Reference Ge, Huang and Wynn ^{84 )}, and identification of putative metabolites that mediate the response to high glucose. The proposed metabolites include: G6P⁽ Reference Li, Chen and Harmancey ^{85 ,} Reference Dentin, Tomas-Cobos and Foufelle ^{86 )}, xylulose 5-P⁽ Reference Uyeda and Repa ^{8 )}, the regulatory metabolite F2,6P₂ ⁽ Reference Arden, Tudhope and Petrie ^{43 ,} Reference Petrie, Al-Oanzi and Arden ^{87 )}, ketone bodies⁽ Reference Nakagawa, Ge and Pawlosky ^{88 )} and the product of the hexosamine pathway UDP-N-acetylglucosamine⁽ Reference Guinez, Filhoulaud and Rayah-Benhamed ^{79 ,} Reference Sakiyama, Fujiwara and Noguchi ^{80 )} which is the substrate for O-GlcNAc-modification of proteins. The induction of ChREBP target genes by high glucose is abolished by glucokinase inhibitors and markedly enhanced by inhibitors of G6P hydrolysis⁽ Reference Arden, Petrie and Tudhope ^{42 ,} Reference Parks, Skokan and Timlin ^{32 ,} Reference van Dijk, van der Sluijs and Wiegman ^{53 )}. These inhibitors abolish and enhance, respectively, the elevation in PE pool in hepatocytes comprising G6P and downstream metabolites⁽ Reference Arden, Petrie and Tudhope ^{42 ,} Reference Parks, Skokan and Timlin ^{32 ,} Reference van Dijk, van der Sluijs and Wiegman ^{53 )}. This rules out an effect of glucose and points to mechanism(s) consequent to PE elevation. Current evidence suggests that a combination of mechanisms is most likely involved in ChREBP-α activation by glucose. For example mutation of residues that are substrates for phosphorylation by cyclic AMP dependent protein kinase and AMP-dependent protein kinase (S196, S626, T666; S566) and for dephosphorylation by glucose⁽ Reference Uyeda and Repa ^{8 )}, did not generate a glucose-insensitive ChREBP indicating involvement of additional mechanisms⁽ Reference Tsatsos and Towle ^{77 )}. Covalent modification of ChREBP by O-GlcNAc which is dependent on UDP-N-acetylglucosamine generated by the hexosamine pathway caused ChREBP protein stabilisation and transcriptional activation⁽ Reference Tsatsos and Towle ^{77 ,} Reference Tsatsos, Davies and O'Callaghan ^{78 )}. However, increased flux through the hexosamine pathway at low glucose does not activate ChREBP target genes⁽ Reference Arden, Tudhope and Petrie ^{43 )}. A role for G6P as the signalling metabolite for ChREBP activation⁽ Reference Li, Chen and Harmancey ^{85 ,} Reference Dentin, Tomas-Cobos and Foufelle ^{86 )} was proposed based on two sets of evidence: (i) a glucose-like effect of 2-deoxyglucose which is phosphorylated to its 6-PE but not further metabolised; (ii) attenuation of the high glucose effect by overexpression of enzymes that metabolise G6P (e.g. G6pc, glucose 6-phosphate dehydrogenase, phosphofructokinase-1, phosphofructokinase-2). However, 2-deoxyglucose does not activate ChREBP target genes in hepatocytes despite accumulation of its 6-PE⁽ Reference Arden, Tudhope and Petrie ^{43 )}. Attenuation of the glucose response by overexpression of enzymes that degrade G6P does not rule out hexosamine pathway involvement, because G6P is in equilibrium with F6P, the substrate for the hexosamine pathway. A role for F2,6P₂ was proposed based on inhibition of recruitment of ChREBP-Mlx when this metabolite was selectively depleted at high glucose with a kinase-deficient bisphosphatase-active variant of phosphofructokinase-2/fructose bisphosphatase-2. However, additive activation of ChREBP target genes by glucose and xylitol which do not have additive effects on F2,6P₂ indicates involvement of additional metabolites or mechanisms⁽ Reference Petrie, Al-Oanzi and Arden ^{87 )}.

On the premise that G6P activates MondoA⁽ Reference Peterson and Ayer ^{40 )} and ChREBP⁽ Reference Li, Chen and Harmancey ^{85 ,} Reference Dentin, Tomas-Cobos and Foufelle ^{86 )}, McFerrin and Atchley⁽ Reference McFerrin and Atchley ^{89 )} conducted a bioinformatic analysis for the putative ‘G6P binding motif’ within the N-terminal conserved ‘glucose sensing module’ based on G6P-binding sites of glucokinase and other enzymes, and identified a ‘low-specificity’ motif (Sx[ST]xx[ST]) similar to that found in glucosephosphate isomerase and glutamine:fructose 6-phosphate amidotransferase⁽ Reference McFerrin and Atchley ^{89 )}. Based on the ‘low-specificity’ of this motif and that F6P rather than G6P is the common substrate of these two enzymes, this outcome favours F6P or multiple PE.