**2.2 Lipogenesis** *de novo*

Hepatic lipogenesis is induced upon ingestion of excess carbohydrates to convert extra carbohydrates to triglyceride for long-time energy storage. Once inside the hepatocyte, glucose enters glycolytic pathway and provides pyruvate, which enters mitochondrion where it is converted into acetyl-CoA by pyruvate dehydrogenase. On the other side, in the cytoplasm glucose is also oxidized through the pentose phosphate pathway and NADPH is obtained. Acetyl-CoA will serve for fatty acid and also cholesterol synthesis. The initial steps for fatty acid synthesis are the transfer of acetyl-CoA from mitochondria to the cytoplasm and its conversion into malonyl-CoA under the action of the enzyme acetyl-CoA carboxylase. Importantly, malonyl-CoA is a regulatory molecule because it inhibits carnitine palmitoyltransferase-1, a rate limiting enzyme in β-oxidation of fatty acids. Therefore, increasing malonyl-CoA favours lipogenesis. Malonyl-CoA is elongated using NADPH under the action of the enzyme fatty acid synthase. Once obtained, fatty acids can be esterified with glycerol to form diglyceride and triglyceride. Most of the triglyceride is produced for export to the adipose tissue, but in order to be secreted, it must be packaged in very low-density lipoprotein (VLDL) particles together with cholesterol, phospholipids and apolipoprotein B (Figure 1).

After a meal rich in carbohydrates, high levels of glucose reach the liver via portal vein. Glucose enters passively the hepatocyte through GLUT-2, a facilitated glucose transporter, and then is rapidly phosphorylated by GK at the sixth carbon to obtain glucose-6-phosphate which cannot escape the cell. From a functional perspective, it is important to recognize that both GLUT2 and glucokinase are expressed in cell types in which glucose metabolism has to vary accordingly to extracellular glucose concentration (glucose sensors). The high Km for glucose of both proteins, and the absence of product inhibition by glucose-6-phosphate, ensure that glucose uptake and phosphorylation in these cells are proportional to

extracellular glucose concentration throughout the physiological range of glycaemia

The product of GK reaction, glucose-6-phosphate, is the gateway to the major pathways of glucose utilization: glycogen synthesis, glycolysis, oxidation of glucose and pentose phosphate pathway. It should be noted that hepatic glycolysis provides pyruvate principally for lipid synthesis rather than for oxidation. As glucose is the main substrate for fatty acid synthesis, hepatic glycolytic enzymes can be considered an extension of the lipogenic pathway. Glucose, insulin and parasympathetic nervous system orchestrate these glucose metabolic pathways in the fed state, with the aim of maintaining normal levels of

Two enzymes, glycogen synthase and glycogen phosphorylase, control glycogen levels. Both enzymes are regulated by phosphorylation and allosteric modulators. Specifically in the fed state, insulin activates glycogen synthase (limiting enzyme for glycogen synthesis) by promoting its dephosphorylation and, at the same time inhibits glycogen phosphorylase (important for glycogen breakdown). Meanwhile, glucose-6-phosphate binding to glycogen synthase favours its dephosphorylation, promoting glycogen synthase activity (Bollen, 1998; Agius, 2008). As a result, glucose coming from bloodstream fills hepatic glycogen stores.

Hepatic lipogenesis is induced upon ingestion of excess carbohydrates to convert extra carbohydrates to triglyceride for long-time energy storage. Once inside the hepatocyte, glucose enters glycolytic pathway and provides pyruvate, which enters mitochondrion where it is converted into acetyl-CoA by pyruvate dehydrogenase. On the other side, in the cytoplasm glucose is also oxidized through the pentose phosphate pathway and NADPH is obtained. Acetyl-CoA will serve for fatty acid and also cholesterol synthesis. The initial steps for fatty acid synthesis are the transfer of acetyl-CoA from mitochondria to the cytoplasm and its conversion into malonyl-CoA under the action of the enzyme acetyl-CoA carboxylase. Importantly, malonyl-CoA is a regulatory molecule because it inhibits carnitine palmitoyltransferase-1, a rate limiting enzyme in β-oxidation of fatty acids. Therefore, increasing malonyl-CoA favours lipogenesis. Malonyl-CoA is elongated using NADPH under the action of the enzyme fatty acid synthase. Once obtained, fatty acids can be esterified with glycerol to form diglyceride and triglyceride. Most of the triglyceride is produced for export to the adipose tissue, but in order to be secreted, it must be packaged in very low-density lipoprotein (VLDL) particles together with cholesterol, phospholipids and

**2. Liver glucose metabolism** 

blood sugar.

**2.1 Glycogen synthesis** 

**2.2 Lipogenesis** *de novo* 

apolipoprotein B (Figure 1).

Fig. 1. Scheme of de novo lipogenesis from glucose. Once inside the hepatocyte, glucose is metabolized on one hand through glycolysis to pyruvate (GK means glucokinase; PFK-2, 6, phosphofructo-2-kinase/fructose-2,6-bisphosphatase; L-PK, liverpyruvate kinase). On the other hand, glucose is oxidized through pentose phosphate pathway to obtain NADPH (G6P-DH means glucose-6-phosphate dehydrogenase; 6-PGDH, 6-phosphogluconate dehydrogenase). Pyruvate enters the mitochondrion to obtain citrate (PDH means, pyruvate dehydrogenase; MDH, malate dehydrogenase and EM, malic enzyme). De novo synthesis of fatty acids starts with citrate (ATP-CL means ATP citrate lyase; ACC, acetyl-CoA carboxylase) and after suffering elongation and desaturation reactions (ELOVL6 means elongase that catalyzes the conversion of palmitate to stearate; SCD-1, stearoyl-coenzyme A desaturase), fatty acids are converted to triglyceride (TAG) (GPAT means glycerol-3-phosphate acyltransferase; DGAT, diacylglycerol acyltransferase). Triglyceride can be stored in the liver but are mostly packaged into VLDL (very low-density lipoprotein) and secreted to bloodstream (MTP means microsomal triglyceride transfer protein). Original artwork.

Liver Glucokinase and Lipid Metabolism 239

*RE*

*FOXO1 LXR SREBP-1c ChREBP* <sup>P</sup>

*ACC FAS SCD-1*

nuclear exclusion and proteasome degradation; consequently, transcription of

**IRE LXRE LXRE SRE ChORE SRE SRE ChORE ChORE**

Fig. 2. Main regulatory mechanisms of hepatic metabolism in fed state. Insulin and glucose direct gene transcription to switch from glucose producing pathways to glucose uptake and storage. Briefly, insulin signaling promotes the phosphorylation of FOXO1 that results in its

gluconeogenic genes is inhibited. Besides, insulin stimulates transcription of lipogenic genes through SREBP-1c activation and probably through LXR-α, as well. Finally, insulin signaling causes activation of glycogen synthase function. Glucose also controls allostericaly glycogen synthesis and promote transcription of lipogenic genes via activation of ChREBP. IR means insulin receptor, IRS, insulin receptor substrate; PI3K, phosphoinositide 3-kinse; AKT, Ser/Thr protein kinase; GSK3-β, glycogen synthase kinase 3.beta; FOXO1, forkhead box O1; PCK, protein kinase C; LXR, liver X receptor; SREBP-1c, sterol regulatory element binding protein 1c; ChREBP, carbohydrate response element binding protein; GS, glycogen synthase; GP, glycogen phosphorylase; GK, glucokinase; PP2A, protein phosphatase 2A); IRE, insulin response element; LXRE liver X receptor response element; SRE, sterol regulatory elements; ChORE, carbohydrate-response elements; PEPCK-C, cytosolic phosphoenolpyruvate

carboxykinase; G6Pase, glucose-6-phosphatase; ACC, Acyl-CoA carboxylase; FAS, fatty acid synthase; SCD-1, stearoyl-CoA desaturase 1; ELOVL , EM, malic enzyme and L-PK, liver-

(+)

P P P

*Cytoplasmic membrane*

*GSK3-<sup>β</sup>* <sup>P</sup>

?

?

Insulin

P

**RI**

P

*IRS-1/IRS-2*

P

*PI3K*

*AKT* <sup>P</sup>

*FOXO1*<sup>P</sup>

(-)

*PEPCK G6Pase*

pyruvate kinase. Original artwork.

*SREBP-1c ChREBP*

*PKCλ*

*Proteasome*

Glucose

*GLUT-2*

Glucose

Glucose-6P *GK*

Xylulose-5P

*Nuclear membrane*

*L-PK*

*ChREBP* P

*ChREBP*

*EM6*

P

*GK ELOVL*

Glucosa-1P

*GS GP (inactive)*

*GS (active)*

*SREBP-1c*

<sup>P</sup> <sup>P</sup> <sup>P</sup>

GLYCOGEN

(-)

*PP2A*

In mammals, hepatic lipogenesis is controlled by several transcription factors, mainly SREBP-1c (sterol regulatory element binding protein 1c) and ChREBP (carbohydrateresponsive element-binding protein), but also by PPAR-γ (peroxisome proliferator-activated receptor gamma), LXR-α (liver X receptor alpha) and XBP1, all of them regulated by nutritional and hormonal conditions.

SREBP-1c plays a major role in the induction of lipogenic genes by insulin. SREBP-1c is a member of the bZIP transcription factor family that was originally identified as a mediator of sterol signaling (Wang, 1994), and is produced as a precursor form that reside in the endoplasmic reticulum in an inactive state. On one hand insulin stimulates SREBP-1c gene transcription, and on the other hand, induces the maturation of SREBP-1c precursor (Shimomura, 1998). Mature SREBP-1c moves to nucleus and activates transcription of several lipogenic genes with SRE (sterol regulatory elements) sequences in their promoters, for instance fatty acid synthase (FAS), stearoyl-Coenzyme A desaturase 1 (SCD-1), etc. (Figure 2) (Foretz, 1999; Ferre, 2010).

Glucose regulates genes of glycolytic and lipogenic pathways by activating ChREBP (Iizuka, 2008). ChREBP is a transcription factor that binds to ChoRE sequences present in the promoter of ACC (acetyl Coenzyme-A carboxylase), fatty acid synthase (FAS), stearoyl-Coenzyme A desaturase 1 (SCD-1), L-pyruvate kinase (L-PK), etc. (Uyeda, 2006). Under basal conditions, ChREBP is phosphorylated at Ser196 and remains in the cytosol. When glycaemia increase, glucose enters the hepatocyte and is metabolized. Therefore there is an increase in some glucose metabolites such as xylulose-5P, which promotes ChREBP dephosphorylation (Kabashima, 2003). Then, ChREBP rapidly moves to the nucleus and will activate transcription of its target genes (Figure 2).

SREBP-1c and ChREBP are also transcriptionally activated by liver X receptor apha (LXR-α), which could be a glucose sensor although it is controversial (Mitro, 2007; Denechaud, 2008). LXR-α is classically activated by oxysterols and it is important for the transcription of some lipogenic genes, a part form SREBP-1c and ChREBP, since their promoters contain LXRE (LXR response element) sequences (Chen, 2004; Cha, 2007).

XBP1, a transcription factor best known as a key regulator of the unfolded protein response (UPR), has been surprisingly associated with *de novo* fatty acid synthesis in the liver. It seems to be induced by diet carbohydrates and its deletion in mice causes hypocholesterolemia and hypotriglyceridemia, attributed to diminished hepatic lipid production (Lee, 2008). But, there are still some questions about its function to answer: what is its binding site in the promoter regions of these genes? Does it act alone or in partnership with other known transcription factors such as SREBP, ChREBP and LXR?

In summary, hepatic lipogenesis is regulated by several transcription factors that may probably work synergistically (Figure 2). With this complex system, carbons from glucose can be directed to fatty acid synthesis only when there is substrate availability and glycogen depots have been replenished. Altered fatty acid synthesis in the liver can lead to changes in lipid secretion, and consequently to dyslipidemia (Ginsberg, 2006).

#### **2.3 Inhibition of hepatic glucose production**

During fasting, liver produces glucose that enters bloodstream in order to maintain glycaemia, ensuring fuel supply for brain and red blood cells. But after a meal, when diet glucose arrives, hepatocytes must switch glucose production to glucose uptake. Insulin and high glucose levels coordinate the inhibition of glycogenolysis and gluconeogenesis (glucose producing pathways).

In mammals, hepatic lipogenesis is controlled by several transcription factors, mainly SREBP-1c (sterol regulatory element binding protein 1c) and ChREBP (carbohydrateresponsive element-binding protein), but also by PPAR-γ (peroxisome proliferator-activated receptor gamma), LXR-α (liver X receptor alpha) and XBP1, all of them regulated by

SREBP-1c plays a major role in the induction of lipogenic genes by insulin. SREBP-1c is a member of the bZIP transcription factor family that was originally identified as a mediator of sterol signaling (Wang, 1994), and is produced as a precursor form that reside in the endoplasmic reticulum in an inactive state. On one hand insulin stimulates SREBP-1c gene transcription, and on the other hand, induces the maturation of SREBP-1c precursor (Shimomura, 1998). Mature SREBP-1c moves to nucleus and activates transcription of several lipogenic genes with SRE (sterol regulatory elements) sequences in their promoters, for instance fatty acid synthase (FAS), stearoyl-Coenzyme A desaturase 1 (SCD-1), etc.

Glucose regulates genes of glycolytic and lipogenic pathways by activating ChREBP (Iizuka, 2008). ChREBP is a transcription factor that binds to ChoRE sequences present in the promoter of ACC (acetyl Coenzyme-A carboxylase), fatty acid synthase (FAS), stearoyl-Coenzyme A desaturase 1 (SCD-1), L-pyruvate kinase (L-PK), etc. (Uyeda, 2006). Under basal conditions, ChREBP is phosphorylated at Ser196 and remains in the cytosol. When glycaemia increase, glucose enters the hepatocyte and is metabolized. Therefore there is an increase in some glucose metabolites such as xylulose-5P, which promotes ChREBP dephosphorylation (Kabashima, 2003). Then, ChREBP rapidly moves to the nucleus and will

SREBP-1c and ChREBP are also transcriptionally activated by liver X receptor apha (LXR-α), which could be a glucose sensor although it is controversial (Mitro, 2007; Denechaud, 2008). LXR-α is classically activated by oxysterols and it is important for the transcription of some lipogenic genes, a part form SREBP-1c and ChREBP, since their promoters contain LXRE

XBP1, a transcription factor best known as a key regulator of the unfolded protein response (UPR), has been surprisingly associated with *de novo* fatty acid synthesis in the liver. It seems to be induced by diet carbohydrates and its deletion in mice causes hypocholesterolemia and hypotriglyceridemia, attributed to diminished hepatic lipid production (Lee, 2008). But, there are still some questions about its function to answer: what is its binding site in the promoter regions of these genes? Does it act alone or in partnership

In summary, hepatic lipogenesis is regulated by several transcription factors that may probably work synergistically (Figure 2). With this complex system, carbons from glucose can be directed to fatty acid synthesis only when there is substrate availability and glycogen depots have been replenished. Altered fatty acid synthesis in the liver can lead to changes in

During fasting, liver produces glucose that enters bloodstream in order to maintain glycaemia, ensuring fuel supply for brain and red blood cells. But after a meal, when diet glucose arrives, hepatocytes must switch glucose production to glucose uptake. Insulin and high glucose levels coordinate the inhibition of glycogenolysis and gluconeogenesis (glucose

nutritional and hormonal conditions.

(Figure 2) (Foretz, 1999; Ferre, 2010).

activate transcription of its target genes (Figure 2).

(LXR response element) sequences (Chen, 2004; Cha, 2007).

with other known transcription factors such as SREBP, ChREBP and LXR?

lipid secretion, and consequently to dyslipidemia (Ginsberg, 2006).

**2.3 Inhibition of hepatic glucose production**

producing pathways).

Fig. 2. Main regulatory mechanisms of hepatic metabolism in fed state. Insulin and glucose direct gene transcription to switch from glucose producing pathways to glucose uptake and storage. Briefly, insulin signaling promotes the phosphorylation of FOXO1 that results in its nuclear exclusion and proteasome degradation; consequently, transcription of gluconeogenic genes is inhibited. Besides, insulin stimulates transcription of lipogenic genes through SREBP-1c activation and probably through LXR-α, as well. Finally, insulin signaling causes activation of glycogen synthase function. Glucose also controls allostericaly glycogen synthesis and promote transcription of lipogenic genes via activation of ChREBP. IR means insulin receptor, IRS, insulin receptor substrate; PI3K, phosphoinositide 3-kinse; AKT, Ser/Thr protein kinase; GSK3-β, glycogen synthase kinase 3.beta; FOXO1, forkhead box O1; PCK, protein kinase C; LXR, liver X receptor; SREBP-1c, sterol regulatory element binding protein 1c; ChREBP, carbohydrate response element binding protein; GS, glycogen synthase; GP, glycogen phosphorylase; GK, glucokinase; PP2A, protein phosphatase 2A); IRE, insulin response element; LXRE liver X receptor response element; SRE, sterol regulatory elements; ChORE, carbohydrate-response elements; PEPCK-C, cytosolic phosphoenolpyruvate carboxykinase; G6Pase, glucose-6-phosphatase; ACC, Acyl-CoA carboxylase; FAS, fatty acid synthase; SCD-1, stearoyl-CoA desaturase 1; ELOVL , EM, malic enzyme and L-PK, liverpyruvate kinase. Original artwork.

Liver Glucokinase and Lipid Metabolism 241

activated by insulin and inhibited by glucagon. Insulin induction of Gck gene expression is through the PI3-kinase/Akt signaling. However, no IRE (insulin response element) has been described in Gck promoter, and it is not clear which transcription factor mediates insulindirected Gck expression. SREBP-1c is a candidate to mediate insulin-directed expression of Gck, although controversial results exist (Foretz, 1999; Gregory, 2006). Probably, SREBP-1c is not essential for rapid induction of GK transcription, but it can have a role for long-term expression. Other candidates to mediate insulin-dependent expression of Gck gene are the complex HIF-α/HNF-4/p300 (Roth, 2004), and ERR-α -estrogen-related receptor alpha-

GK can be modulated by covalent modifications such as nitrosylation and phosphorylation. However, the physiological importance of these modifications is still not determined. More importantly, protein interaction affects GK activity and even intracellular distribution. It has been described that GK in the liver can interact with GKRP (glucokinase regulatory protein), BAD (Bcl-xL/Bcl-2-Associated Death Promoter), PFK-2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase), GKAP (glucokinase-associated protein), etc. (Massa, 2010). From all GK protein partners, GKRP is the best studied and has high physiological relevance in the liver.

GKRP regulation of GK affects both the activity and subcellular localization of the enzyme. GKRP is a competitive inhibitor with respect to glucose. Van Shaftingen *et al* proposed a mechanistic model (Van Shaftingen, 2004); GKRP exists in two conformations, one with low affinity for GK and the other with high affinity. Fructose-1-phosphate and fructose-6 phosphate bind to the same binding site in the GKRP protein. When fructose-1-phosphate is bound to GKRP, GKRP adopts a conformation with low affinity for GK, and on the contrary,

But, Kamata *et al* also described that GK can exist in different conformations with different affinity for glucose (Kamata, 2004); in the absence of glucose, the enzyme exists in a superopen conformation thermodynamically stable and with low affinity for substrate. When glucose binds to it, there is a conformational change to an open form and next to a closed conformation that binds ATP. Then the catalytic cycle completes, after reaction products are released, GK can relax to an open or to a super-open conformation, depending on glucose concentrations (considering that the open conformation has higher affinity for glucose). GK conformation is important for GKRP protein interaction, as it can only take place when GK is in the super-open conformation (Anderka, 2008). From these conformational models of GKRP and GK, one can extrapolate the exquisite influence of carbohydrate concentration in

GKRP also plays a fundamental role in importing GK to the nucleus, as it can be deduced from animals null for GKRP that present GK permanently in the cytosol (Farrelly, 1999). At low glucose concentrations, GKRP binds to GK and the formation of GKRP/GK complex results in entry and sequestration of both proteins in the nucleus of hepatocytes. However, it is still not resolved how GK is translocated to the nucleus. On the other hand, in metabolic states with high glucose concentrations, accompanied or not by high fructose levels, and sufficient ATP, there is the dissociation of the GKRP/GK complex. GK has a nuclear export signal sequence. Therefore, once dissociated from the complex, GK can be exported to the

when the binding of fructose-6-phosphate to GKRP favours its interaction with GK.

regulating GKRP/GK binding and, consequently, GK phosphorylating activity.

cytoplasm (Shiota, 1999). Insulin also favours the dissociation of the complex.

(Zhu, 2010).

**3.1.1 Post-transcriptional regulation by GKRP** 

Insulin directly inhibits the transcription of gluconeogenic genes by promoting the phosphorylation of FOXO1 (forkhead box O), a transcription factor necessary for the induction of gluconeogenesis in conjunction with PGC-1α (PPAR-gamma coactivator 1 alpha) (Puigserver, 2003). In addition, SREBP-1c promotes the inhibition of some gluconeogenic genes. Insulin also represses glycogenolysis by phosphorylating glycogen synthase (Bollen, 1998). On the other hand, insulin regulates hepatic glucose production indirectly: a) it suppresses lipolysis in adipose tissue causing a reduction in glycerol (gluconeogenic substrate) availability; b) it inhibits glucagon secretion in the pancreas; and c) it activates hypothalamic pathways important for glucose homeostasis.

Synergistically with insulin, glucose inhibits glycogenolysis allosterically (Bollen, 1998). Glucose inhibition on gluconeogenesis is mediated by glucose metabolites, specifically fructose-2,6-bisphosphate (Wu, 2001) and xylulose-5-phosphate (Massillon, 1998).
