**3.1 Regulation of GK activity in the liver**

Gck gene has two distinct promoters and one of them directs gene transcription specifically in the liver (Postic, 1995). Hepatic GK expression responds to nutritional changes; it is

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

Synergistically with insulin, glucose inhibits glycogenolysis allosterically (Bollen, 1998). Glucose inhibition on gluconeogenesis is mediated by glucose metabolites, specifically

In order to enter the lipogenic pathway, glucose must be metabolized. The first and ratelimiting step is the phosphorylation of glucose at the 6th carbon to obtain glucose-6 phosphate. This reaction is catalyzed by glucokinase (GK; EC 2.7.1.1), a member of the hexokinase family. However, GK differs from other hexokinases in its particular kinetic properties: affinity for glucose that is within the physiological plasma concentration range (S0.5 for glucose of 8 mM), positive cooperativity for glucose although it is a monomeric

> **Molecular weight** 100 KDa 50 KDa **Substrates** Hexoses Glucose **S0.5 for glucose** < 0.5 mM 8 mM **Kinetic** Hyperbolic Sigmoidal **Product inhibition** Yes No

As a result of its kinetic characteristics, intracellular glucose phosphorylation rate inside the hepatocyte correlates with glycaemia. Hence, GK can be considered an intracellular glucose sensor. Consequently, apart from hepatocytes, GK is expressed in glucosensitive cells of the pancreas, hypothalamus, anterior pituitary gland, and entero-endocrine K and L cells of the gut (Schuit, 2001; Zelent, 2006; Vieira, 2007; Iynedjian, 2009), all of them crucial in the control

Liver contains 99.9% of the body GK. Therefore, is not surprising that this enzyme influences intermediary metabolism and energy storage. GK reaction controls the flux of glucose through several metabolic pathways: glycolysis, glucose oxidization, glycogenesis, triglyceride synthesis, phospholipids and cholesterol synthesis, glycogenolysis and gluconeogenesis. For that reason, GK is an enzyme highly regulated in the liver, both at the

Gck gene has two distinct promoters and one of them directs gene transcription specifically in the liver (Postic, 1995). Hepatic GK expression responds to nutritional changes; it is

**HEXOKINASES 1-3 GLUCOKINASE** 

c) it activates hypothalamic pathways important for glucose homeostasis.

fructose-2,6-bisphosphate (Wu, 2001) and xylulose-5-phosphate (Massillon, 1998).

**3. Glucokinase regulates the fate of glucose carbons in the liver** 

enzyme, and lack of inhibition by glucose-6-phosphate (Table 1).

Table 1. Hexokinase family kinetic properties

of the whole-body glucose homeostasis.

transcriptional and the post-transcriptional level.

**3.1 Regulation of GK activity in the liver** 

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- (Zhu, 2010).

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.

#### **3.1.1 Post-transcriptional regulation by GKRP**

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

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

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

Liver Glucokinase and Lipid Metabolism 243

A liver specific GK knock-out was obtained using the LoxP-Cre system (Postic, 1999). Transgenic mice showed mild hyperglycemia and hyperinsulinemia in basal conditions, without changes in hepatic glycogen, plasma non-esterified fatty acids, triglycerides or βhydroxybutyrate. In hyperglycaemic clamp studies, reduced hepatic glucose uptake and glycogen levels were observed in KO animals; however, results on lipid profile were not

Several liver GK gain-of-function studies, both using transgenic animals and by means of adenovirus gene transfer, have been performed in healthy animals and models of diabetes such as streptozotocin induced type I and type II induced by ingestion of high fat/high carbohydrate diet. Due to heterogeneity, these studies will be examined according to the

a. Overexpression of GK in the liver of fed, healthy animals is summarized in Table 2

**O'Doherty 1999; Scott 2003** 

(Rats 200-250 g)


Transgenic Adenoviral gene transfer

x 2 x 3 x 6.4


FAS, G6Pase. No change in PEPCK-K

PEPCK-C promoter CMV promoter

**Ferre 2003** 

**Animal model** *Mus musculus Rattus norvegicus* 

**Age at analysis** 2 months 12 months 5 days post-injection

↑ L-PK ↓PEPCK-C, GLUT-2, TAT

**Glycaemia** decrease - no change Decrease **Blood lactate** - - increase Increase **Blood triglycerides** ~ increase - ~ increase Increase **NEFA** ~ increase - no change increase **Insulin** decrease increase no change decrease **Hepatic glucose-6-P** increase ~ increase - - **Hepatic glycogen** increase decrease no change no change **Hepatic triglycerides** no change increase - -

Table 2. Hepatic GK overexpression studies in healthy fed animals. Comments: decrease, increase and no change are referred to control group. "~" means no statistically significant; "-", no determined; "CMV", cytomegalovirus; "PEPCK-C", cytosolic phosphoenolpyruvate carboxykinase; "L-PK", liver pyruvate kinase; "TAT", tyrosine aminotransferase; "ACC1", Acetyl-Coenzyme A carboxylase 1; "FAS", fatty acid synthase; and "G6Pase", glucose-6

In these models, enhancing hepatic glucose uptake by GK overexpression results in a direct reduction of glycaemia. As a consequence of lower blood glucose levels, pancreatic β-cell

**3.2.1 Genetic suppression of hepatic GK** 

**3.2.2 Genetic overexpression of GK in the liver** 

(Ferre, 1996a, 1996b, 2003; O'Doherty, 1999; Scott, 2003).

**Study variables Ferre 1996a, 1996b** 

animal model and analysis conditions.

provided.

**GK activity over** 

**Modulation of enzymes and transcription factors** 

phosphatase.

**control** 

The physiological function of GKRP consists of inhibiting GK activity by sequestering it to the nucleus. GKRP binding also serves to stabilize GK protein and protect it from degradation. Thus, thanks to GKRP a big reservoir of GK exists in the nucleus of the hepatocyte at low glucose concentration. After a meal, this reservoir of GK can be rapidly mobilized (translocation is complete within 30 minutes) to the cytosol in order to promote glucose uptake and storage in the liver. This regulation process is much more fast and efficient than the synthesis de novo of GK promoted by insulin. Conversely, when glucose uptake has finished, GK returns to the nucleus in order to save energy because, on one hand, this translocation avoids the futile cycle between glucose and glucose-6-phosphate, and on the other hand, it ends the glucose signal generated by GK activity that activate transcription of glycolytic and lipogenic genes (Figure 3). The consequence of GK translocation to the nucleus in the postabsorptive state is the induction of glycogenolysis and gluconeogenesis.

Fig. 3. Subcellular localization of GK regulated by GKRP. (A) During fasting, GK is sequestered in the nucleus where it remains bound to GKRP and inactive. After a meal, nutritional signals (i.e. insulin, glucose and fructose) induce the dissociation of the GK/GKRP complex and free GK translocates to the cytosol. Original artwork.

To summarize, thanks to its kinetic properties and its subtle regulation, GK enables the liver to adapt its metabolism for glucose uptake or glucose production as required, and consequently to regulate energy homeostasis.

#### **3.2 GK modulation in the liver: impact on carbohydrate and lipid metabolism**

Numerous natural mutations in GK gene have been associated to disease (Gloyn, 2003 & Osbak, 2009), reinforcing the concept that it is a crucial enzyme in the control of whole-body glucose homeostasis. Mutations that cause decrease or loss of GK activity are associated to maturity onset diabetes of the young-2 (MODY-2) or to permanent neonatal diabetes mellitus (PNDM). In diabetes, as a result of impairment in insulin secretion, the capacity of the liver to uptake glucose is diminished. On the other hand, activating mutations of GK cause persistent hyperinsulinemic hypoglycemia in infancy (PHHI). The phenotype of all these pathologies is mainly dominated by GK function in the pancreatic β-cell, where it regulates glucose-dependent insulin secretion. As insulin controls hepatic GK transcription and influences GKRP regulation, it is difficult to elucidate which are the specific consequences of these mutations on hepatic GK independently of insulin.

Some animal models have been developed to study the specific role of liver GK on metabolism.

The physiological function of GKRP consists of inhibiting GK activity by sequestering it to the nucleus. GKRP binding also serves to stabilize GK protein and protect it from degradation. Thus, thanks to GKRP a big reservoir of GK exists in the nucleus of the hepatocyte at low glucose concentration. After a meal, this reservoir of GK can be rapidly mobilized (translocation is complete within 30 minutes) to the cytosol in order to promote glucose uptake and storage in the liver. This regulation process is much more fast and efficient than the synthesis de novo of GK promoted by insulin. Conversely, when glucose uptake has finished, GK returns to the nucleus in order to save energy because, on one hand, this translocation avoids the futile cycle between glucose and glucose-6-phosphate, and on the other hand, it ends the glucose signal generated by GK activity that activate transcription of glycolytic and lipogenic genes (Figure 3). The consequence of GK translocation to the nucleus in the post-

*Nucleus*

Insulin *Hepatocyte*

Legend:

GK GKRP Glucose Fructose

absorptive state is the induction of glycogenolysis and gluconeogenesis.

Fig. 3. Subcellular localization of GK regulated by GKRP. (A) During fasting, GK is sequestered in the nucleus where it remains bound to GKRP and inactive. After a meal, nutritional signals (i.e. insulin, glucose and fructose) induce the dissociation of the GK/GKRP complex and free GK translocates to the cytosol. Original artwork.

**3.2 GK modulation in the liver: impact on carbohydrate and lipid metabolism** 

consequences of these mutations on hepatic GK independently of insulin.

To summarize, thanks to its kinetic properties and its subtle regulation, GK enables the liver to adapt its metabolism for glucose uptake or glucose production as required, and

Numerous natural mutations in GK gene have been associated to disease (Gloyn, 2003 & Osbak, 2009), reinforcing the concept that it is a crucial enzyme in the control of whole-body glucose homeostasis. Mutations that cause decrease or loss of GK activity are associated to maturity onset diabetes of the young-2 (MODY-2) or to permanent neonatal diabetes mellitus (PNDM). In diabetes, as a result of impairment in insulin secretion, the capacity of the liver to uptake glucose is diminished. On the other hand, activating mutations of GK cause persistent hyperinsulinemic hypoglycemia in infancy (PHHI). The phenotype of all these pathologies is mainly dominated by GK function in the pancreatic β-cell, where it regulates glucose-dependent insulin secretion. As insulin controls hepatic GK transcription and influences GKRP regulation, it is difficult to elucidate which are the specific

Some animal models have been developed to study the specific role of liver GK on

*Hepatocyte*

metabolism.

*Nucleus*

**A B**

consequently to regulate energy homeostasis.
