**1.1. The liver and glucose metabolism**

The liver comprises of hepatocytes, biliary epithelial cells, stellate cells (or Ito cells), Kupffer cells, sinusoid endothelial cells, and pit cells [1,2]. Most of the clinically quantifiable liver functions such as metabolic processes and protein synthesis take place within the hepatocytes, while non-hepatocyte cells are responsible for other functions including inflammatory response (Kupffer cells), collagen deposition (Ito cells), and cell orientation [2-5]. Regulation of blood glucose is one of the main functions exerted by the liver. The organ contains a dynamic storage of glycogen that is rapidly dismissed into the circulation as glucose to maintain glycemia and support brain functions. Hence, hepatocytes are enzymatically specialized to switch rapidly between glycogenolysis and glycogenosynthesis based upon hormonal stimuli and metabolic conditions.

Glucose enters the hepatocytes through the low-affinity transporter GLUT2 (Km=15-20 mM, Table 1). At variance of GLUT1 and GLUT4 glucose transporter that possess a Km=1-5mM and are therefore constitutively active near their maximal rate under euglycemic conditions (i.e. between 60 to 100 mg/dl), GLUT2 is maximally activated following a meal. The high Km of GLUT2 (~15-20mM) correlates well with the high Km glucokinase responsible for the conver‐ sion of glucose to glucose 6-phosphate [6].

Glucose 6-phosphate (G6P) can be routed towards glycogenosynthesis, glycolysis, or oxidation by the cytoplasmic glucose 6-phosphate dehydrogenase, *de facto* entering the pentose shunt pathway, an alternative path that generates ribose 5-phosphate utilized in nucleic acid formation (cell cycle) or return as glucose 6-phosphate to be used once again as most conven‐ ient for the cell (Fig.1). Glucose 6-phosphate is also transported into the endoplasmic reticulum

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Irrespective of the modality of entry, the hydrolysis of glucose 6-phosphate by the hydrolytic site of the glucose 6-phosphatase generates two byproducts, glucose and inorganic phosphate (Pi), which are released into the cytoplasm via two additional, specific transport mechanisms [8-11]. The glucose exported out of the ER is either earmarked for glucose output from the hepatocyte into the bloodstream or is converted anew to glucose 6-phosphate by the glucoki‐ nase thereby contributing to the glucose-glucose 6-phosphate *futile* cycle [12]. The inorganic phosphate (Pi) is either exported out of the ER lumen through its specific transporter, or forms a complex with the Ca2+ions that are actively transported into the ER lumen by the SERCA pumps [13]. Far from being static and irreversible, this Ca\*Pi complex promotes an enlarge‐ ment of the reticular Ca2+pool within the hepatocyte, and it can be dynamically reversed to Ca2+and Pi, with both moieties being mobilized out of the ER following IP3–induced Ca2+ release [13]. Thus, this enlargement of the reticular Ca2+pool is an integral part of the hepatic response to hormones such as vasopressin or norepinephrine that tap into the IP3-related Ca2+-

Role of Magnesium in the Regulation of Hepatic Glucose Homeostasis

Further investigation is required to fully elucidate the functional implications of the reticular hexose 6-phosphate dehydrogenase. This enzyme also utilizes the glucose 6-phosphate transported into the E.R., oxidizing it to 6-phosphogluconolactone [7]. Essentially, this enzyme performs the first two steps of the pentose shunt pathway within the E.R. [7.] and is responsible

in various reticular functions including E.R. stress regulation [15]. Presently, it is unknown whether the expression and activity of the hexose 6-phosphate dehydrogenase (H6PD) change

The liver plays a critical role in maintaining blood glucose levels within the normal range during the fed-fast cycle. During early fasting, hepatic glycogenolysis and glucose output from the organ maintains glycemia within a suitable range for brain function and metabolism. As the amount of glycogen stored within the liver (i.e. ~10% of the organ weight) is not sufficient to maintain glycemia over an extended period of time or prolonged fasting, gluconeogenesis becomes essential to synthesize glucose from amino acids, lactate and pyruvate dismissed into the circulation by skeletal muscles through glycogenolysis and glycolysis, and from glycerol

The complex metabolic scenario of fed to fast cycling is maintained through the antagonistic roles of insulin on one side, and glucagon, catecholamine and glucocorticoids on the other side. All these hormones modulate liver metabolism through the glucose to glucose 6-phosphate futile cycle [12], with insulin inhibiting the glucose 6-phosphatase activity and expression, and

During fed and postprandial states, elevation in blood glucose level promptly increases insulin secretion from pancreatic β-cells, which in turn, decreases glucagon release from pancreatic α-cells. The combined effect of these hormonal changes decreases hepatic glucose output and production by suppressing gluconeogenesis and glycogenolysis while increasing glucose storage within skeletal muscles via glycogenosynthesis and adipocytes via lipogenesis. In addition, insulin promotes glucose utilization in peripheral tissues through activation of

) within the E.R. to be utilized

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97

response for metabolic and functional purposes [14].

dismissed by adipose tissue through lipolysis.

the pro-glycemic hormones increasing them.

glycolysis [16].

for maintaining a reduced pyridine nucleotide pool (NADPH/H+

as a result of hormonal stimuli, metabolic conditions, or liver pathologies.

**Table 1.** Glucose Transporters

to undergo hydrolysis via glucose 6-phophatase, or oxidation via the hexose 6-phosphate dehydrogenase, the reticular variant of the glucose 6-phosphate dehydrogenase [7].

**Figure 1.** Cartoon depicting the different destinies of glucose 6–phosphate (G6P) within the hepatocyte.

The modality whereby glucose 6-phosphate enters the hepatic E.R. lumen is still debated. The *substrate-transport* theory postulates that G6P enters the ER lumen via a specific transporter (T1) distinct from the glucose 6 phosphatase. In this model, T1 represents the rate-limiting factor for the G6Pase system [8]. The *conformational flexibility substrate-transport* theory proposes that the G6Pase enzyme possesses a hydrophilic region that spans the E.R. membrane and project into the cytoplasm. This region is specific for substrate binding and is distinct from the hydrolytic site. Upon binding to glucose 6-phosphate this cytoplasmic site of the protein undergoes a conformational change and delivers the substrate to the intra-luminal catalytic site. According to this model, the substrate binding site and a hydrolytic site of the G6Pase are two parts of the same protein, and the enzyme is not specific for a particular substrate [9]. Irrespective of the modality of entry, the hydrolysis of glucose 6-phosphate by the hydrolytic site of the glucose 6-phosphatase generates two byproducts, glucose and inorganic phosphate (Pi), which are released into the cytoplasm via two additional, specific transport mechanisms [8-11]. The glucose exported out of the ER is either earmarked for glucose output from the hepatocyte into the bloodstream or is converted anew to glucose 6-phosphate by the glucoki‐ nase thereby contributing to the glucose-glucose 6-phosphate *futile* cycle [12]. The inorganic phosphate (Pi) is either exported out of the ER lumen through its specific transporter, or forms a complex with the Ca2+ions that are actively transported into the ER lumen by the SERCA pumps [13]. Far from being static and irreversible, this Ca\*Pi complex promotes an enlarge‐ ment of the reticular Ca2+pool within the hepatocyte, and it can be dynamically reversed to Ca2+and Pi, with both moieties being mobilized out of the ER following IP3–induced Ca2+ release [13]. Thus, this enlargement of the reticular Ca2+pool is an integral part of the hepatic response to hormones such as vasopressin or norepinephrine that tap into the IP3-related Ca2+ response for metabolic and functional purposes [14].

to undergo hydrolysis via glucose 6-phophatase, or oxidation via the hexose 6-phosphate

Glut4 3.5-8 mmol/L skeletal muscles, adipocytes

**Transporter Affinity for Glucose (Km) Location** Glut1 1-2 mmol/L ubiquitous Glut2 15-20 mmol/L hepatocytes, β-cells Glut3 1-2 mmol/L ubiquitous

dehydrogenase, the reticular variant of the glucose 6-phosphate dehydrogenase [7].

**Table 1.** Glucose Transporters

96 Glucose Homeostasis

**Figure 1.** Cartoon depicting the different destinies of glucose 6–phosphate (G6P) within the hepatocyte.

The modality whereby glucose 6-phosphate enters the hepatic E.R. lumen is still debated. The *substrate-transport* theory postulates that G6P enters the ER lumen via a specific transporter (T1) distinct from the glucose 6 phosphatase. In this model, T1 represents the rate-limiting factor for the G6Pase system [8]. The *conformational flexibility substrate-transport* theory proposes that the G6Pase enzyme possesses a hydrophilic region that spans the E.R. membrane and project into the cytoplasm. This region is specific for substrate binding and is distinct from the hydrolytic site. Upon binding to glucose 6-phosphate this cytoplasmic site of the protein undergoes a conformational change and delivers the substrate to the intra-luminal catalytic site. According to this model, the substrate binding site and a hydrolytic site of the G6Pase are two parts of the same protein, and the enzyme is not specific for a particular substrate [9].

Further investigation is required to fully elucidate the functional implications of the reticular hexose 6-phosphate dehydrogenase. This enzyme also utilizes the glucose 6-phosphate transported into the E.R., oxidizing it to 6-phosphogluconolactone [7]. Essentially, this enzyme performs the first two steps of the pentose shunt pathway within the E.R. [7.] and is responsible for maintaining a reduced pyridine nucleotide pool (NADPH/H+ ) within the E.R. to be utilized in various reticular functions including E.R. stress regulation [15]. Presently, it is unknown whether the expression and activity of the hexose 6-phosphate dehydrogenase (H6PD) change as a result of hormonal stimuli, metabolic conditions, or liver pathologies.

The liver plays a critical role in maintaining blood glucose levels within the normal range during the fed-fast cycle. During early fasting, hepatic glycogenolysis and glucose output from the organ maintains glycemia within a suitable range for brain function and metabolism. As the amount of glycogen stored within the liver (i.e. ~10% of the organ weight) is not sufficient to maintain glycemia over an extended period of time or prolonged fasting, gluconeogenesis becomes essential to synthesize glucose from amino acids, lactate and pyruvate dismissed into the circulation by skeletal muscles through glycogenolysis and glycolysis, and from glycerol dismissed by adipose tissue through lipolysis.

The complex metabolic scenario of fed to fast cycling is maintained through the antagonistic roles of insulin on one side, and glucagon, catecholamine and glucocorticoids on the other side. All these hormones modulate liver metabolism through the glucose to glucose 6-phosphate futile cycle [12], with insulin inhibiting the glucose 6-phosphatase activity and expression, and the pro-glycemic hormones increasing them.

During fed and postprandial states, elevation in blood glucose level promptly increases insulin secretion from pancreatic β-cells, which in turn, decreases glucagon release from pancreatic α-cells. The combined effect of these hormonal changes decreases hepatic glucose output and production by suppressing gluconeogenesis and glycogenolysis while increasing glucose storage within skeletal muscles via glycogenosynthesis and adipocytes via lipogenesis. In addition, insulin promotes glucose utilization in peripheral tissues through activation of glycolysis [16].

## **1.2. Physiological magnesium homeostasis**

## *1.2.1. Cellular magnesium distribution*

Our body absorbs minerals through food and drinks consumed daily. However, industrial food processing techniques limit to a varying extent the dietary content and intake of minerals and vitamins, making necessary the utilization of supplements. This is indeed the case of the macro mineral magnesium. Overall, Mg2+is the fourth most abundant cation in vertebrates and the second most abundant cation within cells after potassium. In humans, total body magne‐ sium (Mg2+) is found mostly in the bones (60-65% of total content), soft tissues and cells in general [17]. Only 1% of total body magnesium is found in the extracellular fluid, thus making serum magnesium level a poor indicator of total magnesium content and availability in the body. Of the 1% total body Mg2+present in the extracellular fluid, about sixty percent (60%) is free, the reminder (~33%) being bound to proteins, citrate, bicarbonate, ATP1 and phosphate (≤7%) [18].

enzymes or channels by Mg2+is not restricted to the cytoplasm occurring also in the cellular organelles in which Mg2+is compartmentalized. In liver mitochondria, changes in matrix Mg2+content regulate the activity of succinate and glutamate dehydrogenases but not αketoglutarate dehydrogenases [23]. In addition, Mg2+regulates the opening of the inner mitochondrial anion channel (IMAC), the permeability transition pore (PTP), KATP-channels,

solved as to whether Mg2+is required for the adenine nucleotide translocase to operate [23]. At the level of the hepatic rough endoplasmic reticulum (R.E.R.), Mg2+regulates Ca2+uptake via the Ca-ATPase, and its release through the IP3 receptor [27], as well as the rate of protein synthesis and dismissal into the cytoplasm via the translocon [28]. Experimental evidence suggests that Mg2+inversely regulates the rate of glucose 6-phosphate entry into the E.R. lumen, thus providing higher level of substrate to the glucose 6-phosphatase (G6Pase), and the hexose 6-phosphate dehydrogenase (H6PD) under conditions in which cellular Mg2+levels are reduced [29]. In the nucleus, changes in Mg2+content have been associated with inhibition of specific endonucleases and chromatin folding [21]. Less known is the function of Mg2+within the Golgi lumen. The recent localization of a Mg2+transporter in the Golgi cisternae, however, suggests a possible role of the cation in regulating protein glycosylation [23]. As for endosomal and lysosomal vesicles, nothing is known about the Mg2+concentration within these vesicles

Despite its large total concentration within the cell, Mg2+is not a static cation. Major Mg2+fluxes have been detected across the cell membrane of the hepatocyte and other mammalian cells. Various hormones and pharmacological agents modulate total and free Mg2+concentrations within the hepatocyte, supporting the hypothesis that many of the metabolic changes elicited by these agents are attained by changing the concentration of Mg2+within the cells and/or within specific cellular compartments, which then results in the up-or down-regulation in the

The current understanding of Mg2+transport across the hepatocyte cell membrane indicates

which activates the exchanger through phosphorylation [34]. Under conditions in which

mechanism that utilize different cations or anions in counter-transport for or co-transport with

As for Mg2+entry, hepatocytes appear to utilize predominantly the TRPM7 channel [35]. Protein kinase C (PKC) appears to regulate this channel directly via phosphorylation of its C terminus,

Several other Mg2+entry mechanisms have been observed to be present in liver cells [32] but it is still unclear to which extent these mechanism cooperate with TRPM7 in mediating Mg2+entry

gradient is present across the cell membrane, or Na+

by agents like amiloride or imipramine, cellular Mg2+is extruded via a Na+

/Mg2+exchanger [30,31], which functionally depends on

[30,32] and the cellular level of cAMP [33],

transport is inhibited


exchanger, thus regulating the organelle volume [23]. It is still unre‐

Role of Magnesium in the Regulation of Hepatic Glucose Homeostasis

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99

and possibly the H+

/K+

and its role in modulating their physiological processes.

activity of Mg-sensitive enzymes.

that Mg2+exits the liver cell via a Na+

limited inward Na+

Mg2+, respectively [32].

*1.2.2. Cellular magnesium transport mechanisms*

the physiological concentration of extracellular Na+

or indirectly by removing RACK1-inhibition [36].

and in regulating hepatic Mg2+homeostasis.

Whole body Mg2+homeostasis changes overtime. At an early stage, most Mg2+in the bones can readily exchange with serum, representing an optimal store to compensate for occasional dietary deficiency. As age progresses, however, the proportion of readily exchangeable Mg2+in the bones decreases significantly due to a change in bone crystal size with age [19]. In indi‐ viduals consuming Mg2+enriched diet, a positive association between bone mineral density and Mg2+content within the erythrocytes has been reported [20].

At the cellular level, Mg2+is highly compartmentalized within nucleus, endoplasmic or sarcoplasmic reticulum, mitochondria, and cytoplasm [18], the only notable exception being the erythrocytes, in which Mg2+is merely cytoplasmic [21]. In the majority of mammalian cells examined, including the hepatocytes, total cellular Mg2+concentrations range from 15 to 20mM as measured by various techniques including electron X ray microprobe analysis (EXPMA), fluorescent dyes, and scanning fluorescence x-ray microscopy [21]. Total Mg2+concentrations between 15 and 20mM have also been measured within the nucleus, the mitochondria, and the rough endoplasmic reticulum of various cell types by EPXMA [21]. In the cytoplasm, Mg2+is present as a complex with ATP (~4-5mM=Mg\*ATP) and other phosphonucleotides [22]. Consequently, the free Mg2+concentration ([Mg2+]i ) within the cytoplasm and the mitochon‐ drial matrix ranges between 0.5 and 1.2 mM [21,23], i.e. slightly below or at the concentration present in the extracellular environment. These measurements suggest that the majority of mammalian cells are near *zero trans* conditions as far it concerns the cellular distribution of Mg2+.

Despite the large amount of Mg2+present within the majority of mammalian cells, limited information is available about the physiological role of Mg2+for specific cell function. In liver cells, Mg2+controls ATP production by the mitochondria and its utilization by various ATPases including the Na+ /K+ -ATPase [21] and the reticular Ca2+-ATPase [21]. As a result, 90% of cytoplasmic ATP is in the form of a complex with Mg2+[24]. Moreover, in hepatocytes Mg2+is a cofactor for many enzymes involved in energy metabolism, including glycolysis and Krebs cycle [25]. The list of Mg2+-regulated glycolytic enzymes includes hexokinase, phosphofructo‐ kinase, aldolase, phosphoglycerate kinase and pyruvate kinase [26]. The regulation of specific enzymes or channels by Mg2+is not restricted to the cytoplasm occurring also in the cellular organelles in which Mg2+is compartmentalized. In liver mitochondria, changes in matrix Mg2+content regulate the activity of succinate and glutamate dehydrogenases but not αketoglutarate dehydrogenases [23]. In addition, Mg2+regulates the opening of the inner mitochondrial anion channel (IMAC), the permeability transition pore (PTP), KATP-channels, and possibly the H+ /K+ exchanger, thus regulating the organelle volume [23]. It is still unre‐ solved as to whether Mg2+is required for the adenine nucleotide translocase to operate [23]. At the level of the hepatic rough endoplasmic reticulum (R.E.R.), Mg2+regulates Ca2+uptake via the Ca-ATPase, and its release through the IP3 receptor [27], as well as the rate of protein synthesis and dismissal into the cytoplasm via the translocon [28]. Experimental evidence suggests that Mg2+inversely regulates the rate of glucose 6-phosphate entry into the E.R. lumen, thus providing higher level of substrate to the glucose 6-phosphatase (G6Pase), and the hexose 6-phosphate dehydrogenase (H6PD) under conditions in which cellular Mg2+levels are reduced [29]. In the nucleus, changes in Mg2+content have been associated with inhibition of specific endonucleases and chromatin folding [21]. Less known is the function of Mg2+within the Golgi lumen. The recent localization of a Mg2+transporter in the Golgi cisternae, however, suggests a possible role of the cation in regulating protein glycosylation [23]. As for endosomal and lysosomal vesicles, nothing is known about the Mg2+concentration within these vesicles and its role in modulating their physiological processes.

Despite its large total concentration within the cell, Mg2+is not a static cation. Major Mg2+fluxes have been detected across the cell membrane of the hepatocyte and other mammalian cells. Various hormones and pharmacological agents modulate total and free Mg2+concentrations within the hepatocyte, supporting the hypothesis that many of the metabolic changes elicited by these agents are attained by changing the concentration of Mg2+within the cells and/or within specific cellular compartments, which then results in the up-or down-regulation in the activity of Mg-sensitive enzymes.

## *1.2.2. Cellular magnesium transport mechanisms*

**1.2. Physiological magnesium homeostasis**

Our body absorbs minerals through food and drinks consumed daily. However, industrial food processing techniques limit to a varying extent the dietary content and intake of minerals and vitamins, making necessary the utilization of supplements. This is indeed the case of the macro mineral magnesium. Overall, Mg2+is the fourth most abundant cation in vertebrates and the second most abundant cation within cells after potassium. In humans, total body magne‐ sium (Mg2+) is found mostly in the bones (60-65% of total content), soft tissues and cells in general [17]. Only 1% of total body magnesium is found in the extracellular fluid, thus making serum magnesium level a poor indicator of total magnesium content and availability in the body. Of the 1% total body Mg2+present in the extracellular fluid, about sixty percent (60%) is

Whole body Mg2+homeostasis changes overtime. At an early stage, most Mg2+in the bones can readily exchange with serum, representing an optimal store to compensate for occasional dietary deficiency. As age progresses, however, the proportion of readily exchangeable Mg2+in the bones decreases significantly due to a change in bone crystal size with age [19]. In indi‐ viduals consuming Mg2+enriched diet, a positive association between bone mineral density

At the cellular level, Mg2+is highly compartmentalized within nucleus, endoplasmic or sarcoplasmic reticulum, mitochondria, and cytoplasm [18], the only notable exception being the erythrocytes, in which Mg2+is merely cytoplasmic [21]. In the majority of mammalian cells examined, including the hepatocytes, total cellular Mg2+concentrations range from 15 to 20mM as measured by various techniques including electron X ray microprobe analysis (EXPMA), fluorescent dyes, and scanning fluorescence x-ray microscopy [21]. Total Mg2+concentrations between 15 and 20mM have also been measured within the nucleus, the mitochondria, and the rough endoplasmic reticulum of various cell types by EPXMA [21]. In the cytoplasm, Mg2+is present as a complex with ATP (~4-5mM=Mg\*ATP) and other phosphonucleotides [22].

drial matrix ranges between 0.5 and 1.2 mM [21,23], i.e. slightly below or at the concentration present in the extracellular environment. These measurements suggest that the majority of mammalian cells are near *zero trans* conditions as far it concerns the cellular distribution of

Despite the large amount of Mg2+present within the majority of mammalian cells, limited information is available about the physiological role of Mg2+for specific cell function. In liver cells, Mg2+controls ATP production by the mitochondria and its utilization by various ATPases

cytoplasmic ATP is in the form of a complex with Mg2+[24]. Moreover, in hepatocytes Mg2+is a cofactor for many enzymes involved in energy metabolism, including glycolysis and Krebs cycle [25]. The list of Mg2+-regulated glycolytic enzymes includes hexokinase, phosphofructo‐ kinase, aldolase, phosphoglycerate kinase and pyruvate kinase [26]. The regulation of specific


and phosphate

) within the cytoplasm and the mitochon‐

free, the reminder (~33%) being bound to proteins, citrate, bicarbonate, ATP1

and Mg2+content within the erythrocytes has been reported [20].

Consequently, the free Mg2+concentration ([Mg2+]i

/K+

*1.2.1. Cellular magnesium distribution*

(≤7%) [18].

98 Glucose Homeostasis

Mg2+.

including the Na+

The current understanding of Mg2+transport across the hepatocyte cell membrane indicates that Mg2+exits the liver cell via a Na+ /Mg2+exchanger [30,31], which functionally depends on the physiological concentration of extracellular Na+ [30,32] and the cellular level of cAMP [33], which activates the exchanger through phosphorylation [34]. Under conditions in which limited inward Na+ gradient is present across the cell membrane, or Na+ transport is inhibited by agents like amiloride or imipramine, cellular Mg2+is extruded via a Na+ -independent mechanism that utilize different cations or anions in counter-transport for or co-transport with Mg2+, respectively [32].

As for Mg2+entry, hepatocytes appear to utilize predominantly the TRPM7 channel [35]. Protein kinase C (PKC) appears to regulate this channel directly via phosphorylation of its C terminus, or indirectly by removing RACK1-inhibition [36].

Several other Mg2+entry mechanisms have been observed to be present in liver cells [32] but it is still unclear to which extent these mechanism cooperate with TRPM7 in mediating Mg2+entry and in regulating hepatic Mg2+homeostasis.

#### *1.2.3. Regulation of magnesium transport*

The specific modality of operation and regulation of the various Mg2+transport mechanisms have been extensively addressed in recent review articles [32,37-39], and we refer to those reviews for further information. For the purpose of this chapter, we will only mention that in liver cells both Mg2+entry and extrusion are under hormonal control. Hormones like catechol‐ amine and glucagon, which increase cAMP level within the hepatocyte, all promote Mg2+extrusion by phosphorylating the Na+ /Mg2+exchanger mentioned above [40]. Activation of α1-adrenoceptors by catecholamine also induces Mg2+extrusion. Stimulation of this class of adrenergic receptors activates PLCγ, which in turn hydrolyzes phosphatidyl-inositol bisphos‐ phate (PIP2) to diacylglycerol and inositol 1,4,5-trisphosphate (IP3). The IP3-induced Ca2+release from the endoplasmic reticulum and the subsequent capacitative Ca2+entry through the hepatocyte plasma membrane promote Mg2+extrusion from the hepatocyte, most likely by displacing Mg2+for Ca2+from organelle and cytosolic binding sites [41]. This is consistent with the observation that distinct Na+ and Ca2+-dependent Mg2+extrusion mecha‐ nisms operate in the basolateral and the apical portion of the hepatocyte cell membrane, respectively [34,42]. The differential activation of the Na+ -dependent (β-adrenergic receptors and glucagon receptor) and the Ca2+-dependent Mg2+extrusion mechanism (α1-adrenergic receptors) points to the ability of the hepatocytes to activate Mg2+extrusion by different modalities and circumvent possible inhibitory mechanisms. It has to be noted, in fact, that insulin pre-treatment abolishes the Mg2+extrusion mediated by cAMP but not that mediated via α1-adrenoceptor activation [43]. Conversely, hormones or agents that maximize Ca2+release from the ER elicit a time-dependent inhibition of α1-adrenergic receptor mediated Mg2+extrusion that leaves unaffected the extrusion occurring via β-adrenergic receptors stimulation and cellular cAMP elevation [44]. In this contest, it has to be noted that cytoplasmic free [Mg2+]i modulates adenylyl cyclase activation in a variety of cell types including hepato‐ cytes [45]. Under resting conditions, cytoplasmic [Mg2+]i is insufficient to activate the adenylyl cyclase maximally. Following β-adrenoceptor or glucagon receptor stimulation the cytoplas‐ mic Mg2+pool increases markedly but transiently via the release of Mg2+from other cellular pool (namely mitochondria and endoplasmic reticulum) promoting adenylate cyclase activity and cAMP synthesis [45]. Elevation of cytoplasmic [Mg2+]i also inhibits IP3-induced Ca2+-release [27] most likely via a direct modulatory effect of Mg2+on the IP3 receptor subunits.

Both Mg2+extrusion and Mg2+accumulation are quantitatively and timely limited processes [48,49], implying the movement of Mg2+from and to specific cellular compartments. The cytoplasm is but one of the cellular compartments involved in Mg2+transport out of the cell or into the cells [21,22], other compartments being the mitochondria and the endoplasmic reticulum [21]. This notion is supported by the observation that the co-stimulation of hepatic β2-and α1-adrenergic receptors by the mix agonist epinephrine results in a Mg2+extrusion that is quantitatively similar to the sum of the Mg2+amounts mobilized by the stimulation of each adrenoceptor class by specific agonists [40]. However, the mechanisms involved in Mg2+transport in-and-out of these compartments have not been fully elucidated. It is known that mitochondria accumulate Mg2+through Mrs2, a Mg2+-specific channel, the absence of which affects complex I expression and activity [50]. Less certain is whether Mg2+extrusion from the mitochondria occurs via the adenine nucleotide translocase [21]. As for the cytoplasm, this compartment acts as a temporary step-in between the extracellular compartment and the cellular organelles both in the extrusion and in the accumulation of Mg2+due to the high concentration of ATP that buffers Mg2+with a very high Kd (~75µM) and the presence of other phosphonucleotides and binding proteins [22]. The role of ATP is further supported by the observation that pathological conditions that decrease cellular ATP content through dysme‐ tabolic processes (namely diabetes and alcoholic liver disease) ultimately cause Mg2+loss from

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101

Insulin signaling is mediated by a complex and highly integrated signaling network that controls several processes including whole body glucose homeostasis. The liver is the first organ 'seen' by insulin following its release from the β-cells into the portal vein, and is responsible for the clearance of 50% of the released insulin at the first pass. Stimulation of the insulin receptor in liver cells is a key event to regulate hepatic glucose homeostasis. In addition, insulin acts indirectly on hepatic glucose homeostasis in that insulin released from β-cells inhibits glucagon release from pancreatic α-cells thus limiting the drive on hepatic gluconeo‐ genesis. The impairment of both these processes observed in insulin resistance is linked to

Insulin initiates its signaling cascade by interacting with the insulin receptor on the cell surface. Binding of insulin to the extracellular α-subunits of the insulin receptor results in a confor‐ mation change that translates to the intracellular β-subunits of the receptor. The consequent activation of the kinase domain in the β-subunits of the receptor results in the autophosphor‐ ylation of specific tyrosine residues in the intracellular β-subunits. The phosphorylated insulin receptor now recruits the insulin receptor susbstrate (IRS), which upon phosphorylation on tyrosine residues acts as a docking unit for numerous cellular proteins including the phos‐ phatidyl inositol 3-kinase (PI3K) [54]. Recruitment of these proteins to the IRS results in their activation. Activation of PI3K results in the phosphorylation of PIP2 to PIP3 and in the subsequent activation of protein kinase B (PKB or Akt), which then phosphorylates Forkhead box protein O1 (FoxO1), preventing its translocation to the nucleus. In its un-phosphorylated

the cell [51-53].

**2. Insulin signaling in the liver**

major health problems including type 2-diabetes.

Cellular Mg2+accumulation is also under hormonal regulation. Among the hormones involved in the process there are insulin and vasopressin. These hormones either counteract cAMP production by acting at the level of the β-adrenergic receptor (inhibition) or the cytoplasmic phosphodiesterase that converts cAMP to AMP (stimulation), and/or activate PKC signaling, which acts as cAMP *alter ego*. Due to its ubiquitous presence and abundance, the TRPM7 channel is the mechanism most likely responsible for Mg2+accumulation in the hepatocyte [46]. It is presently unclear whether PKC activates the channel by binding RACK1 and removing this protein from a specific site near the C terminus of the channel through which RACK1 inhibits TRPM7 conductance [36], or whether phosphorylation of the channel C-terminus is also required for full activation [46]. In the case of insulin, a direct modulatory effect on the putative Na+ /Mg2+exchanger has also been observed [47].

Both Mg2+extrusion and Mg2+accumulation are quantitatively and timely limited processes [48,49], implying the movement of Mg2+from and to specific cellular compartments. The cytoplasm is but one of the cellular compartments involved in Mg2+transport out of the cell or into the cells [21,22], other compartments being the mitochondria and the endoplasmic reticulum [21]. This notion is supported by the observation that the co-stimulation of hepatic β2-and α1-adrenergic receptors by the mix agonist epinephrine results in a Mg2+extrusion that is quantitatively similar to the sum of the Mg2+amounts mobilized by the stimulation of each adrenoceptor class by specific agonists [40]. However, the mechanisms involved in Mg2+transport in-and-out of these compartments have not been fully elucidated. It is known that mitochondria accumulate Mg2+through Mrs2, a Mg2+-specific channel, the absence of which affects complex I expression and activity [50]. Less certain is whether Mg2+extrusion from the mitochondria occurs via the adenine nucleotide translocase [21]. As for the cytoplasm, this compartment acts as a temporary step-in between the extracellular compartment and the cellular organelles both in the extrusion and in the accumulation of Mg2+due to the high concentration of ATP that buffers Mg2+with a very high Kd (~75µM) and the presence of other phosphonucleotides and binding proteins [22]. The role of ATP is further supported by the observation that pathological conditions that decrease cellular ATP content through dysme‐ tabolic processes (namely diabetes and alcoholic liver disease) ultimately cause Mg2+loss from the cell [51-53].
