MAGNESIUM REGULATION OF AND

METABOLISM IN HEPG2 CELLS

By

ZIENAB ETWEBI

Submitted in partial fulfillment of the requirements

For the degree of Master of Science

Thesis Advisor: Dr. Andrea Romani

Department of Physiology and Biophysics

CASE WESTERN RESERVE UNIVERSITY

August, 2011

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Zienab Etwebi candidate for the Master of Science degree *.

(signed) Dr. William Schilling (chair of the committee)

Dr. George Dubyak

Dr. Margaret Chandler

Dr. John Kirwan

Dr. Colleen Croniger

Dr. Andrea Romani

(date) 05/18/2011

*We also certify that written approval has been obtained for any

proprietary material contained therein.

Dedication

I would like to dedicate this thesis to my very supportive husband, Ghasan Ferjani and my family back home. Without their help, encouragement, and ongoing support I would not be where I am today.

Thank you.

i

Table of Contents

Table of Contents…………………………………………………………………….…..ii

List of Tables………………………………………………………………………...... iii

List of Figures ………………………………………………………………….………...iv

Acknowledgement…………………………………………..…………….……………..vi

Abbreviations used in the Text………………………………………………………...... vii

Abstract……………………………..……………..…………………………………...... ix

Introduction………………………………………………………………………..……..1

Magnesium Distribution…………………………………………………………………..2

Magnesium transport and hormonal regulation………..………………………………….4

Magnesium and ……………………………………..………………….10

Research Objective and Specific Aims…………………………….…………….………24

Materials and Methods………………………………………………………….…….….25

Results……………………………………………………………………………………32

Discussion………………………………………………………………………………..38

Future Directions…………………………………………………………...……………43

Bibliography……………………………………………………………………………..66

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List of Tables

Table 1 A List of Analytical Methods used to Measure Mg2+ Content and Distribution……………………………………………………..………..45

Table 2 Tissues in which a Na+-dependent Mg2+ Efflux has been Observed or Hypothesized.……………………………..……………………………...46

Table 3 Facilitative Isoforms…..….……….………….……47

Table 4 Primers for Quantitative Real-Time PCR ………...………....…………..48

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List of Figures

Figure 1 Cellular Magnisium Profile………………………………………...…….49

Figure 2 Magnesium Transport and Hormonal Regulation………………….…….50

Figure 3 Magnesium Compartmentalization…………………………………....…51

Figure 4 Magnesium’s affect on ATP levels…………………………………….…52

Figure 5A Magnesium’s Effect on Glucose Uptake without …………...…....53

Figure 5B Magnesium’s Effect on Glucose Uptake with insulin………………....…53

Figure 6A Insulin’s Effect on Glucose Uptake in 0.8 mM Mg2+ ………………..…..54

Figure 6B Insulin’s Effect on Glucose Uptake in 0.4 mM Mg2+ ………………..….54

Figure 7 Effect of Magnesium on Glucose Transporters (qPCR) ……………...... 55

Figure 8 Effect of Magnesium on Glucose Transporters (WB) ………………..…56

Figure 9 Effect of Acute [Mg2+]o Change on Glucose Uptake ………………..…57

Figure 10 Effect of Magnesium on SREBP-1c and SREBP2 (qPCR)…………...…58

Figure 11 Effect of Magnesium on SREBP-1c and SREBP2 (WB).…………...…..59

Figure 12 Effect of Magnesium on SCAP and Insig2.…….…………………..…....60

Figure 13A Effect of Magnesium on PPARα ……………………………….....…….61

Figure 13B Effect of Magnesium on PPARγ………………….. …………………….61

Figure 14A Effect of Magnesium on Glucose Transporters in animals (qPCR) …....62

Figure 14B Effect of Magnesium on Glucose Transporters in animals(WB) ….....…62

iv

Figure 15 Effect of Magnesium on SREBP-1c in animals (qPCR) …………….…63

Figure 16A Effect of Magnesium on SREBP-1c in SD rats (WB)………………..…64

Figure 16B Effect of Magnesium on SREBP-1c in B6 mice (WB) ………………...64

Figure 17A Effect of Magnesium on PPARα in Animals ……….………………..…65

Figure 17B Effect of Magnesium on PPARγ in Animals ……….………………..…65

v

Acknowledgements

I would like to extend my appreciation to my thesis advisor Andrea Romani, for he has been very helpful and supportive throughout this process. In addition, I would like to thank my thesis committee members, Dr. Schilling, Dr. Dubyak, Dr. Chandler, Dr.

Kirwan, and Dr. Croniger. I would also like to thank the rest of my family for their continued encouragement and support.

vi

Abbreviations used in the Text

AAS atomic absorbance spectrophotometry

ANOVA analysis of variance

ATP adenosine tri-phosphate cAMP cyclic

DAG diacylglycerol

2,3-DPG 2,3-diphosphoglycerate

EPXMA electron probe X-ray microanalysis

HDL high-density lipoproteins

HepG2 human hepatoma cells

Insig insulin-induced gene

IP3 tri-phosphate

LCAT lecithin acyltransferase

LPL lipoprotein

[Mg2+]i intracellular free Mg2+ concentration

2+ 2+ [Mg ]o extracellular Mg concentration

PIP2 phosphatidylinositol-4,5-bisphosphate

PLC

PKC kinase C

vii

PPAR peroxisome proliferator activated receptors

R.E.R Rough

SCAP SREBP-cleavage activating protein

SD rats Sprague-Dawley rats

SREBP sterol regulatory element binding

TGRLP triacylglycerol-rich lipoprotein

TBS tris-buffered saline

TRP transient receptor potential

VP vasopressin

V1aR vasopressin receptor 1a

viii

Magnesium Regulation of Glucose and in HEPG2 Cells

Abstract

By

Zienab Etwebi

Magnesium (Mg2+) is an important cation for a variety of cell functions such as activity, and protein synthesis, and metabolism. Mg2+

deficiency has been correlated with the onset and progression of several pathological conditions including , insulin resistance, metabolic syndrome and obesity.

Several studies looking at Mg2+ show that Mg2+ homeostasis is associated

with glucose metabolism and is inversely correlated with metabolism. Exposure to

Mg2+ deficient diet results in a noticeable decrease in hepatic glucose accumulation and a two- fold increase in intrahepatic content. In this study, we investigated the

effect of changes in Mg2+ concentrations inside and/or outside the cell on glucose

uptake in the absence and in the presence of insulin stimulation. Our results show that

low extracellular Mg2+ content impairs insulin stimulated glucose uptake, with no significant effects on glucose transporters expression. In the case of ,

low extracellular Mg2+ content or Mg2+ deficiency decreases the levels of SREBP-1c

precursor, an insulin dependent transcription factor, further corroborating the involvement of Mg2+ in modulating hepatic response to insulin. Mg2+ deficiency also

decreases the expression level of PPARα, a transcriptional factor involved in fatty acid

oxidation. Lastly, Mg2+ deficiency upregulates SREBP2, and PPARγ, transcription

ix factors that are both involved in increasing in liver cells. All together our data indicate that extracellular and cellular Mg2+ levels are important in modulating glucose uptake and fatty acids metabolism in liver cells.

x

1. Introduction

Magnesium (Mg2+) is a divalent cation with a relatively small size and a large

hydration shell. It is the second most abundant cation within the cell and is essential for a

number of cell functions such as the transport of calcium and potassium ions, enzyme

activities, nucleic acid and protein synthesis, energy metabolism and cell proliferation

(Flatman, 1984 and Grubbs & Maguire 1987). Intracellular regulation of Mg2+ is very

important to maintain proper cellular functions such as DNA transcription, oxidative

, and (Barbagallo et al., 2009; Bogucka et al., 1976). Mg2+

plays a critical role in the regulation of metabolism, response and cell growth in

many cell systems. Mg2+ is also a in a number of intracellular enzymatic

processes and in stabilizing membrane integrity (Laires et al., 2004). Research has found that cellular Mg2+ content is maintained below the concentration predicted by the

transmembrane electrochemical potential in skeletal, smooth, and cardiac muscle

(Flatman, 1984). This is evidence that cellular Mg2+ content is regulated by precise

control mechanism(s) at the level of influx, efflux, intracellular compartmentation and

buffering (Cole & Quamme, 2000). Although a large body of information on Mg2+

transport has been obtained from and giant cells, and despite the abundance and

importance of cellular Mg2+, limited and often inconsistent data are available in the literature to explain Mg2+ mobilization or Mg2+ intracellular compartmentation in response to external stimuli in mammalian cells. Only in the past few years have some of

the transport and regulatory mechanisms been identified, increasing their physiological

and pathological significance.

1

Magnesium Distribution

Mg2+ is the fourth most abundant cation after Na+, K+ and Ca2+ in the whole

body, and the second most abundant after K+ at the cellular level. Mg2+ plays a major role

in intracellular and biochemical functions (Cowan, 1995). Total cellular Mg2+ levels range between 14 and 20mM (Grubbs & Maguire, 1987 and Romani & Scarpa, 1992b).

These levels were confirmed by Electron Probe X-ray Micro-Analysis (EPXMA), which measures total element content (Na, K, Ca, Mg, P, S and Cl) in whole cells such as hepatocytes (Dalal et al., 1998), cells (Ziegler et al., 1992), cardiac myocytes (Shuman & Somlyo, 1987), skeletal muscles (Somlyo et al., 1985) as well as within distinct cellular . Previous experiments have shown that Mg2+ is highly

compartmentalized within the nucleus, endoplasmic reticulum, mitochondria and

. In the nucleus and endoplasmic reticulum Mg2+ accumulation is favored by its

binding to chromatin, nucleic acids, ribonucleic proteins, and (Bogucka &

Wojtczak, 1976). In mitochondria, Mg2+ predominantly forms a complex to matrix

adenine phosphonucleotides. In the inter-membraneous space the presence of two

putative Mg2+-binding proteins has been postulated (Boguka & Wojtczak, 1976).

Presently, neither protein has been successfully identified. As for the ER, little is known

about Mg2+ transport in and out of the and binding within the lumen. Whether

Mg2+ redistributes among these compartments under specific conditions is still uncertain.

Because of this binding and distribution, only a small fraction of cellular Mg2+ is free in

the lumen of these compartments or within the cytoplasm. It should also be taken into

consideration that limited techniques are available to exactly determine dynamic changes

2

in free versus bound Mg2+ within these compartments under basal conditions and following hormonal stimuli (Romani, 2008).

The cytoplasm also accommodates one of largest cellular Mg2+ pool. There is

about 5mM ATP in the and almost 90% of it is in the form of Mg*ATP complex.

Hence, somewhere between 4-5mM Mg2+ is bound to ATP with very high affinity (Kd

~33µm) (Quamme, 1997), with a free cytosolic Mg2+ concentration ([Mg2+]i) estimated to

be between 0.5 and 1 mM (Romani & Scarpa, 1992) (Fig1). Fluorimetric determinations

and nullpoint titration have determined a similar [Mg2+]i within the mitochondrial matrix

and outside the cell, minimizing the conditions for the development of a transmembrane

gradient (Raju et al., 1989 and Corkey et al., 1986). Another technique used to measure intracellular free Mg2+ utilizes Eriochrome blue in combination with nullpoint

intracellular dialysis titration (Scarpa & Brinley, 1981). This method uses multiple

sensitive wavelength pulse microspetroscopy to measure differential changes in

absorbance of the Mg2+ metallochromic indicator following its microinjection in the

cytosol of large single cells (e.g. fibers of Balanus aquila barnacle muscle in which a free

[Mg2+]i of 4.2 mmol/kg wet wt has been determined using this method) (Scarpa &

Brinley, 1981). Using fluorescent indicators, Jung et al. (1986) and Rutter et al. (1990) have calculated 0.8 and 1.2 mM [Mg2+]i in the matrix of liver and cardiac mitochondria,

respectively. Currently, no estimations of nuclear [Mg2+]i are available. As for intra-

reticular [Mg2+]i, the fluorescent dyes commercially available are not sensitive enough to

properly measure luminal Mg2+ concentration. The affinity of Mag-Fura (or Mag-Indo) for Mg2+ (Km ~1.5mM) is almost two orders of magnitude lower than for Ca2+ (Km

3

~50µM) at front of an intra-organelle Ca2+ concentration of ~3 mM (Bygrave &

Benedetti, 1996).

Despite the mentioned lack of a significant trans-membrane chemical gradient,

there is a noticeable inward electrochemical gradient for Mg2+ due to the negatively

charged inside of the plasma membrane. This results in an equilibrium potential for

intracellular free Mg2+ of ~50 mM whereas the majority of experimental evidence

indicates a free [Mg2+]i of≤ 1 mM (reviewed in Romani & Maguire, 2002). For

example, Sun et al 2009 found that Mg2+ concentration in cerebrospinal fluid (CSF) is

about 0.89 ± 0.11 mM, and it does not change significantly even if extracellular Mg2+

increases, highlighting the presence of a tight control of Mg2+ homeostasis and transport

(Sun et al., 2009).

A list of the analytical methods to measure cell or Mg2+ content and

distribution is reported in (Table 1).

1.1 Magnesium transport and hormonal regulation

The cellular Mg2+ distribution (Fig. 1), however, does not prevent the movement of Mg2+ across the . In his review Flatman (Flatman, 1991) indicated that

intracellular Mg2+ level was maintained within a narrow range in spite of a wide range of

variations in external Mg2+ concentration. This tight regulation implies the existence of a specialized Mg2+ transport system. Under resting conditions, Mg2+ slowly moves across

the cell membrane with a turnover of several hours that varies from cell type to cell type

(reviewed in Romani & Maguire, 2002). However, significant changes in both total

cellular Mg2+ content and the amplitude of Mg2+ fluxes across the plasma membrane have

4

been observed following varying hormonal and non-hormonal stimuli (Romani & Scarpa,

2000), suggesting the operation of well regulated efflux and influx mechanisms in

maintaining Mg2+ homeostasis in mammalian cell types (Figure 2).

During the past two decades experimental evidence has shown that under

physiological conditions mammalian cells have a tight control on Mg2+ transport across

the plasma membrane. Mg2+ can be extruded from the cell via two separate mechanisms,

identified as Na+- dependent and Na+- independent transporters, respectively. The Na+-

dependent transporter is also referred to as a Na+/Mg2+ exchanger, and it is activated by cAMP (Cefaratti et al., 2004 and Romani et al., 1993). This transporter has been found to be present in a large number of tissues including: hepatocytes (Gunther et al., 1991,

Romani et al., 1993, Gunther & Hollriegl, 1993, Tessman & Romani, 1998, and Fagan &

Romani, 2000), erythrocytes (Gunther & Vormann, 1985), cardiac cells (Vorman &

Gunther, 1987, Murphy et al., 1991, Romani et al., 1993, and Handy et al., 1996),

smooth muscle cells (Tashiro & Konishi, 1997), and lymphocytes (Wolf et al., 1997).

Mg2+ extrusion through this exchanger was first observed in rat erythrocytes in which it

was stimulated by increasing extracellular Na+ concentrations (Gunther et al., 1990). This

mechanism can also operate in the reverse direction at least in liver plasma membrane

vesicles, favoring Mg2+ accumulation in exchange for Na+ extrusion (Cefaratti et al.,

2000). This transporter appears to be the main extrusion pathway activated by epinephrine, or in liver cells (Romani & Scarpa, 1990a and

Fagan & Romani, 2000). The Na+-independent pathway, instead, is poorly characterized, in that it exchanges cellular Mg2+ for extracellular cations such as Mn2+ (Feray & Garay,

1987) or Ca2+ (Cefaratti et al., 1998, Romani et al., 1993a), or anions such as Cl-

5

(Gunther, 1993). Table 2 reports a list of the Na+-independent Mg2+ extrusion

mechanisms and tissues in which the operation of the transporters has been observed.

While neither the Na+- dependent nor the Na+- independent transporters have been cloned, several Mg2+ entry mechanisms have been identified. Among these mechanisms

are TRPM6 and TRPM7. These are two members of the melastatin subfamily of the

transient receptor potential (TRP) family. Both channels share a high degree of homology

and posses an α-kinase domain at their C terminus; hence, the term of chanzyme

(Schlingmann et al., 2007). TRPM6, also known as Chak2, was the first channel to be

characterized. This Mg2+ and Ca2+ permeable channel is specifically expressed in the

intestine and kidneys, and mutations in its sequence result in hypomagnesaemia with secondary hypocalcaemia (HSH), and a number of neurological complications including seizures, whose severity and frequency can be reduced by Mg2+ supplementation.

Altogether, these results highlight the importance of TRPM6 for Mg2+ absorption in the

intestine (Hofmann et al., 2010, Schlingmann et al., 2007, and Teramoto et al., 2010).

TRPM7, also known as ChaK1, TRP-PLIK, and LTRPC7, is ubiquitous and it has been shown to be very important for cellular Mg2+ homeostasis as any target deletion or mutation in this channel leads to growth arrest and intracellular Mg2+ deficiency (Schmitz et al., 2003, Schlingmann et al., 2007). Data indicate that this channel has an intracellular ligand gated domain that is involved in the transport of Mg2+ and is sensitive to Mg*ATP

levels (Nadler et al., 2001). Loss of channel activity or expression influences cellular

energy and Mg2+ homeostasis (Schmitz et al., 2003, Schlingmann et al., 2007). Studies by

Fleig’s group (Nadler et al., 2001) and Hofmann et al. (2010) have shown that TRPM channels are essential to control extra- and intra-cellular Mg2+ levels in various cell types

6

(Walder et al., 2002, and Schlingmann et al., 2007). It is unclear, however, how the other

Mg2+ entry mechanisms are regulated. Another mechanism of Mg2+ transport is

represented by a novel family of membrane Mg2+ transporters (MMgT1 and MMgT2)

(Goytain et al., 2008). Experimental data have shown that these transporters are present

in a wide variety of cells. Moreover, immunohistochemistry experiments have found that

these proteins reside in the Golgi complex and post Golgi vesicles. Therefore, both these

transporters can be used for Mg2+ transport and regulation in those organelles (Goytain et

al., 2008). Other Mg2+ entry mechanisms include SLC41-A1 and A2 carriers. The modus operandi of these mechanisms has been reviewed in detail by Schmitz et al. (2007).

The rate of Mg2+ transport through these entry and exit mechanisms and the overall Mg2+ homeostasis appear to be regulated by a variety of , but the specific details have not yet been finalized.

1.2.1 Mg2+ Accumulation

Some of the hormones that regulate Mg2+ uptake include acetylcholine, insulin

and vasopressin. In the case of acetylcholine and vasopressin, the hormones decrease

cellular cAMP level and induce a marked accumulation of Mg2+ in liver cells (Romani et

al., 1992), cardiac myocytes (Romani & Scarpa, 1990b; Romani et al., 1992 and Romani

et al., 2000), and (Takaya et al., 1998). Henquin et al. (1983 & 1986), also

found that in β-cells, substances that stimulate insulin release also stimulate Mg2+ uptake.

In this model, glucose also induces a dose dependent Mg2+ uptake. Agents that activate

C (PKC) such as diacylglycerol (Castagna et al., 1982), derivates of

phorbol 12-myristate 13- (PMA) (Romani et al., 1992), or hormones (e.g.

7

vasopressin) (Romani et al., 1991) also increase cellular Mg2+ content in total terms as

well as within specific compartments. For instance, Bond et al. (1987) found a significant rise in mitochondrial Mg2+ upon stimulation with vasopressin. Accumulation

of Mg2+ via PKC activation is further validated by the observation that Mg2+

accumulation does not occur when PKC signaling is impaired following exposure of cells

to a supramaximal dose of phorbol myristate acetate (Romani et al., 1992), or treatment

with calphostin C (Touyz & Schiffrin, 1996).

1.2.2 Mg2+ Extrusion

As mentioned previously, while mammalian cells under resting conditions move

relatively small amounts of Mg2+ across their biological membranes, large amounts of

Mg2+ are extruded across the plasma membrane and from intracellular stores following

hormonal stimulation (Romani et al., 1991, 1992, 1993b and 2000; Romani & Scarpa,

1990a and 1992a). A number of groups have reported that perfused and hearts release a considerable amount of cellular Mg2+ into the perfusate within a few minutes

from the administration of α- or β-adrenergic agonists, in a dose-dependent fashion

(Gunther et al., 1991, Jakob et al., 1989, Romani & Scarpa, 1990a and 1990b). Other

2+ investigators have confirmed the occurrence Mg extrusion in response to α1- or β-

adrenergic stimulation in several other tissues or cell types (reviewed in Romani &

Scarpa, 2000), including erythrocytes (Matsuura et al., 1993), thymocytes (Gunther &

Vormann, 1992), and primary lymphocytes (Wolf et al., 1997).

Data in intact tissues (Romani & Scarpa, 1990; Romani et al., 1991) as well as in

cells (Romani & Scarpa, 1990 and Romani et al., 1991) and plasma membranes

8

(Cefaratti et al., 2000) have demonstrated a correlation between Mg2+ transport and

cAMP. In heart and liver cells, Mg2+ extrusion is the result of an increase in intracellular

cAMP induced by norepinephrine through β- adrenergic stimulation, forskolin or cAMP

analogues (Romani, 2007). Additionally, , E1 (PGE1), and

PGE2, which also increase cellular cAMP, can induce Mg2+ extrusion from primary

lymphocytes or S49 limphoma cells or Erhlich ascites cells (Wolf et al., 1996 & 1997,

and Henquin et al., 1986) On the other hand, the administration of Rp-cyclic AMP isomer, a stable inhibitor of adenylyl cyclase, fully prevents Mg2+ extrusion (Wolf et al.,

1997). The pretreatment of perfused heart (Romani et al., 2000) or liver (Keenan et al.,

1996) with insulin, which inhibits cAMP production, prevents the Mg2+ extrusion elicited

by isoproterenol or cell permeant cAMP analogs while having no effect on the Mg2+

extrusion elicited by the α1-adrenergic agonist phenylephrine (Keenan et al., 1996). In

summary, these results indicate that cAMP acts as second messenger to activate the Mg2+

extrusion pathway (Gunther & Vormann, 1992), possibly via phosphorylation of the

putative Na+/ Mg2+ exchanger (Gunther & Vormann, 1992).

2+ Mg extrusion can also be elicited through stimulation of α1-adrenergic receptors,

in a cAMP independent manner (Keenan et al., 1996, Jakob et al., 1989). Fagan and

Romani (2001) have investigated in detail the α1- signaling pathway in perfused livers and isolated hepatocytes. They observed that the α1-adrenergic receptor mediated Mg2+ extrusion through activation of phospholipase C (PLC) that cleaves

phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol tris-phosphate (IP3), which

leads to a mobilization of reticular Ca2+ and an increase in cytosolic [Ca2+]. In addition,

these authors have shown that Mg2+ extrusion from the liver following catecholamine

9

administration (3.4 µmol/8 min) correspond to the amounts mobilized by β- (1.1 µmol/8

min) and α1-adrenergic agonist (2.2 µmol/8 min) together. Similar findings were

retrospectively validated from the data published by Keenan et al. (1996), and suggest the

presence of two distinct cellular pools from which α1- and β-adrenergic receptor signaling

pathways can mobilize Mg2+ (Fagan & Romani, 2000). Data from Fagan and Romani

(2000) strongly support that β-adrenergic signaling specifically activates the Na+ -

dependant Mg2+ extrusion pathway. Taken together the available information, indicates

that the Na+/Mg2+ exchanger is the primary pathway involved in Mg2+ extrusion, and that

it can be activated independently or simultaneously by cAMP (via β-adrenergic

2+ receptors) or Ca /calmodulin (via α1-adrenergic receptors) (Fagan & Romani, 2000 and

Keenan et al., 1996). It has to be kept in mind however that hormones have different

effects depending on the tissue. For example, epinephrine and norepinephrine stimulate

Mg2+ uptake in but decrease Mg2+ uptake and/or induced a Mg2+ extrusion in

cardiac or liver cells (Romani, 2007).

1. 3 Magnesium and cell metabolism

Mg2+ is important for many cellular functions. Many studies have focused on how

Mg2+ is involved in regulating different metabolic processes within the body. Several of

these studies have reported that Mg2+ homeostasis has a direct relationship with glucose transport and utilization, and an inverse relationship with lipid metabolism (Rayssiguier et al., 1981, and Paolisso et al., 1990).

10

1.3.1 Magnesium versus Glucose

Glucose is a key metabolic in mammalian cells. It functions as a

precursor for the synthesis of , , and and provides an

important energy source by generating ATP through glycolysis (Olson & Pessin, 1996).

Glucose is obtained from the diet and by synthesis from other substrates

() in organs like liver and kidney. Because glucose is a polar hydrophilic , it does not readily diffuse across the hydrophobic plasma membrane.

Therefore, specific mechanisms exist to facilitate the specific uptake of this sugar across the plasma membranes (Olson & Pessin, 1996).

The glucose transporters can be divided into two structurally and functionally distinct groups: (1) the Na+-dependent glucose co-transporter (SGLT), member of a large

family of Na-dependent transporters), and (2) the facilitative Na+-independent sugar

transporters (GLUT family) (Joost & Thorens, 2001). The Na+-dependent glucose co-

transporters is an energy-dependent transporter (Coady et al., 1990, and Olson & Pessin,

1996) operating in the intestine and in the renal epithelium to absorb dietary glucose or

reabsorb glomerulus filtered glucose, respectively (Dohm et al., 1991).

On the other hand, the facilitative Na+-independent glucose transport across the

cell membrane of other tissues is mediated by a family of 13 different transport proteins

(GLUT) (Wood & Trayhurn, 2003). These facilitative glucose transporters are a highly related group of integral membrane proteins with significant sequence similarities (Olson

& Pessin, 1996). These transporters are characterized by a high degree of selectivity, and

provide a bidirectional transport of glucose, passive diffusion solely occurring down its

concentration gradient. These transporters regulate the movement of glucose between the

11

extracellular and intracellular spaces within the body, thereby maintaining a relatively

constant supply of circulating glucose to cells for metabolic purposes.

In adult hepatocytes, GLUT2 isoform accounts for 90% of the glucose transporter

present in the cell membrane. GLUT2 is a 524- protein that is located on the

basolateral domain of liver cells (Wood & Trayhurn, 2003). GLUT2 is also responsible

for glucose transport in kidney cells, pancreatic β-cells, and, at least in part, for the

movement of glucose into absorptive epithelial cells of the jejunum. GLUT2 is

distinguished from the other GLUT isoforms kinetically, based upon its low-affinity and high turnover rate (Olson & Pessin, 1996).

Aside from GLUT2, hepatocytes also contain a limited amount of GLUT1 isoform in their cell membrane (<10% of total hepatic GLUT transporters). This transporter is specifically highly expressed in all fetal tissues (Shepherd et al., 1993). In adult tissues, GLUTl is widely expressed, and it is most abundant in erythrocytes, endothelial cells, and fibroblasts with low levels of expression in muscle, , and liver (Olson & Pessin, 1996). Zheng et al. (1995) found that the expression of this transporter increases slightly after fasting or insulin deprivation. For a complete list of the glucose transporters expressed in the liver see Table 3. The insulin-sensitive GLUT4 transporter is not expressed in hepatocytes (Bell et al., 1990), and insulin indirectly modulates hepatic glucose transport by up-regulating activity.

Mg2+ plays an important role in glycolysis by affecting key such as

glucokinase and glucose 6 phosphatase. Overnight fasting also results in the depletion of

hepatic glycogen and a 10-12% decrease in total hepatic Mg2+ content (Torres et al.,

2005). Several studies suggest a correlation between glucose transport and Mg2+ in

12

and out of the cell after hormonal stimulation in hepatocytes (Paolisso et al., 1990, and

Henquin et al., 1983), β-pancreatic islets (Henquin et al., 1986), and cardiac cells (Eckel

et al., 1983 and Romani et al., 2000). Yet, despite this evidence, the modality by which

these moieties affect each other is still unclear. For example, Laughlin and Thompson

(1996) studied the effect of [Mg2+]i on glucose utilization in human erythrocytes under conditions in which [Mg2+]i was changed between 0.01 and 1.2 mM using the divalent

ionophore A23187. These authors determined that glucose utilization strongly depends

on [Mg2+]i while the concentration of phosphorylated glycolytic intermediates depends

on [Mg2+]i and [Mg*ATP] (Laughlin & Thompson, 1996). In cardiac cells, instead,

insulin stimulates the parallel accumulation of glucose and Mg2+ into the myocytes

(Romani et al., 2000). Eckel et al. (1983) proposed an involvement of Mg2+ in insulin

signaling due to the observation that EDTA addition completely abolished insulin stimulated glucose entry in cardiac myocytes treated with A23187 (Eckel et al., 1983).

Evidence has shown that Mg2+ deficient animals present a reduction in both

autophosphorylation of insulin receptors and phosphorylation of insulin receptor kinases

(Suarez et al., 1995) as well as a significant change in glycemia and glucose utilization

(Kimura et al., 1996). Furthermore, the administration of glucagon or catecholamine to hepatocytes leads to a parallel mobilization of Mg2+ and glucose from inside the cell to the extracellular compartment. In this experimental design, the presence of glucose transport inhibitors decreased Mg2+ extrusion whereas inhibition of Mg2+ extrusion blocked glucose output (Fagan & Romani, 2000). Similar findings on the effect of extracellular glucose on Mg2+ transport in β-pancreatic islets have been reported by

Henquin and collaborators (Henquin et al., 1983). Altogether, these observations

13

strongly support a role of Mg2+ in modulating insulin response and cellular glucose

utilization.

1.3.1.1 Magnesium and Diabetes Mellitus

Several pathological conditions are characterized by a decreased level of Mg2+

within the cell or the plasma. Among the clinical conditions associated with Mg2+

depletion, the most well known are: prolonged fasting, surgical stress, hypertension,

acute alcoholism, cirrhosis, and diabetes mellitus (see Djurhuus et al., 2001; Kimura et al., 1996; Paolisso & Barbagallo, 1997; Paolisso et al., 1990; White & Campbell, 1993

for reviews)

Diabetes Mellitus (DM) is a metabolic disease that results from defects in the

insulin action and/or the insulin secretion, and is characterized by hyperglycemia.

According to the American Diabetes Association (ADA), this disease is classified into

four classes: 1) Type 1 DM characterized by destruction of β-cells, leading to insulin

deficiency; 2) Type 2 DM, spanning between insulin resistance with relative insulin deficiency and insulin secretory defect with insulin resistance; 3) Gestational DM, the

most common clinical condition affecting pregnancy, characterized by glucose

intolerance, and 4) Other specific types of diabetes such as particular genetic defects of

insulin action or pancreatic β-cells, iatrogenic, exocrine diseases of the pancreas, infectious, uncommon forms of autoimmune diabetes, and other genetic syndromes (e.g.

Down syndrome) (Sales et al., 2006).

14

According to the National Diabetes Statistics, 2011, there are about 18.8 million

Americans currently diagnosed with diabetes with the diagnosis of approximately

800,000 new cases each year. Diabetes represents the sixth leading death related disease

in the United States. Taking to mind the magnitude of the problem and the impact it has

on patients’ health and economic status, there is an urgent need to pinpoint the factors

contributing to diabetes and its severity, and an increasing demand for new potential

therapeutic drugs with new targets.

Hypomagnesemia occurs at an incidence of 13.5 to 47.7% among patients with

type 2 diabetes. Kimura et al., (1996) has shown that fasted and fed blood glucose levels

are decreased in Mg2+-deficient rats as well as the response of blood glucose to sucrose loading. This finding would suggest that Mg2+ deficiency enforces changes on glucose metabolism by impairing glucose absorption in the intestine or by changing glucose uptake in the liver. In addition, a decreased phosphorylation of insulin receptor and downstream signaling has been observed in Mg2+ deficient rats (Suarez et al.,

1995). Ferreira et al. (2004) has reported that Mg2+ transport, via Na+/Mg2+ is

linked to insulin receptor activation. Altogether these results may provide a new rationale

for decreased insulin effectiveness under diabetic conditions.

In the presence of diabetes, it is observed that inadequate metabolic control can

affect the concentrations of Mg2+, developing hypomagnesaemia, which may be still

directly related with some micro- and macrovascular complications observed in diabetes,

as retinopathy, cardiovascular disease, and neuropathy (Sales et al., 2006). Therefore,

supplementation with Mg2+ has been suggested in patients with diabetes mellitus who present hypomagnesaemia and exhibit diabetic complications (Sales et al., 2006).

15

Epidemiological studies had shown a relation between the ingestion of food rich in Mg2+

(such as dairy products, whole grains, most of the green leafy vegetables, nuts, seeds, poultries, meats, dry beans, fishes, peas, lentils, soy and natural ) and the attenuation of diabetes progression and its complications (Sales et al., 2006). A cohort study of postmenopausal women in Iowa and The Framingham Offspring Study have indicated that an increased intake of whole grains and other food sources of Mg2+

significantly reduces the relative risk of diabetes (Sales et al., 2006). Other studies carried out in women and men have also verified an inverse association between the risk of type

2 DM and Mg2+ intake. Barbagallo et al. (2003) demonstrated that Mg2+ supplementation

improved the circulating glucose levels and the oxidation of tissue glucose in patients

with type 2 DM, in addition to favoring the action of peripheral insulin.

Experimental and clinical evidence indicates that a loss of tissue and plasma Mg2+

is observed under both type 1 and type 2 DM (Resnick et al., 1993; Wallach and Verch,

1987). As shown by Fagan et al. (2004), rats rendered diabetic by streptozotocin

injection presented 10% and 20% decreases in total hepatic Mg2+ content at 4 and 8

weeks, respectively, following diabetes onset. In addition, these authors have shown that diabetic hepatocytes did not accumulate Mg2+ following stimulation of protein kinase C

pathway by diacylglycerol analogs, vasopressin, or phorbol 12-myristate 13-acetate

derivates despite the reduced basal content in cellular Mg2+ (Fagan et al., 2004). A defective Mg2+ accumulation has been confirmed by Cefaratti et al. (2004) in diabetic

liver plasma membrane vesicles. While a defective Mg2+ accumulation can explain, at

least in part, the decrease in tissue Mg2+ level observed under diabetic conditions, the

modality by which this defect occurs is largely unclear. Because Mg2+ accumulation is

16

influenced directly or indirectly via PKC signaling (Csermely et al., 1987 and Romani et

al., 1992), one possible explanation would be that under diabetic conditions the signaling

pathway is altered. Tang et al. (1993) have investigated the effect of streptozotocin- induced diabetes on PKC signaling in liver cells and found alterations in the expression and the cytosol/membrane partitioning of 5 PKC isoforms, which were restored by insulin therapy (Tang et al., 1993). However, it remains unclear whether this restoration is associated with Mg2+ reaccumulation, and to what extent a decrease in tissue Mg2+

content contributes to the short- and long-term complications of diabetes.

1.3.2 Magnesium versus Lipid

Lipids are hydrophobic or amphiphilic small molecules. This biochemical family includes fatty acids and their derivatives (mono-, di-, and tri-, and phospholipids), as well as sterol-containing such as cholesterol. are very important as structural components of cell membranes and for a variety of biological functions that encompass energy storage and cellular signaling. Due to their amphiphilic nature, some lipids can form structures such as liposomes, vesicles, or membranes in an

aqueous environment (Fahy et al., 2005). In a recent review Vallim and Salter (2010)

suggest that saturated fatty acids modulate lipid metabolism within liver cells thereby

impacting lipids synthesis, storage and secretion. These effects are mediated, at least in part, through modulating the activity of a number of transcriptional factors, including peroxisome proliferator activated receptors (PPARs), and sterol regulatory element binding proteins (SREBP) family (Vallim & Salter, 2010).

17

PPARs are members of the nuclear fatty acid receptor superfamily of transcription factors (Vallim & Salter, 2010). These transcriptional factors have been implicated in playing crucial roles in a number of metabolic disorders such as insulin resistance, diabetes, and (Terauchi & Kadowaki, 2005). There are three subtypes:

PPARα, PPARβ (also known as δ), and PPARγ, with PPARα being the predominant isoform in liver. Activation of PPARα is associated with fatty acid in the liver, through the up-regulation of a variety of genes associated with fatty acid oxidation and lipoprotein metabolism, such as; , apolipoproteins AI, acyl- oxidase, and carnitine palmitoyl (Vallim and Salter, 2010)

PPARγ is essential for differentiation and hypertrophy, and mediates the activity of the insulin sensitizing thiazolidinediones. PPARγ regulates fatty acid storage and glucose metabolism. The genes activated by PPARγ stimulate lipid uptake and adipogenesis by cells. PPARγ knockout mice fail to generate adipose tissue when fed a high fat diet (Kamon et al., 2003). PPARβ may be important in regulating body weight and lipid metabolism in fat tissues. Thus, revealing the affect of Mg2+ deficiency on these

PPARs will provide insights into the pathogenesis of metabolic diseases and offer valuable information for potential drug (Terauchi & Kadowaki , 2005).

SREBPs are membrane-bound proteins that are part of the basic helix-loop-helix zipper (bHLHLZ) family of transcription factors. SREBP has three isoforms

(SREBP1a, 1c and SREBP2), with SREBP2 as the predominant isoforms in the liver

(Takahashi et al., 2005 and Vallim & Salter, 2010). SREBP-1a activates both fatty acid and cholesterol biosynthetic pathways. SREBP-1c regulates adipocytes differentiation and fatty acid synthesis, and is considered an essential transcription factor for the

18

genomic action of insulin on both lipid metabolism and . SREBP2 is more

specific for cholesterol (Dong et al., 2010). Each of the SREBP proteins is

synthesized in an immature (inactive) form on the endoplasmic reticulum (ER)

membrane where it forms a complex with SREBP-cleavage activating protein (SCAP)

immediately after their synthesis. Through the action of SCAP, the SREBP proteins move

via coatomer II protein (COPII) vesicles to the Golgi complex where they become

activated by a two-step proteolysis process involving S1P and S2P proteases (Dong et al.,

2010). The transcriptionally active SREBP, also known as mature SREBP, is then

translocated to the nucleus where it binds to sterol regulatory elements (SREs) and plays

a major role in up-regulating the expression of genes associated with lipid and lipoprotein

metabolism (Vallim & Salter, 2010). Studies have determined that the SCAP/SREBP complex remains localized in the ER through the action of two proteins, insulin- induced gene (Insig-1 and Insig-2), which cooperate with sterols to inhibit the exit of the

SCAP/SREBP complex from the ER (Dong et al., 2010 and Vallimand & Salter, 2010).

SREBPs have been involved in the development a number of diseases such as obesity, type 2 diabetes, dyslipidemia, atherosclerosis, global syndrome X, and lipodystrophy (Eberlé et al., 2004) all conditions associated with Mg2+ deficiency to a varying extent. Experimental evidence suggests that there may be some differences in the sensitivity of certain tissues to the effect of insulin resistance on SREBP-1 expression.

The mechanism(s) behind these differential sensitivity, however, is not understood. For example, in adipose tissues of obese patients insulin resistance was accompanied by a decrease expression of SREBP-1c (Oberkofler et al., 2002). On the other hand, SREBP-

1c mRNA expression is decreased in muscles of type 2 diabetic patients but not in those

19

of obese patients (Sewter et al., 2002). A significant percentage of individuals and

animals with obesity and insulin resistance also present fatty livers. SREBP-1c levels are

elevated in the fatty livers of obese, insulin-resistant and hyperinsulinemic ob/ob mice

(Eberlé et al., 2004). Shimomura et al. (1999) have suggested that increased levels of nuclear SREBP-1c in liver cells is associated with an elevated rate of hepatic fatty acid synthesis, which causes steatosis in diabetic mice (Shimomura et al., 1999).

Overexpression of the mature form of SREBP-1a in murine adipocytes using the adipocytes specific aP2 promoter (aP2-nSREBP-1a) led to a significant increase in the rate of fatty acid synthesis and fatty acid secretion, promoting the development of a fatty liver (Shimomura et al., 1998). Nevertheless, the regulation of SREBP isoforms activity still remains vague.

Magnesium ions (Mg2+) are an important co-factor in lipid metabolism.

Magnesium regulates the activity of lecithin cholesterol acyltransferase (LCAT) and

lipoprotein lipase (LPL), which lower triglyceride levels and raise HDL-cholesterol levels (Inoue, 2005, and Randell et al., 2008). The complex Mg*ATP also acts as a regulatory factor on 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), the rate-limiting enzyme in cholesterol biosynthesis (Inoue, 2005). It has been suggested that low Mg2+ may impair HMG-CoA reductase inactivation via phosphorylation.

Conversely, high Mg2+ is required for the reactivation of HMG-CoA reductase (Randell et al., 2008).

Previous studies have found an inverse relationship between lipid metabolism and

Mg2+. Underscoring the significance of Mg2+ supplementation, magnesium-deficient animals present an increased synthesis of triglycerides in the liver, which elicits

20

, elevated levels of free cholesterol and decreased level of esterified

cholesterol (Rayssiguier et al., 1981). Following the initial observation, many studies

have correlated Mg2+ deficiency with an increase incidence in insulin resistance, obesity

and hypertension.

Decreased levels of Mg2+ in the diet also lead to an increase in plasma levels of triacylglycerol-rich lipoprotein (TGRLP) and its susceptibility to peroxidation (Gueux et al., 1995), as well as an increase in lipid peroxidation and net deposition of collagen in the myocardium (Kumar et al., 1997). Mg2+ deficiency can intensify and accelerate lipid

deposition and lesions coronary artery in animals. In Mg2+ deficient rats, there was an

increase in the concentrations of , VLDL, and LDL, while the concentration of HDL was lowered (Lynking, 1999).

Rayssiguier et al. (1981) have reported that Mg2+ deficiency significantly

increases triglycerides, α-glycerophosphate and lactate levels while decreasing hepatic

glycogen content. Moreover, Mg2+ deficiency increases the circulating levels of free

cholesterol and triglyceride levels and decreases esterified cholesterol levels in weaning

rats fed a high carbohydrate (sucrose) diet (Rayssiguier et al., 1981 and Gueux et al.,

1991). Previous studies aimed at determining the effects of Mg2+ deficiency on hepatic

apolipoprotein and plasma lipoproteins levels in rats revealed an increase

in TGRLP associated with a significant increase in plasma apolipoprotein B (apo B) and

apo C concentration for VLDL (Gueux et al., 1991). These increases were accompanied by a decrease in high-density lipoproteins (HDL) and a corresponding decrease in plasma apo E as well as hepatic apo E mRNA abundance and biosynthesis. All together, these data suggest a defect in the catabolism rather than the secretion of TGLRP as the major

21

factor underlying the altered plasma lipoprotein profile (Nassir et al., 1995). Conversely,

a study by Randell et al., (2008) has found a positive relationship between lipoprotein

and serum Mg2+ level in a healthy population. Despite these results, the relationship between Mg2+ and lipid metabolism is far from clear and needs further investigation

(Randell et al., 2008).

1.3.2.1 Magnesium and obesity

Obesity is a chronic disease with a high prevalence. In the last 20 years, more than 500 million people have been identified as overweight and 250 million as obese

adults world-wide. Due to such a high prevalence, obesity has become a major health and

economic problem in most industrialized countries. In the United States, in which

approximately 61% of the adult population is overweight or obese, obesity is responsible

for claiming the life of approximately 300,000 people a year, and more than $100 billion

per year are spent as direct and indirect health costs associated with obesity (Samuel et

al., 2002). Data obtained from national population surveys [NHES I] have demonstrated

that the prevalence of obesity has more than doubled since 1960 (Klein et al., 2002), and

it is now increasingly present in children and adolescents. Data from NHANES show an

increase in obesity among preschool children aged 2–5, from 5.0% to 10.4% between

1976–1980 and 2007–2008 and from 5.0% to 18.1% among adolescents aged 12–19

during the same period (Ogden & Caroll, 2010). Obesity-related diseases such as type 2

diabetes mellitus, hyperlipidemia, hypertension, sleep apnea, gallbladder disease,

orthopedic complications, and nonalcoholic steatohepatitis (NASH), which are typically

22

seen in adults are now seen with increasing frequency in children as well (Samuel et al.,

2002).

Diets rich in saturated fatty acids and deficient in Mg2+ have been associated with

elevated plasma cholesterol concentrations and therefore, increased risk of cardiovascular

disease and non-alcoholic . A study by Resnick et al. (1993) proposed a

decrease in dietary Mg2+ as the cause of an abnormal increase in Na+ and Ca+ content within cells. Depending on the cell type this increase in Na+ and Ca+ would be

responsible for the deregulation of insulin release and metabolic changes leading to

insulin resistance and obesity (Resnick, 1993). Serum Mg2+ level has been observed to be

significantly decreased in obese children compared to lean children (obese: 0.748 ± 0.015

vs. lean: 0.801 ± 0.012 mmol/l; P = 0.009) (Huerta et al., 2005) with no significant

changes in serum calcium and potassium levels. magnesium supplementation may be an

important tool in the prevention of type 2 diabetes onset in obese children (Milagros et

al., 2005). Altogether, these data suggests that Mg2+ deficiency is an important factor in

the complications of obesity; and its homeostasis and implications need to be further

investigated.

23

2. Research Objective and Specific Aims

The overall goal of this study was to define the role of Mg2+ in regulating both glucose and lipid metabolism. We hypothesize that a decrease in cellular Mg2+ impairs

glucose uptake in hepatocytes, and as a consequence the hepatocytes will become more

dependent on lipid metabolism for energetic purposes. If supported by experimental data,

our hypothesis would further emphasize the role of Mg2+ as a key regulator of enzymes

and cell functions under physiological conditions. The obtained knowledge will then be

applied to pathological states (e.g. diabetes and obesity) that are characterized by a

reduced cellular Mg2+ content.

AIMS

Aim 1 - To understand the interactions existing between glucose uptake and/or utilization

and changes in Mg2+ concentration, the present study was aimed to determine the relationship between changes in cellular Mg2+ and glucose uptake. In particular, we

investigated the effect of different Mg2+ concentrations inside and/or outside the cell on glucose uptake with and without insulin.

Aim 2- Low Mg2+ diet and low extracellular Mg2+ concentration are associated with

elevated levels of triglycerides and free cholesterol in the liver; metabolic processes

regulated by SREBP-1c and SREBP2. Hence, this study was aimed to determine the how

a decrease in cellular Mg2+ modulates SREBP-1c and SREBP2 activity, and regulates fatty acid metabolism.

24

3. Materials and Methods

HepG2 cells were used to investigate how changes in Mg2+ concentrations inside

and/or outside the liver cell would affect glucose uptake with and without insulin. The

cells were cultured in the presence of 0.4mM (low) and 0.8mM (normal) extracellular

Mg2+ concentration ([Mg2+]o). The cells were used in adhesion and in suspension as needed. Glucose uptake was studied using 14C-glucose to determine the levels of glucose

accumulated in HepG2 cells grown in the presence of different [Mg2+]o. Total cellular

Mg2+ content and compartmentalization was assessed by atomic absorbance

spectrophotometry (AAS). Cellular ATP content was measured by luciferin-luciferase assay. Changes in Glut1, Glut2, SREBP1c, SREBP2, PPARα and PPARγ expression

were assessed by western blot analysis and qPCR, respectively.

3.1 Cell culture: HepG2 (human hepatoma cells) were grown in MEM media (sigma M-

2279) with 10% bovine calf serum, 1% pen-strep, 1% L-glutamine and 0.5% geneticin.

When cells were confluent, the media was removed and the cells were washed with 10ml

phosphate buffered saline (PBS) for 30 seconds. The PBS was then removed and replaced

with 4ml of trypsin. Once the cells began to lift off of the flask, they were removed, and

resuspended in media with different [Mg2+]o (i.e. 0.4mM and 0.8 mM Mg2+). The cells were maintained in an incubator (37°C and 5% CO2) until they were confluent (usually within 5-8 days).

25

3.2 Animal model: C57/B6 male mice (25 g body weight) and male Sprague-Dowley rats

(250 g body weight) were randomly divided into two groups, which received regular

Mg2+ diet (507 mg of Mg2+ per Kg of pellet food) or Mg2+ deficient diet (380 mg of Mg2+

per Kg of pellet food) for two, four, and 8 weeks. Controls (regular diet) and Mg2+- deficient animals were sacrificed the same day by i.p. injection of saturated pentobarbital solution (60 mg/kg bw - rats) or ketamine/xylazine (80 mg/kg/10 mg/kg, respectively –

mice). Following attainment of deep anesthesia, the abdomen was opened and the liver explanted. A fragment of the tissue (~100 mg) was used for mRNA preparation.

Another fragment (~100 mg) was homogenized (10% final concentration, w/v) in 10%

2+ HNO3 for assessment of Mg content by AAS as previously reported (Tessman &

Romani, 1998).

3.3 Cellular Mg2+ Compartmentation: To determine the cellular Mg2+ profile, cells

maintained in both 0.4 mM and 0.8 mM [Mg2+]o were trypsinized as reported previously.

The cells were then transferred into a 15ml centrifuge tube and sedimented at 1000 g for

5 min. The cell pellets was resuspended in 5 ml Mg2+ free buffer and incubated at 37°C.

After cell addition to the incubation buffer, a 500 μL aliquot was withdrawn for protein

concentration. Another 500 μL aliquot was withdrawn at 0 time and sedimented in

microfuge tubes (5000 g x 2 minutes) to determine basal total cellular (pellet) and

extracellular (supernatant) Mg2+ content. Then, 50 μg/mL digitonin were added to the

incubation system followed by 2 μg/mL (FCCP or carbonyl-cyanide-p-trifluoromethoxy-

phenylhydrazone) and 1 μg/mL A23187 at 5 minute intervals. Aliquots of 500 μL of the

incubation mixture were withdrawn prior to the addition of each agent, and sedimented

26 by centrifugation (5000 g x 2 minutes). The supernatants were removed and stored for

2+ analysis. The pellets were digested overnight in 10% HNO3 to determine residual Mg content. Both the supernatants and the pellets were assessed for Mg2+ content by atomic absorbance spectrophotometry (AAS) in a Perkin-Elmer 3100 calibrated with appropriate standards (Romani et al. 1993a).

3.4 Biorad protein assay: Protein content was measured with bovine serum as standard using Bradford protein assay (Bradford, 1976) using an Agilent 8453 spectrophotometer.

3.5 ATP luciferin luciferase assay: To determine the effect of Mg2+ deficiency on cellular ATP levels, cells grown in 0.4mM and 0.8mM Mg2+ were washed with PBS and extracted on ice for 10 minutes with 10% PCA (2ml/100ml dish). The acid mixture was then scraped, sedimented in microfuge tubes. The supernatant was neutralized with 1.75

M KHCO3 (3ml for each 2ml of 10% PCA) and measured for ATP content. The pellet was washed with then digested in 1x NaOH for protein determination. To measure ATP content, 21 µl basal saline solution (BSS) and 4 ul FLAAM were added to

75 µl sample. ATP content was then quantified using a Turner Designs (TD 20/20) luminometer calibrated with known amounts (0.1–10 pmol) of standard ATP.

Luminescence (as relative light units, or RLU) was integrated over a 5-s photon counting, and normalized for protein content.

27

3.6 Radio Isotope flux assay: We used radio-labeled 14C-glucose to determine the levels of glucose accumulated in HepG2 cells grown in the presence of different extracellular

Mg2+ concentrations (0.4 mM or 0.8 mM Mg2+). For each concentration, 2 different experimental conditions were tested on cells in suspension: 1) 0.1mM glucose with

insulin 2) 0.1mM glucose without insulin (6nmols/L). The indicated glucose concentration containing 0.5µCi/ml 14C-glucose as tracer was added to the incubation

mixture after withdrawal of t= 0 min (500 µl), and the incubation continued for 20 min.

Additional aliquots of 500 µL were withdrawn at t= 1 min, 2 min, 5min, 10min and

20min. The samples were diluted 10 fold in 250mM ‘ice-cold’ sucrose containing

100µM phloridzin (as a GLUT2 inhibitor), and filtered onto a Whatman glass filter under vacuum. The filters were air-dried overnight. The next day, scintillation cocktail (Fisher

Scientific) was added as an enhancer and the radioactivity retained within the cells onto the filter was measured in a beta scintillation counter (Beckman LS6000). Twenty (20)

µL of the incubation mixture were removed for total counts whereas 500 µL of the incubation mixture were withdrawn for protein determination.

3.7 Western blot: HepG2 cells were resuspended in lysis buffer in the presence of proteinase inhibitors cocktail (SIGMA, St Louis). Animal tissues were homogenized in

homogenization buffer (200mM sucrose + 20mM HEPES) also in the presence of proteinase inhibitors cocktail (SIGMA, St Louis). Protein content was diluted to a final concentration of 1 mg/ml in Laemmli buffer, and equivalent quantities of solubilized protein (20µg/lane) were fractionated by electrophoresis on a 10% SDS-polyacrylamide gel. Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes

28

via exposure to a constant 100 volts for 75 minutes. Membranes were then incubated with

blocking buffer (5% milk in PBS with 0.1% Tween) for 1 hr at room temperature and

then incubated with primary antibodies in PBS/Tween overnight at 4°C. Secondary

antibodies were applied for an hour. The membranes were washed three times with PBS

(plus 0.1% Tween). The chemiluminescent substrate stable peroxidase and substrate

luminal/enhancer solutions (Amersham Biosciences, RPN2209) were added, and the

immunoreactive proteins visualized on film. The primary antibodies specific for SREBP-

1c, SREBP2, GLUT1, GLUT2 and GAPDH were optimized using a series of progressive

dilutions using the manufacturer’s suggested concentration as a reference.

3.8 mRNA isolation: HepG2 cells cultured in the presence of 0.4 mM or 0.8 mM external

Mg2+ were grown in 6-well plastic culture plates. The cells were washed with 1x PBS, and (1 mL/well Trizol ) in cells, and (100mg tissue/1ml Trizol reagent) in animals

were added for mRNA extraction. Cells were scraped and placed in 1.5 mL DNAase,

RNAase-free tubes and left at room temperature for 5 minutes. 200 µl chloroform were added per each ml of Trizol utilized. The mixture was vigorously shaken for 1-2 minutes.

The tubes were then centrifuged at 12,000 rpm for 15 minutes at 4°C. The top aqueous layer containing the mRNA was removed and transferred to 1.5 mL DNAase, RNAase- free centrifuge tube to which 500 μL isopropanol were added. Tubes were shaken vigorously and incubated at room temperature (RT) for 10 min before being sedimented at 12,000 rpm for 10 minutes at 4°C. Following supernatant removal, the pelleted mRNA was washed with 1 mL 75% ethyl , vortexed, and sedimented again at 12,000 rpm for 5 minutes at 4°C. Supernatant was removed, and the tubes were quickly sedimented at

29

6,000 rpm for 2 minutes at RT to remove any residual supernatant. The pellets were

allowed to air dry, prior to being dissolved in 100-150 µL RNase/DNase free water for 10 minutes at 55°C. Concentrations of mRNA in samples were determined using a

Nanodrop Spectrophotometer 1000, and adjusted to approximately 1 µg/µl.

3.9 cDNA synthesis: In 200 μL PCR tubes the following mixture was added: 1 μg of mRNA combined with enough RNase/DNase free water to bring mRNA to a final volume of 8 μl, 1 μl 10x DNase I buffer, and 1 μl DNase I. Following 15 minutes incubation at RT, 1 μL 25 mM EDTA was added to each tube, and the tubes were incubated at 70°C for 10 minutes to inactivate DNase I. Next, 1 μL Oligo-dt was added to the tube containing DNase treated RNA, and the tube was incubated at 75°C for 2 minutes followed by cooling on ice. The following components were added to each tube:

2 μL 10x NEB RT buffer, 1 μL dNTP mix (10 mM each), 0.5 μL NEB RNase inhibitor, 1

μL NEB MMLV and A volume of 3.5 μL RNase/DNase free water, bringing the volume in each tube to 20 μL. Tubes were then sedimented at 1500 rpm for 2 minutes, incubated at 42°C for 1 hour, then heated to 95°C for 5 minutes to inactivate the MMLV. Tubes were subsequently cooled down on ice and diluted with 180 μL RNase/DNase free water, bringing the final volume in each tube to 200 μL cDNA.

3.10 qPCR: ABgene® SuperPlate™ 96well PCR plates were used for qPCR protocol. To each well the following were added: 1 μL cDNA, 7.5 μL RNase/DNase free water, 0.6

μL forward primer (F primer) or 0.6 μL reverse primer (R primer), and 4.53 μL SYBR®

GreenER™ Universal Dye reagent. Sequences of F and R primers relative to the genes of

30

interest (GOI) were designed through Roche Applied Science and are reported in (Table

4). The plate was sealed using adhesive Sealing strips from ABgene®. After sealing with

the adhesive strip, the plate was sedimented (1500 rpm x 2 minutes) before being placed on SYBR® GreenER™ qPCR machine for qPCR analysis of GOI.

3.11 Statistical analysis: Statistical analysis will be carried out by one and two way

ANOVA (Sigma Stat) followed by analysis of variance by Tukey’s test set at (p < 0.05)

for significance.

31

4. Results

4.1 HepG2 cells

HepG2 cells (human hepatoma cells) were used to investigate how changes in

Mg2+ concentrations inside and/or outside the liver cell would affect cellular metabolic

processes including glucose and lipid metabolism. The cells were cultured in the

presence of 0.4mM (low) and 0.8mM (normal) extracellular Mg2+ concentration

([Mg2+]o).

4.1.1 Mg2+ compartmentalization changes with low [Mg2+]o

Under basal conditions, cellular Mg2+ is highly compartmentalized within

cytoplasm, mitochondria, endoplasmic reticulum, and nucleus. Changes in Mg2+ content

within these cellular compartments in HepG2 cells cultured in the presence of 0.8

(normal), and 0.4 (low) [Mg2+]o were tested. At confluence, the cells were gently washed with 1x PBS, resuspended in Krebs-Henseleit medium devoid of Mg2+ (Young et

al., 2007), and treated sequentially with digitonin, FCCP, and A23187 at the

concentrations reported under Materials and Methods. The addition of these agents

resulted in a mobilization of Mg2+ from cytoplasm, mitochondria, and post-mitochondrial

compartments, respectively, down their concentration gradient (Young et al., 2007).

Although this protocol presents severe technical limitations in terms of Mg2+ binding and

experimental accessibility to cellular components other than mitochondria and cytoplasm, no alternative procedures are available to our laboratory to determine cellular Mg2+

distribution. The results for cellular Mg2+ compartmentalization are shown in (Figure 3).

32

The cytoplasmic Mg2+ content was measured as the net change in Mg2+ extrusion at the time prior to FCCP addition minus the basal levels of Mg2+. the mitochondrial Mg2+

content was the net change in Mg2+ extrusion at the time prior to A23187 addition minus

the cytoplasmic content. The extra-mitochondrial Mg2+ content was assessed as the net

change in Mg2+ extrusion 5 min after adding A23187 minus the mitochondrial and

cytoplasmic values. Compared to HepG2 cells grown in 0.8 mM, cells grown in 0.4 mM

[Mg2+]o showed ~45% less total cellular Mg2+ content (77.7 ± 3.6 to 42.8 ± 3.7 nmol

Mg2+/mg protein). This decreased was observed predominantly in the cytoplasm (59.4 ±

4.8 to 30.1 ± 3.8 nmol Mg2+/mg protein) 50% decrease. The mitochondrial and extra-

mitochondrial compartments, instead, did not present significant chances in Mg2+

content.

4.1.2 The effect of low Mg2+ on ATP levels

Most cellular Mg2+ is bound to ATP and the Mg*ATP complex is involved in the

regulation of several metabolic enzymes. Therefore, we investigated whether the

observed changes in total and cytosolic Mg2+ content affected cellular ATP content.

Using an ATP luciferin-luciferase assay cellular ATP content was measured following 10

min acid extract with perchloric acid (10% final concentration) (Tessman and Romani,

1998). As Figure 4 shows, ATP content in HepG2 cells grown in 0.4 mM [Mg2+]o was significantly decreased (minus 35%) as compared to the content measured in cells maintained in 0.8 mM [Mg2+]o (i.e. 4.96 ± 0.17 vs. 7.37 ± 1.2 nmol ATP/mg protein,

respectively, n= 6, p< 0.05).

33

4.1.3 Effect of Mg2+ on glucose uptake

A number of studies have indicated a concomitance of glucose transport and Mg2+

fluxes in and out of the cell following hormonal stimulation in β-pancreatic islets, hepatocytes, and cardiac cells (Fagan et al., 2004; Henquin et al., 1983; Jacob et al.,

1989; Romani et al., 2000; Torres et al., 2005). However, the exact mechanism behind

this association is still not clear. In an attempt to explain this correlation, we investigated

whether changes in extracellular and cellular Mg2+ content affected the ability of liver cells to accumulate glucose. Radiolabeled 14C-glucose was administered to cells in

culture over a period of time of 20 minutes (i.e. t = 0 min, 1min, 2min, 5min, 10min, and

20min) both in the absence and in the presence of insulin (6nmols/L). Interestingly, in the absence of insulin no significant differences in glucose uptake in HepG2 maintained in 0.4 or 0.8mM [Mg2+ ]o were observed (Figure 5a). In contrast, the presence of insulin

significantly increased glucose uptake in cells grown in 0.8 mM [Mg2+]o starting at t = 5

min as compared to cells maintained and incubated in 0.4 mM [Mg2+]o (Figure 5b). This

increase in glucose accumulation persisted up to the end of our period of incubation. It

has to be noted that insulin administration enhanced glucose uptake in cells grown in 0.8

mM [Mg2+]o as compared to basal glucose uptake in these cells (Figure 6a). This

enhancement was not observed in 0.4 mM [Mg2+]o cells (Figure 6b).

4.1.4 Effect of Mg2+ on glucose transporters

HepG2 cells in culture express predominantly GLUT2 (90%) but also GLUT1

(10%) facilitative glucose transporters. To determine whether changes in Mg2+ had an

effect on the expression level of GLUT1 or GLUT2, mRNA from both HepG2 cell

34

cultures was extracted and assessed by qPCR. Cells grown in 0.4 mM [Mg2+ ]o presented a 2.5 fold increase in GLUT2 mRNA expression when compared to cells grown in 0.8 mM [Mg2+ ]o (Figure 7), but no change in GLUT1 mRNA expression (Figure 7). Western

blot analysis, however, showed no change in the protein level of GLUT1 and GLUT2

transporters (Figure 8). Finally, in an attempt to restore glucose uptake to the levels seen

in normal Mg2+, we incubated the cells grown in 0.4 mM [Mg2+]o in 0.8 mM [Mg2+ ]o

buffer at time of glucose uptake. Paradoxically, the results reported in (Figure 9) show an

impairment of glucose uptake in cells incubated in 0.8 mM Mg2+ versus those incubated in 0.4 mM [Mg2+ ]o.

4.1.5 Effect of Mg2+ on SREBP and PPAR transcription factors

Previous reports have indicated that Mg2+ is an important factor in lipid

metabolism through its effect on a number of enzymes involved in lipid synthesis and

metabolism (Nassir et al., 1995; Gueux et al., 1991). Because the liver plays a major role

in lipid and lipoprotein metabolism, we investigated the effect of Mg2+ deficiency on the

mRNA expression level of some important transcriptional factors in HepG2 cells. Using

qPCR, we observed a significant decrease (~ 70%) in SREBP-1c mRNA in cells grown

in 0.4 mM [Mg2+]o as compared to cells in 0.8 mM [Mg2+]o (Figure 10). On the other hand, 0.4 mM [Mg2+]o cells presented >2 fold increase in the levels of SREBP-2 mRNA.

Western blot analysis for SREBP-1c expression showed, however, that although the

SREBP-1c precursor is low in 0.4 mM [Mg2+ ]o cells as compared to 0.8mM [Mg2+ ]o

cells, the mature form of SREBP-1c is expressed at an equivalent level (Figure 11),

suggesting an active conversion process. In contrast, the bands of mature SREBP2,

35

another member of the SREBP family in liver cells, appeared to be slightly darker and

thicker in 0.4 mM [Mg2+]o cells (Figure 11).

Since SREBP activity depends on two regulatory proteins, SCAP and Insig 2, we

investigated the effect that low Mg2+ has on the mRNA expression of these proteins. The

results reported in (Figure 12) show that cells cultured in the presence of 0.4 mM [Mg2+]o had significantly lower levels of SCAP and Insig (minus 50% and 40%, respectively).

Additional transcriptional factors involved in fatty acid metabolism are the members of the PPAR family. We focused on PPARα, and PPARγ, and observed that

HepG2 maintained in 0.4 mM [Mg2+ ]o presented ~20% less PPARα mRNA than cells in

normal (0.8 mM) [Mg2+ ]o (Figure 13a, p < 0.02). In contrast, mRNA expression for

PPARγ was increased 4.5 fold (Figure 13b).

4.2 Animals

To determine whether the effect of Mg2+ deficiency on glucose transporter and

fatty acid transcriptional factors occurred in the whole animal, we used C57/B6 mice and

Sprague-Dawley rats on Mg2+ deficient diet for varying period of time.

4.2.1 Effect of Mg2+ on glucose transporters

First, we assessed expression of GLUT2 transporters by qPCR (Figure 14a) and

Western blot analysis (Figure 14b). No differences in the level of GLUT2 protein

expression in liver tissue were observed, in good agreement with the data obtained in

HepG2 cells. As expected, GLUT1 expression was nearly undetectable (not shown) in keeping with the very low level of expression of this transporter in the liver of adult

36

animals. On the contrary, there was a significant decrease in the GLUT2 mRNA

expression in SD rat (0.7 ± 0.063 folds). A decrease in GLUT2 expression was also observed in C57/B6 mice, but it was not statistically significant (p = 0.21)

4.2.2 Effect of Mg2+ on SREBP and PPAR transcription factors

In whole animals, the levels of SREBP-1c mRNA presented the same trend as in cell. Mg2+ deficient SD rats presented about 0.7 ± 0.07 fold decrease whereas Mg2+

deficient C57/B6 mice presented a 0.5 ± 0.19 fold decrease. Both these changes were

statistically significant (Figure 15). Western blot analysis provided results consistent with those obtained in cells in that SREBP-1c precursor was decreased in the presence of a similar level of mature SREBP-1c in both Mg2+ deficient animal models (Figures 16a and 16b).

In the case of PPARs, PPARα showed a significant decrease in animals on low

Mg2+ diet (0.8 ± 0.07 fold in SD rats and 0.2 ± 0.045 fold in C57/B6 mice) (Figure 17a).

However, PPARγ presented opposite results when compared with cells in that the mRNA

levels was decreased by 0.9 ± 0.01 fold in SD rats and 0.5 ± 0.16 fold in C57/B6 mice

(Figure 17b).

37

5. Discussion

Mg2+ is an essential cellular cation, playing a crucial role in many physiological functions. It is critical in protein synthesis, in energy-requiring metabolic processes, nervous tissue conduction, neuromuscular excitability, muscle contraction, and hormone secretion (Laires et al., 2004). The small intestine and kidney maintain serum Mg2+

concentration within a narrow range, and under conditions of Mg2+ deprivation operate

by increasing their fractional magnesium absorption and reabsorption, respectively.

Mg2+ is the fourth most abundant cation in the whole body and the second most

common cation within the cell (Voets et al., 2004). Despite its abundance and its role in

various physiological functions and possibly in the genesis of various pathological

conditions (Grubbs & Maguire, 1987; Cameron & Smith, 1989 and O’Rourke, 1993),

many aspects of Mg2+ regulation, especially at cellular level, remain unclear and in some

way conflicting.

Data reported in this thesis confirm the high compartmentalization of Mg2+ in

cellular compartments. Furthermore, results from Figure 3 supports the notion that a

decrease in [Mg2+]o resulted in a significant decrease in total Mg2+ content. This decrease

was more evident in the cytoplasm while limited or no detectable variations were

observed in the mitochondria, and the extra-mitochondrial pools. An appealing but still unproved hypothesis could be that Mg2+ is tightly retained (or redistributed) within these

cellular compartments to maintain proper function of mitochondrial dehydrogenases and

reticular enzymes. More specific experiments need to be designed to determine this

possibility.

38

Interestingly, culturing the cells in 0.4 mM [Mg2+]o resulted also in a significant reduction in total ATP content. Because most of the cytoplasmic ATP forms an Mg*ATP complex, which acts as a very important regulator for a large number of metabolic enzymes, this decrease in ATP content could have some far reaching implications for important cellular processes such as glycolysis and enzyme phosphorylation.

Mg2+ is an important factor in the regulation of a large number of enzymes, most

of which are involved in anaerobic glycolysis. This converts glucose

into pyruvate, which can then be further converted to lactate (anaerobic glycolysis) or

enter the Krebs cycle (aerobic glcolysis). High-energy compounds released in this process are ATP and NADH (reduced nicotinamide adenine dinucleotide) (Garfinkle &

Garfinkle, 1988). We believe that the effect of Mg2+ on glucose uptake as observed in our

study could be explained by the possibility that growing liver cells in low external Mg2+

affects glycolytic enzymes such as hexokinase, , and glucose 6- phosphatase, slowing down the first step of glucose utilization. On the other hand, Mg2+

is known to affect insulin activity or sensitivity, and low cellular and serum Mg2+ levels have been associated with insulin resistance and low insulin sensitivity (Nadler et al.,

1993). Growing liver cells in low Mg2+ would then affect the cell response to insulin.

Because insulin modulates the activity of hepatic hexokinase and , Mg2+ deficiency and the possibly associated decrease in insulin effect on

these enzymes would have major repercussion on hepatic glucose utilization. This could

explain the lack of an effect of insulin on glucose uptake in HepG2 cells grown in 0.4

mM [Mg2+]o as compared to cells maintained in normal (0.8mM) Mg2+.

39

In the absence of insulin, however, no stimulatory effect would take place on the

Krebs cycle, resulting in the absence of significant differences in glucose uptake

irrespective of the external Mg2+ concentration (Figure 5a). The basal glucose uptake observed in these two experimental models also suggest that in the absence of insulin the various steps of glucose uptake, phosphorylation and utilization operate at a similar rate,

and that is why no differences in glucose uptake are observed in HepG2 cells grown in

0.4 mM and 0.8 mM [Mg2+]o. In vitro assessment of enzyme activity and expression in

future investigation should provide supporting evidence for this hypothesis.

The increased expression of GLUT2 mRNA observed in HepG2 cells in low Mg2+

could be explained as a compensatory mechanism to obviate to the reduced ATP content

and perhaps lack of insulin effect. This contrasts with the essentially similar level of

Glut2 protein expressed in the cells irrespective of the external Mg2+ concentration. This

result is also observed in animals. Animals on Mg2+-deficient diet, however, present a decreased expression of GLUT2 mRNA. At present, we do not have a clear explanation for this discrepancy between cells and animals. It has to be kept in mind that any

immortalized cell line presents a specific phenotype, which differs – from the regulatory

point of view - from the phenotype of the native cell type within an animal. Furthermore,

animals present a level of complexity including the presence of circulating neuro-

humoral factors and hormones, and other interacting systems that are virtually absent in

cells in culture.

The second aim of this study was to determine the effect of Mg2+ on lipid

metabolism in particular its effect on SREBP regulation. According to the Randall

hypothesis, glucose and fatty acid metabolism are alternatively utilized by liver cells to

40

maintain proper caloric intake and support bioenergetic processes (Randall et al., 2008).

Hence, it was to be expected that a reduced glucose uptake would result in an increased

expression of gene products involved in fatty acid metabolism. The results provided by

our study support this concept in that a decrease in cellular Mg2+ content appears to modulate SREBP isoforms activity at the transcriptional and activation level. At the transcriptional level, Mg2+ deficiency causes a decrease in SREBP-1c mRNA expression

both in cells (Figure 10) and animals (Figure 15). This is possibly due to the effect of

Mg2+ on insulin signaling since SREBP-1c is reported to be regulated by this hormone,

and it decreases in diabetes and obesity, conditions characterized by insulin resistance

(Eberlé et al., 2004, and Oberkofler et al., 2002). However SREBP2, the predominant

form in liver cells, is not regulated by insulin, and we observed that low Mg2+

significantly increases SREBP2 mRNA expression. This increase could explain the

elevated levels of cholesterol and triglycerides observed in Mg2+ deficiency and reported

in other studies.

The decrease in cellular Mg2+ content also affects the activation level of SREBP.

SREBP isoforms are produced as a precursor that is modulated through a number of steps

involving proteins such as SCAP and Insings to generate the active, mature form (Dong

et al., 2010). In low Mg2+ , SREBP-1c is less present as the precursor but similar levels of

mature SREBP can be detected in both cells and animals (Figure 11 and 16). This would

suggest that in low Mg2+ there is an accelerated activation of the various steps leading to

the formation of the final mature form. This is supported by the observation that both

SCAP and Insig2 appear to be significantly decreased under the same experimental

conditions, causing less retention of SREBP precursor in the ER and more translocation

41

to Golgi to be activated, thus increasing the rate of activation. This is consistent with

data in the literature indicating an increase in mature SREBPs under conditions in which

a decrease in Insig1 and Insig2 mRNAs (Dong et al., 2010).

Further indications that in low Mg2+ lipid metabolism is upregulated as a result of changes in glucose accumulation and utilization are provided by the changes in the expression of other transcriptional factors including PPARα, which is involved in fatty

acid oxidation, and PPARγ, which is involved in and adipocytes storage. The results shown in (Figure 13a and 17a) shows that PPARα is significantly decreased,

leading to less fatty acid oxidation. As for PPARγ, this transcriptional factor – which

promotes lipogenesis - is increased in cells (Figure 13b) but is decreased in animals

(Figure 17b). A possible explanation could be that in animals PPARγ is more abundantly produced in adipose tissue. Although this hypothesis needs further corroboration, preliminary data in our lab indicate a significant increase in PPARγ levels in white adipose tissues (data not shown).

6. Conclusion

Mg2+ plays an important role in maintain normal cell functions and proper glucose

and fatty acid metabolism. Mg2+ deficiency has been associated with a large number of

disorders including diabetes and obesity. Our results demonstrate that low Mg2+ disrupts glucose uptake possibly through its affects on glycolysis enzymes and insulin. Our study also provides supporting evidence that low Mg2+ leads to increase levels of transcriptional factors involved in fatty acid synthesis and decreased fatty acid oxidation. All together

42

our data indicate that extracellular and cellular Mg2+ levels are important for glucose

uptake as well as fatty acid metabolism.

7. Future Directions

7.1 Mg2+ and Glucose

Numerous reports in the literature suggest a role of Mg2+ transport and

homeostasis in regulating glucose transport in and out of hepatocytes and beta islet cells.

The data reported here suggest that normal levels of extracellular and intracellular Mg2+

are required for proper glucose uptake, at least in liver cells. As for the possible

underlying mechanisms, we hypothesize that Mg2+ exerts its effect on enzymes involved

in anaerobic glycolysis and possibly in mediating insulin effect in liver cells (e.g. Krebs

cycle).

Our results, however, fall short from identifying a clear site of action. Targets of

future investigation include:

1 – Changes in total cellular Mg2+ content and subcellular Mg2+ distribution following

stimulation with insulin;

2 - Expression and activity of enzymes like hexokinase, phosphofructokinase, and possibly pyruvate dehydrogenase in HepG2 cells incubated in the presence of varying

[Mg2+]o;

3 - Expression and activity of enzymes like phosphoenol-pyruvate carboxykinase and glucose 6 phosphatase (as key enzymes in gluconeogenesis);

4 – Cellular content of ATP and Mg2+ in HepG2 cells energetically supported by

pyruvate and lactate, both in the absence and in the presence of insulin stimulation. In

43

this set of experiments, accumulation of these energetic substrates within the hepatocytes

will also be determined by radioisotopic distribution.

7.2 Mg2+ and Lipids

Given the wide diversity of Mg2+ requiring processes and the many transcriptional factors affected by Mg2+ levels it is anticipated that several synthetic and oxidative

processes will determine changes in cellular and plasma lipid levels. We will start by

assessing some upstream regulators like adiponectin and insulin since these hormones

regulate numerous genes involved in lipid metabolism including SREBP, PPARs,

FATP/Slc27 protein family members, and CPT1 expression and activity. Based upon the

initial results, another area of interest might be represented by the adipose tissue, which

would provide useful information regarding the effect of Mg2+ on lipid metabolism within

adipose tissue.

44

Table 1: Analytical Methods to Measure Mg2+ Content and Distribution

From: Wolf et al. 2003, Mol Aspects Med 24: 11-26. With permission.

45

Table 2: Tissues in which a Na+-independent Mg2+ Efflux has been Observed or Hypothesized.

Tissue Na+-independent Reference

liver cells Ca2+/Mg2+ (Romani et al. 1993)

Sr2+/Mg2+antiporter (Cefaratti et al. 2000)

erythrocytes Mg2+/Mg2+ antiporter (Gunther & Vormann 1987), (Gunther et al.

Mn2+/Mg2+ antiporter 1990)

(Gunther & Vormann 1987), (Feray & Garay 1987), (Gunther et al. 1990) choline/Mg2+ (Ebel & Gunther 1999) Cl-/Mg2+co-transporter (Gunther & Vormann 1989, 1990) - 2+ HCO3 /Mg

(Gunther & Vormann 1990)

cardiac cells Ca2+/Mg2+ (Romani et al. 1993)

Sr2+/Mg2+antiporter (Romani et al. 1993)

Mg2+/Mg2+ antiporter (Maguire et al. 1984)

46

Table 3: Facilitative Glucose Transporter Isoforms in the Liver.

Transporter Class Expression in Liver% Substrate Km (mM)

GLUT1 I <10 Glucose 18-21

GLUT2 I >85 Glucose; fructose, 1-2

galactose

GLUT9 II Not determined Not determined

_

GLUT10 III Not determined Glucose 0.3

Adapted from: Wood & Trayhurn 2003, Br J Nutri 89: 3-9.

47

Table 4: Primers for Quantitative Real-Time PCR.

Gene Species Forword/reverse primers

GAPDH Human F:agccacatcgctcagacac R: gcccaatacgaccaaatcc Rat F: ttctttttgggtgcagtgct R: caatccctgaatagtccagtttg Mice F: ttctttttgggtgcagtgct R: caatccctgaatagtccagtttg

Β-actin Human F: aacctgcacccagtgctc R: ttgtactgctcgaacatcagc

Glut 1 Human F: aaggttcgagaggccacat R ccattgttagctcatgggttg Rat F: cggaccctgcatctcatt .R: gccacgatactcagataggaca

Mice F: accctgggactgcaggtt R: agggacggagggctactg

Glut2 Human F: ccctgtctgtatccagctttg R: tgtttgctactaacatggctttg Rat F: atcttgatgacggtggctct R: acgatggacacataactcatgg

Mice F: tgtgatccagtgagtctccaa R: ggcgcacatctataatgctct

SREB-1c Human F: cgctcctccatcaatgaca R: tgcgcaagacagcagattta Rat F: acaagattgtggagctcaagg R: tgcgcaagacagcagattta

Mice F: ttcctcagactgtaggcaaatct R: agcctcagtttacccactcct

SREBP2 Human F: caccagctgcacatcacag R: gccatgtgtacatcggaaca

SCAP Human F: gaaccacgtgctgagagaca R: gcagcaggtcggtcactt

Insig2 Human F: ttcctctatgttcgttcttggtt R: tttctgcgataactttacattcgt

PPARα Human F: gcactggaactggatgacag R: tttagaaggccaggacgatct Rat F: tgcggactaccagtacttaggg R: gctggagagagggtgtctgt

Mice F: ctgagaccctcggggaac R: aaacgtcagttcacagggaag

PPARγ Human F: gacaggaaagacaacagacaaatc R: ggggtgatgtgtttgaacttg Rat F: cccaatggttgctgattaca R: ggacgcaggctctactttga Mice F: gaaagacaacggacaaatcacc R: gggggtgatatgtttgaacttg

48

Cellular Magnesium Profile

Figure 1: Schematic diagram showing Mg2+ compartmentalization and distribution within mammalian cell.

49

Magnesium Transport and Hormonal Regulation

Figure 2: Schematic representation of Mg2+ transport and the hormonal regulation responsible for Mg2+ accumulation and extrusion in mammalian cells including hepatocytes.

50

Magnesium Compartmentalization

100

90

80

70

60 50 * 40 * 0.8 mM Mg2+ nmol Mg2+/mg ptn ptn Mg2+/mg nmol 30 0.4mM Mg2+ 20

10

0

Figure 3: Cellular Mg2+ distribution in HepG2 cells grown in 0.4 mM (low) and 0.8 mM normal) [Mg2+]o treated in vitro with digitonin, mitochondrial uncoupler (FCCP), and ionophore (A23187). Data are means ± S.E. of 6 different preparations, each tested in duplicate * = P < 0.05.

51

Magnesium Effect on ATP levels

10 9 8 7 6 5 * 0.4 4 0.8 3

nmol ATP/mg prt 2 1 0 0.4 0.8

Figure 4: ATP concentration in HepG2 cells grown in 0.4 mM [Mg2+]o compared to cells grown in 0.8 mM [Mg2+]o. Data are means ± S.E. of 6 different preparations, each tested in duplicate * (P < 0.05).

52

Magnesium Effect on Glucose Uptake

0.006 A

0.005

0.004

0.003 0.4 mM Mg2+ (-) insulin

0.002 0.8 mM Mg2+ (-) insulin umol glucose/mg prt glucose/mg umol

0.001

0 0 min 1 min 2 min 5 min 10 min 20min

0.006 B

0.005 * * 0.004 * 0.003 0.4 mM Mg2+ (+) insulin 0.8 mM Mg2+ (+) insulin 0.002 umol glucose/mg prt glucose/mg umol 0.001

0 0 min 1 min 2 min 5 min 10 min 20 min

Figure 5: Glucose accumulation in HepG2 cells grown in the two different Mg2+ concentrations reported previously. (A) Show the effect of low Mg2+ on glucose uptake in the absence of insulin. (B) Represents the effect of low Mg2+ on glucose uptake in the presence of insulin. Data are means ± S.E. of 8 different preparations for 0.8 mM [Mg2+]o with insulin, 7 different preparations for 0.4 mM [Mg2+]o with insulin, and 5 different preparations for 0.4 and 0.8 mM [Mg2+]o without insulin. * (P < 0.05).

53

Insulin Effect on Glucose Uptake

A 0.006

0.005 * 0.004 * 0.8 mM Mg2+ (+) insulin 0.003 * 0.8 mM Mg2+ (-) insulin 0.002 umol glucose/mg prt glucose/mg umol 0.001

0 0 min 1 min 2 min 5 min 10 min 20 min

0.006 B 0.005

0.004

0.003 0.4 mM Mg2+ (+) insulin 0.4 mM Mg2+ (-) insulin 0.002 umol glucose/mg prt glucose/mg umol 0.001

0 0 min 1 min 2 min 5 min 10 min 20 min

Figure 6: Glucose accumulation in HepG2 cells grown in two different Mg2+ concentrations. (A) Represents the effect of insulin on glucose uptake in 0.8 mM Mg2+ cells. (B) Show the effect of insulin on glucose uptake in 0.4 mM Mg2+ cells. Data are means ± S.E. of 8 different preparations for 0.8 mM [Mg2+]o with insulin, 7 different preparations for 0.4 mM [Mg2+]o with insulin, and 5 different preparations for 0.8 and 0.4 mM [Mg2+]o without insulin. * (P < 0.05).

54

Effect of Magnesium on Glucose Transporters

3.5

3 *

2.5 ∆∆Ct ) ∆∆Ct - 2 0.8 1.5 0.4

1 fold change (2^ change fold

0.5

0 GLUT-1 GLUT-2

Figure 7: Expression of glucose transporters GLUT-1 and GLUT-2 by qPCR, in HepG2 cells cultured in 0.4mM (low), and 0.8mM (normal) [Mg2+]o. β-actin as a normalizing gene. Data are means ± S.E. of 6 different preparations, each tested in duplicate * (P < 0.05).

55

Effect of Magnesium on Glucose Transporters

Figure 8: Western Blot showing the levels of GLUT2 and GLUT1 transporters in HepG2 cells grown in different Mg2+ concentrations. Each line represents a different preparation. A typical experiment out of 3 is reported.

56

Effect of Acute [Mg2+]o Change on Glucose Uptake

0.006

0.005

0.004 * * 0.003 * 0.4 in 0.4 mM Mg2+ (+) insulin * 0.4 in 0.8 mM Mg2+ (+) insulin

umol glucose/mg prt glucose/mg umol 0.002

0.001

0 0 min 1 min 2 min 5 min 10 min 20 min

Figure 9: Glucose accumulation in HepG2 cells grown in 0.4 mM Mg2+ concentration to see the effect of acutely changing [Mg2+]o in cells grown in 0.4 mM Mg2+ to 0.8 mM Mg2+ in the presence of insulin. Data are means ± S.E. of 7 different preparations for 0.4 mM [Mg2+]o in 0.4 mM [Mg2+]o, and 3 different preparations for 0.4 in 0.8 mM [Mg2+]o. * (P < 0.05).

57

Effect of Magnesium on SREBP-1c and SREBP2

2.5 *

2 ∆∆Ct ) ∆∆Ct

- 1.5

0.8 1 0.4 fold change (2^ change fold * 0.5

0 SREBP-1c SREBP2

Figure 10: Expression of SREBP-1c (left) and SREBP2 (right) by qPCR , in HepG2 cells cultured in 0.4mM (low), and 0.8mM (normal) [Mg2+]o. β-actin as a normalizing gene. Data are means ± S.E. of 6 different preparations, each tested in duplicate * (P < 0.05).

58

Effect of Magnesium on SREBP-1c and SREBP2

SREBP-1c precursor

Mature SREBP -1c

SREBP2

Mature SREBP2

GAPDH

Figure 11: Western Blot showing the levels of SREBP-1c and SREBP2 precursor (126 KD) and mature SREBP-1c (68 KD) and SREBP2 (55-60 KD) in HepG2 cells grown in different Mg2+ concentrations. Each line represents a different preparation. A typical experiment out of 4 is reported.

59

Effect of Magnesium on SCAP and Insig2

1.2

1

0.8 ∆∆Ct) ∆∆Ct) - * * 0.6 0.8 0.4 0.4 fold change (2^ change fold

0.2

0 SCAP Insig2

Figure 12: expression of SCAP (left) and Insig (right) by qPCR, in HepG2 cells cultured in 0.4mM (low), and 0.8mM (normal) [Mg2+]o. β-actin as a normalizing gene. Data are means ± S.E. of 6 different preparations, each tested in duplicate * (P < 0.05).

60

Effect of Magnesium on PPARs in Cells

PPARα A 1.4

1.2 * 1 ∆∆Ct) ∆∆Ct) - 0.8 0.8 0.6 0.4 0.4 fold change (2^ change fold 0.2

0 0.8 0.4

PPARγ B 7 * 6

5 ∆∆Ct) ∆∆Ct) - 4 0.8 3 0.4 2 fold change (2^ change fold 1

0 0.8 0.4

Figure 13: expression of PPARα (A) and PPARγ (B) by qPCR, in HepG2 cells cultured in 0.4mM (low), and 0.8mM (normal) [Mg2+]o. β-actin as a normalizing gene. Data are means ± S.E. of 6 different preparations, each tested in duplicate * (P < 0.05). Figures presented in two separate graphes due to the large scale on the PPARγ figure.

61

Effect of Magnesium on Glucose Transporters in Animals

GLUT2 A

1.2

1 ∆∆Ct) ∆∆Ct) - 0.8

0.6 * Mg2+ (+) 0.4 Mg2+ (-) 0.2 fold change (2^ change fold

0 B6 SD

+ + - + + - + - B

GLUT2 62 KD

GAPDH 37 KD

Figure 14: (A) Expression of glucose transporters Glut2 by qPCR, in b6 mice liver , and SD rats livers(right) in normal Mg2+ diet for control and Mg2+ deficient diet. GABDH as a normalizing gene. Data are means ± S.E. of 6 different preparations, each tested in duplicate * (P < 0.05). (B) Western Blot showing the levels of GLUT2 transporter SD rats livers in normal Mg2+ diet for control and Mg2+ deficient diet. Each line represents a different preparation. A typical experiment out of 2 is reported.

62

Effect of Magnesium on SREBP-1c in Animals

SREBP-1C 1.2

1 *

∆∆Ct) ∆∆Ct) 0.8 - (2^ 0.6 * Mg2+ (+) Mg2+ (-) 0.4 fold Change 0.2

0 B6 SD

Figure 15: Expression of SREBP-1c by qPCR, (left) in b6 mice liver, and SD rats livers (right) in normal Mg2+ diet for control and Mg2+ deficient diet. GABDH as a normalizing gene. Data are means ± S.E. of 6 different preparations, each tested in duplicate * (P < 0.05).

63

Effect of Magnesium on SREBP-1c in Animals

+ - + - + - A

SREBP-1c precursor

Mature SREBP-1c

GAPDH

B

+ - + - + -

SREBP-1c precursor

Mature SREBP-1c

Figure 16: Western Blot showing the levels of SREBP-1c precursor (126 KD) and mature SREBP-1c (68 KD). A typical experiment out of 4 is reported in SD rats livers(A), and b6 mice liver (B) in normal Mg2+ diet for control and Mg2+ deficient diet. Each line represents a different preparation.

64

Effect of Magnesium on PPARs in Animals

PPARα

1.2 A

1 ∆∆Ct) ∆∆Ct)

- 0.8

0.6 Mg2+ (+) * Mg2+ (-) 0.4

fold Change (2^ Change fold 0.2

0 B6 SD

PPARγ B 1.2

1 * ∆∆Ct) ∆∆Ct)

- 0.8 (2^ 0.6 Mg2+ (+) Mg2+ (-) 0.4 *

fold Change 0.2

0 B6 SD

Figure 17: Expression of PPARs by qPCR, (left) in b6 mice liver, and SD rats livers (right) in normal Mg2+ diet for control and Mg2+ deficient diet. (A) PPARα. (B) PPARγ. GABDH as a normalizing gene. Data are means ± S.E. of 6 different preparations, each tested in duplicate * (P < 0.05).

65

8. Bibliography

Barbagallo M, Dominguez LJ, Galioto A, Ferlisi A, Cani C, Malfa L, Pineo A, Busardo A, and Paolisso G. Role of magnesium in insulin action, diabetes and cardio-metabolic syndrome X. Mol Aspects Med 2003; 24: 39-52.

Bell GI, Kayano T, Buse JB, Burant CF, Takeda J, Lin D, Gukumoto H and Seino S. Molecular biology of mammalian glucose transporters. Diabetes Care 1990; 13: 198-208.

Boguka K and Wojtczak L. Binding of magnesium by proteins of the mitochondrial intermembrane compartment. Biochim Biophys Res Commun 1976; 71: 161–167.

Bond M, Vadasz G, Somlyo AV, and Somlyo AP. Subcellular calcium and magnesium mobilization in rat liver stimulated in vivo with vasopressin and glucagon. J Biol Chem 1987; 262: 15630–15636.

Bradford, M.M. "Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding", Anal. Biochem. 1976; 72: 248–254.

Bygrave FL and Benedetti A. What is the concentration of calcium ions in the endoplasmic reticulum? Cell Calcium 1996; 19: 547-551.

Cameron IL and Smith NKR. Cellular concentration of magnesium and other ions in relation to protein synthesis, cell proliferation and cancer. Magnesium 1989; 8: 31- 44.

Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, and Nishizuka Y. Direct activation of calcium-activated, -dependent protein kinase by tumor promoting phorbol esters. JBiol Chem 1982; 257:7847-7851.

Cefaratti C, McKinnis A, and Romani A. Altered Mg2+ transport across liver plasma membrane from streptozotocin-treated rats. Mol Cell Biochem 2004; 262: 145-154. 66

Cefaratti C, Romani A, and Scarpa A. Characterization of two Mg2+ transporters in sealed plasma membrane vesicles from rat liver. Am J Physiol Cell Physiol 1998; 275: C959–C1008.

Cefaratti C, Romani A, and Scarpa A. Differential localization and operation of distinct Mg2+ transporters in apical and basolateral sides of rat liver plasma membrane. J Biol Chem 2000; 275: 3772–3880.

Coady MJ, Pajor AM, Wright EM. Sequence homologies between intestinal and renal Na+/glucose . Am. J. Physiol. 1990; 259:C605-10.

Cole DEC and Quamme GA. Inherited disorders of renal magnesium handling. J Am Soc Nephrol 2000; 11: 1937-1947.

Corkey BE, Duszynski J, Rich TI, Matschinsky B, and Williamson JR. Regulation of free and bound magnesium in rat hepatocytes and isolated mitochondria. J Biol Chem 1986;.261: 2567–2574.

Cowan JA. Introduction to the biological chemistry of magnesium ion. In: Cowan, J.A. (Ed.), The Biological Chemistry of Magnesium. VCH 1995.,pp.1–23,.

Csermely P, Fodor P, and Somogyi J. The tumor promoter tetradecanoylphorbol- 13-acetate elicits the redistribution of heavy metals in subcellular fractions of rabbit thymocytes as measured by plasma emission spectroscopy. Carcinogenesis 1987; 8: 1663-1666.

Dalal P, Romani A, and Scarpa A. Redistribution of Mg2+ in isolated rat hepatocytes upon chemical hypoxia. Biophys J 1998; 74: A193 (abstract),

Djurhuus MS, Klitgaard NA, Pedersen KK, Blaabjerg O, Altura BM, Altura BT, and Henriksen JE. Magnesium reduces insulin-stimulated glucose uptake and serum lipid concentrations in type 1 diabetes. Metabolism 2001; 50: 1409-1417,

Dohm GL. Elton CW, Friedman JE, Pilch PF, Pones WJ, et al. Decreased expression of glucose transporter in muscle from insulin-resistant patients. Am. J. Physiol. 1991; 260S459-63

67

Dong XY, Tang SQ. Insulin-induced gene: a new regulator in lipid metabolism. Peptides. 2010; 31:2145-50.

Eberlé D, Hegarty B, Bossard P, Ferré P, Foufelle F.SREBP transcription factors: master regulators of lipid homeostasis. Biochimie. 2004; 86:839-48.

Eckel J, Pandalis G, Reinauer H. Insulin action on the glucose transport system in isolated cardiocytes from adult rats. Biochem J 1983; 212: 385–392.

Fagan TE, Cefaratti C, and Romani A. Streptozotocin-induced diabetes impairs Mg2+ homeostasis and uptake in rat liver cells. Am J Physiol. 2004; 286: E184-E193.

Fagan TE and Romani A. Activation of Na+- and Ca2+-dependent Mg2+ extrusion by alpha(1)- and beta-adrenergic agonists in rat liver cells. Am J Physiol 2000; 279: G943-G950.

Fahy E, Subramaniam S, Brown HA, Glass CK, Merrill AH Jr, Murphy RC, Raetz CR, Russell DW, Seyama Y, Shaw W, Shimizu T, Spener F, van Meer G, VanNieuwenhze MS, White SH, Witztum JL, Dennis EA. A comprehensive classification system for lipids. J Lipid Res. 2005; 46:839-61.

Feray JC and Garay R. A one-to-one Mg2+:Mn2+ exchange in rat erythrocytes. J Biol Chem 1987;262: 5763-5768.

Ferreira A, Rivera A, and Romero JR. Na+/Mg2+ exchange is functionally coupled to the insulin receptor. J Cell Physiol 2004; 199: 434-440.

Flatman PW. Magnesium transport across cell membranes. J Membr Biol 1984; 80: 1-14.

Flatman PW. Mechanisms of magnesium transport. Annu Rev Physiol 1991; 53: 259-271.

Garfinkel D and Garfinkel L. Magnesium and regulation of at the molecular level. Magnesium 1988; 7: 249-261.

68

Goytain A, Quamme GA. Identification and characterization of a novel family of membrane magnesium transporters, MMgT1 and MMgT2. Am J Physiol 2008; 294:495-502.

Grubbs RD and Maguire ME. Magnesium as a regulatory cation: criteria and evaluation. Magnesium 1987; 6: 113-127.

Gueux E, Mazur A, Cardot P, Rayssiguier Y. Magnesium deficiency affects plasma lipoprotein composition in rats. J Nutr. 1991; 121:1222-7.

Gueux E, Azais-Braesco V, Bussière L, Grolier P, Mazur A, Rayssiguier Y. Effect of magnesium deficiency on triacylglycerol-rich lipoprotein and tissue susceptibility to peroxidation in relation to E content. Br J Nutr. 1995;74:849-56.

Gunther T. Mechanisms and regulation of Mg2+ efflux and Mg2+ influx. Miner Electrolyte Metab 1993; 19: 259-265.

Gunther T and Hollriegl V. Na+- and anion-dependent Mg2+ influx in isolated hepatocytes. Biochim Biophys Acta 1993; 1149: 49–54.

Gunther T and Vormann J. Mg2+ efflux is accomplished by an amiloride-sensitive Na+/Mg2+ antiport. Biochem Biophys Res Commun 1985; 130: 540-545.

Gunther T and Vormann J. Activation of Na+/Mg2+ antiport in thymocytes by cAMP. FEBS Lett. 1992; 297: 132–134.

Gunther T, Vormann J, and Hollriegl V. Noradrenaline-induced Na+-dependent Mg2+ efflux from rat liver. Magnes Bull 1991; 13: 122–124.

Hofmann T, Chubanov V, Chen X, Dietz AS, Gudermann T, Montell C. Drosophila TRPM channel is essential for the control of extracellular magnesium levels. PLoS One. 2010 6;5:e10519.

Henquin JC, Tamagawa T, Nenquin M, Cogneau M. Glucose modulates Mg2+ fluxes in pancreatic islet cells. Nature. 1983 Jan 6;301:73–74.

69

Henquin JC, Nenquin M, Awouters P, Cogneau M. Magnesium uptake by pancreatic islet cells is modulated by stimulators and inhibitors of the B-cell function. Eur J Biochem. 1986;155:653-7.

Huerta MG, Roemmich JN, Kington ML, Bovbjerg VE, Weltman AL, Holmes VF, Patrie JT, Rogol AD, Nadler JL. Magnesium deficiency is associated with insulin resistance in obese children. Diabetes Care. 2005;28:1175-81.

Inoue I. [Lipid metabolism and magnesium]. Clin Calcium. 2005;15:65-76 (Abstract).

Jakob A, Becker J, Schottli G, and Fritzsch G. Alpha 1-adrenergic stimulation causes Mg2+ release from perfused rat liver. FEBS Lett 1989; 246: 127–130.

Joost HG, Thorens B. The extended GLUT-family of sugar/polyol transport facilitators: nomenclature, sequence characteristics, and potential function of its novel members (review). Mol Membr Biol. 2001;18:247-56.

Jung DW and Brierly GP. Matrix magnesium and the permeability of heart mitochondria to potassium ion. J Biol Chem 1986; 261: 6408-6415.

Kamon J, Yamauchi T, Terauchi Y, Kubota N, Kadowaki T.[The mechanisms by which PPARgamma and adiponectin regulate glucose and lipid metabolism]. Nippon Yakurigaku Zasshi. 2003; 122:294-300.

Keenan D, Romani A, and Scarpa A. Regulation of Mg2+ homeostasis by insulin in perfused rat livers and isolated hepatocytes. FEBS Lett 1996; 395: 241-244.

Kimura Y, Murase M, Nagata Y. Change in glucose homeostasis in rats by long- term magnesium deficient diet. J Nutr Sci Vitaminol (Tokyo).1996;42:407– 422.

Klein S, Wadden T, Sugerman HJ AGA technical review on obesity. Gastroenterology. 2002;123:882-932.

Kumar BP, Shivakumar K, Kartha CC. Magnesium deficiency-related changes in lipid peroxidation and collagen metabolism in vivo in rat heart. Int J Biochem Cell Biol. 1997;29:129-34.

70

Laires MJ, Monteiro CP, and Bicho M. Role of cellular magnesium in health and human disease. Front Biosci 2004;9: 262-76.

Laughlin MR and Thompson D. The regulatory role for magnesium in glycolytic flux of the human erythrocyte. J Biol Chem 1996; 271: 28977-28983.

Lynking J. Dietary Magnesium Intake and Atherosclerotic Plaque Development in Rabbits. B.S., University of Illinois at Urbana-Champaign, student thesis 1999

Matsuura T, Kanayama Y, Inoue T, Takeda T, and Morishima I. cAMP- induced changes of intracellular free Mg2+ levels in human erythrocytes. Biochim Biophys Acta 1993;1220: 31-36.

Nadler JL, Buchanan T, Natarajan R, Antonipillai I, Bergman R, Rude R. Magnesium deficiency produces insulin resistance and increased synthesis. Hypertension. 1993;21:1024-9.

Nadler MJ, Hermosura MC, Inabe K, Perraud AL, Zhu Q, Stokes AJ, Kurosaki T, Kinet JP, Penner R, Scharenberg AM, and Fleig A. LTRPC7 is a MgATP- regulated divalent cation channel required for cell viability. Nature 2001; 411: 590- 595.

Nassir F, Mazur A, Giannoni F, Gueux E, Davidson NO, Rayssiguier Y. Magnesium deficiency modulates hepatic lipogenesis and apolipoprotein gene expression in the rat. Biochim Biophys Acta. 1995 13;1257:125-32.

Oberkofler H, Fukushima N, Esterbauer H, Krempler F, Patsch W. Sterol regulatory element binding proteins: relationship of adipose tissue gene expression with obesity in humans. Biochim Biophys Acta. 2002 3;1575:75-81.

Ogden C. & Caroll M., Prevalence of Obesity Among Children and Adolescents: United States, Trends 1963–1965 Through 2007–2008. National Health and Nutrition Examination Survey (NHANES). 2010

Olson AL and Pessin JE. Structure, function, and regulation of the mammalian facilitative glucose transporter gene family. Annu Rev Nutr 1996; 16, 235-256.

71

O’Rourke B. Ion channels as sensors of cellular energy: mechanisms of modulation by magnesium and . Biochem Pharmacol 1993; 46: 1103-1112.

Paolisso G and Barbagallo M. Hypertension, diabetes mellitus, and insulin resistance: the role of intracellular magnesium. Am J Hypertens 1997; 10: 346-355.

Paolisso G, Scheen A, D'Onofrio F, and Lefebvre P. Magnesium and glucose homeostasis. Diabetologia. 1990;33:511–514.

Quamme GA. Renal magnesium handling: New insights in understanding old problems. Kidney Int 1997; 52: 1180-1195.

Randell EW, Mathews M, Gadag V, Zhang H, Sun G. Relationship between serum magnesium values, lipids and anthropometric risk factors. Atherosclerosis. 2008; 196:413-9.

Raju B, Murphy E, Levy LA, Hall RD, and London RE. A fluorescent indicator for measuring cytosolic free magnesium. Am J Physiol 1989; 256: C540–C548.

Rayssiguier Y, Gueux E, Weiser D. Effect of magnesium deficiency on lipid metabolism in rats fed a high carbohydrate diet. J Nutr. 1981;111:1876-83.

Resnick LM. Ionic basis of hypertension, insulin resistance, vascular disease, and related disorders. The mechanism of “syndrome X” Am J Hypertens. 1993;6:123S– 34S.

Resnick LM, Altura BT, Gupta RK, Laragh JH, Alderman MH, and Altura BM. Intracellular and extracellular magnesium depletion in type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia 1993; 36: 767–770.

Romani A. Magnesium homeostasis in mammalian cells. Front Biosci. 2007; 12: 308-331.

Romani AM. Magnesium homeostasis and alcohol consumption. Magnes Res. 2008;21:197-204.

Romani A, Dowell E, and Scarpa A. Cyclic AMP-induced Mg2+ release from rat liver hepatocytes, permeabilized hepatocytes, and isolated mitochondria. J Biol Chem 1991; 266: 24376–24384.

72

Romani AM and Maguire ME. Hormonal regulation of Mg2+ transport and homeostasis in eukaryotic cells. Biometals 2002; 15: 271-283.

Romani A, Marfella C, and Scarpa A. Regulation of Mg2+ uptake in isolated rat myocytes and hepatocytes by protein kinase C. FEBS Lett 1992; 296: 135–140.

Romani A, Marfella C, and Scarpa A. Regulation of magnesium uptake and release in the heart and in isolated ventricular myocytes. Circ Res 1993a; 72: 1139–1148.

Romani A, Marfella C, and Scarpa A. Cell magnesium transport and homeostasis: role of intracellular compartments. Miner Electrolyte Metab 1993c; 19: 282–289.

Romani A, Matthews V, and Scarpa A. Parallel stimulation of glucose and Mg2+ accumulation by insulin in rat hearts and cardiac ventricular myocytes. Circ Res 2000; 86: 326-333.

Romani A and Scarpa A. Norepinephrine evokes a marked Mg2+ efflux from liver cells. FEBS Lett 1990a; 269: 37–40.

Romani A and Scarpa A. Hormonal control of Mg2+ in the heart. Nature 1990b; 346: 841–844.

Romani A and Scarpa A. cAMP control of Mg2+ homeostasis in heart and liver cells. Magnesium Res 1992a; 5: 131–137.

Romani A and Scarpa A. Regulation of cell magnesium. Arch Biochem Biophys 1992b; 298: 1–12.

Romani AM and Scarpa A. Regulation of cellular magnesium. Front Biosci 2000; 5: D720-D734.

Rutter GA, Osbaldeston NJ, McCormack JG, and Denton RM. Measurement of matrix free Mg2+ concentration in rat heart mitochondria by using entrapped fluorescent probes. Biochem J 1990; 271: 627–634.

73

Sales CH, Pedrosa Lde F. Magnesium and diabetes mellitus: their relation. Clin Nutr. 2006; 25:554-62.

Scarpa A and Brinley FJ. In situ measurements of free cytosolic magnesium ions. Fed Proc 1981; 40: 2646–2652.

Schlingmann KP, Waldegger S, Konrad M, Chubanov V, Gudermann T. TRPM6 and TRPM7--Gatekeepers of human magnesium metabolism. Biochim Biophys Acta. 2007; 1772:813-21.

Schmitz C, Deason F, Perraud AL. Molecular components of vertebrate Mg2+- homeostasis regulation. Magnes Res. 2007; 20:6–18.

Schmitz C, Perraud AL, Johnson CO, Inabe K, Smith MK, Penner R, Kurosaki T, Fleig A, and Scharenberg AM. Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7. Cell 2003; 114: 191-200.

Sewter C, Berger D, Considine RV, Medina G, Rochford J, Ciaraldi T, Henry R, Dohm L, Flier JS, O'Rahilly S, Vidal-Puig AJ. Human obesity and type 2 diabetes are associated with alterations in SREBP1 isoform expression that are reproduced ex vivo by tumor necrosis factor-alpha. Diabetes. 2002;51:1035-41.

Shepherd PR, Gnudi L, Tozzo E, Yang H, Leach F, Kahn BB. Adipose cell hyperplasia and enhanced glucose disposal in transgenic mice overexpressing GLUT4 selectively in adipose tissue. J. Biol. Chem. 1993; 268:22243-46

Shimomura I, Bashmakov Y, Horton JD. Increased levels of nuclear SREBP-1c associated with fatty livers in two mouse models of diabetes mellitus. J Biol Chem. 1999 15;274:30028-32.

Shuman H and Somlyo AP. Electron energy loss analysis of neartrace-element concentrations of calcium. Ultramicroscopy 1987; 21: 23–32.

Somlyo AV, McClellan G, Gonzalez-Serratos H, and Somlyo AP. Electron probe X-ray microanalysis of post-tetanic Ca2+ and Mg2+ movements across the sarcoplasmic reticulum in situ. J Biol Chem 1985; 260: 6801–6807.

74

Suarez A, Pulido N, Casla A, Casanova B, Arrieta FJ, Rovira A. Impaired tyrosine-kinase activity of muscle insulin receptors from hypomagnesemic rats. Diabetologia. 1995;38:1262–1270.

Sun L, Kosugi Y, Kawakami E, Piao YS, Hashimoto T, Oyanagi K. Magnesium concentration in the cerebrospinal fluid of mice and its response to changes in serum magnesium concentration. Magnes Res. 2009; 22:266-72.

Takahashi A, Motomura K, Kato T, Yoshikawa T, Nakagawa Y, Yahagi N, Sone H, Suzuki H, Toyoshima H, Yamada N, Shimano H. Transgenic mice overexpressing nuclear SREBP-1c in pancreatic beta-cells. Diabetes. 2005;54:492-9.

Takaya J, Higashino H, Miyazaki R, and Kobayashi Y. Effects of insulin and insulin-like growth factor-1 on intracellular magnesium of platelets. Exp Mol Pathol 1998; 65: 104–109.

Tang EC, Parker PJ, Beattie J, and Houslay MD. Diabetes induces selective alterations in the expression of protein kinase C isoforms in hepatocytes. FEBS 1993; 326: 117-123.

Tashiro M and Konishi M. Na+ gradient-dependent Mg2+ transport in smooth muscle cells of guinea pig tenia cecum. Biophys J 1997; 73: 3371-3384.

Teramoto T, Sternick LA, Kage-Nakadai E, Sajjadi S, Siembida J, et al. Magnesium excretion in C. elegans requires the activity of the GTL-2 TRPM channel. PLoS One 2010; 5:e9589,

Terauchi Y, Kadowaki T. Peroxisome proliferator-activated receptors and insulin secretion. Endocrinology. 2005;146:3263-5.

Tessman PA and Romani A. Acute effect of EtOH on Mg2+ homeostasis in liver cells: evidence for the activation of an Na+/Mg2+ exchanger. Am J Physiol 1998; 275: G1106-G1116.

Tashiro M and Konishi M. Na+ gradient-dependent Mg2+ transport in smooth muscle cells of guinea pig tenia cecum. Biophys J 1997; 73: 3371-3384.

75

Torres LM, Youngner J, Romani A. Role of glucose in modulating Mg2+ homeostasis in liver cells from starved rats. Am J Physiol . 2005;288:G195-206.

Touyz RM and Schiffrin EL. Angiotensin II and vasopressin modulate intracellular free magnesium in vascular smooth muscle cells through Na+-dependent protein kinase C pathways. J Biol Chem 1996; 271: 24353-24358.

Vallim T, Salter AM. Regulation of hepatic gene expression by saturated fatty acids. Leukot Essent Fatty Acids. 2010 82:211-8.

Voets J, Nilius B, Hoefs S, van der Kemp AW, Droogmans G, Bindels RJ, and Hoenderop JG, TRPM6 forms the channel involved in intestinal and renal Mg2+ absorption. J Biol Chem 2004; 279:19–25.

Vorman J and Gunther T. Amiloride sensitive net Mg2+ efflux from isolated perfused rat hearts. Magnesium 1987; 6: 220-224.

Walder RY, Landau D, Meyer P, Shalev H, Tsolia M, Borochowitz Z, Boettger MB, Beck GE, Englehardt RK, Carmi R, Sheffield VC. Mutation of TRPM6 causes familial hypomagnesemia with secondary hypocalcemia. Nat Genet. 2002; 31:171-4.

Wallach S and Verch R. Tissue magnesium content in diabetic rats. Magnesium 1987; 6: 302–306.

White JR Jr and Campbell RK. Magnesium and diabetes: a review. Ann Pharmacother 1993; 27: 775-780.

Wolf FI, Di Francesco A, Covacci V, and Cittadini A. Regulation of intracellular magnesium in ascites cells: involvement of different regulatory pathways. Arch Biochem Biophys 1996; 331: 194-200.

Wolf FI, Di Francesco A, Covacci V, and Cittadini A. Regulation of magnesium efflux from rat spleen lymphocytes. Arch Biochem Biophys 1997; 334: 397–403.

Wood IS and Trayhurn P. Glucose transporters (GLUT and SGLT): expanded families of sugar transport proteins. Br J Nutri 2003; 89: 3-9.

76

Young, A., Berti-Mattera, L., and Romani, A. Effect of Repeated Doses of Ethanol on Hepatic Mg2+ Homeostasis and Mobilization. Alcohol Clin Exp Res 2007; 31: 459- 471.

Zheng Q, Levitsky LL, Mink K, and Rhoads DB. Glucose regulation of glucose transporters in cultured adult and fetal hepatocytes. Metabolism 1995; 44: 1553-1558.

Ziegler A, Somlyo AV, and Somlyo AP. Beta-adrenergic effects on cellular Na+, Mg2+, Ca2+, K+ and Cl- in vascular smooth muscle: Electron probe analysis of rabbit pulmonary artery. Cell Calcium 1992; 13: 593–602.

77