Involvement of regucalcin in lipid metabolism and diabetes

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Metabolism www.metabolismjournal.com

Review

Involvement of regucalcin in lipid metabolism and diabetes Masayoshi Yamaguchi a,⁎, Tomiyasu Murata b a b

Department of Medicine, Division of Diabetes, Endocrinology and Metabolism, Baylor College of Medicine, Houston, TX 77030, USA Department of Analytical Neurobiology, Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan

A R T I C LE I N FO

AB S T R A C T

Article history:

Regucalcin (RGN/SMP30) was originally discovered in 1978 as a unique calcium-binding

Received 12 December 2012

protein that does not contain the EF-hand motif of calcium-binding domain. The regucalcin

Accepted 31 January 2013

gene (rgn) is localized on the X chromosome and is identified in over 15 species consisting the regucalcin family. Regucalcin has been shown to play a multifunctional role in cell regulation;

Keywords:

maintaining of intracellular calcium homeostasis and suppressing of signal transduction,

Regucalcin

translational protein synthesis, nuclear deoxyribonucleic acid (DNA) and ribonucleic acid

RGN

(RNA) synthesis, proliferation, and apoptosis in many cell types. Moreover, regucalcin may

Insulin resistance

play a pathophysiological role in metabolic disorder. The expression of regucalcin is

Lipid metabolic disorder

stimulated through the action of insulin in liver cells in vitro and in vivo and it is decreased

Diabetes

in the liver of rats with type I diabetes induced by streptozotocin administration in vivo. Overexpression of endogenous regucalcin stimulates glucose utilization and lipid production in liver cells with glucose supplementation in vitro. Regucalcin reveals insulin resistance in liver cells. Deficiency of regucalcin induces an impairment of glucose tolerance and lipid accumulation in the liver of mice in vivo. Overexpression of endogenous regucalcin has been shown to decrease triglyceride, total cholesterol and glycogen contents in the liver of rats, inducing hyperlipidemia. Leptin and adiponectin mRNA expressions in the liver tissues are decreased in regucalcin transgenic rats. Decrease in hepatic regucalcin is associated with the development and progression of nonalcoholic fatty liver disease and fibrosis in human patients. Regucalcin may be a key molecule in lipid metabolic disorder and diabetes. © 2013 Elsevier Inc. All rights reserved.

1.

Introduction

Regucalcin was discovered in 1978 as a calcium-binding protein that does not contain EF-hand motif as a calciumbinding domain, which is found in many calcium-binding proteins [1]. The name of regucalcin was proposed for this calcium-binding protein, which can regulate various Ca2+-

dependent enzyme activations in liver cells. [1–5]. The protein named senescence marker protein-30 (SMP30), which is identical to regucalcin, has also been reported after discovery of regucalcin [6,7]. The regucalcin gene (rgn) is localized on the X chromosome [8,9], and regucalcin (RGN) is identified in over 15 species and is highly conserved in vertebrate species [10,11]. The expressions of regucalcin mRNA and protein are

Abbreviations: RGN, Regucalcin; SMP30, senescence marker protein-30; DNA, deoxyribonucleic acid; RNA, ribonucleic acid; STZ, streptozotocin; HepG2, human hepatoma cells; KO, knockout; LDL, low-density lipoprotein; TG, transgenic; PI3K, phosphatidylinositol 3kinase; PPAR-γ, peroxisome proliferator-activated receptor-gamma; HSCs, hepatic stellate cells; NAFLD, nonalcoholic fatty liver disease; HDL, high-density lipoprotein. ⁎ Corresponding author. Department of Medicine, Division of Diabetes, Endocrinology and Metabolism, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Tel.: + 1 713 798 6686; fax: +1 713 798 8764. E-mail address: [email protected] (M. Yamaguchi). 0026-0495/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.metabol.2013.01.023

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regulated through various hormonal stimulation and physiological states [12–16]. Regucalcin has been demonstrated to play a multifunctional role in the regulation in various tissues and type cells (reviewed in Refs. [5,17–21]). Regucalcin plays a pivotal role in maintaining of intracellular Ca2+ homeostasis and in suppression of various signal transductions that are induced after stimulations of many hormones and cytokines. Regucalcin is localized from the cytoplasm to nucleus in cells, and it has been shown to suppress Ca2+-dependent protein kinase and protein phosphatase activities, Ca2+-activated DNA fragmentation, and DNA and RNA synthesis in the nucleus. Moreover, regucalcin has been shown to depress protein synthesis and activate proteolysis, suggesting a role in protein turnover. Overexpression of endogenous regucalcin has been demonstrated to depress cell proliferation and apoptosis, which is mediated through various signal factors, in various type cells. Calcium signal plays a pivotal role in the regulation of many intracellular metabolic pathways including glucose metabolism and diabetes [17,18]. Regucalcin has been shown to have a suppressive effect on Ca2+-increased activity of various enzymes, which are implicated in liver glucose metabolism [2,4,5,19,20], suggesting an involvement in the regulation of gluconeogenesis and glycolysis. Regucalcin has also been demonstrated to stimulate adipogenesis in bone marrow cells and adipocytes [21]. Regucalcin may play a role in the regulation of glucose and lipid metabolism. There is growing evidence that regucalcin may be a key molecule in metabolic disorder including lipid metabolism and diabetes in vivo [22]. This review has been written to outline the recent advances that have been made concerning the role of regucalcin in the regulation of lipid metabolism and will discuss an involvement in diabetes.

2. Insulin stimulates liver regucalcin gene expression Insulin has been shown to stimulate the expression of regucalcin mRNA and protein using the cloned human hepatoma cells (HepG2) in vitro [23]. Regucalcin mRNA was expressed in HepG2 cells, although its expression is lower compared with that of normal rat liver [23]. Regucalcin protein in HepG2 cells was detected using Western blot analysis with a polyclonal rabbit anti-regucalcin antibody [21]. Regucalcin mRNA expression and protein level in HepG2 cells were stimulated culture with insulin (10−8 mol/L) of an effective concentration [21]. Insulin has been found to stimulate regucalcin expression in HepG2 cells in vitro. Insulin has also been shown to stimulate regucalcin expression in the liver of rats in vivo [24]. When rats were fasted for overnight, the hepatic regucalcin mRNA level was reduced about 70% of that in feeding rats [24]. Re-feeding produced a remarkable elevation of hepatic regucalcin mRNA level (about 150%–170% of fasted rats) [24]. Liver regucalcin concentration was increased after re-feeding, although it was not changed after fasting [24]. Oral administration of glucose (2 g/kg body weight) to fasted rats caused a significant

increase in hepatic regucalcin mRNA levels [24], suggesting an involvement of insulin secreted from pancreatic cells after glucose administration. Moreover, hepatic regucalcin mRNA level was clearly elevated after a single subcutaneous administration of insulin (10 and 100 U/kg body weight) to fasted rats in vivo [24]. Thus, insulin has been demonstrated to stimulate regucalcin expression in liver cells in vitro and in vivo, suggesting that regucalcin may be involved in liver metabolism, possibly being a mediator of insulin action.

3.

Regucalcin and insulin resistance

Insulin resistance may be modeled in the cloned rat hepatoma H4-II-E cells in tissue culture with the use of the cytokine tumor necrosis factor-alpha (TNF-α) and insulin [25]. This tissue-culture model nicely mimics insulin resistance in human type 2 diabetic mellitus. H4-II-E cells were cultured with insulin alone, TNF-α alone, and TNF-α plus insulin, as well as a control sample [25]. From the proteome analysis of H4-II-E cells exposed to insulin and TNF-α, regucalcin was identified as a protein which is involved in insulin resistance [25]; 13 other proteins were also identified including eukaryotic translation initiation factor-3, subunit 2, regulator of Gprotein signaling-5, superoxide dismutase, protein disulfide isomerase A6, proteasome subunit-alpha type 3, disulfide isomerase A6, cell-division protein kinase-4, kinogen heavy chain, carbonic anhydrase-7, E 3 ubiquitin protein ligase, UREB1; Rab GDP dissociation inhibitor-beta, Rab GDP dissociation inhibitor-beta2, and MAWDBP [25]. Differentially expressed proteins, which are affected by treatment with insulin or with TNF-α plus insulin, included molecule for regulators of translation, protein degradation, cellular Ca2+ signaling, Gproteins, and free-radical production [25]. The role of regucalcin in the regulation of glucose utilization and lipid production has been shown using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin (transfectants) in vitro [26]. Overexpression of endogenous regucalcin was found to stimulate the production of triglyceride and free fatty acid in H4-II-E cells cultured with or without the supplementation of glucose in the absence of insulin [26], suggesting that regucalcin may stimulate lipid production, which is linked to glucose metabolism in the cells in vitro. Culture with insulin enhanced an increase in medium glucose consumption, triglyceride, and free fatty acid productions in wild-type cells cultured with glucose supplementation [26]. Interestingly, this enhancing effect of insulin was found to depress in the transfectants overexpressing regucalcin [26]. The expression of acetyl-CoA carboxylase, HMG-CoA reductase, glucokinase, pyruvate kinase, and glyceroaldehyde-3-phosphate dehydrogenase mRNAs in wild-type cells was not changed after culture with or without glucose supplementation in the presence of insulin [26]. These gene expressions were not changed in the transfectants [26]. Thus, regucalcin did not have a stimulatory effect on the gene expression of enzymes, which are related to glucose and lipid metabolisms, in H4-II-E cells. However, regucalcin may have a

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regulatory effect on various enzyme activities, which are related to glucose and lipid metabolism, in H4-II-E cells. The expression of glucose transporter 2 (GLUT 2) mRNA has been found to increase in the transfectants as compared with that of wild-type cells [26]. This increase was not changed in the transfectants cultured in the presence of insulin with or without glucose supplementation [26]. The expression of GLUT 2 mRNA in the transfectants may be partly involved in the enhancement of glucose utilization revealed in H4-II-E cells overexpressing endogenous regucalcin. The effect of regucalcin on the gene expression of insulin signaling-related proteins has been examined using H4-II-E cells overexpressing endogenous regucalcin in vitro [27]. Overexpression of regucalcin was found to have a suppressive effect on the expression of rat insulin receptor (Insr) or phosphatidylinositol 3-kinase (PI3K) mRNAs enhanced after culture with glucose supplementation in the presence of insulin, while it did not have a significant effect on Insr, PI3K, or glyceroaldehyde-3phosphate dehydrogenase mRNA expression in the cells cultured in the absence of insulin [27]. This finding suggests that overexpression of endogenous regucalcin suppresses the gene expression of insulin signaling-related proteins. The suppressive effect of regucalcin on Insr and PI3K mRNA expressions may be important in revelation of insulin resistance in H4-II-E cells overexpressing endogenous regucalcin.

4.

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Deficiency of regucalcin may induce an impairment of glucose tolerance. This has been examined using regucalcin knockout (KO) mice treated with L-ascorbic acid, because deficiency of regucalcin induces a decrease in serum vitamin C [29,30]. In an intraperitoneal glucose tolerance test at 15 weeks of age of mice, blood glucose levels were increased and serum insulin levels were decreased in regucalcin KO mice after glucose administration as compared with those of wild-type mice [29]. Interestingly, an insulin tolerance test showed a greater glucose-lowering effect in regucalcin KO mice [29], suggesting that the KO mice reveal enhanced insulin sensitivity regardless of vitamin C status [29]. Moreover, high-fat diet feeding severely worsened glucose tolerance in both wild type and regucalcin KO mice [29]. Morphometric analysis revealed no differences in the degree of high-fat diet-induced compensatory increase in β-cell mass and proliferation [29]. In the static incubation study of islets, insulin secretion in response to glucose or potassium chloride was decreased in regucalcin KO mice [29]. Islet adenosine triphosphate (ATP) content in regucalcin KO mice was similar to that in wild-type mice [29]. These observations suggest that regucalcin deficiency impairs the distal portion of insulin secretion pathway [29]. Deficiency of regucalcin may contribute to the worsening of glucose tolerance through suppressing of insulin secretion [29,30]. Regucalcin in pancreatic cells may regulate insulin secretion in the cells.

Involvement of regucalcin in diabetes

Hepatic regucalcin content has been shown to be suppressed in type-I diabetic state induced by administration of streptozotocin (STZ) [28]. When STZ (60 mg/kg body weight) was subcutaneously administered in rats and 1 or 3 weeks later they were sacrificed by bleeding, the liver regucalcin mRNA levels were not altered in diabetic state as evidenced by Northern blotting [28]. However, based on enzyme-linked immunoadsorbent assay with rabbit-anti-regucalcin IgG, the hepatic regucalcin concentration was found to decrease about 50% of control levels after STZ treatment [28]. Serum regucalcin concentration was not significantly changed after STZ treatment [28]. The decrease in liver regucalcin concentration may not be based on the decrease in liver regucalcin mRNA expression and an increase in serum regucalcin release. Thus, the decrease in hepatic regucalcin concentration was found in STZ-diabetic rats. The mechanism by which liver regucalcin is suppressed in STZ-diabetic state may be complex. The biosynthesis of regucalcin at the translational process may be suppressed in STZ-diabetic state. The metabolic degradation of regucalcin in the liver may also be promoted. Insulin stimulates both regucalcin mRNA and protein expressions [23,24]. The secretion of insulin in pancreatic cells is impaired in STZ diabetic state. The impairment of insulin secretion may be partly related to the decrease in liver regucalcin. In addition, serum transaminases activities were significantly elevated in STZdiabetic state, suggesting that liver injury is induced [28]. The disorder of liver metabolism, which was affected in diabetic state, may result from the decrease in liver regucalcin. Presumably, the decrease in hepatic regucalcin may lead to the disorder of liver metabolism in diabetic state.

5. Involvement of regucalcin in lipid metabolic disorder Regucalcin has been shown to have a regulatory effect on lipid metabolism. Culture with exogenous regucalcin has been found to stimulate adipogenesis in bone marrow cells ex vivo and adipocytes in vitro [21], suggesting an involvement in lipid metabolism. The role of regucalcin in the regulation of lipid metabolism, however, has been poorly understood. Hepatocytes from regucalcin KO mice but not the wild-type mice at 12 months of age have been shown to contain many lipid droplets, abnormally enlarged mitochondria with indistinct cristae, and enlarged lysosomes filled with electrondense bodies in the electron microscope [31]. In liver specimens from regucalcin KO mice, the marked number of lipid droplets visible around the central vein increased notably in size and amount as the animals aged [31]. Biochemical analysis of neutral lipids, total hepatic triglyceride, and cholesterol from regucalcin KO mice showed higher levels as compared with those from age-matched wild-type mice [31]. Moreover, values for total hepatic phospholipids from regucalcin KO mice were higher than those for their wild-type counterparts. Deficiency of regucalcin induced accumulation of neutral lipids and phospholipids including phosphatidylethanolamine, cardiolipin, phosphatidylcholine, phosphatidylserine, and sphingomyelin in the liver of regucalcin KO mice [31]. Regucalcin transgenic (TG) rats with overexpression of endogenous regucalcin has been shown to induce bone loss associated with increase in serum triglyceride and highdensity lipoprotein (HDL)-cholesterol concentrations at the

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age of 36 weeks in vivo [32–35]. Serum free fatty acid, triglyceride, cholesterol or HDL-cholesterol concentration was markedly increased in regucalcin TG male and female rats at 14–50 weeks of age [34]. Moreover, serum calcium concentration was raised in regucalcin TG male and female rats at 50 weeks of age [34]. Also, serum albumin concentration was elevated in regucalcin TG female rats at 25–50 weeks of age [34]. Serum zinc, glucose or urea nitrogen concentration was not altered in TG male and female rats [34]. These findings demonstrate that hyperlipidemia is uniquely induced in regucalcin TG rats with increasing age in vivo. The change in lipid components in the adipose and liver tissues of regucalcin TG rats with increasing age, moreover, has been examined [35]. Regucalcin was expressed in the adipose tissues of normal rats [35]. Regucalcin expression in the adipose tissues was not enhanced in the TG rats [35]. Whether regucalcin stimulates the release of the adipose tissue lipid components into the serum of regucalcin TG rats is unknown. Triglyceride content in the adipose tissues was increased in 50-week-old regucalcin TG rats, while the free fatty acid content was not changed [35]. This increase in serum lipid components may partly result from that increased lipids in the adipose tissues were released in serum. Triglyceride, total cholesterol, free fatty acid, or glycogen content in the liver tissues has been found to decrease in regucalcin TG rats [35]. The expression of regucalcin in the liver tissues was enhanced in regucalcin TG rats [32]. Regucalcin has been shown to have a suppressive effect on the activations of glycogen particulate phosphorylase α [4], cytoplasmic pyruvate kinase [19] and fructose 1,6-diphosphatase [2] by Ca2+ and calmodulin in rat liver. Regucalcin may suppress glycogen synthesis and stimulate glycogenol-

Glycogen Regucalcin

GLUT 2

Glucose

Glucose

Lipids Mitochondria Insulin receptor

Insulin

PI3 kinase Nucleus

Regucalcin

Liver Fig. 1 – Possible mechanism by which overexpression of regucalcin induces hyperlipidemia. Overexpression of regucalcin stimulates glucose utilization and lipid production in the cloned rat hepatoma H4-II-E cells. Regucalcin increases GLUT 2 mRNA expression to enhance glucose utilization in the cells, and it suppresses the gene expressions of insulin receptor or PI3 kinase, which is enhanced after culture with insulin and/or glucose supplementation. Overexpression of regucalcin induces insulin resistance.

ysis in the liver of regucalcin TG rats. As a result, lipid synthesis may be stimulated in the liver tissues of the TG rats in vivo. Leptin and adiponectin are adipokines that are involved in lipid metabolism [36,37]. Leptin mRNA expression in the adipose or liver tissues was found to be decreased in 50week-old regucalcin TG rats [35]. Adiponectin mRNA expression was not changed in the adipose tissues of 50-week-old regucalcin TG rats, while it was decreased in the liver tissues [35]. These decreases may be partly involved in hyperlipidemia induced in regucalcin TG rats. From our findings with in vitro and in vivo experiments in liver cells, the possible mechanism by which overexpression of regucalcin induces hyperlipidemia is summarized in Fig. 1 [26,27,34,35]. The involvement of regucalcin in adipocytes, however, remains to be elucidated. Hyperlipidemia has been shown to induce in the lipoprotein lipase-deficient mice [38], low-density lipoprotein (LDL) receptor-deficient mice [39], apolipoprotein C3-KO mice [40], apolipoprotein C1 TG mice [41], very LDL lipoprotein receptor KO mice [42], cholesterol 7 alpha-hydroxylase-deficient mice [43], apoE-deficient mice [44], and hepatic myr-Akt overexpressing mice [45]. These animal models for hyperlipidemia are involved in molecules that regulate lipid metabolism. Regucalcin TG rats may be significant as an animal model for hyperlipidemia. Regucalcin has been proposed to be a key molecule that regulates lipid metabolism.

6. Regucalcin and nonalcoholic fatty liver disease Hepatic regucalcin expression has been shown to be suppressed in liver disease. The expression of regucalcin mRNA and protein was suppressed after the administration of carbon tetrachloride [46], galactosamine [47], and ethanol [28], which induce liver disorder, in rats in vivo. Serum release of hepatic regucalcin was caused after liver impairment [46,47]. Regucalcin was found to have a potential sensitivity as a marker of chronic liver disease in human subjects [48]. Whether the suppression of liver regucalcin expression is a result or cause of liver injury is unknown. However, regucalcin plays a multifunctional role in liver metabolism [49–51]. Liver disease, which is related to lipid metabolic disorder, may be developed through the decrease in hepatic regucalcin expression. The involvement in regucalcin in liver fibrosis has been examined using regucalcin KO mice. Carbon tetrachloride administration-induced liver fibrosis and the nuclear translocation of p-Smad2/3, which is the immediate downstream of transforming growth factor-β, were found to be inhibited in the liver of regucalcin KO mice as compared with wild-type mice [52]. Regucalcin was not expressed in hepatic stellate cells (HSCs) of both wild type and SMP30 KO mice [53]. Peroxisome proliferator-activated receptor-gamma (PPAR-γ) was up-regulated in the liver of regucalcin KO mice [53]. Numerous HSCs were hypertrophic and contained abundant microvesicular lipid droplets in the liver cytoplasm of aged regucalcin KO mice [53]. The expression of PPAR-γ, which is a protein related to lipid metabolism and HSC quiescence, was

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Table 1 – Involvement of regucalcin in lipid metabolism and diabetes. Condition Liver cell Glucose uptake Lipid production Gene expression

Diabetes

Lipid metabolic disorder

Nonalcoholic fatty liver disease

Materials H4-II-E cell H4-II-E cell H4-II-E cell

STZ rat liver KO mice liver KO mice pancreas KO mice liver TG rat liver TG rat adipose

TG rat Human liver

also found to be increased in hypertrophic HSCs of regucalcin KO mice [53]. Regucalcin has been shown to be a molecule that is related to insulin resistance [25–27]. Insulin resistance in the liver is associated with the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Change in hepatic regucalcin levels may be associated with the development and progression of NAFLD [54]. Patients with NAFLD had a significant lower level of hepatic regucalcin [54]. Hepatic regucalcin levels decreased in a fibrosis stage-dependent manner and were correlated negatively with the homeostasis model assessment of insulin resistance, the net electronegative charge modified-LDL, and type IV collagen 7S [54]. The immune-staining intensity levels of 4-hydroxynonenal in the liver were significantly and inversely correlated with hepatic regucalcin levels [54]. Both serum large very LDL and very small LDL levels were elevated in patients with NAFLD [54]. Whether the decrease in hepatic regucalcin in human patients is a result or a cause of cirrhosis remains to be elucidated, however [54].

7.

Prospect

Regucalcin plays a pathophysiological role in lipid metabolic disorder and diabetes as summarized in Table 1 [26– 29,31,34,35,53,54]. Regucalcin, which is stimulated by insulin, is identified as a molecule that is related to insulin resistance in liver cells. Moreover, deficiency and overexpression of endogenous regucalcin have been demonstrated to induce diabetes and lipid metabolic disorder. Based on these animal experiments, regucalcin may be a key molecule in the understanding of pathophysiology of diabetes and lipid metabolic disorder. At present, human studies of regucalcin are very few. Application to human metabolism of regucalcin will be expected in future studies. Regucalcin may be a target molecule for therapy of lipid metabolic disorder and diabetes in human patients. Specific therapies for regucalcin will be innovated and developed.

Molecule Glucose Triglyceride, Free fatty acid Glucose transporter 2 Insulin receptor Phosphatidylinositol 3-kinase Acetyl-CoA carboxylase HMG-CoA reductase Glucokinase, Pyruvate kinase Regucalcin Glucose tolerance Insulin secretion Lipids, Phospholipids, PPAR-γ Triglyceride, Cholesterol, Fatty acid Triglyceride Leptin Adiponectin Hyperlipidemia Regucalcin

Effect Stimulation Stimulation Stimulation Suppression No change Decrease Impairment Suppression Accumulation Decrease Decrease Increase Decrease No change Decrease

Author contribution and disclosures Masayoshi Yamaguchi and Tomiyasu Murata contributed to the design and conduct of the study, collection, analysis, and interpretation of data, and manuscript writing. All authors state that they have no conflicts of interest.

Funding Regucalcin studies of the author (MY) were supported by a Grant-in-Aid for Scientific Research (C) No. 04671362, No. 06672193 and No. 13672292 from the Ministry of Education, Science, Sports, and Culture, Japan. Also, the author (MY) was awarded the Bounty of Encouragement Foundation in Pharmaceutical Research, Japan and the Bounty of the Yamanouchi Foundation for Research on Metabolic Disorders, Japan. This study was also supported by the Fundation for Biomedical Research on Regucalcin.

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