|Regucalcin was discovered in 1978 as a unique calcium-binding protein that does not contain EF-hand motif of calcium-binding domain [1-3]. The name regucalcin, which was proposed for this calcium-binding protein, suppresses calcium signaling that mediates many intracellular responses, which are amplified through calmodulin and protein kinase C [2,4]. The regucalcin gene, which consists of seven exons and six introns, is localized on the X chromosome and is identified in over 15 species consisting of regucalcin (RGN) family . The protein named as senescene marker protein-30 (SMP30), which is identical to regucalcin, is also reported after discovery of regucalcin . Regucalcin has been demonstrated to play a multifunctional role in the regulation in many tissues and cell types [7-12]. Regucalcin plays a pivotal role in maintaining of intracellular Ca2+ homeostasis and as a suppressor protein in various signal transductions in maintaining of cell homeostasis for stimuli. Moreover, regucalcin, which is localized into the nucleus, has been shown to suppress protein kinase and protein phosphatase activities, Ca2+-activated deoxyribonucleic acid (DNA) fragmentation, and DNA and ribonucleic acid (RNA) synthesis in the nucleus of many cell types. Regucalcin has also been shown to suppress protein synthesis and activate proteolysis, suggesting a role in protein turnover. Overexpression of endogenous regucalcin has been demonstrated to suppress cell proliferation and apoptosis, which is mediated through various signal factors, in the cloned rat hepatoma H4-II-E cells and normal rat kidney tubular epithelial NRK52E cells.
|There is growing evidence that regucalcin may be a key molecule in metabolic disorder including diabetes and lipid metabolism. Insulin has been shown to stimulate the regucalcin gene expression in the liver cells. The hepatic regucalcin mRNA level is reduced after fasting of overnight about 70% of that in feeding rats . Re-feeding produces a remarkable elevation of hepatic regucalcin mRNA and protein levels . Oral administration of glucose to fasted rats caused a significant increase in hepatic regucalcin mRNA levels , 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 to fasted rats . These findings suggest that hepatic regucalcin mRNA expression stimulated by re-feeding is involved in the action of insulin as its stimulating factors. Insulin has also been shown to directly stimulate regucalcin mRNA and protein levels by using the cloned human hepatoma cells (HepG2) in vitro . Thus, insulin stimulates regucalcin expression in liver cells. Regucalcin may play a role in cellular regulation that is related to insulin action in liver cells.
|Hepatic regucalcin levels have been shown to decrease about 50% of control levels at 1 or 3 weeks after a single subcutaneous administration of streptozotocin (STZ) which induces type-I diabetic state , suggesting an involvement of regucalcin in diabetes. The decrease in hepatic regucalcin may lead to the disorder of liver metabolism in diabetic state.
|Deficiency of regucalcin has been demonstrated to induce an impairment of glucose tolerance by using regucalcin knockout (KO) mice treated with L-ascorbic acid because deficiency of regucalcin induces a decrease in serum vitamin C [16,17]. Blood glucose levels in regucalcin KO mice were increased by 25% at 30 min after glucose administration compared with wild-type mice . Serum insulin levels in the KO mice were decreased by 37% at 30 min after glucose compared with wild-type mice . Interestingly, an insulin tolerance test shows a greater glucose-lowering effect in regucalcin KO mice , suggesting that the KO mice reveal enhanced insulin sensitivity regardless of vitamin C status . Moreover, high-fat diet feeding severely worsened glucose tolerance in both wild type and regucalcin KO mice .
|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 . 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 . From the proteome analysis of H4-II-E cells exposed to insulin and TNF-α, regucalcin is identified as a protein which is involved in insulin resistance, although it is identified other 13 proteins including regulators of translation, protein degradation, cellular Ca2+ signaling, G-proteins, and free-radical production . Regucalcin, which is known to be a regulatory protein in Ca2+-dependent signaling pathway [7-9], is a molecule that is related to insulin resistance.
|The role of regucalcin in the regulation of glucose utilization and lipid production is shown using the modeled H4-II-E cells overexpressing endogenous regucalcin (transfectants) in vitro . Overexpression of regucalcin stimulates 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 , suggesting that regucalcin may stimulate lipid production, which is linked to glucose metabolism in the cells in vitro. Interestingly, the effect of insulin, which enhances medium glucose consumption, triglyceride and free fatty acid productions in wild-type cells cultured with glucose supplementation, is found to repress in the transfectants overexpressing regucalcin .
|The expression of acetyl-CoA carboxylase, HMG-CoA reductase, glucokinase and pyruvate kinase mRNAs is not changed in wildtype cells and transfectants after culture with or without glucose supplementation in the presence of insulin , suggesting that regucalcin does not have a stimulatory effect on the gene expression of enzymes, which are related to glucose and lipid metabolism. It is possible; however, that regucalcin has a regulatory effect on various enzyme activities, which are related to glucose and lipid metabolism in liver cells.
|Overexpression of regucalcin is 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, although it did not have a significant effect on Insr and PI3K mRNAs expression in the cells cultured in the absence of insulin . Endogenous regucalcin may suppress the gene expression of insulin signaling-related proteins. The suppressive effect of regucalcin on Insr and PI3K mRNAs expression may be important in causing of insulin resistance in H4-II-E cells overexpressing endogenous regucalcin. Insulin resistance in the liver is associated with the pathogenesis of nonalcoholic fatty liver disease.
|Regucalcin has been shown to have a regulatory effect on lipid metabolism. Hepatocytes from regucalcin KO mice but not the wildtype 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 electron-dense bodies in the electron microscope . Biochemical analysis of neutral lipids, total hepatic triglyceride, and cholesterol from regucalcin KO mice showed approximately 3.6- and 3.3-fold higher levels, respectively, than those from age-matched wild-type mice . Moreover, values for total hepatic phospholipids from regucalcin KO mice were approximately 3.7-fold higher than those for their wild-type counterparts.
|Regucalcin transgenic (TG) rats with overexpression of endogenous regucalcin have been shown to induce a remarkable of bone loss associated with increase in serum triglyceride and high-density lipoprotein (HDL)-cholesterol concentrations at the age of 36 weeks in vivo . Serum free fatty acid, triglyceride, cholesterol or HDLcholesterol concentrations were markedly increased in regucalcin TG male and female rats at 14-50 weeks of age . Hyperlipidemia is uniquely induced in regucalcin TG rats with increasing age. Moreover, the change in lipid components in the adipose and liver tissues of regucalcin TG rats with increasing age is shown in vivo . Regucalcin is expressed in the adipose tissues of normal rats . Triglyceride content in the adipose tissues is increased in 50-week-old regucalcin TG rats . This increase may partly contribute hyperlipidemia.
|Triglyceride, total cholesterol, or free fatty acid content in the liver tissues is decreased in 50-week-old regucalcin TG rats . Liver glycogen content is decreased in the TG rats . The expression of regucalcin in the liver tissues is enhanced in regucalcin TG rats . Regucalcin has been shown to have a suppressive effect on the activations of glycogen particulate phosphorylase a, cytoplasmic pyruvate kinase, and fructose 1,6-diphosphatase by Ca2+ and calmodulin in rat liver [4,8,9]. Regucalcin may suppress glycogen synthesisin the liver and stimulate glycogenolysis in regucalcin TG rats. As the result, lipid synthesis may be stimulated in the liver tissues of the TG rats in vivo.
|Leptin and adiponectine are adipokines that are involved in lipid metabolism [24,25]. Leptin mRNA expression in the adipose or liver tissues is found to decrease in 50-week-old regucalcin TG rats . Adiponectin mRNA expression was not changed in the adipose tissues of the TG rats, while the levels were decreased in the liver tissues . These decreases may be partly involved in hyperlipidemia induced in regucalcin TG rats. Thus, regucalcin may play an important role in the disorder of lipid metabolism in the liver.
|Hyperlipidemia has been shown to induce in the lipoprotein lipasedeficient mice , low-density lipoprotein (LDL) receptor-deficient mice , apolipoprotein C3-KO mice , apolipoprotein C1 TG mice , very LDL lipoprotein receptor KO mice , cholesterol 7 alpha-hydroxylase-deficient mice , apoE-deficient mice , and hepatic myr-Akt overexpressing mice . These animal models for hyperlipidemia are involved in molecules that regulate lipid metabolism. Regucalcin has also been proposed to be a key molecule that regulates lipid metabolism.
|As described above, regucalcin plays a pathophysiological role in diabetes and lipid metabolism. Regucalcin, which is stimulated by insulin, is identified as a molecule that is related to insulin resistance in liver cells. Deficiency of regucalcin impaires glucose tolerance and induces lipid accumulation in the liver of mice in vivo . Overexpression of regucalcin reveals a suppression of glycogen content and lipid components in the liver tissues of rats in vivo . Moreover, hyperlipidemia is induced in regucalcin TG rats in vivo . Regucalcin may be a key molecule in diabetes and lipid metabolic disorder in vivo. Thus, regucalcin may be important as a novel protein molecule in the understanding of pathophysiology in diabetes and lipid metabolic disorder. Regucalcin may be a target molecule for therapy of these diseases. Development of further study is expected.
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