Gamma-glutamyl transferase variability can predict the development of end-stage of renal disease: a nationwide population-based study.

The aim of this study is to investigate whether GGT variability is able to predict the risk of end-stage renal disease (ESRD). The study subjects were Koreans who conducted health exams supported by the Korean National Health Insurance Corporation during 2009-2012 (baseline). After excluding individuals aged < 40 years, heavy alcoholics, or those with histories of chronic liver disease or ESRD, we followed 6,058,995 individuals. We calculated the average successive variability (ASV) of GGT values during the 5 years before the baseline as a parameter of variability. Using Cox proportional analyses, we evaluated the risk of ESRD according to GGT ASV quartiles, defined as the initiation of renal replacement therapy or kidney transplantation, or December 31, 2016. During 38,663,279.3 person-years of follow-up, 12,057 cases of ESRD were identified. Compared with GGT ASV quartile 1, the risk of ESRD was higher in ASV quartiles 3-4 and increased serially, even after adjustment for several metabolic parameters, baseline renal function, presence of comorbidities, low income, and baseline GGT and hemoglobin level. The fully adjusted hazard ratios (95% confidence intervals) of GGT ASV quartiles 3 and 4 were 1.06 (1.01-1.12) and 1.12 (1.06-1.18), respectively. In conclusion, GGT variability is a putative risk factor for ESRD in Koreans.

Author Correction: Apolipoprotein J is a hepatokine regulating muscle glucose metabolism and insulin sensitivity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Apolipoprotein J is a hepatokine regulating muscle glucose metabolism and insulin sensitivity.

Crosstalk between liver and skeletal muscle is vital for glucose homeostasis. Hepatokines, liver-derived proteins that play an important role in regulating muscle metabolism, are important to this communication. Here we identify apolipoprotein J (ApoJ) as a novel hepatokine targeting muscle glucose metabolism and insulin sensitivity through a low-density lipoprotein receptor-related protein-2 (LRP2)-dependent mechanism, coupled with the insulin receptor (IR) signaling cascade. In muscle, LRP2 is necessary for insulin-dependent IR internalization, an initial trigger for insulin signaling, that is crucial in regulating downstream signaling and glucose uptake. Of physiologic significance, deletion of hepatic ApoJ or muscle LRP2 causes insulin resistance and glucose intolerance. In patients with polycystic ovary syndrome and insulin resistance, pioglitazone-induced improvement of insulin action is associated with an increase in muscle ApoJ and LRP2 expression. Thus, the ApoJ-LRP2 axis is a novel endocrine circuit that is central to the maintenance of normal glucose homeostasis and insulin sensitivity.

Melatonin improves insulin resistance and hepatic steatosis through attenuation of alpha-2-HS-glycoprotein.

Melatonin plays an important role in regulating circadian rhythms. It also acts as a potent antioxidant and regulates glucose and lipid metabolism, although the exact action mechanism is not clear. The α2-HS-glycoprotein gene (AHSG) and its protein, fetuin-A (FETUA), are one of the hepatokines and are known to be associated with insulin resistance and type 2 diabetes. The aim of this study was to determine whether melatonin improves hepatic insulin resistance and hepatic steatosis in a FETUA-dependent manner. In HepG2 cells treated with 300 µmol/L of palmitic acid, phosphorylated AKT expression decreased, and FETUA expression increased, but this effect was inhibited by treatment with 10 µmol/L of melatonin. However, melatonin did not improve insulin resistance in FETUA-overexpressing cells, indicating that improvement in insulin resistance by melatonin was dependent on downregulation of FETUA. Moreover, melatonin decreased palmitic acid-induced ER stress markers, CHOP, Bip, ATF-6, XBP-1, ATF-4, and PERK. In addition, in the high-fat diet (HFD) mice, oral treatment with 100 mg/kg/day melatonin for 10 weeks reduced body weight gain to one-third of that of the HFD group and hepatic steatosis. Insulin sensitivity and glucose intolerance improved with the upregulation of muscle p-AKT protein expression. FETUA expression and ER stress markers in the liver and serum of HFD mice were decreased by melatonin treatment. In conclusion, melatonin can improve hepatic insulin resistance and hepatic steatosis through reduction in ER stress and the resultant AHSG expression.

Transcriptional activation of p21(WAF¹/CIP¹) is mediated by increased DNA binding activity and increased interaction between p53 and Sp1 via phosphorylation during replicative senescence of human embryonic fibroblasts.

Although p21(WAF1/CIP1) is known to be elevated during replicative senescence of human embryonic fibroblasts (HEFs), the mechanism for p21 up-regulation has not been elucidated clearly. In order to explore the mechanism, we analyzed expression of p21 mRNA and protein and luciferase activity of full-length p21 promoter. The result demonstrated that p21 up-regulation was accomplished largely at transcription level. The promoter assay using serially-deleted p21 promoter constructs revealed that p53 binding site was the most important site and Sp1 binding sites were necessary but not sufficient for transcriptional activation of p21. In addition, p53 protein was shown to interact with Sp1 protein. The interaction was increased in aged fibroblasts and was regulated by phosphorylation of p53 and Sp1. DNA binding activity of p53 was significantly elevated in aged fibroblasts but that of Sp1 was not. DNA binding activities of p53 and Sp1 were also regulated by phosphorylation. Phosphorylation of p53 at serine-15 and of Sp1 at serines appears to be involved. Taken together, the result demonstrated that p21 transcription during replicative senescence of HEFs is up-regulated by increase in DNA binding activity and interaction between p53 and Sp1 via phosphorylation.

Antidiabetic and Beta cell-protection activities of purple corn anthocyanins.

Antidiabetic and beta cell-protection activities of purple corn anthocyanins (PCA) were examined in pancreatic beta cell culture and db/db mice. Only PCA among several plant anthocyanins and polyphenols showed insulin secretion activity in culture of HIT-T15 cells. PCA had excellent antihyperglycemic activity (in terms of blood glucose level and OGTT) and HbA1c-decreasing activity when compared with glimepiride, a sulfonylurea in db/db mice. In addition, PCA showed efficient protection activity of pancreatic beta cell from cell death in HIT-T15 cell culture and db/db mice. The result showed that PCA had antidiabetic and beta cell-protection activities in pancreatic beta cell culture and db/db mice.

ATM mediates interdependent activation of p53 and ERK through formation of a ternary complex with p-p53 and p-ERK in response to DNA damage.

DNA damage in eukaryotic cells induces signaling pathways mediated by the ATM, p53 and ERK proteins, but the interactions between these pathways are not completely known. To address this issue, we performed a time course analysis in human embryonic fibroblast cells treated with DNA-damaging agents. DNA damage induced the phosphorylation of p53 at Ser 15 (p-p53) and the phosphorylation of ERK (p-ERK). Inhibition of p53 by a dominant negative mutant or in p53(-/-) fibroblast cells abolished ERK phosphorylation. ERK inhibitor prevented p53 phosphorylation, indicating that phosphorylations of p53 and p-ERK are interdependent each other. A time course analysis showed that ATM interacted with p-p53 and p-ERK in early time (0.5 h) and interaction between ATM-bound p-p53 and p-ERK or ATM-bound p-ERK and p-p53 occurred in late time (3 h) of DNA damage. These results indicate that ATM mediates interdependent activation of p53 and ERK through formation of a ternary complex between p-p53 and p-ERK in response to DNA damage to cause growth arrest.

Effect of combined treatment with progesterone and tamoxifen on the growth and apoptosis of human ovarian cancer cells.

Progesterone has a potential protective effect against ovarian carcinoma induced by estrogen. Progesterone is also known to cause apoptosis while tamoxifen induces growth arrest. Therefore, we attempted to determine whether combined treatment with progesterone and tamoxifen has a synergistic effect on anti-cancer activity. Although progesterone is known to cause apoptosis while tamoxifen induces growth arrest in many cancer cells, the detailed action of progesterone and tamoxifen and the anticancer effect of combined treatment have not been tested in ovarian cancer cells. Therefore, we tested the growth and apoptosis activity of progesterone and tamoxifen and the anticancer effect of combined treatment of progesterone and tamoxifen in ovarian cancer cells. Ovarian cancer cells, PA-1, were treated with progesterone, tamoxifen, or a combination of progesterone and tamoxifen. The anti-cancer effects were investigated by use of flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, DNA fragmentation analysis, and Western blot analysis. We found that 100 µM progesterone induced typical apoptosis in PA-1 cells. Treatment of PA-1 cells with 10 µM tamoxifen resulted in an increase in the levels of p21, p27, p16 and phospho-pRb, indicating typical G1 arrest. Co-treatment of PA-1 cells with 100 µM progesterone and 10 µM tamoxifen resulted in typical apoptosis, similar to that induced by treatment with 100 µM progesterone alone. These results indicate that progesterone caused apoptosis and tamoxifen induced G1 arrest. Combined treatment with tamoxifen and progesterone caused apoptosis similar to that induced by treatment with progesterone alone and had no additional anti-cancer effect in ovarian cancer cells.

Higher DNA repair activity is related with longer replicative life span in mammalian embryonic fibroblast cells.

Since the detailed comparison of DNA repair activities among mammalian embryonic fibroblast cells with different replicative life spans has not been investigated, we tested DNA repair activities in embryonic fibroblast cells derived from mammals including human, dog, rat, and mouse. The cell viability after treatment of four DNA damage agents appeared to be decreased in the order of human embryonic fibroblasts (HEFs) > dog embryonic fibroblasts (DEFs) > rat embryonic fibroblasts (REFs) > mouse embryonic fibroblasts (MEFs) although statistical significance was lacking. The amounts of strand breaks and AP (apurinic/apyrimidinic) sites also appear to be decreased in the order of HEFs > DEFs > REFs ≥ MEFs after treatment of DNA damage agents. The DNA repair activities and rates including base excision repair (BER), nucleotide excision repair (NER) and double-strand break repair (DSBR) including non-homologous end-joining (NHEJ) decreased again in the order of HEFs > DEFs > REFs ≥ MEFs. BER and NHEJ activities in 3% O(2) also decreased in the order of HEFs > DEFs > REFs > MEFs. This order in DNA repair activity appears to be coincident with that of replicative life span of fibroblasts and that of life span of mammals. These results indicate that higher DNA repair activity is related with longer replicative life span in embryonic fibroblast cells.

Calorie restriction (CR) reduces age-dependent decline of non-homologous end joining (NHEJ) activity in rat tissues.

Even though CR has shown to enhance base excision repair (BER) and nucleotide excision repair (NER) capacities, it has not been reported whether CR can enhance non-homologous end joining (NHEJ) activity. To examine the effect of CR on NHEJ activity, ad libitum (AL)- and calorie restricted (CR)-dieted rats were used. Age-dependent decline of NHEJ activity was apparent in the lung, liver, and kidney and appeared to be slightly decreased in spleen. CR reduced age-dependent decline of NHEJ activity in all tissues, even though the extent of recovery was variable among tissues. Moreover, CR appeared to reduce age-dependent decline of XRCC4 protein level. These results suggest that CR could reduce age-dependent decline of NHEJ activity in various tissues of rats possibly through up-regulation of XRCC4.

ERK mediates anti-apoptotic effect through phosphorylation and cytoplasmic localization of p21Waf1/Cip1/Sdi in response to DNA damage in normal human embryonic fibroblast (HEF) cells.

Since anti-apoptotic effect of ERK has not been elucidated clearly in DNA-damage-induced cell death, the role of ERK was examined in normal HEF cells treated with mild DNA damage using etoposide or camptothecin. ERK was activated by DNA damage in HEF cells. PD98059 increased apoptosis and reduced DNA-damage-induced p21Waf1/Cip1/Sdi level. Depletion of p21Waf1/Cip1/Sdi induced cell death and PD98059 induced additional cell death. DNA-damage-induced increase in cytoplasmic localization and phosphorylation of threonine residues of p21Waf1/Cip1/Sdi was reversed by PD98059. Thus, the results suggest that ERK pathway mediates anti-apoptotic effects through phosphorylation and cytoplasmic localization of p21Waf1/Cip1/Sdi in response to mild DNA damage.

Nitric oxide is an essential mediator for neuronal differentiation of rat primary cortical neuron cells.

Nitric oxide (NO) regulates proliferation, differentiation and survival of neurons. Although NO is reported to involve in NGF-induced differentiation of PC12 cells, the role of NO has not been characterized in primary neuron cells. Therefore, we investigated the role of NO in neuronal differentiation of primary cortical neuron cells. Primary cortical neuron cells were prepared from rat embryos of embryonic day 18 and treated with NMMA (NOS inhibitor) or PTIO (NO scavenger). Neurite outgrowth of neuron cells was counted and the mRNA levels of p21, p27, c-jun and c-myc were measured by RT-PCR. Neurite outgrowth of primary cortical neuron cells was inhibited a little by NOS inhibitor and completely by NO scavenger. The mRNA levels of p21 and p27, differentiation-induced growth arrest genes were increased during differentiation, but they were decreased by NOS inhibitor or NO scavenger. On the other hand, the level of c-jun mRNA was not changed and the level of c-myc mRNA was increased during differentiation differently from previously reported. The levels of these mRNA were reversed in NOS inhibitor- or NO scavenger-treated cells. The level of nNOS protein was not changed but NOS activity was inhibited largely by NOS inhibitor or NO scavenger. These results suggest that NO is an essential mediator for neuronal differentiation of primary cortical neuron cells.

Nicotinamide reduces dopamine in postnatal hypothalamus and causes dopamine-deficient phenotype.

Dopamine is an important neurotransmitter in the human central nervous system and also plays a key role in the development of postnatal brains. We previously reported that nicotinamide, a SIRT1 inhibitor, regulates tyrosine hydroxylase (TH) expression in vitro. To investigate the effect of nicotinamide-mediated TH regulation in vivo, nicotinamide was chronically injected into neonatal mice. Interestingly, nicotinamide-treated mice were smaller in size, and their locomotor activity was reduced. L-DOPA treatment caused hypersensitive locomotor activity that indicates a dopamine-depleted state. These changes seemed to be associated with dopamine metabolism in hypothalamus, since dopamine in hypothalamus was reduced but not in striatum. The present study suggests that the regulation of dopamine metabolism during the postnatal development is important and the underlying molecular mechanisms may be associated with SIRT1 signaling.

SIRT1 regulates tyrosine hydroxylase expression and differentiation of neuroblastoma cells via FOXO3a.

To examine the function of SIRT1 in neuronal differentiation, we employed all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells. Nicotinamide inhibited neurite outgrowth and tyrosine hydroxylase (TH) expression. Inhibition of PARP or histone deacetylase did not inhibit TH expression, showing the effect to be SIRT1 specific. Expression of FOXO3a and its target proteins were increased during the differentiation and reduced by nicotinamide. FOXO3a deacetylation was increased by ATRA and blocked by nicotinamide. SIRT1 and FOXO3a siRNA inhibited ATRA-induced up-regulation of TH and differentiation. Taken together, these results indicate that SIRT1 is involved in ATRA-induced differentiation of neuroblastoma cells via FOXO3a.

Preparation and properties of collagen/modified hyaluronic acid hydrogel for biomedical application.

Hydrogels composed of collagen and hyaluronic acid are types of crosslinked water-swellable polymers and possess vast potential for applications in the medical industry. Collagen (Co) is the major structural protein of connective tissues such as skin, tendon and cartilage. Hyaluronic acid (HA) is a non-immunogenic, non-adhesive glycosaminoglycan that has a high water absorption property and plays significant roles in several cellular processes. The purpose of this study is to prepare a collagen (Co)-modified hyaluronic acid (MHA) hydrogel and investigate its potential utility for biomedical products such as wound dressing materials. Collagen (Co, type I) was obtained from pig skin and mucopolysaccharide-HA was modified by a poly (ethylene glycol) diglycidyl ether (PEGDGE) crosslinker. Thermal stability, swelling behavior, and mechanical strength of Co-MHA hydrogel according to different mass ratios of Co and MHA in hydrogel networks were investigated. The physical properties of the hydrogel were measured by SEM, Differential Scanning Calorimetry (DSC), Thermal Gravity Analysis (TGA), and a Universal Testing Machine (UTM). The cell viability of Co-MHA hydrogel was also evaluated using an in vitro MTT assay.

The role of tonicity responsive enhancer sites in the transcriptional regulation of human hsp70-2 in response to hypertonic stress.

The inducible 70 kDa heat shock proteins (Hsp70) in mice are encoded by two almost identical genes, hsp70.1 and hsp70.3. Studies have found that only hsp70.1 is induced by hypertonic stress while both hsp70.1 and hsp70.3 genes are expressed in response to heat shock stress. It is unclear if the human counterparts, hsp70-2 and hsp70-1, are differentially regulated by heat shock and osmotic stress. This study found that only hsp70-2 was induced by hypertonic stress in human embryonic kidney epithelial cells and fibroblasts, while heat shock stress induced both hsp70-1 and hsp70-2. The human hsp70-2 promoter region contains three TonE (tonicity-responsive enhancer) sites, which were reported to play an important role in the response to hypertonicity. When the reporter plasmids containing different parts of the 5' flanking region of hsp70-2 were transfected into human embryonic kidney epithelial cells or fibroblasts, one TonE site at -135 was found to play a key role in the response to hypertonicity. The inactivation of the TonE site using site-directed mutagenesis led to the complete loss of induction by hypertonicity, which demonstrates the essential role of the TonE site. This suggests that the TonE site and the TonEBP (TonE binding protein) are the major regulators for the cellular response against high osmolarity in human kidney tissue.

COX-2 regulates p53 activity and inhibits DNA damage-induced apoptosis.

We have previously shown that p53 induces cyclooxygenase-2 (COX-2) expression and COX-2 inhibits p53- or genotoxic stress-induced apoptosis. However, the COX-2 effects have been demonstrated indirectly by the use of a selective inhibitor, NS-398, and the molecular mechanisms by which COX-2 inhibits apoptosis have not been identified. In the present study, we demonstrated that COX-2 inhibits genotoxic stress-induced apoptosis by using an adenoviral COX-2 overexpression system. In addition, we found that COX-2 regulates the transcription function of p53 as evidenced by suppression of p53 target gene induction by COX-2 cotransfection. Furthermore, COX-2 interacted with p53 in vitro and in vivo, which was inhibited by the treatment with NS-398. Taken together, these results suggest a novel function of COX-2 that inhibits DNA damage-induced apoptosis through direct regulation of p53 function.