Role and mechanism of REG2 depletion in insulin secretion augmented by glutathione peroxidase-1 overproduction.

We previously reported a depletion of murine regenerating islet-derived protein 2 (REG2) in pancreatic islets of glutathione peroxidase-1 (Gpx1) overexpressing (OE) mice. The present study was to explore if and how the REG2 depletion contributed to an augmented glucose stimulated insulin secretion (GSIS) in OE islets. After we verified a consistent depletion (90%, p < 0.05) of REG2 mRNA, transcript, and protein in OE islets compared with wild-type (WT) controls, we treated cultured and perifused OE islets (70 islets/sample) with REG2 (1 µg/ml or ml · min) and observed 30-40% (p < 0.05) inhibitions of GSIS by REG2. Subsequently, we obtained evidences of co-immunoprecipitation, cell surface ligand binding, and co-immunofluorescence for a ligand-receptor binding between REG2 and transmembrane, L-type voltage-dependent Ca2+ channel (CaV1.2) in beta TC3 cells. Mutating the C-type lectin binding domain of REG2 or deglycosylating CaV1.2 removed the inhibition of REG2 on GSIS and(or) the putative binding between the two proteins. Treating cultured OE and perifused WT islets with REG2 (1 µg/ml or ml · min) decreased (p < 0.05) Ca2+ influx triggered by glucose or KCl. An intraperitoneal (ip) injection of REG2 (2 µg/g) to OE mice (6-month old, n = 10) decreased their plasma insulin concentration (46%, p < 0.05) and elevated their plasma glucose concentration (25%, p < 0.05) over a 60 min period after glucose challenge (ip, 1 g/kg). In conclusion, our study identifies REG2 as a novel regulator of Ca2+ influx and insulin secretion, and reveals a new cascade of GPX1/REG2/CaV1.2 to explain how REG2 depletion in OE islets could decrease its binding to CaV1.2, resulting in uninhibited Ca2+ influx and augmented GSIS. These findings create new links to bridge redox biology, tissue regeneration, and insulin secretion.

Dietary selenium deficiency partially rescues type 2 diabetes-like phenotypes of glutathione peroxidase-1-overexpressing male mice.

This study was conducted to determine whether dietary Se deficiency precluded overproduction of glutathione peroxidase-1 (GPX1) activity in mice overexpressing (OE) this gene and thus rescued their type 2 diabetes-like phenotypes. A total of 20 male OE and wild-type (WT) mice were fed an Se-deficient (<0.02 mg/kg) diet or an Se-supplemented (0.3 mg/kg as sodium selenite) diet from 1 to 5 mo of age. Dietary Se deficiency eliminated or attenuated (P < 0.05) genotype differences in concentrations of blood glucose, plasma insulin, and/or hepatic lipids, insulin sensitivity, and glucose-stimulated insulin secretion at the end of the study. Dietary Se deficiency decreased (P < 0.05) OE islet mRNA levels of 2 key transcriptional activators (Beta2 and Foxa2) and removed genotype differences in islet mRNA levels of 7 genes (Beta2, Cfos, Foxa2, Pregluc, Ins1, p53, and Sur1) related to insulin synthesis and secretion. Compared with those of the Se-adequate OE mice, the Se-deficient OE mice had lower (P < 0.05) hepatic mRNA levels of 2 key rate-limiting enzymes for lipogenesis (Acc1) and glycolysis (Gk1), along with lower (P < 0.05) activities of hepatic glucokinase and muscle phosphoenolpyruvate carboxykinase. Dietary Se deficiency also decreased (P < 0.05) blood glucose and hepatic lipid concentrations in the WT mice. In conclusion, dietary Se deficiency precluded the overproduction of GPX1 in full-fed OE mice and partially rescued their metabolic syndromes. This alleviation resulted from modulating the expression and/or function of proinsulin genes, lipogenesis rate-limiting enzyme genes, and key glycolysis and gluconeogenesis enzymes in islets, liver, and muscle.

Knockouts of SOD1 and GPX1 exert different impacts on murine islet function and pancreatic integrity.

Metabolic subtlety and clinical relevance of different forms of reactive oxygen species in diabetes remain unclear. Using single knockout of Cu,Zn-superoxide dismutase (SOD1(-/-)) or Se-glutathione peroxidase-1 (GPX1(-/-)) and their double-knockout (DKO) mouse models, we determined if elevating endogenously-derived superoxide and hydroperoxide exerted distinct impacts and mechanisms on body glucose homeostasis. Whereas the three knockout groups displayed decreased plasma insulin concentrations and islet ß-cells mass, only SOD1(-/-) showed decreased body weight, increased blood glucose, and blocked glucose-stimulated insulin secretion. Null of SOD1 and GPX1 elevated respective islet superoxide and hydroperoxide production, and upregulated p53 phosphorylation. Knockout of SOD1 downregulated the foxhead box A2/pancreatic and duodenal homeobox 1 pathway in a superoxide-dependent fashion at epigenetic, mRNA, and protein levels in islets, but improved insulin signaling in liver and muscle. The SOD1(-/-) mice showed more apparent pancreatitis than the GPX1(-/-) mice that were more susceptible to the cerulein-induced amylase increase. Knockout of SOD1 impaired islet function, pancreas integrity, and body glucose homeostasis more than that of GPX1. Simultaneous ablation of both enzymes did not result in additive or aggravated metabolic outcomes.

Impacts of dietary selenium deficiency on metabolic phenotypes of diet-restricted GPX1-overexpressing mice.

We previously reported a spontaneous development of type 2 diabetes-like phenotypes in glutathione peroxidase-1 (GPX1)-overexpressing (OE) mice. Diet restriction of these mice rescued all their phenotypes, except for hyperinsulinemia and hypersecretion of insulin. This study was to determine whether dietary Se deficiency eliminated these two primary effects of GPX1 overproduction. Forty-seven male OE and wild-type (WT) mice were fed an Se-adequate (0.4 mg Se/kg) or deficient (<0.02 mg Se/kg) diet at 2 to 3 g (full-fed = 5 g) per day from 4 to 12 weeks of age. Although dietary Se deficiency did not rescue the primary phenotypes of the diet-restricted OE mice, it exerted a strong effect (p < 0.05) on mRNA or protein levels (or both) of 14 molecules involved in islet insulin synthesis and secretion and hepatic lipogenesis. Dietary Se deficiency exhibited a hypoinsulinemic trend in OE mice and a strong hypolipidemic effect (p < 0.05) in the liver of WT mice. Hepatic lipogenesis was attenuated in OE compared with WT mice. In conclusion, diet restriction might be too overwhelming to allow a demonstration of a dietary Se-depletion effect on the OE phenotypes. Full-fed animals could offer a better chance to illustrate such effects and the underlying mechanisms.

Supplemental dietary inulin influences expression of iron and inflammation related genes in young pigs.

We have previously shown improved hemoglobin (Hb) repletion efficiency by supplementing a 50:50 mixture of short (P95) and long-chain (HP) inulin (Synergy 1, BENEO-Orafti) into a corn-soybean meal-basal diet (BD) for young pigs. In this study, weanling pigs (5 or 6 wk old) were fed the BD or the BD + 4% of P95, HP, or Synergy 1 (50:50 mixtures of HP and P95) for 5-7 wk. Blood Hb concentrations of pigs were measured weekly and digesta samples were collected at the end of the trial. In a replicate experiment, total RNA was isolated from the liver and mucosa of duodenum, ileum, cecum, and colon of all pigs at the end of the trial. Relative mRNA expression of 27 genes, including iron and inflammation-related genes, was quantified using real-time quantitative-PCR. Although all 3 types of inulin resulted in similar improvements (P < 0.05) in blood Hb concentration and liver ferritin protein amount, neither type of inulin was detectable in the digesta of cecum or colon. Supplemental inulin enhanced the expression of iron-storing protein genes but decreased that of inflammation-related genes. Such effects were more pronounced (P < 0.05) in the mucosa of the lower than the upper gut and were seen on 7 genes in liver. In conclusion, all 3 types of inulin shared similar efficacy and possibly similar modes of action in improving dietary iron utilization by young pigs. Suppressing inflammation-induced genes that can negatively influence iron metabolism might help explain the benefit of inulin.

Role of copper,zinc-superoxide dismutase in catalyzing nitrotyrosine formation in murine liver.

The only known function of Cu,Zn-superoxide dismutase (SOD1) is to catalyze the dismutation of superoxide anion into hydrogen peroxide. Our objective was to determine if SOD1 catalyzes murine liver protein nitration induced by acetaminophen (APAP) and lipopolysaccharide (LPS). Liver and plasma samples were collected from young adult SOD1 knockout mice (SOD1-/-) and wild-type (WT) mice at 5 or 6 h after an ip injection of saline, APAP, or LPS. Hepatic nitrotyrosine formation was induced by APAP and LPS only in the WT mice. The diminished hepatic protein nitration in the SOD1-/- mice was not directly related to plasma nitrite and nitrate concentrations. Similar genotype differences were seen in liver homogenates treated with a bolus of peroxynitrite. Adding only the holo-, and not the apo-, SOD1 enzyme into the liver homogenates enhanced the reaction in an activity-dependent fashion and nearly eliminated the genotype difference at the high doses. Mass spectrometry showed four more nitrotyrosine residues in bovine serum albumin and 10 more nitrated protein candidates in the SOD1-/- liver homogenates by peroxynitrite with added SOD1. In conclusion, the diminished hepatic protein nitration mediated by APAP or LPS in the SOD1-/- mice is due to the lack of SOD1 activity per se.

Mice deficient in Cu,Zn-superoxide dismutase are resistant to acetaminophen toxicity.

Although antioxidants are used to treat an overdose of the analgaesic/antipyretic drug APAP (acetaminophen), roles of antioxidant enzymes in APAP-induced hepatotoxicity remain controversial. Our objective was to determine impacts of knockout of SOD1 (superoxide dismutase; Cu,Zn-SOD) alone or in combination with selenium-dependent GPX1 (glutathione peroxidase-1) on APAP-induced hepatotoxicity. All SOD1-null (SOD1-/-) and SOD1- and GPX1-double-knockout mice survived an intraperitoneal injection of 600 mg of APAP per kg of body mass, whereas 75% of WT (wild-type) and GPX1-null mice died within 20 h. Survival time of SOD1-/- mice injected with 1200 mg of APAP per kg of body mass was longer than that of the WT mice (934 compared with 315 min, P<0.05). The APAP-treated SOD1-/- mice had less (P<0.05) plasma ALT (alanine aminotransferase) activity increase and attenuated (P<0.05) hepatic glutathione depletion than the WT mice. The protection conferred by SOD1 deletion was associated with a block of the APAP-mediated hepatic protein nitration and a 50% reduction (P<0.05) in activity of a key APAP metabolism enzyme CYP2E1 (cytochrome P450 2E1) in liver. The SOD1 deletion also caused moderate shifts in the APAP metabolism profiles. In conclusion, deletion of SOD1 alone or in combination with GPX1 greatly enhanced mouse resistance to APAP overdose. Our results suggest a possible pro-oxidant role for the physiological level of SOD1 activity in APAP-mediated hepatotoxicity.

Development of insulin resistance and obesity in mice overexpressing cellular glutathione peroxidase.

Insulin resistance, a hallmark of type 2 diabetes, is associated with oxidative stress. However, the role of reactive oxygen species or specific antioxidant enzymes in its development has not been tested under physiological conditions. The objective of our study was to investigate the impact of overexpression of glutathione peroxidase 1 (GPX1), an intracellular selenoprotein that reduces hydrogen peroxide (H(2)O(2)) in vivo, on glucose metabolism and insulin function. The GPX1-overexpressing (OE) and WT male mice (n = 80) were fed a selenium-adequate diet (0.4 mg/kg) from 8 to 24 weeks of age. Compared with the WT, the OE mice developed (P < 0.05) hyperglycemia (117 vs. 149 mg/dl), hyperinsulinemia (419 vs. 1,350 pg/ml), and elevated plasma leptin (5 vs. 16 ng/ml) at 24 weeks of age. Meanwhile, these mice were heavier (37 vs. 27 g, P < 0.001) and fatter (37% vs. 17% fat, P < 0.01) than the WT mice. At 30-60 min after an insulin challenge, the OE mice had 25% less (P < 0.05) of a decrease in blood glucose than the WT mice. Their insulin resistance was associated with a 30-70% reduction (P < 0.05) in the insulin-stimulated phosphorylations of insulin receptor (beta-subunit) in liver and Akt (Ser(473) and Thr(308)) in liver and soleus muscle. Here we report the development of insulin resistance in mammals with elevated expression of an antioxidant enzyme and suggest that increased GPX1 activity may interfere with insulin function by overquenching intracellular reactive oxygen species required for insulin sensitizing.

Low levels of glutathione peroxidase 1 activity in selenium-deficient mouse liver affect c-Jun N-terminal kinase activation and p53 phosphorylation on Ser-15 in pro-oxidant-induced aponecrosis.

Low levels of hepatic selenium (Se)-dependent glutathione peroxidase 1 (GPX1) activity have been shown to protect against oxidative liver injury in Se-deficient mice. The objective of the present study was to determine if the GPX1 protection was associated with phosphorylations of c-Jun N-terminal kinase (JNK) and p53 on Ser-15, two key signalling events in oxidative-stress-mediated cell death. Both Se-deficient GPX1 knockout (GPX1(-/-)) and wild-type (WT) mice ( n =64) were pretreated with an intraperitoneal injection of Se (as sodium selenite, 50 microg/kg body weight) 6 h before an intraperitoneal injection of paraquat (12.5 mg/kg). Liver aponecrosis, a mixed form of cell death sharing apoptosis and necrosis, was induced by paraquat in both groups of mice. However, its appearance was remarkably delayed and the severity was decreased by the repletion of hepatic GPX1 activity to <4% of the normal level by the Se injection in the WT mice, compared with that in the GPX1(-/-) mice. Consistently, the WT mice had lower levels of hepatic phospho-JNK, p53 and phospho-p53 (Ser-15) when compared with the GPX1(-/-) mice at 1-10 h after paraquat injection. Incubating liver homogenates with antibodies raised against JNK or phospho-JNK resulted in co-immunoprecipitation of phospho-p53 (Ser-15), and the amounts of the precipitated phospho-p53 were greater in the GPX1(-/-) mice when compared with that in the WT mice. The co-precipitated complex by the anti-phospho-JNK antibody was capable of phosphorylating intrinsic or extrinsic p53 on Ser-15. In conclusion, phospho-JNK may catalyse phosphorylation of p53 on Ser-15 in Se-deficient mouse liver under moderate oxidative stress, and attenuation of that cascade by low levels of GPX1 activity is associated with its protection against the pro-oxidant-induced liver aponecrosis.

Immunization with surface antigen vaccine alone and after treatment with 1-(2-fluoro-5-methyl-beta-L-arabinofuranosyl)-uracil (L-FMAU) breaks humoral and cell-mediated immune tolerance in chronic woodchuck hepatitis virus infection.

Woodchucks chronically infected with the woodchuck hepatitis virus (WHV) were treated with the antiviral drug 1-(2-fluoro-5-methyl-beta-L-arabinofuranosyl)-uracil (L-FMAU) or placebo for 32 weeks. Half the woodchucks in each group then received four injections of surface antigen vaccine during the next 16 weeks. Vaccination alone elicited a low-level antibody response to surface antigen in most carriers but did not affect serum WHV DNA and surface antigen. Carriers treated first with L-FMAU to reduce serum WHV DNA and surface antigen and then vaccinated had a similar low-level antibody response to surface antigen. Following vaccinations, cell-mediated immunity to surface antigen was demonstrated in both groups, independent of serum viral and antigen load, but was significantly enhanced in woodchucks treated with L-FMAU and was broadened to include other viral antigens (core, e, and x antigens and selected core peptides). Cell-mediated immunity and antibody responses to surface antigen were observed after drug discontinuation in half of the carriers that received L-FMAU alone. Surface antigen vaccine alone or in combination with drug broke humoral and cell-mediated immune tolerance in chronic WHV infection, but the combination with drug was more effective. This suggested that a high viral and antigen load in carriers is important in maintaining immunologic tolerance during chronicity. The humoral and cellular immunity associated with the combination of L-FMAU and vaccine resembled that observed in self-limited WHV infection. Such combination therapy represents a potentially useful approach to the control of chronic hepatitis B virus infection in humans.

Deficiencies in the acute-phase cell-mediated immune response to viral antigens are associated with development of chronic woodchuck hepatitis virus infection following neonatal inoculation.

In vitro proliferation of peripheral blood mononuclear cells was used to measure virus-specific cell-mediated immunity (vCMI) following neonatal woodchuck hepatitis virus (WHV) infection. Fifteen neonates were inoculated with the W8 strain of WHV. In 11, infection was resolved, and 4 became chronic carriers. Nineteen neonates were inoculated with the W7 strain and all became chronic carriers. Seven age-matched uninfected woodchucks served as controls. Virologic and vCMI profiles among the W8 and W7 infections were compared and related to the outcome of infection. Resolving woodchucks had robust, acute-phase vCMI to WHV antigens (core, surface, and x) and to several nonoverlapping core peptides. The acute-phase vCMI was associated temporally with the clearance of viral DNA and of surface antigen from serum at 14 to 22 weeks postinfection. In contrast, in approximately half of the W8 and W7 infections that progressed to chronicity, no significant acute-phase vCMI was detected. In the remaining carriers, acute-phase vCMI was observed, but it was less frequent and incomplete compared to that of resolved woodchucks. Serum viral load developed less rapidly in those carriers that had evidence of acute-phase vCMI, but it was still increased compared to that of resolving woodchucks. Thus, vigorous and multispecific acute-phase vCMI was associated with resolution of neonatal WHV infection. Absent or incomplete acute-phase vCMI was associated with the progression to chronic infection. By analogy, these results suggest that the onset of chronic hepatitis B virus (HBV) infection in humans may be associated with deficiencies in the primary T-cell response to acute HBV infection.

Immunogenic effects of woodchuck hepatitis virus surface antigen vaccine in combination with antiviral therapy: breaking of humoral and cellular immune tolerance in chronic woodchuck hepatitis virus infection.

OBJECTIVE: A rational treatment strategy for chronic hepatitis B virus (HBV) infection might involve the modulation of immunity after the reduction of viremia and antigenemia. This strategy was tested in woodchucks chronically infected with the woodchuck hepatitis virus (WHV) by combining antiviral treatment with 1-(2-fluoro-5-methyl-beta-L-arabinofuranosyl)-uracil (L-FMAU) and therapeutic vaccination with WHV surface antigen (WHsAg). METHODS: Chronic WHV carriers were treated with L-FMAU or placebo for 32 weeks. Half the woodchucks in each group then received four injections of a conventional WHsAg vaccine during the next 16 weeks. RESULTS: Vaccination alone elicited low-level antibody to WHsAg (anti-WHs) in most carriers but did not affect serum WHV DNA, WHsAg or liver enzyme responses. Carriers treated first with L-FMAU to reduce WHV DNA and WHsAg and then vaccinated developed similar low-level anti-WHs and normalized liver enzymes. Following vaccinations, WHsAg-specific cell-mediated immunity (CMI) was demonstrated in both groups, but was significantly enhanced in carriers treated with L-FMAU, and was broadened to include WHV core antigen (WHcAg) and selected peptide epitopes of WHcAg and WHsAg. Anti-WHs and associated CMI to WHcAg and WHsAg were observed after drug discontinuation in half of the carriers that received L-FMAU alone. CONCLUSIONS: Vaccination with WHsAg following treatment with L-FMAU disrupted virus-specific humoral and cell-mediated immune tolerance in chronic WHV infection and enhanced the immune response profiles beyond those seen with monotherapies alone. The combination therapy resulted in immune response profiles that resembled those observed during resolution of WHV infection. The results in woodchucks demonstrate the feasibility of using such a combination therapy for the control of chronic HBV infection in humans.