Effect of Dietary Processed Sulfur on the Meat Quality in Pork under Aging.

The present study was performed to determine the effect of dietary supplementation with processed sulfur on the quality and stability of vacuum packaged pork during aging time. All groups were designated into two groups; NP, a group fed basal diet and SP, a group fed basal diet and processed sulfur, 3 g/kg feed. Following vacuum packaging, Longissimus dorsi muscles were vacuum-packaged and stored under refrigerated condition (1-2℃) for 21 d. Weight loss of the SP group was lower (p<0.05) than that of the NP group. Interaction effect of shear force and cooking loss was observed (p<0.05). Redness values of the SP group at 14 and 21 d after storage were higher than those of the NP group (p<0.05). Lipid oxidation and volatile basic nitrogen (VBN) levels in the SP group were retarded (p<0.05) compared to that of the NP group during storage. Aspartic and glutamic acid in SP were higher than in NP (p<0.1). There was no significant (p>0.05) difference in TPC between the both groups during storage. Therefore, vacuum packaged pork from pigs fed processed sulfur had better aging yield and storage stability than pork from pigs fed basal diet.

Toll-like receptor 4-induced endoplasmic reticulum stress contributes to impairment of vasodilator action of insulin.

Impairment of vasodilator action of insulin is associated with endothelial dysfunction and insulin resistance. Activation of Toll-like receptor 4 (TLR4) induces proinflammatory response and endoplasmic reticulum (ER) stress. Saturated fatty acids (SFA) activate TLR4, which induces ER stress and endothelial dysfunction. Therefore, we determined whether TLR4-mediated ER stress is an obligatory step mediating SFA-induced endothelial dysfunction. Palmitate stimulated proinflammatory responses and ER stress, and this was suppressed by knockdown of TLR4 in primary human aortic endothelial cells (HAEC). Next, we examined the role of TLR4 in vasodilatory responses in intact vessels isolated from wild-type (WT, C57BL/6) and TLR4-KO mice after feeding high-fat (HFD) or normal chow diet (NCD) for 12 wk. Arterioles isolated from HFD WT mice exhibited impaired insulin-stimulated vasodilation compared with arterioles isolated from NCD WT mice. Deficiency of TLR4 was protective from HFD-induced impairment of insulin-stimulated vasodilation. There were no differences in acetylcholine (Ach)- or sodium nitroprusside (SNP)-stimulated vasodilation between the two groups. Furthermore, we examined whether ER stress is involved in SFA-induced impairment of vasodilator actions of insulin. Infusion of palmitate showed the impairment of vasodilatory response to insulin, which was ameliorated by coinfusion with tauroursodeoxycholic acid (TUDCA), an ER stress suppressor. Taken together, the results suggest that TLR4-induced ER stress may be an obligatory step mediating the SFA-mediated endothelial dysfunction.

Effects of the green tea polyphenol epigallocatechin-3-gallate on high-fat diet-induced insulin resistance and endothelial dysfunction.

Insulin resistance, a hallmark of metabolic disorders, is a risk factor for diabetes and cardiovascular disease. Impairment of insulin responsiveness in vascular endothelium contributes to insulin resistance. The reciprocal relationship between insulin resistance and endothelial dysfunction augments the pathophysiology of metabolism and cardiovascular functions. The most abundant green tea polyphenol, epigallocatechin-3-gallate (EGCG), has been shown to have vasodilator action in vessels by activation of endothelial nitric oxide synthase (eNOS). However, it is not known whether EGCG has a beneficial effect in high-fat diet (HFD)-induced endothelial dysfunction. Male C57BL/6J mice were fed either a normal chow diet (NCD) or HFD with or without EGCG supplement (50 mg·kg(-1)·day(-1)) for 10 wk. Mice fed a HFD with EGCG supplement gained less body weight and showed improved insulin sensitivity. In vehicle-treated HFD mice, endothelial function was impaired in response to insulin but not to acetylcholine, whereas the EGCG-treated HFD group showed improved insulin-stimulated vasodilation. Interestingly, EGCG intake reduced macrophage infiltration into aortic tissues in HFD mice. Treatment with EGCG restored the insulin-stimulated phosphorylation of eNOS, insulin receptor substrate-1 (IRS-1), and protein kinase B (Akt), which was inhibited by palmitate (200 µM, 5 h) in primary bovine aortic endothelial cells. From these results, we conclude that supplementation of EGCG improves glucose tolerance, insulin sensitivity, and endothelial function. The results suggest that EGCG may have beneficial health effects in glucose metabolism and endothelial function through modulating HFD-induced inflammatory response.

Epigallocatechin gallate (EGCG) stimulates autophagy in vascular endothelial cells: a potential role for reducing lipid accumulation.

Epigallocatechin gallate (EGCG) is a major polyphenol in green tea that has beneficial effects in the prevention of cardiovascular disease. Autophagy is a cellular process that protects cells from stressful conditions. To determine whether the beneficial effect of EGCG is mediated by a mechanism involving autophagy, the roles of the EGCG-stimulated autophagy in the context of ectopic lipid accumulation were investigated. Treatment with EGCG increased formation of LC3-II and autophagosomes in primary bovine aortic endothelial cells (BAEC). Activation of calmodulin-dependent protein kinase kinase ß was required for EGCG-induced LC3-II formation, as evidenced by the fact that EGCG-induced LC3-II formation was significantly impaired by knockdown of calmodulin-dependent protein kinase kinase ß. This effect is most likely due to cytosolic Ca(2+) load. To determine whether EGCG affects palmitate-induced lipid accumulation, the effects of EGCG on autophagic flux and co-localization of lipid droplets and autophagolysosomes were examined. EGCG normalized the palmitate-induced impairment of autophagic flux. Accumulation of lipid droplets by palmitate was markedly reduced by EGCG. Blocking autophagosomal degradation opposed the effect of EGCG in ectopic lipid accumulation, suggesting the action of EGCG is through autophagosomal degradation. The mechanism for this could be due to the increased co-localization of lipid droplets and autophagolysosomes. Co-localization of lipid droplets with LC3 and lysosome was dramatically increased when the cells were treated with EGCG and palmitate compared with the cells treated with palmitate alone. Collectively, these findings suggest that EGCG regulates ectopic lipid accumulation through a facilitated autophagic flux and further imply that EGCG may be a potential therapeutic reagent to prevent cardiovascular complications.

Toll-like receptor 2 mediates high-fat diet-induced impairment of vasodilator actions of insulin.

Obesity is characterized by a chronic proinflammatory state that leads to endothelial dysfunction. Saturated fatty acids (SFA) stimulate Toll-like receptors (TLR) that promote metabolic insulin resistance. However, it is not known whether TLR2 mediates impairment of vascular actions of insulin in response to high-fat diet (HFD) to cause endothelial dysfunction. siRNA knockdown of TLR2 in primary endothelial cells opposed palmitate-stimulated expression of proinflammatory cytokines and splicing of X box protein 1 (XBP-1). Inhibition of unfolding protein response (UPR) reduced SFA-stimulated expression of TNFα. Thus, SFA stimulates UPR and proinflammatory response through activation of TLR2 in endothelial cells. Knockdown of TLR2 also opposed impairment of insulin-stimulated phosphorylation of eNOS and subsequent production of NO. Importantly, insulin-stimulated vasorelaxation of mesenteric arteries from TLR2 knockout mice was preserved even on HFD (in contrast with results from arteries examined in wild-type mice on HFD). We conclude that TLR2 in vascular endothelium mediates HFD-stimulated proinflammatory responses and UPR that accompany impairment of vasodilator actions of insulin, leading to endothelial dysfunction. These results are relevant to understanding the pathophysiology of the cardiovascular complications of diabetes and obesity.

Activation of mTOR/p70S6 kinase by ANG II inhibits insulin-stimulated endothelial nitric oxide synthase and vasodilation.

Elevated tissue levels of angiotensin II (ANG II) are associated with impairment of insulin actions in metabolic and cardiovascular tissues. ANG II-stimulated activation of mammalian target of rapamycin (mTOR)/p70 S6 kinase (p70S6K) in cardiovascular tissues is implicated in cardiac hypertrophy and vascular remodeling. However, the role of ANG II-stimulated mTOR/p70S6K in vascular endothelium is poorly understood. In the present study, we observed that ANG II stimulated p70S6K in bovine aortic endothelial cells. ANG II increased phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser(636/639) and inhibited the insulin-stimulated phosphorylation of endothelial nitric oxide synthase (eNOS). An inhibitor of mTOR, rapamycin, attenuated the ANG II-stimulated phosphorylation of p70S6K and phosphorylation of IRS-1 (Ser(636/639)) and blocked the ability of ANG II to impair insulin-stimulated phosphorylation of eNOS, nitric oxide production, and mesenteric-arteriole vasodilation. Moreover, point mutations of IRS-1 at Ser(636/639) to Ala prevented the ANG II-mediated inhibition of insulin signaling. From these results, we conclude that activation of mTOR/p70S6K by ANG II in vascular endothelium may contribute to impairment of insulin-stimulated vasodilation through phosphorylation of IRS-1 at Ser(636/639). This ANG II-mediated impairment of vascular actions of insulin may help explain the role of ANG II as a link between insulin resistance and hypertension.

[A case of pneumatosis cystoids intestinalis with polymyositis].

Pneumatosis cystoides intestinalis (PCI), characterized by presence of intramural gas cyst in the intestinal wall is associated with various medical condition. Polymyosistis, however, is rarely associated with PCI. Few cases are reported in the world, and none has not been reported previously in Korea. A 67-year-old woman with polymyositis developed mild abdominal pain and abdominal distension during treatment with steroid and azathioprine. Radiographic findings including CT scan showed intraperitoneal free gas and intramural air, compatible with PCI. The patient's symptom and clinical findings improved after the treatment with antibiotics and high-dose oxygen therapy.

Atherosclerosis induced by a high-fat diet is alleviated by lithium chloride via reduction of VCAM expression in ApoE-deficient mice.

Endothelial cell dysfunction may play an important role in the development of various vascular diseases, including atherosclerosis. Here we investigated whether lithium chloride (LiCl), an inhibitor of glycogen synthase kinase-3ß (GSK-3ß), could counteract atherosclerosis induced by a high-fat diet in ApoE⁻/⁻ mice. Ten-week-old male mice were randomly divided into four groups: normal chow diet, high-fat diet (i.e., 20% fat and 0.5% cholesterol), high-fat diet with LiCl treatment for 6 weeks and high-fat diet with LiCl treatment for 14 weeks. Examination of plasma profiles indicated that blood glucose levels were significantly decreased by LiCl treatment. Supplementation with LiCl dramatically reduced atherosclerotic lesion formation in the aorta and aortic root. LiCl treatment also decreased vascular cell adhesion molecule (VCAM)-1 expression and macrophage infiltration into atherosclerotic lesion areas within the aortic valve. In addition, inhibition of GSK-3ß by TDZD-8, SB216763, and LiCl, as well as adenoviral transduction with a catalytically inactive GSK-3ß, reduced palmitate-induced VCAM-1 expression through inhibition of JNK activity and degradation of Iκ-Bα in human umbilical vein endothelial cells (HUVECs). The results of the present study suggest that LiCl alleviates palmitate-induced cell adhesion molecule expression in HUVECs and decreases atherosclerosis induced by a high-fat diet in ApoE⁻/⁻ mice. Thus, GSK-3ß may be involved in the development of atherosclerosis induced by a high-fat diet in ApoE⁻/⁻ mice.

Classical PKC is not associated with defective insulin signaling in patients with impaired glucose tolerance.

BACKGROUND AND AIM: To investigate the role of insulin signaling defects in impaired glucose tolerance (IGT), we assessed the functionality of the insulin signaling cascade before and after insulin stimulation in both IGT group and control group. METHODS: Ten IGT subjects and 15 control subjects were recruited for this study. Whole-body insulin-mediated glucose uptake was determined using a euglycemic hyperinsulinemic clamp test. Muscle biopsies were obtained from the vastus lateralis muscle before and after insulin stimulation, to assess the insulin signaling cascade. RESULTS: The insulin-stimulated incremental changes in phosphorylated IR-beta, IRS, Akt, and GSK-3 beta and in the membrane-associated PKC-zeta protein level were reduced in the IGT group compared with those in the control group (p<0.05). The membrane-associated PKC-lambda protein level was also reduced in the IGT group, but not significantly so (p=0.08). The incremental changes in the protein levels of PKC-alpha, -beta, and -theta were not significantly different between the two groups. CONCLUSION: The subjects with IGT showed decreased membrane-associated PKC-zeta/lambda activity in response to insulin stimulation, as well as defects in early insulin signaling. Our results suggest that membrane-associated PKC-alpha and -beta may not be associated with insulin resistance in IGT.

A chemical chaperone 4-PBA ameliorates palmitate-induced inhibition of glucose-stimulated insulin secretion (GSIS).

Free fatty acids (FFAs) are believed to be a stimulus to elicit beta cell dysfunction. The present study was undertaken to determine whether endoplasmic reticulum (ER) stress was involved in palmitate-induced inhibition of glucose-stimulated insulin secretion (GSIS) and whether reduction of ER stress using a chemical chaperone restored the GSIS-inhibition. Treatment of INS-1 cells with 300 microM palmitate for 24h elicited ER stress, showing increased levels of phospho-eIF2alpha, Bip and spliced XBP, and also induced GSIS-inhibition without reduction of cell viability. Replenishment with 4-phenyl butyric acid (4-PBA) as a chemical chaperone reduced the palmitate-induced-ER stress and significantly reversed the palmitate-induced GSIS-inhibition. Furthermore, 4-PBA ameliorated palmitate-induced GSIS-inhibition in primary rat islet cells. These data suggested that ER stress was involved in FFA-induced GSIS-inhibition and that the FFA-induced beta cell dysfunction could be ameliorated by treatment with a chemical chaperone.

Involvement of Ca2+-mediated apoptotic signals in palmitate-induced MIN6N8a beta cell death.

The extracellular Ca(2+) chelator EGTA and L-type Ca(2+) channel blockers, such as, nifedipine and nimodipine were found to have a protective effect on palmitate-induced MIN6N8a beta cell apoptosis, whereas the Ca(2+) channel opener, Bay K8644, enhanced the apoptotic process. Moreover, the phospho-form of Bad, in conjunction with phospho-Akt, was reduced in response to palmitate and the palmitate-induced dephosphorylations of Akt and Bad were dependent on Ca(2+) influx. The transient expression of catalytically active Akt prevented MIN6N8a cells from palmitate-induced apoptosis. Deltamethrin, an inhibitor of Ca(2+)-activated phosphatase, delayed Akt and Bad dephosphorylations, and then protected MIN6N8a cells from palmitate-induced apoptosis. On the other hand, palmitate was found to induce CHOP, an apoptotic transcription factor in response to ER stress, and this induction was enhanced by Ca(2+) influx. Our studies suggested that Ca(2+) influx and subsequent Ca(2+)-mediated apoptotic signals are involved in palmitate-induced beta cell death.

Involvement of glycogen synthase kinase-3beta in palmitate-induced human umbilical vein endothelial cell apoptosis.

BACKGROUND/AIMS: The death of endothelial cells may play a critical role in the development of various vascular diseases, including atherosclerosis. While free fatty acids (FFAs) may stimulate endothelial apoptosis, the molecular and cellular mechanisms of this effect have not been studied intensively. To elucidate the mechanisms involved in FFA-induced endothelial cell apoptosis, we investigated the effect of different pharmacological inhibitors on palmitate-induced apoptosis in human umbilical vein endothelial cells (HUVECs). Interestingly, lithium, a glycogen synthase kinase-3 (GSK-3) inhibitor, showed a strong protective effect. METHODS AND RESULTS: To examine the involvement of GSK-3beta in palmitate-induced HUVEC apoptosis, its dephosphorylation at Ser9 and enzymatic activation in response to palmitate treatment were monitored by immunoblotting and in vitro kinase assays, respectively. GSK-3beta was dephosphorylated and its enzymatic activity increased in palmitate-treated HUVECs. In addition, pretreatment with other GSK-3beta inhibitors, e.g. SB216763 or TDZD-8, as well as adenoviral transduction with a catalytically inactive GSK-3beta had significant protective effects against palmitate-induced HUVEC apoptosis. CONCLUSION: These results demonstrate that the GSK-3beta signalling pathway is involved in palmitate-induced HUVEC apoptosis.

Involvement of Ca2+, CaMK II and PKA in EGb 761-induced insulin secretion in INS-1 cells.

EGb 761, a standardized form of Ginkgo biloba L. (Ginkgoaceae) leaf extract, was recently reported to increase pancreatic beta-cell function. To determine whether EGb 761 elicits insulin secretion directly, we treated INS-1 rat beta cells with EGb 761 and then measured insulin release. Treatment of EGb 761 (50 microg/ml) significantly stimulated insulin secretion in INS-1 cells, compared with untreated control (p<0.05) and the stimulatory effect of EGb 761 on insulin secretion was dose-dependent. To elucidate the mechanism of EGb 761-induced insulin secretion, we investigated the involvement of calcium. The treatment with nifedipine, an L-type calcium channel blocker, prevented EGb 761-induced insulin secretion and furthermore, EGb 761 itself elevated [Ca(2+)](i), suggesting the involvement of calcium in this process. To identity the protein kinases involved in EGb 761-induced insulin secretion, INS-1 cells were treated with different kinase inhibitors and their effects on EGb 761-induced secretion were investigated. KN62 and H89, calium/calmodulin kinase (CaMK) II and protein kinase A (PKA) inhibitor, respectively, significantly reduced EGb 761-induced insulin secretion. Immunoblotting studies showed an increase in the phosphorylated-forms of CaMK II and of PKA substrates after EGb 761 treatment. Our data suggest that EGb 761-induced insulin secretion is mediated by [Ca(2+)](i) elevation and subsequent activation of CaMK II and PKA.

p38 and ERK MAP kinase mediates iron chelator-induced apoptosis and -suppressed differentiation of immortalized and malignant human oral keratinocytes.

Iron is essential for neoplastic cell growth, and iron chelators have been tested for potential anti-proliferative and anti-cancer effects, but the effects of iron chelators on oral cancer have not been clearly elucidated. To determine the mechanism of cell death induced by iron chelators, we explored the pathways of the three structurally related mitogen-activated protein (MAP) kinase subfamilies during iron chelator-induced apoptosis and differentiation of immortalized human oral keratinocytes (IHOK) and oral cancer cells (HN4). The iron chelator deferoxamine (DFO) exerted potent time- and dose-dependent inhibitory effects on the growth and apoptosis of IHOK and HN4 cells. DFO strongly activates p38 MAP kinase and extracellular signal-regulated kinase (ERK), but does not activate c-Jun N-terminal kinase/stress-activated protein kinase. Of the three MAP kinase blockers used, the selective p38 MAP kinase inhibitor SB203580 and ERK inhibitor PD98059 protected IHOK and HN4 cells against iron chelator-induced cell death, which indicates that the p38 and ERK MAP kinase is a major mediator of apoptosis induced by this iron chelator. Interestingly, treatment of IHOK and HN4 cells with SB203580 and PD98059 abolished cytochrome c release, as well as the activation of caspase-3 and caspase-8. DFO suppressed the expression of epithelial differentiation markers such as involucrin, CK6, and CK19, and this suppression was blocked by p38 and ERK MAP kinase inhibitors. Collectively, these data suggested that p38 and ERK MAP kinase plays an important role in iron chelator-mediated cell death and in the suppression of differentiation of oral immortalized and malignant keratinocytes, by activating a downstream apoptotic cascade that executes the cell death pathway.