Yanhua Peng

Role of SIRT1 in regulation of LPS- or two ethanol metabolites-induced TNF-α production in cultured macrophage cell lines

Dysregulation of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) has been implicated in the pathogenesis of alcoholic liver injury. Sirtuin 1 (SIRT1) is an NAD(+)-dependent class III protein deacetylase that is known to be involved in regulating production of proinflammatory cytokines including TNF-alpha. In the present study, we examined the role of SIRT1 signaling in TNF-alpha generation stimulated by either lipopolysaccharide (LPS), acetaldehyde (AcH), or acetate (two major metabolites of ethanol) in two cultured macrophage cell lines. In both rat Kupffer cell line 1 (RKC1) and murine RAW 264.7 macrophages, treatment with either LPS, AcH, or acetate caused significant decreases in SIRT1 transcription, translation, and activation, which essentially demonstrated an inverse relationship with TNF-alpha levels. LPS, AcH, and acetate each provoked the release of TNF-alpha from RKC1 cells, whereas coincubation with resveratrol (a potent SIRT1 agonist) inhibited this effect. Conversely, addition of sirtinol (a known SIRT1 inhibitor) or knocking down SIRT1 by the small silencing SIRT1 plasmid (SIRT1shRNA) augmented TNF-alpha release, suggesting that impairment of SIRT1 may contribute to TNF-alpha secretion. Further mechanistic studies revealed that inhibition of SIRT1 by LPS, AcH, or acetate was associated with a marked increase in the acetylation of the RelA/p65 subunit of nuclear transcription factor (NF-kappaB) and promotion of NF-kappaB transcriptional activity. Taken together, our findings suggest that SIRT1-NF-kappaB signaling is involved in regulating LPS- and metabolites-of-ethanol-mediated TNF-alpha production in rat Kupffer cells and in murine macrophages. Our study provides new insights into understanding the molecular mechanisms underlying the development of alcoholic steatohepatitis.

Deletion of toll-like receptor-4 downregulates protein kinase C-ζ and attenuates liver injury in experimental pancreatitis

BACKGROUND Toll-like receptor-4 (TLR4) and protein kinase C-zeta (PKC-zeta) play a role in macrophage activation. We hypothesized that deletion of TLR4 downregulates PKC-zeta and attenuates liver cell apoptosis in experimental pancreatitis. METHODS Acute pancreatitis was induced by choline-deficient ethionine diet in C57/BL6 (TLR4+/+ and TLR4-/-) mice. RESULTS During pancreatitis, staining for TLR4 and PKC-zeta, which colocalized in Kupffer cells but not in hepatocytes, increased in TLR4+/+ mice and decreased in TLR4-/- mice. In TLR4+/+ mice, pancreatitis increased TLR4 protein and mRNA and PKC-zeta protein and activity, nuclear factor (NF)-kappaB, ERK1/2, caspase-3 cleavage, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining; all P < .01 versus controls. In TLR4-/- mice with pancreatitis, PKC-zeta mRNA and activity were reduced, ERK1/2 and caspase-3 did not increase, and NF-kappaB and TUNEL (mostly in hepatocytes) increased mildly (all P < .01 vs control). PKC-zeta did not interact directly with NF-kappaB; however, during pancreatitis, coimmunoprecipitation of PKC-zeta with ERK1/2 was increased in TLR4+/+ mice and was attenuated in TLR4-/- mice (all P < .01 vs control), indicating that PKC-zeta interacts with ERK1/2. CONCLUSION Acute pancreatitis upregulates TLR4, PKC-zeta, NF-kappaB, and ERK1/2, and increases apoptosis in mice livers. PKC-zeta induces nuclear translocation of NF-kappaB via ERK1/2-dependent mechanisms. Deletion of TLR4 downregulates PKC-zeta, NF-kappaB, and ERK1/2, and attenuates pancreatitis-induced liver cell apoptosis.

The Role of p65 NF-κB/RelA in Pancreatitis-Induced Kupffer Cell Apoptosis

Acute pancreatitis induces liver injury by upregulating Kupffer cell-derived Fas/FasL; on the other hand, acute pancreatitis induces apoptosis of Kupffer cells via NF-kB-dependent pathways. The balance between upregulation of Fas/FasL and Fas/FasL-induced apoptosis of its originator cell may determine the severity of pancreatitis-related liver injury. The aim of our study was to determine the role of p65 NF-kB/RelA in pancreatitis-induced Kupffer cell apoptosis. Acute pancreatitis was induced in NIH Swiss mice by a choline-deficient ethionine-supplement (CDE) diet. In vitro mouse Kupffer cell line was transfected with p65 siRNA and treated with pancreatic elastase to mimic pancreatitis. CDE pancreatitis upregulated nuclear translocation of p65 NF-kB/RelA, Fas/FasL, caspase-3, and DNA fragmentation in mice livers (all P<0.001). In vitro, pancreatic elastase mimicked CDE-pancreatitis by upregulating nuclear translocation of p65 NF-kB/RelA, Fas/FasL, caspase-3, DNA fragmentation, and apoptosis in Kupffer cells (all P<0.001). Transfection with p65 siRNA attenuated the elastase-induced nuclear translocation of p65 NF-kB/RelA, upregulation of Fas/FasL, caspase-3, DNA fragmentation, and apoptosis in Kupffer cells (all P<0.001). Acute pancreatitis activates p65 NF-kB/RelA and induces apoptosis of Kupffer cells. Inhibition of p65NF-kB/RelA attenuates elastase-induced upregulation of proapoptotic pathways and apoptosis in Kupffer cells. The ability of Kupffer cells to autoregulate their stress response by inducing self-apoptosis warrants further investigation.

Fas/FasL play a central role in pancreatitis-induced hepatocyte apoptosis.

Liver injury is a clinical prognostic indicator in acute pancreatitis (AP). We have demonstrated that Kupffer cell-derived FasL mediates liver injury during AP and sought to determine its role in AP-induced hepatocyte apoptosis. AP was induced in National Institutes of Health (NIH) Swiss mice, C57/C57, and Fas-/-, FasL-/-mice by a choline-deficient ethionine-supplement diet. Liver Fas, FasL, p38-mitogen activated phosphokinase (p38-MAPK), poly-ADP ribose polymerase (PARP), and cytochrome C were measured by immunoblotting. Apoptosis was assessed by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and DNA fragmentation (ELISA). AP upregulated liver FasL (4280 ± 580 vs. 733 ± 336), Fas (2866 ± 595 vs. 649 ± 111), cytochrome C (6980 ± 237 vs. 903 ± 156), and PARP (6393 ± 591 vs. 466 ± 261) as well as increased TUNEL staining (40 ± 2 vs. 14 ± 1) and DNA fragmentation (all P < 0.03 vs. control). In FasL-/- and Fas-/- mice, AP-induced upregulation of p38-MAPK, PARP, and cytochrome C was significantly attenuated (all P < 0.01 compared to C57/C57 control). In addition, AP-induced DNA fragmentation was reduced 60% in Fas-/- and FasL-/- mice (P < 0.01 vs. C57/C57). AP induces apoptosis by transcriptional activation of Fas/FasL. AP-induced apoptosis was significantly reduced in Fas and FasL knockout mice along with downregulation of p38-MAPK, PARP, and cytochrome C, thereby suggesting a central role for Fas/FasL in hepatocyte apoptosis. The manipulation of interactions between Kupffer cell-derived FasL and hepatocytes may have important therapeutic implications.

Roux-en-Y gastric bypass improves hepatic mitochondrial function in obese rats

BACKGROUND Obesity-related fatty liver disease is linked to mitochondrial dysfunction and oxidative stress. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) regulates mitochondrial function and is a transcriptor of multiple genes that produce antioxidants. Because Roux-en-Y gastric bypass (RYGB) improves fatty liver and decreases the oxidative stress in the liver, we hypothesized that RYGB activates Nrf2 and increases cytochrome C oxidase subunit II (COX-II) in the liver of obese rats. METHODS Sprague-Dawley rats were fed a high-fat diet for 16 weeks. The obese rats underwent either RYGB (n = 20) or a sham operation (n = 20). The tissues were harvested 13 weeks postoperatively. The nuclear fraction and mitochondrial extracts were used for protein analysis with immunoblotting. Immunostaining was done on liver sections for COX-II, Nrf2, and the macrophage marker ED2 and F4/80. The gels were quantified using densitometry; P ≤ .05 was considered significant. RESULTS RYGB increased COX-II expression in the liver sections (3330 ± 56 versus 2056 ± 37 for RYGB versus sham, P < .001). The total (nuclear and cytoplasmic) Nrf2 expression was high in the obese sham-operated control (2456 ± 45 versus 4352 ± 76, RYGB versus sham, P < .001). However, the nuclear fraction of Nrf2 was significantly increased in the RYGB liver (2341 ± 46 versus 1352 ± 35, RYGB versus sham, P < .001). Furthermore, Nrf2 protein co-localized with the molecular markers of Kupffer cells. CONCLUSIONS Diet-induced fatty liver is associated with mitochondrial dysfunction. RYGB increases COX-II, which is involved in mitochondrial respiration, and increases the nuclear translocation of the Nrf2 transcriptional factor, which is involved in mitochondrial biogenesis and function. Taken together, these data suggest that surgically induced weight loss is associated with improved mitochondrial function in obese rats.

Roux-en-Y gastric bypass alters tumor necrosis factor-α but not adiponectin signaling in immediate postoperative period in obese rats

BACKGROUND Adiponectin has anti-inflammatory properties and is increased with weight loss. Tumor necrosis factor (TNF)-α is a pro-inflammatory cytokine that negatively regulates adiponectin. Previously, we have demonstrated that Roux-en-Y gastric bypass (RYGB) induces weight loss and improves steatosis in obese rats. We hypothesized that RYGB would alter the interplay of TNF-α and adiponectin signaling in the postoperative period. METHODS Obese Sprague-Dawley male rats that had undergone RYGB (n = 5) or sham (n = 4) were euthanatized at 9 weeks postoperatively. The adiponectin levels from serial serum samples were measured by enzyme-linked immunosorbent assay. Adiponectin, adiponectin receptor 2, and TNF-α mRNA from adipose and liver samples were quantified by reverse transcriptase-polymerase chain reaction. Data are presented as mean ± standard deviation; using a t test, P <.05 was significant. RESULTS RYGB did not change the serum adiponectin, adipose tissue adiponectin mRNA, or hepatic adiponectin receptor 2 levels compared with the levels in the sham-operated rats (P >.05). However, the TNF-α mRNA levels had decreased in the adipose tissue (P >.05) but remained unchanged in the liver compared with the sham controls (P >.05). CONCLUSION Surgically-induced weight loss in a rat model of RYGB did not increase adiponectin signaling in the immediate postoperative period but was associated with decreased pro-inflammatory signaling in the adipose tissue. During this period, pro-inflammatory signaling might play a more important role than adiponectin. Additional studies with longer follow-up are necessary to determine whether adiponectin plays a role in weight loss and improvement of steatosis after RYGB.

Roux-en-Y gastric bypass improves glucose homeostasis, reduces oxidative stress and inflammation in livers of obese rats and in Kupffer cells via an AMPK-dependent pathway

Background. Oxidative stress and inflammation are implicated in the pathogenesis of steatohepatitis. We hypothesize that Roux‐en‐Y gastric bypass reduces oxidative stress and inflammation in the liver of obese rats via activation of AMPK‐&agr;. Methods. Obese Sprague‐Dawley male rats underwent either sham operation or Roux‐en‐Y gastric bypass. Hepatic TNF‐&agr;, NF‐&kgr;B, IRS‐2, PI3 kinase, PKC‐&zgr;, NOX2, and AMPK‐&agr; were measured. Mechanistic studies were done in a rat Kupffer cell line (RKC1) that was treated with free fatty acids to mimic lipotoxicity and then transfected with AMPK‐&agr; siRNA. Reactive oxygen species, TNF‐&agr;, NF‐&kgr;B, AMPK‐&agr;, p‐AMPK‐&agr;, PPAR‐&ggr;, and NOX2 were measured. A t test was used. Results. Roux‐en‐Y gastric bypass lowered nonfasting serum glucose, improved the glucose tolerance test, and induced IRS2/PI3 kinase interaction. Additionally, Roux‐en‐Y gastric bypass decreased hepatic NOX2, PKC‐&zgr;, TNF‐&agr; expression and activation of NF‐&kgr;B. Free fatty acids increased reactive oxygen species, TNF‐&agr; protein, NOX2 protein, and activated NF‐&kgr;B. Rosiglitazone attenuated the free fatty acids–induced increase in reactive oxygen species, TNF‐&agr;, NOX2, and NF‐&kgr;B; blocking AMPK‐&agr; by siRNA abolished the effects of rosiglitazone. Conclusion. Roux‐en‐Y gastric bypass exhibits antidiabetic properties and is associated with downregulation of proinflammation genes and oxidative stress in the liver and within Kupffer cells via activation of AMPK‐&agr;.