Impact of nonalcoholic fatty liver disease on clinical outcomes in patients with COVID-19 among persons living with HIV: A multicenter research network study.

BACKGROUND: People living with human immunodeficiency virus (PLWH) are at an increased risk of nonalcoholic fatty liver disease (NAFLD) but how these patients react to COVID-19 infection is unclear. We examined the clinical characteristics and outcomes of patients with and without nonalcoholic fatty liver disease (NAFLD) among people living with human immunodeficiency virus (PLWH) diagnosed with COVID-19. METHODS: A multicenter, retrospective cohort study was conducted using TriNetX. Participants diagnosed with COVID-19 between January 20, 2020, and October 31, 2021, in PLWH were identified and divided into cohorts based on preexisting NAFLD. The primary outcome was all-cause mortality, and secondary outcomes were hospitalization, severe disease, critical care, need for mechanical ventilation, and acute kidney injury(AKI). Propensity score matching (PSM) mitigated the imbalance among group covariates. Risk ratios (RR) with 95 % confidence intervals (CI) were calculated. RESULTS: Of the 5012 PLWH identified with confirmed COVID-19 during the study period, 563 had a diagnosis of NAFLD. After PSM, both groups were well-matched with 561 patients. The primary outcome did not differ between the cohorts at 30-days, even after a fully adjusted analysis, and the risk of all-cause mortality did not differ at 60 and 90 days. NAFLD had a significantly higher risk for hospitalization rates (RR 1.32; 95 % CI, 1.06-1.63) and AKI (RR 2.55; 95 % CI 1.42-4.57) than the non-NAFLD group at 30 days. No other differences were detected in other secondary outcome measures. CONCLUSIONS: Preexisting NAFLD is associated with an increased risk for hospitalization and AKI among PLWH infected with COVID-19. The potential role of NAFLD in developing severe COVID-19 among PLWH remains to be elucidated in future studies. Still, this study indicates the need for careful monitoring of this at-risk population.

Changes in Gut Microbiome after Bariatric Surgery Versus Medical Weight Loss in a Pilot Randomized Trial.

BACKGROUND: Gut microbiota likely impact obesity and metabolic diseases. We evaluated the changes in gut microbiota after surgical versus medical weight loss in adults with diabetes and obesity. METHODS: We performed 16S rRNA amplicon sequencing to identify the gut microbial composition at baseline and at 10% weight loss in adults with diabetes who were randomized to medical weight loss (MWL, n = 4), adjustable gastric banding (AGB, n = 4), or Roux-en-Y gastric bypass (RYGB, n = 4). RESULTS: All participants were female, 75% reported black race with mean age of 51 years. At similar weight loss amount and glycemic improvement, the RYGB group had the most number of bacterial species (10 increased, 1 decreased) that significantly changed (p < 0.05) in relative abundance. Alpha-diversity at follow-up was significantly lower in AGB group compared to MWL and RYGB (observed species for AGB vs. MWL, p = 0.0093; AGB vs. RYGB, p = 0.0093). The relative abundance of Faecalibacterium prausnitzii increased in 3 participants after RYGB, 1 after AGB, and 1 after MWL. CONCLUSIONS: At similar weight loss and glycemic improvement, the greatest alteration in gut microbiota occurred after RYGB with an increase in the potentially beneficial bacterium, F. prausnitzii. Gut microbial diversity tended to decrease after AGB and increase after RYGB and MWL. Future studies are needed to determine the impact and durability of gut microbial changes over time and their role in long-term metabolic improvement after bariatric surgery in adults with type 2 diabetes. CLINICAL TRIAL REGISTRATION: NCTDK089557- ClinicalTrials.gov.

The Role of Laboratory Testing in Differentiating Type 1 Diabetes from Type 2 Diabetes in Patients Undergoing Bariatric Surgery.

BACKGROUND: It may be difficult to distinguish between adults with type 1 diabetes and type 2 diabetes by clinical assessment. In patients undergoing bariatric surgery, it is critical to correctly classify diabetes subtype to prevent adverse perioperative outcomes including diabetic ketoacidosis. This study aimed to determine whether testing for C-peptide and islet cell antibodies during preoperative evaluation for bariatric surgery could improve the classification of type 1 versus type 2 diabetes compared to clinical assessment alone. METHODS: This is a retrospective analysis of the Improving Diabetes through Lifestyle and Surgery trial, which randomized patients with clinically diagnosed type 2 diabetes and BMI 30-40 kg/m2 to medical weight loss or bariatric surgery; one participant was discovered to have type 1 diabetes after experiencing postoperative diabetic ketoacidosis. Using blood samples collected prior to study interventions, we measured islet cell antibodies and fasting/meal-stimulated C-peptide in all participants. RESULTS: The participant with type 1 diabetes was similar to the 11 participants with type 2 diabetes in age at diagnosis, adiposity, and glycemic control but had the lowest C-peptide levels. Among insulin-treated participants, fasting and stimulated C-peptide correlated strongly with the C-peptide area-under-the-curve on mixed meal tolerance testing (R = 0.86 and 0.88, respectively). Three participants, including the one with type 1 diabetes, were islet cell antibody positive. CONCLUSIONS: Clinical characteristics did not correctly identify type 1 diabetes in this study. Preoperative C-peptide testing may improve diabetes classification in patients undergoing bariatric surgery; further research is needed to define the optimal C-peptide thresholds.

Activation of liver X receptor/retinoid X receptor pathway ameliorates liver disease in Atp7B(-/-) (Wilson disease) mice.

UNLABELLED: Wilson disease (WD) is a hepatoneurological disorder caused by mutations in the copper-transporter, ATP7B. Copper accumulation in the liver is a hallmark of WD. Current therapy is based on copper chelation, which decreases the manifestations of liver disease, but often worsens neurological symptoms. We demonstrate that in Atp7b(-/-) mice, an animal model of WD, liver function can be significantly improved without copper chelation. Analysis of transcriptional and metabolic changes in samples from WD patients and Atp7b(-/-) mice identified dysregulation of nuclear receptors (NRs), especially the liver X receptor (LXR)/retinoid X receptor heterodimer, as an important event in WD pathogenesis. Treating Atp7b(-/-) mice with the LXR agonist, T0901317, ameliorated disease manifestations despite significant copper overload. Genetic markers of liver fibrosis and inflammatory cytokines were significantly decreased, lipid profiles normalized, and liver function and histology were improved. CONCLUSIONS: The results demonstrate the major role of an altered NR function in the pathogenesis of WD and suggest that modulation of NR activity should be explored as a supplementary approach to improving liver function in WD. (Hepatology 2016;63:1828-1841).

Effects of vitamin D3 stimulation of thioredoxin-interacting protein in hepatocellular carcinoma.

AIM: Thioredoxin-interacting protein (TXNIP) promotes oxidative stress by inactivating thioredoxin (TXN). This protein is involved in diverse disease processes, including insulin resistance, atherosclerosis and carcinogenesis. The aim of the present study was to measure the expression and function of TXNIP in in vitro models of liver disease, as well as in primary human hepatocellular carcinoma (HCC) tissue specimens. In addition, we wanted to determine the effects of vitamin D3-induced TXNIP stimulation in HCC-derived cell lines. METHODS: TXNIP expression was measured by quantitative reverse transcription polymerase chain reaction and western blots. TXNIP expression was stimulated by vitamin D exposure and by transfection. Cell proliferation, apoptosis and reactive oxygen species were determined by standard assays. RESULTS: TXNIP expression levels were low in HCC cell lines, and vitamin D3 stimulated TXNIP expression in vitro. In HCC cells transfected with a TXNIP expression vector or treated with exogenous vitamin D3, there was a reduction in cell proliferation and an increase in apoptosis. Cells expressing TXNIP were markedly susceptible to oxidative injury induced by cobalt chloride or bacterial lipopolysaccharide. TXNIP expression was reduced or absent in a majority of primary human HCC specimens relative to matching, non-cancerous liver tissue. CONCLUSION: TXNIP expression is low or absent in human HCC specimens and HCC-derived cell lines. Vitamin D3 stimulates TXNIP expression, resulting in diminished proliferation and enhanced apoptosis. Liver cells expressing TXNIP are primed for oxidative injury. These findings suggest that stimulation of TXNIP expression, by factors such as vitamin D3, may attenuate carcinogenesis in patients with chronic liver disease.

Elevated ALT and GGT predict all-cause mortality and hepatocellular carcinoma in Taiwanese male: a case-cohort study.

PURPOSE: Evidence indicates a positive association between liver enzymes and the risk of death in Western countries; however, the evidence in Asian populations is scarce. We investigated the association between liver enzymes and total, cardiovascular (CVD), cancer and hepatocellular carcinoma (HCC) mortality in a cohort of Taiwanese male free of cancer at baseline. METHODS: From 1996 to 2003, 54,751 Taiwanese male aged 40-80 years without cancer completed a health screening and were followed through 2005 (5.8 ± 2.5 years of follow-up). A random cohort of 3,961 male was selected to compare to 1,864 male who died. We used Cox proportional hazards regression models to assess the risk of all-cause, cardiovascular and cancer mortality associated with alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma glutamyl transferase (GGT). RESULTS: In this population, higher levels of ALT, AST and GGT were significantly associated with all-cause mortality [hazard ratio (HR) 1.2, 1.8 and 1.6 for ALT, AST and GGT, respectively; all p < 0.05], cancer mortality (HR 1.8-2.8) and HCC mortality (HR 5.5-36.1). GGT was significantly associated with CVD mortality (HR 1.2). CONCLUSIONS: In Taiwanese male free of cancer at baseline, elevations of ALT, AST and GGT were associated with future risk of all-cause death, all cancer and HCC mortality, independent of conventional risk factors, and could be used to identify male who would benefit from HCC screening.

Yes-associated protein regulates the hepatic response after bile duct ligation.

UNLABELLED: Human chronic cholestatic liver diseases are characterized by cholangiocyte proliferation, hepatocyte injury, and fibrosis. Yes-associated protein (YAP), the effector of the Hippo tumor-suppressor pathway, has been shown to play a critical role in promoting cholangiocyte and hepatocyte proliferation and survival during embryonic liver development and hepatocellular carcinogenesis. Therefore, the aim of this study was to examine whether YAP participates in the regenerative response after cholestatic injury. First, we examined human liver tissue from patients with chronic cholestasis. We found more-active nuclear YAP in the bile ductular reactions of primary sclerosing cholangitis and primary biliary cirrhosis patient liver samples. Next, we used the murine bile duct ligation (BDL) model to induce cholestatic liver injury. We found significant changes in YAP activity after BDL in wild-type mice. The function of YAP in the hepatic response after BDL was further evaluated with liver-specific Yap conditional deletion in mice. Ablating Yap in the mouse liver not only compromised bile duct proliferation, but also enhanced hepatocyte necrosis and suppressed hepatocyte proliferation after BDL. Furthermore, primary hepatocytes and cholangiocytes isolated from Yap-deficient livers showed reduced proliferation in response to epidermal growth factor in vitro. Finally, we demonstrated that YAP likely mediates its biological effects through the modulation of Survivin expression. CONCLUSION: Our data suggest that YAP promotes cholangiocyte and hepatocyte proliferation and prevents parenchymal damage after cholestatic injury in mice and thus may mediate the response to cholestasis-induced human liver disease.

Coordinated effects of microRNA-494 induce G2/M arrest in human cholangiocarcinoma.

MicroRNA (miRs) have emerged as salient regulators in cancer homeostasis and, recently, as putative therapeutics. Cholangiocarcinomas (CCA) are aggressive cancers with survival usually measured in months. mRNA arrays followed by pathway analysis revealed that miR-494 is a major modulator of the cell cycle progression from gap 2 (G2) to mitosis (M). We performed fluorescence activated cell sorting (FACS) as well as differential interference contrast (DIC) microscopy, and confirmed that miR-494 induces a significant arrest in G2/M in CCA cells. Furthermore, we verified that miR-494 modulates the protein level of six genes involved in the G2/M transition: Polo-like Kinase 1 (PLK1), pituitary tumor-transforming gene 1 (PTTG1), Cyclin B1 (CCNB1), cell-division cycle 2 (CDC2), cell-division cycle 20 (CDC20) and topoisomerase II α (TOP2A). Next, we identified direct binding of miR-494 to the open reading frame (ORF) and downregulation of PTTG1 and TOP2A. In summary, our findings suggest that miR-494 has a global regulatory role in cell cycle progression, exerted by concerted effects on multiple proteins involved in gap 1 (G1) to synthesis (S), as described previously, as well as G2 to M progression. Therefore, it appears that the simultaneous effects of a single miR species on multiple targets along the same canonical pathway is advantageous for the usage of miRs as therapeutics. In addition, our data suggest that miRs act within a narrow range. miR expression above the upper threshold does not appear to induce further effects, which is reassuring in terms of off-target effects of miR surrounding noncancerous tissue.

Inositol pyrophosphates inhibit Akt signaling, thereby regulating insulin sensitivity and weight gain.

The inositol pyrophosphate IP7 (5-diphosphoinositolpentakisphosphate), formed by a family of three inositol hexakisphosphate kinases (IP6Ks), modulates diverse cellular activities. We now report that IP7 is a physiologic inhibitor of Akt, a serine/threonine kinase that regulates glucose homeostasis and protein translation, respectively, via the GSK3ß and mTOR pathways. Thus, Akt and mTOR signaling are dramatically augmented and GSK3ß signaling reduced in skeletal muscle, white adipose tissue, and liver of mice with targeted deletion of IP6K1. IP7 affects this pathway by potently inhibiting the PDK1 phosphorylation of Akt, preventing its activation and thereby affecting insulin signaling. IP6K1 knockout mice manifest insulin sensitivity and are resistant to obesity elicited by high-fat diet or aging. Inhibition of IP6K1 may afford a therapeutic approach to obesity and diabetes.

Histone deacetylase inhibition suppresses the transforming growth factor beta1-induced epithelial-to-mesenchymal transition in hepatocytes.

UNLABELLED: Transforming growth factor beta1 (TGFbeta1) plays a crucial role in the induction of the epithelial-to-mesenchymal transition (EMT) in hepatocytes, which contributes to the pathogenesis of liver fibrosis. The inhibition of the TGFbeta1 cascade suppresses EMT and the resultant fibrosis. In this study, we focus on EMT-induced fibrosis in hepatocytes and the epigenetic regulation of the type I collagen gene. Histone acetylation is an important, major epigenetic mechanism that modulates gene transcription. We evaluated the epigenetic regulation of type I collagen in alpha mouse liver 12 hepatocytes (an untransformed mouse cell line) that had undergone EMT after treatment with TGFbeta1. The histone deacetylase inhibitor trichostatin A (TSA) inhibited EMT; this was reflected by the preservation of epithelial markers and function (E-cadherin and albumin). Fibrosis, the ultimate outcome of EMT, was abolished by TSA; this was indicated by the inhibition of type I collagen deposition. TSA exerted its anti-EMT effects by deactivating the mothers against decapentaplegic homolog 3 (Smad3)/Smad4 transcription complex and by interfering with p300, a coactivator of the type I collagen promoter, and preventing its binding to Smad3. TSA also restored Friend leukemia virus integration 1, an inhibitor of the type I collagen gene. TGFbeta1-induced EMT and its inhibition by TSA were replicated in human primary hepatocytes. CONCLUSION: Histone deacetylase inhibition abrogates TGFbeta1-induced EMT in hepatocytes and reverses EMT-induced fibrosis by epigenetic modulation of type I collagen.

Deficiency of nicotinamide adenine dinucleotide phosphate, reduced form oxidase enhances hepatocellular injury but attenuates fibrosis after chronic carbon tetrachloride administration.

UNLABELLED: Reactive oxygen species (ROS) activate hepatic stellate cells and enhance fibrogenesis. This study determined the role of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase deficiency in the development of hepatocellular necrosis, inflammation, and apoptosis in relation to fibrosis produced by chronic carbon tetrachloride (CCl(4)) administration. Wild-type (WT) mice or mice with deficiency of the gp91(phox) subunit of NADPH complex (gp91(phox(-/-) )) were subjected to biweekly CCl(4) injections over 8 weeks, whereas controls were given isovolumetric injections of olive oil. Serum aspartate aminotransferase (AST) was higher after CCl(4) administration in gp91(phox(-/-) ) than in WT mice, correlating with increased necrosis on liver histology. By contrast, more hepatocyte apoptosis was found after CCl(4) in the WT than in the gp91(phox(-/-) ) mice, which was associated with changes in components of the mitochondrial pathway of apoptosis, namely, an increase in the pro-apoptotic BAX protein in the WT, but not in the gp91(phox(-/-) ) mice and also a lower cytosolic cytochrome c in the gp91(phox(-/-) ) mice. There were fewer stellate cells and less fibrosis after CCl(4) in the gp91(phox(-/-) ) as compared with the WT mice. The increase in alpha(1)(I) collagen messenger RNA (mRNA), however, was greater after CCl(4) in the gp91(phox(-/-) ) mice. Matrix metalloproteinase-2 (MMP-2) and MMP-9 mRNA increased more in the gp91(phox(-/-) ) than in WT mice after CCl(4.) Tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2 increased after CCl(4) only in the gp91(phox(-/-) ) mice. CONCLUSION: Decreased hepatic fibrosis after chronic CCl(4) administration in mice with NADPH oxidase deficiency occurs in the setting of greater necrosis and inflammation but decreased apoptosis.

Myc stimulates nuclearly encoded mitochondrial genes and mitochondrial biogenesis.

Although several genes involved in mitochondrial function are direct Myc targets, the role of Myc in mitochondrial biogenesis has not been directly established. We determined the effects of ectopic Myc expression or the loss of Myc on mitochondrial biogenesis. Induction of Myc in P493-6 cells resulted in increased oxygen consumption and mitochondrial mass and function. Conversely, compared to wild-type Myc fibroblasts, Myc null rat fibroblasts have diminished mitochondrial mass and decreased number of normal mitochondria. Reconstitution of Myc expression in Myc null fibroblasts partially restored mitochondrial mass and function and normal-appearing mitochondria. Concordantly, we also observed in primary hepatocytes that acute deletion of floxed murine Myc by Cre recombinase resulted in diminished mitochondrial mass in primary hepatocytes. Our microarray analysis of genes responsive to Myc in human P493-6 B lymphocytes supports a role for Myc in mitochondrial biogenesis, since genes involved in mitochondrial structure and function are overrepresented among the Myc-induced genes. In addition to the known direct binding of Myc to many genes involved in mitochondrial structure and function, we found that Myc binds the TFAM gene, which encodes a key transcriptional regulator and mitochondrial DNA replication factor, both in P493-6 lymphocytes with high ectopic MYC expression and in serum-stimulated primary human 2091 fibroblasts with induced endogenous MYC. These observations support a pivotal role for Myc in regulating mitochondrial biogenesis.

Effects of acetaldehyde and TNF alpha on the inhibitory kappa B-alpha protein and nuclear factor kappa B activation in hepatic stellate cells.

AIMS: Increased plasma tumour necrosis alpha (TNFalpha) and elevated monocyte nuclear factor kappa B (NF-kappaB) are associated with liver injury and inflammation in models of alcoholic liver disease and are found to be elevated in monocytes of patients with alcoholic hepatitis. Acetaldehyde enhances, whereas TNFalpha inhibits, transcription of the type I collagen promoters and type I collagen production. NF-kappaB, an inhibitor of the type I collagen promoters, is increased by both acetaldehyde and TNFalpha. This study determined the effects of acetaldehyde in comparison to the effects of TNFalpha on inhibitory kappa B-alpha (IkappaB-alpha) protein and NF-kappaB activation in hepatic stellate cells. METHODS: Activated rat hepatic stellate cells in culture were exposed to acetaldehyde or TNFalpha for short periods of time, following which the cells were harvested for the determination of IkappaB-alpha protein, IkappaB-alpha kinase activity and nuclear NF-kappaB. RESULTS: Acetaldehyde increased IkappaB-alpha kinase activity and decreased IkappaB-alpha after 10 min of exposure, with recovery towards control levels at 20 min. In contrast, TNFalpha resulted in higher IkappaB-alpha kinase activity at 20 min than at 10 min, and similar low IkappaB-alpha at 10 and 20 min. Both acetaldehyde and TNFalpha enhanced nuclear NF-kappaB (p65), but acetaldehyde alone also increased NF-kappaB (p50). CONCLUSIONS: TNFalpha and acetaldehyde independently activate NF-kappaB by rapid enhancement of IkappaB-alpha kinase activity and degradation of IkB-alpha protein. Increased TNFalpha is the principal mechanism for the elevation of NF-kappaB in severe alcoholic hepatitis. The elevation of NF-kappaB due to TNFalpha enhance liver injury, but inhibit fibrogenesis. In contrast, the effect of acetaldehyde in activating NF-kappaB is associated with increases in both liver injury and fibrogenesis, indicating that the effects of acetaldehyde on fibrogenesis are mediated by cytokines and by trans-acting factors other than NF-kappaB.

Identification of a novel NF-kappaB-binding site with regulation of the murine alpha2(I) collagen promoter.

Hepatic fibrosis is due to the increased synthesis and deposition of type I collagen. Acetaldehyde activates type I collagen promoters. Nuclear factor kappaB (NF-kappaB) was previously shown to inhibit expression of murine alpha(1)(I) and human alpha(2)(I) collagen promoters. The present study identifies binding of NF-kappaB, present in nuclear extracts of stellate cells, to a region between -553 and -537 of the murine alpha(2)(I) collagen promoter. The NF-kappaB (p65) expression vector inhibited promoter activity. Mutation of the promoter at the NF-kappaB-binding site increased basal promoter activity and abrogated the activating and inhibitory effects of transforming growth factor beta and tumor necrosis factor alpha, respectively, on promoter activity. Acetaldehyde increased IkappaB-alpha kinase activity and phosphorylated IkappaB-alpha, NF-kappaB nuclear protein, and its binding to the promoter. However, the activating effect of acetaldehyde was not affected by the mutation of the promoter. In conclusion, although acetaldehyde increases the binding of NF-kappaB to the murine alpha(2)(I) collagen promoter, this binding does not mediate the activating effect of acetaldehyde on promoter activity. The effects of acetaldehyde in increasing the translocation of NF-kappaB to the nucleus with increased DNA binding activity may be important in mediating the effects of acetaldehyde on other genes.

Leptin deficiency reduces but does not eliminate the development of hepatic fibrosis in mice infected with Schistosoma mansoni.

AIMS/BACKGROUND: Leptin, a product of the obese (ob) gene, was demonstrated previously in activated hepatic collagen-producing stellate cells, but not in quiescent retinol-storing stellate cells. The role of leptin in fibrogenesis is unknown. This study investigated the possible influence of leptin in the pathogenesis of fibrosis by determination of the amount of fibrosis produced by Schistosoma mansoni infection in leptin deficient male ob/ob mice as compared to control mice. METHODS: The mice were infected percutaneously with cercaria of Schistosoma mansoni and the amount of liver fibrosis determined 12 weeks after infection. The amount of hepatic collagen deposited was quantified by morphometric analysis of liver sections stained with sirius red and by hydroxyproline content. RESULTS: The amount of histologically detectable fibrosis was greater in the infected controls than in the infected ob/ob mice. In the infected control mice, but not in the ob/ob mice, the fibrosis surrounding the granuloma was broad and extended beyond the portal tracts into the lobule with the formation of fibrous septa. CONCLUSIONS: This study shows that leptin is a potentiating, but not an essential factor, for the development of hepatic fibrosis, because leptin deficiency reduces but does not prevent the development of hepatic fibrosis.