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Obesity represents the main contributor to nonalcoholic fatty liver disease (NAFLD) and adipose tissue is strongly interlaced with the liver in the disease pathogenesis and progression. Previous studies were restricted to investigate the hepatic transcriptome across the entire spectrum of NAFLD, whereas the transcriptomic changes which occur in white adipose tissue (WAT) in relation to liver damage have been poorly investigated. Therefore, we aimed to compare hepatic and adipose tissue transcriptome in NAFLD patients with the purpose to identify shared biomarkers useful for the diagnosis of advanced liver damage. We performed high-throughput RNA-sequencing in 167 hepatic samples from obese patients and in a subset of 79 matched adipose tissues. Patients were subdivided in normal liver, mild and severe NAFLD according to histology. Circulating cathepsin D (CTSD) was assessed by ELISA. We identified a specific transcriptomic signature that may discriminate patients with severe NAFLD and isolated steatosis, including 424 deregulated genes in liver and 209 in adipose tissue. According to pathway and network analyses, inflammation, ECM remodeling and mitochondrial dysfunction were upregulated whereas oxidative phosphorylation was downregulated in both tissues. We highlighted 13 genes commonly deregulated in both tissues and among them, CTSD showed the most robust diagnostic accuracy in discriminating mild and severe NAFLD. In 52 obese subjects and in a validation cohort of 432 histologically-characterized NAFLD patients, serum CTSD progressively increased from normal liver to severe NAFLD and it was associated with steatosis, necroinflammation, fibrosis, steatohepatitis (NASH) and NAS>5. The area under the curve (AUC) weighted for transaminases to foresee severe NAFLD versus mild and normal liver was 0.78 and 0.87, respectively. CTSD may be a possible biomarker of severe NAFLD since its hepatic/adipose tissue expression as well as circulating levels correlated with liver damage thus allowing to discriminate advanced disease. [ABSTRACT FROM AUTHOR]

β-Klotho (KLB) gene encodes the hepatic co-receptor of fibroblast growth factor receptor 4 (FGFR4). We previously reported that the rs17618244 G>A KLB gene variant dampened KLB hepatic and plasma levels, leading to inflammation, ballooning and fibrosis both in pediatric and adult patients with nonalcoholic fatty liver disease (NAFLD). Hepatic stellate cells (HSCs) are directly involved in the fibrotic processes, playing a crucial role in the switching to severe forms of NAFLD. To generate a new cell line of hepatic stellate cells (HSCs), responsible for hepatic fibrosis, which are deleted for KLB , by using Crispr/Cas9 and to investigate the impact of its deficiency on their pro-fibrotic phenotype, inflammation and oxidative stress. We stably silenced KLB gene in LX2 cells (referred to as HSCs KLB−/−) by Crispr-Cas9 technology. Then, markers of activation, cellular stress, inflammation and proliferation have been investigated. KLB mRNA and protein levels were reduced in HSC KLB −/- cells (p<0.05). Markers of activation (i.e., aSMA, COL1A1/3A1/4A1, TGFb, PDGFRB, BAMBI) were increased in KLB−/− cells at both gene and protein levels. Accordingly, retinol synthesis (RALDH) was reduced (p<0.05), suggesting a more pro-fibrotic phenotype. Moreover, KLB deletion strongly induced oxidative stress, enhancing ROS/reactive nitrite species (RNS) and lactate production, aldehyde derivate concentrations (MDA) (p<0.05). These events triggered the release of pro-inflammatory cytokines (TNFα, IL1β and IL6) into the KLB −/- cultured media. Finally, KLB −/- cells also showed an elevated proliferative rate (p<0.05). We generated, for the first time, a stable knock-out of KLB in HSCs. Our preliminary results outlined that KLB deficiency induced an enhanced pro-fibrotic phenotype with increased proliferation, inflammation and oxidative stress, thus possibly explaining the association between the rs17618244 KLB at-risk variant and more severe forms of liver damage. Altogether, these observations pointed KLB a novel target to prevent severe NAFLD. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction is a key player in the transition from NASH to HCC. The knock-out (KO) of MBOAT7 and/or TM6SF2 hampers the mitochondrial dynamics in Hepg2 cells resulting in an enrichment of misshapen and failed mitochondria. The overexpression of MBOAT7 and/or TM6SF2 wild-type genes in KO models through lentiviral vectors decreases the number of damaged-globular mitochondria, while increases the normo-shaped ones. To deepen the impact of PNPLA3/MBOAT7/TM6SF2 loss-of-function mutations on mitochondrial aberrances, we investigated the mitochondrial lifecycle and activity in KO cells overexpressed for the wild-type forms of MBOAT7 and/or TM6SF2. Mitochondrial lifecycle and activity were assessed through RT-PCR, Western Blot, Seahorse assay and immunohistochemistry. The overexpression of MBOAT7, TM6SF2 or both decreased PGC1α, the master regulator of mitobiogenesis, which was activated in KO cells in response to fusion-fission unbalance, boosted the expression of Mfn1/Mfn2 (mitochondrial outer membranes proteins involved in fusion) and OPA1 (inner mitochondrial membranes fusion protein) whereas decreased FIS1 and DRP1 (fission proteins) levels, thus re-establishing the mitochondrial turnover. Consistently, the mitophagy pathways (PINK/PARKIN/BNIP3/BNIP3-L/LC3/phospho-UBIQUITIN) increased after the MBOAT7 and/or TM6SF2 overexpression, prompting the disruption of damaged mitochondria. The balance of mitochondrial biogenesis is essential for organelles' homeostasis and activity. Indeed, the overexpressed models augmented the COX-I/SDHA ratio, COX-III, and citrate synthase activity alongside the oxygen consumption rate, recovering the OXPHOS capacity and Krebs cycle which were impaired in KO cells. Finally, lactate levels decreased after the upregulation of MBOAT7 and/or TM6SF2 wild-type genes together with the glycolytic/extracellular acidification rate, thus inhibiting the switch to anerobic glycolysis which was promoted in KO cells to trigger tumorigenesis. Genetics impacts on mitochondrial maladaptation during NAFLD and the overexpression of MBOAT7 and/or TM6SF2 wild-type genes in KO HepG2 cells re-balances the mitochondrial lifecycle and turnover, thus ensuring the organelles' function and possibly reversing hepatocellular damage. [ABSTRACT FROM AUTHOR]

Mitochondrial (mt) dysfunction is a hallmark of progressive NAFLD. MtDNA copy number (mtDNA-CN) and cell-free circulating mtDNA (ccf-mtDNA), which reflect mt-mass and mt-dysfunction, respectively, are gaining attention for NAFLD non-invasive assessment. We demonstrated that PNPLA3, MBOAT7 and TM6SF2 deficiency in HepG2 cells increased mt-mass, mtDNA-CN and ccf-mtDNA. To assess the genetic contribution on mt-dynamics, mtDNA-CN and ccf-mtDNA in 1) primary mouse hepatocytes silenced for PNPLA3 / MBOAT7/TM6SF2 genes; 2) Discovery (n=28) and Validation (n=773) cohorts, including biopsied NAFLD patients, stratified according to number of risk variants (NRV=3). Mt-morphology was assessed by TEM. mtDNA-CN was measured in the entire Validation cohort (n=773), while ccf-mtDNA in a subgroup (n=300) with available serum samples. mtDNA-CN and mt-related genes were evaluated in liver biopsies. Primary mouse hepatocytes challenged with fat overload or PNPLA3/TM6SF2 / MBOAT7 co-silencing lowered mt-fusion paralleled by higher mt-fission and ccf-mtDNA release, suggesting that lipid accumulation and genetics may independently unbalance mt-dynamics. In the Discovery cohort, NRV=3 patients showed the highest mtDNA-CN compared to those with 1-2 or no variants. At TEM, NRV=3 carriers increased mt-mass and presented an elevated pattern of mt-morphological alterations (swollen shapes, double membranes rupture). In the Validation cohort, mtDNA-CN associated with the NAFLD histological spectrum and NRV=3 at multivariate analyses, supporting that both NAFLD severity and genetics may modulate mt-dynamics. In liver biopsies, mtDNA-CN was higher in NRV=3 patients together with reduction of mt-fusion and activation of mt-fission, resembling what observed in hepatocytes. Ccf-mtDNA was augmented in NRV=3 patients with low-moderate/severe NAFLD, thereby sustaining that this effect was amenable to the 3 at-risk polymorphisms. ROC curves showed that mtDNA-CN discriminated NAFLD subjects vs controls (AUC: 0.71), while ccf-mtDNA was highly predictive of NAFLD-HCC vs NALFD (AUC: 0.79). mtDNA-CN and ccf-mtDNA may have pathological and predictive significance in NAFLD patients at high-risk, especially in those genetically-predisposed. [ABSTRACT FROM AUTHOR]