NASH Patients Research Articles

Hepatic PEMT Expression Decreases with Increasing NAFLD Severity.

Choline deficiency causes hepatic fat accumulation, and is associated with a higher risk of nonalcoholic fatty liver disease (NAFLD) and more advanced NAFLD-related hepatic fibrosis. Reduced expression of hepatic phosphatidylethanolamine N-methyltransferase (PEMT), which catalyzes the production of phosphatidylcholine, causes steatosis, inflammation, and fibrosis in mice. In humans, common PEMT variants impair phosphatidylcholine synthesis, and are associated with NAFLD risk. We investigated hepatic PEMT expression in a large cohort of patients representing the spectrum of NAFLD, and examined the relationship between PEMT genetic variants and gene expression. Hepatic PEMT expression was reduced in NAFLD patients with inflammation and fibrosis (i.e., nonalcoholic steatohepatitis or NASH) compared to participants with normal liver histology (β = −1.497; p = 0.005). PEMT levels also declined with increasing severity of fibrosis with cirrhosis < incomplete cirrhosis < bridging fibrosis (β = −1.185; p = 0.011). Hepatic PEMT expression was reduced in postmenopausal women with NASH compared to those with normal liver histology (β = −3.698; p = 0.030). We detected a suggestive association between rs7946 and hepatic fibrosis (p = 0.083). Although none of the tested variants were associated with hepatic PEMT expression, computational fine mapping analysis indicated that rs4646385 may impact PEMT levels in the liver. Hepatic PEMT expression decreases with increasing severity of NAFLD in obese individuals and postmenopausal women, and may contribute to disease pathogenesis in a subset of NASH patients.

Circulating TREM2 as a noninvasive diagnostic biomarker for NASH in patients with elevated liver stiffness.

Reliable noninvasive biomarkers are an unmet clinical need for the diagnosis of NASH. This study investigates the diagnostic accuracy of the circulating triggering receptor expressed on myeloid cells 2 (plasma TREM2) as a biomarker for NASH in patients with NAFLD and elevated liver stiffness. We collected cross-sectional, clinical data including liver biopsies from a derivation ( n = 48) and a validation cohort ( n = 170) of patients with elevated liver stiffness measurement (LSM ≥ 8.0 kPa). Patients with NAFLD activity scores (NAS) ≥4 were defined as having NASH. Plasma TREM2 levels were significantly elevated in patients with NASH of the derivation cohort, with an area under the receiver operating characteristics curve (AUROC) of 0.92 (95% confidence interval [CI], 0.84-0.99). In the validation cohort, plasma TREM2 level increased approximately two-fold in patients with NASH, and a strong diagnostic accuracy was confirmed (AUROC, 0.83; 95% CI, 0.77-0.89; p < 0.0001). Plasma TREM2 levels were associated with the individual histologic features of NAS: steatosis, lobular inflammation, and ballooning ( p < 0.0001), but only weakly with fibrosis stages. Dual cutoffs for rule-in and rule-out were explored: a plasma TREM2 level of ≤38 ng/ml was found to be an optimal NASH rule-out cutoff (sensitivity 90%; specificity 52%), whereas a plasma TREM2 level of ≥65 ng/ml was an optimal NASH rule-in cutoff (specificity 89%; sensitivity 54%). Plasma TREM2 is a plausible individual biomarker that can rule-in or rule-out the presence of NASH with high accuracy and thus has the potential to reduce the need for liver biopsies and to identify patients who are eligible for clinical trials in NASH.

FRI516 - Derivation of machine learning histologic scores correlating with portal pressures and the development of varices in NASH patients with cirrhosis

FRI516 - Derivation of machine learning histologic scores correlating with portal pressures and the development of varices in NASH patients with cirrhosis

FRI193 - Quantitative assessment of liver fibrosis in NASH patients

FRI193 - Quantitative assessment of liver fibrosis in NASH patients

SAT114 - Efx treatment improved histopathology and non-invasive markers of liver injury and fibrogenesis to a similar extent in NASH patients with high-risk PNPLA3 genotypes, compared to those with lowest genetic risk: a post-hoc analysis of balanced study

SAT114 - Efx treatment improved histopathology and non-invasive markers of liver injury and fibrogenesis to a similar extent in NASH patients with high-risk PNPLA3 genotypes, compared to those with lowest genetic risk: a post-hoc analysis of balanced study

OS125 - Safety, pharmacokinetics and efficacy of the novel pan-phosphodiesterase inhibitor ZSP1601 in 36 NASH patients: a double-blinded, placebo-controlled, multiple-dose escalation phase Ib study

OS125 - Safety, pharmacokinetics and efficacy of the novel pan-phosphodiesterase inhibitor ZSP1601 in 36 NASH patients: a double-blinded, placebo-controlled, multiple-dose escalation phase Ib study

EPH141 Assessing Cardiovascular Disease (CVD) Risk By Fibrosis Stage Defined By Fibrosis-4 (FIB-4) Among Patients with Non-Alcoholic Steatohepatitis (NASH) Using Real World Data (RWD)

Accumulating evidence has shown that NASH is related to increased risk of death caused by CVD, but little is known about the association between fibrosis stage and CVD. This study assessed CVD risk in NASH patients by fibrosis stage using a large real-world database.

African genetic ancestry is associated with lower frequency of PNPLA3 G allele in non-alcoholic fatty liver in an admixed population

Introduction and objectivesPNPLA3 (rs738409) and TM6SF2 (rs58542926) variants, interindividual and ethnic differences may be risk factors for non-alcoholic fatty liver disease (NAFLD). The PNPLA3 G allele is associated with worse NAFLD evolution in Hispanics and Caucasians. TM6SF2 is associated with hypertriglyceridemia, NAFLD, and cardiovascular disease. We aimed to evaluate the association between genetic ancestry by Ancestry Informative Markers (AIM), PNPLA3 and TM6SF2 polymorphisms in patients with biopsy-proven NAFLD in an admixed population. MethodsWe included adults with biopsy-proven NAFLD and excluded patients with the presence of other chronic liver disease, alcohol intake >100g/week, HIV, drug-induced fatty liver disease, or liver transplantation. We classified NAFLD using the Non-Alcoholic Steatohepatitis Clinical Research Network (NASH-CRN) histological scoring system. The PNPLA3 (rs738409 c.444C>G) and TM6SF2 (rs58542926 c.449C>T) genotyping were performed by RT-PCR. Genetic ancestry was determined using 46 insertion-deletion AIM; α<0.05 was considered significant. ResultsA total of 248 patients with NAFLD were enrolled [34 with simple steatosis (NAFL); 214 with NASH]. Overall, we detected a greater European ancestry contribution (0.645), followed by African (0.173), Amerindian (0.095), and East Asian (0.087) ancestry contribution, without differences between NAFL and NASH patients. However, we found a higher African genetic ancestry contribution among patients with NAFL who had the PNPLA3 C/C genotype than those with the G allele (0.216 ± 0.205 versus 0.105 ± 0.101, respectively; p=0.047). Ancestry contributions did not differ among TM6SF2 genotypes. ConclusionAmong NAFL patients, greater African genetic ancestry was associated to a lower frequency of the PNPLA3 G allele, demonstrating a possible NASH ancestry-related protective factor.

Plasminogen Activator Inhibitor-1 Levels in Non-alcoholic Fatty Liver Disease: A Systematic Review and Meta-Analysis.

Several studies have investigated the role of multiple proteins in nonalcoholic fatty liver disease (NAFLD); one that has recently gained attention is plasminogen activator inhibitor-1 (PAI-1). However, studies evaluating PAI-1 levels in NAFLD demonstrated conflicting results. Our objective was to understand the role of PAI-1 in NAFLD more clearly by carrying out a systematic review and meta-analysis. We gathered evidence by performing a systematic search on PubMed, EMBASE, and Cochrane Library, through using a predefined search string. The included studies diagnosed NAFLD through either liver biopsy, ultrasonography, computed tomography (CT), magnetic resonance spectroscopy, or using one of the latter methods with blood parameters. Studies had to fulfill predefined inclusion and exclusion criteria. To assess the quality of the studies included, we used the NHLBI quality assessment tools. The main summary outcome was the mean difference (MD) in serum PAI-1 levels reported as ng/mL Results: 33 articles involving 10,840 subjects fulfilled our inclusion criteria and were systematically reviewed. 11 studies were included in our meta-analyses. We found a significant MD in PAI-1 levels in NAFLD patients vs. controls [17.147 (95%CI: 7.720-26.574)]. Moreover, subgroup analysis evaluating PAI-1 levels in biopsy- proven NAFLD vs. controls remained significant [24.086 (95%CI: 3.812-44.361)], as well as in CT-diagnosed NAFLD [15.523 (95%CI: 7.163-23.883)]. However, no significant MD in PAI-1 levels was found in ultrasound- diagnosed NAFLD patients vs. controls [10.394 (95%CI: -13.335-34.123)]. No significant MD in PAI-1 levels in NASH patients vs. controls was observed [26.835 (95%CI: -0.879-54.549)]. In summary, elevated serum PAI-1 levels are associated with adult NAFLD (biopsy-proven and CT-diagnosed). However, no significant difference was found in ultrasound-diagnosed NAFLD and NASH patients. Nonetheless, the included studies have methodological variance, dictating that the obtained results should be carefully interpreted.

Promotion of nonalcoholic steatohepatitis by RNA N6-methyladenosine reader IGF2BP2 in mice

AbstractNonalcoholic steatohepatitis (NASH) has emerged as the major cause of end-stage liver diseases. However, an incomplete understanding of its molecular mechanisms severely dampens the development of pharmacotherapies. In the present study, through systematic screening of genome-wide mRNA expression from three mouse models of hepatic inflammation and fibrosis, we identified IGF2BP2, an N6-methyladenosine modification reader, as a key regulator that promotes NASH progression in mice. Adenovirus or adeno-associated virus-mediated overexpression of IGF2BP2 could induce liver steatosis, inflammation, and fibrosis in mice, at least in part, by increasing Tab2 mRNA stability. Besides, hepatic overexpression of IGF2BP2 mimicked gene expression profiles and molecular pathways of human NASH livers. Of potential clinical significance, IGF2BP2 expression is significantly upregulated in the livers of NASH patients. Moreover, knockdown of IGF2BP2 substantially alleviated liver injury, inflammation, and fibrosis in diet-induced NASH mice. Taken together, our findings reveal an important role of IGF2BP2 in NASH, which may provide a new therapeutic target for the treatment of NASH.

272-OR: Weight Loss with Time-Restricted Feeding or Treatment with Obeticholic Acid/Semaglutide Combination Have a Different Impact on Nonalcoholic Steatohepatitis in Diet-Induced Obese Mice

Introduction: Combination of therapies is a promising strategy for the treatment of NASH and liver fibrosis. Here we evaluated whether the Farnesoid X Receptor agonist obeticholic acid (OCA) combined with the glucagon-like peptide-1 (GLP-1) receptor agonist semaglutide (SEMA) would show superior effects than weight loss induction with time-restricted feeding (TRF) in mice. Methods: Mice were fed a 60% high fat/2% cholesterol diet with 10% fructose supplemented drinking water (HFCF diet) for 25 weeks to induce obesity and NASH/liver fibrosis. After the diet-induction period, mice were kept on HFCF diet and treated with vehicle (control) or OCA 30mg/kg p.o. QD + SEMA 0.06mg/kg s.c. QD, or placed on TRF from the last 3 hours of the dark cycle till the end of the light cycle, without access to food, but free access to normal drinking water (i.e. without fructose) every day, for 6 weeks. Results: OCA+SEMA induced a 20% lower caloric intake, which led to a 27% lower body weight (p&amp;lt;0.0vs. control) . TRF had a weaker effect on caloric intake (-9%) but also reduced body weight (-11%, p&amp;lt;0.vs. control) . Both OCA+SEMA and TRF reduced the HOMA-IR index of insulin resistance and significantly reduced plasma ALT and AST levels, but these effects were more pronounced with OCA+SEMA. TRF, and to a greater extent OCA+SEMA, significantly reduced liver weight, hepatic fatty acids, triglycerides, and total cholesterol levels. Both OCA+SEMA and TRF led to significantly lower NAFLD activity score. However, OCA+SEMA did not alter liver fibrosis, while TRF showed a clear anti-fibrotic effect in the liver, with lower % Sirius Red labelling (p&amp;lt;0.vs. control) . Conclusion: OCA+SEMA combination reduced body weight and NAFLD activity score but did not improve hepatic fibrosis, while TRF improved both NASH and liver fibrosis. These TRF benefits should be further investigated in obese NASH patients. Disclosure F.Briand: None. E.Grasset: None. N.Breyner: None. T.Sulpice: None.

A Class Effect Network Meta-analysis of Lipid Modulation in Non-alcoholic Steatohepatitis for Dyslipidemia.

Pharmaceutical therapy for NASH is associated with lipid modulation, but the consensus on drug treatment is limited and lacks comparative analysis of effectiveness. A network meta-analysis was conducted to compare NASH drug classes in lipid modulation. Online databases were searched for randomized controlled trails (RCTs) evaluating NASH treatments in biopsy-proven NASH patients. Treatments were classified into four groups: (1) inflammation, (2) energy, (3) bile acids, and (4) fibrosis based on the mechanism of action. A Bayesian network analysis was conducted with outcome measured by mean difference (MD) with credible intervals (Crl) and surface under the cumulative ranking curve (SUCRA). Forty-four RCTs were included in the analysis. Bile acid modulating treatments (MD: 0.05, Crl: 0.03-0.07) were the best treatment for improvement in high-density lipid (HDL) cholesterol, followed by treatments modulating energy (MD: 0.03, Crl: 0.02-0.04) and fibrosis (MD: 0.01, Crl: -0.12 to 0.14) compared with placebo. The top three treatments for reduction in triglycerides were treatments modulating energy (MD: -0.46, Crl: -0.49 to -0.43), bile acids (MD: -0.22, Crl: -0.35 to -0.09), and inflammation (MD: -0.08, Crl: -0.13 to -0.03) compared with placebo. SUCRA found treatment modulating fibrosis (MD: -1.27, Crl: -1.76 to -0.79) was the best treatment for reduction in low-density lipid (LDL) cholesterol followed by treatment modulating inflammation (MD: -1.03, Crl: -1.09 to -0.97) and energy (MD: -0.37, Crl: -0.39 to -0.34) compared with placebo, but LDL cholesterol was worsened by treatments modulating bile acids. Network analysis comparing the class effects of dyslipidemia modulation in NASH found that treatment targets can include optimization of atherogenic dyslipidemia. Future studies are required to evaluate the cardiovascular outcomes.

Serum Mir-34A and Mir-122 as an early non-invasive diagnostic marker for simple steatosis and NASH in comparison to FIB-4

Early detection of simple steatosis and differentiating it from NASH is an important turning point for evaluation and management. Liver biopsy is the gold standard method, but it is unpractical for many reasons and not accepted by most patients. So, many noninvasive methods have been used including microRNAs and fibrosis score (Fib-4). Our study aimed to validate the usefulness of these biomarkers for the early diagnosis of simple steatosis, differentiating it from NASH and evaluating their performance in comparison to FIB-4.Sixty two patients who met the inclusion criteria and thirty healthy controls were evaluated by Routine laboratory tests, miRNA (122,34a), Fib-4 and Liver Stiffness Measurement (LSM). The diagnostic value of miRNAs and its correlations to clinical and laboratory data in NASH and simple steatosis were analyzed and compared with FIB-4. A simple model was developed, combining microRNA levels and hs-CRP. miR-122 and -34a levels showed differential values between simple steatosis and NASH patients (p&lt;0.01) and correlated with hepatic histology. Combined microRNA expression profiles with hs-CRP had a higher potential of simple steatosis and NASH compared to other risk biomarkers (AUC &gt; 0,990). We concluded that circulating miR-122 and -34a may act as a disease-specific noninvasive biomarker for NASH diagnosis. miRNA along with hs-CRP assigning a prediction panel with improved diagnostic value superior to FIB-4 in early recognition of NASH to tailor efficient therapeutic interventions.

Higher hepatic advanced glycation end products and liver damage markers are associated with nonalcoholic steatohepatitis

Advanced glycation end products (AGEs) may be associated with nonalcoholic fatty liver disease (NAFLD) from stimulation of oxidative stress, inflammation, and fibrosis. We hypothesized that patients with NAFLD would have a lower concentration of soluble AGEs receptor and higher quantity of serum and liver AGEs and an increase in hepatic smooth muscle actin alpha (α-SMA) and transforming growth factor beta 1 (TGF-β1) compared with a control group. We compared the presence of hepatic and serum AGEs, AGE soluble receptor (sRAGE), and markers associated with hepatic damage between NAFLD patients and controls without disease. Histological characteristics, plasma biochemical parameters, serum AGEs, serum receptor sRAGE, and liver proteins (α-SMA, TGF-β1, AGEs, immunohistochemistry) were assessed in participants aged 18 to 65 years, with NAFLD (simple steatosis [SS]: n = 7; steatohepatitis [NASH]: n = 15) and controls (n = 11). NASH patients presented higher glycated hemoglobin levels (%) (5.7; 5.4-6.3) compared with SS (5.4; 5.2-5.7) and controls (5.4; 5.3-5.5). The NAFLD activity score (NAS) for NASH patients was 4.9 ± 1.3; for SS patients, 2.0 ± 1.0. NASH patients showed higher hepatic AGEs, TGF-β1, and α-SMA compared with SS and control groups. The NAS score indicates that patients with 5 to 8 had higher hepatic AGEs, TGF-β1, and α-SMA compared with a NAS of 1 to 4 and 0. For α-SMA, a NAS of 1 to 4 was higher than NAS 0. No difference was found in serum AGEs and sRAGE between groups. Higher hepatic AGEs, TGF-β1, and α-SMA were observed with increasing disease severity (according to NAS); therefore, endogenous liver AGEs may participate in hepatic damage progression.

Discovery of an insulin-induced gene binding compound that ameliorates nonalcoholic steatohepatitis by inhibiting sterol regulatory element-binding protein-mediated lipogenesis.

NASH is associated with high levels of cholesterol and triglyceride (TG) in the liver; however, there is still no approved pharmacological therapy. Synthesis of cholesterol and TG is controlled by sterol regulatory element-binding protein (SREBP), which is found to be abnormally activated in NASH patients. We aim to discover small molecules for treating NASH by inhibiting the SREBP pathway. Here, we identify a potent SREBP inhibitor, 25-hydroxylanosterol (25-HL). 25-HL binds to insulin-induced gene (INSIG) proteins, stimulates the interaction between INSIG and SCAP, and retains them in the endoplasmic reticulum, thereby suppressing SREBP activation and inhibiting lipogenesis. In NASH mouse models, 25-HL lowers levels of cholesterol and TG in serum and the liver, enhances energy expenditure to prevent obesity, and improves insulin sensitivity. 25-HL dramatically ameliorates hepatic steatosis, inflammation, ballooning, and fibrosis through down-regulating the expression of lipogenic genes. Furthermore, 25-HL exhibits both prophylactic and therapeutic efficacies of alleviating NASH and atherosclerosis in amylin liver NASH model diet-treated Ldlr-/- mice, and reduces the formation of cholesterol crystals and associated crown-like structures of Kupffer cells. Notably, 25-HL lowers lipid contents in serum and the liver to a greater extent than lovastatin or obeticholic acid. 25-HL shows a good safety and pharmacokinetics profile. This study provides the proof of concept that inhibiting SREBP activation by targeting INSIG to lower lipids could be a promising strategy for treating NASH. It suggests the translational potential of 25-HL in human NASH and demonstrates the critical role of SREBP-controlled lipogenesis in the progression of NASH by pharmacological inhibition.

The prognostic value of HVPG-response to non-selective beta-blockers in patients with NASH cirrhosis and varices.

Non-alcoholic steatohepatitis has become a leading cause of cirrhosis. The prognostic value of (HVPG)-guided NSBB prophylaxis remains to be investigated in the setting of NASH cirrhosis. Patients with NASH cirrhosis and varices undergoing HVPG-guided NSBB therapy were included. HVPG-response to NSBBs was evaluated within a median 52 (IQR:28-71) days after baseline measurement. HVPG-Response was defined as a decrease of ≥10% from baseline or below <12mmHg. The composite endpoint was defined as variceal bleeding, decompensation, and liver-related death. Thirtyeight patients were included: Child-A/B:33(87%), Child-C:5(13%) median HVPG:19.7±4.7mmHg. 21(55.3%) patients achieved HVPG-response to NSBB. Presence of diabetes(aOR:0.16, p=0.038) and arterial blood pressure (aOR:1.07, p=0.044) were independently associated with NSBB-response. While NSBB-HVPG-responders showed fewer decompensations within 90 days (n=1(5%) vs. n=3(29%), p=0.172), only Child-Pugh stage B/C (p=0.001), MELD≥15(p=0.021) and HVPG≥20mmHg(p=0.011) predicted the composite endpoint at 90 days. Similarly, after 2years of follow-up, only Child-Pugh stage (B:p=0.001, C:p<0.001), MELD≥15 (p=0.021), HVPG≥20mmHg (p=0.011) predicted the composite endpoint. Importantly, all bleeding events occurred in HVPG-NSBB non-responders. HVPG-response to NSBB was achieved in 55.3% of NASH patients with varices and this seemed to protect from variceal bleeding. However, only baseline HVPG≥20mmHg, Child-Pugh stage B/C and MELD≥15 were predictors of decompensation/death in patients with NASH cirrhosis and varices.

Biomarkers, imaging and safety in resmetirom 52 week non-cirrhotic NASH phase 3 clinical trial, completed open-label arm of maestro-NAFLD-1

Background: MAESTRO-NASH (NCT03900429) and MAESTRO-NAFLD-1 (NCT04197479) are 52-week Phase 3 registrational double-blind placebo controlled clinical trials to study the effect of resmetirom, a selective thyroid receptor beta (THR-β) agonist in NASH patients. A goal of MAESTRO-NAFLD-1, a 1200 patient “real life” NASH study is to identify non-invasive markers that correlate with patient response to resmetirom treatment. The 171 patient 100 mg open label arm completed the 52-week study in July 2021.

Procollagen C-Proteinase Enhancer-1 (PCPE-1) deficiency in mice reduces liver fibrosis but not NASH progression.

Background and aimsNonalcoholic Steatohepatitis (NASH) is a major cause of end-stage liver diseases such as cirrhosis and hepatocellular carcinoma resulting ultimately in increased liver-related mortality. Fibrosis is the main driver of mortality in NASH. Procollagen C-Proteinase Enhancer-1 (PCPE-1) plays a key role in procollagen maturation and collagen fibril formation. To assess its role in liver fibrosis and NASH progression, knock-out mice were evaluated in a dietary NASH model.MethodsGlobal constitutive Pcolce-/- and WT male mice were fed with a Choline Deficient Amino acid defined High Fat Diet (CDA HFD) for 8 weeks. Liver triglycerides, steatosis, inflammation and fibrosis were assessed at histological, biochemical and gene expression levels. In addition, human liver samples from control and NASH patients were used to evaluate the expression of PCPE-1 at both mRNA and protein levels.ResultsPcolce gene deficiency prevented diet-induced liver enlargement but not liver dysfunction. Furthermore, liver triglycerides, steatosis and inflammation were not modified in Pcolce-/- male mice compared to WT under CDA HFD. However, a significant decrease in liver fibrosis was observed in Pcolce-/- mice compared to WT under NASH diet, associated with a decrease in total and insoluble collagen content without any significant modifications in the expression of genes involved in fibrosis and extracellular matrix remodeling. Finally, PCPE-1 protein expression was increased in cirrhotic liver samples from both NASH and Hepatitis C patients.ConclusionsPcolce deficiency limits fibrosis but not NASH progression in CDA HFD fed mice.

Nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease affecting millions of people worldwide with potentially serious complications that may lead to mortality and constitute a significant socioeconomic burden. Although modern lifestyle, obesity and insulin resistance play leading roles in the pathophysiology of the disease, various metabolic aberrations, as well as genetic predisposition participate in the development and progression of NAFLD. The diagnosis of NAFLD, especially the early recognition of NASH and fibrosis, is vital for the early management of the disease. Many diagnostic methods have been developed for the disease, ranging from liver biopsy to imaging techniques and serum biomarkers, each one followed by advantages and disadvantages. Despite the extensive research in this field, there is to-date no licensed pharmacological treatment. However, several medications have been investigated and provided promising results; in this regard, the results of some phase III clinical trials in NASH patients are expected. Until the approval of specific for the disease medications, the cornerstone of its management remains lifestyle modifications, i.e. dietary changes and increase in physical activity.

CYP450 drug inducibility in NAFLD via an in vitro hepatic model: Understanding drug-drug interactions in the fatty liver

Drug-drug-interactions (DDIs) occur when a drug alters the metabolic rate, efficacy, and toxicity of concurrently used drugs. While almost 1 in 4 adults now use at least 3 concurrent prescription drugs in the United States, the Non-alcoholic fatty liver disease (NAFLD) prevalence has also risen over 25%. The effect of NALFD on DDIs is largely unknown. NAFLD is characterized by lipid vesicle accumulation in the liver, which can progress to severe steatohepatitis (NASH), fibrosis, cirrhosis, and hepatic carcinoma. The CYP450 enzyme family dysregulation in NAFLD, which might already alter the efficacy and toxicity of drugs, has been partially characterized. Nevertheless, the drug-induced dysregulation of CYP450 enzymes has not been studied in the fatty liver. These changes in enzymatic inducibility during NAFLD, when taking concurrent drugs, could cause unexpected fatalities through inadvertent DDIs. We have, thus, developed an in vitro model to investigate the CYP450 transcriptional regulation in NAFLD. Specifically, we cultured primary human hepatocytes in a medium containing free fatty acids, high glucose, and insulin for seven days. These cultures displayed intracellular macro-steatosis after 5 days and cytokine secretion resembling NAFLD patients. We further verified the model’s dysregulation in the transcription of key CYP450 enzymes. We then exposed the NAFLD model to the drug inducers rifampicin, Omeprazole, and Phenytoin as activators of transcription factors pregnane X receptor (PXR), aryl hydrocarbon receptor (AHR) and constitutive androstane receptor (CAR), respectively. In the NAFLD model, Omeprazole maintained an expected induction of CYP1A1, however Phenytoin and Rifampicin showed elevated induction of CYP2B6 and CYP2C9 compared to healthy cultures. We, thus, conclude that the fatty liver could cause aggravated drug-drug interactions in NAFLD or NASH patients related to CYP2B6 and CYP2C9 enzymes.