Impact of age on NIS2+™ and other non-invasive blood tests for the evaluation of liver disease and detection of at-risk MASH.

Background & Aims: Robust performance of non-invasive tests (NITs) across ages is critical to assess liver disease among patients with metabolic dysfunction-associated liver disease (MASLD). We evaluated the impact of age on the performance of NIS2+™ vs. other NITs. Methods: An analysis cohort (N = 1,926) with biopsy-proven MASLD was selected among individuals screened for the phase III RESOLVE-IT clinical trial and divided into ≤45, 46-55, 56-64, and ≥65 years groups. To avoid potential confounding effects, a well-balanced cohort (n = 708; n = 177/age group) was obtained by applying a propensity score-matching algorithm to the analysis cohort. Baseline values of biomarkers and NITs were compared across age groups using one-way ANOVA, and the impact of age and histology were compared through three-way ANOVA. The impact of age on NIT performance for the detection of at-risk metabolic dysfunction-associated steatohepatitis (MASH; MASLD activity score [MAS] ≥4 and fibrosis stage [F] ≥2) was also evaluated. Results: Age did not affect the distributions of NIS2+™ and APRI (aspartate aminotransferase-to-platelet ratio index), but significantly (p <0.0001) impacted those of NFS (NAFLD fibrosis score), FIB-4 (Fibrosis-4 index), and Enhanced Liver Fibrosis (ELF™) score. NIS2+™ was the only NIT on which fibrosis and MAS exerted a moderate to large effect. While the impact of fibrosis on APRI was moderate, that of MAS was low. The impact of age on FIB-4 and NFS was larger than that of fibrosis. NIS2+™ exhibited the highest AUROC values for detecting at-risk MASH across age groups, with stable performances irrespective of cut-offs. Conclusions: NIS2+™ was not significantly impacted by age and was sensitive to both fibrosis and MAS grade, demonstrating a robust performance to rule in/out at-risk MASH with fixed cut-offs. Impact and Implications: While metabolic dysfunction-associated steatotic liver disease (MASLD) can affect individuals of all ages, patient age could represent an important confounding factor when interpreting non-invasive test (NIT) results, highlighting the need for reliable and efficient NITs that are not impacted by age and that could be interpreted with fixed cut-offs, irrespective of patient age. We report the impact of age on different well-established NITs - among those tested, only two panels, NIS2+™ and APRI, were not impacted by age and can be used and interpreted independently of patient age. NIS2+™ was also sensitive to both fibrosis and MAS, further confirming its efficiency for the detection of the composite endpoint of at-risk MASH and its potential as a valuable candidate for large-scale implementation in clinical practice and clinical trials.

NIS2+TM as a screening tool to optimize patient selection in metabolic dysfunction-associated steatohepatitis clinical trials.

BACKGROUND & AIMS: Strategies to reduce liver biopsy (LB) screen failures through better patient selection are needed for clinical trials. Standard fibrosis biomarkers were not derived to detect "at-risk" metabolic dysfunction-associated steatohepatitis (MASH; MASH with metabolic dysfunction-associated steatotic liver disease score ≥4 and fibrosis stage ≥2). We compared the performance of screening pathways that incorporate NIS2+™, an optimized version of the blood-based NIS4® technology designed to identify at-risk MASH, with those incorporating fibrosis (FIB)-4 within the RESOLVE-IT clinical trial (NCT02704403), aiming for optimized selection of patients for LB. METHODS: A retrospective simulation analysis was conducted in the RESOLVE-IT screening pathway (RSP) cohort. LB failure rate (LBFR), number of patients needed to screen, and overall cost estimations of different pathways were calculated for a range of NIS2+™ and FIB-4 cut-offs and compared with those of the RSP, which relied on investigators' local practices. An analysis of potential recruitment bias based on histology, sex, age, or comorbidities was performed. RESULTS: The analysis cohort included 1,929 patients, 765 (40%) with at-risk MASH. The NIS2+™ pathway resulted in a significantly lower LBFR (39%) compared with the FIB-4 pathway (58%) or the RSP (60%) when using cost-optimized cut-offs (NIS2+™, 0.53; FIB-4, 0.58). For every 1,000 inclusions, NIS2+™ significantly reduced unnecessary LBs (632 vs. 1,522; -58%) and screening costs (US$12.7 million vs. US$15.0 million) vs. the RSP, while the number of patients needed to screen increased moderately (3,220 to 4,033). NIS2+™ alone is better than FIB-4 alone or combined with FIB-4. CONCLUSIONS: This analysis demonstrated that patient selection for LB using NIS2+™ significantly reduced unnecessary biopsies and screening costs, which could greatly improve the feasibility of MASH clinical trials. IMPACT AND IMPLICATIONS: Simple and accurate non-invasive strategies to optimize the selection of patients who should be referred for liver biopsy for inclusion in MASH clinical trials is critical to reduce the high liver biopsy failure rates. While the use of the Fibrosis-4 index alone did not lead to a significant improvement of the screening process, selecting patients using NIS2+™, a recently developed optimization of the NIS4® technology for the detection of at-risk MASH, showed improved performance by simultaneously reducing liver biopsy failure rates and the overall cost of the trial, while maintaining the number of patients needed to screen at a manageable level and not generating any bias in included patients' characteristics. This makes NIS2+™ an accurate and reliable screening tool that could improve the recruitment of patients in future MASH clinical trials, and would lead to increased patient comfort and security, ensuring timely and cost-efficient trial completion.

NIS2+™, an optimisation of the blood-based biomarker NIS4® technology for the detection of at-risk NASH: A prospective derivation and validation study.

BACKGROUND & AIMS: NIS4® is a blood-based non-invasive test designed to effectively rule in/rule out at-risk non-alcoholic steatohepatitis (NASH), defined as non-alcoholic fatty liver disease activity score ≥4 and significant fibrosis (stage ≥2), among patients with metabolic risk factors. Robustness of non-invasive test scores across characteristics of interest including age, type 2 diabetes mellitus, and sex, and optimised analytical aspects are critical for large-scale implementation in clinical practice. We developed and validated NIS2+™, an optimisation of NIS4®, specifically designed to improve score robustness. METHODS: A well-balanced training cohort (n = 198) included patients from the GOLDEN-505 trial. The validation (n = 684) and test (n = 2,035) cohorts included patients from the RESOLVE-IT trial. Well-matched subgroups were created to avoid potential confounding effects during modelling and analysis of score robustness. Models were trained using logistic regressions for at-risk NASH detection and compared using Bayesian information criteria. Performance of NIS2+™ was compared with that of NIS4®, Fibrosis-4, and alanine aminotransferase using area under the receiver operating characteristic curve, and robustness was analysed through score distribution. RESULTS: Using the training cohort to compare all combinations of NIS4® biomarkers, NIS2 (miR-34a-5p, YKL-40) was identified as the best combination of parameters. To correct for the sex effect on miR-34a-5p (validation cohort), sex and sex ∗ miR-34a-5p parameters were added, creating NIS2+™. In the test cohort, NIS2+™ exhibited a statistically higher area under the receiver operating characteristic curve (0.813) vs. NIS4® (0.792; p = 0.0002), Fibrosis-4 (0.653; p <0.0001), and alanine aminotransferase (0.699; p <0.0001). NIS2+™ scores were not affected by age, sex, BMI, or type 2 diabetes mellitus status, providing robust clinical performances irrespective of patient characteristics. CONCLUSION: NIS2+™ constitutes a robust optimisation of NIS4® technology for the detection of at-risk NASH. IMPACT AND IMPLICATIONS: The development of non-invasive tests for accurate, large-scale detection of patients with at-risk non-alcoholic steatohepatitis (NASH; defined as NASH with non-alcoholic fatty liver disease activity score ≥4 and fibrosis stage ≥2) - who are at higher risk for disease progression and for developing liver-related life-threatening outcomes - is critical for identifying this patient population in the clinical setting and improving the screening process of NASH clinical trials. We report the development and validation of NIS2+™, a diagnostic test designed as an optimisation of NIS4® technology, a blood-based panel currently used to detect at-risk NASH in patients with metabolic risk factors. NIS2+™ showed improved performance for the detection of at-risk NASH compared with NIS4® and other non-invasive liver tests that was not impacted by patients' characteristics of interest, such as age, sex, type 2 diabetes mellitus, BMI, dyslipidaemia, and hypertension. This makes NIS2+™ a robust and reliable tool for the diagnosis of at-risk NASH among patients with metabolic risk factors, and an effective candidate for large-scale implementation in clinical practice and clinical trials.

A blood-based biomarker panel (NIS4) for non-invasive diagnosis of non-alcoholic steatohepatitis and liver fibrosis: a prospective derivation and global validation study.

BACKGROUND: Non-invasive tests that can identify patients with non-alcoholic steatohepatitis (NASH) at higher risk of disease progression are lacking. We report the development and validation of a blood-based diagnostic test to non-invasively rule in and rule out at-risk NASH (defined as non-alcoholic fatty liver disease [NAFLD] activity score [NAS] ≥4 and fibrosis stage ≥2). METHODS: In this prospective derivation and global validation study, blood samples, clinical data, and liver biopsy results from three independent cohorts with suspected NAFLD were used to develop and validate a non-invasive blood-based diagnostic test, called NIS4. Derivation was done in the discovery cohort, which comprised 239 prospectively recruited patients with biopsy-confirmed NASH (NAFLD NAS ≥3; fibrosis stage 0-3) from the international GOLDEN-505 phase 2b clinical trial. A complete matrix based on 23 variables selected for univariate association with the presence of at-risk NASH and avoiding high multi-collinearity was used to derive the model in a bootstrap-based process that minimised the Akaike information criterion. The overall diagnostic performance of NIS4 was externally validated in two independent cohorts: RESOLVE-IT diag and Angers. The RESOLVE-IT diag cohort comprised the first 475 patients screened for potential inclusion into the RESOLVE-IT phase 3 clinical trial. Angers was a retrospective cohort of 227 prospectively recruited patients with suspected NAFLD and clinical risk factors for NASH or fibrosis stage 2 or more according to abnormal elastography results or abnormal liver biochemistry. Both external validation cohorts were independently analysed and were combined into a pooled validation cohort (n=702) to assess clinical performance of NIS4 and other non-invasive tests. FINDINGS: The derived NIS4 algorithm comprised four independent NASH-associated biomarkers (miR-34a-5p, alpha-2 macroglobulin, YKL-40, and glycated haemoglobin; area under the receiver operating characteristics curve [AUROC] 0·80, 95% CI 0·73-0·85), and did not require adjustment for age, sex, body-mass index (BMI), or aminotransferase concentrations. Clinical cutoffs were established within the discovery cohort to optimise both rule out and rule in clinical performance while minimising indeterminate results. NIS4 was validated in the RESOLVE-IT diag cohort (AUROC 0·83, 95% CI 0·79-0·86) and the Angers cohort (0·76, 0·69-0·82). In the pooled validation cohort, patients with a NIS4 value less than 0·36 were classified as not having at-risk NASH (ruled out) with 81·5% (95% CI 76·9-85·3) sensitivity, 63·0% (57·8-68·0) specificity, and a negative predictive value of 77·9% (72·5-82·4), whereas those with a NIS4 value of more than 0·63 were classified as having at-risk NASH (ruled in) with 87·1% (83·1-90·3) specificity, 50·7% (45·3-56·1) sensitivity, and a positive predictive value of 79·2% (73·1-84·2). The diagnostic performance of NIS4 within the external validation cohorts was not influenced by age, sex, BMI, or aminotransferase concentrations. INTERPRETATION: NIS4 is a novel blood-based diagnostic that provides an effective way to non-invasively rule in or rule out at-risk NASH in patients with metabolic risk factors and suspected disease. Use of NIS4 in clinical trials or in the clinic has the potential to greatly reduce unnecessary liver biopsies in patients with lower risk of disease progression. FUNDING: Genfit.

Elafibranor, an Agonist of the Peroxisome Proliferator-Activated Receptor-α and -δ, Induces Resolution of Nonalcoholic Steatohepatitis Without Fibrosis Worsening.

BACKGROUND & AIMS: Elafibranor is an agonist of the peroxisome proliferator-activated receptor-α and peroxisome proliferator-activated receptor-δ. Elafibranor improves insulin sensitivity, glucose homeostasis, and lipid metabolism and reduces inflammation. We assessed the safety and efficacy of elafibranor in an international, randomized, double-blind placebo-controlled trial of patients with nonalcoholic steatohepatitis (NASH). METHODS: Patients with NASH without cirrhosis were randomly assigned to groups given elafibranor 80 mg (n = 93), elafibranor 120 mg (n = 91), or placebo (n = 92) each day for 52 weeks at sites in Europe and the United States. Clinical and laboratory evaluations were performed every 2 months during this 1-year period. Liver biopsies were then collected and patients were assessed 3 months later. The primary outcome was resolution of NASH without fibrosis worsening, using protocol-defined and modified definitions. Data from the groups given the different doses of elafibranor were compared with those from the placebo group using step-down logistic regression, adjusting for baseline nonalcoholic fatty liver disease activity score. RESULTS: In intention-to-treat analysis, there was no significant difference between the elafibranor and placebo groups in the protocol-defined primary outcome. However, NASH resolved without fibrosis worsening in a higher proportion of patients in the 120-mg elafibranor group vs the placebo group (19% vs 12%; odds ratio = 2.31; 95% confidence interval: 1.02-5.24; P = .045), based on a post-hoc analysis for the modified definition. In post-hoc analyses of patients with nonalcoholic fatty liver disease activity score ≥4 (n = 234), elafibranor 120 mg resolved NASH in larger proportions of patients than placebo based on the protocol definition (20% vs 11%; odds ratio = 3.16; 95% confidence interval: 1.22-8.13; P = .018) and the modified definitions (19% vs 9%; odds ratio = 3.52; 95% confidence interval: 1.32-9.40; P = .013). Patients with NASH resolution after receiving elafibranor 120 mg had reduced liver fibrosis stages compared with those without NASH resolution (mean reduction of 0.65 ± 0.61 in responders for the primary outcome vs an increase of 0.10 ± 0.98 in nonresponders; P < .001). Liver enzymes, lipids, glucose profiles, and markers of systemic inflammation were significantly reduced in the elafibranor 120-mg group vs the placebo group. Elafibranor was well tolerated and did not cause weight gain or cardiac events, but did produce a mild, reversible increase in serum creatinine (effect size vs placebo: increase of 4.31 ± 1.19 µmol/L; P < .001). CONCLUSIONS: A post-hoc analysis of data from trial of patients with NASH showed that elafibranor (120 mg/d for 1 year) resolved NASH without fibrosis worsening, based on a modified definition, in the intention-to-treat analysis and in patients with moderate or severe NASH. However, the predefined end point was not met in the intention to treat population. Elafibranor was well tolerated and improved patients' cardiometabolic risk profile. ClinicalTrials.gov number: NCT01694849.

Pan-PPAR modulation effectively protects APP/PS1 mice from amyloid deposition and cognitive deficits.

Alzheimer's disease (AD) is a neurodegenerative condition that leads to neuronal death and memory dysfunction. In the past, specific peroxisome proliferator-activated receptor (PPAR)γ-agonists, such as pioglitazone, have been tested with limited success to improve AD pathology. Here, we investigated the therapeutic efficacy of GFT1803, a novel potent PPAR agonist that activates all the three PPAR isoforms (α/δ/γ) in the APP/PS1 mouse model in comparison to the selective PPARγ-agonist pioglitazone. Both compounds showed similar brain/plasma partitioning ratios, although whole body and brain exposure to GFT1803 was significantly lower as compared to pioglitazone, at doses used in this study. Oral treatment of APP/PS1 mice with GFT1803 decreased microglial activation, amyloid ß (Aß) plaque area, Aß levels in sodium dodecyl sulfate- and formic acid-soluble fractions in a concentration-dependent manner. With a single exception of Aß38 and Aß40 levels, measured by ELISA, these effects were not observed in mice treated with pioglitazone. Both ligands decreased glial fibrillary acidic protein (GFAP) expression to similar extent and did not affect ApoE expression. Finally, GFT1803 increased insulin-degrading enzyme expression. Analysis of spatial memory formation in the Morris water maze demonstrated that both compounds were able to partially revert the phenotype of APP/PS1 mice in comparison to wild-type mice with GFT1803 being most effective. As compared to pioglitazone, GFT1803 (pan-PPAR agonist) produced both quantitatively superior and qualitatively different therapeutic effects with respect to amyloid plaque burden, insoluble Aß content, and neuroinflammation at significantly lower whole body and brain exposure rates.

The dual peroxisome proliferator-activated receptor alpha/delta agonist GFT505 exerts anti-diabetic effects in db/db mice without peroxisome proliferator-activated receptor gamma-associated adverse cardiac effects.

We report here the efficacy and safety of GFT505, a novel liver-targeted peroxisome proliferator-activated receptor alpha/delta (PPARα/δ) agonist, in the db/db mouse model of diabetes. Mice were treated with vehicle, GFT505, PPARγ agonist rosiglitazone or dual-PPARα/γ agonist aleglitazar for up to 8 weeks. All compounds comparably reduced fasting glycaemia and HbA1c and improved insulin sensitivity. The glucose-lowering effect of GFT505 was associated with decreased hepatic gluconeogenesis, correlating with reduced expression of gluconeogenic genes. In contrast with the PPARγ-activating drugs, treatment with GFT505 did not affect heart weight and did not increase plasma adiponectin concentrations. This absence of cardiac effects of GFT505 was confirmed after 12 months of administration in cynomolgus monkeys, by the absence of echocardiographic and histological findings. Moreover, long-term GFT505 administration to monkeys induced no change in haematological parameters or in bone marrow differential cell counts. Compared to PPARγ-activating drugs, the dual-PPARα/δ agonist GFT505 therefore shows an improved benefit/risk ratio, treating multiple features of type 2 diabetes without inducing the cardiac side-effects associated with PPARγ activation.

The identification of naturally occurring neoruscogenin as a bioavailable, potent, and high-affinity agonist of the nuclear receptor RORα (NR1F1).

Plants represent a tremendous structural diversity of natural compounds that bind to many different human disease targets and are potentially useful as starting points for medicinal chemistry programs. This resource is, however, still underexploited due to technical difficulties with the identification of minute quantities of active ingredients in complex mixtures of structurally diverse compounds upon raw phytomass extraction. In this work, we describe the successful identification of a novel class of potent RAR-related orphan receptor alpha (RORα or nuclear receptor NR1F1) agonists from a library of 12,000 plant extract fractions by using an optimized, robust high-throughput cell-free screening method, as well as an innovative hit compound identification procedure through further extract deconvolution and subsequent structural elucidation of the active natural compound(s). In particular, we demonstrate that neoruscogenin, a member of the steroidal sapogenin family, is a potent and high-affinity RORα agonist, as shown by its activity in RORα reporter assays and from its effect on RORα target gene expression in vitro and in vivo. Neoruscogenin represents a universal pharmacological tool for RORα research due to its specific selectivity profile versus other nuclear receptors, its excellent microsomal stability, good bioavailability, and significant peripheral exposure in mouse.

Hepatoprotective effects of the dual peroxisome proliferator-activated receptor alpha/delta agonist, GFT505, in rodent models of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis.

UNLABELLED: Nonalcoholic fatty liver disease (NAFLD) covers a spectrum of liver damage ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. To date, no pharmacological treatment is approved for NAFLD/NASH. Here, we report on preclinical and clinical data with GFT505, a novel dual peroxisome proliferator-activated receptor alpha/delta (PPAR-α/δ) agonist. In the rat, GFT505 concentrated in the liver with limited extrahepatic exposure and underwent extensive enterohepatic cycling. The efficacy of GFT505 was assessed in animal models of NAFLD/NASH and liver fibrosis (Western diet [WD]-fed human apolipoprotein E2 [hApoE2] transgenic mice, methionine- and choline-deficient diet-fed db/db mice, and CCl4 -induced fibrosis in rats). GFT505 demonstrated liver-protective effects on steatosis, inflammation, and fibrosis. In addition, GFT505 improved liver dysfunction markers, decreased hepatic lipid accumulation, and inhibited proinflammatory (interleukin-1 beta, tumor necrosis factor alpha, and F4/80) and profibrotic (transforming growth factor beta, tissue inhibitor of metalloproteinase 2, collagen type I, alpha 1, and collagen type I, alpha 2) gene expression. To determine the role of PPAR-α-independent mechanisms, the effect of GFT505 was assessed in hApoE2 knock-in/PPAR-α knockout mice. In these mice, GFT505 also prevented WD-induced liver steatosis and inflammation, indicating a contribution of PPAR-α-independent mechanisms. Finally, the effect of GFT505 on liver dysfunction markers was assessed in a combined analysis of four phase II clinical studies in metabolic syndrome patients. GFT505 treatment decreased plasma concentrations of alanine aminotransferase, gamma-glutamyl transpeptidase, and alkaline phosphatase. CONCLUSION: The dual PPAR-α/δ agonist, GFT505, is a promising liver-targeted drug for treatment of NAFLD/NASH. In animals, its protective effects are mediated by both PPAR-α-dependent and -independent mechanisms.

Does endoplasmic reticulum stress participate in APD-induced hepatic metabolic dysregulation?

Metabolic side effects caused by certain antipsychotic drugs (APDs), in particular clozapine and olanzapine, are now clinically well-documented. However, the potential mechanisms implicated in the metabolic disturbances of these drugs on peripheral tissues remain obscure. Here, we investigated the effects of five frequently prescribed APDs on the Sterol Regulatory Element Binding Protein (SREBP) transcription factor pathways which control lipogenesis and cholesterogenesis, using the Immortalized Human Hepatocyte cell model (IHH). First, clozapine, haloperidol, olanzapine and risperidone activated, at different levels, SREBP-1 activity reflected by an increased expression of SREBP-1 target genes involved in fatty acid biosynthesis (SREBP-1, FAS and/or SCD1) resulting in an accumulation of intracellular lipids. Second, clozapine and haloperidol also stimulated the SREBP-2 pathway associated with an increase in HMGCoAR expression. In contrast, quetiapine did not affect either the SREBP-1 or -2 pathways, but induced a slight accumulation of intracellular lipids. Interestingly, clozapine, haloperidol and olanzapine induced Endoplasmic Reticulum (ER) stress and, more precisely, initiation of the ER stress-activated eIF2α kinase (PERK) branch of the Unfolded Protein Response (UPR). Furthermore, treatment with thapsigargin, which increases intracellular calcium release, induced both ER stress and SREBP-1 and -2 pathway activation, whereas Ca(2+) chelation by BAPTA completely reversed the lipogenic effects and ER stress induction produced by clozapine. Based on these results, we propose that certain APDs induce ER stress via changes in Ca(2+) homeostasis in hepatocytes. This phenomenon potentially underlies a part of their known undesirable hepatic metabolic side effects. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

Overweight induced by chronic risperidone exposure is correlated with overexpression of the SREBP-1c and FAS genes in mouse liver.

Weight gain and metabolic disturbances, such as dyslipidemia and hyperglycaemia, are common side effects of most antipsychotic drugs, including risperidone. The aim of this study was to investigate the effects of chronic treatment with risperidone on body weight, fat accumulation, liver weight, and hepatic expression of key genes involved in lipid metabolism in female mice. We also addressed the mechanism of risperidone induction of metabolic side effects by exploring its effect on lipid and cholesterol metabolism in primary cultures of rat hepatocytes. Eleven weeks of treatment with long-acting risperidone (12.5 mpk/week) resulted in a significant weight gain associated with an increase of liver and adipose tissue weight. These effects were positively correlated with hepatic mRNA induction of two key genes involved in lipogenesis: sterol regulatory element binding protein-1c (SREBP-1c) and fatty acid synthase (FAS). Furthermore, in line with these in vivo results, risperidone elicited significant inductions of SREBP-1 maturation and FAS mRNA expression in primary cultures of rat hepatocytes associated with an increase of free fatty acid, triacylglycerol, and phospholipid synthesis as assessed by acetate incorporation. The current investigations underscore the usefulness of a mouse model to study the weight gain observed with risperidone treatment in humans. This study shows that risperidone induces similar effects in the liver (in vivo) and in hepatocyte cell cultures (in vitro) on the expression of key genes and/or proteins that control lipid metabolism. This suggests that risperidone could alter lipid metabolism in the liver and induce weight gain in a way that is partly independent of its action on the central nervous system.

Antipsychotic drug action on SREBPs-related lipogenesis and cholesterogenesis in primary rat hepatocytes.

The use of some of antipsychotic drugs (APDs) in humans has been hampered by the induction of metabolic disorders such as weight gain, dyslipidemia, and diabetes. In primary rat hepatocytes, we investigated the actions of several APDs on lipid and cholesterol metabolism using [(14)C]acetate incorporation, quantitative reverse transcription-polymerase chain reaction, and western blotting. Clozapine and olanzapine, known to have significant metabolic side effects in man, strongly increased de novo lipid and cholesterol synthesis in rat hepatocytes. Haloperidol, which has less impact in metabolic disorders, enhanced lipogenesis without altering cholesterol production. By contrast, quetiapine, which exhibits few metabolic side effects in man, did not affect lipid and cholesterol synthesis. Interestingly, aripiprazole, which has not yet been reported to induce metabolic disorders in humans, strongly decreases cholesterol synthesis. Furthermore, these inductions of lipid and cholesterol synthesis observed with clozapine and olanzapine were also associated with up-regulation of the transcription factors sterol regulatory element-binding protein (SREBP)-1 and/or SREBP-2 and their associated target genes. Part of the APD-induced metabolic disorders in humans may be due to direct effects on liver metabolism. Our model may also be of interest to assess the action of future drugs on metabolic parameters.

Phosphorylation of farnesoid X receptor by protein kinase C promotes its transcriptional activity.

The farnesoid X receptor (FXR, NR1H4) belongs to the nuclear receptor superfamily and is activated by bile acids such as chenodeoxycholic acid, or synthetic ligands such as GW4064. FXR is implicated in the regulation of bile acid, lipid, and carbohydrate metabolism. Posttranslational modifications regulating its activity have not been investigated yet. Here, we demonstrate that calcium-dependent protein kinase C (PKC) inhibition impairs ligand-mediated regulation of FXR target genes. Moreover, in a transactivation assay, we show that FXR transcriptional activity is modulated by PKC. Furthermore, phorbol 12-myristate 13-acetate , a PKC activator, induces the phosphorylation of endogenous FXR in HepG2 cells and PKCalpha phosphorylates in vitro FXR in its DNA-binding domain on S135 and S154. Mutation of S135 and S154 to alanine residues reduces in cell FXR phosphorylation. In contrast to wild-type FXR, mutant FXRS135AS154A displays an impaired PKCalpha-induced transactivation and a decreased ligand-dependent FXR transactivation. Finally, phosphorylation of FXR by PKC promotes the recruitment of peroxisomal proliferator-activated receptor gamma coactivator 1alpha. In conclusion, these findings show that the phosphorylation of FXR induced by PKCalpha directly modulates the ability of agonists to activate FXR.

Safety issues and prospects for future generations of PPAR modulators.

Because of their wide range of actions on glucose homeostasis, lipid metabolism and vascular inflammation, peroxisome proliferator-activated receptors (PPARs) are promising targets for the development of new drugs for the treatment of metabolic disorders such as diabetes, dyslipidemia and atherosclerosis. In clinical practice, PPARalpha agonists, such as the already available fibrates, improve dyslipidemia, while PPARgamma agonists, such as thiazolidinediones, improve insulin resistance and diabetes. The complementary action of simultaneous activation of each PPAR in patients suffering from metabolic syndrome and type 2 diabetes has led to new pharmacological strategies focused on the development of agonists targeting more than one receptor such as the dual PPARalpha/gamma agonists. However, despite the proven benefits of targeting PPARs, safety concerns have recently led to late stage development failures of various PPAR agonists including novel specific PPARgamma agonists and dual PPARalpha/gamma agonists. These safety concerns include potential carcinogenicity in rodents, signs of myopathy and rhabdomyolysis, increase in plasma creatinine and homocysteine, weight gain, fluid retention, peripheral edema and potential increased risk of cardiac failure. Although the discontinued compounds shared common side effects, the reason for discontinuation was always compound specific and the toxicological or adverse effects which have motivated the discontinuation could be either due to the activation of PPARgamma, PPARalpha or both (class effect) or due to a PPAR unrelated effect. Thus, the risk evaluation of each adverse effect should be viewed on a case by case basis considering both the PPAR profile of the drug, its absorption/distribution profile, the nature of the side effect and the putative PPAR-related mechanism of action. This review mainly focuses on the preclinical and clinical adverse events of PPAR agonists that could be of concern when considering the development of new PPAR agonists. The selective modulation of PPAR activities is a promising approach to develop new drugs with preserved efficacy but diminished adverse effects.

TReP-132 is a novel progesterone receptor coactivator required for the inhibition of breast cancer cell growth and enhancement of differentiation by progesterone.

The sex steroid progesterone is essential for the proliferation and differentiation of the mammary gland epithelium during pregnancy. In relation to this, in vitro studies using breast carcinoma T47D cells have demonstrated a biphasic progesterone response, consisting of an initial proliferative burst followed by a sustained growth arrest. However, the transcriptional factors acting with the progesterone receptor (PR) to mediate the progesterone effects on mammary cell growth and differentiation remain to be determined. Recently, it has been demonstrated that the transcriptional regulating protein of 132 kDa (TReP-132), initially identified as a regulator of steroidogenesis, is also a cell growth suppressor. Similar to progesterone-bound PR, TReP-132 acts by inducing the gene expression of the G1 cyclin-dependent kinase inhibitors p21WAF1/Cip1 (p21) and p27Kip1 (p27). The putative interaction between TReP-132 and progesterone pathways in mammary cells was therefore analyzed in the present study. Our results show that TReP-132 interacts in vitro and in T47D cells with progesterone-activated PR. TReP-132 synergizes with progesterone-bound PR to trans activate the p21 and p27 gene promoters at proximal Sp1-binding sites. Moreover, TReP-132 overexpression and knockdown, respectively, increased or prevented the induction of p21 and p27 gene expression by progesterone. As a consequence, TReP-132 knockdown also resulted in the loss of the inhibitory effects of progesterone on pRB phosphorylation, G1/S cell cycle progression, and cell proliferation. Furthermore, the knockdown of TReP-132 expression also prevented the induction of both early and terminal markers of breast cell differentiation which had been previously identified as progesterone target genes. As well, the progesterone-induced accumulation of lipid vacuoles was inhibited in the TReP-132-depleted cells. Finally, TReP-132 gene expression levels increased following progesterone treatment, indicating the existence of a positive auto-regulatory loop between PR and TReP-132. Taken together, these data identify TReP-132 as a coactivator of PR mediating the growth-inhibitory and differentiation effects of progesterone on breast cancer cells.

TReP-132 controls cell proliferation by regulating the expression of the cyclin-dependent kinase inhibitors p21WAF1/Cip1 and p27Kip1.

The transcriptional regulating protein of 132 kDa (TReP-132) has been identified in steroidogenic tissues, where it acts as a coactivator of steroidogenic factor 1 (SF-1). We show here that TReP-132 plays a role in the control of cell proliferation. In human HeLa cells, TReP-132 knockdown by using small interfering RNA resulted in increased G(1)-->S cell cycle progression. The growth-inhibitory effects of TReP-132 was further shown to be mediated by induction of G(1) cyclin-dependent kinase inhibitors p21(WAF1) (p21) and p27(KIP1) (p27) expression levels. As a consequence, G(1) cyclin/cyclin-dependent kinase activities and pRB phosphorylation were markedly reduced, and cell cycle progression was blocked in the G(1) phase. The stimulatory effect of TReP-132 on p21 and p27 gene transcription involved interaction of TReP-132 with the transcription factor Sp1 at proximal Sp1-binding sites in their promoters. Moreover, in different breast tumor cell lines, endogenous TReP-132 expression was positively related with a lower proliferation rate. In addition, TReP-132 knockdown resulted in enhanced cell proliferation and lowered p21 and p27 mRNA levels in the steroid-responsive and nonresponsive T-47D and MDA-MB-231 cell lines, respectively. Finally, a statistic profiling of human breast tumor samples highlighted that expression of TReP-132 is correlated with p21 and p27 levels and is associated with lower tumor incidence and aggressiveness. Together, these results identify TReP-132 as a basal cell cycle regulatory protein acting, at least in part, by interacting with Sp1 to activate the p21 and p27 gene promoters.

Transcriptional regulation of apolipoprotein A5 gene expression by the nuclear receptor RORalpha.

OBJECTIVE: The newly identified apolipoprotein A5 (APOA5), selectively expressed in the liver, is a crucial determinant of plasma triglyceride levels. Because elevated plasma triglyceride concentrations constitute an independent risk factor for cardiovascular diseases, it is important to understand how the expression of this gene is regulated. In the present study, we identified the retinoic acid receptor-related orphan receptor-alpha (RORalpha) as a regulator of human APOA5 gene expression. METHODS AND RESULTS: Using electromobility shift assays, we first demonstrated that RORalpha1 and RORalpha4 proteins can bind specifically to a direct repeat 1 site present at the position -272/-260 in the APOA5 gene promoter. In addition, using transient cotransfection experiments in HepG2 and HuH7 cells, we demonstrated that both RORalpha1 and RORalpha4 strongly increase APOA5 promoter transcriptional activity in a dose-dependent manner. Finally, adenoviral overexpression of hRORalpha in HepG2 cells led to enhanced hAPOA5 mRNA accumulation. We show that the homologous region in mouse apoa5 promoter is not functional. Moreover, we show that in staggerer mice, apoa5 gene is not affected by RORalpha. CONCLUSIONS: These findings identify RORalpha1 and RORalpha4 as transcriptional activators of human APOA5 gene expression. These data suggest an additional important physiological role for RORalpha in the regulation of genes involved in lipid homeostasis and probably in the development of atherosclerosis.

Farnesoid X receptor represses hepatic lipase gene expression.

The farnesoid X receptor (FXR) is a nuclear receptor that regulates gene expression in response to bile acids (BAs). FXR plays a central role in BA, cholesterol, and lipoprotein metabolism. Here, we identify HL, an enzyme involved in the metabolism of remnant and high density lipoproteins, as a novel FXR-regulated gene. The natural FXR ligand, chenodeoxycholic acid (CDCA), downregulates HL gene expression in a dose- and time-dependent manner in human hepatoma HepG2 cells. The nonsteroidal synthetic FXR agonist GW4064 also decreases HL mRNA levels in HepG2 cells and in primary human hepatocytes. Moreover, the decrease of HL mRNA levels after treatment with FXR agonists was associated with a significant decrease in secreted enzymatic activity. In addition, FXR-specific gene silencing using small interfering RNAs demonstrated that CDCA- and GW4064-mediated downregulation of HL transcript levels occurs via an FXR-dependent mechanism. Finally, using transient transfection experiments, it is shown that FXR represses transcriptional activity of a reporter driven by the -698/+13 bp human HL promoter. Taken together, these results identify HL as a new FXR-regulated gene in human liver cells. In view of the role of HL in plasma lipoprotein metabolism, our results further emphasize the central role of FXR in lipid homeostasis.

The liver X receptor ligand T0901317 down-regulates APOA5 gene expression through activation of SREBP-1c.

Alterations in the expression of the recently discovered apolipoprotein A5 gene strongly affect plasma triglyceride levels. In this study, we investigated the contribution of APOA5 to the liver X receptor (LXR) ligand-mediated effect on plasma triglyceride levels. Following treatment with the LXR ligand T0901317, we found that APOA5 mRNA levels were decreased in hepatoma cell lines. The observation that no down-regulation of APOA5 promoter activity was obtained by LXR-retinoid X receptor (RXR) co-transfection prompted us to explore the possible involvement of the known LXR target gene SREBP-1c (sterol regulatory element-binding protein 1c). In fact, we found that co-transfection with the active form of SREBP-1c down-regulated APOA5 promoter activity in a dose-dependent manner. We then scanned the human APOA5 promoter sequence and identified two putative E-box elements that were able to bind specifically SREBP-1c in gel-shift assays and were shown to be functional by mutation analysis. Subsequent suppression of SREBP-1 mRNA through small interfering RNA interference abolished the decrease of APOA5 mRNA in response to T0901317. Finally, administration of T0901317 to hAPOA5 transgenic mice revealed a significant decrease of APOA5 mRNA in liver tissue and circulating apolipoprotein AV protein in plasma, confirming that the described down-regulation also occurs in vivo. Taken together, our results demonstrate that APOA5 gene expression is regulated by the LXR ligand T0901317 in a negative manner through SREBP-1c. These findings may provide a new mechanism responsible for the elevation of plasma triglyceride levels by LXR ligands and support the development of selective LXR agonists, not affecting SREBP-1c, as beneficial modulators of lipid metabolism.

The farnesoid X receptor induces very low density lipoprotein receptor gene expression.

The farnesoid X receptor (FXR) is a nuclear receptor activated by bile acids (BAs). In response to ligand-binding, FXR regulates many genes involved in BA, lipid, and lipoprotein metabolism. To identify new FXR target genes, microarray technology was used to profile total RNA extracted from HepG2 cells treated with the natural FXR agonist chenodeoxycholic acid (CDCA). Interestingly, a significant increase of transcript level of the very low density lipoprotein receptor (VLDLR) was observed. Our data, resulting from selective FXR activation, FXR RNA silencing and FXR-deficient mice, clearly demonstrate that BAs up-regulate VLDLR transcript levels via a FXR-dependent mechanism in vitro in human and in vivo in mouse liver cells.