The PPAR α/γ Agonist Saroglitazar Improves Insulin Resistance and Steatohepatitis in a Diet Induced Animal Model of Nonalcoholic Fatty Liver Disease

Insulin resistance and hepatic lipid accumulation constitute the metabolic underpinning of nonalcoholic steatohepatitis (NASH). We tested the hypothesis that saroglitazar, a PPAR α/γ agonist would improve NASH in the diet-induced animal model of NAFLD. Mice received chow diet and normal water (CDNW) or high fat western diet and ad lib sugar water (WDSW). After 12 weeks, WDSW fed mice were randomized to receive (1) WDSW alone, (2) WDSW + vehicle, (3) WDSW + pioglitazone or (4) WDSW + saroglitazar for an additional 12 weeks. Compared to mice on WDSW and vehicle controls, mice receiving WDSW + saroglitazar had lower weight, lower HOMA-IR, triglycerides, total cholesterol, and ALT. Saroglitazar improved steatosis, lobular inflammation, hepatocellular ballooning and fibrosis stage. NASH resolved in all mice receiving saroglitazar. These effects were at par with or superior to pioglitazone. Molecular analyses confirmed target engagement and reduced oxidative stress, unfolded protein response and fibrogenic signaling. Transcriptomic analysis further confirmed increased PPAR-target expression and an anti-inflammatory effect with saroglitazar. Lipidomic analyses demonstrated that saroglitazar also reduced triglycerides, diglycerides, sphingomyelins and ceramides. These preclinical data provide a strong rationale for developing saroglitazar for the treatment of NASH in humans.


SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1. Study design to evaluate the efficacy of saroglitazar in DIAMOND mice DIAMOND mice (B6/129 mice) were fed chow diet (CDNW) or high fructose/glucose, high fat western diet (WDSW) for up to 12 weeks to develop fatty liver and steatohepatitis. Mice were administered pioglitazone, saroglitazar or vehicle control along with CDNW or WDSW for an additional 12 weeks. At the end of 24 weeks following initiation of dietary intervention and treatment, mice were fasted overnight, weighed and euthanized for blood collection and tissue harvesting.

Supplementary Figure 2. Effect of saroglitazar treatment on serum levels of adiponectin and TNFα in DIAMOND mice
DIAMOND mice fed on CDNW or WDSW were treated with saroglitazar for 12 weeks. Following treatment, adiponectin and TNFα levels were measured in the serum of DIAMOND mice. Data are expressed as the mean ± SEM for 6-12 mice per group. # p<0.05 compared to CDNW; **p<0.001 compared to WDSW, vehicle control. TNFα, tumor necrosis factor alpha.

Supplementary Figure 3. Saroglitazar reduces oxidative stress and inflammatory markers in DIAMOND mice
The relative expression of hepatic mRNA levels of SOD1, TNF-α and IL1-β were measured using qRT-PCR. The experiments were carried out in triplicates and β-actin was used as endogenous control for normalizing the mRNA levels. Data are expressed as the mean ± SEM for 6-12 mice per group. # p<0.05, ## p<0.001 compared to CDNW; **p<0.001 compared to WDSW, vehicle control. SOD1, superoxide dismutase1; TNFα, tumor necrosis factor alpha; IL1-β, interleukin 1-beta.

Supplementary Figure 4. Saroglitazar reduces hepatic fibrosis markers in DIAMOND mice
The mRNA expression levels of col6a3 and αSMA (A and B) were measured in the liver tissue of DIAMOND mice treated with saroglitazar fed on CDNW or WDSW by qRT-PCR. The experiments were carried out in triplicates and β-actin was used as endogenous control for normalizing the mRNA levels. Morphometric analysis of collagen proportionate area (CPA) of DIAMOND mice treated with vehicle control, pioglitazone or saroglitazar for 12 weeks along with CDNW or WDSW. Data are expressed as the mean ± SEM for 6-12 mice per group. # p<0.05 compared to CDNW; *p<0.05 compared to WDSW, vehicle control. col6a3, collagen type VI alpha 3 chain; αSMA, alpha smooth muscle actin.

Supplementary Figure 5. Activation of PPARα and PPARγ target genes by saroglitazar
DIAMOND mice fed on CDNW or WDSW were treated with saroglitazar for 12 weeks. Hepatic mRNA levels of PPARα target genes (ACOX1, CPT1A and LPIN1) and PPARγ target genes (UCP2 and CD36) were measured by qRT-PCR. The experiments were carried out in triplicates and Calnexin was used as endogenous control for normalizing the mRNA levels. Data are expressed as the mean ± SEM for 6-12 mice per group. *p<0.05, **p<0.001 compared to WDSW, vehicle control. ACOX1, acyl coenzyme A oxidase 1; CPT1A, carnitine palmitoyltransferase 1A; UCP2, uncoupling protein 2; CD36, cluster of differentiation 36.

Supplementary Figure 6. Top maps (sorted by statistically significant maps) in mice treated/untreated with saroglitazar fed on WDSW
The top (A) scored map (map with the lowest p-value) and second (B) scored map (map with the second lowest p-value) based on the enrichment distribution sorted by 'Statistically significant Maps' set. Experimental data from all files is linked to and visualized on the maps as thermometer-like figures. Up-ward thermometers have red color and indicate up-regulated signals and down-ward (blue) ones indicate down-regulated expression levels of the genes. Analyzed using MetaCore pathway analysis-version 6.5 (Clarivate Analytics, New York, NY-https://portal.genego.com).

Supplementary Figure 7. Top scored networks in mice treated/untreated with saroglitazar fed on WDSW
The top (A) scored (by the number of pathways) and second scored (by the number of pathways) AN network in mice treated/untreated with saroglitazar fed on WDSW gene list. Thick cyan lines indicate the fragments of canonical pathways. Up-regulated genes are marked with red circles; down-regulated with blue circles. The 'checkerboard' color indicates mixed expression for the gene between files or between multiple tags for the same gene. Biological pathway interactions of mRNA expressions were analyzed using MetaCore pathway analysis (version 6.5) of differentially expressed genes (Clarivate Analytics, New York, NY-https://portal.genego.com) with p <0.05 and greater than two-fold change.

Supplementary Table 1. Differential gene expression in saroglitazar treated vs. untreated mice
Mice fed on WDSW were either treated or untreated with saroglitazar. Differentially expressed hepatic mRNA were quantified (FDR<0.05) and the genes upregulated or down regulated listed.