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. Author manuscript; available in PMC: 2025 Nov 1.
Published in final edited form as: Lipids. 2024 Jul 16;59(6):221–231. doi: 10.1002/lipd.12408

Obeticholic Acid’s Effect on HDL function in MASH Varies by Diabetic Status

Chunki Kim 1, Tsung-Heng Tsai 2, Rocio Lopez 3, Arthur McCullough 4, Takhar Kasumov 1,4
PMCID: PMC11560728  NIHMSID: NIHMS2007512  PMID: 39014264

Abstract

Inflammation and oxidative stress are the key factors in the pathogenesis of both metabolic dysfunction-associated steatohepatitis (MASH) and atherosclerosis. Obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist, improves hepatic inflammation and fibrosis in patients with MASH. However, it also reduces HDL cholesterol, suggesting that OCA may increase CVD risk in patients with MASH. We assessed HDL cholesterol efflux function, antioxidant (paraoxonase and ceruloplasmin activity), pro-inflammatory index, and particle sizes in a small group of patients with and without diabetes (n=10/group) at baseline and after 18 months of OCA treatment. Patients on lipid-lowering medications (statins, fibrates) were excluded.

At baseline, ferritin levels were higher in patients with MASH without diabetes (336.5[157.0, 451.0] vs. 83[36.0, 151.0] ng/mL, P<0.005). Markers of HDL functions were similar in both groups. OCA therapy significantly improved liver histology and liver enzymes but increased alkaline phosphatase levels in non-diabetic patients with MASH (P<0.05). However, it didn’t have any significant effect on cholesterol efflux and the antioxidant paraoxonase functions. In non-diabetics, ceruloplasmin (CP) antioxidant activity decreased (P<0.005) and the pro-inflammatory index of HDL increased (P<0.005) due to OCA therapy. In contrast, in diabetics, OCA increased levels of pre-β-HDL -the HDL particles enhanced protective capacity (P=0.005) with no alteration in HDL functionality.

In all patients, serum glucose levels were negatively correlated with OCA-induced change in pro-inflammatory function in HDL (P<0.001), which was primarily due to diabetes (P=0.05). These preliminary results suggest a distinct effect of OCA therapy on diabetic and non-diabetic patients with MASH and warrant a future large-scale study.

Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD), also called nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and the most common chronic liver disease in the United States and developed countries worldwide1. One-third of MASLD patients develop metabolic dysfunction-associated steatohepatitis (MASH, formerly NASH) with increased prevalence to progress to cirrhosis and hepatocarcinoma, which became the leading indication for liver transplantation. Type 2 diabetes mellitus (T2DM) is the critical risk factor for MASLD, and more than half of T2DM patients develop MASLD2. Diabetic MASLD patients have more progressive fibrosis in MASH and three times higher mortality than non-diabetic MASLD patients. Cardiovascular disease (CVD) is the major complication of both MASH and T2DM3. However, no effective therapies exist to treat both MASH and associated CVD complications in diabetic patients with MASH.

Hepatic inflammation and oxidative stress are critical factors in the disease progression from steatosis to MASH4. Inflammation is also involved in atherosclerosis, which is the leading cause of CVD5. The anti-inflammatory synthetic farnesoid X receptor (FXR) agonist obeticholic acid (OCA) is highly effective in treating MASH6. OCA reduces steatosis, improves insulin sensitivity, and importantly inhibits inflammatory and fibrogenic responses in the liver7. Mechanistically, OCA-induced activation of FXR reduces hepatic lipogenesis and inhibits VLDL secretion via suppression of microsomal triglyceride transfer protein and apolipoprotein genes8. The FXR activation also inhibits cholesterol metabolism to bile acid, the principal pathway of cholesterol disposal. The phase 2 FXR Ligand OCA in MASH Treatment (FLINT) clinical trial indicates that the OCA also increases LDL cholesterol and reduces HDL cholesterol, known risk factors of CVD7,9. In a study of MASLD patients with T2DM, OCA therapy for six weeks didn’t increase total cholesterol levels but reduced HDL cholesterol levels. The interim analysis from the most recent Randomized Global Phase 3 Study to Evaluate the Impact on MASH With Fibrosis of OCA Treatment (REGENERATE) showed that OCA treatment increased LDL cholesterol levels which were resolved after a month of therapy10. The recent Combination OCA and Statin monitoring of lipids (CONTROL) trial in patients with MASH showed that OCA-induced increases in LDL cholesterol were mitigated with atorvastatin11. However, atorvastatin failed to affect the OCA-induced decline in HDL cholesterol levels, raising concern that OCA may potentially increase CVD risk in patients with MASH. The FDA restricted the use of OCA for treating MASH due to safety concerns and the need for additional efficacy data. Serious risks, including increased liver injury and CVD, raised doubts about the drug’s overall benefit. Older adults may be more sensitive to the medication and experience more pronounced side effects. Most clinical trial participants were white women, limiting the understanding of differences in efficacy and side effects among racial groups and sexes. The FDA emphasized the need to complete the long-term outcomes phase of the REGENERATE study before resubmission, requiring more extensive data to assess OCA’s long-term clinical outcomes and safety for MASH treatment.

Despite the overwhelming observational data on the role of higher HDL levels in CVD protection12, recent studies questioned the HDL hypothesis because the genetic and pharmacological mechanisms that raise plasma HDL cholesterol levels failed to show beneficial cardiovascular outcomes13–15. In contrast, dysfunctional HDL is associated with an increased risk of CVD16. These studies indicate that static HDL levels have limitations in CVD protection, as they don’t reflect HDL function and underscore the importance of HDL functionality studies. HDL has several cardiovascular protective functions, including reverse cholesterol transport (RCT) from the periphery to the liver for disposal, preventing inflammation, oxidation, platelet activation, and maintaining endothelial function17. Insulin resistance-related disorders such as T2DM are associated with impaired HDL functions18, although MASLD patients without MASH may show increased cholesterol efflux19. Inflammation further contributes to HDL dysfunction and transforms HDL to pro-oxidant and pro-inflammatory particles20,21.

Paraoxonase 1 (PON1) and ceruloplasmin (CP) are HDL-associated proteins with anti-oxidant and anti-inflammatory functions22,23. PON1 has esterase/lipolactonase activity and prevents the accumulation of lipid peroxides in LDL24. PON1 also inactivates bioactive oxidized phospholipids and enhances cholesterol efflux25. CP is an endogenous inhibitor of myeloperoxidase (MPO)26, a neutrophil-derived protein linked to oxidative stress and inflammation in MASLD and atherosclerosis27. CP binds to the heme pocket of MPO and inhibits its peroxidase activity responsible for generating reactive oxygen species (ROS) in circulation28. In addition, the ferroxidase activity of CP prevents iron-induced oxidative stress. In this reaction, reactive ferric iron (Fe2+) is oxidized to stable ferrous iron (Fe3+) that incorporates transferrin for iron delivery to organs and tissues29. Therefore, CP also plays an important role in iron metabolism with dysmetabolic iron overload implicated in the pathogenesis of MASLD, T2DM, and CVD. As an acute phase response protein, CP levels are increased in the early stages of MASLD but decreased in MASH30,31, suggesting that reduced CP could be involved in altered iron status in patients with MASH. It is unknown how OCA therapy affects anti-oxidant, anti-inflammatory, and other functions of HDL and whether PON1 and CP are involved in these changes. In this proof-of-concept study, we used samples from the FLINT trial to evaluate the effect of OCA therapy on HDL functions in biopsy-proven patients with MASH with and without T2DM. We characterized ex vivo HDL functions and particle sizes in serum samples from a subset of patients with MASH who were not taking lipid-lowering medications.

Methods

Study population

In this retrospective study, serum samples were obtained from the Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) 7, which has completed the clinical FLINT trial (ClinicalTrials.gov number: NCT01265498) sponsored by the National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK). The patients with MASH (18–65 years of age and 25–40 kg/min2 of BMI) were diagnosed based on histological criteria established by the NASH CRN 7. Other causes of liver disease (viral hepatitis, autoimmune hepatitis, sclerosing cholangitis, primary biliary cirrhosis, hemochromatosis, Wilson’s disease, and alpha-1-antitrypsin deficiency) were excluded. All subjects had normal renal and thyroid function. The eligible patients with MASH with or without T2DM (n=10/group) were eventually treated with 25 mg/daily of OCA for 18 months, and samples were collected before (baseline) and after therapy (post-treatment). Subjects taking medications that could impact hepatic cholesterol metabolism (statins, fibrates) within the previous three months were excluded from this study. Clinical data were obtained from the NASH CRN database, including the characteristics of the study population, histological features, liver enzymes, biochemical concentrations, and metabolic factors.

Analytical Methods

HDL isolation and particle size.

HDL was isolated using an anti-HDL immunoaffinity column (GenWay Biotech, Inc. San Diego, CA). Briefly, 50 μL of serum (~ 5 mg protein) was diluted with 450 μL of Tris-Buffered Saline (TBS) and loaded onto the spin column. The column was washed two times with TBS and TBS containing 0.05% Tween-20 to remove the unbound proteins. HDL was eluted with stripping buffer (0.1 M Glycine-HCl, pH 2.5). After neutralization, the eluted HDL solution was concentrated and desalted, and the protein concentration of the immunocaptured HDL fraction was measured by the bicinchoninic acid (BCA) protein assay method.

HDL particles were separated by size using non-denaturing polyacrylamide gel electrophoresis (ND-PAGE) on a 4–20% gradient Mini-Protean TGX Precast gel (BioRad) as described 18,19. The gels were stained with a Pierce Silver staining kit (Thermo Scientific), and HDL particles were quantified by the NIH imaging software ImageJ (NIH, Bethesda, MD).

Cholesterol efflux with HDL.

Cholesterol efflux assays were performed in ApoB-depleted serum in triplicate as described19. ApoB-containing particles were removed by mixing the 20 μL serum with the 8 μL polyethylene glycol 6000 (20% in water, PEG 6000, Sigma Aldrich, St. Louis, MO) incubated for 20 min at room temperature 32. After centrifugation at 10,000 g for 30 minutes at 4°, the supernatant was transferred to new tubes and stored until use at −80°C.

Murine RAW 264.7 cells (American Type Culture Collection, Manassas, Virginia) were loaded with 0.5 μCi/mL radiolabeled cholesterol (either [3H]-cholesterol, Perkin Elmer, Boston, MA) maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% FBS (DMRM, tissue culture core facility, Cleveland Clinic) for 24 h at 37°C and incubated with ApoB-depleted serum (2%, vol/vol) for 4 h to measure total efflux. In parallel, a second set of cells were pretreated with 0.2 mM 8Br-cAMP in 0.2% BSA-DMEM media for 16 hours at 37°C to induce ABCA1-dependent efflux. The radioactivity of media and cells was measured by liquid scintillation counting. The percentage of cholesterol efflux activity was calculated as the radioactivity in the media divided by total radioactivity (media plus cell extracts) in a sample. The ABCA1-dependent efflux was calculated as the difference between efflux in the presence of 8-Br-cAMP (total efflux) and the absence of 8-Br-cAMP (ABCA1- independent efflux).

Ceruloplasmin activity assay.

CP has oxidase activity toward several substrates, including Fe2+ and amino compounds. Since the amino oxidase and the ferroxidase activities of CP are strongly correlated (r = 0.99)33, we used the oxidase activity as a surrogate of ferroxidase activity of CP. The oxidase activity was measured using the o-dianisidine dihydrochloride as a substrate 34,35. Briefly, 10 μL of serum was incubated with 60 μL of 0.1 M sodium acetate (pH 5.0) and 60 μL of 2.5 mg/mL o-dianisidine in a 96-well microplate at 37°C. The reaction was stopped by adding 170 μL of 18 N sulfuric acid at either 15- or 45-minute intervals. The absorbance of oxidized o-dianisidine by CP was measured at 540 nm.

Preparation of the oxidized LDL (Ox-LDL).

To assess pro-inflammatory index of HDL, we prepared oxidized LDL (Ox-LDL) as described 19. In brief, 1 mg protein/mL of LDL was added to the 5 μL of 4 mM CuSO4 solution to obtain a final concentration of 20 nmol CuSO4/mg LDL and incubated for 24 hours at 37°C. The oxidation reaction was stopped with 40 μL of 2 mM butylated hydroxytoluene in 0.1 M EDTA. Ox-LDL was purified by dialysis against phosphate-buffered saline (PBS) containing 0.5 mM EDTA at 4°C for 48 hours. After dialysis, the diluted Ox-LDL was lyophilized and stored at −80°C.

Pro-inflammatory index of HDL.

HDL pro-inflammatory index was measured using the dichlorofluorescein (DCF)-based cell-free assay as described 36,37. This assay measures the ability of apoB-depleted serum to inhibit or enhance LDL oxidation. ApoB-depleted serum was incubated with Ox-LDL and 2’,7’-dichlorodihydrofluorescein in a 96-well plate at 37 °C. Fluorescent emission with 530 nm wavelength was measured after serial excitation at 485 nm.

Myeloperoxidase (MPO) activity assay.

MPO activity of serum was measured using described method 38 using 3,3′,5,5′-Tetramethylbenzidine (TMB, Sigma Aldrich, St. Louis, MO) as a substrate. The absorbance was monitored at 650 nm for 10 minutes every 30 seconds at 37°C using a spectrophotometer. MPO activity was calculated using an extinction efficiency of 3.9 × 104 M−1 cm−1, and the units were expressed as mUnit/L.

Paraoxonase1 (PON1) activity assay.

Serum PON1 activity was assayed using paraoxon (Sigma Aldrich, St. Louis, MO) substrate as described39. Para-nitrophenol (ε= 17,000 M−1 × cm−1) absorbance was measured at 405 nm for 1 hour every 1 minute at 37°C.

Statistical analysis.

Continuous variables were evaluated for normality using the Shapiro-Wilk test. Normally distributed continuous measures were summarized using means and standard deviations (SDs) and were compared between diabetes groups using multiple comparison analysis of variance (ANOVA) with Tukey adjustment. Non-normally distributed continuous and ordinal measures were summarized using medians, 25th and 75th percentiles, and were compared between diabetes groups using Kruskal-Wallis tests. Categorical factors were compared using Fisher’s exact test. Pre- and post-OCA labs and measures of HDL functionality were compared using paired t-tests for normally distributed continuous variables and paired signed-rank tests for non-normally distributed continuous or ordinal measures. This was done for all subjects combined as well as stratified by diabetes. The correlations between continuous factors were assessed using Spearman correlation. The statistical analyses were performed using SAS (version 9.4, The SAS Institute, Cary, NC) and R (version 4.2.1) and a significance level of 0.05 was considered.

Results.

Study population.

Table 1 displays baseline characteristics of all study subjects, including a comparison between diabetic and non-diabetic patients with MASH. All patients with MASH had obesity. Non-diabetic and diabetic patients were matched by body mass index (BMI) with diabetic patients with MASH being relatively older, albeit not significantly, compared to patients with MASH without T2DM (45.4±14 vs. 55.2±5.6, P=0.053). Study subjects were not matched by sex due to strict exclusion criteria (no hypolipidemic therapy), except for one non-diabetic male in the non-diabetic group, all other patients were female. Diabetic patients with MASH had higher fasting blood glucose (P<0.05) and HbA1c (P<0.001) levels due to group definitions. Histologically, diabetic patients with MASH exhibited significantly higher fibrosis (P<0.05). In addition, subjects with diabetes showed lower lobular inflammation grades, though not reaching statistical significance (P<0.089). Non-diabetic patients with MASH had significantly higher ferritin and LDL cholesterol levels (P<0.05). No differences were observed in other baseline biochemical variables, including triglycerides, total cholesterol, and HDL cholesterol levels.

Table 1.

Baseline characteristics of the study population.

Factor Total (N=20) No T2DM (N=10) T2DM (N=10) p-value
Demographics
Proportion female 19(95.0) 9(90.0) 10(100.0) 0.99d
Age (years) 50.3±11.5 45.4±14.0 55.2±5.6 0.053a
BMI (kg/m2) 34.0[31.2,44.5] 37.1[32.2,45.6] 32.9[30.2,44.5] 0.26b
Liver tests
Bilirubin (total) (mg/dL) 0.50[0.40,0.70] 0.55[0.40,0.70] 0.50[0.40,0.90] 0.91b
AST (U/L) 51.5[34.5,84.5] 46.0[36.0,99.0] 60.5[33.0,70.0] 0.82b
ALT (U/L) 67.5[47.0,115.5] 62.0[50.0,123.0] 78.0[42.0,108.0] 0.82b
Alkaline Phosphatase (U/L) 81.2±23.3 74.1±18.6 88.2±26.3 0.18a
Lipids
Total cholesterol (mg/dL) 199.0[169.0,222.0] 207.5[175.0,222.0] 176.0[142.0,222.0] 0.19b
HDL (mg/dL) 41.5[37.5,46.0] 40.0[37.0,47.0] 42.0[39.0,45.0] 0.43b
LDL (mg/dL) 120.7[75.0,161.2] 126.2[80.1,191.0] 99.2[68.1,162.0] 0.03 b
Triglycerides (mg/dL) 160.0[129.0,166.0] 160.0[143.0,165.0] 156.5[115.0,181.0] 0.82b
Insulin sensitivity
Fasting serum glucose (mg/dL) 106.5[87.5,139.0] 96.5[84.0,106.0] 139.0[111.0,157.0] 0.013 b
Insulin (uU/mL) 25.8[17.9,41.2] 26.4[20.1,37.1] 25.6[16.3,59.0] 0.94b
HOMA 7.6[4.8,11.0] 6.1[4.3,9.0] 9.2[5.3,24.6] 0.24b
HbA1c (%) 6.6±1.1 5.8±0.63 7.3±1.04 <0.001 a
Chemistries
White blood cell count (cells/μl) 7.9±2.0 7.6±2.3 8.2±1.8 0.56a
Creatinine (mg/dL) 0.73±0.14 0.77±0.13 0.68±0.14 0.193a
Uric acid (mg/dL) 6.6±1.6 6.8±1.9 6.3±1.2 0.586a
Albumin (g/dL) 4.2±0.48 4.1±0.50 4.3±0.48 0.488a
Iron status
Iron (ug/dL) 69.5[60.5,100.0] 85.0[66.0,102.0] 64.0[54.0,75.0] 0.17b
Total iron binding capacity (ug/dL) 381.5±84.1 385.5±83.3 377.4±89.1 0.84a
Ferritin (ng/mL) 154.0[79.5,336.5] 336.5[157.0,451.0] 83.0[36.0,151.0] 0.004 b
Liver histology
 Steatosis grade 2.0[1.5,3.0] 2.0[2.0,3.0] 2.5[1.00,3.0] 0.94b
Lobular inflammation grade 2.0[1.00,2.0] 2.0[2.0,2.0] 1.5[1.00,2.0] 0.089b
Ballooning grade 2.0[2.0,2.0] 2.0[1.00,2.0] 2.0[2.0,2.0] 0.28b
Fibrosis stage 2.0[1.00,3.0] 1.00[1.00,2.0] 3.0[2.0,3.0] 0.032 b
NAFLD Activity Score (NAS) 6.0[5.0,7.0] 6.0[5.0,7.0] 6.0[5.0,6.0] 0.53b
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Statistics presented as Mean ± SD, Median [P25, P75] or N (column %).

p-values:

a

=ANOVA

b

=Kruskal-Wallis test

d

=Fisher’s Exact test.

Effect of OCA on liver histology, enzymes, and plasma biochemistry.

Consistent with prior findings from all patients in the FLINT study7, OCA therapy significantly decreased plasma ALT and AST and improved liver histology, including steatosis, lobular inflammation, ballooning, and overall NAFLD activity score in this subset of patients with MASH (Table 2). OCA tends to improve the fibrosis endpoint, though the differences didn’t reach significance (P=0.053), presumably due to a small number of patients. OCA also significantly reduced plasma uric acid levels (P<0.05). However, OCA therapy increased alkaline phosphatase (P<0.05). In this subpopulation of patients with MASH, OCA had a modest effect on plasma lipids, slightly reducing triglycerides, but the differences didn’t reach significance (P=0.09). Moreover, no change was observed in total cholesterol, LDL cholesterol, and HDL cholesterol levels due to OCA therapy.

Table 2.

OCA therapy-induced change (Post-Pre) from baseline to 18 months.

Factor All subjects N=20 P-value No T2DM (N=10) P-value T2DM (N=10) P-value
BMI (kg/m2) −0.2[−1.3,1.1] 0.48b −0.3[−1.0,2.1] 1.00b −0.2[−1.5,0.4] 0.43b
Bilirubin (total) (mg/dL) −0.1[−0.2,0.1] 0.18b 0.0[−0.1,0.1] 1.00b −0.2[−0.3,0.0] 0.10b
AST (U/L) −19.0[−44.0,−10.0] <0.001 b −19.0[−56.0,−10.0] 0.006 b −19.0[−27.0,−10.0] 0.008 b
ALT (U/L) −28.5[−80.5,−12.0] <0.001 b −29.5[−93.0,−26.0] 0.006 b −26.5[−68.0,−10.0] 0.006 b
Alkaline Phosphatase (U/L) 12.5±26.0 0.045 a 7.2±10.1 0.05 a 17.8±35.6 0.15a
Uric acid (mg/dL) −0.7±1.1 0.013 a −0.6±1.0 0.08a −0.70±1.2 0.10a
Triglycerides (mg/dL) −23.5[−48.0,19.5] 0.09b −41.5[−53.0,−21.0] 0.01 b 9.0[−34.0,34.0] 1.00b
Total cholesterol (mg/dL) 4.0[−9.5,25.0] 0.40b 11.0[−14.0,22.0] 0.51b 0.0[−5.0,28.0] 0.84b
HDL (mg/dL) −0.5[−5.0,3.0] 0.55b −0.5[−4.0,3.0] 0.68b 0.5[−6.0,3.0] 0.54b
LDL (mg/dL) 9.6[−10.4,25.8] 0.18b 19.7[−12.8,30.4] 0.28b 6.5[−8.0,24.8] 0.49b
Glucose (mg/dL) 9.0[−11.0,20.0] 0.23b 10.5[1.0,16.0] 0.08b 0.5[−18.0,53.0] 0.54b
Insulin (uU/mL) 1.8[−5.5,13.0] 0.29b 4.3[−7.3,11.2] 0.70b 1.8[−4.0,14.8] 0.43b
HOMA 1.0[−2.1,5.2] 0.19b 1.1[−1.6,4.0] 0.56b 1.0[−2.5,6.5] 0.32b
HbA1c (%) 0.2±0.7 0.36a 0.0±0.6 0.85a 0.3±0.8 0.34a
Steatosis grade −1.0[−2.0,0.0] 0.005 b −0.5[−2.0,0.0] 0.09b −1.0[−2.0,0.0] 0.03 b
Lobular inflammation grade −1.0[−1.0,0.0] 0.008 b −1.0[−1.0,0.0] 0.02 b −0.5[−1.0,0.0] 0.30b
Ballooning grade −1.0[−1.0,0.0] 0.001 b −1.0[−1.0,0.0] 0.03 b −1.0[−1.0,0.0] 0.02 b
Fibrosis stage −0.5[−1.5,0.0] 0.053b 0.0[−1.0,0.0] 0.17b −1.0[−2.0,0.0] 0.20b
NAFLD Activity Score (NAS) −2.0[−4.0,−0.5] <0.001 b −2.0[−3.0,−1.0] 0.01 b −2.5[−4.0,0.0] 0.02 b
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Statistics presented as Mean ± SD or Median [P25, P75].

p-values:

a

=Paired t-test

b

=Paired signed-rank test.

Effect of OCA on HDL particle sizes and functions.

Since HDL is a mixture of heterogeneous particles and HDL properties depends on the sizes of each subpopulation, we characterized HDL particle sizes before and after OCA therapy. For this purpose, we isolated HDL using the anti-HDL immunoaffinity column and loaded the same amount of HDL proteins onto ND-PAGE to separate HDL particles by their sizes. This method separated small pre-β1 HDL, mid-sized HDL3, and large HDL2 particles (Supplementary Figure 1A). Although the mid-sized HDL3 and large HDL2 particles (Supplementary Figure 1B) did not show any significant change in HDL particles due to OCA therapy, the normalization of the data to HDL cholesterol levels revealed increased small pre-β1 HDL - the HDL particles with enhanced protective capacity (P=0.017) (Figure 1A). Next, to assess the effect of OCA therapy on the anti-oxidant function of HDL, we measured the ferroxidase activity of CP and acyltransferase activity of PON1, the essential HDL proteins with anti-oxidant activities involved in the prevention of LDL oxidation24. OCA treatment resulted in reduced CP activity (P<0.001) (Figure 1B) without any effect on anti-oxidant PON1 activity (Supplementary Figure 2A). OCA therapy also didn’t affect MPO, a neutrophil-derived pro-inflammatory enzyme regulated by CP (Supplementary Figure 2B). A pro-oxidant enzyme MPO catalyzes the reaction of hydrogen peroxide and halide ions to form cytotoxic intermediates, and CP is an endogenous inhibitor of MPO26. To further evaluate the consequence of OCA therapy on overall HDL ant-oxidant functions, we measured the pro-inflammatory index of HDL. This test estimates the capacity of HDL to either inhibit or worsen Cu2+-induced lipid peroxidation. OCA therapy didn’t alter pro-inflammatory index of HDL (P=0.165). OCA also didn’t have significant effect in total and ABCA1-dependent cholesterol efflux functions of HDL in all subjects (Supplementary Figure 3A).

Figure. 1.

Figure. 1.

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Effect of OCA therapy on HDL properties in patients with MASH (n=20, all subjects). A: Relative abundances of small pre-β1 HDL particles. B: Ferroxidase activity of ceruloplasmin (CP) in serum. C: Pro-inflammatory HDL activity.

OCA therapy has distinct effects on diabetic and non-diabetic patients with MASH.

To assess the effect of diabetes on OCA-induced changes in HDL properties, patients with MASH were stratified into diabetic and non-diabetic MASH groups (Table 1), according to fasting blood glucose and HbA1c levels. OCA therapy significantly reduced ALT and AST in both groups (Table 2). OCA marginally increased alkaline phosphatase in the non-diabetic individuals (P=0.05), with no significant impact on diabetic patients with MASH (P=0.15). While OCA therapy notably decreased ballooning grade and NAFLD score in diabetic and non-diabetic groups, its effects on hepatic steatosis and inflammation varied. OCA significantly ameliorated hepatic steatosis in diabetic patients (P<0.05) but not in non-diabetics. Conversely, it improved hepatic inflammation solely in non-diabetic patients with MASH (P<0.05). Except for triglycerides, which decreased solely in the non-diabetic group (P<0.01), OCA had no significant impact on total, LDL, and HDL cholesterol levels.

While both the diabetic and nondiabetic groups had similar baseline HDL functionality and ferroxidase CP activity measures, OCA therapy had distinct effects on these parameters (Figure 2). OCA therapy increased expression of HDL normalized pre-β1 HDL particles exclusively in diabetic patients with MASH (Figure 2A). The OCA-induced decrease in anti-oxidant CP activity was specific to nondiabetics only (Figure 2B). The OCA-induced decrease in CP activity was also reflected in a significantly increased pro-inflammatory index of HDL in nondiabetic patients (P<0.05, Figure 2C). Separating patients into diabetic and nondiabetic groups revealed that OCA marginally increased total cholesterol efflux only in nondiabetic patients with MASH (P=0.047), with no OCA-induced effect on total or ABCA-dependent cholesterol efflux in diabetic patients with MASH (Supplementary Figure 3B & C) or on the activities of PON1 and MPO (not shown).

Figure. 2.

Figure. 2.

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Impact of T2DM and OCA therapy on HDL functionality in patients with MASH. The data from all patients with MASH presented in Figure 1 were stratified into diabetic and non-diabetic groups (n=10/group) based on the presence of diabetes. A: Relative abundances of small pre-β1 HDL particles. B: Ferroxidase activity of ceruloplasmin in serum. C: Pro-inflammatory HDL activity.

Correlations of OCA-induced changes with other pre-OCA variables.

In all patients with MASH, an OCA-induced change in CP activity was positively associated with HbA1c (rho=0.61, P=0.004; Supplementary Table 1), and patients with a lower level of HbA1c tended to have a greater decrease in CP (Figure 3A). Interestingly, this change in CP was only in non-diabetics (Figure 2B) and was positively associated with hemoglobin (Supplementary Figure 4A) and hematocrit (Supplementary Figure 4B). An OCA-induced increase in pro-inflammatory HDL was inversely associated with fasting blood glucose in all subjects (Figure 3B), an association mainly driven by diabetes (Supplementary Table 1). Furthermore, in diabetic patients, the increase in pro-inflammatory HDL was also inversely related to alkaline phosphatase (Supplementary Table 1), a known predictor of liver damage.

Figure. 3.

Figure. 3.

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Significant associations of OCA-induced changes with other pre-OCA variables in all patients with MASH. A: Positive association of OCA-induced change in CP with pre-OCA HbA1c. B: Negative association of change in pro-inflammatory HDL activity with pre-OCA glucose.

Discussion.

This study aimed to assess the effect of OCA on HDL functions in a subset of patients with MASH with and without T2DM from the FLINT study. The significant new finding of the study was that OCA therapy decreased the anti-oxidant activity of HDL-associated CP and increased pro-inflammatory HDL in non-diabetic patients with MASH. However, these harmful effects of OCA were not observed in diabetic patients with MASH. Interestingly, OCA therapy boosted pre-β1 HDL particle expression, vital for cholesterol efflux from peripheral cells, without significantly affecting cholesterol efflux in diabetic patients with MASH.

Until the recent accelerated approval of Rezdiffra40, no other therapy for MASH existed, making lifestyle modifications such as exercise and diet major management options. Rezdiffra induces vomiting, abdominal pain, urticaria, and rash40, underscoring the need for alternative MASH therapies with fewer side effects. OCA is a promising hepatoprotective FXR agonist that enhances insulin sensitivity, and lipid metabolism, and exerts anti-inflammatory properties. As an anti-inflammatory agent OCA also dose-dependently inhibited aortic plaque formation in ApoE−/− mice following 12 weeks of treatment (10mg/kg daily)41. However, OCA also reduced HDL cholesterol levels. Observational studies show that each 1 mg/dL increase in HDL cholesterol levels is associated with ~2–3% cardiovascular risk decline12. While the reduction in HDL levels caused by OCA is concerning, studies have shown that increasing HDL levels alone, as seen with niacin and CETP inhibitors do not reduce cardiovascular events42,43. This suggests that enhancing HDL function, including RCT, might be a more effective strategy for reducing cardiovascular disease risk rather than merely raising HDL levels. Previous studies have shown that FXR agonism with WAY-362450 reduces plasma cholesterol levels in all lipoprotein particles, including VLDL, LDL, and HDL, in mouse models of dyslipidemia44. The follow-up studies demonstrated that this could be related to increases in transhepatic RCT via SR-B1-mediated hepatic uptake of HDL45. Under a hyperlipidemic state, FXR agonism with OCA in liver-specific SR-B1 deficient mice promotes intestinal cholesterol excretion, suggesting OCA-induced increased transintestinal cholesterol efflux.

In contrast to rodent studies, OCA therapy in humans, in addition to reducing HDL levels, also increased LDL cholesterol7,9. A recent survey of larger OCA-treated patient populations demonstrated that OCA-induced alterations in HDL cholesterol were due to reductions in large- and medium-sized HDL particles9. While the discrepancy in cholesterol metabolism between rodent and human studies is not apparent, animal studies suggest that HDL cholesterol measurements in humans do not necessarily reflect the heterogeneity of HDL functions, including RCT. Our results showed that in diabetic patients with MASH, OCA therapy increased pre-β1 HDL particles, small size HDL with enhanced cholesterol efflux capacity. While OCA inhibits cholesterol metabolism to bile acids, OCA may increase SR-B1-specific hepatic uptake of HDL cholesterol, leading to increased fecal excretion of free cholesterol. Future studies are warranted to assess the effect of OCA on RCT and HDL flux in patients with MASH.

The beneficial effect of OCA therapy in diabetic patients with MASH in our study mirrors findings from the initial phase 2 study, which demonstrated that six weeks of OCA reduced liver inflammation and fibrosis markers while improving TG metabolism and insulin sensitivity6. Insulin resistance alters lipoprotein particle sizes and functions, including HDL metabolism, while the pro-inflammatory state in T2DM further impacts lipid and lipoprotein metabolism and HDL composition. Anti-inflammatory OCA improved glucose metabolism and systemic inflammation, potentially increasing preβ HDL particles in diabetic patients. In our study, pro-inflammatory HDL in T2DM was linked to glucose levels. While OCA therapy reduced glucose, the difference was insignificant due to the small sample size. In contrast to diabetic patients with MASH, OCA therapy caused detrimental effects on HDL functions in non-diabetic patients with MASH. To understand the distinct effects of OCA in patients with MASH with and without diabetes, we need to consider differences in the baseline parameters of these two populations. In addition to anticipated high fasting blood glucose and HbA1c in diabetic patients with MASH, non-diabetic patients with MASH displayed high serum ferritin levels (Table 1). As an iron storage protein, serum ferritin represents a proxy of stored iron in the liver, where the excess body iron accumulates. Ferritin levels increased in response to iron overload and inflammation46. Many epidemiological studies have shown a strong positive association between ferritin and oxidative stress markers. Mechanistically, ferritin may induce oxidative stress by providing Fe2+ to the Fenton reaction, where Fe2+ generates hydroxide radical (HO•) from hydrogen peroxide (H2O2). In contrast to ferritin, a copper-containing acute-phase protein CP has anti-oxidant properties. In addition to binding and securing pro-oxidant copper transport, CP’s ferroxidase activity protects from Fe2+-induced oxidative stress. In this study, ferritin levels were measured at the baseline but not after OCA therapy which prevented us from assessing the effect of OCA on iron metabolism. Although, due to the small sample size in our study, the regression analysis failed to reveal an association between baseline CP activity and ferritin levels, it is plausible that high ferritin-induced oxidative stress in non-diabetic patients with MASH impairs the ferroxidase activity of CP. Notably, an inverse association of CP and ferritin, iron oxidative stress markers have been reported in a larger cohort (n=389) of healthy Japanese population47. Future longitudinal studies with larger sample sizes must test for the reciprocal relationship between ferritin and CP.

Consistent with the reduction of CP activity in non-diabetic patients with MASH, we also observed that OCA therapy increased the pro-inflammatory index of HDL as measured based on HDL’s capacity to prevent or aggravate Cu2+-induced LDL oxidation. In addition to the reduced anti-oxidant activity of CP, OCA-induced pro-inflammatory remodeling of HDL could be related to the direct pro-oxidant property of OCA, a bile acid derivative. It has been shown that hydrophobic bile acids, including taurochenodeoxycholic acid (TCDC) and taurocholic acid (TC), are responsible for the generation of reactive oxygen species (ROS) in isolated hepatocytes or hepatic mitochondria48. Bile acids also increase ROS release by polymorphic leukocytes49. A semi-synthetic bile acid analog OCA may result in mitochondrial derangements due to superoxide formation following hepatic OCA accumulation. Thus, we speculate that OCA therapy may potentiate high ferritin-induced oxidative stress in non-diabetic patients with MASH.

This study has several limitations. It did not elucidate the effect of OCA therapy on HDL proteome composition that determines HDL functions. In addition to anti-oxidant PON1 and CP, HDL contains more than 100 proteins with distinct functions. The role of other HDL proteins on OCA-induced changes in HDL functions was not perceived. The small sample size limited our ability to detect differences in HDL functions between diabetic and non-diabetic groups. In this proof-of-concept study, we could not assess the effect of OCA on HDL metabolism across different ages, genders, and racial groups. Age and racial disparities can influence OCA’s effects on HDL due to differences in liver metabolism and genetic factors. Although OCA appears effective for treating NASH in both men and women, given the notable gender differences in lipid metabolism and OCA’s side effect profile, OCA may have distinct effects on HDL metabolism and function. Excluding patients on lipid-lowering medication prevented assessment of OCA’s gender-dependent effects on HDL metabolism and function. In addition, the analysis did not include placebo samples. Nevertheless, this proof-of-concept study revealed that OCA therapy has a divergent effect on HDL functions in diabetic and non-diabetic patients with MASH. It increases protective small pre-β1 HDL particles in patients with MASH with diabetes but not those without diabetes. In contrast, OCA therapy increases pro-inflammatory HDL and reduces the anti-oxidant activity of HDL-bound CP in patients with MASH without diabetes. These results suggest the potential beneficial role of OCA therapy in HDL functions regardless of changes in HDL levels of diabetic patients with MASH, but not in patients with MASH with diabetes. These findings need to be confirmed in a future study including a larger population of male and female patients with MASH with and without diabetes.

Supplementary Material

SUP INFO 1
SUP INFO 2

Acknowledgments:

The authors would like to extend their appreciation to Jonathan Smith for the cholesterol efflux assay. The MASH CRN is partially supported by grants (U01DK061732, UL1TR000439, U01DK061730, U24DK061730). T.K. was supported by grants (R21 AA029784 and 1R21AG085590-01). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The biospecimens from the MASH CRN reported on here were supplied by the NIDDK Central Repository. The authors also thank the MASH CRN investigators and the Ancillary Studies Committee for providing clinical samples and relevant data from the FLINT trial. The FLINT trial was conducted by the MASH CRN and supported in part by a Collaborative Research and Development Agreement (CRADA) between NIDDK and Intercept Pharmaceuticals. This manuscript was not prepared in collaboration with the NIDDK Central Repository and Intercept Pharmaceuticals, Inc., and does not necessarily reflect their opinions or official views.

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