Kaempferol Improves Cardiolipin and ATP in Hepatic Cells: A Cellular Model Perspective in the Context of Metabolic Dysfunction-Associated Steatotic Liver Disease

Abstract

Targeting mitochondrial function is a promising approach to prevent metabolic dysfunction-associated steatotic liver disease (MASLD). Cardiolipin (CL) is a unique lipid comprising four fatty acyl chains localized in the mitochondrial inner membrane. CL is a crucial phospholipid in mitochondrial function, and MASLD exhibits CL-related anomalies. Kaempferol (KMP), a natural flavonoid, has hepatoprotective and mitochondrial function-improving effects; however, its influence on CL metabolism in fatty liver conditions is unknown. In this study, we investigated the effects of KMP on mitochondrial function, focusing on CL metabolism in a fatty liver cell model (linoleic-acid-loaded C3A cell). KMP promoted mitochondrial respiratory functions such as ATP production, basal respiration, and proton leak. KMP also increased the gene expression levels of CPT1A and PPARGC1A, which are involved in mitochondrial β-oxidation. Comprehensive quantification of CL species and related molecules via liquid chromatography/mass spectrometry showed that KMP increased not only total CL content but also CL72:8, which strongly favors ATP production. Furthermore, KMP improved the monolysocardiolipin (MLCL)/CL ratio, an indicator of mitochondrial function. Our results suggest that KMP promotes energy production in a fatty liver cell model, associated with improvement in mitochondrial CL profile, and can serve as a potential nutrition factor in preventing MASLD.

Keywords: ATP; lipidomics; liquid chromatography/mass spectrometry; metabolic dysfunction-associated steatohepatitis; metabolic dysfunction-associated steatotic liver disease; monolysocardiolipin.

Figure 1

Effects of kaempferol on cell viability in C3A cells. Cell viability was measured using the WST-1 assay. Values are presented as means ± standard deviations (SDs) (n = 6 per group). * p < 0.05, **** p < 0.0001 vs. 0 µM of kaempferol using one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test.

Figure 2

Effects of kaempferol on mitochondrial function in C3A cells. (A) Mitochondrial respiratory profile. Oxygen consumption rate (OCR) was measured over time (pmol/min) using an extracellular flux analyzer. Each reagent of the Mito Stress Test Kit (oligomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone [FCCP], or rotenone and antimycin A [Rot/AA]) was injected into each well stepwise. (B) The parameters were calculated according to each OCR value on mitochondrial oxidative phosphorylation, including (a) basal respiration, (b) ATP production, (c) proton leak, (d) maximal respiration, (e) spare respiration capacity, and (f) non-mitochondrial respiration. Values are presented as means ± standard deviations (SDs) (n = 4–5 per group). * p < 0.05, unpaired t-test.

Figure 3

Effect of kaempferol on the expression of mitochondrial metabolism-related genes in C3A cells. (a) mRNA expression levels of mitochondrial biogenesis-related genes determined by real-time PCR. β-actin was used as a housekeeping control for each target gene. (b) mRNA expression levels of β-oxidation-related genes determined by real-time PCR. β-actin was used as a housekeeping control. (c) Mitochondrial DNA copy number determined by real-time PCR. GAPDH expression level was used as a control for nuclear DNA. All values are indicated as the relative expression compared with control group values, which were set to 1.0 (mean ± standard deviation [SD], n = 8 per group). * p < 0.05, ** p < 0.01, one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test.

Figure 4

Efficacy of kaempferol in improving cardiolipin (CL) profiles in the linoleic acid (LA)-loaded fatty liver cell model. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test.

Figure 5

Efficacy of kaempferol in improving total CL level. Total CL was determined as the sum of the levels of all CL species determined in this study. * p < 0.05, ** p < 0.01, one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test.

Figure 6

Heat map of cardiolipin (CL) species in the linoleic acid (LA)-loaded fatty liver cell model supplemented with or without KMP. With respect to color intensities, the mean values of each CL species in the control group are expressed as 1.0. Individual results for the samples are represented (n = 6 for each group). The cross mark means undetected CL species.

Figure 7

Efficacy of kaempferol in improving monolysocardiolipin (MLCL) profiles in the linoleic acid (LA)-loaded fatty liver cell model. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test.

Figure 8

Efficacy of kaempferol in improving total MLCL levels. Total MLCL was determined as the sum of the levels of all MLCL species determined in this study. * p < 0.05, one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test.

Figure 9

Efficacy of kaempferol in improving the balance of the Total MLCL/Total CL ratio in the LA-loaded fatty liver cell model. * p < 0.05, one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test.

Figure 10

The hypothetical mechanism by which kaempferol enhances hepatocellular mitochondrial energetic metabolism, which is associated with improved cardiolipin profiles. KMP, kaempferol; FA, fatty acids; LA, linoleic acid; CPT1A, carnitine palmitoyltransferase 1A; MLCL, monolysocardiolipin; CL, cardiolipin; MLCL/CL, total MLCL/total CL ratio; mCL, mature CL; I, II, III, IV, V, Complex I-V; ETC, electron transport chain; ATP, adenosine triphosphate; PL, phospholipids.