Alnustone Ameliorates Metabolic Dysfunction-Associated Steatotic Liver Disease by Facilitating Mitochondrial Fatty Acid β-Oxidation via Targeting Calmodulin

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD), including its more severe manifestation metabolic dysfunction-associated steatohepatitis (MASH), poses global public health threats with limited therapeutics. Here, the role of alnustone is explored, a natural compound derived from the traditional Chinese herb Alpinia katsumadai Hayata, in the treatment of MASLD and MASH. It is shown that alnustone administration potently reduces serum triacylglycerol levels, reverses liver steatosis, and alleviates insulin resistance in both male and female MASLD mice. It also effectively ameliorates established fibrosis in MASH mice without any side effects. Mechanistically, hepatic lipidome profiling and energy metabolic assays reveal that alnustone facilitates mitochondrial fatty acid β-oxidation. Employing limited proteolysis-mass spectrometry (LiP-SMap) and further validation, calmodulin is identified as a direct molecular target of alnustone. Alnustone interacts with the Ca²⁺-binding site of calmodulin, leading to increased cytosolic and mitochondrial Ca²⁺ levels and enhanced mitochondrial function, whereas liver-specific calmodulin knockdown abrogates alnustone's therapeutic effects. Moreover, calmodulin is downregulated in human livers of patients with MASLD and MASH, and is genetically associated with reduced MASLD risk. These findings establish alnustone as a promising natural compound and highlight calmodulin as a target for treating MASLD.

Keywords: alnustone; calmodulin; fatty acid β‐oxidation; metabolic dysfunction‐associated steatohepatitis; metabolic dysfunction‐associated steatotic liver disease.

Figure 1

Alnustone alleviates hepatic steatosis and insulin resistance in male and female MASLD mice induced by high‐fat diet. a) Schematic illustration of intraperitoneal alnustone injection to MASLD mice induced by HFD. b) Serum triacylglycerol levels of male (n = 6) and female (n = 9) mice fed with HFD and administrated with vehicle/alnustone for 2 weeks. c) Hepatic triacylglycerol levels of male (n = 6) and female (vehicle: n = 7, alnustone: n = 8) mice fed with HFD and administrated with vehicle/alnustone for 2 weeks. Triacylglycerol contents were normalized by hepatic protein levels. d‐e) H&E (d) and Oil Red O (e) staining were performed in liver sections from vehicle/alnustone‐treated mice fed with HFD. H&E fat cavitation area, ORO staining area were quantitatively compared. Scale bar: 100 µm. (n = 6). f) Glucose tolerance test and insulin tolerance test were performed on male (n = 6) and female (n = 6‐8) mice administrated with alnustone or vehicle for 1 weeks and area under curve (AUC) was calculated and compared. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; significance is assessed by two‐tailed unpaired Student's t test.

Figure 2

Oral administration of alnustone effectively ameliorates hepatic steatosis in HFD‐induced MASLD mice. a) Schematic illustration of oral alnustone administration to MASLD mice induced by HFD. b) Serum triacylglycerol levels of male (n = 6) and female (n = 8) mice fed with HFD and administrated with vehicle/alnustone for 2 weeks. c) Hepatic triacylglycerol levels of male (n = 6) and female (n = 6‐7) mice fed with HFD and administrated with vehicle/alnustone for 2 weeks. Triacylglycerol contents were normalized by hepatic protein levels. d‐e) H&E (d) and Oil Red O (e) staining were performed in liver sections from vehicle/alnustone‐treated mice fed with HFD. H&E fat cavitation area, ORO staining area were quantitatively compared. Scale bar: 100 µm. (n = 6). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001; significance is assessed by two‐tailed unpaired Student's t test.

Figure 3

Alnustone alleviates hepatic steatosis, inflammation, and fibrosis in MCD‐induced MASH mice. a) Schematic illustration of intraperitoneal alnustone injection to MASH mice induced by MCD. b) Serum ALT levels of mice fed with MCD diets and administrated with vehicle/alnustone for 2 weeks. (vehicle: n = 6, alnustone: n = 7). c) Serum AST levels of mice fed with MCD diets and administrated with vehicle/alnustone for 2 weeks. (vehicle: n = 6, alnustone: n = 7). d) Serum triacylglycerol levels of mice fed with MCD diets and administrated with vehicle/alnustone for 2 weeks. (vehicle: n = 6, alnustone: n = 7). e) Hepatic triacylglycerol levels of mice fed with MCD diets and administrated with vehicle/alnustone for 2 weeks. Triacylglycerol contents were normalized by hepatic protein levels. (vehicle: n = 6, alnustone: n = 7). f‐j) H&E (f), Oil Red O (g), F4/80 (h), Masson (i), and Sirius red (j) staining were performed in liver sections from vehicle/alnustone‐treated mice fed with MCD diet. H&E fat cavitation area, ORO staining area, F4/80 staining area, Masson staining area, and Sirius red staining area were quantitatively compared. Scale bar: 100 µm. (vehicle: n = 6, alnustone: n = 7). k) Liver hydroxyproline content of mice fed with MCD diets and administrated with vehicle/alnustone for 2 weeks. (n = 6). l) NAFLD activity score and inflammation score were evaluated by comparing scores from liver sections. (vehicle: n = 6, alnustone: n = 7). m‐n) RNA was extracted from liver tissues of MCD diets‐induced MASH mice administrated with alnustone or vehicle and expression of inflammation (m) and fibrosis‐related (n) genes was determined by RT‐qPCR with β‐actin as an internal control. (n = 6). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; significance is assessed by two‐tailed unpaired Student's t test or rank sum test.

Figure 4

Alnustone alleviates hepatic steatosis, inflammation, and fibrosis in AMLN‐induced MASH mice. a) Schematic illustration of intraperitoneal alnustone injection to MASH mice induced by AMLN. b) Serum ALT levels of mice fed with AMLN diets and administrated with vehicle/alnustone for 2 weeks. (n = 6). c) Serum AST levels of mice fed with AMLN diets and administrated with vehicle/alnustone for 2 weeks. (n = 6). d) Serum triacylglycerol levels of mice fed with AMLN diets and administrated with vehicle/alnustone for 2 weeks. (n = 6). e) Hepatic triacylglycerol levels of mice fed with AMLN diets and administrated with vehicle/alnustone for 2 weeks. Triacylglycerol contents were normalized by hepatic protein levels. (n = 6). f‐k) H&E (f), Oil Red O (g), F4/80 (h), TUNEL (i), Masson (j), and Sirius red (k) staining were performed in liver sections from vehicle/alnustone‐treated mice fed with AMLN diet. H&E fat cavitation area, ORO staining area, F4/80 staining area, TUNEL positive cell percentage, Masson staining area, and Sirius red staining area were quantitatively compared. Scale bar: 100 µm. (n = 6). l) Liver hydroxyproline content of mice fed with AMLN diets and administrated with vehicle/alnustone for 2 weeks. (n = 6). m) NAFLD activity score was evaluated by comparing scores from liver sections. (n = 6). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; significance is assessed by two‐tailed unpaired Student's t test or rank sum test.

Figure 5

Alnustone protects mouse and human hepatocytes from lipid accumulation. a) AML12 cells were treated with 0.2 mM palmitic acid (PA) in the presence of vehicle or alnustone at the dose of 5, 10, 20 µM. After 24 h, cellular triacylglycerol levels were assayed. (n = 6). b) AML12 cells were treated with 0.2 mM palmitic acid for 24 h, and then alnustone was added to the medium for 2 h, 6 h, 12 h, 24 h. Cellular triacylglycerol levels were then assayed. (n = 6). c) Analysis of cellular triacylglycerol levels in mouse primary hepatocytes, AML12, and HepG2 cells treated with 0.2 mM palmitic acid and 10 µM alnustone for 24 h. (n = 6). d‐e) Cells under the indicated conditions in (c) were stained with Oil Red O (d), and concentrations of oil red were extracted by isopropanol and quantified (e). Scale bar: 20 µm. (n = 6‐8). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; significance is assessed by two‐tailed unpaired Student's t test and one‐way ANOVA with Tukey's multiple comparisons post‐hoc test.

Figure 6

Alnustone reduces hepatic lipid metabolites by promoting mitochondrial fatty acid β‐oxidation. a) Principal component analysis (PCA) of liver lipidome from HFD‐induced mice treated with vehicle (n = 3) or alnustone (n = 4). b) Counts of lipid classes identified in liver lipidome. TAG: triacylglycerol; DAG: diacylglycerol; PA: palmitic acid. c) Relative abundance of the TAG, DAG, and PA class identified in liver lipidome. d) Volcano plot showing changes in lipid metabolites after alnustone treatment. e) Heatmap illustrating the effects of alnustone on individual TAG, DAG, and PA metabolites identified in liver lipidome. f‐i) Mitochondrial oxygen consumption rate (OCR) was measured by Seahorse assay in AML12 cells induced by 0.2 mM palmitic acid with or without 10 µM alnustone for 24 h (f). *, vehicle versus PA; #, PA versus PA+Alnustone. Basal respiration (g), maximal respiration (h), and spare respiratory capacity (i) were calculated. (n = 6). j) ATP content of AML12 cells induced by 0.2 mM palmitic acid with or without 10 µM alnustone for 24 h. (n = 8). Data are presented as mean ± SEM. *, #P < 0.05, **P < 0.01; significance is assessed by two‐tailed unpaired Student's t test.

Figure 7

Alnustone directly binds to calmodulin resulting in increased cytosolic and mitochondrial Ca²⁺ levels. a) Flow chart depicting the LiP‐SMap assay. Freshly prepared AML12 whole‐cell lysates after exposed to palmitic acid for 24 h were treated with or without alnustone followed by proteinase K digestion and mass spectrometry (MS) analysis. The binding of alnustone prevents proteinase digestion, leading to the differential MS peptide profiling. b) KEGG enrichment of differentially proteins corresponding to differentially abundant peptides identified by LiP‐SMap. c) Schematic diagram illustrating the screening process of peptides corresponding to alnustone bound proteins. d) STRING network of the candidate targets of alnustone. Protein terms showing significant enrichment (P adj < 0.05) were identified and those with strength > 0.01 are ranked in figure. e) Representative images of autodocking for alnustone and CaM. The CaM protein is represented as a slate cartoon model, ligand is shown as a cyan stick, and their binding sites are shown as magentas stick structures. The hydrogen bond, ionic interactions, and hydrophobic interactions are depicted as yellow, magentas and green dashed lines, respectively. f) SPR analysis of the interactions between calmodulin and alnustone. g) Binding affinity of alnustone to calmodulin was detected by MST. h) Representative images of Fluo‐4 Ca²⁺ fluorescence after treatment with vehicle or 10 µM alnustone in the presence of palmitic acid. Scale bar: 10 µm. Fluorescence intensity of the calcium indicator Fluo‐4/AM was detected with fluorescence microscopy. (n = 6). i) Representative images of Rhod‐2 Ca²⁺ fluorescence after treatment with vehicle or 10 µM alnustone in the presence of palmitic acid. Scale bar: 10 µm. Fluorescence intensity of the calcium indicator Rhod‐2/AM was detected with fluorescence microscopy. (n = 6). j) Analysis of cellular triacylglycerol levels in AML12 cells treated with 1 µM, 1.5 µM, 2 µM, and 2.5 µM CaCl₂. (n = 6). k) Representative images of Fluo‐4 Ca²⁺ and Rhod‐2 Ca²⁺ fluorescence after treatment with or without Calm1‐3 knockdown in the presence of palmitic acid. Scale bar: 10 µm. Fluorescence intensity was detected with fluorescence microscopy. (n = 6). l) Representative images of Fluo‐4 Ca²⁺ and Rhod‐2 Ca²⁺ fluorescence after treatment with or without Calm1 overexpression in the presence of palmitic acid. Scale bar: 10 µm. Fluorescence intensity was detected with fluorescence microscopy. (n = 6). m) Analysis of cellular triacylglycerol levels in AML12 cells with or without Calm1 overexpression in the presence of palmitic acid. (n = 6). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; significance is assessed by two‐tailed unpaired Student's t test and one‐way ANOVA with Tukey's multiple comparisons post‐hoc test.

Figure 8

Calm1‐3 knockdown abrogates the therapeutic effects of alnustone on hepatic steatosis and mitochondrial fatty acid β‐oxidation. a) Representative images of Fluo‐4 Ca²⁺ fluorescence in AML12 cells treated with PA, PA with alnustone, or PA with alnustone after Calm1, Calm2 and Calm3 knockdown. Scale bar: 10 µm. Fluorescence intensity of the calcium indicator Fluo‐4/AM was detected with fluorescence microscopy. (n = 6). b) Representative images of Rhod‐2 Ca²⁺ fluorescence in AML12 cells treated with PA, PA with alnustone, or PA with alnustone after Calm1, Calm2 and Calm3 knockdown. Scale bar: 15 µm. Fluorescence intensity of the calcium indicator Rhod‐2/AM was detected with fluorescence microscopy. (n = 6). c‐d) Mitochondrial oxygen consumption rate (OCR) was measured by Seahorse assay in AML12 cells treated with PA, PA with alnustone, or PA with alnustone after Calm1, Calm2, and Calm3 knockdown (c). *, PA versus PA+A; #, PA+A versus PA+A+KD. Basal respiration, maximal respiration, spare respiratory capacity, and ATP production were calculated (d). (n = 6‐8). e‐g) Analysis of cellular triacylglycerol levels in AML12 cells treated with vehicle, PA, PA with alnustone, or PA with alnustone after Calm1, Calm2, and Calm3 knockdown. Cells under the indicated conditions in (e) were stained with Oil Red O (f), pictured using an inverted phase contrast microscope, and quantified concentrations of oil red extracted by isopropanol (g). Scale bar: 20 µm. h) Schematic overview for Calm1, Calm2 and Calm3 knockdown mice experiments. i‐j) Hepatic triacylglycerol levels (i) and total cholesterol levels (j) of HFD‐induced mice injected with Ad‐sh‐GFP or Ad‐sh‐Calm1‐3 and administrated with vehicle/alnustone for 2 weeks. Triacylglycerol contents were normalized by hepatic protein levels. (n = 6). k‐l) H&E (k) and Oil Red O (l) staining were performed in liver sections from mice under the indicated conditions. Fat cavitation area and ORO staining area were quantitatively compared. Scale bar: 100 µm. m) NAS was compared by measuring scores from biopsy liver sections. (n = 6). Data are presented as mean ± SEM. *, #P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; significance is assessed by two‐tailed unpaired Student's t test or rank sum test.

Figure 9

Calmodulin expression is decreased in human livers of MASLD and is genetically associated with reduced risk of human MASLD. a) Relative mRNA expressions of calmodulin in the livers of mice fed a normal chow diet (NCD) or HFD for 12weeks. (n = 12). b) Linear regression analysis of Calm1 mRNA levels and serum triacylglycerol levels in the livers of NCD and HFD‐induced mice. (n = 12). c) Relative mRNA expressions of calmodulin in the livers of normal control subjects and MASLD patients. (n = 10). d) Relative mRNA expressions of calmodulin in the livers of normal control subjects and MASH patients from the GEO GSE164760 dataset. e) Representative immunohistochemistry images of human liver sections from normal control subjects, MASLD patients, and MASH patients. Scale bar: 100 µm. Average optical density was quantitatively compared. (n = 6). f) Linear regression analysis of CALM1 mRNA levels and NAS in normal control subjects and MASLD patients. (n = 10). g) Schematic illustration of genetic causal association between CALM1‐3 and the risk of MASLD. The expression quantitative trait loci (eQTL) of CALM1‐3 were used as exposures and the summary GWAS data from 6623 MASLD patients and 26318 controls were employed as outcome. h) Genetic causal associations of CALM1, CALM2, and CALM3 with human MASLD in summary data‐based Mendelian randomization analysis. SNP, single nucleotide polymorphisms; OR, odds ratio; CI, confidence interval; HEIDI, heterogeneity in the dependent instrument. i) Model depicting the role of alnustone in the treatment of MASLD: Alnustone increases cytosolic and mitochondrial Ca²⁺ concentration via binding to calmodulin, which in turn promotes mitochondrial fatty acid β‐oxidation, thereby contributing to lower hepatic triacylglycerol and ameliorating MASLD/MASH. The upward arrow denotes protein activated by binding to alnustone or augmentation of the corresponding process. Image was created by Figdraw. TCA: tricarboxylic acid cycle. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; significance is assessed by two‐tailed unpaired Student's t test and one‐way ANOVA with Tukey's multiple comparisons post‐hoc test.