A high-fat diet supplemented with medium-chain triglycerides ameliorates hepatic steatosis by reducing ceramide and diacylglycerol accumulation in mice

Abstract

Non-alcoholic fatty liver disease (NAFLD) is projected to be the most common chronic liver disease worldwide and is closely linked to obesity, insulin resistance and type 2 diabetes. Currently, no pharmacological treatments are available to treat NAFLD, and lifestyle modification, including dietary interventions, is the only remedy. Therefore, we conducted a study to determine whether supplementation with medium-chain triglycerides (MCTs), containing a mixture of C8 and C10 (60/40), attenuates NAFLD in obese and insulin-resistant mice. To achieve that, we fed C57BL/6 male mice a high-fat diet (HFD) for 12 weeks to induce obesity and hepatic steatosis, after which obese mice were assigned randomly either to remain on the HFD or to transition to an HFD supplemented with MCTs (HFD + MCTs) or a low-fat diet (LFD) for 6 weeks as another dietary intervention model. Another group of mice was kept on an LFD throughout the study and used as a lean control group. Obese mice that transitioned to HFD + MCTs exhibited improvement in glucose and insulin tolerance tests, and the latter improvement was independent of changes in adiposity when compared with HFD-fed mice. Additionally, supplementation with MCTs significantly reduced hepatic steatosis, improved liver enzymes and decreased hepatic expression of inflammation-related genes to levels similar to those observed in obese mice transitioned to an LFD. Importantly, HFD + MCTs markedly lowered hepatic ceramide and diacylglycerol content and prevented protein kinase C-ε translocation to the plasma membrane. Our study demonstrated that supplementation with MCTs formulated mainly from C8 and C10 effectively ameliorated NAFLD in obese mice.

Keywords: ceramide; diacylglycerol; hepatic steatosis; inflammation; medium-chain triglycerides.

FIGURE 1

High‐fat diet (HFD) supplemented with medium‐chain triglycerides (MCTs) improves glucose tolerance and insulin sensitivity. (a) Study design. (b–d) Body weight, inguinal white adipose tissue (iWAT) weight and random blood glucose during the 6 week dietary intervention. (e) Glucose tolerance test (GTT) and area under the curve (AUC). (f) Plasma insulin levels during GTT. (g) Insulin tolerance test (ITT). (h) Pyruvate tolerance test (PTT) and AUC. In scatter plots with a bar, one circle represents data from one mouse, and n = 6–8 mice per group. Values are presented as means ± SD. Statistical significance was determined using one‐ or two‐way ANOVA. ^* P < 0.05, HFD + MCTs group versus HFD group. ^† P < 0.05, HFD‐to‐LFD group versus HFD group.

FIGURE 2

High‐fat diet (HFD) supplemented with medium‐chain triglycerides (MCTs) decreases hepatic steatosis and inflammation. (a, b) Representative images showing Haematoxylin and Eosin (H&E)‐stained liver sections along with histological liver fat quantification and non‐alcoholic fatty liver disease score (NAS). Scale bars: 50 μm. (c) Liver weights. (d, e) Liver triglyceride (TG) and cholesterol content. (f–h) Plasma levels of TG, cholesterol and non‐esterified fatty acids (NEFAs). (i, j) Serum alanine transaminase (ALT) and aspartate transaminase (AST) levels. (k) Hepatic expression of mRNAs encoded by inflammatory‐related genes. In scatter plots with a bar, one circle represents data from one mouse, and n = 6–8 mice per group. Values are presented as means ± SD. Statistical significance was determined using one‐way ANOVA. ^* P < 0.05, HFD + MCTs group versus HFD group. ^† P < 0.05, HFD‐to‐LFD group versus HFD group. Abbreviations: Ccl2, C‐C motif chemokine ligand 2; Ccl3, C‐C motif chemokine ligand 3; Ccl5, C‐C motif chemokine ligand 5; Il1β, interleukin‐1 beta; Tnfα, tumor necrosis factor alpha.

FIGURE 3

High‐fat diet (HFD) supplemented with medium‐chain triglycerides (MCTs) reduces hepatic diacylglycerol and ceramide content. (a, b) Liver ceramide and diacylglycerol (DAG) content. (c) Representative Western blot bands along with quantification (bottom) showing protein kinase C‐ε (PKCε) protein expression level in membrane (Memb) normalized to Na⁺,K⁺‐ATPase intensity, cytoplasmic (Cyto) normalized to heat shock protein 90 (HSP90) intensity, and whole liver protein kinase C‐ζ (PKCζ) protein expression normalized to GAPDH. (d) Schematic representation of de novo ceramide synthesis pathway (light yellow box), sphingomyelin hydrolysis pathway (light blue box) and salvage pathway (light pink box). This schematic diagram was created using BioRender. (e) Hepatic expression of mRNAs encoded by de novo ceramide synthesis‐related genes. (f) Hepatic expression of mRNAs encoded by sphingomyelin hydrolysis‐related genes. (g) Hepatic expression of mRNAs encoded by salvage pathway‐related genes. In scatter plots with a bar, one circle represents data from one mouse, and n = 6–8 mice per group. Values are presented as means ± SD. Statistical significance was determined using one‐way ANOVA. ^* P < 0.05, HFD + MCTs group versus HFD group. ^† P < 0.05, HFD‐to‐LFD group versus HFD group. Abbreviations: Asah1–3, acid ceramidase 1–3; Cers1–6, ceramide synthase 1–6; Degs1 and 2, dihydroceramide desaturase 1 and 2; Sgms1 and 2, sphingomyelin synthase 1 and 2; Smpd1–3, sphingomyelin phosphodiesterase 1–3; Sptlc1 and 2, serine palmitoyltransferase long chain base subunit 1 and 2.

FIGURE 4

High‐fat diet (HFD) supplemented with medium‐chain triglycerides (MCTs) does not increase hepatic fatty acid oxidation and ketogenesis. (a) β‐Hydroxybutyrate (βOHB) levels during the 6 week dietary intervention. (b) β‐Hydroxybutyrate levels in fed and fasting states. (c, d) Hepatic expression of mRNAs encoded by fatty acid oxidation and ketogenesis‐related genes. (e) Whole‐body oxygen consumption (VO₂), carbon dioxide production (VCO₂), respiratory exchange ratio (RER) and energy expenditure (EE) values collected during 48 h. (f, g) Average food consumption and spontaneous activity during 48 h. In scatter plots with a bar, one circle represents data from one mouse, and n = 6–8 mice per group. Values are presented as means ± SD. Statistical significance was determined using one‐ or two‐way ANOVA. ^* P < 0.05, HFD + MCTs group versus HFD group. ^† P < 0.05, HFD‐to‐LFD group versus HFD group. Abbreviations: Acaa2, acetyl‐CoA acyltransferase 2; Acadl, acyl‐CoA dehydrogenase long chain; Acadm, acyl‐CoA dehydrogenase medium chain; Acads, acyl‐CoA dehydrogenase short chain; Acat1, acetyl‐CoA acetyltransferase 1; Bdh1, 3‐hydroxybutyrate dehydrogenase 1; Bdh2, 3‐hydroxybutyrate dehydrogenase 2; Crot, carnitine O‐octanoyltransferase; Hadh, hydroxyacyl‐CoA dehydrogenase; Hmgcs2, 3‐hydroxy‐3‐methylglutaryl‐CoA synthase 2; Hmgcl, 3‐hydroxymethyl‐3‐methylglutaryl‐CoA lyase.

FIGURE 5

Model depicting the mechanism by which supplementation with medium‐chain triglycerides (MCTs) mitigates hepatic steatosis in obese mice. A high‐fat diet (HFD) supplemented with MCTs, primarily composed of C8 and C10: (1) decreases intrahepatic triglyceride accumulation; (2) lowers hepatic ceramide and diacylglycerol content, which prevents protein kinase C‐ε (PKCε) translocation; (3) reduces hepatic inflammation; and (4) enhances whole‐body glucose tolerance and insulin sensitivity independent of adiposity. This figure was created using BioRender.