Hepatic Energy Metabolism Underlying Differential Lipidomic Responses to High-Carbohydrate and High-Fat Diets in Male Wistar Rats

Background Low-carbohydrate diets are suggested to exert metabolic benefits by reducing circulating triacylglycerol (TG) concentrations, possibly by enhancing mitochondrial activity. Objective We aimed to elucidate mechanisms by which dietary carbohydrate and fat differentially affect hepatic and circulating TG, and how these mechanisms relate to fatty acid composition. Methods Six-week-old, ∼300 g male Wistar rats were fed a high-carbohydrate, low-fat [HC; 61.3% of energy (E%) carbohydrate] or a low-carbohydrate, high-fat (HF; 63.5 E% fat) diet for 4 wk. Parameters of lipid metabolism and mitochondrial function were measured in plasma and liver, with fatty acid composition (GC), high-energy phosphates (HPLC), carnitine metabolites (HPLC-MS/MS), and hepatic gene expression (qPCR) as main outcomes. Results In HC-fed rats, plasma TG was double and hepatic TG 27% of that in HF-fed rats. The proportion of oleic acid (18:1n–9) was 60% higher after HF vs. HC feeding while the proportion of palmitoleic acid (16:1n–7) and vaccenic acid (18:1n–7), and estimated activities of stearoyl-CoA desaturase, SCD-16 (16:1n–7/16:0), and de novo lipogenesis (16:0/18:2n–6) were 1.5–7.5-fold in HC vs. HF-fed rats. Accordingly, hepatic expression of fatty acid synthase (Fasn) and acetyl-CoA carboxylase (Acaca/Acc) was strongly upregulated after HC feeding, accompanied with 8-fold higher FAS activity and doubled ACC activity. There were no differences in expression of liver-specific biomarkers of mitochondrial biogenesis and activity (Cytc, Tfam, Cpt1, Cpt2, Ucp2, Hmgcs2); concentrations of ATP, AMP, and energy charge; plasma carnitine/acylcarnitine metabolites; or peroxisomal fatty acid oxidation. Conclusions In male Wistar rats, dietary carbohydrate was converted into specific fatty acids via hepatic lipogenesis, contributing to higher plasma TG and total fatty acids compared with high-fat feeding. In contrast, the high-fat, low-carbohydrate feeding increased hepatic fatty acid content, without affecting hepatic mitochondrial fatty acid oxidation.publishedVersio

A fatty acid analogue targeting mitochondria exerts a plasma triacylglycerol lowering effect in rats with impaired carnitine biosynthesis.

L-carnitine is important for the catabolism of long-chain fatty acids in the mitochondria. We investigated how the triacylglycerol (TAG)-lowering drug 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA) influenced lipid metabolism in carnitine-depleted, 3-(2,2,2-trimethylhydrazinium)propionate dehydrate (Mildronate; meldonium)-treated male Wistar rats. As indicated, carnitine biosynthesis was impaired by Mildronate. However, TAG levels of both plasma and liver were decreased by 1-triple TTA in Mildronate-treated animals. This was accompanied by increased gene expression of proteins involved in mitochondrial activity and proliferation and reduced mRNA levels of Dgat2, ApoB and ApoCIII in liver. The hepatic energy state was reduced in the group of Mildronate and 1-triple TTA as reflected by increased AMP/ATP ratio, reduced energy charge and induced gene expression of uncoupling proteins 2 and 3. The increase in mitochondrial fatty acid oxidation was observed despite low plasma carnitine levels, and was linked to strongly induced gene expression of carnitine acetyltransferase, translocase and carnitine transporter, suggesting an efficient carnitine turnover. The present data suggest that the plasma TAG-lowering effect of 1-triple TTA in Mildronate-treated rats is not only due to increased mitochondrial fatty acid oxidation reflected by increased mitochondrial biogenesis, but also to changes in plasma clearance and reduced TAG biosynthesis

Hepatic gene expression in Wistar male rats after three weeks of treatment.

<p>Hepatic gene expression in Wistar male rats after three weeks of treatment.</p

Hepatic β-oxidation and enzyme activities involved in lipid catabolism in male Wistar rats after three weeks of treatment.

<p>(A) Total β-oxidation of palmitoyl-CoA in liver; (B) Total β-oxidation of palmitoyl-CoA with addition of malonyl-CoA in liver; (C) Enzyme activity of acyl-CoA synthetase; (D) Enzyme activity of carnitine palmitoyltransferase (CPT) 2; (E) Enzyme activity of 3-ketothiolase; (F) Enzyme activity of malonyl-CoA decarboxylase (MCD); (G) Enzyme activity of acyl-CoA oxidase (ACOX); (H) Enzyme activity of citrate synthase. Values are shown as means ± SD (n = 6–8). One-Way ANOVA with p<0.05 was followed by Fisher LSD to determine significant difference between all three groups: Different letters above bars indicate significant difference between group mean values, p<0.05; same letters above bars indicate no significant difference between group mean values p>0.05. C–Control, M–Mildronate (550 mg/kg body weight), MT–combination of Mildronate (550 mg/kg body weight) and 1-triple TTA (100 mg/ kg body weight).</p

Hepatic energy parameters in male Wistar rats after three weeks of treatment.

<p>(A) Energy charge (ATP + 0.5 ADP)/(AMP + ADP + ATP). (B) Ratio of AMP and ATP. Values are shown as mean ± SD (n = 6–8). One-Way ANOVA with p<0.05 was followed by Fisher LSD to determine significant difference between all three groups: Different letters above bars indicate significant difference between group mean values, p<0.05; same letters above bars indicate no significant difference between group mean values p>0.05. C–Control, M–Mildronate (550 mg/kg body weight), MT–combination of Mildronate (550 mg/kg body weight) and 1-triple TTA (100 mg/ kg body weight).</p

Hepatic enzyme activities involved in lipid anabolism in male Wistar rats after three weeks of treatment.

<p>(A) Enzyme activity of acetyl-CoA carboxylase (ACC). (B) Enzyme activity of fatty acid synthase (FAS). (C) Enzyme activity of citrate-ATP lyase. (D) Enzyme activity of glycerol-3-phosphate transferase (GPAT). Values are shown as mean± SD (n = 6–8). One-Way ANOVA with p<0.05 was followed by Fisher LSD to determine significant difference between all three groups: Different letters above bars indicate significant difference between group mean values, p<0.05; same letters above bars indicate no significant difference between group mean values p>0.05. C–Control, M–Mildronate (550 mg/kg body weight), MT–combination of Mildronate (550 mg/kg body weight) and 1-triple TTA (100 mg/ kg body weight).</p

Plasma levels of carnitine derivatives and carnitine precursors in male Wistar rats after three weeks of treatment.

<p><b>A.</b> Plasma L-carnitine; <b>B.</b> Plasma acetylcarnitine; <b>C.</b> Plasma γ-butyrobetaine; <b>D.</b> Plasma trimethyllysine; <b>E.</b> Protein expression of carnitine translocase (CACT). Values are shown as mean ± SD (n = 6–8). One-Way ANOVA with p<0.05 was followed by Fisher LSD to determine significant difference between all three groups. Different letters above bars indicate significant difference between group mean values, p<0.05; same letters above bars indicate no significant difference between group mean values p>0.05. C–Control, M–Mildronate (550 mg/kg body weight), MT–combination of Mildronate (550 mg/kg body weight) and 1-triple TTA (100 mg/ kg body weight).</p

Plasma and liver lipids in male Wistar rats after three weeks of treatment.

<p>Plasma and liver lipids in male Wistar rats after three weeks of treatment.</p

Hepatic Energy Metabolism Underlying Differential Lipidomic Responses to High-Carbohydrate and High-Fat Diets in Male Wistar Rats

Background Low-carbohydrate diets are suggested to exert metabolic benefits by reducing circulating triacylglycerol (TG) concentrations, possibly by enhancing mitochondrial activity. Objective We aimed to elucidate mechanisms by which dietary carbohydrate and fat differentially affect hepatic and circulating TG, and how these mechanisms relate to fatty acid composition. Methods Six-week-old, ∼300 g male Wistar rats were fed a high-carbohydrate, low-fat [HC; 61.3% of energy (E%) carbohydrate] or a low-carbohydrate, high-fat (HF; 63.5 E% fat) diet for 4 wk. Parameters of lipid metabolism and mitochondrial function were measured in plasma and liver, with fatty acid composition (GC), high-energy phosphates (HPLC), carnitine metabolites (HPLC-MS/MS), and hepatic gene expression (qPCR) as main outcomes. Results In HC-fed rats, plasma TG was double and hepatic TG 27% of that in HF-fed rats. The proportion of oleic acid (18:1n–9) was 60% higher after HF vs. HC feeding while the proportion of palmitoleic acid (16:1n–7) and vaccenic acid (18:1n–7), and estimated activities of stearoyl-CoA desaturase, SCD-16 (16:1n–7/16:0), and de novo lipogenesis (16:0/18:2n–6) were 1.5–7.5-fold in HC vs. HF-fed rats. Accordingly, hepatic expression of fatty acid synthase (Fasn) and acetyl-CoA carboxylase (Acaca/Acc) was strongly upregulated after HC feeding, accompanied with 8-fold higher FAS activity and doubled ACC activity. There were no differences in expression of liver-specific biomarkers of mitochondrial biogenesis and activity (Cytc, Tfam, Cpt1, Cpt2, Ucp2, Hmgcs2); concentrations of ATP, AMP, and energy charge; plasma carnitine/acylcarnitine metabolites; or peroxisomal fatty acid oxidation. Conclusions In male Wistar rats, dietary carbohydrate was converted into specific fatty acids via hepatic lipogenesis, contributing to higher plasma TG and total fatty acids compared with high-fat feeding. In contrast, the high-fat, low-carbohydrate feeding increased hepatic fatty acid content, without affecting hepatic mitochondrial fatty acid oxidation