Lipoic acid improves hypertriglyceridemia by stimulating triacylglycerol clearance and downregulating liver triacylglycerol secretion

Abstract

Elevated blood triacylglycerol (TG) is a significant contributing factor to the current epidemic of obesity-related health disorders, including type-2 diabetes, nonalcoholic fatty liver disease, and cardiovascular disease. The observation that mice lacking the enzyme sn-glycerol-3-phosphate acyltransferase are protected from insulin resistance suggests the possibility that the regulation of TG synthesis be a target for therapy. Five-week-old Zucker Diabetic Fatty (ZDF) rats were fed a diet containing (R)-alpha-lipoic acid (LA, approximately 200mg/kg body weight per day) for 5 weeks. LA offset the rise in blood and liver TG by inhibiting liver lipogenic gene expression (e.g. sn-glycerol-3-phosphate acyltransferase-1 and diacylglycerol O-acyltransferase-2), lowering hepatic TG secretion, and stimulating clearance of TG-rich lipoproteins. LA-induced TG lowering was not due to the anorectic properties of LA, as pair-fed rats developed hypertriglyceridemia. Livers from LA-treated rats exhibited elevated glycogen content, suggesting dietary carbohydrates were stored as glycogen rather than becoming lipogenic substrate. Although AMP-activated protein kinase (AMPK) reportedly mediates the metabolic effects of LA in rodents, no change in AMPK activity was observed, suggesting LA acted independently of this kinase. The hepatic expression of peroxisome proliferator activated receptor alpha (PPARalpha) target genes involved in fatty acid beta-oxidation was either unchanged or decreased with LA, indicating a different mode of action than for fibrate drugs. Given its strong safety record, LA may have potential clinical applications for the treatment or prevention of hypertriglyceridemia and diabetic dyslipidemia.

Fig. 1

Hepatic triglyceride synthesis. Mitochondrial and microsomal sn-glycerol-3-phosphate acyltransferases (mtGPAT and msGPAT, respectively) catalyze the acylation of sn-glycerol-3-phosphate (glycerol-3-P) with acyl-coenzyme A (acyl-CoA) generating lysophosphatidic acid (LPA), which is the rate-controlling step in de novo triglyceride (TG) synthesis. Subsequent reactions are catalyzed by LPA acyltransferase (LPAAT) generating phosphatidic acid (PA), PA phosphatase (PAP) and diacylglycerol O-acyltransferase (DGAT) generating diacylglycerol (DAG) and finally TG. Newly synthesized TG is either deposited in intracellular lipid vacuoles or exported in very low-density lipoprotein (VLDL) particles. Triglyceride hydrolase (TGH), a glycoprotein distributed to the endoplasmic reticulum particularly in regions surrounding mitochondria helps mobilize TG stored in lipid droplets prior to incorporation in VLDL particles by catalyzing the initial lipolysis followed by re-esterification by DGAT.

Fig. 2

Lipoic acid improves hypertriglyceridemia in ZDF rats. (A) Appearance of 2-h fasted blood plasma from 10-week old ZDF rats fed ± LA for 5 weeks. Blood plasma of ZDF rats pair fed the control diet was as lipemic as the plasma of rats fed the control diet ad libitum (group `noLA') (data not shown). Time-courses of blood plasma TG (B), HDL-cholesterol (C), and total cholesterol (D) in 2-h fasted ZDF rats fed ± LA and lean Zucker rats fed chow. Data are shown as the mean ± SEM for 5–6 ZDF rats/group and 4 lean Zucker rats. Lower case letters denote statistical analysis, where the Area Under the Curves (AUC) not sharing a common letter are significantly different (TG and HDL-cholesterol, P < 0.02). Error bars may lie within the symbols. (E) Overnight (16 h)-fasted blood plasma TG (VLDL-TG) in ZDF rats fed with LA or pair-fed the control diet for 5 weeks (n = 5 rats/group). Data represent the mean + SEM (*P < 0.02).

Fig. 3

Lipoic acid stimulates clearance of chylomicron-like particles. (A) TG content associated with chylomicrons (white column) or VLDL (black column) in 2-h fasted blood plasma of ZDF rats fed ± LA (n = 4–5 rats/group). Data represent the mean + SEM. Values not sharing a common letter within plasma fraction are significantly different (P < 0.05). (B) Intravenous lipid tolerance test carried out in 16-h fasted ZDF rats fed ± LA for 5 weeks. A bolus of Liposyn II 20% (0.8 ml/kg body weight) was injected at t = 0 min. Plasma chylomicron-like TG was calculated by subtracting non-chylomicron TG from whole plasma TG. Insert, the Area Under the Curve (AUC, corrected for baseline TG) is significantly different between LA-fed and pair-fed rats (−42%, *P < 0.007, n = 7 rats/group).

Fig. 4

Lipoic acid represses the hepatic expression of lipogenic genes and decreases in vivo hepatic secretion of VLDL-TG. (A) Liver mRNA levels of enzymes involved in TG synthesis; GPAT-1, DGAT-1, and DGAT-2, and de novo fatty acid synthesis; ACC-1, ACC-2, and FAS. Livers were obtained from 10-week old ZDF rats fed ± LA for 5 weeks. mRNA levels were quantified by real-time PCR as described under “Materials and methods” and expressed as % of control group `noLA' (mean + SEM for 5 rats/group). Values not sharing a common letter are significantly different (GPAT-1, DGAT-2, and ACC-1, P < 0.001; DGAT-1; P < 0.002; ACC-2, P < 0.02; FAS, P < 0.03). (B) Blood plasma VLDL-TG concentrations prior (t = −20 min) and following (t = 30, 50, 70, 90, 100, and 130 min) i.v. administration of Triton WR-1339 (n = 5 rats/group, mean ± SEM.). ZDF rats were purchased at 4 weeks of age, acclimated for a week, then fed LA or pair-fed the control diet for 5 weeks. Experiments debuted after an overnight (16 h) fast. Note that fasting tryglyceridemia (t = −20 min) was significantly decreased in LA-fed versus pair-fed rats (*P < 0.02) and remained significantly lower throughout the experiment. (C) Hepatic VLDL-TG secretion rate was decreased (−31%, *P < 0.03, n = 5 rats/group) in LA-fed rats versus pair-fed rats. To determine VLDL-TG secretion rate (μM/min), curves were fitted by linear least-squares regression using a coefficient of determination (r²) > 0.97. Only data collected within the first 90 min post Triton WR-1339 injection were included in the regression analysis.

Fig. 5

Effects of lipoic acid on the hepatic content and activity of ChREBP and SREBP-1c. (A) ChREBP and precursor SREBP-1c content in total liver extract were measured by immunoblotting as described under “Materials and methods”. (B) ChREBP and precursor SREBP-1c content in hepatic cytoplasmic fraction (immunoblotting). (C) ChREBP and mature SREBP-1c content in hepatic nuclear fraction (immunoblotting). (D) Transcriptional activity of ChREBP toward liver-type pyruvate kinase, and SREBP-1c toward its own gene. Gene expression was determined by real-time PCR as described under “Materials and methods” and expressed as % of control group `noLA'. Data are shown as the mean + SEM for 5 rats/group (real-time PCR) or 4 rats/group (immunoblotting). β-Actin was used as a loading control of liver and cytoplasmic proteins, and histone H3 as a loading control of nuclear proteins. Values not sharing a common letter are significantly different (P < 0.05).

Fig. 6

Lipoic acid does not stimulate liver or skeletal muscle AMPK. Immunoblots and densitometry depicting the liver (A) and soleus muscle (B) contents of phosphorylated AMPKα (pAMPKα, Thr 172), total AMPKα (sum of AMPKα1 and AMPKα2), phosphorylated ACC (pACC, Ser 79), and total ACC (sum of ACC-1 and ACC-2). Uncoupling protein 3 (UCP3) content was determined as a mitochondrial protein loading control. β-Actin was used as an overall loading control of liver proteins, and α-tubulin as an overall loading control of muscle proteins. Data represent the mean + SEM for 4 rats/group. Values not sharing a common letter are significantly different (P < 0.05).

Fig. 7

Effects of lipoic acid on the mRNA levels of PPARα and PPARα target genes in liver and skeletal muscle. Gene expression was determined by real-time PCR as described under “Materials and methods” and expressed as % of control group `noLA'. (A) Liver mRNA levels of PPARα, CPT1α, ACO-1, and BIFEZ. Values not sharing of common letter are significantly different (PPARα and CPT1α, P < 0.001; ACO-1, P < 0.003; BIFEZ, P ≤ 0.01). (B) Vastus lateralis mRNA levels of PPARα, CPT1β, ACO-1, and BIFEZ. Values not sharing of common letter are significantly different (ACO-1, P < 0.05). Liver and muscle data represent the mean + SEM for 5 rats/group.