Lipoic acid suppresses portal endotoxemia-induced steatohepatitis and pancreatic inflammation in rats

Abstract

Aim: To examine the effect of α-lipoic acid (LA) on mild portal endotoxemia-induced steatohepatitis and associated pancreatic abnormalities in fructose-fed rats.

Methods: Rats were randomly assigned into two groups with a regular or 60% fructose-enriched diet for 8 wk. After fructose feeding for 4 wk, rats were further divided into four subgroups: with intraportal saline (FPV), with intraportal saline plus administration of LA (FPV + LA), with lipopolysaccharide (LPS) infusion (FPLPS), and with LPS infusion plus administration of LA (FPLPS + LA). Rats were treated with LPS using intraportal infusion while LA was administered orally. Metabolite levels, superoxide levels, inflammatory markers, malondialdehyde content, glutathione content and toll-like receptor 4 (TLR4) gene expression were all measured using standard biochemical techniques. Pancreatic insulin secretion was evaluated by a hyperglycemic clamp technique. Histology of liver and pancreas tissues were evaluated using hematoxylin and eosin staining and immunohistochemistry.

Results: Fructose-induced elevation in plasma C-reactive protein, amylase, superoxide, white blood cell count as well as in hepatic and pancreatic contents of malondialdehyde, tumor necrosis factor alpha and interleukin-6 were increased in animals treated with LPS and reversed with LA administration. The augmented hepatic gene expression of TLR4 in fructose-fed rats was further increased in those with intraportal LPS infusion, which was partially reversed by LA administration. Pathological examination showed inflammatory changes and leukocyte infiltration in hepatic and pancreatic islets of animals treated with LPS but were rarely observed in those with LA treatment. In addition to affects on the liver, impaired pancreatic insulin secretion seen in fructose-fed rats was deteriorated in with LPS treatment and partially reversed with LA administration.

Conclusion: These data suggest LA could significantly suppress mild portal-endotoxemia but not fructose-induced liver and pancreatic abnormalities in a rodent model for metabolic syndrome.

Keywords: Fructose; Insulin secretion; Lipoic acid; Oxidative stress; Portal endotoxemia; Steatohepatitis.

Figure 1

Effects of α-lipoic acid on hepatic triglyceride contents. Hepatic triglyceride levels were measured from the blood of each group of fructose-fed rats and the control group. Values are expressed as mean ± SE, n = 6 per group, ^aP < 0.05 vs C_PV. C: Regular diet; F: High-fructose enriched diet; LA: Lipoic acid; LPS: Lipopolysaccharides.

Figure 2

Effect of α-lipoic acid on portal endotoxemia-induced changes in hepatic oxidative and inflammatory markers. The following parameters were measured from liver of each group of fructose-fed rats and the control group: (A) Malondialdehyde (MDA) content; (B) Glutathione (GSH) content; (C) Toll-like receptor 4 (TLR4) gene expression; (D) Tumor necrosis factor alpha (TNF-α) protein; and (E) Interleukin-6 (IL-6) protein. Values are expressed as mean ± SE, n = 6 per group. ^aP < 0.05 vs C_PV; ^cP < 0.05 vs F_PV; ^eP < 0.05 vs F_PLPS. C: Regular diet; F: High-fructose enriched diet; LA: Lipoic acid; LPS: Lipopolysaccharides.

Figure 3

Histopathological examination of liver in rats with regular or high-fructose feeding. Lipid accumulation in: (A) C_PV rats; (B) F_PV rats; (C)F_{PV + LA}; (D) F_PLPS; and (E) F_{PLPS + LA} rats. CD-68 positive cell infiltration in: (F) C_PV rats; (G) F_PV rats; (H) F_{PV + LA}; (I) F_PLPS; and (J) F_{PLPS + LA} rats. Slides were stained with hematoxylin and eosin (A-E) and immunostained with an anti-CD68 antibody (F-J). Arrows indicate CD68 positive cells. C: Regular diet; F: High-fructose enriched diet; LA: Lipoic acid; LPS: Lipopolysaccharides; CV: Central vein.

Figure 4

Effect of α-lipoic acid on portal endotoxemia-induced changes in the pancreas. The pancreata from experimental animals were harvested and tested for: (A) Malondialdehyde (MDA) content; (B) Tumor necrosis factor alpha (TNF-α) protein level; and (C) Interleukin-6 (IL-6) protein level. Values are expressed as mean ± SE, n = 6 per group. ^aP < 0.05 vs C_PV; ^cP < 0.05 vs F_PV; ^eP < 0.05 vs F_PLPS. C: Regular diet; F: High-fructose enriched diet; LA: Lipoic acid; LPS: Lipopolysaccharides.

Figure 5

Histopathologicalexamination of pancreatic islets. A: Tissue samples from each subset of animals were analyzed by hematoxylin and eosin (HE) staining (a-C_PV, b-F_PV, c-F_{PV + LA}, d-F_PLPS, e-F_{PLPS + LA}) and immunostaining with an antibody against CD68 (f-C_PV, g-F_PV, h-F_{PV + LA}, i-F_PLPS, j-F_{PLPS + LA}); This data was then quantitated as (B) percentage of islets infiltrated and (C) number of CD68+ cells per islet. Arrows indicated CD68-positive cells. I: Islet. A total of 100 ± 25 islets per experimental group were blindly scored for CD68-positive cells around the periphery and/or within islets from 5 to 6 different animals. Islet area was measured by the area of islet capsule in pancreatic section with HE stain and computed using AxioVision LE 4.8.2.0 software. Values are expressed as mean ± SE, n = 6 per group. Line bar: 50 μm. ^aP < 0.05 vs C_PV; ^cP < 0.05 vs F_PV; ^eP < 0.05 vs F_PLPS. C: Regular diet; F: High-fructose enriched diet; LA: Lipoic acid; LPS: Lipopolysaccharides.

Figure 6

Effect of α-lipoic acid on plasma glucose and insulin levels. A hyperglycemic clamp technique was used to evaluate glucose-stimulated insulin secretion. A: Plasma glucose levels during the clamp period; B: Change in insulin levels during clamp period; C: The insulin level averages during the first phase (0-10 min) and the second phase (10-240 min) of the clamp period. Values are expressed as mean ± SE, n = 6 per group. ^aP < 0.05 vs C_PV; ^cP < 0.05 vs F_PV; ^eP < 0.05 vs F_PLPS. C: Regular diet; F: High-fructose enriched diet; LA: Lipoic acid; LPS: Lipopolysaccharides.