The our major findings of the present study are as follows: 1) The administration of GLE had preventive effects against hepatic lipid accumulation and ameliorated hepatic insulin resistance; 2) The mechanism for this action was the increase of β-oxidation by increasing adiponectin receptors.
SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP/ZF rats) were established as a model of metabolic syndrome. Specifically, they showed alterations in lipogenesis, such as SCD-1, FAS and SREBP-1 in the liver. Abnormality of these genes caused the accumulation of lipid droplets in hepatocytes , and thus, they may be a good tool for the study of fatty liver. Using these model rats, we demonstrated that 6-week GLE treatment reduced body weight, liver weight and fat weight. The most interesting finding in our study was that GLE treatment ameliorated hepatic steatosis and decreased the accumulation of triglyceride (TG) in the liver of SHRSP/ZF rats. This was shown histologically and biochemically.
Alanine transaminase (ALT) is an important enzyme found predominately in the liver. Significantly elevated activity of ALT in serum often suggests its leakage from damaged hepatocytes and reflects hepatic damage. Aspartate aminotransferase (AST) is similar to ALT in that it is another enzyme associated with the liver . A slight rise of AST and the ALT can be appeared for the fatty liver because of cellular membrane defect from FFA, and they are used as diagnostic criteria of the fatty liver. The decrease of serum ALT and AST by GLE also accounts for the improvement of the liver histology and less fat infiltration of hepatocytes.
In visceral obesity, adipocytokines such as TNFα, resistin, IL-6 and FFA are secreted by enlarged fat cells of the internal organs as adipose tissue and induce insulin resistance [19–21]. As a result of insulin resistance, glucose and fat metabolism decrease with the muscle, and the release of fatty acid is promoted with adipose tissue. Glucose and fatty acid are then supplied to the liver excessively. Furthermore, the biosynthesis of fatty acid is aggravated in the liver, and fat over-accumulates in hepatocytes in conjunction with fatty acid supplied from adipose tissue. On the other hand, β-oxidation, which is the fatty acid metabolism system in hepatocytes, decreases. As a result, a fatty liver develops.
Adiponectin is one such adipocytokine secreted by adipose tissue that has recently attracted much attention, because it has lipid metabolism promotion effects and improves insulin resistance . In the liver, adiponectin stimulate glucose utilization and fatty acid combustion via AMPK. Adiponectin also increases the expression levels of PPARα and activates PPARα ligand, thereby stimulating the expression enzyme involved in fatty acid oxidation and decreasing tissue TG content . Expression of adiponectin is induced by the differentiation of fat cells. In non-obese small fat cells, there is a high expression of adiponectin; however, its transcription is inhibited by obesity when a fat cell is enlarged. In our first approach to find a possible mechanism, we focused on adiponectin. We predicted that secretion of adiponectin might increase in the GLE group compared with the control group in rat adipose tissue from the results of decreased body weight and fat weight with GLE treatment; however, the serum adiponectin level did not increase with GLE treatment (data not shown). According to a past report, obesity decreases not only plasma adiponectin but also adiponectin receptor expression due to hyperinsulinemia . We have already confirmed that SHRSP/ZF rat expression of adiponectin receptors is decreased compared with normal rats. In our study, GLE increased the expression of adiponectin receptor 1 and 2, with the activity of AMPK and mRNA expression of PPARα, which is a downstream adiponectin receptor.
Fatty acid β-oxidation takes place in two cellular organelles, mitochondria and peroxisomes . In liver cells, it is proposed that mitochondrial oxidation might have the largest impact on total β-oxidation . Activation of AMPK leads to a decrease in intracellular malonyl-CoA, which is a precursor of the biogenesis of fatty acid and a potent inhibitor of carnitine palmitoyltransferase (CPT) [27, 28]. CPT is a rate-limiting enzyme of mitochondrial β-oxidation and plays an important role in the transport of fatty acid to inside the mitochondria . On the other hand, PPARα controls the transcription of many genes involved in lipid catabolism, such as medium-chain acyl-CoA dehydrogenase (MCAD), acyl-CoA oxidase (ACO) and also CPT. In our study, it was demonstrated that CPT activity in the GLE group increased compared with the control. In addition, we evaluated mRNA expression; MCAD as a marker of enzyme mitochondrial β-oxidation, and ACO as a marker of point to control peroxisomal one . In the GLE group, both were higher than in the control group. Our results showed that GLE treatment increases energy expenditure by increasing fatty acid β-oxidation in liver cells. On the other hand, SREBP-1 and FAS mRNA levels, which are factors in lipogenesis and downstream AMPK, showed no difference between them (data not shown).
Dysregulation of adiponectin and the decrease its receptors play causal roles in the development of insulin resistance . A correlation has been reported that adiponectin receptor gene expression was decreased in type 2 diabetic patients. Moreover, a previous study demonstrated that AdipoR1/R2 knockout mice exhibited aggravated insulin resistance through increased TG content, inflammation and oxidative stress . On the other hand, adipocyte hypertrophy with the development of obesity, subsequently resulting in secretion abnormalities of FFA and adipocytokines such as TNF-α and IL-6, which are involved in insulin resistance . In this study, Akt activity in the liver in the GLE group was significantly higher than in the control group. Akt is an important factor in insulin signaling and in the control of gluconeogenesis. Moreover, the mRNA level of PEPCK, which is controlled by Akt activity and rate-limiting enzyme of gluconeogenesis, was also decreased compared with the control group. In our other group study, Guo et al. demonstrated that SHRSP/ZF rats treated with GLE had improvement glucose metabolism without enhanced insulin secretion. These results might suggest that GLE has an insulin resistance-improving effect by improving fatty liver and reduced adipose tissue due to a decreased adipocytokines.