Type 2 diabetes is a metabolic syndrome with diverse pathological manifestations and is often associated with abnormal glucose and lipid metabolism. Previously, we reported that the supplementation of PL that is rich in fiber and phenolic compounds suppressed the body weight gain and lowered plasma and hepatic lipid levels in rats fed a high-fat diet 
. Furthermore, several in vitro
studies showed the possible beneficial effect of PL on glucose regulation. For example, PL inhibited enzymes involved in the digestion of carbohydrates (α-amylase and α-glucosidase) and the regulation of insulin signaling (protein tyrosine phosphatase 1B) 
. However, no report has been published on the in vivo
anti-diabetic effects of PL in type 2 diabetes. In this study, we firstly demonstrated that dietary supplementation of PL for 5 weeks ameliorated hyperglycemia, dyslipidemia and hepatic steatosis in type 2 diabetic mice, at least in part, by regulation of hepatic glucose and lipid metabolism and antioxidant status.
mice are a widely used genetic model of type 2 diabetes since they exhibit most of the human characteristics of type 2 diabetes, including hyperglycemia, dyslipidemia and insulin resistance 
. A recent study suggests that defects in hepatic insulin signaling contribute to the development of diabetes in C57BL/KsJ-db/db
. The liver is a major insulin-sensitive organ responsible for maintaining glucose and lipid homeostasis. A failure of insulin to increase hepatic glucose utilization and to suppress hepatic endogenous glucose production is a major factor contributing to hyperglycemia in diabetes 
. The key enzyme responsible for the regulation of glucose utilization is GK that catalyzes glucose phosphorylation as the first step of storage of glucose as glycogen and glucose disposal by glycolysis 
. Conversely, PEPCK and G6Pase are rate-controlling enzymes of gluconeogenesis in the liver 
. We found that in the db/db
mice, the supplementation of PL significantly decreased the fasting blood glucose level and HOMA-IR index, which primarily reflects hepatic insulin resistance 
. These changes were accompanied by decreases in hepatic G6Pase and PEPCK activity and increases in hepatic GK activity along with glycogen content. Thus, these observations suggest that PL can promote hepatic insulin sensitivity and thus effectively regulate the activity of enzymes involved in hepatic glucose homeostasis, leading to lower blood glucose level in db/db
The regulation of GK activity is primarily due to changes in the transcription of its gene 
. We also found that the change in GK activity by PL was accompanied by its increased transcriptional level. In contrast, the gene expression of hepatic G6Pase and PEPCK was not affected. Similarly, a lack of regulation of G6Pase and PEPCK gene expression was reported following treatment with some phenolic compounds in rat hepatocytes, despite a significant reduction in glucose production and enhanced hepatic GK mRNA expression 
. In addition, it is known that the suppression of hepatic glucose production by metformin results from the inhibition of G6Pase activity along with an increase in glycogen stores with minimal effects on the gluconeogenic gene expression in the livers of rats 
. Thus, we think that PL may act mainly by suppressing the activity of hepatic gluconeogenic enzymes independent of the transcriptional repression of gluconeogenic genes. Since elevated GK expression led to a reduced endogenous glucose production in the liver 
, it is possible that the observed decrease in activity of gluconeogenic enzymes in PL-supplemented db/db
mice is related to the inhibition of the substrate flux through GK activation.
On the other hand, some studies have raised concerns about the manipulation of GK activator for diabetes treatment since a decline in glucose level in response to hepatic GK overexpression is accompanied by an increase in circulating lipids and hepatic lipogenesis 
. However, several lines of evidence suggest that hepatic GK activation does not alter plasma and hepatic lipid metabolism in normal and high-fat fed animals 
. The present results also showed that PL induced a marked decrease in triglyceride and cholesterol accumulation in the liver, together with the inhibition of activity of hepatic lipogenic enzymes involved in the synthesis and esterification of fatty acid (FAS, PAP) or cholesterol (HMGR, ACAT), which may subsequently reduce the formation of lipid droplets within hepatocytes and the secretion of triglycerides and cholesterol into the blood. Simultaneously, this effect could be related to the down-regulated expression of several lipogenic genes (ACL, SCD1, PAP, DGAT) as well as key transcription factor (PPARγ) in the liver. Normally, PPARγ is expressed at very low levels in the liver, but its expression is dramatically increased in animal model with insulin resistance and hepatic steatosis such as db/db
. The genetic deletion of hepatic PPARγ protected against hepatic steatosis in high fat diet-induced obese mice 
. We also found that PL appeared to facilitate fecal excretion of triglycerides as well as cholesterol in the db/db
mice, in accordance with our previous data on high-fat fed rats 
. Accordingly, PL seemed to lower plasma and hepatic lipid accumulation by decreasing hepatic lipogenesis and increasing fecal lipids. Since inhibition of FAS induces an increase in hepatic malonyl-CoA which is a potent inhibitor of CPT 
, a key enzyme involved in mitochondrial fatty acids uptake for oxidation 
, the reduced activity of hepatic fatty acid oxidation and CPT could be a secondary consequence of the decrease in hepatic FAS activity. Also, the reduced fatty acid oxidation might be associated with activated glucose utilization and a reduction in glucose production in the liver 
In addition to its role in regulating lipogenesis, it is known that PPARγ is required for transcription of the PEPCK gene in adipocytes 
. PPARγ agonists such as pioglitazone and rosiglitazone were potent inducer of PEPCK gene transcription and enzymatic activity in adipose tissue of obese Zucker rats 
. Moreover, hepatocyte specific PPARγ-knockout mice showed reduced serum glucose level and PEPCK mRNA expression 
. However, liver-specific disruption of PPARγ in leptin-deficient mice dramatically increased basal endogenous glucose production 
. Also, PPARγ agonist, troglitazone, inhibited the expression of PEPCK gene by a PPARγ-independent, antioxidant-related mechanism, and other PPARγ agonists, including rosiglitazone and ciglitazone, had little effect on PEPCK gene expression in hepatocyte 
, suggesting that the regulation of PEPCK by PPARγ is cell-specific 
. We also observed that PEPCK mRNA expression was not altered by PL supplementation, although the PL down-regulated hepatic PPARγ and its target lipogenic genes expression. In fact, the regulation of PEPCK gene transcription coordinated by the action of a number of transcriptional factors and various hormones, including insulin, glucocorticoids, retinoic acid, thyroid hormone, and cyclic AMP 
. Thus, it is possible that the PEPCK gene expression in PL-supplemented db/db
mice was controlled by cooperative interaction of multiple transcription factors and hormones involved in PEPCK gene regulation.
Oxidative stress has been implicated in the pathogenesis of diabetes and other metabolic syndrome, including fatty liver disease and cardiovascular disease. In particular, the mitochondria is a major source of reactive oxygen species (ROS), and the ROS generated from the mitochondria damages proteins, DNA, and lipids in the membrane components, which results in mitochondrial dysfunction 
. Under normal physiological conditions, ROS are continuously produced, and oxidative damage induced by ROS can be prevented by antioxidant enzymes, where superoxide anion is rapidly converted by SOD into hydrogen peroxide, which is eliminated by CAT and GPX. However, an imbalance between ROS production and antioxidant capacity can induce cell damage associated with diabetes. Increased ROS levels were observed in the liver of db/db
and the expression of the antioxidant gene was down-regulated in the liver of type 2 diabetic rats 
. In contrast, overexpression of SOD or CAT protected against hepatic oxidative injury in the livers of db/db
or HepG2 cells 
. We found that PL up-regulated the mRNA expression of SOD and CAT in the liver. In parallel with the enhanced antioxidant gene expression, hepatic SOD and CAT activity was increased in the PL-supplemented db/db
mice, suggesting that the elevated SOD and CAT expression may be regulated at the transcriptional level. Thus, the decreased level of mitochondria hydrogen peroxide in the liver of PL-supplemented mice may be attributed to the improved hepatic antioxidant capacity, which may contribute to the decreased hepatic lipid peroxidation and provide protection against hepatic oxidative stress in type 2 diabetes.
We also found that PL significantly increased the plasma PON activity as well as the plasma HDL-cholesterol and adiponectin levels. Plasma PON is another antioxidant enzyme contained in plasma HDL, which protects LDL and HDL from oxidation by ROS, and possesses other multiple anti-atherogenic activities 
. Serum PON activity is low in patients with diabetes and it has potential as a marker for atherosclerosis in diabetes 
. Along with this enzyme activity, HDL-cholesterol is an independent predictor of atherosclerotic cardiovascular complications. Furthermore, the adiponectin concentration is positively correlated with HDL cholesterol and negatively associated with HOMA-IR, independent of age and BMI, in type 2 diabetic subjects 
. Taken together, our findings suggest the potential protective effects of PL on atherosclerotic cardiovascular complications in type 2 diabetes.
In conclusion, our results show that dietary PL improved hyperglycemia by alterations in activity and/or mRNA expression of hepatic enzymes involved in glucose utilization and glucose production (). Furthermore, PL ameliorated dyslipidemia and hepatic steatosis through a combined decrease in hepatic lipogenesis and an increase in the excretion of fecal lipids, which seemed to be related to the enhanced responsiveness of the liver to insulin (). The beneficial metabolic effects were also related to decreased plasma and hepatic oxidative stress as well as increased adiponectin secretion (). Thus, we believe that PL is a promising anti-diabetic compound that will be helpful for improving type 2 diabetes, although further study is required to identify its active components that mediate the hypoglycemic, hypolipidemic and hepatoprotective effects of PL.
Proposed mechanism of PL on the glucose and lipid lowering action in C57BL/KsJ-db/db mice.