Our data suggest that BBR inhibits glucose production in liver. The inhibition is related to suppression of PEPCK and G6Pase expression. This is a new mechanism for BBR action in the regulation of glucose metabolism. In literature, BBR is reported to enhance insulin sensitivity in adipocytes and muscle cells. Activation of the AMPK pathway is a mechanism of enhanced glucose uptake in skeletal muscle 
. However, the role of AMPK pathway is controversial for the metabolic activities of BBR. The AMPK activity was reported in muscle (L6) and hepatocytes (HepG2) 
, but the activity was not observed in 3T3-L1 adipocytes 
. In HepG2 cells, AMPK was reported to mediate BBR activity in the inhibition of cholesterol and triglyceride synthesis 
. However, AMPK was not tested in BBR action in the regulation of hepatic gluconeogenesis. In the current study, we addressed this issue by examining AMPK and gluconeogenesis in liver. We observed that BBR improved AMPK activity and inhibited gluconeogenic gene expression at the same time. In the diabetic rats, BBR treatment restored AMPK activity to the level of non-diabetic rats. The change in AMPK activity is associated with a reduction in PEPCK and G6Pase expression. AMPK activation is proposed as a result of mitochondria inhibition by BBR 
. Here, our data suggest that mitochondria inhibition is also a mechanism for suppression of hepatic gluconeogenesis and lipogenesis. The inhibition leads to suppression of gene expression and catalytic inactivation of related enzymes in liver through depletion of ATP. Recent literature supports that BBR increases glucagon-like peptide-1 (GLP-1) level in vivo and in vitro 
. This activity of BBR may contribute to AMPK activation by BBR as well.
We demonstrated that insulin signaling was not significantly modified by BBR in liver. BBR was reported to enhance glucose uptake through GLUT4 translocation in skeletal muscle 
. However, this mechanism is controversial as the GLUT4 activity was not confirmed in other studies 
. Our data suggest that even this AMPK-GLUT4 interaction is a mechanism for BBR action in vivo, its contribution to glucose metabolism in whole body is limited. Our data do not support the mechanism in vivo as the insulin sensitivity was not significantly improved by BBR in the diabetic rats. This conclusion is supported by a recent study, in which BBR was shown to cause muscle atrophy through induction of protein degradation 
. This report argues against the BBR activity in insulin sensitization in muscle since insulin is known to prevent muscle atrophy 
. In the current study, we examined the insulin signaling pathway in liver by determining Akt phosphorylation status. We did not observe a significant change in the insulin signaling pathway following the BBR treatment. The current study suggests that BBR may regulate glucose metabolism independently of insulin signaling pathway in liver.
Inhibition of mitochondria ATP production may contribute to the suppression of hepatic gluconeogenesis and hepatic steatosis. We observed that oxygen consumption was reduced and the AMP/ATP ratio was increased by BBR in hepatocytes. The data provide evidence for this new action of BBR in mitochondria suppression in hepatocytes. This observation is consistent with BBR activity in the purified mitochondria, in which BBR inhibited NAD-linked respiration in mitochondria 
. In hepatocytes, we observed that BBR enhanced lactate release 
, a sign of the mitochondria inhibition. Here, we demonstrated that oxygen consumption was reduced in hepatocytes by BBR, and this reduction was associated with an increase in AMP/ATP ratio. Though ATP production pathway is switched from mitochondrial oxidative phosphorylation to glycolysis in the presence of BBR, the glycolysis-generated ATP is not sufficient to maintain the intracellular ATP level in hepatocytes. The ATP depletion should account for inhibition of PEPCK and G6Pase expression through inactivation of transcription factors, whose activities are dependent on ATP.
The mitochondrial inhibition is a mechanism for inhibition of lipid accumulation in liver. We observed that BBR reduced hepatic steatosis in the diabetic rats. The mechanism is likely inhibition of lipogenesis in liver. BBR was shown to reduce plasma lipid and reduce plasma hepatic enzymes (ALT, AST, GGT) 
. This BBR activity was confirmed in the current study. In term of mechanism, BBR is reported to inhibit cholesterol synthesis and induce LDLR expression 
. In the current study, we observed that BBR inhibited fatty acid synthesis in liver. BBR inhibited FAS gene expression. Expression of lipogenic transcription factors (SREBP and ChREBP) was also reduced, which provides a mechanism for the FAS inhibition. Given that ATP is required for expression and function of the lipogenic transcription factors, the ATP depletion provides an answer to the lipogenic inhibition by BBR. The lipogenic inhibition may contribute to body weight loss in the BBR-treated rats observed in this study. BBR did not reduce food intake in our study. It has been reported that BBR does not affect expression of the appetite-regulating neuropeptides (POMC and NPY) in the hypothalamus in rodents 
. Therefore, the reduction in hepatic steatosis is not a consequence of alteration in calorie intake.
Pharmacokinetics of BBR is different among tissues 
. This character explains the long lasting AMPK activation that is observed in the current study. Recent studies suggest that: 1) BBR has a long half life in the liver. The BBR clearance rate is quite different among tissues, such as hippocampus VS plasma 
. According to a recent report, there is a 70-fold difference in the BBR clearance rate in liver VS plasma in rats 
. The studies suggest that BBR may have a longer half life in liver for the AMPK activation; 2) GLP-1 may mediate BBR's long-standing effect. BBR increases L cell activity in expression of GLP-1 in vivo 
. The improved L cell activity may remain in the absence of BBR for the elevated GLP-1 expression. If this is the case, GLP-1 will mediate BBR activity in the AMPK activation in liver after BBR withdrawn. In the future, we will test this possibility by examining GLP-1 level in our models.
In summary, we conclude here that BBR reduces fasting blood glucose, attenuates gluconeogenesis and hepatic steatosis in type 2 diabetic rats (). Suppression of hepatic gluconeogenesis provides an excellent mechanism for the reduction in blood glucose. Inhibition of PEPCK and G6Pase expression is associated with decreased expression of transcription factors (FoxO1, SREBP1 and ChREBP) in the liver. BBR suppresses mitochondria function in hepatocytes to reduce ATP level. ATP depletion is a potential mechanism for BBR action in the inhibition of gluconeogenesis and lipogenesis in liver. The study suggests that inhibition of mitochondrial function in liver is responsible for the metabolic activities of BBR.
Schematic model of BBR signaling pathway.