Table 1.
Characteristics of the experimental animals at the end of intervention study (n = 9).
Figure 1.
BBR decreased fasting blood glucose without improvement in insulin sensitivity.
(A) Effects of BBR treatment on OGTT in diabetic rats. Oral glucose tolerance test (OGTT) was conducted with glucose 2 g.kg−1 body wt after 5 week BBR treatment (380 mg.kg−1 day−1) (n = 6). (B) Insulin release during the OGTT (n = 6). (C) ITT test (n = 6). The test was conducted after 8 hour fasting with insulin (0.75 U.kg−1 body wt). Nor, normal rats without obesity; Diab+Veh: Diabetic rats treated with vehicle; Diab+BBR, diabetic rats treated with BBR. # P<0.05, compared with Diab+Veh. (D) Insulin signaling. Phosphorylation of Akt (Ser473). Rats were challenged with insulin (0.75 U.kg−1, intraperitoneal injection) and liver was collected in 30 minutes. The Akt assay was performed in a Western blot. Loading control is GAPDH. (E) Expression and phosphorylation of AMPK (Thr172). # P<0.05, compared with Diab+Veh group. * P<0.05, compared with normal group (Nor).
Figure 2.
Expression of gluconeogenic genes in liver.
(A) Protein levels of PEPCK and G6Pase. Total protein was made from liver tissue and used in a Western blot. (B) mRNA in fasting condition. Total RNA was extracted in from liver tissue and used in qRT-PCR. The signal was normalized with actin mRNA. (C) mRNA in fed condition. The mRNA data are presented as mean ± SEM (n = 6). # P<0.05, compared with Diab+Veh group. * P<0.05, compared with normal group (Nor).
Figure 3.
(A) FoxO1 protein in liver. Nuclear and cytoplasmic proteins were extracted from liver and analyzed in a Western blot. TBP (TATA-binding protein) and GAPDH proteins are loading controls in the nuclear and cytoplasmic proteins. (B) Immunohistostaining of FoxO1 protein in liver. DAB dye (brown) was used to indicate the FoxO1 protein signal; (C) mRNA expression of FoxO1. mRNA was quantified in real time RT-PCR (n = 6). # P<0.05, compared with Diab+Veh group; * P<0.05, compared with normal group (Nor).
Figure 4.
(A) Hematoxylin and eosin (H & E) staining. (B) Oil Red O staining. (C) Sudan III staining. Pictures were taken under a microscopy with ×20 object lenses.
Figure 5.
(A) Lipogenic transcription factor proteins. Liver total protein was used in the Western blot. SREBP1, SREBP2 and ChREBP were detected in the fasted liver with specific antibodies. Beta-actin is a loading control. (B) FAS protein. The protein was detected in a Western blot in the fasted and fed liver, respectively. (C) FAS mRNA. mRNA was detected by qRT-PCR and normalized with beta-actin mRNA (n = 6). # P<0.05, compared with Diab+Veh group; * P<0.05, compared with normal group (Nor).
Figure 6.
Inhibition of mitochondrial function by BBR in hepatocytes.
(A) BBR decreased oxygen consumption in H4IIE hepatocytes. The cells were plated in DMEM culture medium supplemented with 10% FBS. Oxygen consumption was determined for 12 hrs after BBR (20 µM) treatment. (B) AMP/ATP ratio in H4IIE cells. The cells were treated with BBR in serum-free medium for 16 hours. AMP and ATP levels were determined using HPLC. The data are presented as mean ± SEM (n = 5). * P<0.05.
Figure 7.
Schematic model of BBR signaling pathway.
BBR inhibits mitochondria function and decrease intracellular ATP. This leads to a reduction in gluconeogenic and lipogenic transcription factors (FoxO1, SREBP1, and ChREBP). As a result, expression of gluconeogenic genes (PEPCK and G6Pase) and lipogenic gene (FAS) are decreased. These molecular changes represent a signaling pathway for improvement of fasting glucose and liver steatosis in the BBR-treated diabetic rats.