Figure 1.
Biglycan expression in adipose tissue.
Biglycan mRNA expression in adipose tissue of C57BL6/J wild-type mice fed either LFD or HFD (A). Biglycan expression is elevated in all the adipose depots of HFD mice; however, only mesenteric, brown, and epididymal adipose depots had a significantly higher level. Student’s t-test were performed between LFD and HFD samples (*p<0.05). Sample sizes: brown fat (n = 6), mesenteric adipose (n = 4), epididymal adipose (n = 8), retroperitoneal adipose (n = 8), subcutaneous adipose (n = 6), liver (n = 6), gastrocnemius muscle (n = 4). B) Biglycan staining in EWAT sections from bgn+/0 and bgn−/0 mice. Magnification 200x, green: biglycan staining, blue: DAPI stain. C) Western blot of biglycan core protein in EWAT. Each lane represents a separate sample. 30 µg of total protein were loaded per lane. D) Expression of biglycan mRNA in primary adipocytes and primary SVC cells from from EWAT of HFD fed wild type mice. PPARγ2 (adipocyte-specific) and CD68 (macrophage-specific) expression levels are used as cell fraction controls. Biglycan expression was not significantly different between cell types (n = 3). For all graphs in figure 1, results are expressed as mean±SE.
Table 1.
Mouse characteristics.
Figure 2.
Adiponectin expression in bgn−/0 mice.
A) Adiponectin mRNA levels in EWAT of bgn+/0 and bgn−/0 mice on LFD and HFD (n = 9–12). *p<0.05, bgn−/0 vs. bgn+/0. B) Western blot analysis of protein levels of adiponectin in EWAT in bgn+/0 and bgn−/0 mice fed a high fat diet. Adiponectin levels are normalized to β-actin. Each lane represents one mouse. C) Serum adiponectin levels measured by ELISA (n = 9–12 mice per treatment group), *p<0.05, bgn−/0 vs. bgn+/0. For all graphs in figure 2, results are expressed as mean±SE.
Figure 3.
Fasting blood was collected from mice that were fasted for 6 hrs. A) Basal insulin and B) basal glucose of bgn+/0 and bgn−/0 mice (n = 9–12) *p<0.05 bgn−/0 vs. bgn+/0. C) HOMA-IR values calculated from basal insulin and basal glucose. Results are expressed as mean±SE.
Figure 4.
Knockdown of biglycan in 3T3-L1 adipocytes.
A) Biglycan expression from 3T3-L1 mature adipocytes treated with siRNA against biglycan (“si biglycan”) or nontargeting siRNA (“scrambled”). Results are from RT-PCR and western blot for core biglycan protein (representative blot). (n = 3 replicates) B) Adiponectin mRNA and secreted measurements from siRNA treated 3T3-L1 adipocytes. (n = 3 replicates) C) PPARγ and FAS expression measured by RT-PCR. (n = 3 replicates) D) Concentration of LDH in the medium of siRNA treated 3T3-L1 adipocytes. For all graphs in figure 4, results are expressed as mean±SE, *p<0.05 target vs. scrambled.
Figure 5.
Biglycan treated RAW264.7 macrophages and 3T3-L1 adipocytes treated with macrophage conditioned medium (MCM).
A) Diagram of treatment scheme. RAW 264.7 macrophages were treated with combinations of treatment media (control), biglycan, or LPS. Some treatment groups were primed with LPS for two hours. Macrophage conditioned medium (MCM) from the treatments was used to treat 3T3-L1 adipocytes. B) Adiponectin mRNA in 3T3-L1 adipocytes treated with MCM. Eight days after differentiation, 3T3-L1 adipocytes were treated with MCM from RAW 264.7 macrophages for 24 hours. (n = 3 replicates) Statistically different means were determined by Tukey means separation after ANOVA and are denoted by different letters above bars in the graph. C) Adiponectin mRNA from 3T3-L1 adipocytes (8 days post-differentiation) after treatment with biglycan for 24 hours. (n = 3 replicates) n.s. = not significant D) mRNA levels of TNFα, IL-6 and IL-1β from macrophages treated with combinations of LPS and biglycan (outlined in figure 5A). All genes measured in figure 5D are expressed relative to 18 S. (n = 3 replicates) Statistically different means were determined by Tukey means separation after ANOVA and are denoted by different letters above bars in the graph. For all graphs in figure 5, results are expressed as mean±SE.