Small leucine-rich proteoglycans and their role in adipose tissue

During obesity, adipose tissue expands in response to increased energy storage. One of the physiological changes that occur during adipose tissue expansion is extracellular matrix (ECM) remodeling. The contents of this dissertation describe three studies that examine the expression of ECM proteins in obese mice and the impact of small leucine-rich proteoglycans (SLRPs) on adipose tissue function. In the first study, the expression of ECM proteins was measured in the epididymal and subcutaneous adipose tissue of mice fed a low fat or high fat diet over time (4, 8, and 12 weeks of dietary intervention). The results of this study indicate that the gene expression pattern of some ECM proteins is adipose depot specific. In a second study, 3T3-L1 preadipocytes were plated on cell culture dishes coated with biglycan and decorin. Treatment with biglycan and decorin resulted in significantly decreased preadipocyte cell number. It was further demonstrated that biglycan and decorin increased apoptosis in 3T3-L1 preadipocytes. The third study examines the effect of biglycan absence on adipose tissue function using whole-body biglycan knockout mice. Biglycan knockout mice had decreased expression of epididymal adipose tissue IL-6 transcript along with increased circulating levels of adiponectin. However, siRNA knockdown of biglycan in 3T3-L1 adipocytes yielded lower levels of adiponectin. Differences in extracellular environments could explain the discrepancy in results observed in the in vivo and in vitro systems. In summary, the studies contained in this document indicate a possible role for SLRPs in adipose tissue formation, function and inflammation

Biglycan deletion alters adiponectin expression in murine adipose tissue and 3T3-L1 adipocytes.

Obesity promotes increased secretion of a number of inflammatory factors from adipose tissue. These factors include cytokines and very lately, extracellular matrix components (ECM). Biglycan, a small leucine rich proteoglycan ECM protein, is up-regulated in obesity and has recently been recognized as a pro-inflammatory molecule. However, it is unknown whether biglycan contributes to adipose tissue dysfunction. In the present study, we characterized biglycan expression in various adipose depots in wild-type mice fed a low fat diet (LFD) or obesity-inducing high fat diet (HFD). High fat feeding induced biglycan mRNA expression in multiple adipose depots. Adiponectin is an adipokine with anti-inflammatory and insulin sensitizing effects. Due to the importance of adiponectin, we examined the effect of biglycan on adiponectin expression. Comparison of adiponectin expression in biglycan knockout (bgn(-/0)) and wild-type (bgn(+/0)) reveals higher adiponectin mRNA and protein in epididymal white adipose tissue in bgn(-/0) mice, as well higher serum concentration of adiponectin, and lower serum insulin concentration. On the contrary, knockdown of biglycan in 3T3-L1 adipocytes led to decreased expression and secretion of adiponectin. Furthermore, treatment of 3T3-L1 adipocytes with conditioned medium from biglycan treated macrophages resulted in an increase in adiponectin mRNA expression. These data suggest a link between biglycan and adiponectin expression. However, the difference in the pattern of regulation between in vivo and in vitro settings reveals the complexity of this relationship

Adipose tissue biglycan as a potential anti-inflammatory target of sodium salicylate in mice fed a high fat diet

<p>Abstract</p> <p>Background</p> <p>Inflammation in adipose tissue (AT) during obesity causes impaired AT function. Although multiple extracellular matrix (ECM) proteins are expressed in AT their potential role in adipose tissue inflammation is unclear. Biglycan, a pro-inflammatory ECM gene, is highly enriched in adipose tissue. However, whether it is correlated with adipose tissue inflammation is unknown. We provide evidence in support of a strong association between biglycan expression and inflammatory status of adipose tissue.</p> <p>Methods</p> <p>C57BL6 mice were fed either a control (10% fat calories) or a high fat diet (HFD) (60% fat calories) for 8 weeks. Adipose tissue was analyzed for the expression of biglycan, IL-6 and TNFα. Biglycan knockout or wild type were also fed a high fat diet for 8 weeks and the expression of inflammatory genes in the mesenteric adipose tissue was examined. To test anti-inflammatory treatment on biglycan expression, a group of mice were fed either the low fat or high fat diet for eight weeks supplemented with either saline or sodium salicylate @ 25mg/100ml in their drinking water.</p> <p>Results</p> <p>Mice on HFD had an increase in ECM genes (BGN and COL1A1), inflammatory genes (IL-6 and TNFα) in both the subcutaneous and epididymal depots. However, correlation analysis only shows a positive correlation between biglycan, IL-6 and TNFα expression. In addition, lower expression of IL-6 and CD68 was found in the mesenteric adipose tissue of biglycan knockout mice compared to the wild type. Sodium salicylate treatment reduced subcutaneous adipose tissue expression of BGN, COL1A1, and COL6A1 and a concurrent downregulation of TNFα and IL-6 and TLR4 expression. Salicylate also lowered the serum TGFβ1 levels.</p> <p>Conclusion</p> <p>Biglycan expression correlates with adipose tissue inflammation, especially in the subcutaneous depot compared to the epididymal depot. This is supported by the greater effect of sodium salicylate in attenuating both inflammatory and ECM gene expression the subcutaneous adipose depot compared to the epididymal depot. These results show that inflammatory state may explain the induction of biglycan, and perhaps, other ECM genes in adipose tissue.</p

Knockdown of biglycan in 3T3-L1 adipocytes.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050554#pone-0050554-g004" target="_blank">figure 4</a>, results are expressed as mean±SE, *p<0.05 target vs. scrambled.</p

Biglycan treated RAW264.7 macrophages and 3T3-L1 adipocytes treated with macrophage conditioned medium (MCM).

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050554#pone-0050554-g005" target="_blank">figure 5A</a>). All genes measured in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050554#pone-0050554-g005" target="_blank">figure 5D</a> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050554#pone-0050554-g005" target="_blank">figure 5</a>, results are expressed as mean±SE.</p

Adiponectin expression in bgn^−/0 mice.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050554#pone-0050554-g002" target="_blank">figure 2</a>, results are expressed as mean±SE.</p

Biglycan expression in adipose tissue.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050554#pone-0050554-g001" target="_blank">figure 1</a>, results are expressed as mean±SE.</p

Mouse characteristics.

<p>Lengths of individual mice were measured from the nose to anus. Organ weights were divided by total body weight to express organs as a percentage of body weight. Results are represented as mean ± SE. EWAT = epididymal white adipose tissue, SWAT = subcutaneous white adipose tissue, RWAT = retroperitoneal white adipose tissue. When a significant diet by genotype interaction was present, means were separated by Tukey analysis and superscript letters are used to indicate significantly different means. P-values less than 0.05 are deemed significant.</p