Deficiency of the Transcriptional Repressor B Cell Lymphoma 6 (Bcl6) Is Accompanied by Dysregulated Lipid Metabolism

<div><p>The transcriptional repressor B-cell Lymphoma 6 (Bcl6) was recently identified in a profile of genes regulated in adipocytes, suggesting a relationship between Bcl6 and metabolic regulation. As a representative target gene repressed by Bcl6, Suppressor of Cytokine Signaling (Socs) 2 expression was elevated in Bcl6 deficient (KO) mice, including metabolic tissues liver, adipose tissue and muscle, as well as in spleen and thymus. Bcl6 occupied the Socs2 promoter in wild-type, but not Bcl6 KO mice, suggesting direct regulation of Socs2 by Bcl6 <i>in vivo</i>. Mice deficient in Bcl6 were found to exhibit multiple features of dysregulated lipid metabolism. Adipose tissue mass was dramatically reduced or absent in Bcl6 KO mice. Further, hepatic and serum triglycerides were low. Bcl6 deficiency was accompanied by decreased hepatic expression of Stearoyl-CoA desaturase 1 (Scd1) and Fatty acid synthase (Fasn) genes which encode lipogenic enzymes. Expression of the gene for the transcription factor Carbohydrate-Responsive Element Binding Protein (Chrebp), which regulates expression of lipogenic genes, was also reduced in liver of Bcl6 KO mice. Bcl6 deficiency disrupted fasting-induced increases in hepatic triglyceride deposition, but not decreases in lipogenic gene expression. Taken together, these findings suggest that in addition to its well-recognized roles in immune regulation, Bcl6 plays a role in regulatory events of lipid metabolism, and that in the absence of Bcl6, lipid metabolism in liver and adipose tissue is dysregulated.</p></div

Endogenous Socs2 expression is elevated in cells and tissues from Bcl6-deficient mice.

<p><b>A</b>- Mesenchymal stem cells (MSC) were isolated from the ears of WT and Bcl6 KO male mice and analyzed by qpcr. Bars show the mean+SE of triplicate cell preparations from a mouse of each genotype; similar results were obtained in two independent experiments. <b>B</b> - RNA isolated from adipose tissue, liver, and muscle of WT and Bcl6 KO male mice was analyzed using qpcr. mRNA expression is shown as the mean+SE for 4 mice of each genotype. Bcl6 KO are indicated by black bars and WT by grey bars, in this and subsequent figures. Significant differences from WT are designated by * (p<0.05), ** (p<0.005), and *** (p<0.0005). <b>C</b> – Lysates from livers of 3 WT and 3 KO mice were subjected to immunoblotting with anti-SOCS2. Arrowhead indicates migration of upper band as SOCS2. The lower band is non-specific. Tubulin served as a loading control. <b>D</b> - Nuclei from the livers of 2 WT and 2 Bcl6 KO mice were analyzed individually by ChIP, using antibody against Bcl6, with primers for the Bcl6-binding sequence in the Socs2 promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097090#pone.0097090-Chen1" target="_blank">[13]</a>. IgG served as a negative control; 1% input is shown.</p

Bcl6 deficiency decreases expression of lipogenic genes and proteins in liver.

<p><b>A</b>- Hepatic RNA was extracted from the liver of Bcl6 KO or WT mice, and expression of Fasn and Scd1 was measured by qpcr. Gene expression was calculated as fold change relative to WT mice. Bars show the mean+SE for 5 Bcl6 KO and 9 WT mice. <b>B</b> - Liver lysates were analyzed by immunoblotting using antibodies against FAS and SCD1 protein individually for 3 Bcl6 KO or 3 WT mice. α-tubulin served as a loading control. <b>C</b> - Quantification of immunoblots for FAS and SCD1 in B, normalized to α-tubulin, shown as mean+SE. Significant differences are designated by * (p<0.05), ** (p<0.005) and *** (p<0.0005).</p

Fasting-induced decreases of lipogenic gene expression are unimpaired in Bcl6 KO mice.

<p>Bcl6 KO (black bars) and WT male mice (gray bars) were either provided with food <i>ad libitum</i> (fed) or subjected to a 16 hr fast. Expression of hepatic (<b>A</b>) Fasn, (<b>B</b>) Scd1, (<b>C</b>) Chrebp, and (<b>D</b>) Srebp1c mRNA were measured using qpcr. Gene expression in fasted mice was calculated compared to fed mice, with fed state set to 1 for fed WT. Measurements are expressed as the mean+SE for n = fed: 9 WT, 5 KO, fasted: 5 WT, and 3 KO mice. Fasting decreased Scd1 expression in KO by 78%. Significant differences from fed mice of the same genotype are designated by * (p<0.05), and *** (p<0.0005).</p

Expression of genes for fatty acid oxidation.

<p>mRNA from liver of Bcl6 KO and WT mice was analyzed by qpcr for expression of Acox, Cpt1, and Pparα. Bars show the mean+SE for 4 mice of each genotype. Gene expression was calculated as the fold-change relative to WT mice. Asterisk (*) designates significant (p<0.05) decrease in Bcl6 KO.</p

Bcl6 deficiency decreases accumulation of liver triglycerides during fasting.

<p>Triglycerides were measured in livers of fed or fasted (16 hr) Bcl6 KO and WT male mice. Bars show the mean+SE for n = 3 mice/genotype and nutritional status. Brackets indicate significant differences between bracketed bars: * (p<0.05), *** (p<0.0005), or not significant (NS).</p

Insulin Stimulates Interleukin-6 Expression and Release in LS14 Human Adipocytes through Multiple Signaling Pathways

IL-6 is an important cytokine that regulates both immune and metabolic functions. Within adipose tissue, preadipocytes produce significant amounts of IL-6, but little is known about the factors or mechanisms that regulate IL-6 production in these cells. Using LS14, a newly developed human adipocyte cell line, our objective was to determine the mechanisms by which insulin stimulates IL-6 production and release in preadipocytes. Insulin increased IL-6 gene expression and secretion in a time- and dose-dependent manner. Insulin decreased cyclic AMP (cAMP) but increased cyclic GMP (cGMP) levels, and IL-6 expression/release was stimulated by a cGMP analog. The stimulatory effect of insulin and cGMP was abrogated by a specific inhibitor of protein kinase G (cyclic GMP-dependent protein kinase). Both insulin and cGMP rapidly induced phosphorylation of cAMP response element binding protein. Insulin also activated the MAPK signaling pathway, and its blockade prevented the insulin-stimulated increases in IL-6 cell content and release, but not IL-6 gene expression. Although inhibition of the proteosome increased IL-6 cell content and release, proteosome activity was unaffected by insulin. These data suggest that the stimulatory effects of insulin on IL-6 release involve several interrelated components: transcription, intracellular releasable pool, and secretion, which are differentially regulated and, thus, determine the size of the releasable pool of IL-6. Insulin-induced IL-6 gene expression is mediated by cGMP/cyclic GMP-dependent protein kinase/cAMP response element binding protein, whereas MAPK is involved in the insulin-stimulated IL-6 synthesis/release