The silencing mediator of retinoid and thyroid hormone receptors (SMRT) regulates adipose tissue accumulation and adipocyte insulin sensitivity in vivo.

The silencing mediator of retinoid and thyroid hormone receptors (SMRT) serves as a corepressor for nuclear receptors and other factors. Recent evidence suggests that SMRT is an important regulator of metabolism, but its role in adipocyte function in vivo remains unclear. We generated heterozygous SMRT knock-out (SMRT(+/-)) mice to investigate the function of SMRT in the adipocyte and the regulation of adipocyte insulin sensitivity. We show that SMRT(+/-) mice are normal weight on a regular diet, but develop increased adiposity on a high-fat diet (HFD). The mechanisms underlying this phenotype are complex, but appear to be due to a combination of an increased number of smaller subcutaneous adipocytes as well as decreased leptin expression, resulting in greater caloric intake. In addition, adipogenesis of mouse embryonic fibroblasts (MEFs) derived from these mice was increased. However, adipocyte insulin sensitivity, measured by insulin-induced Akt phosphorylation and insulin-mediated suppression of lipolysis, was enhanced in SMRT(+/-) adipocytes. These finding suggest that SMRT regulates leptin expression and limits the ability of fat mass to expand with increased caloric intake, but that SMRT also negatively regulates adipocyte insulin sensitivity.

Enhanced glycogen metabolism in adipose tissue decreases triglyceride mobilization.

Adipose tissue is a primary site for lipid storage containing trace amounts of glycogen. However, refeeding after a prolonged partial fast produces a marked transient spike in adipose glycogen, which dissipates in coordination with the initiation of lipid resynthesis. To further study the potential interplay between glycogen and lipid metabolism in adipose tissue, the aP2-PTG transgenic mouse line was utilized since it contains a 100- to 400-fold elevation of adipocyte glycogen levels that are mobilized upon fasting. To determine the fate of the released glucose 1-phosphate, a series of metabolic measurements were made. Basal and isoproterenol-stimulated lactate production in vitro was significantly increased in adipose tissue from transgenic animals. In parallel, basal and isoproterenol-induced release of nonesterified fatty acids (NEFAs) was significantly reduced in transgenic adipose tissue vs. control. Interestingly, glycerol release was unchanged between the genotypes, suggesting that enhanced triglyceride resynthesis was occurring in the transgenic tissue. Qualitatively similar results for NEFA and glycerol levels between wild-type and transgenic animals were obtained in vivo during fasting. Additionally, the physiological upregulation of the phosphoenolpyruvate carboxykinase cytosolic isoform (PEPCK-C) expression in adipose upon fasting was significantly blunted in transgenic mice. No changes in whole body metabolism were detected through indirect calorimetry. Yet weight loss following a weight gain/loss protocol was significantly impeded in the transgenic animals, indicating a further impairment in triglyceride mobilization. Cumulatively, these results support the notion that the adipocyte possesses a set point for glycogen, which is altered in response to nutritional cues, enabling the coordination of adipose glycogen turnover with lipid metabolism.

Natural angiogenesis inhibitor signals through Erk5 activation of peroxisome proliferator-activated receptor gamma (PPARgamma).

Erk-5, a member of the MAPK superfamily, has a catalytic domain similar to Erk1/2 and a unique C-terminal domain enabling binding with transcription factors. Aberrant vascularization in the Erk5-null mice suggested a link to angiogenesis. Ectopic expression of constitutively active Erk5 blocks endothelial cell morphogenesis and causes HIF1-alpha destabilization/degradation. However the mechanisms by which endogenous Erk5 regulates angiogenesis remain unknown. We show that Erk5 and its activating kinase MEK5 are the upstream mediators of the anti-angiogenic signal by the natural angiogenesis inhibitor, pigment epithelial-derived factor (PEDF). We demonstrate that Erk5 phosphorylation allows activation of PPARgamma transcription factor by displacement of SMRT co-repressor. PPARgamma, in turn is critical for NFkappaB activation, PEDF-dependent apoptosis, and anti-angiogenesis. The dominant negative MEK5 mutant and Erk5 shRNA diminished PEDF-dependent apoptosis, inhibition of the endothelial cell chemotaxis, and angiogenesis. This is the first evidence of Erk5-dependent transduction of signals by endogenous angiogenesis inhibitors.

Altering PPARgamma ligand selectivity impairs adipogenesis by thiazolidinediones but not hormonal inducers.

Peroxisome proliferator-activated receptor gamma (PPARgamma) acts as a ligand-dependent transcription factor with a key role in mediating adipocyte differentiation and insulin sensitivity. Recently, we and others have shown that PPARgamma recruits the nuclear corepressors NCoR and silencing mediator for retinoid and thyroid hormone receptors (SMRT) to modulate adipogenesis. While the synthetic ligands for PPARgamma, the thiazolidinediones (TZD), are widely used in the treatment of type 2 diabetes mellitus, the biologically relevant endogenous PPARgamma ligand involved in adipogenesis remains unidentified. To further understand the role of ligand binding and corepressor interaction in PPARgamma-mediated adipogenesis, a mutation was introduced in the ligand-binding domain (LBD) of murine PPARgamma. PPARgammamut was created via two amino acid substitutions known to be major determinants of ligand selectivity among PPAR isotypes, H323Y and R288M. These mutations alter PPARgamma to the corresponding residues of the PPARalpha. Characterizing the in vitro functional properties of this mutant, we show that PPARgammamut preferentially responds to the PPARalpha agonist, WY-14643, over the TZD, pioglitazone. When expressed in 3T3-L1 preadipocytes using recombinant adenovirus, wild-type PPARgamma leads to adipocyte formation with both hormonal and TZD treatment. PPARgammamut blocks the upregulation of adipocyte-specific proteins by TZD, but surprisingly, not by standard hormonal inducers. Our data suggest that TZDs and the purported endogenous ligand do not interact in the same way with the PPARgamma LBD. We propose that the endogenous ligand has distinct properties that allow for promiscuity within the hydrophobic PPAR ligand-binding pocket, yet fosters appropriate cofactor recruitment and release to allow adipogenesis to proceed.

SMRT recruitment by PPARgamma is mediated by specific residues located in its carboxy-terminal interacting domain.

The silencing mediator of retinoid and thyroid hormone receptors (SMRT) has been shown to play an important role in adipogenesis and PPARgamma transcriptional activity. SMRT contains two interacting domains that mediate interactions with nuclear receptors. Interestingly, SMRT is recruited to PPARgamma via its C-terminal interacting domain, and mutation of the proximal interacting domain does not interfere with recruitment via PPARgamma. To understand how the distal interacting domain mediates recruitment by PPARgamma, we have now mutated residues in this domain to the corresponding amino acids found in the proximal domain. We show that specific residues in this distal domain are vital for interactions with PPARgamma, but not for a related receptor, RARalpha. Furthermore, naturally occurring SMRT isoforms that differ in interacting domain sequences have different effects on PPARgamma as opposed to RARalpha recruitment. These data suggest that PPARgamma and RARalpha interact with SMRT via distinct mechanisms. These differences will be important as ligands are designed that lead to specific patterns of nuclear receptor recruitment of corepressors.

SMRT recruitment by PPARgamma is mediated by specific residues located in its carboxy-terminal interacting domain.

The silencing mediator of retinoid and thyroid hormone receptors (SMRT) has been shown to play an important role in adipogenesis and PPARgamma transcriptional activity. SMRT contains two interacting domains that mediate interactions with nuclear receptors. Interestingly, SMRT is recruited to PPARgamma via its C-terminal interacting domain, and mutation of the proximal interacting domain does not interfere with recruitment via PPARgamma. To understand how the distal interacting domain mediates recruitment by PPARgamma, we have now mutated residues in this domain to the corresponding amino acids found in the proximal domain. We show that specific residues in this distal domain are vital for interactions with PPARgamma, but not for a related receptor, RARalpha. Furthermore, naturally-occuring SMRT isoforms that differ in interacting domain sequences have different effects on PPARgamma as opposed to RARalpha recruitment. These data suggest that PPARgamma and RARalpha interact with SMRT via distinct mechanisms. These differences will be important as ligands are designed that lead to specific patterns of nuclear receptor recruitment of corepressors.