Obesity increases risk for type 2 diabetes, hypertension, and cardiovascular diseases. Accordingly, much research effort currently targets identification of molecular targets and the development of drugs that reduce fat mass and improve insulin sensitivity. We examined the effect of Smad3 deficiency on adiposity in mice. By using Smad3-KO mice, we showed that Smad3 deficiency resulted in decreased adiposity associated with improved glucose tolerance and insulin responsiveness. Despite decreased physical activity, Smad3-KO mice were resistant to HFD-induced obesity. The deficiency in Smad3 also conferred resistance to the development of obesity-associated glucose intolerance, insulin resistance, and liver steatosis, thus providing an insight into the role of Smad3 in the pathogenesis of obesity and type 2 diabetes.
Previous in vitro studies showed that Smad3 inhibits the transactivation potential of C/EBPs and consequently abolishes the stimulation of the adipogenic master regulator PPARγ2 (17
). In fact, an increase in adiposity might be anticipated in the absence of the inhibitory effect of Smad3 on C/EBPs, as in the case of Smad3 deficiency. We found that Smad3-KO mice displayed decreased adiposity resulting from reduced adipocyte number and size, suggesting defective adipogenesis and altered lipid accumulation. Gene expression profiling of the Smad3-KO WAT revealed reduced expression of PPARγ2 mRNA, with increased expression of the preadipocyte-specific marker Pref-1. PPARγ2 not only participates in adipogenesis and survival but also promotes lipid storage (33
). Lipid accumulation relies on lipogenesis, which involves de novo synthesis of FA and glycerol, FA uptake, and synthesis of triglycerides. Notably, the expression of rate-limiting enzymes involved in these processes, such as FAS, PEPCK, and diacylglycerol acyltransferase, was downregulated in the Smad3-KO WAT (34
). The reduced lipogenesis is exacerbated by concomitant downregulation of enzymes participating in the pentose phosphate pathway required for de novo synthesis of biomolecules. Lending support, knockout or adipose-specific deletion of some examined genes in mice has resulted in similar phenotypic features in Smad3-KO mice. Pref-1-null mice, for instance, display obesity and increased serum lipid metabolites (35
). Adipose-specific knockout of PEPCK results in a fraction of mice developing lipodystrophy (36
). As a central enzyme in lipogenesis, FAS has been identified as a candidate gene for determining body fat (37
). Smad3 deficiency also resulted in increased FA β-oxidation as evidenced by the reduced plasma FFA level, offering another plausible explanation for the reduced adipocyte size in the Smad3-KO mice. Altogether, these findings indicate that Smad3 deficiency leads to impaired adipogenesis and lipogenesis in mice.
It has been proposed that adipose lipid storage functions to prevent peripheral lipotoxicity (5
). Particularly evident in HFD-induced obesity, the chronic accumulation of FFA in skeletal muscles and liver eventually dampens their insulin responsiveness (5
). Indeed, the ablation of PPARγ2 in obese mouse models results in reduced fat mass but leads to severe insulin resistance, β-cell failure, and dyslipidemia because of deposition of toxic reactive lipid species in the peripheral organs (38
). However, this reported insulin resistance and β-cell failure were not observed in the Smad3-KO mice, which also exhibited reduced expression of adipose PPARγ2. This outcome may partly be the result of elevated plasma insulin levels and insulin hypersensitivity, which caused hypoglycemia in these mice. Indeed, our results also revealed enhanced insulin responsiveness in peripheral organs, such as skeletal muscle and WAT. Insulin activation of phosphatidylinositol-3-kinase/protein kinase B (PKB) (Akt) can inhibit TGF-β1 signaling via the formation of a PKB-Smad3 complex. This interaction does not inhibit PKB activity, but inhibits Smad3-mediated gene regulation (39
). A recent study showed that Smad3 occupies the insulin gene promoter to repress its expression and increases glucose-stimulated insulin secretion because of enhanced insulin signaling in β-cell islets (21
). Furthermore, the expression of adipocytokines, such as visfatin and resistin, can influence glucose uptake and metabolism (41
). An increase in glucose uptake and glycolysis with the effects of these adipocytokines and a decreased plasma FFA level as observed in the Smad3-KO mice may partially contribute to the improved glucose tolerance and enhanced insulin sensitivity in these animals. Altogether, these findings place Smad3-PKB at a point of convergence in the crosstalk between TGF-β/Smad3 and insulin signaling pathways and provide insights into their roles in the development of obesity and insulin resistance. Our findings that Smad3-KO mice displayed increased insulin-stimulated whole-body (peripheral insulin sensitivity) and tissue-specific (skeletal muscle and WAT) glucose uptake underscored the relevance of this crosstalk in vivo.
The skeletal muscle is the largest energy consumer in mice and plays an important role in lowering plasma FFA and glycerol (42
); however, we found that Smad3-KO mice exhibited reduced physical activity associated with muscular atrophy, in concordance with a role of Smad3 in β2
-adrenergic–induced muscle hypertrophy (44
). Thus, it is unlikely that increased energy expenditure in the skeletal muscles could account for the reduced adiposity in these mice. Although adipose tissue is not a major energy consumer compared with the skeletal muscle, increased β-oxidation in WAT can have a profound impact on adiposity and insulin sensitivity (41
). Comparative gene expression analysis of WT and Smad3-KO WAT revealed upregulation of PPARβ/δ, UCP2, UCP3, and acyl-CoA oxidase 1, all of which are involved in energy dissipation and peroxisomal FA oxidation. The activation of PPARβ/δ in obese mice has been shown to selectively induce expression of an array of genes required for adipose FA catabolism and thermogenesis but not genes involved in lipogenesis and fat storage, which are controlled by PPARγ2 (14
). Metabolic rate remained unchanged in Smad3-KO mice, reflecting a balance between reduced skeletal muscle activity and increased adipose lipid oxidation. Many regulatory mechanisms may influence the expression of PPARγ and PPARβ/δ in adipocytes. The relative proportion of homodimeric or heterodimeric C/EBP is likely to play important role. Although it is essential for PPARγ2 expression, C/EBPβ inhibits PPARβ/δ promoter activity. CHOP-10, which exhibited elevated expression in Smad3-KO WAT, acts as a dominant-negative inhibitor of C/EBP by preventing its binding to DNA (45
); this function adds to the complexity of regulating the expression of these two PPAR isotypes with apparent opposing functions in the adipocyte. The reason for the differential regulation is unclear but may be attributed to different promoter context. The functional AP-1 site driving induction of the PPARβ/δ promoter is in close proximity to the identified C/EBP binding region. Smad3 may interact with c-JUN (AP-1), inhibiting c-JUN activity that is necessary for AP-1–stimulated PPARβ/δ expression, as reported in keratinocytes (46
In summary, we have found that Smad3 is a multifaceted regulator in glucose and lipid metabolism, as well as in the pathogenesis of obesity and type 2 diabetes, thus identifying Smad3 as a potential target for the treatment of obesity and its associated disorders.