Circadian clocks in adipose tissue are known to regulate adipocyte biology. Although circadian dysregulation is associated with development of obesity, the underlying mechanism has not been established. Here we report that disruption of the clock gene, brain and muscle Arnt-like 1 (Bmal1), in mice led to increased adipogenesis, adipocyte hypertrophy, and obesity, compared to wild-type (WT) mice. This is due to its cell-autonomous effect, as Bmal1 deficiency in embryonic fibroblasts, as well as stable shRNA knockdown (KD) in 3T3-L1 preadipocyte and C3H10T1/2 mesenchymal stem cells, promoted adipogenic differentiation. We demonstrate that attenuation of Bmal1 function resulted in down-regulation of genes in the canonical Wnt pathway, known to suppress adipogenesis. Promoters of these genes (Wnt10a, β-catenin, Dishevelled2, TCF3) displayed Bmal1 occupancy, indicating direct circadian regulation by Bmal1. As a result, Wnt signaling activity was attenuated by Bmal1 KD and augmented by its overexpression. Furthermore, stabilizing β-catenin through Wnt ligand or GSK-3β inhibition achieved partial restoration of blunted Wnt activity and suppression of increased adipogenesis induced by Bmal1 KD. Taken together, our study demonstrates that Bmal1 is a critical negative regulator of adipocyte development through transcriptional control of components of the canonical Wnt signaling cascade, and provides a mechanistic link between circadian disruption and obesity.

SummaryThe circadian clock network is an evolutionarily conserved mechanism that imparts temporal regulation to diverse biological processes. Brain and muscle Arnt-like 1 (Bmal1), an essential transcriptional activator of the clock, is highly expressed in skeletal muscle. However, whether this key clock component impacts myogenesis, a temporally regulated event that requires the sequential activation of myogenic regulatory factors, is not known. Here we report a novel function of Bmal1 in controlling myogenic differentiation through direct transcriptional activation of components of the canonical Wnt signaling cascade, a major inductive signal for embryonic and postnatal muscle growth. Genetic loss of Bmal1 in mice leads to reduced total muscle mass and Bmal1-deficient primary myoblasts exhibit significantly impaired myogenic differentiation accompanied by markedly blunted expression of key myogenic regulatory factors. Conversely, forced expression of Bmal1 enhances differentiation of C2C12 myoblasts. This cell-autonomous effect of Bmal1 is mediated by Wnt signaling as both expression and activity of Wnt components are markedly attenuated by inhibition of Bmal1, and activation of the Wnt pathway partially rescues the myogenic defect in Bmal1-deficient myoblasts. We further reveal direct association of Bmal1 with promoters of canonical Wnt pathway genes, and as a result of this transcriptional regulation, Wnt signaling components exhibit intrinsic circadian oscillation. Collectively, our study demonstrates that the core clock gene, Bmal1, is a positive regulator of myogenesis, which may represent a temporal regulatory mechanism to fine-tune myocyte differentiation.

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Aberrant mitochondrial energy transfer underlies prevalent chronic health conditions, including cancer, cardiovascular, and neurodegenerative diseases. Mitochondrial transplantation represents an innovative strategy aimed at restoring favorable metabolic phenotypes in cells with dysfunctional energy metabolism. While promising, significant barriers to in vivo translation of this approach abound, including limited cellular uptake and recognition of mitochondria as foreign. The objective is to functionalize isolated mitochondria with a biocompatible polymer to enhance cellular transplantation and eventual in vivo applications. Herein, it is demonstrated that grafting of a polymer conjugate composed of dextran with triphenylphosphonium onto isolated mitochondria protects the organelles and facilitates cellular internalization compared with uncoated mitochondria. Importantly, mitochondrial transplantation into cancer and cardiovascular cells has profound effects on respiration, mediating a shift toward improved oxidative phosphorylation, and reduced glycolysis. These findings represent the first demonstration of polymer functionalization of isolated mitochondria, highlighting a viable strategy for enabling clinical applications of mitochondrial transplantation.