Uncoupling Protein 1 (UCP1) plays a central role in non-shivering thermogenesis in brown fat; however, its role in beige fat remains unclear. Here we report a robust UCP1-independent thermogenic mechanism in beige fat that involves enhanced ATP-dependent Ca2+ cycling by sarco/endoplasmic reticulum Ca2+-ATPase2b (SERCA2b) and ryanodine receptor 2 (RyR2). Inhibition of SERCA2b impairs UCP1-independent beige fat thermogenesis in humans and mice, as well as in pigs, a species that lacks a functional UCP1 protein. Conversely, enhanced Ca2+ cycling by the activation of α1/β3-adrenergic receptors or the SERCA2b-RyR2 pathway stimulates UCP1-independent thermogenesis. In the absence of UCP1, beige fat dynamically expends glucose through enhanced glycolysis, tricarboxylic acid metabolism, and pyruvate dehydrogenase activity for ATP-dependent thermogenesis by the SERCA2b pathway; beige fat thereby functions as a “glucose-sink” and improves glucose tolerance independent of body-weight loss. Our study uncovers a non-canonical thermogenic mechanism by which beige fat controls whole-body energy homeostasis through Ca2+ cycling.

Beige adipocytes gained much attention as an alternative cellular target in anti-obesity therapy. While recent studies have identified a number of regulatory circuits that promote beige adipocyte differentiation, the molecular basis of beige adipocyte maintenance remains unknown. Here, we demonstrate that beige adipocytes progressively lose their morphological and molecular characteristics after withdrawing external stimuli, and directly acquire white-like characteristics bypassing an intermediate precursor stage. The beige-to-white adipocyte transition is tightly coupled to a decrease in mitochondria, increase in autophagy, and activation of MiT/TFE transcription factor-mediated lysosome biogenesis. The autophagy pathway is crucial for mitochondrial clearance during the transition; inhibiting autophagy by UCP1+-adipocyte-specific deletion of Atg5 or Atg12 prevents beige adipocyte loss after withdrawing external stimuli, maintenaning high thermogenic capacity and protecting against diet-induced obesity and insulin resistance. The present study uncovers a fundamental mechanism by which autophagy-mediated mitochondrial clearance controls beige adipocyte maintenance, thereby providing new opportunities to counteract obesity.

First identified in Drosophila and highly conserved in mammals, the Hippo pathway controls organ size. Lats2 is one of the core kinases of the Hippo pathway and plays major roles in cell proliferation by interacting with the downstream transcriptional cofactors YAP and TAZ. Although the function of the Hippo pathway and Lats2 is relatively well understood in several tissues and organs, less is known about the function of Lats2 and Hippo signaling in adipose development. Here, we show that Lats2 is an important modulator of adipocyte proliferation and differentiation via Hippo signaling. Upon activation, Lats2 phosphorylates YAP and TAZ, leading to their retention in the cytoplasm, preventing them from activating the transcription factor TEAD in the nucleus. Because TAZ remains in the cytoplasm, PPARγ regains its transcriptional activity. Furthermore, cytoplasmic TAZ acts as an inhibitor of Wnt signaling by suppressing DVL2, thereby preventing β-catenin from entering the nucleus to stimulate TCF/LEF transcriptional activity. The above effects contribute to the phenotype of repressed proliferation and accelerated differentiation in adipocytes. Thus, Lats2 regulates the balance between proliferation and differentiation during adipose development. Interestingly, our study provides evidence that Lats2 not only negatively modulates cell proliferation but also positively regulates cell differentiation.

Cell-penetrating peptides (CPPs) have been increasingly used to deliver various molecules, both in vitro and in vivo. However, there are no reports of CPPs being used in porcine fetal fibroblasts (PFFs). The increased use of transgenic pigs for basic research and biomedical applications depends on the availability of technologies for efficient genetic-modification of PFFs. Here, we report that three CPPs (CPP5, TAT, and R9) can efficiently deliver active Cre recombinase protein into PFFs via an energy-dependent endocytosis pathway. The three CPP–Cre proteins can enter PFFs and subsequently perform recombination with different efficiencies. The recombination efficacy of CPP5–Cre was found to be nearly 90%. The rate-limiting step for CPP–Cre-mediated recombination was the step of endosome escape. HA2 and chloroquine were found to improve the recombination efficiency of TAT–Cre. Furthermore, we successfully obtained marker-free transgenic pigs using TAT–Cre and CPP5–Cre. We provide a framework for the development of CPP-based farm animal transgenic technologies that would be beneficial to agriculture and biomedicine.