Summary Brown fat can increase energy expenditure and protect against obesity through a specialized program of uncoupled respiration. We show here by in vivo fate mapping that brown but not white fat cells arise from precursors that express myf5, a gene previously thought to be expressed only in the myogenic lineage. Notably, the transcriptional regulator, PRDM16 controls a bidirectional cell fate switch between skeletal myoblasts and brown fat cells. Loss of PRDM16 from brown fat precursors causes a loss of brown fat characteristics and promotes muscle differentiation. Conversely, ectopic expression of PRDM16 in myoblasts induces their differentiation into brown fat cells. PRDM16 stimulates brown adipogenesis by binding to PPARγ and activating its transcriptional function. Finally, PRDM16-deficient brown fat displays an abnormal morphology, reduced thermogenic gene expression and elevated expression of muscle-specific genes. Taken together, these data indicate that PRDM16 specifies the brown fat lineage from a progenitor that expresses myoblast markers and is not involved in white adipogenesis.

Brown fat cells are specialized to dissipate energy and can counteract obesity; however, the transcriptional basis of their determination is largely unknown. We show here that the zinc-finger protein PRDM16 is highly enriched in brown fat cells compared to white fat cells. When expressed in white fat cell progenitors, PRDM16 activates a robust brown fat phenotype including induction of PGC-1alpha, UCP1, and type 2 deiodinase (Dio2) expression and a remarkable increase in uncoupled respiration. Transgenic expression of PRDM16 at physiological levels in white fat depots stimulates the formation of brown fat cells. Depletion of PRDM16 through shRNA expression in brown fat cells causes a near total loss of the brown characteristics. PRDM16 activates brown fat cell identity at least in part by simultaneously activating PGC-1alpha and PGC-1beta through direct protein binding. These data indicate that PRDM16 can control the determination of brown fat fate.

Skeletal and cardiac muscle depend on high turnover of ATP made by mitochondria in order to contract efficiently. The transcriptional coactivator PGC-1alpha has been shown to function as a major regulator of mitochondrial biogenesis and respiration in both skeletal and cardiac muscle, but this has been based only on gain-of-function studies. Using genetic knockout mice, we show here that, while PGC-1alpha KO mice appear to retain normal mitochondrial volume in both muscle beds, expression of genes of oxidative phosphorylation is markedly blunted. Hearts from these mice have reduced mitochondrial enzymatic activities and decreased levels of ATP. Importantly, isolated hearts lacking PGC-1alpha have a diminished ability to increase work output in response to chemical or electrical stimulation. As mice lacking PGC-1alpha age, cardiac dysfunction becomes evident in vivo. These data indicate that PGC-1alpha is vital for the heart to meet increased demands for ATP and work in response to physiological stimuli.

The transcriptional coactivator peroxisome proliferator-activated receptor ␥ coactivator 1␣ (PGC-1␣) is a key integrator of neuromuscular activity in skeletal muscle. Ectopic expression of PGC-1␣ in muscle results in increased mitochondrial number and function as well as an increase in oxidative, fatigue-resistant muscle fibers. Whole body PGC-1␣ knock-out mice have a very complex phenotype but do not have a marked skeletal muscle phenotype. We thus analyzed skeletal muscle-specific PGC-1␣ knock-out mice to identify a specific role for PGC-1␣ in skeletal muscle function. These mice exhibit a shift from oxidative type I and IIa toward type IIx and IIb muscle fibers. Moreover, skeletal muscle-specific PGC-1␣ knock-out animals have reduced endurance capacity and exhibit fiber damage and elevated markers of inflammation following treadmill running. Our data demonstrate a critical role for PGC-1␣ in maintenance of normal fiber type composition and of muscle fiber integrity following exertion.Skeletal muscle has an enormous capacity to adapt to motor neuron activity. Many changes in gene expression are controlled by motor neuron-induced calcium signaling (1-3). The transcriptional coactivator peroxisome proliferator-activated receptor ␥ coactivator 1␣ 3 is at the nexus of this signaling and subsequently regulates the expression of gene programs needed for skeletal muscle adaptations to increased work load (4, 5). By coactivating the myocyte enhancer factor 2 members MEF2C and MEF2D, PGC-1␣ potently drives transcription of myofibrillar genes typical of oxidative muscle fibers (6). Interestingly, MEF2 and PGC-1␣ also control PGC-1␣ gene transcription in an autoregulatory loop (7,8). Metabolic genes, including those responsible for mitochondrial oxidative phosphorylation, are induced by a transcriptional cascade with coactivation of the estrogen-related receptor ␣ (ERR␣, official nomenclature NR3B1), the nuclear respiratory factor 2 (NRF-2, alternatively called GA-binding protein, GABP), and the nuclear respiratory factor 1 (NRF-1) by PGC-1␣ and subsequent increase in the levels of mitochondrial transcription factor A (TFAM) and mitochondrial transcription specificity factors TFB1M and TFB2M (9 -13). Recently, we have found that activity-induced remodeling of the postsynaptic side of neuromuscular junctions involves a complex between PGC-1␣, GABP, and host cell factor that assembles upon phosphorylation of PGC-1␣ and GABP in post-synaptic nuclei (14).Data obtained from muscle-specific PGC-1␣ transgenic animals underline the importance of PGC-1␣ in skeletal muscle in vivo (6). These mice have increased number and function of mitochondria accompanied by a higher number of type IIa and type I oxidative, slow twitch, high endurance muscle fibers (6). Furthermore, even in the absence of functional motor nerve signaling following hind leg denervation, ectopically expressed PGC-1␣ maintains skeletal muscle function and blunts skeletal muscle atrophy that normally occurs in the absence of motor neuron signaling (15). Thus, PGC-...

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene that regulates chloride and water transport across all epithelia and affects multiple organs including the lungs. Here we report an in vitro directed differentiation protocol for generating functional CFTR-expressing airway epithelia from human embryonic stem cells. Carefully timed treatment by exogenous growth factors that mimic endoderm developmental pathways in vivo followed by air-liquid interface culture results in maturation of patches of tight junction-coupled differentiated airway epithelial cells that demonstrate active CFTR transport function. As a proof-of-concept, treatment of CF patient induced pluripotent stem cells (iPSC)-derived epithelial cells with a novel small molecule compound to correct for the common CF-processing mutation resulted in enhanced plasma membrane localization of mature CFTR protein. Our study provides a method for generating patient-specific airway epithelial cells for disease modeling and in vitro drug testing.

The coactivator PGC-1␣ mediates key responses of skeletal muscle to motor nerve activity. We show here that neuregulin-stimulated phosphorylation of PGC-1␣ and GA-binding protein (GABP) allows recruitment of PGC-1␣ to the GABP complex and enhances transcription of a broad neuromuscular junction gene program. Since a subset of genes controlled by PGC-1␣ and GABP is dysregulated in Duchenne muscular dystrophy (DMD), we examined the effects of transgenic PGC-1␣ in muscle of mdx mice. These animals show improvement in parameters characteristic of DMD, including muscle histology, running performance, and plasma creatine kinase levels. Thus, control of PGC-1␣ levels in skeletal muscle could represent a novel avenue to prevent or treat DMD.[Keywords: PGC-1; neuromuscular junction; GA-binding protein; Duchenne muscular dystrophy; transcriptional regulation] Supplemental material is available at http://www.genesdev.org.

Brown fat is a specialized tissue that can dissipate energy and counteract obesity through a pattern of gene expression that greatly increases mitochondrial content and uncoupled respiration. PRDM16 is a zinc-finger protein that controls brown fat determination by stimulating brown fat-selective gene expression, while suppressing the expression of genes selective for white fat cells. To determine the mechanisms regulating this switching of gene programs, we purified native PRDM16 protein complexes from fat cells. We show here that the PRDM16 transcriptional holocompex contains C-terminal-binding protein-1 (CtBP-1) and CtBP-2, and this direct interaction selectively mediates the repression of white fat genes. This repression occurs through recruiting a PRDM16/CtBP complex onto the promoters of white fat-specific genes such as resistin, and is abolished in the genetic absence of CtBP-1 and CtBP-2. In turn, recruitment of PPAR-␥-coactivator-1␣ (PGC-1␣) and PGC-1␤ to the PRDM16 complex displaces CtBP, allowing this complex to powerfully activate brown fat genes, such as PGC-1␣ itself. These data show that the regulated docking of the CtBP proteins on PRDM16 controls the brown and white fat-selective gene programs.[Keywords: PRDM16; adipogenesis; CtBP; PGC-1; PPAR-␥; resistin] Supplemental material is available at http://www.genesdev.org.

The transcriptional coactivator PPARγ coactivator 1α (PGC-1α) is a strong activator of mitochondrial biogenesis and oxidative metabolism. While expression of PGC-1α and many of its mitochondrial target genes are decreased in the skeletal muscle of patients with type 2 diabetes, no causal relationship between decreased PGC-1α expression and abnormal glucose metabolism has been established. To address this question, we generated skeletal muscle-specific PGC-1α knockout mice (MKOs), which developed significantly impaired glucose tolerance but showed normal peripheral insulin sensitivity. Surprisingly, MKOs had expanded pancreatic β cell mass, but markedly reduced plasma insulin levels, in both fed and fasted conditions. Muscle tissue from MKOs showed increased expression of several proinflammatory genes, and these mice also had elevated levels of the circulating IL-6. We further demonstrated that IL-6 treatment of isolated mouse islets suppressed glucose-stimulated insulin secretion. These data clearly illustrate a causal role for muscle PGC-1α in maintenance of glucose homeostasis and highlight an unexpected cytokine-mediated crosstalk between skeletal muscle and pancreatic islets.