3FTFBSDIBSUJDMF "COPSNBMHMVDPTFIPNFPTUBTJT JOTLFMFUBMNVTDMFmTQFDJGJD1($α LOPDLPVUNJDFSFWFBMTTLFMFUBM NVTDMFmQBODSFBUJDβDFMMDSPTTUBML Christoph Handschin,1,2 Cheol Soo Choi,3 Sherry Chin,1 Sheene Kim,3 Dan Kawamori,4 Amarnath J. Kurpad,4 Nicole Neubauer,4 Jiang Hu,4 Vamsi K. Mootha,5,6 Young-Bum Kim,7 Rohit N. Kulkarni,4 Gerald I. Shulman,3 and Bruce M. Spiegelman1 1Dana-Farber Cancer Institute and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA. 2Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland. 3Howard Hughes Medical Institute and Departments of Internal Medicine and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, USA. 4Joslin Diabetes Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA. 5Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. 6Center for Human Genetic Research, Massachusetts General Hospital, and Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA. 7Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA. 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 expres- sion 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. *OUSPEVDUJPO ing pathways by which PGC-1α controls OXPHOS gene expres- PPARγ coactivator 1α (PGC-1α) is a coactivator protein that binds sion in skeletal muscle have been elucidated (3, 7–13). Notably, to a number of transcription factors and augments their ability to PGC-1α dysregulation is also observed in pre-diabetic individuals, induce target gene expression (1, 2). A central feature of PGC-1α suggesting that reduction of PGC-1α levels is an early event in the is its potential to boost mitochondrial biogenesis and increase development of the disease (7, 8). oxidative metabolism (3). In addition, PGC-1α regulates certain Mitochondrial dysfunction has been closely associated with skel- tissue-specific gene programs in vivo, such as increased myofibril- etal muscle insulin resistance in several studies (14–16). Decreased lar gene expression associated with oxidative muscle fibers (4). PGC-1α levels could theoretically trigger a pathway of pathology PGC-1α is regulated in many tissues under a variety of physiologi- starting with reduced OXPHOS gene expression, decreased fatty acid cal stimuli, including cold, exercise, and fasting (1, 2). In addition, oxidation, and subsequent lipid accumulation in skeletal muscle dysregulation of PGC-1α mRNA expression has been observed in and culminating in insulin resistance and type 2 diabetes (8, 15, 16). many disease states (1, 2). For example, PGC-1α expression is However, the causality of PGC-1α dysregulation in skeletal mus- increased in the liver and islets of animal models of type 1 and cle to the development of abnormalities in glucose homeostasis, type 2 diabetes (5, 6). In contrast, mRNA levels of PGC-1α, PGC- particularly insulin resistance and type 2 diabetes, has never to 1β, and genes encoding for proteins involved in mitochondrial our knowledge been directly tested in a loss-of-function model in oxidative phosphorylation (OXPHOS) are reduced in skeletal vivo. Mice lacking PGC-1α in all tissues have been made, but these muscle of human type 2 diabetics, at least in some populations animals have many systemic abnormalities, including complete (7, 8). The OXPHOS genes that are dysregulated in type 2 diabetic resistance to diet-induced obesity and hyperactivity secondary to patients are under transcriptional control of PGC-1α, and signal- central nervous system abnormalities (17, 18). This has made a serious examination of the role of skeletal muscle PGC-1α in sys- temic glucose homeostasis impossible in these animals (17, 18). Nonstandard abbreviations used: Cox, cytochrome c oxidase; GABP, GA-binding These mice also have constitutively active AMP-activated protein protein; MKO, skeletal muscle–specific PGC-1α knockout mouse; OXPHOS, oxida- tive phosphorylation; PGC-1α, PPARγ coactivator 1α; SDH, succinate dehydrogenase; kinase in skeletal muscle, which might compensate for the loss of SKM-Het, skeletal muscle–specific PGC-1α heterozygous mouse. PGC-1α in this tissue (17, 18). Therefore, to assess the importance Conflict of interest: The authors have declared that no conflict of interest exists. of PGC-1α in peripheral tissues, tissue-specific knockout animals Citation for this article: J. Clin. Invest. doi:10.1172/JCI31785. have to be generated. Proof of principle stems from liver-specific The Journal of Clinical Investigation http://www.jci.org SFTFBSDIBSUJDMF 'JHVSF (FOFSBUJPOBOEBOBMZTJTPG.,0T " .,0TXFSFHFOFSBUFECZDSPTTJOHNJDFXJUIBGMPYFE1($αBMMFMFUPBOJNBMTUIBUUSBOTHFOJDBMMZFYQSFTT DSFSFDPNCJOBTFVOEFSUIFDPOUSPMPGUIFNZPHFOJOQSPNPUFSBOE.&'$FOIBODFS .ZP$SF # 1($αQSPUFJOMFWFMTJOHBTUSPDOFNJVT NVTDMFXFSFEFUFSNJOFECZ8FTUFSOCMPU $ 3FMBUJWF1($αBOE1($βN3/"FYQSFTTJPOXBTNFBTVSFEJOEJGGFSFOUUJTTVFTCZSFBMUJNF 1$37BMVFTBSFNFBO4&. 1 .,0WFSTVTDPOUSPMOQFSHSPVQ PGC-1α knockout mice, which, in contrast to whole-body knock- compensatory adaptations in PGC-1β levels (Figure 1C). In terms out mice, show the expected reduction of gene expression involved of muscle function, MKOs has a lower expression of myosin heavy in gluconeogenesis and heme biosynthesis (19). chains normally expressed in oxidative fibers (type I and IIa) and We thus generated skeletal muscle–specific PGC-1α knockout exhibits reduced voluntary physical activity (21). mice (MKOs) and studied the impact of aberrant PGC-1α levels in Increase in mitochondrial function and oxidative metabolism are core this tissue on glucose homeostasis and insulin sensitivity. Interest- functions of PGC-1α. We first studied the expression of metabolic ingly, while the loss of PGC-1α expression caused significant glu- genes and their corresponding transcription factors by real-time cose intolerance, this intolerance was not associated with insulin PCR. Two of the early PGC-1α target genes, estrogen-related recep- resistance in peripheral tissues per se. Rather, we found an unex- tor α (ERRα) and GA-binding protein A (GABPA), the DNA-bind- pected link between absence or reduction of PGC-1α expression in ing subunit of GABP, showed reduced expression in MKOs com- skeletal muscle and decreased insulin secretion in vivo. These data pared with control mice (Figure 2A). These transcription factors suggest an important crosstalk between skeletal muscle and pan- are essential in controlling PGC-1α–induced OXPHOS expression creatic islets, perhaps via secretion of proinflammatory cytokines in skeletal muscle (9, 10). Transcript levels of the more downstream such as IL-6 by skeletal muscle. transcription factors nuclear respiratory factor 1 and mitochon- drial transcription factor A were unaltered in MKOs (Figure 2A). 3FTVMUT Nevertheless, expression of a number of OXPHOS and other Generation and characterization of MKOs. MKOs were generated by mitochondrial genes was significantly reduced in MKOs, includ- breeding animals harboring a floxed PGC-1α allele (17) with mice ing the 1β subcomplex 5 and Fe-S protein 1 subunits of NADH- that transgenically express cre recombinase under the control of ubiquinone oxidoreductase, cytochrome c, cytochrome c oxidase the myogenin promoter and the MEF2C enhancer (20) (Figure 1A). (Cox) subunit 5b, and ATP synthase o subunit. These genes have The transcript and protein levels of PGC-1α were subsequently been found to be directly regulated by ERRα, GABP, and PGC-1α monitored by real-time PCR and Western blots, respectively. In gas- (9, 10). Other OXPHOS genes did not differ in their expression trocnemius muscle, protein expression of PGC-1α was almost com- between MKOs and controls, e.g., the NADH-ubiquinone oxido- pletely abolished (Figure 1B). Similarly, PGC-1α mRNA expression reductase flavoprotein 2. Finally, non-OXPHOS mitochondrial was drastically reduced in gastrocnemius and soleus muscles of genes that are known to be regulated by PGC-1α (19) were reduced MKOs (Figure 1C). Other known PGC-1α–expressing tissues such in MKOs, for example the first enzyme in heme biosynthesis, as heart, liver, brown adipose tissue, and brain were examined, and 5-aminolevulinate synthase (Figure 2A). Notably, the reduction none of those had significantly altered PGC-1α transcript levels of mitochondrial