Paul Schaeffer

PGC-1α deficiency causes multi-system energy metabolic derangements: Muscle dysfunction, abnormal weight control and hepatic steatosis

The gene encoding the transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) was targeted in mice. PGC-1α null (PGC-1α−/−) mice were viable. However, extensive phenotyping revealed multi-system abnormalities indicative of an abnormal energy metabolic phenotype. The postnatal growth of heart and slow-twitch skeletal muscle, organs with high mitochondrial energy demands, is blunted in PGC-1α−/− mice. With age, the PGC-1α−/− mice develop abnormally increased body fat, a phenotype that is more severe in females. Mitochondrial number and respiratory capacity is diminished in slow-twitch skeletal muscle of PGC-1α−/− mice, leading to reduced muscle performance and exercise capacity. PGC-1α−/− mice exhibit a modest diminution in cardiac function related largely to abnormal control of heart rate. The PGC-1α−/− mice were unable to maintain core body temperature following exposure to cold, consistent with an altered thermogenic response. Following short-term starvation, PGC-1α−/− mice develop hepatic steatosis due to a combination of reduced mitochondrial respiratory capacity and an increased expression of lipogenic genes. Surprisingly, PGC-1α−/− mice were less susceptible to diet-induced insulin resistance than wild-type controls. Lastly, vacuolar lesions were detected in the central nervous system of PGC-1α−/− mice. These results demonstrate that PGC-1α is necessary for appropriate adaptation to the metabolic and physiologic stressors of postnatal life.

PGC-1α Coactivates PDK4 Gene Expression via the Orphan Nuclear Receptor ERRα: a Mechanism for Transcriptional Control of Muscle Glucose Metabolism

ABSTRACT The transcriptional coactivator PGC-1α is a key regulator of energy metabolism, yet little is known about its role in control of substrate selection. We found that physiological stimuli known to induce PGC-1α expression in skeletal muscle coordinately upregulate the expression of pyruvate dehydrogenase kinase 4 (PDK4), a negative regulator of glucose oxidation. Forced expression of PGC-1α in C2C12 myotubes induced PDK4 mRNA and protein expression. PGC-1α-mediated activation of PDK4 expression was shown to occur at the transcriptional level and was mapped to a putative nuclear receptor binding site. Gel shift assays demonstrated that the PGC-1α-responsive element bound the estrogen-related receptor α (ERRα), a recently identified component of the PGC-1α signaling pathway. In addition, PGC-1α was shown to activate ERRα expression. Chromatin immunoprecipitation assays confirmed that PGC-1α and ERRα occupied the mPDK4 promoter in C2C12 myotubes. Additionally, transfection studies using ERRα-null primary fibroblasts demonstrated that ERRα is required for PGC-1α-mediated activation of the mPDK4 promoter. As predicted by the effects of PGC-1α on PDK4 gene transcription, overexpression of PGC-1α in C2C12 myotubes decreased glucose oxidation rates. These results identify the PDK4 gene as a new PGC-1α/ERRα target and suggest a mechanism whereby PGC-1α exerts reciprocal inhibitory influences on glucose catabolism while increasing alternate mitochondrial oxidative pathways in skeletal muscle.

The Lectin-like Domain of Thrombomodulin Confers Protection from Neutrophil-mediated Tissue Damage by Suppressing Adhesion Molecule Expression via Nuclear Factor κB and Mitogen-activated Protein Kinase Pathways

Thrombomodulin (TM) is a vascular endothelial cell (EC) receptor that is a cofactor for thrombin-mediated activation of the anticoagulant protein C. The extracellular NH2-terminal domain of TM has homology to C-type lectins that are involved in immune regulation. Using transgenic mice that lack this structure (TMLeD/LeD), we show that the lectin-like domain of TM interferes with polymorphonuclear leukocyte (PMN) adhesion to ECs by intercellular adhesion molecule 1–dependent and –independent pathways through the suppression of extracellular signal–regulated kinase (ERK)1/2 activation. TMLeD/LeD mice have reduced survival after endotoxin exposure, accumulate more PMNs in their lungs, and develop larger infarcts after myocardial ischemia/reperfusion. The recombinant lectin-like domain of TM suppresses PMN adhesion to ECs, diminishes cytokine-induced increase in nuclear factor κB and activation of ERK1/2, and rescues ECs from serum starvation, findings that may explain why plasma levels of soluble TM are inversely correlated with cardiovascular disease. These data suggest that TM has antiinflammatory properties in addition to its role in coagulation and fibrinolysis.

A Role for the Transcriptional Coactivator PGC-1α in Muscle Refueling

The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) has been identified as an inducible regulator of mitochondrial function. Skeletal muscle PGC-1α expression is induced post-exercise. Therefore, we sought to determine its role in the regulation of muscle fuel metabolism. Studies were performed using conditional, muscle-specific, PGC-1α gain-of-function and constitutive, generalized, loss-of-function mice. Forced expression of PGC-1α increased muscle glucose uptake concomitant with augmentation of glycogen stores, a metabolic response similar to post-exercise recovery. Induction of muscle PGC-1α expression prevented muscle glycogen depletion during exercise. Conversely, PGC-1α-deficient animals exhibited reduced rates of muscle glycogen repletion post-exercise. PGC-1α was shown to increase muscle glycogen stores via several mechanisms including stimulation of glucose import, suppression of glycolytic flux, and by down-regulation of the expression of glycogen phosphorylase and its activating kinase, phosphorylase kinase α. These findings identify PGC-1α as a critical regulator of skeletal muscle fuel stores.

Transcriptional coactivators PGC-1α and PGC-lβ control overlapping programs required for perinatal maturation of the heart

Oxidative tissues such as heart undergo a dramatic perinatal mitochondrial biogenesis to meet the high-energy demands after birth. PPARgamma coactivator-1 (PGC-1) alpha and beta have been implicated in the transcriptional control of cellular energy metabolism. Mice with combined deficiency of PGC-1alpha and PGC-1beta (PGC-1alphabeta(-/-) mice) were generated to investigate the convergence of their functions in vivo. The phenotype of PGC-1beta(-/-) mice was minimal under nonstressed conditions, including normal heart function, similar to that of PGC-1alpha(-/-) mice generated previously. In striking contrast to the singly deficient PGC-1 lines, PGC-1alphabeta(-/-) mice died shortly after birth with small hearts, bradycardia, intermittent heart block, and a markedly reduced cardiac output. Cardiac-specific ablation of the PGC-1beta gene on a PGC-1alpha-deficient background phenocopied the generalized PGC-1alphabeta(-/-) mice. The hearts of the PGC-1alphabeta(-/-) mice exhibited signatures of a maturational defect including reduced growth, a late fetal arrest in mitochondrial biogenesis, and persistence of a fetal pattern of gene expression. Brown adipose tissue (BAT) of PGC-1alphabeta(-/-) mice also exhibited a severe abnormality in function and mitochondrial density. We conclude that PGC-1alpha and PGC-1beta share roles that collectively are necessary for the postnatal metabolic and functional maturation of heart and BAT.

Angiotensin AT1 receptor antagonist irbesartan decreases lesion size, chemokine expression, and macrophage accumulation in apolipoprotein E-deficient mice.

&NA; Recent data suggest that angiotensin II AT1 receptor antagonists may be beneficial in the treatment of atherosclerosis. To clarify how AT1 receptor antagonists reduce atherosclerosis, the effect of irbesartan on atherosclerotic lesion development was determined in low‐fat, chow‐fed apolipoprotein (Apo) E‐deficient mice. Irbesartan (50 mg/kg per day) strongly decreased lesion development after a 12‐week treatment period (lesion size: irbesartan treated, 20,524 ± 4,200 &mgr;m2 vs. control, 99,600 ± 14,500; 79.4% inhibition, p < 0.001). This effect was not due to an effect of irbesartan on lipoprotein levels because irbesartan slightly increased total cholesterol levels and decreased the ratio of Apo A‐I relative to Apo B levels. Immunochemical analysis of the atherosclerotic lesions using the mac3 monoclonal antibody showed the presence of macrophages in the lesions of control mice, whereas sections from irbesartan‐treated animals only showed occasional labeling in the lesion area. These data suggest that irbesartan inhibits monocyte/macrophage influx into the vessel wall. Therefore, expression levels of monocyte chemoattractant protein‐1 (MCP‐1), as well as other chemokines involved in macrophage infiltration into the lesion area, were measured in the aortic sinus of control and irbesartan‐treated animals. Irbesartan treatment strongly decreased MCP‐1 mRNA levels as well as MCP‐1 immunostaining in the lesion area. This effect of irbesartan on MCP‐1 occurred without an effect on CCR2, the receptor of MCP‐1. Expression of macrophage inflammatory protein (MIP)‐1&agr;, another CC chemokine expressed in atherosclerotic lesions, was also reduced after irbesartan treatment, without effect on CCR3 and CCR5, the receptors of MIP‐1&agr;. Concomitantly, the expression of the angiogenic chemokines KC and MIP‐2, which are functionally related to interleukin‐8, were downregulated, whereas their shared receptor CXCR2 was upregulated. These data suggest that inhibition of the inflammatory component of lesion progression plays an important role in the inhibitory effect of AT1 receptor antagonists on atherosclerotic lesion formation.

Human umbilical vein endothelial cells express high affinity receptors for factor Xa.

The binding of [125I]‐factor Xa to human umbilical vein endothelial cell (HUVEC) monolayers was studied. At 7°C, [125I]‐factor Xa bound to a single class of binding sites with a dissociation constant value of 6.6 ± 0.8 nM and a binding site density of 57,460 ± 5,200 sites/cell (n = 3). Association and dissociation kinetics were of a pseudo‐first order and gave association and dissociation rate constant values of 0.15 × 106 M‐1 s‐1 and 4.0 × 10‐4 s‐1, respectively. [125I]‐factor Xa binding was inhibited by factor Xa but was not affected by factor X, thrombin or monoclonal antibodies against factor V, antithrombin‐III or tissue factor pathway inhibitor (TFPI) but was inhibited by an antibody specific for the effector cell protease receptor‐1 (EPR‐1), a well‐known receptor of factor Xa on various cell types. [125I]‐factor Xa binding to HUVEC was not affected by various inhibitors of factor Xa such as DX 9065, pentasaccharide‐antithrombin‐III or TFPI. Factor Xa increased intracellular free calcium levels and phosphoinositide turnover in endothelial cells and, when added to HUVEC in culture, factor Xa was a potent mitogen, stimulating an increase in cell number at a 0.3 to 100 nM concentration. HUVEC‐bound factor Xa promoted prothrombin activation in the presence of factor Va only. This effect was inhibited by both indirect and direct inhibitors of factor Xa. These findings indicate that HUVEC express functional high affinity receptors for factor Xa, related to EPR‐1, which may be of importance in the regulation of coagulation and homeostasis of the vascular wall. J. Cell. Physiol. 172:36–43, 1997.

Impaired contractile function and calcium handling in hearts of cardiac-specific calcineurin b1-deficient mice

To define the necessity of calcineurin (Cn) signaling for cardiac maturation and function, the postnatal phenotype of mice with cardiac-specific targeted ablation of the Cn B1 regulatory subunit (Ppp3r1) gene (csCnb1(-/-) mice) was characterized. csCnb1(-/-) mice develop a lethal cardiomyopathy, characterized by impaired postnatal growth of the heart and combined systolic and diastolic relaxation abnormalities, despite a lack of structural derangements. Notably, the csCnb1(-/-) hearts did not exhibit diastolic dilatation, despite the severe functional phenotype. Myocytes isolated from the mutant mice exhibited reduced rates of contraction/relaxation and abnormalities in calcium transients, consistent with altered sarcoplasmic reticulum loading. Levels of sarco(endo) plasmic reticulum Ca-ATPase 2a (Atp2a2) and phospholamban were normal, but phospholamban phosphorylation was markedly reduced at Ser(16) and Thr(17). In addition, levels of the Na/Ca exchanger (Slc8a1) were modestly reduced. These results define a novel mouse model of cardiac-specific Cn deficiency and demonstrate novel links between Cn signaling, postnatal growth of the heart, pathological ventricular remodeling, and excitation-contraction coupling.

Chronic cold acclimation increases thermogenic capacity, non-shivering thermogenesis and muscle citrate synthase activity in both wild-type and brown adipose tissue deficient mice.

The purpose of this study was to determine whether chronic cold exposure would increase the aerobic capacity of skeletal muscle in UCP-dta mice, a transgenic line lacking brown adipose tissue (BAT). Wild type and UCP-dta mice were acclimated to either warm (23 °C), or cold (4 °C) conditions. Cold increased muscle oxidative capacity nearly equivalently in wild-type and UCP-dta mice, but did not affect the respiratory function of isolated mitochondria. Summit metabolism ( ̇V O2summit) and norepinephrine-induced thermogenesis ( ̇V O2NST) were significantly lower in UCP-dta mice relative to wild-type mice regardless of temperature treatment, but both were significantly higher in cold relative to warm acclimated mice. BAT mass was significantly higher in the cold relative to warm acclimated wild-type mice, but not in cold acclimated UCP-dta mice. BAT citrate synthase activity was lower in transgenic animals regardless of acclimation temperature and BAT citrate synthase activity per depot was significantly higher only in the cold acclimated wild-type mice. Muscle citrate synthase activity was increased in both genotypes. As defects in muscle oxidative function have been observed with obesity and type 2 diabetes, these results suggest that chronic cold exposure is a useful intervention to drive skeletal muscle oxidative capacity in mouse models of obesity.

Life-threatening thrombosis in mice with targeted Arg48-to-cys mutation of the heparin-binding domain of antithrombin

Abstract— Antithrombin (AT) inhibits thrombin and some other coagulation factors in a reaction that is dramatically accelerated by binding of a pentasaccharide sequence present in heparin/heparan-sulfate to a heparin-binding site on AT. Based on the involvement of R47 in the heparin/AT interaction and the frequent occurrence of R47 mutations in AT deficiency patients, targeted knock-in of the corresponding R48C substitution in AT in mice was performed to generate a murine model of spontaneous thrombosis. The mutation efficiently abolished the effect of heparin-like molecules on coagulation inhibition in vitro and in vivo. Mice homozygous for the mutation (ATm/m mice) developed spontaneous, life-threatening thrombosis, occurring as early as the day of birth. Only 60% of the ATm/m offspring reached weaning age, with further loss at different ages. Thrombotic events in adult homozygotes were most prominent in the heart, liver, and in ocular, placental, and penile vessels. In the neonate, spontaneous death invariably was associated with major thrombosis in the heart. This severe thrombotic phenotype underlines a critical function of the heparin-binding site of antithrombin and its interaction with heparin/heparan-sulfate moieties in health, reproduction, and survival, and represents an in vivo model for comparative analysis of heparin-derived and other antithrombotic molecules.