Skeletal muscle plays important roles in whole-body glucose and fatty acid metabolism. However, muscle also secretes cytokines and growth factors (collectively termed myokines) that can potentially act in an autocrine, a paracrine, and/or an endocrine manner to modulate metabolic, inflammatory, and other processes. Here, we report the identification and characterization of myonectin, a novel myokine belonging to the C1q/TNF-related protein (CTRP) family. Myonectin transcript was highly induced in differentiated myotubes and predominantly expressed by skeletal muscle. Circulating levels of myonectin were tightly regulated by the metabolic state; fasting suppressed, but refeeding dramatically increased, its mRNA and serum levels. Although mRNA and circulating levels of myonectin were reduced in a diet-induced obese state, voluntary exercise increased its expression and circulating levels. Accordingly, myonectin transcript was up-regulated by compounds (forskolin, epinephrine, ionomycin) that raise cellular cAMP or calcium levels. In vitro, secreted myonectin forms disulfide-linked oligomers, and when co-expressed, forms heteromeric complexes with other members of the C1q/TNF-related protein family. In mice, recombinant myonectin administration reduced circulating levels of free fatty acids without altering adipose tissue lipolysis. Consistent with this, myonectin promoted fatty acid uptake in cultured adipocytes and hepatocytes, in part by up-regulating the expression of genes (CD36, FATP1, Fabp1, and Fabp4) that promote lipid uptake. Collectively, these results suggest that myonectin links skeletal muscle to lipid homeostasis in liver and adipose tissue in response to alterations in energy state, revealing a novel myonectin-mediated metabolic circuit.

Adipose tissue-derived adipokines play important roles in controlling systemic insulin sensitivity and energy balance. Our recent efforts to identify novel metabolic mediators produced by adipose tissue have led to the discovery of a highly conserved family of secreted proteins, designated as C1q/TNF-related proteins 1–10 (CTRP1 to -10). However, physiological functions regulated by CTRPs are largely unknown. Here we provide the first in vivo functional characterization of CTRP3. We show that circulating levels of CTRP3 are inversely correlated with leptin levels; CTRP3 increases with fasting, decreases in diet-induced obese mice with high leptin levels, and increases in leptin-deficient ob/ob mice. A modest 3-fold elevation of plasma CTRP3 levels by recombinant protein administration is sufficient to lower glucose levels in normal and insulin-resistant ob/ob mice, without altering insulin or adiponectin levels. The glucose-lowering effect in mice is linked to activation of the Akt signaling pathway in liver and a marked suppression of hepatic gluconeogenic gene expression. Consistent with its effects in mice, CTRP3 acts directly and independently of insulin to regulate gluconeogenesis in cultured hepatocytes. In humans, alternative splicing generates two circulating CTRP3 isoforms differing in size and glycosylation pattern. The two human proteins form hetero-oligomers, an association that does not require interdisulfide bond formation and appears to protect the longer isoform from proteolytic cleavage. Recombinant human CTRP3 also reduces glucose output in hepatocytes by suppressing gluconeogenic enzyme expression. This study provides the first functional evidence linking CTRP3 to hepatic glucose metabolism and establishes CTRP3 as a novel adipokine.

Spatial compartmentalization of signaling pathway components generally defines the specificity and enhances the efficiency of signal transduction. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is known to be compartmentalized within plasma membrane microdomains; however, the underlying mechanisms and functional impact of this compartmentalization are not well understood. Here, we show that phosphoinositide-dependent kinase 1 is activated in membrane rafts in response to growth factors, whereas the negative regulator of the pathway, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), is primarily localized in nonraft regions. Alteration of this compartmentalization, either by genetic targeting or ceramide-induced recruitment of PTEN to rafts, abolishes the activity of the entire pathway. These findings reveal critical steps in raft-mediated PI3K/Akt activation and demonstrate the essential role of membrane microdomain compartmentalization in enabling PI3K/Akt signaling. They further suggest that dysregulation of this compartmentalization may underlie pathological complications such as insulin resistance.

We previously described the adipokine CTRP1, which has up-regulated expression following exposure to the anti-diabetic drug rosiglitazone and increased circulating levels in adiponectin-null mice (Wong, G. W., Krawczyk, S. A., Kitidis-Mitrokostas, C., Revett, T., Gimeno, R., and Lodish, H. F. (2008) Biochem. J. 416, 161–177). Although recombinant CTRP1 lowers blood glucose in mice, its physiological function, mechanisms of action, and roles in metabolic stress remain unknown. Here, we show that circulating levels of CTRP1 are strikingly reduced in diet-induced obese mice. Overexpressing CTRP1 in transgenic mice improved insulin sensitivity and decreased high-fat diet-induced weight gain. Reduced adiposity resulted from enhanced fatty acid oxidation and energy expenditure, effects mediated by AMP-activated protein kinase (AMPK). In skeletal muscle of transgenic mice, AMPKα and its downstream target, acetyl-CoA carboxylase (ACC), were hyperphosphorylated, indicative of AMPK activation and ACC inhibition. Inactivation of ACC promotes mitochondrial fat oxidation. Consistent with the direct effect of CTRP1 on AMPK signaling, recombinant CTRP1 administration acutely stimulated muscle AMPKα and ACC phosphorylation in vivo. In isolated soleus muscle, recombinant CTRP1 activated AMPK signaling to increase fatty acid oxidation ex vivo, an effect abrogated by an AMPK inhibitor. These results provide the first in vivo evidence that CTRP1 is a novel regulator of fatty acid metabolism.

Despite the prevalence of insulin resistance and type 2 diabetes mellitus, their underlying mechanisms remain incompletely understood. Many secreted endocrine factors and the intertissue cross-talk they mediate are known to be dysregulated in type 2 diabetes mellitus. Here, we describe CTRP12, a novel adipokine with anti-diabetic actions. The mRNA and circulating levels of CTRP12 were decreased in a mouse model of obesity, but its expression in adipocytes was increased by the anti-diabetic drug rosiglitazone. A modest rise in circulating levels of CTRP12 by recombinant protein administration was sufficient to lower blood glucose in wild-type, leptin-deficient ob/ob, and diet-induced obese mice. A short term elevation of serum CTRP12 by adenovirus-mediated expression improved glucose tolerance and insulin sensitivity, normalized hyperglycemia and hyperinsulinemia, and lowered postprandial insulin resistance in obese and diabetic mice. CTRP12 improves insulin sensitivity in part by enhancing insulin signaling in the liver and adipose tissue. Further, CTRP12 also acts in an insulin-independent manner; in cultured hepatocytes and adipocytes, CTRP12 directly activated the PI3K-Akt signaling pathway to suppress gluconeogenesis and promote glucose uptake, respectively. Collectively, these data establish CTRP12 as a novel metabolic regulator linking adipose tissue to whole body glucose homeostasis through insulin-dependent and independent mechanisms.

Members of the C1q/TNF family play important and diverse roles in the immune, endocrine, skeletal, vascular, and sensory systems. Here, we identify and characterize CTRP13, a new and extremely conserved member of the C1q/TNF family. CTRP13 is preferentially expressed by adipose tissue and the brain in mice and predominantly by adipose tissue in humans. Within mouse adipose tissue, CTRP13 is largely expressed by cells of the stromal vascular compartment. Due to sexually dimorphic expression patterns, female mice have higher transcript and circulating CTRP13 levels than males. CTRP13 transcript and circulating levels are elevated in obese male mice, suggesting a potential role in energy metabolism. The insulin-sensitizing drug rosiglitazone also increases the expression of CTRP13 in adipocytes, which correlates with the insulin-sensitizing action of CTRP13. In a heterologous expression system, CTRP13 is secreted as a disulfide-linked oligomeric protein. When co-expressed, CTRP13 forms heteromeric complexes with a closely related family member, CTRP10. This heteromeric association does not involve conserved N-terminal Cys residues. Functional studies using purified recombinant protein demonstrated that CTRP13 is an adipokine that promotes glucose uptake in adipocytes, myotubes, and hepatocytes via activation of the AMPK signaling pathway. CTRP13 also ameliorates lipid-induced insulin resistance in hepatocytes through suppression of the SAPK/JNK stress signaling that impairs the insulin signaling pathway. Further, CTRP13 reduces glucose output in hepatocytes by inhibiting the mRNA expression of gluconeogenic enzymes, glucose-6-phosphatase and the cytosolic form of phosphoenolpyruvate carboxykinase. These results provide the first functional characterization of CTRP13 and establish its importance in glucose homeostasis.