FAM3A belongs to a novel cytokine-like gene family, and its physiological role remains largely unknown. In our study, we found a marked reduction of FAM3A expression in the livers of db/db and high-fat diet (HFD)-induced diabetic mice. Hepatic overexpression of FAM3A markedly attenuated hyperglycemia, insulin resistance, and fatty liver with increased Akt (pAkt) signaling and repressed gluconeogenesis and lipogenesis in the livers of those mice. In contrast, small interfering RNA (siRNA)-mediated knockdown of hepatic FAM3A resulted in hyperglycemia with reduced pAkt levels and increased gluconeogenesis and lipogenesis in the livers of C57BL/6 mice. In vitro study revealed that FAM3A was mainly localized in the mitochondria, where it increases adenosine triphosphate (ATP) production and secretion in cultured hepatocytes. FAM3A activated Akt through the p110α catalytic subunit of PI3K in an insulin-independent manner. Blockade of P2 ATP receptors or downstream phospholipase C (PLC) and IP3R and removal of medium calcium all significantly reduced FAM3A-induced increase in cytosolic free Ca2+ levels and attenuated FAM3A-mediated PI3K/Akt activation. Moreover, FAM3A-induced Akt activation was completely abolished by the inhibition of calmodulin (CaM). Conclusion: FAM3A plays crucial roles in the regulation of glucose and lipid metabolism in the liver, where it activates the PI3K-Akt signaling pathway by way of a Ca2+/CaM-dependent mechanism. Up-regulating hepatic FAM3A expression may represent an attractive means for the treatment of insulin resistance, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD). (Hepatology 2014;59:1779–1790)
Type 2 diabetes has become one of the most prevalent and debilitating chronic diseases, with a global prevalence 6.4%, affecting about 285 million adults in the year 2010. Hepatic insulin resistance and fatty liver play a crucial role in the development and progression of type 2 diabetes. Liver is the key tissue regulating release of glucose into circulation during the fasting state, and hepatic insulin resistance is a decisive factor causing fasting hyperglycemia and type 2 diabetes. The liver is also one of the major organs regulating triglyceride (TG) and cholesterol (CHO) metabolism. Hepatic insulin resistance is mainly described as the failure of insulin to repress the expression of gluconeogenic genes through the PI3K/Akt signaling pathway and is closely associated with the dysregulation of glucose and lipid metabolism in the liver. Although the underlying mechanisms remain largely unknown, increasing evidence points to a close association between reduced hepatic adenosine triphosphate (ATP) synthesis and the progression of hepatic insulin resistance, steatosis, and type 2 diabetes in both human and rodents.
The FAM3 gene family is a novel cytokine-like gene family, which includes four members designated as FAM3A, FAM3B, FAM3C, and FAM3D. Due to the high abundance of FAM3B in pancreatic islets, it is also called pancreatic-derived factor (PANDER). We and others have previously shown that PANDER negatively regulates pancreatic β cell function and hepatic insulin sensitivity.[5-9] More recently, liver-derived PANDER is found to be involved in the progression of nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes.[10, 11] Increased hepatic PANDER expression in insulin-resistant mice promotes gluconeogenesis and lipogenesis in the liver by way of the inhibition of Akt and adenosine monophosphate kinase (AMPK) and the activation of FOXO1. In contrast, PANDER deficiency protects against high-fat diet (HFD)-induced hyperglycemia in mice. Collectively, these findings suggest that dysregulated PANDER may promote the pathogenesis of type 2 diabetes by decreasing pancreatic β cell function and hepatic insulin sensitivity.
To date, the physiological role of FAM3A remains largely unknown. In the present study, we report that FAM3A is constitutively expressed in the liver, where its expression is significantly decreased in db/db and HFD-induced diabetic mice. Hepatic overexpression of FAM3A significantly attenuates hyperglycemia, insulin resistance, and fatty liver in those mice by way of insulin-independent, Ca2+/CaM-mediated activation of PI3K/Akt signaling. These findings reveal a vital role of FAM3A in the regulation of glucose and lipid metabolism in the liver.
Since the discovery of the FAM3 gene family, the roles of FAM3B (PANDER) in glucose and lipid metabolism have been extensively investigated.[4, 5] However, the biological function of FAM3A remains poorly understood. In this study, we found FAM3A expression was reduced in the livers of patients with NAFLD and obese diabetic mice. Physical exercise, which attenuated hyperglycemia and fatty liver in db/db mice, increased hepatic FAM3A expression levels. These findings implicate that deregulated FAM3A expression might be associated with the pathogenesis of fatty liver and hepatic insulin resistance.
Regarding possible mechanisms underlying the connections between exercise and FAM3A, we have recently demonstrated that FAM3A expression in liver cells was down-regulated by fatty acids, but up-regulated by PPARγ activation. We also found that chronic exposure to high concentrations of insulin repressed FAM3A expression in HepG2 cells (data not shown). Therefore, swimming exercise may restore FAM3A expression in diabetic mouse livers by way of reducing hepatic lipid accumulation and attenuating hyperinsulinemia.
OGTT, pyruvate tolerance test, and hyperinsulinemic-euglycemic clamp assays indicated that hepatic overexpression of FAM3A markedly attenuated hyperglycemia and insulin resistance, suggesting suppressed hepatic gluconeogenesis and improved insulin signaling may be responsible for metabolic improvement in db/db mice. FAM3A overexpression also attenuated hepatic steatosis, possibly by way of repressing fatty acid synthesis and increasing fatty acid oxidation and VLDL-TG secretion. Hepatic overexpression of FAM3A improved global insulin sensitivity and attenuated fatty liver with increased pAkt levels in the livers of diabetic mice. In contrast, siRNA-mediated knockdown of hepatic FAM3A resulted in a fasting hyperglycemia and increased hepatic lipid accumulation, with reduced pAkt levels and increased gluconeogenesis in the livers of normal mice. Mechanistically, FAM3A increases mitochondrial ATP production and secretion, which activates the PI3K-Akt signaling pathway by way of an insulin-independent, ATP receptor-mediated mechanism. FAM3A may thus represent an attractive target for the treatment of insulin resistance, type 2 diabetes, and NAFLD. In addition to blocking gluconeogenesis and glycogenolysis, Akt has also been found to play a critical role in the regulation of liver lipid metabolism. Activation of Akt represses lipogenesis and prevents excessive lipid deposition in the liver by way of the inactivation of GSK-3, PGC-1α, and FOXO1[11, 20-22] and increased hepatic pAkt levels in the liver is associated with the amelioration of steatosis in diabetic mice. As expected, hepatic overexpression of FAM3A significantly attenuated steatosis in db/db mice and HFD-induced diabetic mice. In addition, hepatic FAM3A overexpression increases, while hepatic knockdown of FAM3A reduces, AdipoR1 expression, suggesting FAM3A may attenuate gluconeogenesis and lipogenesis and increase fatty acid oxidation by way of activation of AdipoR/AMPK signaling in the liver.
In the present study, we found that FAM3A activates AMPK activity regardless of increased ATP production. Multiple mechanisms are involved in AMPK activation. It has been recently demonstrated that AdipoR1 also plays an important role in the regulation of AMPK activation. AdipoR1 gene-deficient mouse livers exhibit blunted AMPK activation induced by adiponectin, while overexpression of AdipoR1 increases AMPK activity in db/db mouse livers. Given the important role of AdipoR1 in the activation of AMPK, it is likely that up-regulation of AdipoR1 contributes to FAM3A-induced AMPK activation in diabetic mouse livers.
We further showed that FAM3A activates Akt by way of a PI3K p110α-dependent pathway in liver cells, which is consistent with recent report that p110α predominantly mediates Akt activation in the liver, where selective inhibition of p110α increases lipid deposition. Interestingly, FAM3A can activate Akt in the absence of insulin, with little effect on pIRS-1 levels and the association of p85 with IRS-1, suggesting the FAM3A-activated p110α/Akt signaling pathway is insulin-independent. In support, hepatic FAM3A overexpression also activated Akt and attenuated of hyperglycemia and hepatic gluconeogenesis in type 1 diabetic mice.
For the first time, we found that FAM3A protein is present in the mitochondria of mouse hepatocytes and HepG2 cells. Furthermore, FAM3A protein was increased in the mitochondria of mouse livers and HepG2 cells treated with Ad-FAM3A. These observations demonstrate that FAM3A is a novel mitochondrial protein. In vivo, hepatic FAM3A overexpression increased, while hepatic FAM3A knockdown reduced ATP levels in mouse livers. In vitro, FAM3A overexpression increased intracellular and extracellular ATP levels in both HepG2 cells and primary cultured mouse hepatocytes. These findings strongly support the possibility that increased expression of mitochondrial FAM3A enhances ATP production, thereby increasing intracellular and extracellular levels of ATP in hepatocytes. However, the mechanism by which FAM3A increases mitochondrial ATP synthesis warrants further investigation.
It has been previously reported that ATP can be released and function as a signaling molecule to activate the PI3K/Akt signaling pathway by way of ATP receptors in many organs or cell types.[18, 28, 29] The receptors mediating such action include P2 purinoceptors, i.e., P2X and P2Y receptors. P2X receptors are ligand-gated ion channels that are permeable to calcium, while P2Y receptors are G-protein-coupled receptors that stimulate PLC to increase IP3 production, resulting in calcium release from internal stores. As a result of increased cytosolic free calcium level upon extracellular ATP treatment, CaM is activated, leading to the activation of the PI3K/Akt pathway.[30, 31] In the present study, we confirmed that treatment of HepG2 cells with exogenous ATP elevated cytosolic free calcium and activated Akt in a p110α-dependent manner, which was attenuated by the inhibition of the P2 receptors or their downstream signaling molecules, including PLC and IP3R. FAM3A-induced intracellular calcium level increases and Akt activation were significantly blocked by P2 receptor antagonists, PLC inhibitor, IP3R antagonist, and depletion of extracellular calcium in HepG2 cells. Furthermore, the finding that the CaM antagonist CPZ completely abolished FAM3A-induced Akt activation reveals a critical role of increased cellular calcium level in this process. Collectively, these findings demonstrate that both P2 receptor isoforms are involved in FAM3A-induced increases in cytosolic free calcium and Akt activation in liver cells. Interestingly, although blockage of the P2 receptors completely inhibited Akt activation induced by exogenous ATP, it did not completely abolish FAM3A-induced Akt activation, suggesting that other minor mechanism(s) may also be involved. One possibility is that FAM3A-elicited increase in intracellular ATP level leads to the closure of the KATP channels, resulting in increased calcium influx through L-type calcium channel.
The findings that exogenous ATP can elicit intracellular calcium signaling and Akt activation through P2 receptors in liver cells suggests a novel mechanism involved in hepatic glucose and lipid homeostasis regulation (Fig. 7). Increasing evidence has revealed that impaired energy metabolism in the liver could be an early defect in the pathogenesis of type 2 diabetes in patients.[32, 33] It has been also reported that reduced hepatic ATP levels are associated with the development of NAFLD in rats. These observations support a possibility that defective ATP synthesis and P2 receptor-mediated signaling may underlie hepatic insulin resistance and elevated liver lipid contents.
In summary, the present study demonstrates that FAM3A plays a crucial role in hepatic glucose and lipid metabolism. FAM3A enhances ATP synthesis and elevates extracellular ATP levels, which activate P2 receptors to increase cytosolic free calcium level, leading to activation PI3K-Akt signaling pathway in a CaM-dependent manner and suppression of hepatic gluconeogenesis and lipogenesis. In addition, FAM3A may also activate the AdipoR1-AMPK signaling pathway and enhance fatty acid β oxidation in the liver by way of a yet unknown mechanism. In conclusion, to increase hepatic ATP synthesis and signaling by way of up-regulation of FAM3A might represent a promising means for treatment of insulin resistance and NAFLD.