TGF 1 / Smad 3 signaling regulates hepatic gluconeogenesis 1 TGF-/ Smad 3 pathway targets PP 2 A-AMPK-FoxO 1 to regulate hepatic gluconeogenesis

Maintenance of glucose homeostasis is essential for normal physiology. Deviation from normal glucose levels, in either direction, increases susceptibility to serious medical complications such as hypoglycemia or diabetes. Maintenance of glucose homeostasis is achieved via functional interactions amongst various organs – liver, skeletal muscle, adipose tissue, brain and the endocrine pancreas. The liver is the primary site for endogenous glucose production, especially during states of prolonged fasting. However, enhanced gluconeogenesis is also a signature feature of type 2 diabetes (T2D). Thus, elucidating the signaling pathways that regulate hepatic gluconeogenesis would allow better insight into the process of normal endogenous glucose production as well as how this process is impaired in T2D. Here we demonstrate that the TGF-/Smad3 signaling pathway promotes hepatic gluconeogenesis, both upon prolonged fasting and during T2D. In contrast, genetic and pharmacological inhibition of TGF-/Smad3 signals suppressed endogenous glucose production. TGF- and Smad3 signals achieve this effect via targeting key regulators of hepatic gluconeogenesis, the protein phosphatase 2A (PP2A), AMPK and FoxO1 proteins. Specifically, TGF- signaling suppressed the LKB1-AMPK axis thereby facilitating the nuclear translocation of FoxO1 and activation of key gluconeogenic genes, glucose-6phosphatase and phosphoenol-pyruvate carboxy kinase. These findings underscore an important role of TGF-/Smad3 signaling in hepatic gluconeogenesis, both in normal physiology and in the pathophysiology of metabolic diseases such as diabetes, and are thus of significant medical relevance. Glucose homeostasis is achieved via the integration of physiological processes occurring in multiple organs. Glycolysis and gluconeogenesis represent two competing processes that regulate blood glucose levels (1). Hepatic gluconeogenesis is a critical mechanism for endogenous glucose production during periods of prolonged fasting. However, deregulated hepatic gluconeogenesis is a common feature in patients with type 2 diabetes (T2D) (2). Expression of phosphoenol pyruvate-carboxy kinase (PEPCK) and glucose-6-phosphatases http://www.jbc. org/cgi/doi/10.1074/jbc.M116.764910 The latest version is at JBC Papers in Press. Published on January 9, 2017 as Manuscript M116.764910 Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Jauary 3, 2017 hp://w w w . jb.org/ D ow nladed from TGF1/Smad3 signaling regulates hepatic gluconeogenesis 2 (G6Pase) (3), key rate limiting enzymes for hepatic gluconeogenesis, increase abnormally in T2D (4). Likewise, the transgenic over expression of these enzymes in mice results in hyperglycemia, hyperinsulinemia and insulin resistance (5,6). In contrast, deletion of PEPCK reverses hyperglycemia and insulin resistance in diabetes prone db/db mice (7). The AMP-activated protein kinase (AMPK) signaling pathway, among others, regulates the expression of PEPCK and G6Pase (8). AMPK is a heterotrimeric protein, consisting of 1/2-, 1/2and 1/2/3subunits, that regulates glucose and fatty acid metabolism (9). Phosphorylation of Thr172 in the 1-subunit is important for AMPK activation, which is in turn regulated by two upstream kinases, LKB1 and Ca2+calmodulin dependent protein kinases  and  (10,11). Further, AMPK is a serine/threonine kinase and activation of hepatic AMPK suppresses gluconeogenesis via substrate phosphorylation (12). The commonly prescribed anti-diabetic drug, Metformin, and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) both activate AMPK via LKB1-dependent mechanisms to suppress hepatic gluconeogenesis (13). In addition, AMPK suppresses gluconeogenesis via downregulation of FoxO1 target genes (14), through increasing FoxO1 phosphorylation (15). FoxO1 promotes hepatic gluconeogenesis via transcriptional activation of PEPCK and G6Pase (16,17). The transforming growth factor-beta (TGF-) signaling pathway regulates biological processes including cell proliferation, differentiation and death (18). Canonical TGF- signaling involves ligand binding to the TGF- receptor II (TRII) that promotes phosphorylation and activation of TGF- receptor I (TRI), which in turn phosphorylates and activates the downstream Smad transcription factors. Smad3 is the preferred downstream transcription factor for TGF- isoforms. Once activated Smad3 complexes with Smad4 and translocates to the nucleus to bind target promoters. In addition to the SMAD pathway, TGF-signaling is known to interact with non-SMAD signaling nodes (19). For example, TGF- signaling interacts with insulin receptor signaling in the nematode Caenorhabditis elegans (C. elegans). TGF-/daf-7 activates the TGFreceptor I and II kinases (daf-1 and daf-4) and Smad transcription factor (daf-8 and daf-14) network and interacts with FoxO1 (daf-16), LKB1 (par-4) and AMPK (aak-1/aak-2) to regulate metabolic and nutrient sensory pathways and glucose metabolism (20) (21). Along those lines, we have illustrated the key role of TGF- signaling in regulating the function of diverse cell types tasked with maintenance of glucose homeostasis (22-24). Here, we investigated the role of TGF- signaling in regulating hepatic gluconeogenesis, in normal physiology and during the pathogenesis of metabolic disease. We found that TGF-/Smad promotes hepatic gluconeogenesis via targeting the PP2A-AMPK-FoxO1 pathways during fasting/feeding and during pathologic conditions like insulin resistance and diabetes.