Unraveling the Regulation of Hepatic Gluconeogenesis

Abstract

Hepatic gluconeogenesis, de novo glucose synthesis from available precursors, plays a crucial role in maintaining glucose homeostasis to meet energy demands during prolonged starvation in animals. The abnormally increased rate of hepatic gluconeogenesis contributes to hyperglycemia in diabetes. Gluconeogenesis is regulated on multiple levels, such as hormonal secretion, gene transcription, and posttranslational modification. We review here the molecular mechanisms underlying the transcriptional regulation of gluconeogenesis in response to nutritional and hormonal changes. The nutrient state determines the hormone release, which instigates the signaling cascades in the liver to modulate the activities of various transcriptional factors through various post-translational modifications like phosphorylation, methylation, and acetylation. AMP-activated protein kinase (AMPK) can mediate the activities of some transcription factors, however its role in the regulation of gluconeogenesis remains uncertain. Metformin, a primary hypoglycemic agent of type 2 diabetes, ameliorates hyperglycemia predominantly through suppression of hepatic gluconeogenesis. Several molecular mechanisms have been proposed to be metformin's mode of action.

Keywords: AMPK; acetylation; gluconeogenesis; hormone; metformin; methylation; transcription factor.

Figure 1

Schematic overview of major enzymes and metabolites involved in the regulation of gluconeogenesis. Pyruvate derived from lactate and alanine enters the mitochondrion, where it is carboxylated to oxaloacetate (OAA) by pyruvate carboxylase (PC). OAA is then reduced to malate to be shuttled to the cytoplasm where it is reoxidized to OAA, which is decarboxylated and then phosphorylated to phosphoenolpyruvate (PEP) by PEP carboxykinase (PEPCK). PEP then enters the gluconeogenic cycle. After several steps of reverse glycolysis, the yield fructose 1,6-bisphosphate (F1,6BP) is dephosphorylated by fructose 1,6-bisphosphatase (FBPase) to form fructose 6-phosphate, which is then converted to glucose-6-phosphate (G6P) by phosphoglucoisomerase. G6P is finally converted to glucose via dephosphorylation by glucose-6-phosphatase (G6Pase). Other gluconeogenic amino acids (Asp/Asn and Glu/Gln) are converted into alanine or specific intermediates in the tricarboxylic acid (TCA) cycle for gluconeogenesis. Glycerol is converted to dihydroxyacetone phosphate (DHAP), which is then converted to glyceraldehyde-3-phosphate (Glyceral-3-P) or F1,6BP entering the gluconeogenic pathway. ALT, alanine aminotransaminase; AST, aspartate aminotransaminase; GK, glucokinase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase. Gluconeogenic and glycolytic enzymes are highlighted in red and green, respectively.

Figure 2

Hepatic gluconeogenesis is regulated by various transcriptional factors in response to the hormonal changes. Under feeding conditions (right part), insulin-dependent activation of the AKT signaling stimulate FoxO1 phosphorylation and cytoplasmic retention. Meanwhile, CREB-regulated transcriptional coactivator-2 (CRTC2) undergoes rapid phosphorylation by salt-inducible kinases (SIKs), which results in its sequestration in the cytoplasm via interaction with 14-3-3 adaptor protein. Transcription factor 7-like 2 (TCF7L2) blocks gene expression through occupying transcriptional recognition sites. Small heterodimer partner-interacting leucine zipper protein (SMILL) and small heterodimer partner (SHP)/DAX1 directly bind to transcriptional factors to interfere with transcription. Under fasting conditions (left part), binding of glucagon or thyroid stimulating hormone (TSH) to its cognate G-protein-coupled receptor, respectively, stimulate adenylyl cyclase (AC), which converts ATP to cAMP. The cAMP in turn stimulates protein kinase A (PKA) to phosphorylate cAMP response element binding protein (CREB). Phosphorylated CREB transfers into the nucleus where it interacts with its coactivator CRTC2 and CBP/300 to form a complex, which binds to the CRE in the gluconeogenic promoters. CREB-CRTC-CBP/300 complex also binds to the promoters of PGC1-1α and FoxO1 and stimulates their transcriptions. Nuclear factor-Y (NF-Y) induced by cAMP interacts with CREB to enhance the expression of PEPCK and G6Pase genes. Glucocorticoid and thyroid hormone (TH) bind gluconeogenic promoters in a hormone-receptor complex manner. Yin Yang 1 (YY1) induced by CREB promotes the GR transcription. C/EBP and HNF4α directly bind to gene promoter. Steroid receptor coactivator 2 (SRC2) functions as a coactivator of receptor-related orphan receptor alpha (RORα), which directly binds to the promoters of PEPCK and G6Pase.

Figure 3

Protein arginine methyltransferases (PRMTs) mediate hepatic gluconeogenesis. PRMT1 catalyzes asymmetric methylation of FoxO1, repressing phosphorylation, and promoting its nuclear translocation. PRMT4 and 5 are recruited to the gluconeogenic promoters by interacting with CREB and CRTC2, respectively, where they mediate symmetric dimethylation of histone H3 at arginine 2 (H3R2), leading to enhanced chromatin accessibility at promoters and promoting CREB-dependent transcription. On the other hand, PRMT4 also can mediate arginine methylation in the KIX domain of CBP, leading its dissociation from CREB; PRMT5 can directly promote arginine methylation of SHP, which in turn recruits other chromatin-modifying repressive cofactors (Brm, Sin3A, and HDAC1) to repress gluconeogenic gene expression. PRMT6 can directly mediate asymmetric dimethylation of CTRC2 to enhance the interaction between CRTC2 and CREB.

Figure 4

Hepatic gluconeogenesis is modulated by histone deacetylases (HDACs). In fasting state, increased glucagon/cAMP/PKA signaling pathway induces class IIa HDACs dephosphorylation and nuclear translocation. In nucleus, they recruit class I HDAC3 and form a complex that promotes deacetylation and activation of FoxO1. Class IIb HDAC6 promotes glucocorticoid deacetylation and nuclear translocation. Class III HDACs SIRT1 mediates deacetylation and activation FoxO1 and PGC-1α. During prolonged starvation, Sirt1 can repress glucagon-induced gluconeogenesis through deacetylation and subsequent ubiquitin-dependent degradation of CRTC2.

Figure 5

Mechanisms of metformin suppressing hepatic gluconeogenesis. (Left) Pharmacologic metformin concentrations activates AMPK by promoting the formation of the AMPK heterotrimeric complex or the phosphorylation of serine/threonine kinase 11 (STK11/LKB1) or through a lysosomal mechanism requiring Axin and late endosomal/lysosomal adaptor, MAPK and mTOR activator 1 (LAMTOR1). Activated AMPK reduces hepatic lipogenesis and increases insulin sensitivity. Activated AMPK also phosphorylates and activates the cAMP-specific phosphodiesterase 4B (PDE4B), which triggers cAMP breakdown. (Middle) Supra-pharmacologic metformin concentrations inhibit mitochondrial complex I, as does the inhibition of AMP deaminase, preventing mitochondrial ATP production and increasing cytoplasmic AMP levels. Reduction of cellular ATP levels leads to the suppression of hepatic gluconeogenesis that is an energy demanding process. Elevated AMP levels not only block the cAMP-PKA pathway by the inhibition of adenylyl cyclase activity, also inhibit the gluconeogenic rate-controlling enzyme FBPase and activate AMPK. (Right) Metformin inhibits mitochondrial glycerol 3-phosphate dehydrogenase (G3PDH, also named GPD2), resulting in an increase in cytosolic NADH levels and a suppression of lactate utilization and a consequent decreased gluconeogenesis.