The liver plays a central role in fuel metabolism, and thus regulates dynamic catabolic and anabolic processes to maintain energy homeostasis of organisms. Breakdown products of carbohydrate and lipid (i.e. glucose and fatty acids) are common energy sources which are converted to adenosine-triphosphate (ATP) in mitochondria. In addition, mitochondria have many other metabolic functions, such as regulation of membrane potential, cellular metabolism, calcium signaling (including calcium-induced apoptosis), and apoptosis. During the process of catabolism, the mitochondrion serves as the main source of energy for the cell because it converts nutrients into energy via cellular respiration. Most of the oxygen delivered to cells or organs is consumed by mitochondria for ATP generation. When the energy is excessive in the cell, mitochondrial energy production is inhibited so that glucose and free fatty acids can be stored as glycogen and fat through anabolic processes.
AMPK signaling pathways for fuel metabolism
AMPK: AMPK is a heterotrimer complex that consists of a catalytic subunit (α1/2) and two regulatory subunits (β1/2 and γ1/2/3), and functions as a serine/threonine protein kinase; AMPK activation is mediated by phosphorylation of threonine-172 in the catalytic domain of the α subunit. The activity of AMPK can be regulated by upstream kinases, which include liver kinase B1 (LKB1), Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) β, and transforming growth factor β-activated kinase-1. Both LKB1 and CaMKK increase the AMPK activity through direct phosphorylation of threonine-172 in the α subunit. In addition, LKB1 is constitutively active as a major upstream kinase. The upstream signaling molecules of LKB1 may include protein kinase C (PKC)-ζ, protein kinase A, and p90 kDa ribosomal S6 kinase. The fact that the calcium/calmodulin complex regulates CaMKK suggests that AMPK may be involved in Ca2+ modulation in cells.
AMPK regulates energy homeostasis in various organs through response to hormones and nutrient signals. AMPK physiologically responds to the change in the AMP:ATP ratio, and thus serves as an intracellular sensor for energy homeostasis. In addition to ATP production with switching off from anabolic processes in tissues, the activation of AMPK affects whole body fuel utilization and induces fatty acid oxidation and glucose uptake in skeletal muscle and heart, but inhibits lipogenesis and adipocyte differentiation[6-7]. In the liver, AMPK inhibits gluconeogenesis and synthesis of glycogen, fatty acid and cholesterol. Since AMPK plays a key role in metabolic regulation, it is recognized as an important target for metabolic disorders such as obesity, diabetes, and metabolic liver diseases.
S6K1: S6K1 is a mitogen-activated serine/threonine protein kinase that is associated with growth and cell cycle progression. In translational processes, S6K1 phosphorylates the S6 protein of the 40S ribosomal subunit. Phosphoinositide-3 kinase (PI3K)-the mammalian target of rapamycin (mTOR) regulates S6K1 as a distinct pathway from the Ras/mitogen-activated protein kinase cascade. S6K1 signaling suppresses catabolic events such as lipolysis in adipose tissue and fatty acid oxidation in muscle, both of which stimulate ATP generation. Since S6K1 is sensitive to nutrients including amino acids, nutrients negatively regulate insulin signaling by phosphorylating insulin receptor substrate-1 (IRS1) through S6K1 activation. Thus, S6K1 may also affect the regulation of nutrient and hormone signaling pathways under normal and pathological conditions (e.g. obesity, diabetes, and cancer). Moreover, S6K1 may play a role in the balance between survival and death in tissues including the liver. It is noteworthy that AMPK activation leads to inhibition of the mTOR/S6K1 pathway through tuberous sclerosis protein 2 (TSC2) phosphorylation. The regulation of S6K1 by AMPK is now recognized as an important regulatory step, by which cells maintain energy metabolism.
AMPK as a target for metabolic diseases
Nonalcoholic fatty liver disease (NAFLD) is defined as a common liver disease ranging from steatosis to nonalcoholic steatohepatitis, and cirrhosis. Moreover, NAFLD is considered as a main hepatic component of metabolic syndrome. The characteristics of metabolic syndrome are obesity, insulin resistance, and cardiovascular disorders. In obese people mostly with insulin resistance, excessive fat is deposited in the liver and the raised hepatic lipid amount is closely associated with pathogenic processes of the syndrome[18,19].
Hepatic steatosis by liver X receptor-α-sterol regulatory element, binding protein-1c: A variety of conditions such as excess delivery of fatty acids, decreased oxidation of hepatic fatty acid and/or impaired synthesis or secretion of very low-density lipoprotein increase the sources of hepatic lipids, leading to fatty liver disease. The amount of accumulated fat is also increased by lipogenesis; emerging evidence supports the importance of de novo lipogenesis in abnormal hepatic fat accumulation in NAFLD patients[20,21]. Lipogenesis is transcriptionally regulated by the membrane-bound sterol regulatory element, binding protein-1c (SREBP-1c), which belongs to the basic helix-loop-helix-leucine zipper family. In the nucleus, SREBP-1c activates transcription of genes involved in lipogenesis, as supported by the finding that the overexpression of SREBP-1c in transgenic mice promotes the development of fatty liver. In animal models of insulin-resistant diabetes and obesity, the increased synthesis of fatty acids contributes to the development of hepatic steatosis.
Liver X receptor-α (LXRα), a transcriptional nuclear receptor, is a key regulator of lipogenic genes encoding for the enzymes that promote hepatic fat accumulation (e.g. fatty acid synthase, FAS; acetyl-CoA carboxylase, ACC; and stearoyl-CoA desaturase-1, SCD-1)[22,23]. Ligand activation of LXRα promotes induction of the lipogenic genes through SREBP-1c, causing increases in fatty acid synthesis and progression to steatosis, hypertriglyceridemia, and steatohepatitis. Thus, SREBP-1c is an important target gene of LXRα. Since the LXRα-SREBP-1c pathway activates lipogenesis in the liver, it is an attractive target for the treatment of hepatic steatosis and hepatitis. In clinical situations, the expression of SREBP-1c and lipogenic genes including ACC and FAS is enhanced in NAFLD patients[24,25]. In addition, increases in LXRα target gene expression (e.g. ACC and FAS) were observed in the patients with fatty liver, which was accompanied by SREBP-1c activation, but not activation of carbohydrate responsive element-binding protein.
The AMPK-S6K1 pathway is involved in the regulation of LXRα-SREBP-1c and thus in LXRα-induced lipogenesis; chemical activation of AMPK in conjunction with its inhibition of S6K1 leads to the intervention of hepatic steatosis (Figure 1). As an example, AMPK activation by oltipraz treatment inhibits S6K1 activity, which inhibits the activity of LXRα and prevents the ability of activated LXRα to bind the LXR binding site upstream of the genes including SREBP-1c and CYP7A1. Therefore, the consequent repression of SREBP-1c expression contributes to decreased synthesis of fat in the liver.
Figure 1 Adenosine monophosphate-activated protein kinase pathway in hepatic fuel metabolism.
Adenosine monophosphate-activated protein kinase (AMPK), a metabolic energy sensor, negatively regulates protein synthesis through inhibition of the mammalian target of rapamycin (mTOR)-S6 kinase-1 (S6K1) pathway. The inhibitory effect of AMPK on liver X receptor-α (LXRα)-dependent triglyceride synthesis is opposed by the action of S6K1. AMPK also shuts down glycogen synthesis via inhibitory phosphorylation of glycogen synthase. AMPK as a fuel sensor induces glucose transport and fat oxidation in response to metabolic stress such as energy deprivation, and also increases mitochondrial biogenesis. AMPK counteracts energy depletion by stimulating energy production and limiting energy utilization. Endocannabinoids such as 2-arachidonoylglycerol derived from hepatic stellate cells decrease AMPK phosphorylation resulting in downregulation of lipogenic action. 2-AG: 2-arachidonoylglycerol; CB1R: Cannabinoid 1 receptor; HSC: Hepatic stellate cell; IR: Insulin receptor; Raptor: Regulatory-associated protein of mTOR; IRS1: Insulin receptor substrate-1; PI3K: Phosphoinositide-3 kinase; TSC1: Tuberous sclerosis complex 1; pS: Phospho-serine; pT: Phospho-threonine; SREBP-1c: Sterol regulatory element binding protein-1c.
Repeated alcohol consumption decreases the production of adiponectin secreted from adipocytes. Adiponectin increases hepatic fatty acid oxidation through AMPK activation. Therefore, it is tempting to speculate that AMPK activity is repressed as hepatic function deteriorates in alcoholic patients. Similarly, AMPK activity was decreased in animals which consumed alcohol for 4 wk. As a compensatory response, alcohol consumption increased lipogenesis in the liver, which may also result from the reduced rate of fatty acid oxidation. Thus, pharmacological activation of AMPK may be of help in treating hepatic steatosis. Peroxisome proliferator-activated receptors (PPARs) play a role in sensing nutrient levels and regulating lipid and glucose metabolism. Thiazolidinediones (TZDs) and fibrates that activate PPARγ and PPARα, respectively, are prescribed for patients with diabetes and/or dyslipidemia. In those taking PPARγ agonists, insulin-mediated adipose tissue uptake and storage of free fatty acids are augmented with the inhibition of hepatic fatty acid synthesis, which may result in part from indirect activation of AMPK[32,33].
Hepatic insulin resistance: Insulin signaling is important in maintaining homeostasis of glucose, lipid, and protein metabolism, and thus induces anabolism in tissues. In addition, it has effects on normal growth and development. Insulin receptor and its associated protein IRS1 relay signal transmission to the PI3K-Akt pathway, which consequently increases mTOR-S6K activity. Activation of the mTOR-S6K1 pathway by insulin may lead to fat accumulation in adipose tissue, hypertrophy of skeletal muscle, growth of pancreatic β cells, and protein synthesis. Therefore, the control of insulin signaling is tightly regulated by a negative feedback mechanism. In fact, the downstream components of the insulin receptor give inhibitory autoregulatory signals to upstream molecules along the insulin-signaling pathway or through unrelated pathways that cause insulin resistance. In particular, phosphorylation of IRS proteins on serine residues is a key step in the processes of physiological and pathological conditions. So, the kinases that phosphorylate IRS1/2 have been extensively studied.
Hepatic steatosis alone, or to a greater degree in combination with endotoxin challenge, makes the liver susceptible to oxidative damage and thus facilitates the pathologic process of hepatitis. The cytokines produced by accumulated fat with or without endotoxin cause insulin resistance. In particular, tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) lead to insulin resistance through multiple mechanisms. These include c-Jun N-terminal kinase 1 (JNK1)-mediated serine phosphorylation of IRS-1, IκB kinase-dependent nuclear factor-κB activation, and suppressors of cytokine signaling-3 (SOCS-3) induction[34-36]. Since TNFα increases insulin resistance in peripheral organs, inhibition of TNFα activity and/or its decreased expression would be of help to overcome insulin resistance. However, IL-6 displays pleiotropic functions in a tissue-specific and time-dependent manner. IL-6 confers insulin resistance in hepatocytes through activation of SOCS protein through the Jak/Stat pathway to inhibit tyrosine phosphorylation of IRS1, while IL-6 increases insulin sensitivity by stimulating basal glucose transport in 3T3-L1 adipocytes, smooth muscle and chondrocytes. Acute treatment with IL-6 increases insulin sensitivity due to AMPK activation, while chronically elevated IL-6 leads to impaired insulin signaling and cellular insulin resistance via activating SOCS-3 and reducing the expression of the adiponectin, GLUT4, IRS1 mRNA, IRS-1 protein and its tyrosine phosphorylation[41,42].
Glucose is overproduced in the liver of patients with type 2 diabetes. Because AMPK serves as an energy-saving mechanism, its activation decreases hepatic gluconeogenesis. The experimental results using gene knockouts, pharmacological means, or adenoviral activation of AMPK support the role of AMPK in the regulation of glucose production in the liver. Consistently, hepatic glucose production increased to show hyperglycemia and glucose intolerance in liver-specific AMPKα2 deficient mice. Hence, it is highly likely that the hepatic AMPKα2 isoform is critical for repressing hepatic glucose production and maintaining fasting blood glucose levels in the physiological range. Consistently, AMPK activation by adenovirus expressing a constitutively active form of AMPKα2 as well as by 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR, a direct AMPK activator) or metformin reduced glucose output[45-47].
Activation of S6K1 exerts a negative feedback action on insulin signaling. As an example, TNFα secreted by non-parenchymal cells activates S6K1 in pathologic states. The important role of S6K1 on insulin resistance was proven by a study using S6K1-null mice[48,49]. A key role for mTOR-S6K1 regulation of insulin resistance was also supported by the finding that rapamycin blocked IRS1 phosphorylation[50,51], confirming the importance of S6K1 activity in inducing insulin resistance. Hence, insulin resistance induced by abnormal conditions such as hyperinsulinemia, obesity and excess nutrient availability is accompanied by an increase in S6K1 activity[48,52]. The result of a study using a knockout model proved the critical role of S6K1 and its physiological feedback importance to IRS1/2 and PI3K for insulin resistance. In an experimental model, the inhibitory effect of high-fat diet consumption on the insulin receptor-PI3K pathway is also mediated by S6K1. In our laboratory, it was found that the inhibitory modulation of S6K1 activity by beneficial candidates reversed insulin resistance and hyperglycemia. In particular, oltipraz treatment inhibited S6K1 through AMPK activation. Consistently, a dominant negative mutant of AMPK abrogated S6K1 phosphorylation. AMPK activation by other drugs like metformin and rosiglitazone also contribute to insulin sensitivity enhancement[47,53]. Similarly, other agents that inhibit insulin resistance also antagonize S6K1 activation downstream of AMPK. So, these agents have the effects of improving insulin sensitivity through a mechanism involving AMPK-mediated S6K1 inhibition in hepatocytes.
JNK1 is activated by various stress signals such as cytokines or oxidative stress, and thus the activity of JNK1 increases under prediabetic or diabetic conditions. This important kinase is also implicated in the phosphorylation of IRS1/2[54-56], interfering with insulin action. The JNK pathway is stimulated by oxidative stress conditions, increased flux of free fatty acids and TNFα production, which contributes to developing insulin resistance. The importance of JNK activation is supported by the finding that a deficiency of JNK1 prevented insulin resistance in an experimental model. Moreover, JNK mediates dysfunction of insulin secretion from β cells. Hence, inhibition of JNK by chemical means may help improve insulin resistance and ameliorate hepatic energy metabolism[54,58]. For example, isoliquiritigenin from various natural herbs including licorice has a JNK-inhibitory effect. Thus, isoliquiritigenin is capable of repressing lipogenesis in the liver and protecting hepatocytes from oxidative injury inflicted by fat accumulation through a novel JNK-dependent pathway that acts as an upstream component of LXRα (unpublished data).