The Mechanism Of Cortisol Regulates Glucose Metabolism Via Redox In Hepatic Endoplasmic Reticulum

Background and objectivesThe liver serves as a central organ in the maintaining of glucose homeostasis, and is responsible for about 80% of circular glucose in the postabsorptive state. Fifty percent of the released glucose in the circulation derived from glycogenolysis, while the reminder is caused by the gluconeogenesis. Normally, the kiney contains little glycogen, releases glucose via gluconeogenesis. During fasting, a condition of nutrient deficiency, hepatic gluconeogenesis is becoming main pathway to increase glucose level. The proportion caused by gluconeogenesis increases along with the duration of fasting and depletion of glycogen. It accounts for approximately 70% of all glucose released into circulation by 24 hours fasting, and over 90% by 48 hours. Therefore, both glycogenolysis and gluconeogenesis, particular the latter one, the capability of live for de novo glucose production, is a prerequisite for the maintenance of stable blood glucose concentrations, and result in proper energy availability for the brain and renal. The terminal reaction of glycogenolysis and gluconeogenesis, from glucose-6-phosphate (G6P) to glucose, is catalysed by glucose-6-phospahatase (G6Pase) in the endoplasmic reticulum (ER).The neuroendocrine system contributes to the regulation of glucose homeostasis. As the main hormone that can increase glucose concentrations, glucocorticoid (GC) plays a critical role in the stress, diabetes, insulin resistance, obesity and other metabolic dysfunction diseases. GC in the circulation derived from two resources:1. Adrenal cortex:under control of the hypothalamo-pituitary-adrenal (HPA) axis; 2. Peripheral tissue:liver and adipose. The liver contributes approximate 1/3 while extrahepatic splanchnic tissue contributes 2/3. Because the enzyme 11β-hydroxy steroid dehydrogenase type 1 (11β-HSD1) is high expressed in the liver and adipose. This enzyme regenerates the active glucocorticoid cortisol from its inactive 11-keto metabolite cortisone in the ER.At the cellular level, GC mediates its function through binding with glucocorticoids receptor (GR), which belongs to the nuclear receptor transcription factor family. The two isoforms of GR are GRa and GRβ. Generally, GR located in the cytosol in form of dimerization with a heat shock protein 90 (HSP90). Upon GC bingding to the GR in the cytosol, the GR is released from HSP90 and migrates into the cell nucleus, mediates the target gene transcription via either binding to glucocorticoid response element (GRE) or direct hormone-receptor interactions with other transcriptional regulators. However, there are examples of non-genomic control of cell biological function through the GR. Moreover, tissue-specific intracellular activation of glueocorticoids amplifies glucocorticoid receptor (GR) activation independently of the level of cortisol in blood.Redox states are key elements of the metabolic sensor function of the ER. Recent experimental observations have indicated that a high luminal NADPH/NADP+ratio is essential for the proper function of ER, as well as required for efficient 11β-HSD1 reductase activity. The pyridine nucleotide is dictated by the concentration of G6P, and mediated by the coordinated activities of two enzymes, hexose-6-phosphate dehydrogenase (H6PDH) and 11β-HSD1. And G6P, transported from cytoplasm into ER lumen by membrane-bound G6P transporter (G6PT) also is a well-known substrate for G6Pase in the terminal step of glycogenolysis and gluconeogenesis. Therefore, glucose metabolism is tightly linked to intracellular glucocorticoids through the enzymatic reactions in the ER.Based on our previous findings and recently research background, we reasoned that a tacit G6P pool exists in the ER and is equally accessible to both H6PDH and G6Pase, altering of luminal redox could result in different availability to the two enzymes and further affect glucose production. The aim of this study is to investigate the relationship and potential mechanisms among GC, pyridine nucleotides and glucose metabolism in the ER, which includes the following aspects:(1) To observe the changes of glucose and CO2 production upon several G6Pase inhibitors under various concentration of G6P, by using kinetic analysis to investigate whether the two aforesaid enzymes share the G6P pool equally; (2) To explore changes of glucose production upon different concentration of cortisol, corticosterone; (3) Using different concentration of cortisol, corticosterone, with or without inhibitor of 11β-HSD1 (Metyropone), to observe level of NADPH production. Because whether NADP could across the intact ER membrane is still debatable, we also to observe the crossing of NADP under different time incubation. In addition, deductive luminal concentration of G6P was 0.02 mM and it is unclear as to the hepatic intracellular G6P concentrations during prolonged fasting. But reported G6P in hepatocytes are consistently below 0.1 mM. All our experiments were conducted under four different concentrations. The purpose of this study is to elucidate the stimulating effect of cortisol on endoplasmic glucose production via alteration of luminal NADPH/NADP ratio during prolonged fasting, this latent mechanism provides more evidence for deepening the understanding of the regulation of the neuroendocrine system on the glucose metabolism.The main results:1. G6P forms a pool after being transported by G6PT from cytoplasm into the ER, these two aforesaid luminal enzymes, H6PD and G6Pase, do share the G6P pool in the ER, but not equally. Based on kinetic modeling of G6P flux, the ER transporter for G6P (T1) preferentially delivers this substrate to G6Pase, hence, the luminal enzymes do not share G6P equally.2. NADP can across the intact ER membrane slowly with long time incubation (30 min), although some previous studies reported the ER membrane is impermeable to NADP.3. The redox shift in the NADP-NADPH pool can be altered by cortisol via the 11β-HSD1, which can be reflected by changes of luminal NADPH/NADP+ratio.4. As a result of alteration of luminal redox, H6PDH pathway was inhibited by active GC, which let more G6P available to G6Pase, and further increase glucose production.Conclusions:During fasting status, increased active GC (cortisol) acting through 11β-HSD1, begets a more reduced pyridine redox ratio. By altering this luminal redox ratio, G6P flux through H6PDH is restrained, allowing more G6P for the competing enzyme G6Pase. And, at low G6P concentrations in the ER lumen, which occur during fasting, this acute cortisol-induced redox adjustment promotes glucose production. This reproducible cortisol-driven mechanism has been heretofore unrecognized. And provides evidence to study of the relationship between glucose metabolism and regulation of neuroendocrine system.