Investigating The Role Of Fyn In Regulating Mice Hepatic Glucose Metabolism

Excessive Hepatic glucose production is the major contributor of fasting hyperglycemiaresulted from insulin resistance in type2diabets mellitus (T2DM). Therefore, identificationof the regulation mechanism of hepatic glucose production might provide potential newtargets for therapeutic intervention in the treatment of T2DM. The regulation of glucosehomeostasis is a complex integrative response between multiple tissues that dynamicallyrespond to metabolic and nutritional states. Classically, glucose metabolism is predominantlycontrolled through the counter-regulatory actions of insulin and glucagon. In the fed state,insulin signalling in peripheral tissues (skeletal muscle and adipose tissue) increases glucoseuptake and in the liver, insulin drives glycogen synthesis and suppresses hepatic glucoseproduction. In the fasted state, glucagon stimulates hepatic glucose release into the circulationto ensure a relatively constant glucose supply for peripheral tissues. As such, circulatingblood glucose levels are tightly regulated to remain constant irrespective of dietary nutritionalinput. Net hepatic glucose release occurs when dietary carbohydrates are unavailable resultingfrom two tightly regulated pathways: glycogenolysis and de novo synthesis of glucose(gluconeogenesis). Although the exact contribution of each process to glucose production isstill controversial, gluconeogenesis has a greater importance for prolonged fasting periods inmice, since glycogen stores are likely to be nearly depleted after the first few hours followingfood withdrawal. To ensure that glucose production matches the whole-body requirements,gluconeogenesis must be accurately regulated.Fyn is a member of Src nonreceptor tyrosine kinases family. The Src-family kinases areregulated by various extracellular signals and, in turn, control diverse cell and tissue functionsincluding cell migration, proliferation and differentiation, cell cycle entry, immune responseand tumorigenesis. Although Fyn knockout (FynKO) mice have been available for severalyears, most studies focused on those fields, the metabolic phenotype of this animal model hasnot been investigated. Our previous studies have demonstrated that glucose disposal isincreased in the FynKO mice due to increased peripheral tissue (adipose tissue and skeletalmuscle) insulin sensitivity. FynKO mice also display fasting hypoglycaemia despite decreased insulin levels, which suggested that hepatic glucose production was unable tocompensate for the increased basal glucose utilization, and the improved glucose tolerancewas not due to alterations in pancreatic cell function.Thus, the present study investigates the basis for the reduction in plasma glucose levelsand the reduced ability for the liver to produce glucose in response to gluconeogenicsubstrates. We used cellular and molecular biotechnology, including Real-time PCR, RT2Profiler PCR array assay, western blotting, construction and transfection of plasmid vectors,and isolation of primary hepatocytes, hepatic glucose/glycogen assay,pyruvate/lactate/glycerol tolerance tests, stable isotope assessment of HGP, hydrophilicmetabolites analysis as well as LC□MS assay. The results are described as follow：1. The Mouse Glucose Metabolism RT2Profiler PCR Array was used to analyze theexpression of a focused panel of genes involved in glucose metabolism in fasted FynKO miceliver. The results indicated dramatically reduced expression of gluconeogenic genes includingfructose1,6-bisphosphatase (Fbp1), glucose-6-phosphatase (G6pc), phosphoenolpyruvatecarboxykinase (PEPCK) and pyruvate dehydrogenase kinase4(pdk4) in FynKO mice, as wellas glycogen branching enzyme (Gbe1), compared to WT mice. Meantime, the expressionlevels of glycolytic gene glucokinase (Gck) and glycogen degradation genes includingglucanotransferase (Agl) and glycogen phosphorylase (Pygl) were all upregulated.2. FynKO mice had a5-fold reduction in PEPCK gene and protein expression and amarked reduction in pyruvate, pyruvate/lactate-stimulated glucose output. Remarkably, denovo glucose production was also blunted using gluconeogenic substrates that bypass thePEPCK step.3. The above findings were confirmed in FAO cells transfected with pcDNA3.1-Fyn-CA(Y527F)-V5and isolated primary hepatocytes from WT and FynKO mice. The glucoseproduction induced by Lactate/Pyruvate or glycerol in the presence of cAMP/Dexamethasonewas increased in Fyn-CA transfected FAO cells, which was reduced in isolated primaryhepatocytes from FynKO mice compared to WT. The expression of PEPCK has the samepattern as in vivo. In addtion, aquaporin9mRNA level was significantly lower in FynKOprimary hepatocytes, which functions in glycerol uptake by liver during starvation.4. Impaired conversion of glycerol to glucose was observed in both glycerol tolerancetest and determination of the conversion of13C-glycerol to glucose in the fasted state. α-glycerol phosphate levels were reduced but glycerol kinase protein expression levels were notchanged. 5. Fructose-driven glucose production was also diminished without alteration offructokinase expression levels. The normal levels of dihydroxyacetone phosphate andglyceraldehyde-3-phosphate observed in the FynKO liver extracts suggested normal triosekinase function. Fructose-bisphosphate aldolase (aldolase) mRNA or protein levels werenormal in the Fyn-deficient livers, however, there was a large reduction in liver fructose-6-phosphate (30-fold) and fructose-1,6-bisphosphate (7-fold) levels as well as a reduction inglucose-6-phosphate (2-fold) levels. These data suggest a mechanistic defect in the allostericregulation of aldolase activity.6. Phosphoenolpyruvate levels were decreased in liver extracts. In addition to reducedPEPCK activity, decreased pyruvate dehydrogenase kinase (PDK)2and PDK4geneexpression resulted in an unexpected increase in TCA cycle activity, supported by reducedlactate levels and increased TCA cycle intermediates (citrate, isocitrate and a-ketoglutarate(aKG)). And metabolite profiling supported that shuttles of high-energy electrons from thecytosol to the mitochondria were impaired. The unexpected lack of oxaloacetate and malateaccumulation, along with increased aKG and aspartate levels suggested a defect in the malate-aKG transporter.7. Lastly, normal levels of pyruvate but reduced levels of alanine and a-glycerolphosphate content suggested that due to the increased TCA activity, these substrates weredirected from pyruvate to oxidation via pyruvate dehydrogenase (PDH) rather than togluconeogenesis via pyruvate carboxylase (PC) and PEPCK, and as such, glucose productionwas impaired.In conclusion, we have identified a profound alteration of the gluconeogenic pathway inthe FynKO mice, associated with reduced PEPCK expression levels. Importantly, we alsoidentified that accentuated TCA cycle activity due to de-repressed PDH complex and thealteration of the malate-aspartate shuttle have a major contribution in the inhibition of theglucose production in the FynKO mice. Indeed, due to the increased TCA cycle activity, allgluconeogenic substrates were diverted from their original fate to funnel from the pyruvatepool, predominantly via PDH, into the TCA cycle. These combined alterations could alsoexplain the reported low levels of triglycerides and non-esterified fatty acids in the liver of theFynKO mice despite low PEPCK levels. Further studies are now needed to identify theallosteric regulators of aldolase B in the liver and to determine how Fyn kinase regulates theirlevels along with aldolase B activity, and also to investigate the upstream mechanisms bywhich PEPCK and other key enzymes of the gluconeogenic pathway and TCA cycle regulation are regulated in the FynKO mice.