The Role And Mechanism Of Long Non-coding RNA Gm10768 In Liver Gluconeogenesis

The liver plays a critical role in controlling glucose homeostasis though glycogenolysis and gluconeogenesis using lactate,pyruvate,glycerol,and amino acids as precursors,and is the the major place for gluconeogenesis.During pathological state,increased rates of hepatic glucose production(HGP)in patients with T2DM,significantly impair glucose homeostasis and further contribute to hyperglycemia.In addition to response to nutrition signals and hormone levels,the hepatic gluconeogenesis is strictly regulated by transcription factors of key enzymes controling hepatic gluconeogenesis.Therefore,revealing the transcriptional regulators of hepatic gluconeogenesis to inhibit the rates of HGP are considered as potential therapeutic targets for the treatment of type 2 diabetes.Long non-coding RNAs(lncRNAs)are transcripts larger than 200 nt in length and have no coding potential.Accumulating results reveal that IncRNAs act through diverse mechanisms in pre-transcription,post-transcription and epigenetic levels,and exert discrete effects on numerous biological processes.For example,lncRNA Meg3 overexpression increases hepatic glucose production.In contrast,liver-specific knocked down of Meg3 reverses the impairment of glucose homeostasis in diabetic mice.Another lncRNA,lncLGR,which is induced by fasting,has been shown to suppress glucokinase expression and glycogen storage by binding to heterogenous nuclear ribonucleoprotein L.But in general,the physiological function of lncRNAs in maintaining glucose homeostasis in liver remains largely unexplored.Part I Screen lncRNAs involving in gluconeogenesis using high-throughput RNA sequencingTo identify lncRNAs potentially involved in hepatic gluconeogenesis,we compared the expression levels of lncRNAs in the liver of mice under fasting-refeeding cycles using high-throughput RNA sequencing.As a result,28,312 and 31,947 lncRNAs were identified in ad libitum feeding group and 16h-fasting group.We then set three criteria to further screen these lncRNAs:1.folds>3 in response to 16-h fasting;2.relatively high abundance in the mouse liver(counting reads>500);3.already annotation as authentic lncRNAs in GenBank.Our results showed that four lncRNAs,namely Gm 15441,483341 lC07Rik,Gm10768,and 4931408D14Rik,met all the given criteria.To confirm these results,an independent reverse transcription quantitative PCR(RT-qPCR)assay was performed.We found these four lncRNAs were induced upon fasting and they were quickly decreased by the refeeding at 8 h.We further validated these lncRNAs in the liver of diabetic db/db mice with over-activated hepatic gluconeogenesis and found that only two lncRNAs(4833411C07Rik and Gm10768)were increased.Finally,we chose RNA interference method in vitro to screen out functional IncRNAs affecting gluconeogenesis.Our data indicated that Gm10768,but not 4833411C07Rik,was positively correlated with the glucose production in mouse primary hepatocytes.These findings helped us to quickly narrow down our research target and selected Gm 10768 as a promising regulator involved in hepatic gluconeogenesis.Part II Function and mechanism studies on Gm10768 during hepatic gluconeogenesisWe characterized a long non-coding RNA,Gm 10768,as a positive regulator involved in hepatic gluconeogenesis both in vitro and in vivo.When overexpression Gm 10768 in vitro,the glucose production was specifically acticated in mouse primary hepatocytes,and a significant increase in the expression levels of PEPCK and G6Pase was observed.Consistently,Gm10768 overexpression in vivo markedly activated hepatic gluconeogenesis and elevated the serum levels of glucose and insulin when compared to the control mice.Glucose tolerance test and insulin tolerance test indicated that forced expression of Gm 10768 in mice exhibited an impairment of glucose disposal and insulin sensitivity.Oppositely,knockdown of Gm 10768 led to a remarkable decrease in the mRNA and protein expression levels of PEPCK and G6Pase in mouse primary hepatocytes.The rate of hepatic gluconeogenesis was robustly decreased,when specifically knocked down Gm 10768 expression in the liver of normal C57BL/6J mice.Of note,serum levels of glucose and insulin were significantly reduced in mice with liver-specific Gm 10768 knockdown.These mice also showed improved glucose and insulin tolerance.Adenovirus-mediated knockdown of Gm 10768 in the liver improved hyperglycemia and insulin resistance in diabetic db/db mice.Mechanistically,Gm10768 sequestered miR-214 and recovered the expression and function of activating transcription factor 4(ATF4),which is a miR-214 target gene and is an activator of hepatic gluconeogenesis.