A Study Of Metformin’s Suppression On Hepatic Gluconeogenesis Using Transgenic AMPK Mice

BackgroundDiabetes mellitus is a chronic disease featured with disturbed glucose metabolism.Its prominent clinical symptom is elevated fasting glucose level,which mainly results from increased hepatic gluconeogenesis.Metformin,world widely prescribed as the first-line oral anti-diabetic drug,has proven efficacy to decrease fasting glucose level by suppressing hepatic gluconeogenesis.However,the underlying mechanism of how the suppression works remains unclear.Understanding of the mechanism can provide useful guidance for clinical therapy,drug design optimization and even new drug synthesis,which ultimately can bring benefits to numerous patients in the clinic.To present scientists have mainly proposed four theories trying to explain how metformin inhibits hepatic gluconeogenesis:the liver AMPK-dependent theory,the mitochondria respiration chain nucleotide theory,the mitochondria G3PDH dependent theory and the gut-brain-liver neuron theory.In general,the following factors can be enhanced:(1)the metformin concentration matters,most studies used superpharmacological concentration,especially in vitro cellular experiments,leading to potentially different results from using normal pharmacological concentration and even toxic effect;(2)the animal model problem,non-hyperglycemia or non-diabetic mice models used in some studies do not match well with the disease model of diabetes;(3)acute verse chronic assessment,some studies assessed metformin’s acute effects within several hours of its intake,whereas in the clinic metformin chronically show its effectiveness after long term intake;(4)most experiments were performed on cellular levels and thus can be improved by using animal models.So the classic liver AMPK-dependent theory is being challenged due to flaws in inconsistent experiment designs,such as metformin dosage,disease model induction and evaluation.Our hypothesis is consistent with the first theory,metformin inhibit hepatic gluconeogenesis through AMPK-dependent pathway.ObjectivesBased on our hypothesis,we’ll overcome design defects and take use of the AMPK transgenic mice to explore the mechanism of metformin’s suppression of hepatic gluconeogenesis.Contents1.Construct multigene transgenic mice models.1.To study the role of AMPK in metformin’s suppression on hepatic gluconeogenesis based on glucose metabolism changes after conditional knock out of AMPK.2.To identify metformin’s target on AMPK by investigating the function of its two isoforms αl and α2.3.To study whether the gut-brain-liver neuron regulation influence metformin’s effects using a chronic transgenic mice model.Materials and Methods1.Multigene transgenic mice models were constructed by multi-breeding and genotyping.2.We carried out our animal studies in a controlled,randomized and repeatable manner.We studied the connection between metformin and AMPK by monitoring glucose metabolism levels in several types of transgenic mice models.Liver AMPK of these mice was knocked out by injecting the AAV8-TBG-Cre virus.Some groups of mice were fed with HFD to induce hyperglycemia and provided with metformin water for treatment.Particularly,we also investigated the role of two AMPK a isoforms in metformin’s suppression on hepatic gluconeogenesis in an effort to locate the target site of metformin.3.Glucose metabolism evaluation:first perform the glucose related tolerance tests to assess pyruvate tolerance,glucose tolerance and insulin tolerance,then use Western Blot and mRNA quantification to check gene expression levels.4.Statistical analysis:Excel and Graph Pad Prism were used for data analysis and plotting.At least 3 repeated values were used for analysis and quantitative values of all experiments were expressed as the mean+/-standard deviation(SD).The quantitative values between groups were analyzed through two-tailed independent-samples T test.Differences were considered of significant if P value<0.05.Innovations1.Conducted experiments on transgenic and in vivo animals rather than in vitro cellular conditions.2.Improved the disease model by using high fat diet induced mice and normal metformin dose.3.Conducted the first investigation to study the function of two hepatic AMPK a isoforms.Results and Conclusions1.Liver specific knock out of AMPK attenuated metformin’s suppression on hepatic gluconeogenesis,so AMPK is required and mediated by metformin to suppress hepatic gluconeogenesis.2.For both normal diet and high fat diet induced mice,liver specific knock out of AMPK had no evident impact on general glucose metabolism,including liver gluconeogenesis,glucose tolerance and insulin sensitivity.3.Knocking out either one of the two AMPK a isoforms did not affect metformin’s suppression on hepatic gluconeogenesis.When mediating metformin to suppress the gluconeogenesis,hepatic AMPK α1 and α2 isoforms have comparable functions and compensate for each other.More studies are required to explore the possible special role of α1.4.In the chronic mice model,metformin managed to inhibit hepatic gluconeogenesis even when the gut-brain-liver neuron pathway was blocked.This acute theory failed to illuninate metformin’s function on the chronic mice model.Defects and ProspectIn our study,we failed to see the increase of fasting glucose level on AMPK knock out mice with metformin treatment.The HFD induction period and metformin treatment period after AMPK knock out maybe factors to optimize.And ideally,our conclusion would be tested on transgenic AMPK diabetes mice in the future.Whether metformin interacts directly with AMPK and locate the interaction site,both questions are key points worth researchers’ attention.We may also spend efforts to check the long term effect on lipid and glucose metabolism after AMPK is knocked out.Further study is required to explore the typical function of AMPK α1 and α2.Meanwhile,verifying theory 2 on our improved transgenic mice model is also a good topic.