Differential effects of metformin glycinate and hydrochloride in glucose production, AMPK phosphorylation and insulin sensitivity in hepatocytes from non-diabetic and diabetic mice

Abstract

The liver is a main target tissue of the biguanide metformin which activates AMP-activated protein kinase (AMPK). We have previously reported that administration of metformin glycinate showed a greater decrease in glycated hemoglobin A1c than placebo in patients with type 2 diabetes mellitus (T2DM). Herein, we have compared the effects of metformin hydrochloride, the oral antidiabetic drug of first choice, with those of metformin glycinate in hepatocytes from non-diabetic, diabetic mice and humans. Both formulations were equally potent in the reduction of basal and glucagon-induced glucose production and mRNA levels of gluconeogenic enzymes (Pck1 and G6pc) in hepatocytes from C57/Bl6 mice and humans. In contrast, phosphorylation of AMPK and its substrate acetyl CoA carboxylase (ACC) was more rapid in hepatocytes treated with metformin glycinate. Likewise, in hepatocytes from obese/diabetic db/db mice we found stronger reduction of glucagon-induced glucose output and more sustained AMPK phosphorylation by the treatment with metformin glycinate. Importantly, insulin sensitization regarding phosphorylation of AKT (Ser473) was enhanced in hepatocytes from db/db mice or humans pretreated with metformin glycinate. Altogether, our data point out preclinical mechanistic evidences in hepatocytes suggesting that metformin glycinate might represent a superior alternative therapy against insulin resistance during obesity and/or T2DM. Response to Reviewers: Point-by-point response to the Reviewer´s comments. Reviewer #1: This manuscript compares the effects of metformin hydrochloride and metformin glycinate in reducing basal and induced glucose production as well as the regulation of gluconeogenesis. The authors use hepatocytes from mice, db/db mice as well as from humans for their study. The experimental data reported by the authors suggests that metformin glycinate can effect some facets of glucose regulation at a INSTITUTO DE INVESTIGACIONES BIOMÉDICAS "ALBERTO SOLS", IIBM http://www.iib.uam.es C/Arturo Duperier, nº 4 28029 MADRID ESPAÑA PHO: (34) 91 585 4400 FAX: (34) 91 585 4401 Dear Editor: Find enclosed the electronic submission of the revised manuscript referenced as FCT-D-18-01715 entitled "Differential effects of metformin glycinate and hydrochloride in glucose production, AMPK phosphorylation and insulin sensitivity in hepatocytes from non-diabetic and diabetic mice" to be considered for publication in Food and Chemical Toxicology. In this revised manuscript we have addressed all the minor issues raised by the Reviewers (highlighted in red font). We also provide a detailed point-by-point response letter to the Reviewer´s comments.

The Metformin Mechanism on Gluconeogenesis and AMPK Activation: The Metabolite Perspective

“…This enzyme also has a crucial role in regenerating inorganic phosphate in conditions of raised G6P [51]. Gene expression of G6pc like Pck1 (encoding phosphoenolpyruvate carboxykinase) is induced several-fold (>10-fold) by cell permeable cAMP-analogues and this induction is attenuated by metformin [11,105,106]. The lower mRNA levels for both G6pc and Pck1 in hepatocytes from a mouse model with a gain of function mutation in the AMPK-γ1 subunit (D316A) with a~2.5-fold increase in AMPK activity supports a role for AMPK activation in the repression of both G6pc and Pck1 in basal non-stimulated conditions [107].…”

“…AMPK activation counteracts cAMP-mediated induction of both G6pc and Pck1 [105,106]. However, there is also evidence for repression of G6pc but not Pck1 by metformin through AMPK-independent mechanism(s) [11].…”

“…Third, G6pc and Pck1 are oppositely regulated by high glucose [110,111], and also by gluconeogenic precursors such as dihydroxyacetone and glycerol, which like high glucose induce G6pc but repress Pck1 [111,115]. Because G6pc and Pck1 are regulated co-ordinately by cAMP-linked mechanisms and likewise by AMPK activation [105,106], but oppositely by nutrient excess, irrespective of whether the substrate is glucose or gluconeogenic precursors [110,115], measurement of both genes may help discriminate between AMPK-dependent and metabolite linked mechanisms which are independent of AMPK [49]. Type 2 diabetes is commonly associated with nutrient excess which predicts compromised intracellular homeostasis.…”

Stevia Improves the Antihyperglycemic Effect of Metformin in Streptozotocin-Induced Diabetic Rats: A Novel Strategy in Type 2 Diabetes Mellitus

“…They found that AMPK 2 activity increased by 52% after 4 weeks and by 80% after 10 weeks of treatment with metformin 27 . Very recently, in a differential study Rada et al, (2019) reported that metformin could activate AMPK, inhibit glucose production and increase insulin sensitivity 28 . This study also documented increased insulin sensitivity by metformin but in diabetic rats.…”

Integrated or Independent Actions of Metformin in Target Tissues Underlying Its Current Use and New Possible Applications in the Endocrine and Metabolic Disorder Area

“…To this regard, metformin can reduce the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase (PEPCK), two key-enzymes encoded by G6pc and Pck1 , respectively. There is evidence for a role of AMPK activation in the downregulation of G6pc and Pck1 expression in basal non-stimulated conditions [ 39 , 75 , 76 , 77 ], as well as in the reversal of the cAMP-induced expression of both genes downstream of glucagon signaling pathway [ 78 , 79 ]. Indeed, the transcriptional complex composed by cyclic AMP response element binding protein (CREB), CREB binding protein (CBP), and CREB-regulated transcription factor 2 (CRTC2, also known as TORC2) increases the expression of both genes.…”