Imeglimin prevents human endothelial cell death by inhibiting mitochondrial permeability transition without inhibiting mitochondrial respiration.

Imeglimin is the first in a new class of oral glucose-lowering agents, having recently completed its phase 2b trial. As Imeglimin did show a full prevention of ß-cell apoptosis, and since angiopathy represents a major complication of diabetes, we studied Imeglimin protective effects on hyperglycemia-induced death of human endothelial cells (HMEC-1). These cells were incubated in several oxidative stress environments (exposure to high glucose and oxidizing agent tert-butylhydroperoxide) which led to mitochondrial permeability transition pore (PTP) opening, cytochrome c release and cell death. These events were fully prevented by Imeglimin treatment. This protective effect on cell death occurred without any effect on oxygen consumption rate, on lactate production and on cytosolic redox or phosphate potentials. Imeglimin also dramatically decreased reactive oxygen species production, inhibiting specifically reverse electron transfer through complex I. We conclude that Imeglimin prevents hyperglycemia-induced cell death in HMEC-1 through inhibition of PTP opening without inhibiting mitochondrial respiration nor affecting cellular energy status. Considering the high prevalence of macrovascular and microvascular complications in type 2 diabetic subjects, these results together suggest a potential benefit of Imeglimin in diabetic angiopathy.

Cellular and molecular mechanisms involved in insulin's potentiation of glycogen synthase activity by metformin.

By taking advantage of the Xenopus oocyte model, we recently confirmed the in vitro enhancing effect of metformin (MET) on glycogen synthase (GS) activity when induced by insulin (INS). We now investigated some mechanistic aspects of its modulatory role upon the hormonal regulation of this rate-limiting enzyme. The action of 20 microM MET (approximately 3.3 microg/mL) was measurable at early steps in the intracellular metabolic pathway: the amount of adenosine 3',5'-cyclic monophosphate (cAMP) was markedly decreased in the presence of the biguanide plus 50 nM INS (to about 60% of control vs 25% with INS alone). The injection of tyrphostin B46, a potent inhibitor of insulin receptor (IR)-associated tyrosine kinase activity, led to a drastic reduction in MET-stimulated GS activity in the presence of INS. MET failed to increase the activity of type 2 protein phosphatases whether INS was present or not. However, a specific inhibitor of type 1 phosphatases, when microinjected, blocked both the hormonal effect on GS and its potentiation by MET. The salient feature of this study was that there was almost no accumulation of radiolabeled MET in oocytes: less than 0.1% was found in the cytosol of cells which had been exposed to MET at a therapeutic dose (10 microM) for up to 16 hr. Moreover, a lack of detectable intracellular MET after a 60-min incubation nevertheless correlated with its sustained action on INS-regulated GS activity. From these results, it could be inferred that the major site of MET action may reside within some membrane components of a signaling complex most likely linked to the IR, but in any case located upstream of the branching of reactions which tightly control GS activity.

Metformin interaction with insulin-regulated glucose uptake, using the Xenopus laevis oocyte model expressing the mammalian transporter GLUT4.

The primary goal of this work was to better define, in molecular terms, the impact of metformin on hexose carriers. The methodology consisted of determining the zero-trans kinetics of 2-deoxy-D-glucose uptake for the mammalian insulin-sensitive glucose transporter (GLUT4) expressed in Xenopus laevis oocytes. These cells possessed the specialized protein and, when treated with insulin (2 microM) plus metformin (20 microM), showed a markedly enhanced hexose transport activity (2.4-fold increase over basal) as compared to that of cells incubated in the presence of insulin alone (1.8-fold increase over basal). Kinetic analysis of this process revealed that insulin induced a similar response to that observed for the native carrier, i.e., a higher Vmax. When metformin was added together with insulin, we mainly recorded a significant decrease in apparent Km for the sugar transported, Vmax being only marginally modified. Parathyroid hormone (PTH), which is known to impair the intrinsic activity of GLUT4, prevented the stimulatory effect of metformin in both kinds of oocytes whereas cytochalasin D, which interferes with the translocation of carriers, was without effect. These results suggest that metformin combined with insulin can maintain glucose homeostasis by increasing the catalytic activity of some hexose carriers or by improving the affinity of GLUT4 for glucose.

Mitochondrial metabolism and type-2 diabetes: a specific target of metformin.

Several links relate mitochondrial metabolism and type 2 diabetes or chronic hyperglycaemia. Among them, ATP synthesis by oxidative phosphorylation and cellular energy metabolism (ATP/ADP ratio), redox status and reactive oxygen species (ROS) production, membrane potential and substrate transport across the mitochondrial membrane are involved at various steps of the very complex network of glucose metabolism. Recently, the following findings (1) mitochondrial ROS production is central in the signalling pathway of harmful effects of hyperglycaemia, (2) AMPK activation is a major regulator of both glucose and lipid metabolism connected with cellular energy status, (3) hyperglycaemia by inhibiting glucose-6-phosphate dehydrogenase (G6PDH) by a cAMP mechanism plays a crucial role in NADPH/NADP ratio and thus in the pro-oxidant/anti-oxidant cellular status, have deeply changed our view of diabetes and related complications. It has been reported that metformin has many different cellular effects according to the experimental models and/or conditions. However, recent important findings may explain its unique efficacy in the treatment of hyperglycaemia- or insulin-resistance related complications. Metformin is a mild inhibitor of respiratory chain complex 1; it activates AMPK in several models, apparently independently of changes in the AMP-to-ATP ratio; it activates G6PDH in a model of high-fat related insulin resistance; and it has antioxidant properties by a mechanism (s), which is (are) not completely elucidated as yet. Although it is clear that metformin has non-mitochondrial effects, since it affects erythrocyte metabolism, the mitochondrial effects of metformin are probably crucial in explaining the various properties of this drug.

Potentiating effect of metformin on insulin-induced glucose uptake and glycogen metabolism with Xenopus oocytes.

Xenopus laevis oocytes were chosen as the in vitro model for this study with the aim of reconsidering metformin action on the main insulin-responsive glucose pathway. Metformin alone, when present at a therapeutic dose (20 micromol/l) in the incubation medium, did not alter the basal rate of glucose uptake or of glycogen synthesis as measured by [U-14C] D-glucose incorporation. The drug had no effect on the main rate-limiting enzyme implicated in this pathway, i.e. glycogen synthase. In contrast, when combined with 2 micromol/l insulin, metformin led to a specific rise of both free and stored glucose, by 42.4 and 102.3% respectively. Moreover, a short-term preincubation of mature oocytes with metformin, but in the absence of glucose, enhanced significantly the amount of synthase a when stimulated by 50 nmol/l insulin (basal 17.4 +/- 5.7%, metformin 21.3 +/- 4.1%, insulin 31.2 +/- 4.6%, metformin together with insulin 62.7 +/- 4.2%, p < 0.005, n = 5). Interestingly, the microinjection of this biguanide, at a final concentration of 20 nmol/l, allowed a similar biochemical response. These data clearly suggest that metformin could act primarily at postreceptor steps which are thought to be key sites in controlling the cellular glucose homeostasis.