The mechanism for how metformin activates AMPK (AMPactivated kinase) was investigated in isolated skeletal muscle L6 cells. A widely held notion is that inhibition of the mitochondrial respiratory chain is central to the mechanism. We also considered other proposals for metformin action. As metabolic pathway markers, we focused on glucose transport and fatty acid oxidation. We also confirmed metformin actions on other metabolic processes in L6 cells. Metformin stimulated both glucose transport and fatty acid oxidation. The mitochondrial Complex I inhibitor rotenone also stimulated glucose transport but it inhibited fatty acid oxidation, independently of metformin. The peroxynitrite generator 3-morpholinosydnonimine stimulated glucose transport, but inhibited fatty acid oxidation. Addition of the nitric oxide precursor arginine to cells did not affect glucose transport. These studies differentiate metformin from inhibition of mitochondrial respiration and from active nitrogen species. Knockdown of adenylate kinase also failed to affect metformin stimulation of glucose transport. Hence, any means of increase in ADP appears not to be involved in the metformin mechanism. Knockdown of LKB1, an upstream kinase and AMPK activator, did not affect metformin action. Having ruled out existing proposals, we suggest a new one: metformin might increase AMP through inhibition of AMP deaminase (AMPD). We found that metformin inhibited purified AMP deaminase activity. Furthermore, a known inhibitor of AMPD stimulated glucose uptake and fatty acid oxidation. Both metformin and the AMPD inhibitor suppressed ammonia accumulation by the cells. Knockdown of AMPD obviated metformin stimulation of glucose transport. We conclude that AMPD inhibition is the mechanism of metformin action.

Background: Metformin is widely believed to inhibit mitochondrial respiration. Results: Metformin increased phosphocreatine recovery from dinitrophenol or azide in intact cells, increased MTT reduction, left ATP levels unchanged, and increased free AMP. Conclusion: Metformin stimulated mitochondrial energy production. Significance: Distinct mechanisms for metformin other than mitochondrial inhibition, such as the inhibition of breakdown of AMP proposed in our work, need to be pursued.

While enzyme inhibition is a widely taught subject across chemical and biochemical disciplines, it remains poorly understood. A mental image is presented to facilitate the understanding of inhibition types other than competitive. Subsequently, enzyme inhibition is developed using V max/K m in place of K m. Interpretation of direct (initial velocity vs substrate concentration) plots makes clear the meanings of competitive, noncompetitive, and mixed inhibition in a manner entirely distinct from current textbook treatments. The effects of inhibitors on enzymes can be seen to be reduced to a simple consideration of actions at zero and infinite substrate concentrations, corresponding to V max/K m and V max, respectively.

We examined capacitative calcium entry (CCE) in Jurkat and in L6 skeletal muscle cells. We found that extracellular Ca 2؉ can enter the endoplasmic reticulum (ER) of both cell types even in the presence of thapsigargin, which blocks entry into the ER from the cytosol through the CaATPase. Moreover, extracellular Ca 2؉ entry into the ER was evident even when intracellular flow of Ca 2؉ was in the direction of ER to cytosol due to the presence of caffeine. ER Ca 2؉ content was assessed by two separate means. First, we used the Mag-Fura fluorescent dye, which is sensitive only to the relatively high concentrations of Ca 2؉ found in the ER. Second, we transiently expressed an ER-targeted derivative of aequorin, which reports Ca 2؉ by luminescence. In both cases, the Ca 2؉ concentration in the ER increased in response to extracellular Ca 2؉ after the ER had been previously depleted despite blockade by thapsigargin. We found two differences between the Jurkat and L6 cells. L6, but not Jurkat cells, inhibited Ca 2؉ uptake at very high Ca 2؉ concentrations. Second, ryanodine receptor blockers inhibited the appearance of cytosolic Ca 2؉ during CCE if added before Ca 2؉ in both cases, but the L6 cells were much more sensitive to ryanodine. Both of these can be explained by the known difference in ryanodine receptors between these cell types. These findings imply that the origin of cytosolic Ca 2؉ during CCE is the ER. Furthermore, kinetic data demonstrated that Ca 2؉ filled the ER before the cytosol during CCE. Our results suggest a plasma membrane Ca 2؉ channel and an ER Ca 2؉ channel joined in tandem, allowing Ca 2؉ to flow directly from the extracellular space to the ER. This explains CCE; any decrease in ER [Ca 2؉ ] relative to extracellular [Ca 2؉ ] would provide the gradient for refilling the ER through a mass-action mechanism.Ca 2ϩ is a critical regulator for a large number of cells, and it is known that for many, the key signaling event is the release of this ion from the ER 1 (1, 2). After release most of the Ca 2ϩ is re-sequestered to the ER through the CaATPase, although some is lost through the plasma membrane to the cell exterior. Maintaining the ER pool of Ca 2ϩ requires re-entry from outside the cell. Casteels and Droogmans (3) first reported a correlation between depletion of ER [Ca 2ϩ ] and entry of Ca 2ϩ from extracellular sources into the cell. This phenomenon was then extensively studied by Putney (4) and named "store-operated" or capacitative calcium entry (CCE).The mechanism for CCE remains unknown, although two general models have been proposed. One, borrowed from the known association of the skeletal muscle plasma membrane potential sensor (L channel) and ER protein (ryanodine receptor), posits a direct connection in which Ca 2ϩ depletion within the ER is sensed by an ER protein, transmitted by proteinprotein interaction to the plasma membrane protein, which then allows entry of Ca 2ϩ into the cytosol. A second, based on second messenger signaling systems such as that for cyclic AMP, posits a mess...

We have previously established that L6 skeletal muscle cell cultures display capacitative calcium entry (CCE), a phenomenon established with other cells in which Ca2+ uptake from outside cells increases when the endoplasmic reticulum (sarcoplasmic reticulum in muscle, or SR) store is decreased. Evidence for CCE rested on the use of thapsigargin (Tg), an inhibitor of the SR CaATPase and consequently transport of Ca2+ from cytosol to SR, and measurements of cytosolic Ca2+. When Ca2+ is added to Ca2+-free cells in the presence of Tg, the measured cytosolic Ca2+ rises. This has been universally interpreted to mean that as SR Ca2+ is depleted, exogenous Ca2+ crosses the plasma membrane, but accumulates in the cytosol due to CaATPase inhibition. Our goal in the present study was to examine CCE in more detail by measuring Ca2+ in both the SR lumen and the cytosol using established fluorescent dye techniques for both. Surprisingly, direct measurement of SR Ca2+ in the presence of Tg showed an increase in luminal Ca2+ concentration in response to added exogenous Ca2+. While we were able to reproduce the conventional demonstration of CCE—an increase of Ca2+ in the cytosol in the presence of thapsigargin—we found that this process was inhibited by the prior addition of ryanodine (Ry), which inhibits the SR Ca2+ release channel, the ryanodine receptor (RyR). This was also unexpected if Ca2+ enters the cytosol first. When Ca2+ was added prior to Ry, the later was unable to exert any inhibition. This implies a competitive interaction between Ca2+ and Ry at the RyR. In addition, we found a further paradox: we had previously found Ry to be an uncompetitive inhibitor of Ca2+ transport through the RyR during excitation-contraction coupling. We also found here that high concentrations of Ca2+ inhibited its own uptake, a known feature of the RyR. We confirmed that Ca2+ enters the cells through the dihydropyridine receptor (DHPR, also known as the L-channel) by demonstrating inhibition by diltiazem. A previous suggestion to the contrary had used Mn2+ in place of direct Ca2+ measurements; we showed that Mn2+ was not inhibited by diltiazem and was not capacitative, and thus not an appropriate probe of Ca2+ flow in muscle cells. Our findings are entirely explained by a new model whereby Ca2+ enters the SR from the extracellular space directly through a combined channel formed from the DHPR and the RyR. These are known to be in close proximity in skeletal muscle. Ca2+ subsequently appears in the cytosol by egress through a separate, unoccupied RyR, explaining Ry inhibition. We suggest that upon excitation, the DHPR, in response to the electrical field of the plasma membrane, shifts to an erstwhile-unoccupied receptor, and Ca2+ is released from the now open RyR to trigger contraction. We discuss how this model also resolves existing paradoxes in the literature, and its implications for other cell types.