Amino acid homeostasis is a target of metformin therapy.

OBJECTIVE: Unexplained changes in regulation of branched chain amino acids (BCAA) during diabetes therapy with metformin have been known for years. Here we have investigated mechanisms underlying this effect. METHODS: We used cellular approaches, including single gene/protein measurements, as well as systems-level proteomics. Findings were then cross-validated with electronic health records and other data from human material. RESULTS: In cell studies, we observed diminished uptake/incorporation of amino acids following metformin treatment of liver cells and cardiac myocytes. Supplementation of media with amino acids attenuated known effects of the drug, including on glucose production, providing a possible explanation for discrepancies between effective doses in vivo and in vitro observed in most studies. Data-Independent Acquisition proteomics identified that SNAT2, which mediates tertiary control of BCAA uptake, was the most strongly suppressed amino acid transporter in liver cells following metformin treatment. Other transporters were affected to a lesser extent. In humans, metformin attenuated increased risk of left ventricular hypertrophy due to the AA allele of KLF15, which is an inducer of BCAA catabolism. In plasma from a double-blind placebo-controlled trial in nondiabetic heart failure (trial registration: NCT00473876), metformin caused selective accumulation of plasma BCAA and glutamine, consistent with the effects in cells. CONCLUSIONS: Metformin restricts tertiary control of BCAA cellular uptake. We conclude that modulation of amino acid homeostasis contributes to therapeutic actions of the drug.

Metformin selectively targets redox control of complex I energy transduction.

Many guanide-containing drugs are antihyperglycaemic but most exhibit toxicity, to the extent that only the biguanide metformin has enjoyed sustained clinical use. Here, we have isolated unique mitochondrial redox control properties of metformin that are likely to account for this difference. In primary hepatocytes and H4IIE hepatoma cells we found that antihyperglycaemic diguanides DG5-DG10 and the biguanide phenformin were up to 1000-fold more potent than metformin on cell signalling responses, gluconeogenic promoter expression and hepatocyte glucose production. Each drug inhibited cellular oxygen consumption similarly but there were marked differences in other respects. All diguanides and phenformin but not metformin inhibited NADH oxidation in submitochondrial particles, indicative of complex I inhibition, which also corresponded closely with dehydrogenase activity in living cells measured by WST-1. Consistent with these findings, in isolated mitochondria, DG8 but not metformin caused the NADH/NAD+ couple to become more reduced over time and mitochondrial deterioration ensued, suggesting direct inhibition of complex I and mitochondrial toxicity of DG8. In contrast, metformin exerted a selective oxidation of the mitochondrial NADH/NAD+ couple, without triggering mitochondrial deterioration. Together, our results suggest that metformin suppresses energy transduction by selectively inducing a state in complex I where redox and proton transfer domains are no longer efficiently coupled.

Anti-Inflammatory Effects of Metformin Irrespective of Diabetes Status.

RATIONALE: The diabetes mellitus drug metformin is under investigation in cardiovascular disease, but the molecular mechanisms underlying possible benefits are poorly understood. OBJECTIVE: Here, we have studied anti-inflammatory effects of the drug and their relationship to antihyperglycemic properties. METHODS AND RESULTS: In primary hepatocytes from healthy animals, metformin and the IKKß (inhibitor of kappa B kinase) inhibitor BI605906 both inhibited tumor necrosis factor-α-dependent IκB degradation and expression of proinflammatory mediators interleukin-6, interleukin-1ß, and CXCL1/2 (C-X-C motif ligand 1/2). Metformin suppressed IKKα/ß activation, an effect that could be separated from some metabolic actions, in that BI605906 did not mimic effects of metformin on lipogenic gene expression, glucose production, and AMP-activated protein kinase activation. Equally AMP-activated protein kinase was not required either for mitochondrial suppression of IκB degradation. Consistent with discrete anti-inflammatory actions, in macrophages, metformin specifically blunted secretion of proinflammatory cytokines, without inhibiting M1/M2 differentiation or activation. In a large treatment naive diabetes mellitus population cohort, we observed differences in the systemic inflammation marker, neutrophil to lymphocyte ratio, after incident treatment with either metformin or sulfonylurea monotherapy. Compared with sulfonylurea exposure, metformin reduced the mean log-transformed neutrophil to lymphocyte ratio after 8 to 16 months by 0.09 U (95% confidence interval, 0.02-0.17; P=0.013) and increased the likelihood that neutrophil to lymphocyte ratio would be lower than baseline after 8 to 16 months (odds ratio, 1.83; 95% confidence interval, 1.22-2.75; P=0.00364). Following up these findings in a double-blind placebo controlled trial in nondiabetic heart failure (trial registration: NCT00473876), metformin suppressed plasma cytokines including the aging-associated cytokine CCL11 (C-C motif chemokine ligand 11). CONCLUSION: We conclude that anti-inflammatory properties of metformin are exerted irrespective of diabetes mellitus status. This may accelerate investigation of drug utility in nondiabetic cardiovascular disease groups. CLINICAL TRIAL REGISTRATION: Name of the trial registry: TAYSIDE trial (Metformin in Insulin Resistant Left Ventricular [LV] Dysfunction). URL: https://www.clinicaltrials.gov. Unique identifier: NCT00473876.

Investigation of salicylate hepatic responses in comparison with chemical analogues of the drug.

Anti-hyperglycaemic effects of the hydroxybenzoic acid salicylate might stem from effects of the drug on mitochondrial uncoupling, activation of AMP-activated protein kinase, and inhibition of NF-κB signalling. Here, we have gauged the contribution of these effects to control of hepatocyte glucose production, comparing salicylate with inactive hydroxybenzoic acid analogues of the drug. In rat H4IIE hepatoma cells, salicylate was the only drug tested that activated AMPK. Salicylate also reduced mTOR signalling, but this property was observed widely among the analogues. In a sub-panel of analogues, salicylate alone reduced promoter activity of the key gluconeogenic enzyme glucose 6-phosphatase and suppressed basal glucose production in mouse primary hepatocytes. Both salicylate and 2,6 dihydroxybenzoic acid suppressed TNFα-induced IκB degradation, and in genetic knockout experiments, we found that the effect of salicylate on IκB degradation was AMPK-independent. Previous data also identified AMPK-independent regulation of glucose but we found that direct inhibition of neither NF-κB nor mTOR signalling suppressed glucose production, suggesting that other factors besides these cell signalling pathways may need to be considered to account for this response to salicylate. We found, for example, that H4IIE cells were exquisitely sensitive to uncoupling with modest doses of salicylate, which occurred on a similar time course to another anti-hyperglycaemic uncoupling agent 2,4-dinitrophenol, while there was no discernible effect at all of two salicylate analogues which are not anti-hyperglycaemic. This finding supports much earlier literature suggesting that salicylates exert anti-hyperglycaemic effects at least in part through uncoupling.

Evidence that the capacity of nongenotoxic carcinogens to induce oxidative stress is subject to marked variability.

Many drugs and environmental chemicals which are not directly mutagenic have the capacity to increase the incidence of tumors in the liver and other tissues. For this reason, such compounds are known as nongenotoxic carcinogens. The mechanisms underlying their effects remain unclear; however, their capacity to induce oxidative stress is considered to be a critical step in the carcinogenic process, although the evidence that this is actually the case remains equivocal and sparse. We have exploited a novel heme oxygenase-1 reporter mouse to evaluate the capacity of nongenotoxic carcinogens with different mechanisms of action to induce oxidative stress in the liver in vivo. When these compounds were administered at doses reported to cause liver tumors, marked differences in activation of the reporter were observed. 1,4-Dichlorobenzene and nafenopin were strong inducers of oxidative stress, whereas phenobarbital, piperonyl butoxide, cyproterone acetate, and WY14,643 were, at best, only very weak inducers. In the case of phenobarbital and thioacetamide, the number of LacZ-positive hepatocytes increased with time, and for the latter also with dose. The data obtained demonstrate that although some nongenotoxic carcinogens can induce oxidative stress, it is not a dominant feature of the response to these compounds. Therefore in contrast to the current models, these data suggest that oxidative stress is not a key determinant in the mechanism of nongenotoxic carcinogenesis but may contribute to the effects in a compound-specific manner.

The anti-neurodegenerative agent clioquinol regulates the transcription factor FOXO1a.

Many diseases of aging including AD (Alzheimer's disease) and T2D (Type 2 diabetes) are strongly associated with common risk factors, suggesting that there may be shared aging mechanisms underlying these diseases, with the scope to identify common cellular targets for therapy. In the present study we have examined the insulin-like signalling properties of an experimental AD 8-hydroxyquinoline drug known as CQ (clioquinol). The IIS [insulin/IGF-1 (insulin-like growth factor-1) signalling] kinase Akt/PKB (protein kinase B) inhibits the transcription factor FOXO1a (forkhead box O1a) by phosphorylating it on residues that trigger its exit from the nucleus. In HEK (human embryonic kidney)-293 cells, we found that CQ treatment induces similar responses. A key transcriptional response to IIS is the inhibition of hepatic gluconeogenic gene expression, and, in rat liver cells, CQ represses expression of the key gluconeogenic regulatory enzymes PEPCK (phosphoenolpyruvate carboxykinase) and G6Pase (glucose-6-phosphatase). The effects on FOXO1a and gluconeogenic gene expression require the presence of Zn2+ ions, reminiscent of much earlier studies examining diabetogenic properties of 8-hydroxyquinolines. Comparative investigation of the signalling properties of a panel of these compounds demonstrates that CQ alone exhibits FOXO1a regulation without diabetogenicity. Our results suggest that Zn2+-dependent regulation of FOXOs and gluconeogenesis may contribute to the therapeutic properties of this drug. Further investigation of this signalling response might illuminate novel pharmacological strategies for the treatment of age-related diseases.

Zinc-dependent effects of small molecules on the insulin-sensitive transcription factor FOXO1a and gluconeogenic genes.

Metal-binding compounds have recently been reported to have anti-hyperglycaemic properties in vivo. In the current study, we have investigated the ability of these compounds and related structures to induce insulin-like signal transduction to downstream effectors such as the transcription factor FOXO1a and the key gluconeogenic regulatory enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase). Our results indicate that ß-thujaplicin, diethyldithiocarbamate (DEDTC) and its clinically-used dimer disulfiram, induce insulin-like dose-dependent effects on signalling to FOXO1a in a manner that is strictly dependent on the presence of zinc ions, as other ions including aluminium, cobalt, copper, lithium and manganese cannot substitute. The most potent compound tested on gluconeogenesis is disulfiram, which in the presence of 10 µM zinc, inhibited both PEPCK and G6Pase with an IC50 of 4 µM. Our results demonstrate that metal-binding compounds with diverse structures can induce zinc-dependent insulin-like effects on signal transduction and gene expression.

Black tea polyphenols mimic insulin/insulin-like growth factor-1 signalling to the longevity factor FOXO1a.

In vertebrates and invertebrates, relationships between diet and health are controlled by a conserved signalling pathway responsive to insulin-like ligands. In invertebrate models for example, forkhead transcription factor family O (FOXO) transcription factors in this pathway regulate the rate of aging in response to dietary cues, and in vertebrates, obesity and age-induced deficits in the same pathway are thought to contribute to dysregulation of hepatic gluconeogenesis through genes such as phosphoenolpyruvate carboxykinase (PEPCK). Recently, we have begun to screen for dietary constituents capable of regulating this pathway in our cell culture model. Here, we identify three black tea theaflavins, theaflavin 3-O-gallate, theaflavin 3'-O-gallate, theaflavin 3,3'di-O-gallate and thearubigins as novel mimics of insulin/IGF-1 action on mammalian FOXO1a, PEPCK and moreover we provide evidence that the effects on this pathway of the green tea constituent (-)-epigallocatechin gallate depend on its ability to be converted into these larger structures. With the exception of water, tea is the most popular drink globally, but despite this, little is known about the biological availability of black tea polyphenols in vivo or the molecular target(s) mediating the effects presented here. Further investigation in these two areas might provide insight into how age-related metabolic disease may be deferred.