Metformin regulates global DNA methylation via mitochondrial one-carbon metabolism.

The anti-diabetic biguanide metformin may exert health-promoting effects via metabolic regulation of the epigenome. Here we show that metformin promotes global DNA methylation in non-cancerous, cancer-prone and metastatic cancer cells by decreasing S-adenosylhomocysteine (SAH), a strong feedback inhibitor of S-adenosylmethionine (SAM)-dependent DNA methyltransferases, while promoting the accumulation of SAM, the universal methyl donor for cellular methylation. Using metformin and a mitochondria/complex I (mCI)-targeted analog of metformin (norMitoMet) in experimental pairs of wild-type and AMP-activated protein kinase (AMPK)-, serine hydroxymethyltransferase 2 (SHMT2)- and mCI-null cells, we provide evidence that metformin increases the SAM:SAH ratio-related methylation capacity by targeting the coupling between serine mitochondrial one-carbon flux and CI activity. By increasing the contribution of one-carbon units to the SAM from folate stores while decreasing SAH in response to AMPK-sensed energetic crisis, metformin can operate as a metabolo-epigenetic regulator capable of reprogramming one of the key conduits linking cellular metabolism to the DNA methylation machinery.

AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B.

Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation.

AMPK maintains energy homeostasis and survival in cancer cells via regulating p38/PGC-1α-mediated mitochondrial biogenesis.

Cancer cells exhibit unique metabolic response and adaptation to the fluctuating microenvironment, yet molecular and biochemical events imprinting this phenomenon are unclear. Here, we show that metabolic homeostasis and adaptation to metabolic stress in cancer cells are primarily achieved by an integrated response exerted by the activation of AMPK. We provide evidence that AMPK-p38-PGC-1α axis, by regulating energy homeostasis, maintains survival in cancer cells under glucose-limiting conditions. Functioning as a molecular switch, AMPK promotes glycolysis by activating PFK2, and facilitates mitochondrial metabolism of non-glucose carbon sources thereby maintaining cellular ATP level. Interestingly, we noted that AMPK can promote oxidative metabolism via increasing mitochondrial biogenesis and OXPHOS capacity via regulating expression of PGC-1α through p38MAPK activation. Taken together, our study signifies the fundamental role of AMPK in controlling cellular bioenergetics and mitochondrial biogenesis in cancer cells.

Differential effects of AMPK agonists on cell growth and metabolism.

As a sensor of cellular energy status, the AMP-activated protein kinase (AMPK) is believed to act in opposition to the metabolic phenotypes favored by proliferating tumor cells. Consequently, compounds known to activate AMPK have been proposed as cancer therapeutics. However, the extent to which the anti-neoplastic properties of these agonists are mediated by AMPK is unclear. Here we examined the AMPK dependence of six commonly used AMPK agonists (metformin, phenformin, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), 2-deoxy-D-glucose (2DG), salicylate and A-769662) and their influence on cellular processes often deregulated in tumor cells. We demonstrate that the majority of these agonists display AMPK-independent effects on cell proliferation and metabolism with only the synthetic activator, A-769662, exerting AMPK-dependent effects on these processes. We find that A-769662 promotes an AMPK-dependent increase in mitochondrial spare respiratory capacity. Finally, contrary to the view of AMPK activity being tumor suppressive, we find that A-769662 confers a selective proliferative advantage to tumor cells growing under nutrient deprivation. Our results indicate that many of the antigrowth properties of these agonists cannot be attributed to AMPK activity in cells, and thus any observed effects using these agonists should be confirmed using AMPK-deficient cells. Ultimately, our data urge caution not only regarding the type of AMPK agonist proposed for cancer treatment but also the context in which they are used.

Lipoprotein internalisation induced by oncogenic AMPK activation is essential to maintain glioblastoma cell growth.

AIM OF THE STUDY: Metabolic adaptations are essential during tumour growth to maintain the high proliferation levels exhibited by cancer cells. In this study, we examined the transformations that occurred in the lipid metabolism in astrocytic tumours, and the possible role of the fuel-sensing enzyme AMPK. Metabolic targets might help design new and effective drugs for cancer. METHODS: To accomplish this objective, we studied both mice and human astrocytic tumours. We first used a mouse model of astrocytoma driven by oncogenic H-RasV12 and/or with PTEN deletion based on the common constitutive activation of the Raf/MEK/ERK and PI3K/AKT cascades in human astrocytomas. We then confirmed the results in human glioblastoma cell lines and in glioblastoma tissue samples from patients. RESULTS: We show that the high levels of activated AMPK, observed in astrocytic tumours, increase extracellular lipid internalisation and reduce energy expenditure by inhibiting 'de novo' fatty acid (FA) synthesis, which allows tumour cells to obtain building blocks and energy to be able to create new organelles and new cells. CONCLUSIONS: Our findings demonstrate that AMPK plays a crucial role in glioblastoma cell growth and suggest that blocking lipoprotein receptors could potentially be used as a plausible therapeutic approach for these and other type of tumours with high levels of AMPK.

Interleukin-6 deletion in mice driven by aP2-Cre-ERT2 prevents against high-fat diet-induced gain weight and adiposity in female mice.

AIM: Interleukin-6 (IL-6) is a major cytokine controlling body weight and metabolism, but because many types of cells can synthesize and respond to IL-6 considerable uncertainty still exists about the mechanisms underlying IL-6 effects. Therefore, the aim of this study was to analyse the effects of tissue-specific deletion of IL-6 using a fatty acid binding protein (aP2) promoter-Cre inducible system (aP2-Cre-ERT2). METHODS: Tissue-specific IL-6 KO mice (aP2-IL-6 KO mice) were produced upon tamoxifen administration and were fed a high-fat diet (HFD, 58.4% kcal from fat) or a control diet (18%) for 14 weeks. RESULTS: aP2-IL-6 KO female mice on a HFD gained less weight and adiposity than littermate wild-type mice, but these effects were not observed in males. Hypothalamic factors such as NPY and AgRP showed a pattern of expression consistent with this sex-specific phenotype. PGC-1α expression was increased in several tissues in aP2-IL-6 KO female mice, which is compatible with increased energy expenditure. Serum leptin, insulin, glucose, cholesterol and triglycerides levels were increased by HFD, and in females IL-6 deficiency reversed this effect in the case of insulin and cholesterol. HFD induced impaired responses to insulin and glucose tolerance tests, but no significant differences between genotypes were observed. CONCLUSION: The present results demonstrate that deletion of IL-6 driven by aP2-Cre regulates body weight, body fat and metabolism in a sex-specific fashion.

The Ca(2+) /calmodulin-dependent kinase kinase ß-AMP-activated protein kinase-α1 pathway regulates phosphorylation of cytoskeletal targets in thrombin-stimulated human platelets.

BACKGROUND: Platelet activation requires sweeping morphologic changes, supported by contraction and remodeling of the platelet actin cytoskeleton. In various other cell types, AMP-activated protein kinase (AMPK) controls the phosphorylation state of cytoskeletal targets. OBJECTIVE: To determine whether AMPK is activated during platelet aggregation and contributes to the control of cytoskeletal targets. RESULTS: We found that AMPK-α1 was mainly activated by thrombin, and not by other platelet agonists, in purified human platelets. Thrombin activated AMPK-α1 ex vivo via a Ca(2+) /calmodulin-dependent kinase kinase ß (CaMKKß)-dependent pathway. Pharmacologic inhibition of CaMKKß blocked thrombin-induced platelet aggregation and counteracted thrombin-induced phosphorylation of several cytoskeletal proteins, namely, regulatory myosin light chains (MLCs), cofilin, and vasodilator-stimulated phosphoprotein (VASP), three key elements involved in actin cytoskeletal contraction and polymerization. Platelets isolated from mice lacking AMPK-α1 showed reduced aggregation in response to thrombin, and this was associated with defects in MLC, cofilin and VASP phosphorylation and actin polymerization. More importantly, we show, for the first time, that the AMPK pathway is activated in platelets of patients undergoing major cardiac surgery, in a heparin-sensitive manner. CONCLUSION: AMPK-α1 is activated by thrombin in human platelets. It controls the phosphorylation of key cytoskeletal targets and actin cytoskeletal remodeling during platelet aggregation.

The AMPK-SIRT signaling network regulates glucose tolerance under calorie restriction conditions.

AIMS: SIRT1 and AMP-activated protein kinase (AMPK) share common activators, actions and target molecules. Previous studies have suggested that a putative SIRT1-AMPK regulatory network could act as the prime initial sensor for calorie restriction-induced adaptations in skeletal muscle-the major site of insulin-stimulated glucose disposal. Our study aimed to investigate whether a feedback loop exists between AMPK and SIRT1 in skeletal muscle and how this may be involved glucose tolerance. MAIN METHODS: To investigate this, we used skeletal muscle-specific AMPKα1/2 knockout mice (AMPKα1/2(-/-)) fed ad libitum (AL) or a 30% calorie restricted (CR) diet and L6 rat myoblasts incubated with SIRT1 inhibitor (EX527). KEY FINDINGS: CR-AMPKα1/2(-/-) displayed impaired glucose tolerance (*p<0.05), in association with down-regulated SIRT1 and PGC-1α expression (<300% vs. CR-WT, (±±)p<0.01). Moreover, AMPK activity was decreased following SIRT1 inhibition in L6 cells (~0.5-fold vs. control, *p<0.05). SIGNIFICANCE: This study demonstrates that skeletal muscle-specific AMPK deficiency impairs the beneficial effects of CR on glucose tolerance and that these effects may be dependent on reduced SIRT1 levels.

The anti-diabetic drug metformin does not affect bone mass in vivo or fracture healing.

SUMMARY: The present study shows no adverse effects of the anti-diabetic drug metformin on bone mass and fracture healing in rodents but demonstrates that metformin is not osteogenic in vivo, as previously proposed. INTRODUCTION: In view of the increased incidence of fractures in patients with type 2 diabetes mellitus (T2DM), we investigated the effects of metformin, a widely used T2DM therapy, on bone mass and fracture healing in vivo using two different rodent models and modes of metformin administration. METHODS: We first subjected 12-week-old female C57BL/6 mice to ovariectomy (OVX). Four weeks after OVX, mice received either saline or metformin administered by gavage (100 mg/kg/daily). After 4 weeks of treatment, bone micro-architecture and cellular activity were determined in tibia by micro-CT and bone histomorphometry. In another experiment, female Wistar rats aged 3 months were given only water or metformin for 8 weeks via the drinking water (2 mg/ml). After 4 weeks of treatment, a mid-diaphyseal osteotomy was performed in the left femur. Rats were sacrificed 4 weeks after osteotomy and bone architecture analysed by micro-CT in the right tibia while fracture healing and callus volume were determined in the left femur by X-ray analysis and micro-CT, respectively. RESULTS: In both models, our results show no significant differences in cortical and trabecular bone architecture in metformin-treated rodents compared to saline. Metformin had no effect on bone resorption but reduced bone formation rate in trabecular bone. Mean X-ray scores assessed on control and metformin fractures showed no significant differences of healing between the groups. Fracture callus volume and mineral content after 4 weeks were similar in both groups. CONCLUSIONS: Our results indicate that metformin has no effect on bone mass in vivo or fracture healing in rodents.

Role of AMPK in UVB-induced DNA damage repair and growth control.

Skin cancer is the most common cancer in the United States, while DNA-damaging ultraviolet B (UVB) radiation from the sun remains the major environmental risk factor. Reducing skin cancer incidence is becoming an urgent issue. The energy-sensing enzyme 5'-AMP-activated protein kinase (AMPK) has a key role in the regulation of cellular lipid and protein metabolism in response to stimuli such as exercise and changes in fuel availability. However, the role of AMPK in the response of skin cells to UVB damage and in skin cancer prevention remains unknown. Here we show that AMPK activation is reduced in human and mouse squamous cell carcinoma as compared with normal skin, and by UVB irradiation, suggesting that AMPK is a tumor suppressor. At the molecular level, AMPK deletion reduced the expression of the DNA repair protein xeroderma pigmentosum C (XPC) and UVB-induced DNA repair. AMPK activation by its activators AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) and metformin (N',N'-dimethylbiguanide), the most widely used antidiabetic drug, increased the expression of XPC and UVB-induced DNA repair in mouse skin, normal human epidermal keratinocytes, and AMPK wild-type (WT) cells but not in AMPK-deficient cells, indicating an AMPK-dependent mechanism. Topical treatment with AICAR and metformin not only delayed onset of UVB-induced skin tumorigenesis but also reduced tumor multiplicity. Furthermore, AMPK deletion increased extracellular signal-regulated kinase (ERK) activation and cell proliferation, whereas AICAR and metformin inhibited ERK activation and cell proliferation in keratinocytes, mouse skin, AMPK WT and AMPK-deficient cells, suggesting an AMPK-independent mechanism. Finally, in UVB-damaged tumor-bearing mice, both topical and systemic metformin prevented the formation of new tumors and suppressed growth of established tumors. Our findings not only suggest that AMPK is a tumor suppressor in the skin by promoting DNA repair and controlling cell proliferation, but also demonstrate previously unknown mechanisms by which the AMPK activators prevent UVB-induced skin tumorigenesis.

Mice lacking AMP-activated protein kinase α1 catalytic subunit have increased bone remodelling and modified skeletal responses to hormonal challenges induced by ovariectomy and intermittent PTH treatment.

AMP-activated protein kinase (AMPK) is a key regulator of cellular and body energy homeostasis. We previously demonstrated that AMPK activation in osteoblasts increases in vitro bone formation while deletion of the Ampkα1 (Prkaa1) subunit, the dominant catalytic subunit expressed in bone, leads to decreased bone mass in vivo. To investigate the cause of low bone mass in the Ampkα1(-/-) mice, we analysed bone formation and resorption in the tibia of these mice by dynamic histomorphometry and determined whether bone turnover can be stimulated in the absence of the Ampkα1 subunit. We subjected 12-week-old Ampkα1(+)(/)(+) and Ampkα1(-/-) mice to ovariectomy (OVX), intermittent PTH (iPTH) administration (80 µg/kg per day, 5 days/week) or both OVX and iPTH hormonal challenges. Tibiae were harvested from these mice and bone micro-architecture was determined by micro-computed tomography. We show for the first time that Ampkα1(-/-) mice have a high bone turnover at the basal level in favour of bone resorption. While both Ampkα1(+)(/)(+) and Ampkα1(-/-) mice lost bone mass after OVX, the bone loss in Ampkα1(-/-) mice was lower compared with controls. iPTH increased trabecular and cortical bone indexes in both ovariectomised Ampkα1(+)(/)(+) and Ampkα1(-/-) mice. However, ovariectomised Ampkα1(-/-) mice showed a smaller increase in bone parameters in response to iPTH compared with Ampkα1(+)(/)(+) mice. By contrast, non-ovariectomised Ampkα1(-/-) mice responded better to iPTH treatment than non-ovariectomised Ampkα1(+)(/)(+) mice. Overall, these data demonstrate that Ampkα1(-/-) mice are less affected by changes in bone turnover induced by OVX but respond better to the anabolic challenge induced by iPTH. These results suggest that AMPKα1 activation may play a role in the hormonal regulation of bone remodelling.

AMP-activated protein kinase pathway and bone metabolism.

There is increasing evidence that osteoporosis, similarly to obesity and diabetes, could be another disorder of energy metabolism. AMP-activated protein kinase (AMPK) has emerged over the last decade as a key sensing mechanism in the regulation of cellular energy homeostasis and is an essential mediator of the central and peripheral effects of many hormones on the metabolism of appetite, fat and glucose. Novel work demonstrates that the AMPK signaling pathway also plays a role in bone physiology. Activation of AMPK promotes bone formation in vitro and the deletion of α or ß subunit of AMPK decreases bone mass in mice. Furthermore, AMPK activity in bone cells is regulated by the same hormones that regulate food intake and energy expenditure through AMPK activation in the brain and peripheral tissues. AMPK is also activated by antidiabetic drugs such as metformin and thiazolidinediones (TZDs), which also impact on skeletal metabolism. Interestingly, TZDs have detrimental skeletal side effects, causing bone loss and increasing the risk of fractures, although the role of AMPK mediation is still unclear. These data are presented in this review that also discusses the potential roles of AMPK in bone as well as the possibility for AMPK to be a future therapeutic target for intervention in osteoporosis.

Metformin activates AMP-activated protein kinase in primary human hepatocytes by decreasing cellular energy status.

AIM/HYPOTHESIS: The glucose-lowering drug metformin has been shown to activate hepatic AMP-activated protein kinase (AMPK), a master kinase regulating cellular energy homeostasis. However, the underlying mechanisms remain controversial and have never been investigated in primary human hepatocytes. METHODS: Hepatocytes isolated from rat, mouse and human livers were treated with various concentrations of metformin. Isoform-specific AMPKα abundance and activity, as well as intracellular adenine nucleotide levels and mitochondrial oxygen consumption rates were determined at different time points. RESULTS: Metformin dose- and time-dependently increased AMPK activity in rat and human hepatocytes, an effect associated with a significant rise in cellular AMP:ATP ratio. Surprisingly, we found that AMPKα2 activity was undetectable in human compared with rat hepatocytes, while AMPKα1 activities were comparable. Accordingly, metformin only increased AMPKα1 activity in human hepatocytes, although both AMPKα isoforms were activated in rat hepatocytes. Analysis of mRNA expression and protein levels confirmed that only AMPKα1 is present in human hepatocytes; it also showed that the distribution of ß and γ regulatory subunits differed between species. Finally, we demonstrated that the increase in AMP:ATP ratio in hepatocytes from liver-specific Ampkα1/2 (also known as Prkaa1/2) knockout mice and humans is due to a similar and specific inhibition of the mitochondrial respiratory-chain complex 1 by metformin. CONCLUSIONS/INTERPRETATION: Activation of hepatic AMPK by metformin results from a decrease in cellular energy status owing to metformin's AMPK-independent inhibition of the mitochondrial respiratory-chain complex 1. The unique profile of AMPK subunits found in human hepatocytes should be considered when developing new pharmacological agents to target the kinase.

AMP-activated protein kinase (AMPK) activation regulates in vitro bone formation and bone mass.

Adenosine 5'-monophosphate-activated protein kinase (AMPK), a regulator of energy homeostasis, has a central role in mediating the appetite-modulating and metabolic effects of many hormones and antidiabetic drugs metformin and glitazones. The objective of this study was to determine if AMPK can be activated in osteoblasts by known AMPK modulators and if AMPK activity is involved in osteoblast function in vitro and regulation of bone mass in vivo. ROS 17/2.8 rat osteoblast-like cells were cultured in the presence of AMPK activators (AICAR and metformin), AMPK inhibitor (compound C), the gastric peptide hormone ghrelin and the beta-adrenergic blocker propranolol. AMPK activity was measured in cell lysates by a functional kinase assay and AMPK protein phosphorylation was studied by Western Blotting using an antibody recognizing AMPK Thr-172 residue. We demonstrated that treatment of ROS 17/2.8 cells with AICAR and metformin stimulates Thr-172 phosphorylation of AMPK and dose-dependently increases its activity. In contrast, treatment of ROS 17/2.8 cells with compound C inhibited AMPK phosphorylation. Ghrelin and propranolol dose-dependently increased AMPK phosphorylation and activity. Cell proliferation and alkaline phosphatase activity were not affected by metformin treatment while AICAR significantly inhibited ROS 17/2.8 cell proliferation and alkaline phosphatase activity at high concentrations. To study the effect of AMPK activation on bone formation in vitro, primary osteoblasts obtained from rat calvaria were cultured for 14-17days in the presence of AICAR, metformin and compound C. Formation of 'trabecular-shaped' bone nodules was evaluated following alizarin red staining. We demonstrated that both AICAR and metformin dose-dependently increase trabecular bone nodule formation, while compound C inhibits bone formation. When primary osteoblasts were co-treated with AICAR and compound C, compound C suppressed the stimulatory effect of AICAR on bone nodule formation. AMPK is a alphabetagamma heterotrimer, where alpha is the catalytic subunit. RT-PCR analysis of AMPK subunits in ROS17/2.8 osteoblastic cells and in mouse tibia showed that the AMPKalpha1 subunit is the dominant isoform expressed in bone. We analysed the bone phenotype of 4month-old male wild type (WT) and AMPKalpha1-/- KO mice using micro-CT. Both cortical and trabecular bone compartments were smaller in the AMPK alpha1-deficient mice compared to the WT mice. Altogether, our data support a role for AMPK signalling in skeletal physiology.

Ablation of AMP-activated protein kinase alpha1 and alpha2 from mouse pancreatic beta cells and RIP2.Cre neurons suppresses insulin release in vivo.

AIMS/HYPOTHESIS: AMP-activated protein kinase (AMPK) is an evolutionarily conserved enzyme and a target of glucose-lowering agents, including metformin. However, the precise role or roles of the enzyme in controlling insulin secretion remain uncertain. METHODS: The catalytic alpha1 and alpha2 subunits of AMPK were ablated selectively in mouse pancreatic beta cells and hypothalamic neurons by breeding Ampkalpha1 [also known as Prkaa1]-knockout mice, bearing floxed Ampkalpha2 [also known as Prkaa2] alleles (Ampkalpha1 ( -/- ),alpha2( fl/fl ),), with mice expressing Cre recombinase under the rat insulin promoter (RIP2). RIP2 was used to express constitutively activated AMPK selectively in beta cells in transgenic mice. Food intake, body weight and urinary catecholamines were measured using metabolic cages. Glucose and insulin tolerance were determined after intraperitoneal injection. Beta cell mass and morphology were analysed by optical projection tomography and confocal immunofluorescence microscopy, respectively. Granule docking, insulin secretion, membrane potential and intracellular free Ca(2+) were measured with standard techniques. RESULTS: Trigenic Ampkalpha1 ( -/- ),alpha2( fl/fl ) expressing Cre recombinase and lacking both AMPKalpha subunits in the beta cell, displayed normal body weight and increased insulin sensitivity, but were profoundly insulin-deficient. Secreted catecholamine levels were unchanged. Total beta cell mass was unaltered, while mean islet and beta cell volume were reduced. AMPK-deficient beta cells displayed normal glucose-induced changes in membrane potential and intracellular free Ca(2+), while granule docking and insulin secretion were enhanced. Conversely, betaAMPK transgenic mice were glucose-intolerant and displayed defective insulin secretion. CONCLUSIONS/INTERPRETATION: Inhibition of AMPK activity within the beta cell is necessary, but not sufficient for stimulation of insulin secretion by glucose to occur. AMPK activation in extrapancreatic RIP2.Cre-expressing cells might also influence insulin secretion in vivo.

AMP-activated protein kinase in the regulation of hepatic energy metabolism: from physiology to therapeutic perspectives.

As the liver is central in the maintenance of glucose homeostasis and energy storage, knowledge of the physiology as well as physiopathology of hepatic energy metabolism is a prerequisite to our understanding of whole-body metabolism. Hepatic fuel metabolism changes considerably depending on physiological circumstances (fed vs. fasted state). In consequence, hepatic carbohydrate, lipid and protein synthesis/utilization are tightly regulated according to needs. Fatty liver and hepatic insulin resistance (both frequently associated with the metabolic syndrome) or increased hepatic glucose production (as observed in type 2 diabetes) resulted from alterations in substrates oxidation/storage balance in the liver. Because AMP-activated protein kinase (AMPK) is considered as a cellular energy sensor, it is important to gain understanding of the mechanism by which hepatic AMPK coordinates hepatic energy metabolism. AMPK has been implicated as a key regulator of physiological energy dynamics by limiting anabolic pathways (to prevent further ATP consumption) and by facilitating catabolic pathways (to increase ATP generation). Activation of hepatic AMPK leads to increased fatty acid oxidation and simultaneously inhibition of hepatic lipogenesis, cholesterol synthesis and glucose production. In addition to a short-term effect on specific enzymes, AMPK also modulates the transcription of genes involved in lipogenesis and mitochondrial biogenesis. The identification of AMPK targets in hepatic metabolism should be useful in developing treatments to reverse metabolic abnormalities of type 2 diabetes and the metabolic syndrome.

Targeting AMP-activated protein kinase as a novel therapeutic approach for the treatment of metabolic disorders.

In the light of recent studies in humans and rodents, AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, has been described as an integrator of regulatory signals monitoring systemic and cellular energy status. AMP-activated protein kinase (AMPK) has been proposed to function as a 'fuel gauge' to monitor cellular energy status in response to nutritional environmental variations. Recently, it has been proposed that AMPK could provide a link in metabolic defects underlying progression to the metabolic syndrome. AMPK is a heterotrimeric enzyme complex consisting of a catalytic subunit alpha and two regulatory subunits beta and gamma. AMPK is activated by rising AMP and falling ATP. AMP activates the system by binding to the gamma subunit that triggers phosphorylation of the catalytic alpha subunit by the upstream kinases LKB1 and CaMKKbeta (calmodulin-dependent protein kinase kinase). AMPK system is a regulator of energy balance that, once activated by low energy status, switches on ATP-producing catabolic pathways (such as fatty acid oxidation and glycolysis), and switches off ATP-consuming anabolic pathways (such as lipogenesis), both by short-term effect on phosphorylation of regulatory proteins and by long-term effect on gene expression. As well as acting at the level of the individual cell, the system also regulates food intake and energy expenditure at the whole body level, in particular by mediating the effects of insulin sensitizing adipokines leptin and adiponectin. AMPK is robustly activated during skeletal muscle contraction and myocardial ischaemia playing a role in glucose transport and fatty acid oxidation. In liver, activation of AMPK results in enhanced fatty acid oxidation as well as decreased glucose production. Moreover, the AMPK system is one of the probable targets for the anti-diabetic drugs biguanides and thiazolidinediones. Thus, the relationship between AMPK activation and beneficial metabolic effects provide the rationale for the development of new therapeutic strategies in metabolic disorders.

Iron overload in Hepc1(-/-) mice is not impairing glucose homeostasis.

Diabetes Mellitus is found with increasing frequency in iron overload patients with hemochromatosis. In these conditions, the pancreas shows predominant iron overload in acini but also islet beta-cells. We assess glucose homeostasis status in iron-overloaded hepcidin-deficient mice. These mice presented with heavy pancreatic iron deposits but only in the acini. The beta-cell function was found unaffected with a normal production and secretion of insulin. The mutant mice were not diabetic, responded as the control group to glucose and insulin challenges, with no alteration of insulin signalling in the muscle and the liver. These results indicate that, beta-cells iron deposits-induced decreased insulin secretory capacity might be of primary importance to trigger diabetes in hemochromatosic patients.

A possible role for AMP-activated protein kinase in exercise-induced glucose utilization: insights from humans and transgenic animals.

Exercise-induced glucose uptake in skeletal muscle is mediated by an insulin-independent mechanism, but the actual signals to glucose transport in response to muscle contraction have not been identified. The 5'-AMP-activated protein kinase (AMPK) has emerged as a putative mediator of contraction-induced glucose transport, although no conclusive evidence has been provided so far. Recent experiments in AMPK transgenic mice suggest that glucose transport induced by 5-amino-4-imidazolecarboxamide riboside (AICAR) or hypoxia is mediated by AMPK. In contrast, contraction-induced glucose transport in rodent skeletal muscle induced by electrical stimulation in vitro or in situ is not influenced or is only partially reduced by abolishing both or one of the catalytic AMPK subunits. This is compatible with exercise studies done in humans, where no tight correlation is found between AMPK activity and glucose uptake during exercise. Taken together, these results question an essential role of AMPK in exercise-induced glucose uptake and imply that one or more additional pathways are involved in mediating glucose transport in skeletal muscle during exercise.