HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Brunmair, B. Articles by Fürnsinn, C. Search for Related Content PubMed PubMed Citation Articles by Brunmair, B. Articles by Fürnsinn, C. Social Bookmarking                What's this?

Thiazolidinediones, Like Metformin, Inhibit Respiratory Complex I

A Common Mechanism Contributing to Their Antidiabetic Actions?

¹ Department of Medicine III, Division of Endocrinology & Metabolism, University of Vienna, Vienna, Austria
² Basic Research in Pharmacology and Toxicology, Veterinary University Vienna, Vienna, Austria
³ Department of Transplant Surgery, Clinical and Interdisciplinary Bioenergetics, University of Innsbruck, Innsbruck, Austria

	ABSTRACT

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Metformin and thiazolidinediones (TZDs) are believed to exert their antidiabetic effects via different mechanisms. As evidence suggests that both impair cell respiration in vitro, this study compared their effects on mitochondrial functions. The activity of complex I of the respiratory chain, which is known to be affected by metformin, was measured in tissue homogenates that contained disrupted mitochondria. In homogenates of skeletal muscle, metformin and TZDs reduced the activity of complex I (30 mmol/l metformin, -15 ± 2%; 100 µmol/l rosiglitazone, -54 ± 7; and 100 µmol/l pioglitazone, -12 ± 4; P < 0.05 each). Inhibition of complex I was confirmed by reduced state 3 respiration of isolated mitochondria consuming glutamate + malate as substrates for complex I (30 mmol/l metformin, -77 ± 1%; 100 µmol/l rosiglitazone, -24 ± 4; and 100 µmol/l pioglitazone, -18 ± 5; P < 0.05 each), whereas respiration with succinate feeding into complex II was unaffected. In line with inhibition of complex I, 24-h exposure of isolated rat soleus muscle to metformin or TZDs reduced cell respiration and increased anaerobic glycolysis (glucose oxidation: 270 µmol/l metformin, -30 ± 9%; 9 µmol/l rosiglitazone, -25 ± 8; and 9 µmol/l pioglitazone, -45 ± 3; lactate release: 270 µmol/l metformin, +84 ± 12; 9 µmol/l rosiglitazone, +38 ± 6; and 9 µmol/l pioglitazone, +64 ± 11; P < 0.05 each). As both metformin and TZDs inhibit complex I activity and cell respiration in vitro, similar mitochondrial actions could contribute to their antidiabetic effects.

Although metformin has been in clinical use for decades, the early steps that mediate its antidiabetic action are still not clear. It is established that metformin inhibits the enzymatic activity of complex I of the respiratory chain and thereby impairs both mitochondrial function and cell respiration (2–4). Mitochondrial disruption on the level of complex I is held responsible for elevated plasma lactate concentrations in metformin-treated patients (5,6), which in very rare instances can aggravate to lactic acidosis (7). There is evidence that complex I inhibition could also be the cause of metformin’s beneficial antihyperglycemic action (2,8), but this matter awaits final clarification.

At variance to the uncertainties about metformin’s initial molecular target, the antidiabetic effects of TZDs are unanimously attributed to their agonistic action on peroxisome proliferator–activated receptor (PPAR)-, which is predominantly expressed in fat cells (9,10) and is not addressed by metformin (11). Via activation of this nuclear receptor, TZDs modulate gene expression, trigger adipocyte differentiation, and induce remodeling of adipose tissue, which is associated with changes in adipocyte signal output (9,10,12). It is believed that adipose tissue–derived signals including free fatty acids and peptide hormones (adiponectin, resistin, leptin, and tumor necrosis factor-) mediate the TZD-induced improvement of skeletal muscle glucose disposal (9,10,12).

TZD action via PPAR- and adipose tissue, however, does not exclude contributions by other mechanisms. In vitro, TZDs reduce fuel oxidation and elevate lactate release by direct action on skeletal muscle, which is obviously a group effect and independent of PPAR-–mediated gene expression (13,14). Such a PPAR-–independent shift from aerobic to anaerobic fuel utilization has likewise been described in other TZD-exposed tissues and cells and hints at an inhibition of cell respiration (15–17). Taken together, these findings suggest that TZDs could share metformin’s inhibitory influence on mitochondrial function.

The present study aimed to define better the mechanisms underlying TZD action on mitochondrial function and cell respiration in vitro and to compare directly the effects of TZDs with those induced by metformin. Parallels in the pharmacological actions of TZDs and metformin are shown and hint at a possible involvement of similar mitochondrial mechanisms in their metabolic effects. In a wider context, our findings are in line with the hypothesis that changes in the cellular energy charge could have a basic role in the pharmacological and physiological modulation of insulin sensitivity.

	RESEARCH DESIGN AND METHODS

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Male Sprague-Dawley rats were purchased from the breeding facilities of the University of Vienna (Himberg, Austria). They were kept at an artificial 12-h light:dark cycle at constant room temperature and, unless stated otherwise, were provided with conventional laboratory diet and tap water ad libitum. Food but not water was withdrawn overnight before rats were killed by cervical dislocation between 8:30 and 9:30 A.M. for the preparation of tissues. All experiments were performed according to local law and to the principles of good laboratory animal care.

Test compounds.
Agents examined were rotenone (a specific and irreversible inhibitor of respiratory complex I; Sigma, St. Louis, MO), metformin (1,1-dimethylbiguanide hydrochloride, inhibitory action on complex I; Sigma), troglitazone and pioglitazone (PPAR- agonistic TZDs; provided by Sankyo, Tokyo, Japan), rosiglitazone and RWJ-241947 (PPAR- agonistic TZDs, the latter also known as MCC-555; provided by Johnson & Johnson, Raritan, NJ), GI-262570 and RWJ-348260 (PPAR- agonists without TZD structure; provided by Roche, Basel, Switzerland, and Johnson & Johnson, respectively), PD-68235 and bisphenol A diglycidyl ether (PPAR- antagonists, the latter also known as BADGE; provided by Pfizer, Ann Arbor, MI, and purchased from Fluka, Buchs, Switzerland, respectively), YM440 (an oxadiazolidinedione with a structure similar to TZDs but without major PPAR- activity; provided by Yamanouchi, Tsukuba, Japan), glimepiride (a sulfonylurea; provided by Aventis, Frankfurt am Main, Germany), and dexlipotam [R-(+)--lipoic acid; provided by Viatris, Frankfurt am Main, Germany].

Complex I activity in tissue homogenates.
Immediately after killing the 5- to 7-week-old rats (140 g body wt), samples of gastrocnemius muscle (red part) and liver were prepared, weighed, frozen in liquid nitrogen, and stored at -70°C. For analysis, tissue specimens were thawed, cut into small pieces, and brought into 0.1 mmol/l K phosphate buffer (30 mg tissue/ml, 0°C, pH adjusted to 7.4 with KOH) containing 0.3% wt/vol fatty acid–free BSA (Roche). The tissues were then homogenized for 1 min with a Polytron homogenizer (Kriens, Switzerland) and sonicated to disrupt all cells and mitochondria (70 pulses, Labsonic U; Braun, Melsungen, Germany).

Complex I is part of the electron transport chain, which is located in the inner mitochondrial membrane, and is responsible for electron transfer from NADH to ubiquinone. Its activity was determined by a spectrophotometric assay based on the enzymatic reaction NADH + H⁺ + ubiquinone-1 NAD⁺ + dihydroubiquinone-1. A mixture of 1.8 ml of K phosphate buffer (see above), 4 µl of KCN (0.5 mol/l in water), 4 µl of NaN₃ (1 mol/l in water), 50 µl of tissue homogenate, and 4 µl of DMSO containing an appropriate concentration of the respective test compound was equilibrated at 30°C for 10 min. DMSO (0.2%, vol/vol) did not affect the rates of NADH conversion (data not shown) and was also added to control solutions used for comparison. DMSO was omitted when water soluble compounds were examined (metformin and dexlipotam). The suspension was then transferred into a quartz cuvette, and the reaction was started by the admixture of the reaction substrates: 40 µl of NADH (15 mmol/l in water; Fluka) and 80 µl of ubiquinone-1 (2.5 mmol/l in ethanol; Sigma). Photometric measurement was started immediately thereafter, and the decrease in NADH, which absorbs at 340 nm, was determined over 2 min. Specificity of the spectrophotometric assay was confirmed by complete abrogation of NADH conversion in the absence of tissue homogenate or ubiquinone-1. Furthermore, 1 µmol/l rotenone blocked NADH conversion by muscle homogenates (-96 ± 2%), which indicates quantitative dependence on complex I. In liver homogenates, approximately one-third of NADH conversion seemed to be rotenone insensitive (-64 ± 4% inhibition by 1 µmol/l rotenone).

Oxygen consumption by isolated mitochondria.
Liver mitochondria were prepared from 4-month-old rats (420 g body wt; 1 animal/preparation). After cervical dislocation, rats were decapitated and livers quickly excised and plunged into ice-cold isolation buffer (0.25 mol/l sucrose, 20 mmol/l triethanolamine, 1 mmol/l EDTA, pH 7.4, 4°C). The tissue was chopped into small pieces with scissors and washed several times with buffer to remove the contaminating blood. Livers were then homogenized (0.25 g/ml isolation buffer) in a 60-ml capacity Potter-Elvehjem tissue homogenizer (electrically driven Teflon pestle). The homogenate was centrifuged at 2,500 rpm for 10 min (SS34 rotor, Sorvall RC26 Plus centrifuge). The supernatant was decanted through two layers of cheesecloth and spun down at 9,000 rpm for 10 min. The resulting pellets were carefully resuspended using a manually driven 15-ml Potter-Elvehjem homogenizer and centrifuged for 10 min at 9,000 rpm. This procedure was repeated twice (last resuspension with a 5-ml homogenizer). The final mitochondrial suspension was carefully prepared with a 2-ml homogenizer and stored at 4°C in Eppendorf tubes. The protein content of the mitochondrial suspension (40–50 g/l) was determined by the Biuret method with BSA as a standard.

Oxygen consumption was measured in air-saturated mitochondrial isolation buffer (pH 7.4, 25°C) with a Clark-type oxygen electrode. Mitochondria (1 g protein/l) were preincubated for 3 min at 25°C in isolation buffer additionally containing 0.3% BSA (wt/vol) and the indicated concentrations of metformin, rosiglitazone, pioglitazone, or KCl (the last used as an osmotic control). Buffer with TZDs as well as the appropriate controls contained 0.2% DMSO (vol/vol), which did not affect the rates of oxygen consumption (data not shown).

For stimulating mitochondrial respiration, 4 mmol/l inorganic phosphate was then added together with 5 mmol/l glutamate + 5 mmol/l malate (substrates for complex I) or, alternatively, with 10 mmol/l succinate (substrate for complex II) + 4.5 µmol/l rotenone. After 3 min, mitochondrial respiration was accelerated by the addition of 200 µmol/l ADP allowing ATP synthesis, and the rates of oxygen consumption were measured in state 3 (i.e., in the presence of ADP). After the quantitative consumption of added ADP, the rates of oxygen consumption were measured in state 4 (i.e., in the absence of ADP). The energy-conserving capacity of mitochondria was determined by respiratory control index (state 3/state 4). As a measure of the efficiency of mitochondrial ATP synthesis, the ADP-to-oxygen ratio was calculated (total amount of ADP added to amount of oxygen consumed during state 3 respiration) (18). All measurements were done in duplicate.

Fuel metabolism of isolated skeletal muscle.
Immediately after 5- to 7-week-old rats were killed, two longitudinal soleus muscle strips per leg (i.e., four strips/rat) were prepared, weighed (25 mg/strip), and tied under tension on stainless steel clips as described previously (19). According to procedures used previously (13), muscles were immediately put into 50-ml Erlenmeyer flasks coated with BlueSlick solution (Serva, Heidelberg, Germany) and were placed into a shaking waterbath (three strips per flask, 37°C, 130 cycles/min). Each flask contained 20 ml of Cell Culture Medium 199 (pH 7.35, 5.5 mmol/l glucose; Sigma, cat. no. M-4530) with additions of 0.3% wt/vol BSA, 5 mmol/l HEPES, 25,000 units/l penicillin G, 25 mg/l streptomycin, and 0.2 mg/l ciprofloxacine. Palmitate was dissolved in ethanol and added to the medium to give final concentrations of 300 µmol/l palmitate and 0.25% vol/vol ethanol. Stock solutions of the test compounds (metformin in saline, all others in DMSO) were added to the medium to give the indicated final concentrations of the respective agent. The final DMSO concentration of 0.1% vol/vol did not affect metabolic rates in this experimental setting (data not shown) and was also added to the control medium. An atmosphere of 95% O₂/5% CO₂ was continuously provided within the flasks. Muscles were incubated for 24 h in the absence of insulin and were then transferred for 1 h into 25-ml flasks (one strip per flask) containing 3 ml of medium additionally supplemented with 100 nmol/l human insulin (Actrapid; Novo, Bagsvaerd, Denmark) and trace amounts of D-[U-¹⁴C]glucose, D-[U-¹⁴C]palmitic acid, or 2-deoxy-D-[2,6-³H]glucose plus D-[U-¹⁴C]sucrose (all from Amersham, Amersham, U.K.). At the end of the experiment, muscles were quickly removed from the flasks, blotted, and frozen in liquid nitrogen.

Later, frozen muscle strips were lysed in 1 mol/l KOH at 70°C. The net rate of glucose incorporation into glycogen is referred to as glycogen synthesis and was calculated from the conversion of [¹⁴C]glucose into [¹⁴C]glycogen as described previously (19). Rates of CO₂ production from glucose (referred to as glucose oxidation) and CO₂ production from palmitate were calculated from the conversion of [¹⁴C]glucose or [¹⁴C]palmitate into ¹⁴CO₂, which was trapped with a solution containing methanol and phenethylamine (1:1) (20). For the measurement of muscle glycogen content, glycogen in the muscle lysate was degraded to glucose with amyloglucosidase (20) and glucose was then measured enzymatically by a commercial kit (Human, Taunusstein, Germany).

Statistics.
Absolute values and variability under the experimental conditions used are listed in Table 1 (control experiments). In the text and the figures, the effects of drug treatment are given in percentage of an intraindividual control value (=100%) as means ± SE. P values were calculated by paired two-tailed t test, with a P < 0.05 considered as significant.

	RESULTS

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

View larger version (40K): [in this window] [in a new window]   FIG. 1. Dose-dependent effects of rotenone and various agents, to which antidiabetic potentials have been ascribed, on the enzymatic activity of respiratory complex I in homogenates of the red part of rat gastrocnemius muscle (top) and rat liver (bottom), which were sonicated to disrupt cell and mitochondrial membranes. Data are given as a percentage of an intraindividual control (=100%; ) and are means ± SE; n = 6–24 each. 1TZD, 2PPAR- agonist, 3PPAR- antagonist; *P < 0.05; P < 0.01; P < 0.001 vs. control.

The results were very similar in homogenates of skeletal muscle and liver, except that inhibition seemed to be less pronounced in liver homogenates. This obviously reflected that approximately one-third of NADH conversion was rotenone insensitive in liver homogenates (i.e., presumably not catalyzed by complex I).

Oxygen consumption by isolated mitochondria.
In the presence of substrates for complex I (glutamate + malate), metformin (3–30 mmol/l) dose-dependently and markedly reduced states 3 and 4 respiration of isolated mitochondria (Fig. 2). With substrate for complex II (succinate), the highest concentration of metformin used (30 mmol/l) failed to affect state 3 respiration, and a minor decrease in state 4 respiration was much less pronounced than metformin’s effect in the presence of glutamate + malate (-12 ± 4 vs. -57 ± 1%; P < 0.0001). As 30 mmol/l KCl did not share any effect exerted by metformin, an osmotic mechanism was obviously not responsible for metformin action.

View larger version (59K): [in this window] [in a new window]   FIG. 2. Effects of metformin, rosiglitazone, and pioglitazone on respiratory functions of isolated mitochondria from rat liver. KCl () was used to control for osmotic effects. Rates of oxygen consumption in states 3 and 4 (i.e., in the presence and absence of ADP, respectively), the respiratory control index (ratio of rates of oxygen consumption in state 3 to state 4), and the ADP-to-oxygen ratio (P/O) are shown in the presence of glutamate + malate or succinate + rotenone. Data are given as a percentage of an intraindividual control (=100%; ) and are means ± SE; n = 4–6 each. *P < 0.05; P < 0.01; P < 0.001 vs. control.

Furthermore, metformin reduced respiratory control and the ADP-to-oxygen ratio in the presence of glutamate + malate but not in the presence of succinate. Substrate specificity suggests that this was caused by the inhibition of complex I. At variance to metformin, TZDs reduced respiratory control and the ADP-to-oxygen ratio independent of the substrate provided (although this was only a trend for ADP to oxygen in the presence of glutamate + malate). Reductions in respiratory control and ADP-to-oxygen ratio are both indicative of an increased contribution of uncoupled respiration without ATP production. TZDs therefore seem to exert uncoupling properties, which at least in part are not related to complex I inhibition and which could explain the increase in state 4 respiration with succinate.

Fuel metabolism of isolated muscle strips.
In freshly isolated native specimens of rat skeletal muscle, rotenone, metformin, and three different TZDs (rosiglitazone, pioglitazone, and RWJ-241947) dose-dependently reduced CO₂ production from both glucose and palmitate and increased anaerobic glycolysis (lactate release), which suggests disruption of mitochondrial function and cell respiration (Fig. 3). On a concentration basis, rotenone was much more efficient than the TZDs, which in turn were much more efficient than metformin. Reduced mitochondrial fuel oxidation induced by rotenone, metformin, and TZDs was accompanied by increased glucose transport and blunted glycogen storage, which is obviously due to higher carbohydrate requirements for anaerobic than aerobic ATP production.

View larger version (44K): [in this window] [in a new window]   FIG. 3. Effects of TZDs, metformin, and rotenone on cell respiration in isolated muscle. Dose-dependent effects of 24-h exposure to rotenone, metformin, RWJ-241947, pioglitazone, and rosiglitazone on insulin-stimulated fuel metabolism of rat soleus muscle strips. Data are given as a percentage of an intraindividual control value (=100%; ) and are means ± SE; n = 6–21 each. *P < 0.05; P < 0.01; P < 0.001 vs. control.

	DISCUSSION

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

In parallel to what was found for PPAR- ligands, metformin inhibited complex I in sonicated tissue homogenates that contained disrupted mitochondria. Although this is not proof that metformin directly interacts with the enzyme complex, it is in line with previously described effectiveness of metformin in submitochondrial particles (2) but contrasts with the view that its mitochondrial actions require intact cells and extramitochondrial signal transduction (3,4).

Metformin- and TZD-induced inhibition of complex I was confirmed by the impairment of respiratory parameters of isolated mitochondria in the presence of glutamate + malate (complex I substrates). In contrast to metformin, which did not influence succinate-stimulated respiration (complex II substrate), rosiglitazone and pioglitazone in addition to their complex I inhibition led to decreased respiratory control and ADP-to-oxygen ratios with succinate as a result of increased mitochondrial oxygen consumption in state 4. TZDs therefore not only inhibit complex I activity but also uncouple oxidative phosphorylation, both effects resulting in an impaired mitochondrial energy conservation. A decade ago, other TZDs likewise were found to inhibit complex I and to induce uncoupling in isolated mitochondria, but these relevant data were published only in an abstract (22).

Consistent with complex I–dependent impairment of cell respiration, both TZDs and metformin affected fuel metabolism of isolated muscle strips in a rotenone-like manner. The conclusion that complex I inhibition underlies the observed impairment of cell respiration is corroborated by parallel relative efficacies on complex I activities in homogenates and on cell respiration in muscle strips (rotenone >> TZDs >> metformin). Because similar metabolic effects as in muscle have been described in isolated hepatocytes and perfused rat liver (8,17,23), TZDs and metformin seem to impair mitochondrial function in both of their major target tissues (1). These findings obviously challenge the paradigm that TZDs and metformin act via completely different mechanisms.

Ranking efficacies of individual TZDs on a molar basis shows that rosiglitazone is more potent than pioglitazone in disrupted mitochondria (where it has direct access to complex I), equally potent in intact mitochondria (where it has to pass the mitochondrial membrane), and less potent in intact tissue (where it additionally has to pass the plasmalemma). Relative efficacies of individual TZDs thus seem to be influenced by the presence of cytoplasmic enzymes and/or by membranes that modulate access to the site of action.

The striking parallels in the mitochondrial effects of TZDs and metformin in vitro make it tempting to speculate that complex I–dependent impairment of cell respiration could contribute to their therapeutic actions. In this context, it should be considered that TZDs are highly lipo-philic and therefore could accumulate in the mitochondrial membrane, whereas metformin is a positively charged drug, which because of the mitochondrial membrane potential has been estimated to accumulate slowly 1,000-fold within the mitochondrial matrix (2). Reliable data on drug concentrations in these relevant fractions are not available, but progressive accumulation in the vicinity of complex I would be in line with our earlier finding that troglitazone becomes a progressively more efficient inhibitor of cell respiration when the treatment period is extended (13,14). This time-dependent increase in efficacy also explains why much lower drug concentrations were effective in muscle strips that were preexposed to the drugs for 24 h than in isolated mitochondria, which do not withstand long-term incubation and, hence, were exposed for a few minutes only.

Analyzing via which cascade of events the inhibition of cell respiration could principally result in antidiabetic action; it is of note that the rapid as well as the delayed effects of TZDs and metformin to some extent resemble those of exercise and 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR). Like muscle contractions, metformin and TZDs immediately reduce the ratios of ATP to AMP, ATP to ADP, and phosphocreatine to creatine in vitro (3,8,24,25), which can obviously be attributed to their impact on cell respiration. AICAR is a pharmacological tool to mimic an increase in cellular AMP (26,27), which activates the enzyme AMP-activated protein kinase (AMPK) and causes catabolic responses on the short term and insulin sensitization on the long term (26–32). Reduction of cellular energy availability could therefore be a common mechanism by which muscle contractions, TZDs, and metformin activate AMPK (5,25,28,33–35); stimulate glucose transport, glycolysis, and glycogenolysis on the short term (26–28,35; and this study); and ameliorate hyperglycemia and insulin resistance on the long term (1,9,26) (Fig. 4).

View larger version (38K): [in this window] [in a new window]   FIG. 4. The "energy charge hypothesis." The hypothesis holds that exercise, metformin, and TZDs decrease the cellular energy charge as a result of increased ATP consumption (exercise), complex I–dependent disruption of cell respiration (metformin and TZDs), and/or adipocyte-derived adiponectin (TZDs). Similar effects can be triggered by AICAR, which mimics loss of energy availability by giving rise to the AMP analog ZMP. Changes in the energy charge are known to acutely affect many enzymes (e.g., AMPK), which could contribute to the rapid and delayed metabolic responses common to exercise, TZDs, metformin, and AICAR. Hence, changes in the cellular energy charge could be a unifying early event in the physiological and pharmacological modulation of insulin sensitivity.

Taken together, we show that TZDs, like metformin, directly inhibit complex I and cell respiration in target tissues of insulin in vitro. In a wider context, our findings support the hypothesis that changes in cellular energy availability could be a unifying early event in the physiological and pharmacological modulation of insulin sensitivity. Studies on the metabolic effects of TZDs in humans and animals in vivo will be necessary to determine to what extent the pharmacological inhibition of complex I, described here, contributes to the antidiabetic actions of these drugs.

	ACKNOWLEDGMENTS

 
This work was supported by the Austrian Science Fund (grant no. P16352-B08).

We thank the following companies for generously providing compounds: Sankyo (Tokyo, Japan), Johnson & Johnson (Raritan, NJ), Roche (Basel, Switzerland), Pfizer (Ann Arbor, MI), Yamanouchi (Tsukuba, Japan), Aventis (Frankfurt am Main, Germany), and Viatris (Frankfurt am Main, Germany). We also thank the staff at the Biomedical Research Centre, University of Vienna, for taking care of the rats.

Address correspondence and reprint requests to Clemens Fürnsinn, PhD, Department of Medicine III, Division of Endocrinology & Metabolism, Währinger Gürtel 18-20, A-1090 Vienna, Austria. E-mail: clemens.fuernsinn{at}akh-wien.ac.at

Received for publication October 14, 2003 and accepted in revised form December 23, 2003

Abbreviations: AICAR, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside; AMPK,AMP-activated protein kinase; PPAR, peroxisome proliferator–activated receptor; TZD, thiazolidinedione

	REFERENCES

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Inzucchi S, Maggs DG, Spollett GR, Page SL, Rife FS, Walton V, Shulman GI: Efficacy and metabolic effects of metformin and troglitazone in type II diabetes mellitus. N Engl J Med338 :867 –872,1998[Abstract/Free Full Text]
2. Owen MR, Doran E, Halestrap AP: Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochem J348 :607 –614,2000
3. El-Mir M-Y, Nogueira V, Fontaine E, Avéret N, Rigoulet M, Leverve X: Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I. J Biol Chem275 :223 –228,2000[Abstract/Free Full Text]
4. Detaille D, Guigas B, Leverve X, Wiernsperger N, Devos P: Obligatory role of membrane events in the regulatory effect of metformin on the respiratory chain function. Biochem Pharmacol63 :1259 –1272,2002[Medline]
5. Musi N, Hirshman MF, Nygren J, Svanfeldt M, Bavenholm P, Rooyackers O, Zhou G, Williamson JM, Ljunqvist O, Efendic S, Moller DE, Thorell A, Goodyear LJ: Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes. Diabetes51 :2074 –2081,2002[Abstract/Free Full Text]
6. Davis TM, Jackson D, Davis WA, Bruce DG, Chubb P: The relationship between metformin therapy and the fasting plasma lactate in type 2 diabetes: the Fremantle Diabetes Study. Br J Clin Pharmacol52 :137 –144,2001[Medline]
7. Luft FC: Lactic acidosis update for critical care clinicians. J Am Soc Nephrol12 :S15 –S19,2001[Abstract/Free Full Text]
8. Jalling O, Olsen C: The effects of metformin compared to the effects of phenformin on lactate production and the metabolism of isolated parenchymal rat liver cells. Acta Pharmacol Toxicol54 :327 –332,1984[Medline]
9. Fürnsinn C, Waldhäusl W: Thiazolidinediones: metabolic actions in vitro. Diabetologia45 :1211 –1223,2002[Medline]
10. Rosen ED, Spiegelman BM: PPAR: a nuclear regulator of metabolism, differentiation, and cell growth. J Biol Chem41 :37731 –37734,2001
11. Lenhard JM, Kliewer SA, Paulik MA, Plunket KD, Lehmann JM, Weiel JE: Effects of troglitazone and metformin on glucose and lipid metabolism. Biochem Pharmacol54 :801 –808,1997[Medline]
12. Yamauchi T, Kamon J, Waki H, Murakami K, Motojima K, Komeda K, Ide T, Kubota N, Terauchi Y, Tobe K, Miki H, Tsuchida A, Akanuma Y, Nagai R, Kimura S, Kadowaki T: The mechanisms by which both heterozygous peroxisome proliferator-activated receptor (PPAR) deficiency and PPAR agonist improve insulin resistance. J Biol Chem44 :41245 –41254,2001
13. Brunmair B, Gras F, Neschen S, Roden M, Wagner L, Waldhäusl W, Fürnsinn C: Direct thiazolidinedione action on isolated rat skeletal muscle fuel handling is independent of peroxisome proliferator–activated receptor-–mediated changes in gene expression. Diabetes50 :2309 –2315,2001[Abstract/Free Full Text]
14. Fürnsinn C, Brunmair B, Neschen S, Roden M, Waldhäusl W: Troglitazone directly inhibits CO₂ production from glucose and palmitate in isolated rat skeletal muscle. J Pharmacol Exp Ther293 :487 –493,2000[Abstract/Free Full Text]
15. Coates G, Nissim I, Battarbee H, Welbourne T: Glitazones regulate glutamine metabolism by inducing a cellular acidosis in MDCK cells. Am J Physiol283 :E729 –E737,2002
16. Dello Russo C, Gavrilyuk V, Weinberg G, Almeida A, Bolanos JP, Palmer J, Pelligrino D, Galea E, Feinstein DL: Peroxisome proliferator-activated receptor thiazolidinedione agonists increase glucose metabolism in astrocytes. J Biol Chem278 :5828 –5836,2003[Abstract/Free Full Text]
17. Preininger K, Stingl H, Englisch R, Fürnsinn C, Graf J, Waldhäusl W, Roden M: Acute troglitazone action in isolated perfused rat liver. Br J Pharmacol126 :372 –378,1999[Medline]
18. Gnaiger E, Méndez G, Hand SC: High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc Natl Acad Sci U S A97 :11080 –11085,2000[Abstract/Free Full Text]
19. Crettaz M, Prentki M, Zanietti D, Jeanrenaud B: Insulin resistance in soleus muscle from obese Zucker rats: involvement of several defective sites. Biochem J186 :525 –534,1980[Medline]
20. Fürnsinn C, Ebner K, Waldhäusl W: Failure of GLP-1(7-36)amide to affect glycogenesis in rat skeletal muscle. Diabetologia38 :864 –867,1995[Medline]
21. Shimaya A, Kurosaki E, Nakano R, Hirayama R, Shibasaki M, Shikama H: The novel hypoglycemic agent YM440 normalizes hyperglycemia without changing body fat weight in diabetic db/db mice. Metabolism49 :411 –417,2000[Medline]
22. Slieker L, Sundell KL, Bush KA, Sage SW, Fitch L, Schmiegel K: Thiazolidinediones stimulate 2-deoxyglucose uptake into G9 myocytes secondary to inhibition of oxidative phosphorylation (Abstract). Diabetes43 (Suppl. 1) :A126 ,1994
23. Radziuk J, Zhang Z, Wiernsperger N, Pye S: Effects of metformin on lactate uptake and gluconeogenesis in the perfused rat liver. Diabetes46 :1406 –1413,1997[Abstract]
24. Fischer Y, Thomas J, Rösen P, Kammermeier H: Action of metformin on glucose transport and glucose transporter GLUT1 and GLUT4 in heart muscle cells from healthy and diabetic rats. Endocrinology136 :412 –420,1995[Abstract]
25. Fryer LGD, Parbu-Patel A, Carling D: The anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways. J Biol Chem277 :25226 –25232,2002[Abstract/Free Full Text]
26. Hardie DG, Hawley SA: AMP-activated protein kinase: the energy charge hypothesis revisited. Bioassays23 :1112 –1119,2001[Medline]
27. Young M, Radda G, Leighton B: Activation of glycogen phosphorylase and glycogenolysis in rat skeletal muscle by AICAR: an activator of AMP-activated protein kinase. FEBS Lett382 :43 –47,1996[Medline]
28. Hayashi T, Hirshman MF, Fujii N, Habinowski SA, Witters LA, Goodyear LJ: Metabolic stress and altered glucose transport: activation of AMP-activated protein kinase as a unifying coupling mechanism. Diabetes49 :527 –531,2000[Abstract]
29. Buhl ES, Jessen N, Pold R, Ledet T, Flyvbjerg A, Pedersen SB, Pedersen O, Schmitz O, Lund S: Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying features of the insulin resistance syndrome. Diabetes51 :2199 –2206,2002[Abstract/Free Full Text]
30. Fiedler M, Zierath JR, Selen G, Wallberg-Henriksson H, Liang Y, Sakariassen KS: 5-aminoimidazole-4-carboxy-amide-1-beta-D-ribofuranoside treat-ment ameliorates hyperglycemia and hyperinsulinemia but not dyslipidemia in KKAy-CETP mice. Diabetologia44 :2180 –2186,2001[Medline]
31. Fryer LGD, Foufelle F, Barnes K, Baldwin SA, Woods A, Carling D: Characterization of the role of the AMP-activated protein kinase in the stimulation of glucose transport in skeletal muscle cells. Biochem J363 :167 –174,2002[Medline]
32. Mu J, Brozinick JT, Valladares O, Bucan M, Birnbaum MJ: A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle. Mol Cell7 :1085 –1094,2001[Medline]
33. Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, Wu M, Ventre J, Doebber T, Fujii N, Musi N, Hirshman MF, Goodyear LJ, Moller DE: Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest108 :1167 –1174,2001[Medline]
34. Hawley SA, Gadalla AE, Olsen GS, Hardie DG: The antidiabetic drug metformin activates the AMP-activated protein kinase cascade via an adenine nucleotide-independent mechanism. Diabetes51 :2420 –2425,2002[Abstract/Free Full Text]
35. Dean DJ, Ruderman NB: Role of AMP kinase and malonyl CoA in exercise-stimulated skeletal muscle metabolism and insulin action. In Muscle Metabolism. Zierath JR, Wallberg-Henriksson H, Eds. New York, Taylor & Francis,2002 , p.211 –226
36. Phillips SA, Ciaraldi TP, Kong APS, Bandukwala R, Aroda V, Carter L, Baxi S, Mudaliar SR, Henry RR: Modulation of circulating and adipose tissue adiponectin levels by antidiabetic therapy. Diabetes52 :667 –674,2003[Abstract/Free Full Text]
37. Yamauchi T, Kamon J, Waki H, Terauchi Y, Kubota N, Hara K, Mori Y, Ide T, Murakami K, Tsuboyama-Kasaoka N, Ezaki O, Akanuma Y, Gavrilova O, Vinson C, Reitman ML, Kagechika H, Shudo K, Yoda M, Nakano Y, Tobe K, Nagai R, Kimura S, Tomita M, Froguel P, Kadowaki T: The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med7 :941 –946,2001[Medline]
38. Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Waki H, Uchida S, Yamashita S, Noda M, Kita S, Ueki K, Eto K, Akanuma Y, Froguel P, Foufelle F, Ferre P, Carling D, Kimura S, Nagai R, Kahn BB, Kadowaki T: Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat Med8 :1288 –1295,2002[Medline]
39. Tomas E, Tsao T-S, Saha AK, Murrey HE, Zhang CC, Itani SI, Lodish HF, Ruderman NB: Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation. Proc Natl Acad Sci U S A99 :16309 –16313,2002[Abstract/Free Full Text]

CiteULike

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				N. Turner, J.-Y. Li, A. Gosby, S. W.C. To, Z. Cheng, H. Miyoshi, M. M. Taketo, G. J. Cooney, E. W. Kraegen, D. E. James, et al. Berberine and Its More Biologically Available Derivative, Dihydroberberine, Inhibit Mitochondrial Respiratory Complex I: A Mechanism for the Action of Berberine to Activate AMP-Activated Protein Kinase and Improve Insulin Action Diabetes, May 1, 2008; 57(5): 1414 - 1418. [Abstract] [Full Text] [PDF]

				N. P.E. Kadoglou, F. Iliadis, C. D. Liapis, D. Perrea, N. Angelopoulou, and M. Alevizos Beneficial Effects of Combined Treatment With Rosiglitazone and Exercise on Cardiovascular Risk Factors in Patients With Type 2 Diabetes Diabetes Care, September 1, 2007; 30(9): 2242 - 2244. [Full Text] [PDF]

				J. M. Perez-Ortiz, P. Tranque, M. Burgos, C. F. Vaquero, and J. Llopis Glitazones Induce Astroglioma Cell Death by Releasing Reactive Oxygen Species from Mitochondria: Modulation of Cytotoxicity by Nitric Oxide Mol. Pharmacol., August 1, 2007; 72(2): 407 - 417. [Abstract] [Full Text] [PDF]

				D. K. Kramer, L. Al-Khalili, B. Guigas, Y. Leng, P. M. Garcia-Roves, and A. Krook Role of AMP Kinase and PPAR{delta} in the Regulation of Lipid and Glucose Metabolism in Human Skeletal Muscle J. Biol. Chem., July 6, 2007; 282(27): 19313 - 19320. [Abstract] [Full Text] [PDF]

				R. Scatena, P. Bottoni, G. Botta, G. E. Martorana, and B. Giardina The role of mitochondria in pharmacotoxicology: a reevaluation of an old, newly emerging topic Am J Physiol Cell Physiol, July 1, 2007; 293(1): C12 - C21. [Abstract] [Full Text] [PDF]

				L. D. Marroquin, J. Hynes, J. A. Dykens, J. D. Jamieson, and Y. Will Circumventing the Crabtree Effect: Replacing Media Glucose with Galactose Increases Susceptibility of HepG2 Cells to Mitochondrial Toxicants Toxicol. Sci., June 1, 2007; 97(2): 539 - 547. [Abstract] [Full Text] [PDF]

				M. Soller, S. Drose, U. Brandt, B. Brune, and A. von Knethen Mechanism of Thiazolidinedione-Dependent Cell Death in Jurkat T Cells Mol. Pharmacol., June 1, 2007; 71(6): 1535 - 1544. [Abstract] [Full Text] [PDF]

				M. Krebs, B. Brunmair, A. Brehm, M. Artwohl, J. Szendroedi, P. Nowotny, E. Roth, C. Furnsinn, M. Promintzer, C. Anderwald, et al. The Mammalian Target of Rapamycin Pathway Regulates Nutrient-Sensitive Glucose Uptake in Man Diabetes, June 1, 2007; 56(6): 1600 - 1607. [Abstract] [Full Text] [PDF]

				S. Ghosh, N. Patel, D. Rahn, J. McAllister, S. Sadeghi, G. Horwitz, D. Berry, K. X. Wang, and R. H. Swerdlow The Thiazolidinedione Pioglitazone Alters Mitochondrial Function in Human Neuron-Like Cells Mol. Pharmacol., June 1, 2007; 71(6): 1695 - 1702. [Abstract] [Full Text] [PDF]

				F. Turturro, R. Oliver III, E. Friday, I. Nissim, and T. Welbourne Troglitazone and pioglitazone interactions via PPAR-{gamma}-independent and -dependent pathways in regulating physiological responses in renal tubule-derived cell lines Am J Physiol Cell Physiol, March 1, 2007; 292(3): C1137 - C1146. [Abstract] [Full Text] [PDF]

				D. Safiulina, N. Peet, E. Seppet, A. Zharkovsky, and A. Kaasik Dehydroepiandrosterone Inhibits Complex I of the Mitochondrial Respiratory Chain and is Neurotoxic In Vitro and In Vivo at High Concentrations Toxicol. Sci., October 1, 2006; 93(2): 348 - 356. [Abstract] [Full Text] [PDF]

				D. G. Hardie, S. A. Hawley, and J. W. Scott AMP-activated protein kinase - development of the energy sensor concept J. Physiol., July 1, 2006; 574(1): 7 - 15. [Abstract] [Full Text] [PDF]

				N. K. LeBrasseur, M. Kelly, T.-S. Tsao, S. R. Farmer, A. K. Saha, N. B. Ruderman, and E. Tomas Thiazolidinediones can rapidly activate AMP-activated protein kinase in mammalian tissues Am J Physiol Endocrinol Metab, July 1, 2006; 291(1): E175 - E181. [Abstract] [Full Text] [PDF]

				D. G. Hardie and K. Sakamoto AMPK: A Key Sensor of Fuel and Energy Status in Skeletal Muscle Physiology, February 1, 2006; 21(1): 48 - 60. [Abstract] [Full Text] [PDF]

				D. Detaille, B. Guigas, C. Chauvin, C. Batandier, E. Fontaine, N. Wiernsperger, and X. Leverve Metformin Prevents High-Glucose-Induced Endothelial Cell Death Through a Mitochondrial Permeability Transition-Dependent Process Diabetes, July 1, 2005; 54(7): 2179 - 2187. [Abstract] [Full Text] [PDF]

				D. G. Hardie The AMP-activated protein kinase pathway - new players upstream and downstream J. Cell Sci., November 1, 2004; 117(23): 5479 - 5487. [Abstract] [Full Text] [PDF]

				B. Brunmair, A. Lest, K. Staniek, F. Gras, N. Scharf, M. Roden, H. Nohl, W. Waldhausl, and C. Furnsinn Fenofibrate Impairs Rat Mitochondrial Function by Inhibition of Respiratory Complex I J. Pharmacol. Exp. Ther., October 1, 2004; 311(1): 109 - 114. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF)