Enhancement of muscle cell glucose uptake by medicinal plant species of Canada's native populations is mediated by a common, Metformin-like mechanism

Abstract Aim The purpose of the present study was to elucidate the mechanisms of action mediating enhancement of basal glucose uptake in skeletal muscle cells by seven medicinal plant products recently identified from the pharmacopeia of native Canadian populations ( Spoor et al., 2006 ). Methods Activity of the major signaling pathways that regulate glucose uptake was assessed by western immunoblot in C2C12 muscle cells treated with extracts from these plant species. Effects of extracts on mitochondrial function were assessed by respirometry in isolated rat liver mitochondria. Metabolic stress induced by extracts was assessed by measuring ATP concentration and rate of cell medium acidification in C2C12 myotubes and H4IIE hepatocytes. Extracts were applied at a dose of 15–100 μg/ml. Results The effect of all seven products was achieved through a common mechanism mediated not by the insulin signaling pathway but rather by the AMP-activated protein kinase (AMPK) pathway in response to the disruption of mitochondrial function and ensuing metabolic stress. Disruption of mitochondrial function occurred in the form of uncoupling of oxidative phosphorylation and/or inhibition of ATPsynthase. Activity of the AMPK pathway, in some instances comparable to that stimulated by 4 mM of the AMP-mimetic AICAR, was in several cases sustained for at least 18 h post-treatment. Duration of metabolic stress, however, was in most cases in the order of 1 h. Conclusions The mechanism common to the seven products studied here is analogous to that of the antidiabetic drug Metformin. Of interest is the observation that metabolic stress need not be sustained in order to induce important adaptive responses. The results support the use of these products as culturally adapted treatments for insulin resistance and hyperglycemia in susceptible aboriginal populations where adherence to modern diabetes pharmaceuticals is an issue. The mechanism reported here may be widespread and mediate the antidiabetic activity of traditional remedies from various other cultures. Keywords Traditional medicine Type II diabetes Intracellular signaling pathways AMP-activated protein kinase Mitochondrial energy transduction Metabolic stress 1 Introduction Aboriginal populations the world over are predisposed to the development of type II diabetes ( Yu and Zinman, 2007 ). When this susceptibility is compounded by the cultural disconnection of modern pharmaceuticals, the prevalence rates of diabetic complications can reach alarming levels. These complications more than diabetes itself impact quality of life and longevity and represent important social and economic burdens. In native populations of Canada, such as the Cree of Iiyiyiu Istchii (James Bay region), the prevalence rate of type II diabetes is 3–5 higher than in non-native populations ( Kuzmina and Dannenbaum, 2004; Kuzmina et al., 2008; Young et al., 2000 ) and, due to cultural disconnection to modern treatment options, the prevalence rate of complications is disproportionately high. Our team has taken a novel approach to address this specific situation; in close collaboration with the Cree of Iiyiyiu Istchii, we are attempting to improve diabetes treatments by developing culturally adapted complementary and alternative options that are based on products of this population's own pharmacopeia. Plant products with hyperglycemia-normalizing activity are common in the traditional medicine of cultures throughout the world ( Haddad et al., 2006; Marles and Farnsworth, 1995 ). The efficacy of several of these products has been demonstrated in clinical and animal studies. In some cases, active principles have also been identified. These natural products represent a rich source of alternatives and complements to the limited number of antidiabetic medications currently on the market. Furthermore, in populations with limited access to modern pharmaceuticals or with a cultural preference for traditional remedies, these products often represent the main treatment option for diabetes. Perhaps the most important testimony to the importance and efficacy of antidiabetic natural products comes from the widely used antidiabetic drug Metformin and the other members of the biguanide class. Indeed, these drugs are based on naturally occurring guanidines isolated in the 1920s from Galega officinalis (French lilac), a plant used for centuries to treat diabetes ( Cavaliere, 2007; Witters, 2001 ). The biguanides act not by increasing insulin secretion, but rather by decreasing gluconeogenesis and increasing glucose uptake ( Giannarelli et al., 2003 ). While the biguanides have been on the market since the 1950s, the major breakthrough in understanding their mode of action occurred in 2001 when the enzyme adenosine-monophosphate-activated protein kinase (AMPK) was found to play a central role in their effects ( Zhou et al., 2001 ). This highly conserved enzyme is exquisitely sensitive to disruptions in cellular energy homeostasis and is now recognized as both a master regulatory enzyme of metabolism and an important therapeutic target for diabetes and metabolic diseases ( Misra, 2008; Viollet et al., 2009 ). Activation of AMPK triggers cytoprotective responses to acutely restore energy homeostasis and to chronically protect against future perturbations, including stimulation of transport and oxidation of glucose and fats, increased expression of key genes of glucose and fat metabolism, and mitochondrial biogenesis ( Reznick and Shulman, 2006; Winder, 2001 ). In recent years, many plant products and naturally occurring compounds have been shown to activate AMPK ( Ahn et al., 2008; Collins et al., 2007; Hayashi et al., 2000; Hwang et al., 2005; Lee et al., 2007, 2006; Liu et al., 2007; Mooney et al., 2008; Park et al., 2007; Zang et al., 2006 ). In many, if not all cases, a site of action of these products is the mitochondrial inner membrane or a protein thereof ( Dorta et al., 2005; Lee et al., 2006; Polya, 2003; Trumbeckaite et al., 2006 ), with the effect of disrupting oxidative phosphorylation and decreasing the capacity for aerobic ATP synthesis. If mitochondrial energy production becomes insufficient to meet the cellular energy demand, energy homeostasis is perturbed and AMPK is activated. Targeting of energy transduction pathways is consistent with the defensive antimicrobial and insecticidal functions of plant secondary metabolites ( Polya, 2003 ). Mild and transient disruption of mitochondrial function is believed to be the primary mechanism of action of Metformin, shown to inhibit oxidative phosphorylation at complex I of the electron transport chain ( El-Mir et al., 2000; Owen et al., 2000 ). As aerobic energy production is compromised, the cell must increasingly rely on ATP synthesized anaerobically through glycolysis. Consequently, the primary danger associated with this mode of action is metabolic acidosis. Indeed, acidosis is the most important complication of biguanide use ( Luft et al., 1978 ). Furthermore, consumption of Galega officinalis by grazing livestock can result in this condition as well ( Cavaliere, 2007 ). Our team has recently conducted a successful screening project of medicinal plant species of the Canadian boreal forest stemming from the traditional medicine of the Cree of Iiyiyiu Istchii ( Leduc et al., 2006; Spoor et al., 2006 ). Seven of eight ethnobotanically selected species were found to enhance glucose uptake in skeletal muscle cells in the absence of insulin by 10–45% ( Spoor et al., 2006 ), an effect similar to that of Metformin ( Klip et al., 1992; Spoor et al., 2006 ). This activity had not previously been ascribed to these species. The goal of the present work was to determine the molecular events through which these effects are mediated. Our findings indicate that all seven plant products act through the AMPK pathway and that activation of this pathway results from a transient disruption of mitochondrial function. These findings further support the use of these products as culturally adapted alternative anti-hyperglycemic therapies. These findings also suggest that this mode of action may be widespread among medicinal plant species and natural health products with antidiabetic activity. 2 Materials and methods 2.1 Plant materials The following eight products of Canadian boreal forest medicinal plant species were the objects of this study: inner bark of Abies balsamea (L.) Mill.; inner bark of Alnus incana ssp. Rugosa (Du Roi) R.T. Clausen; inner bark of Larix laricina K. Koch; cones of Picea mariana ; cones of Pinus banksiana Lamb.; leaves of Rhododendron groenlandicum (Oeder) Kron & Judd; leaves of Sarracenia purpurea L.; inner bark of Sorbus decora C.K. Schneid. The collection, handling, and extraction of plant material have previously been described ( Spoor et al., 2006 ). Briefly, plant material was collected in 2003 in Mistissini, QC, Canada. Material was collected from several specimens and pooled. Species were identified by a plant taxonomist (Dr. Alain Cuerrier, Montreal Botanical Garden, Montreal, QC) and voucher specimens were deposited at the Marie-Victorin Herbarium of the Montreal Botanical Garden (specimens # Mis03-1, Mis03-2, Mis03-4, Mis03-5, Mis03-8, Mis03-12, Mis03-14, and Mis03-15). Plant material was extracted at the University of Ottawa. Material from each species was dried in a fruit dryer at 35 °C, cleaned, and ground using a Wiley Mill with a mesh size of 2 mm. Ground material was extracted twice for 24 h in 80% ethanol (10 ml/g dry material) on a mechanical shaker at ambient temperature and then filtered through Whatman #1 paper. The supernatants of first and second extractions were pooled, evaporated to dryness in a rotary evaporator (RE500; Yamamoto Scientific Co., Ltd., Tokyo, Japan) with a water bath temperature of 40 °C, and lyophilized in a freeze dryer (Super Moduylo; Thermo Fisher, Ottawa, ON) to produce a crude ethanol extract (referred to as crude extract from this point on). Extraction yields (%) were as follows: Abies balsamea 15; Alnus incana 26; Larix laricina 24; Picea mariana 21; Pinus banksiana 9; Rhododendron groenlandicum 31; Sarracenia purpurea 25; Sorbus decora 9 ( Spoor et al., 2006 ). Dried crude extracts were preserved at 4 °C in a dessicator and protected from light until further use. Extracts were solubilized in dimethyl sulfoxide (DMSO; Sigma–Aldrich, Oakville, ON) at 1000× their final working concentration, filter sterilized, aliquoted, and stored at −20 °C. On the day of an experiment, an aliquot was thawed and diluted in culture medium at 1:1000. Final working concentrations in microgram crude extract/ml were as follows: Abies balsamea 50; Alnus incana 50; Larix laricina 25; Picea mariana 10; Pinus banksiana 15; Rhododendron groenlandicum 75; Sarracenia purpurea 100; Sorbus decora 15. These concentrations have previously been determined to be optimal and non-toxic for C2C12 muscle cells over a treatment duration of 24 h ( Spoor et al., 2006 ). Final DMSO concentration was 0.1% in all experiments. A preliminary phytochemical characterization of the extracts, including determination of total concentration of phenolics and identification and quantification of marker compounds, has been reported elsewhere ( Spoor et al., 2006 ). 2.2 Cell culture C2C12 murine skeletal myoblasts and H4IIE rat hepatocytes were obtained from American Type Culture Collection (ATCC; Manassas, VA). Cells were cultured at 37 °C in a humidified 5% CO 2 /95% air atmosphere. Culture reagents were purchased from Wisent (St-Bruno, QC). As previously described ( Benhaddou-Andaloussi et al., 2008; Martineau et al., 2006; Spoor et al., 2006; Vuong et al., 2007 ), myoblasts were proliferated to confluence in high-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS), 10% horse serum (HS; Sigma–Aldrich), and antibiotics. These cells were then differentiated into multinucleated myotubes over a 7-day period in high-glucose DMEM supplemented with 2% HS. Experiments with C2C12 cells were always timed to end on day 7 of differentiation. Hepatocytes were proliferated to confluence in high-glucose DMEM supplemented with 10% FBS and antibiotics. H4IIE experiments were performed between post-confluence days 1 and 3. 2.3 Western immunoblot studies of phospho-Akt, -AMPK, and -ACC contents Content of phosphorylated protein kinase Akt (Ser 473) was measured by western immunoblot in order to assess activation of the insulin signaling pathway. Similarly, contents of phosphorylated AMPKalpha (catalytic subunit; Thr 172) and phosphorylated acetyl-coA carboxylase (ACC; Ser 79) were measured as markers of activation of the AMPK pathway. Reagents were purchased from Sigma–Aldrich unless otherwise noted. Primary antibodies were purchased from Cell Signaling Technologies Inc. (cat. # 9271, 2531, 3661; Danvers, MA). Horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody was purchased from Jackson ImmunoResearch Laboratories Inc. (cat. # 111-035-144; West Grove, PA). C2C12 cells were seeded in 6-well plates and proliferated and differentiated as above. Cells were treated with extract or vehicle (DMSO) 18, 6, or 1 h before lysis on day 7 of differentiation. 5-Aminoimidazole-4-carboxamide-1β-riboside (AICAR; Toronto Research Chemicals Inc., North York, ON) was used as a positive control for the AMPK pathway; AICAR was dissolved in water and applied at a final concentration of 4 mM to a subgroup of vehicle control wells 30 min prior to lysis. At the end of the treatment period, plates were placed on ice and cells were rinsed twice with ice-cold phosphate-buffered saline (PBS; 8.1 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 , pH 7.4, 2.68 mM KCl, 0.137 M NaCl) and covered with 250 μl/well of HEPES lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EGTA, 2 mM MgCl 2 , 5% glycerol, 1% Triton X-100, 1% Na deoxycholate, 0.1% Na dodecyl sulphate (SDS)) containing a cocktail of protease inhibitors (Complete-Mini EDTA-free; Roche Diagnostics, Laval, QC; supplemented with 1 mM phenylmethylsulphonyl fluoride) and phosphatase inhibitors (10 mM NaF, 100 μM Na 3 VO 4 , 1 mM Na 4 P 2 O 7 ). Cells were scraped and transferred to microcentrifuge tubes. The tubes were vortexed and kept on ice for 30 min with frequent vortexing. Tubes were then centrifuged at 600 × g for 10 min at 4 °C. The supernatants were decanted into new tubes, and these lysates were frozen at −80 °C until analysis. The protein content of the cell lysates was measured using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer's instructions. An equal amount of total protein from each sample was denatured by boiling 5 min in reducing sample buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 5% β-mercaptoethanol, 1% SDS) ( Laemmli, 1970 ). One hundred micrograms of each sample in a 100 μl volume were resolved by SDS-polyacrylamide gel electrophoresis ( Shapiro et al., 1967 ) using a Protean IIxi apparatus (Bio-Rad Laboratories, Hercules, CA). The resolving gel was composed of a 6.5% acrylamide phase over a 10% acrylamide phase, and the stacking gel was 5% acrylamide. Electrophoresis was performed at 4 °C in migration buffer (25 mM Tris, pH 8.3, 192 mM glycine, 0.1% SDS) at 50 mA for 3 h followed by 25 mA for 14 h. Resolved samples were then electrotransferred ( Towbin et al., 1979 ) to Immobilon-P polyvinylidene fluoride membrane (Millipore Corp., Billerica, MA) using a Trans-Blot cell (Bio-Rad Laboratories) in transfer buffer (25 mM Tris, pH 8.3, 192 mM glycine, 10% methanol, 0.02% SDS) at 4 °C, 900 mA, for 1.5 h. Membranes were stained with Ponceau Red to confirm equal loading, then blocked for 1.5 h in 5% bovine serum albumin (BSA) dissolved in Tris-buffered saline (TBS) plus Triton X-100 (TBST; 50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Triton X-100). Blocked membranes were incubated overnight at 4 °C with constant agitation in primary antibody solution (antibody at 1:1000 in TBST plus 1% BSA and 0.5% NaN 3 ). Membranes were rinsed in TBST and incubated 1.5 h at ambient temperature in secondary antibody solution (antibody at 1:100 000 in TBST plus 0.5% BSA). Membranes were then thoroughly washed in TBST and TBS, and treated for 1 min with ECL reagent (Amersham/GE Healthcare, Baie d’Urfé, QC). Membranes were exposed to blue-light sensitive ECL film (Amersham/GE Healthcare) for the appropriate duration for maximal signal without film saturation. Films were developed manually using D-19 developer and RapidFixer (Eastman Kodak Co., Rochester, NY). Developed films were scanned using a Hewlett Packard 6100 flatbed scanner (HP; Palo Alto, CA) with HP DeskScan II software. Densitometry analysis was then performed using Image 1.63 software (National Institutes of Health, Bethesda, MD). Three replicates, each from a different cell passage, were performed for each condition. For each series of replicates, all samples were simultaneously subjected to electrophoresis and transferred to a single membrane. Data from the densitometric analysis of each replicate series were normalized to the vehicle control of that series. Normalized data from the three series were then pooled. 2.4 Mitochondrial respiration studies The effects of plant extracts on oxygen consumption of isolated mitochondria were assessed by oxygraphy with a Clark-type oxygen microelectrode system. Mitochondria were isolated from the liver of male Wistar rats weighing between 200 and 225 g. All animal manipulations were sanctioned by the Animal Ethics Committee of the Université de Montréal and respected the guidelines from the Canadian Council for the Care and Protection of Animals. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight) and underwent laparotomy. The portal vein was cannulated and the hepatic artery and the infrahepatic inferior vena cava were ligated. The liver was flushed with 100 ml of Krebs–Henseleit buffer (25 mM NaHCO 3 , 1.2 mM KH 2 PO 4 , pH 7.4, 250 mM NaCl, 4.8 mM KCl, 2.1 mM CaCl 2 , 1.2 mM MgSO 4 ) at 22 °C prior to excision. Mitochondria were isolated from 2 g of liver by the method of Johnson and Lardy ( Johnson and Lardy, 1967 ). Briefly, tissue was homogenized on ice using a Teflon potter homogenizer in ice-cold Tris-sucrose buffer (10 mM Tris, pH 7.2, 250 mM sucrose, 1 mM EGTA). The homogenate was centrifuged at 600 × g for 10 min at 4 °C. The supernatant was centrifuged at 15 000 × g for 5 min at 4 °C. The pellet from this centrifugation was delicately washed once in the same buffer, centrifuged, washed once in EGTA-free buffer, and centrifuged again at 15 000 × g . The final pellet, containing viable mitochondria, was suspended in EGTA-free homogenizing buffer and kept on ice. Protein content of the homogenate was determined by Lowry's protein assay ( Lowry et al., 1951 ). O 2 consumption was measured at 25 °C in a Hansatech Oxygraph apparatus (Norfolk, UK) with a 1 ml reaction chamber, as previously described ( Ligeret et al., 2008; Morin et al., 2001 ). Briefly, 1 mg of mitochondrial protein was added to respiration buffer (5 mM KH 2 PO 4 , pH 7.2, 250 mM sucrose (ultra pure), 5 mM MgCl 2 , 1 mM EGTA, and 2 μM of the complex I inhibitor rotenone) at 25 °C in the reaction chamber, for a final volume of 990 μl. Mitochondrial respiration was initiated by the injection of 6 mM (final concentration) of the complex II substrate succinate, and the rate of basal oxygen consumption per mg mitochondrial protein (RBOC or state 4 respiration) was determined. One microliter of 1000× concentrated plant extract or 1 μl of DMSO was then injected and its effect on RBOC was assessed. Basal respiration was allowed to proceed for at least 30 additional seconds. Oxidative phosphorylation (state 3 respiration) was induced by the addition of 200 μM (final concentration) ADP and the rate of ADP-stimulated O 2 consumption per mg mitochondrial protein (RASOC) was determined. Extracts were tested in three different experimental sessions, with at least two replicate experiments per mitochondrial preparation. DMSO-vehicle control experiments were conducted at the beginning and end of each experimental session in order to establish the session-normal RBOC and RASOC and to ensure no loss in mitochondrial viability over the duration of the session, typically less than 4 h from the end of the isolation protocol. DMSO was confirmed to have no effect on the basal rate of O 2 consumption. The effect of each plant extract was evaluated as: (1) the increase in the RBOC (a measure of the magnitude of the uncoupling effect); (2) the decrease in functional capacity (FC) per mg protein (a measure of the magnitude of the uncoupling effect plus any additional inhibitory effect), where FC was defined as the difference of the RASOC (maximal functional rate of consumption) and the RBOC (rate of consumption driven by proton leak and not contributing to ATP synthesis). Calculations were as follows: the average FC per mg protein of the vehicle control experiments for a given session was calculated by subtracting the average RBOC from the average RASOC. For (1) above, the absolute increase in RBOC measured in a given experiment was expressed as a percentage of the average control FC for the session. For (2) above, the FC measured in a given experiment was expressed as a percentage of the average control FC for the session to give the % residual FC. The compound 2,4-dinitrophenol (Sigma–Aldrich) was used at 5 μM as a reference uncoupler, whereas oligomycin A (Sigma–Aldrich) was used at 0.5 μM as a reference ATPsynthase inhibitor. 2.5 Rate of acidification of extracellular medium A spectrophotometric assay of change in cell culture medium pH over time, quantitative between pH 7.2 and 6.4, was developed based on similar assays ( Schornack and Gillies, 2003; Yang and Balcarcel, 2003 ). The assay medium consisted of Dulbecco's PBS containing Phenol Red as a pH indicator and modified to contain a total of 2 mM phosphate (for approx. 20% of the buffering capacity of Dulbecco's original recipe) while keeping other ion concentrations within the physiological range of extracellular fluid. The composition of this modified Dulbecco's PBS (mD-PBS) was: 1.5 mM Na 2 HPO 4 , 0.5 mM KH 2 PO 4 , 137 mM NaCl, 25 mM glucose, 4 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 , Phenol Red 0.1 mM (Sigma–Aldrich Phenol Red 0.5% solution), and deionized ultra-filtered water (Fisher Scientific, Ottawa, ON). This formulation resulted in a pH of 7.1, which was adjusted to 7.20 at ambient temperature with NaOH immediately prior to the assay using an Accumet pH meter with calomel electrode (Fisher Scientific). Absorbance of 100 μl samples of medium transferred to 96-well plates (Sarstedt Inc., Montreal, QC) was measured at ambient temperature at 530 and 450 nm using a Wallac Victor 2 plate reader (Perkin-Elmer, St-Laurent, QC) and the ratio of abs 530/abs 450 was calculated. The relationship between pH and the log of this ratio was observed to be linear over the range of pH of 6.4–7.4, in agreement with a pKa of 6.9 for balanced-salt phosphate buffers. The following function was used to model the relationship between pH and absorbance over the pH range of 6.4–7.2: pH = 0.765 × ln(abs 530/abs 450) + 7.61 ( R 2 = 0.99). A titration experiment was performed to determine the buffering capacity of mD-PBS. This capacity was observed to be nearly linear between pH 6.2 and 7.2 and was calculated to be 1.075 mM equivalents per pH units between 6.3 and 7.1. Experiments were performed on 7-day differentiated C2C12 muscle cells and on 1-day post-confluent H4IIE liver cells grown in 12 well plates. On the day of the experiment, cells were gently rinsed twice with mD-PBS, and then allowed to equilibrate in exactly 1.0 ml of mD-PBS for 30 min at 37 °C in a humidified air atmosphere. The assay was started by gently mixing pre-warmed 3× concentrated treatments in a 500 μl volume of mD-PBS to the 1.0 ml volume of mD-PBS already present, for a final volume of exactly 1.5 ml and treatments at their final working concentration. After the rapid addition of treatments to all the wells of a single plate, an initial 100 μl sample of medium, corresponding to time 0, was transferred to a microtiter plate for spectrophotometric analysis. Cells were then incubated at 37 °C in a humidified air atmosphere for the duration of the experiment. At times 20, 40, 60, 120, 180, and 240 min, plates were stirred and a 100 μl sample of medium was transferred to a microtiter plate for analysis. Calculations of rate of acidification and cumulative secretion of acid equivalents over time accounted for the decreasing experimental volume with each sampling. Because DMSO was observed to stimulate acidification, as noted by others ( Gerber et al., 1996 ), extracts were solubilized in 80% ethanol at 1000× their final concentration, for a final ethanol concentration of 0.08%. The addition of extracts or controls affected the pH of mD-PBS and therefore all treatments were adjusted to pH 7.2 separately immediately prior to the assay. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; Sigma–Aldrich) solubilized in ethanol was used at 5 μM as a positive control. Results were expressed as cumulative secretion of acid equivalents (micromoles) for four to five replicates per condition per time point. 2.6 Cytosolic ATP assays Total cytosolic ATP was measured in cell lysates by luminescence using the ATPlite assay kit (Perkin-Elmer, Waltham, MA), as per the manufacturer's protocol. Briefly, C2C12 myotubes in 24-well plates or H4IIE hepatocytes in 96-well plates were treated in parallel for 1, 3, or 6 h with extract or DMSO. FCCP was used at 5 μM as a positive control. Cells were rinsed in PBS and lysed with supplied lysis buffer for 5 min with orbital shaking. Supplied substrate solution was then added and cells were shaken for 5 min. Plates were then covered for 10 min prior to measurement of luminescence in a plate reader. An ATP standard curve was prepared in parallel using the supplied ATP solution. Results were expressed as % ATP content of vehicle-treated wells. Three to four replicates per condition per time point were performed. 2.7 Statistical analysis Results are reported as mean ± SEM, with the number of replicates and number of independent experiments indicated. Data were analyzed by one-way analysis of variance with a Fisher post hoc test using StatView 5.0 software (SAS Institute, Cary, NC) for the Macintosh platform. Statistical significance was set at p ≤ 0.05. Area under the curve calculations performed with Prism 4.0 software (GraphPad Software Inc., La Jolla, CA) for the Macintosh platform. 3 Results 3.1 The AMPK pathway, but not the insulin receptor pathway, is activated in C2C12 muscle cells following an 18 h treatment with extracts In our earlier screening study ( Spoor et al., 2006 ), it was observed that deoxyglucose uptake in the absence of insulin stimulation (basal glucose uptake) was increased in C2C12 muscle cells by extracts of three of the eight species studied here following a 1 h treatment, and by seven of the eight species following an 18 h treatment. Metformin, used as a positive control at 400 μM, had little effect after 1 h but increased uptake by approximately 30% after 18 h. At rest, glucose uptake in skeletal muscle is mediated by GLUT1 glucose transporters constitutively expressed in the plasma membrane ( Klip and Marette, 1992 ). In response to stimulation of the insulin receptor signaling pathway or of the AMPK pathway, uptake can be transiently increased by the translocation of GLUT4 and GLUT1 transporters to the plasma membrane ( Abbud et al., 2000; Fujii et al., 2004; Krook et al., 2004 ), and possibly by the release of inhibition of the intrinsic activity of these transporters ( Michelle Furtado et al., 2003 ). Activation of AMPK can also upregulate transport capacity ( McGee and Hargreaves, 2006, 2008 ). To determine whether the effects of the active plant products were mediated through the insulin signaling pathway or through the AMPK pathway, C2C12 cells were treated for 1, 6, or 18 h with respective extracts and cell lysates were analyzed by western immunoblot for increased content of serine 473-phosphorylated Akt (protein kinase B), a well-recognized marker of stimulation of the insulin signaling pathway, and for increased content of threonine 172-phosphorylated AMPKalpha. The content of phospho-Akt ( Fig. 1 ) was not increased by more than 25% above the respective vehicle control levels by any extract following either a 1 or 18 h treatment, and content was even decreased relative to control by some conditions at the later time point. As a reference, stimulation with 1 nM of insulin for 15 min results in an increase in phospho-Akt content of approximately 150%, and 45 min later this content remains elevated by approximately 75% ( Benhaddou-Andaloussi et al., 2009 ). In contrast to the small fluctuations in content of phospho-Akt, phospho-AMPK content ( Fig. 2 ) underwent more important increases. Threonine 172-phosphorylated AMPKalpha content was increased by all eight extracts at the end of the 1 h treatment by 60–170% above vehicle control levels. At the end of the 6 h treatment, content remained elevated by 50% or more by six extracts, and in four cases, content was actually higher at 6 h than at 1 h. Treatment with the Sarracenia purpurea extract was notable in that it resulted in a 2.5-fold increase in phospho-AMPK content at this time point, an effect of similar magnitude as that of the AICAR positive control applied for 30 min. At the end of the 18 h treatment, phospho-AMPK contents were in all conditions lower than at the 6 h time point. However, in cells treated with Sarracenia purpurea , Sorbus decora , or Alnus incana extracts, phospho-AMPK remained 2-fold or more above the level of the vehicle control at the 18 h mark. The same samples were also analyzed for serine 79-phosphorylated ACC content ( Fig. 3 ), a marker of activation of the AMPK pathway. The same six extracts observed to increase phospho-AMPK content at all measured time points produced a similar pattern of increase in phospho-ACC content. In keeping with its downstream position relative to AMPK, ACC phosphorylation was observed to be generally more sustained than AMPK activation; in cells treated with Sarracenia purpurea , Sorbus decora , Alnus incana , or Rhododendron groenlandicum extracts, contents measured at 18 h were similar to or even greater than those measured at 6 h. 3.2 A relationship between increased phospho-AMPK content and glucose uptake Seven of the eight species studied here have previously been shown to increase glucose uptake in muscle cells in the absence of insulin stimulation following an 18 h treatment ( Spoor et al., 2006 ). The area under the curve of phospho-AMPK content over 18 h, as defined by contents measured here at 1, 6, and 18 h (above), was observed to be well correlated ( R 2 = 0.86) to the enhancement of basal glucose uptake previously reported ( Fig. 4 ). 3.3 Extracts instantaneously disrupt function of isolated mitochondria AMPK is highly sensitive to metabolic stress and perturbations of energy homeostasis. As many plant defensive metabolites exert disruptive effects on well-conserved energy transduction pathways ( Polya, 2003 ), we next addressed the hypothesis that the extracts found to activate the AMPK pathway would also disrupt mitochondrial function. Succinate-supported rates of basal and ADP-stimulated oxygen consumption were measured in isolated rat liver mitochondria treated with either vehicle or with respective plant extracts at the same concentration used to induce glucose uptake and stimulation of AMPK. An increase in RBOC reflects an uncoupler-type disruption of mitochondrial function, as can be induced with the reference compound 2,4-dinitrophenol ( Fig. 5 A ). A decrease in RASOC, occurring in the absence of any change in RBOC, typically reflects an inhibition of ATP synthase, as can be induced with the reference compound oligomycin A ( Fig. 5 A). Concurrent uncoupling and inhibition of ATP synthase can occur, especially when testing complex mixtures possibly containing multiple active principles. However, concurrent effects on RBOC and RASOC can also indicate other types of disruption, such as an inhibition of substrate transport across the inner mitochondrial membrane. Of the eight plant products tested here, six induced a concurrent increase in RBOC and decrease in RASOC, whereas one, the Rhododendron groenlandicum extract, affected RASOC only, and one, the Picea mariana extract, had no effect on mitochondrial function. The seven products found to affect mitochondrial function were therefore the same seven previously observed to enhance glucose uptake ( Spoor et al., 2006 ). These disruptions, whether concurrent and therefore cumulative, or of a single type, resulted in reductions in mitochondrial capacity for ATP synthesis ranging from 11% to 67% ( Table 1 ). All disruptions of mitochondrial function observed were instantaneous. Representative oxygen consumption tracings are shown for the extract of Rhododendron groenlandicum ( Fig. 5 B) affecting only RASOC, and for the extract of Abies balsamea ( Fig. 5 C), the product that induced the greatest disruptions (23% and 46% reductions in mitochondrial capacity by uncoupling and inhibitory effects, respectively). 3.4 Extracts stimulate the rate of acidification of the cell culture medium When mitochondrial capacity for ATP synthesis is reduced to the point where it can no longer meet the energy needs of the cell, dependence on anaerobic glycolysis for energy production is increased. By measuring flux through glycolysis, it is therefore possible to obtain information concerning the degree of metabolic stress induced by the plant products as well as information about their rapidity and duration of action. Furthermore, these experiments allow for the comparison of effects across different cell types. As lactic acid is the end product of anaerobic glycolysis, flux through this pathway was measured as the rate of acidification of cell culture medium. Extracts were applied to cells for 4 h, over which time the medium was periodically sampled and pH determined spectrophotometrically using Phenol Red as indicator. Results were expressed in the form of cumulative secretion of acid equivalents over time ( Fig. 6 ). All extracts were observed to increase the rate of acidification by 2–3-fold over the first 20 min of the assay in C2C12 cells. However, beyond this time, effects varied greatly between the different extracts. A sustained increase in the rate of acidification over the 4 h experimental period was produced only by the Larix laricina and Abies balsamea extracts, the two extracts that most severely disrupted function in isolated mitochondria. The Alnus incana extract produced the smallest effect, which lasted no longer than 20 min. The Picea mariana extract, despite having no observable effect in isolated liver mitochondria, produced the largest increase in rate of acidification; however, this increase was not sustained beyond the first hour. The Sorbus decora , the Rhododendron groenlandicum , and the Sarracenia purpurea extracts produced short-lived effects. Interestingly, the latter two demonstrated a tendency to depress anaerobic metabolism below the vehicle control rate of acidification after the first hour of treatment. In H4IIE hepatocytes, slightly different effects were observed. Notably, the Picea mariana extract had no effect in this cell line. Also, the magnitude of effect of the Abies balsamea was much greater in hepatocytes than muscle cells, surpassing the effect of the positive control FCCP. Finally, in some instances, rate of acidification was observed to be paradoxically depressed rather than stimulated. 3.5 Extracts induce a depression of cytosolic ATP content Similarly to the acidification assay above, the concentration of cytosolic ATP was assessed in order to gain insight into magnitude, rapidity of onset, and duration of the metabolic stress induced by the plant extracts in whole cells. In C2C12 cells, all species, with the exception of Rhododendron groenlandicum , were observed to induce a depression of cytosolic ATP content 1 h into the treatment ( Fig. 7 ). In the case of the Larix laricina and Abies balsamea extracts, this depression was sustained over the 6 h experimental period, concordant with their sustained increase in anaerobic metabolism observed in the acidification assay. The effect of the Sorbus decora extract was the shortest lived. The effects of other extracts were normalized by the 6 h time point. The Rhododendron groenlandicum and Sorbus decora extracts tended to induce supranormal ATP levels, possibly due to the overcompensation of ATP synthesis in response to metabolic stress and activation of AMPK. Supranormal ATP content by these extracts coincided with a tendency for inhibition of Akt activity ( Fig. 1 ). As in the acidification assay, cell-type differences were observed. Notably, the magnitude of effect of the extracts was smaller in H4IIE hepatocytes; only the Larix laricina extract induced a depression between 1 and 6 h ( Fig. 7 ). However, the overcompensation phenomenon described above was observed to be induced by five extracts as well as by the FCCP positive control. The two most important overcompensations, those induced by the Sarracenia purpurea and Sorbus decora extracts, concord with the observed depression of anaerobic metabolism induced by these products ( Fig. 6 ), which may indicate allosteric inhibition of glycolysis by surplus ATP. 4 Discussion Medicinal plant products with anti-hyperglycemic activity are common in the traditional medicine of cultures throughout the world, and the efficacy of many has been rigorously studied ( Haddad et al., 2006; Marles and Farnsworth, 1995 ). The cellular and molecular mechanisms of action of these, however, have generally not been elucidated. The present study focuses on plant products selected from the pharmacopeia of an aboriginal Canadian population, the Cree of Iiyiyiu Istchii, and was undertaken for further evaluation of these products as promising and culturally adapted treatment options for diabetes care in susceptible aboriginal populations. Specifically, the present study addressed the mechanism of action through which these products enhance the rate of basal glucose uptake in a model skeletal muscle cell line, an activity first observed in our recent screening study of the antidiabetic potential of these products ( Spoor et al., 2006 ). Our results demonstrate that the selected medicinal plant products act through a common mechanism involving the AMPK signaling pathway, rather than through the insulin receptor pathway, to increase basal glucose uptake in C2C12 skeletal muscle cells following 18 h of treatment. Indeed, whereas no significant increases in the content of phosphorylated Akt were observed throughout the 18 h period, phosphorylation of AMPK and ACC were increased up to 2.5- and 3.5-fold, respectively. The AMPK pathway converges with the insulin receptor pathway at the level of AS160 and in this way can induce the translocation of GLUT4 glucose transporters to the sarcolemma and promote an increase in the rate of uptake ( Cartee and Wojtaszewski, 2007; Thong et al., 2007 ). Direct effects of the AMPK pathway on the intrinsic activity of constitutively expressed GLUT1 transporters and of GLUT4 transporters have also been reported ( Michelle Furtado et al., 2003 ). However, increase in the expression of glucose transporters and of other effectors of the insulin receptor pathway that are mediated by AMPK signaling ( McGee and Hargreaves, 2008 ) likely play a more important role in the long-lasting effects of the products studied herein. This was supported by the observation that some plant products enhanced basal glucose uptake to levels surpassing the maximal acute stimulatory effect of insulin ( Spoor et al., 2006 ). This is now also supported by an excellent correlation between the enhancement of glucose uptake after the 18 h treatment and the activation of AMPK over the entire treatment period ( Fig. 5 ). Arguments against residual acute stimulation of uptake at 18 h, such as the observation that the effect of insulin applied acutely was additive to the effect induced by 18 h treatment with plant products ( Spoor et al., 2006 ), now also include the finding that AMPK activation was either declining or had returned to control levels at the 18 h time point. This activation of the AMPK pathway can be explained by the induction of a metabolic stress through a mild and transient disruption of mitochondrial energy transduction ( Hayashi et al., 2000 ). Indeed, all seven plant products previously shown to stimulate glucose uptake were here found to induce in isolated mitochondria an inhibitory-type disruption of oxidative phosphorylation or a combination of inhibition and uncoupling-type disruptions. Metabolic stress was confirmed in intact cells as increased flux through anaerobic glycolysis and depression of cytosolic ATP concentrations. Both inhibitory and uncoupling effects additively contribute to a reduction of mitochondrial capacity. When capacity is no longer sufficient to meet energy demand, energy homeostasis is disturbed and activation of AMPK then results from the simultaneous drop in ATP concentration and rise in AMP concentration ( Winder and Thomson, 2007 ). In the face of such metabolic stress, the function of AMPK is to restore energy homeostasis as well as to protect the cell against future metabolic stress. This is accomplished by acutely increasing glucose and fat uptake to support a coordinated stimulation of carbohydrate and lipid oxidation, while at the same time transiently inhibiting energy-consuming synthetic processes ( Winder and Thomson, 2007 ). The adaptive effects of AMPK include an increase in mitochondrial capacity through mitochondrial biogenesis and an increase in substrate uptake capacity ( McGee and Hargreaves, 2008; Reznick and Shulman, 2006; Winder, 2001 ). Induction of metabolic stress through disruptions of mitochondrial function has long been known to produce cytoprotective effects. For example, the classical uncoupler 2,4-dinitrophenol has been shown to increase glucose uptake in muscle cells ( Bashan et al., 1993 ), and sodium azide, a cytochrome oxidase inhibitor, has been shown to rapidly upregulate GLUT1 expression by several folds in liver cells ( Behrooz and Ismail-Beigi, 1997; Shetty et al., 1993 ). The effects of the commonly prescribed hypoglycemic drug Metformin are believed to be mediated similarly through inhibition of complex I of the electron transport chain ( El-Mir et al., 2000; Owen et al., 2000 ) and subsequent activation of AMPK ( Hayashi et al., 2000 ). It is remarkable that products from such a wide variety of plant species, as are represented here, share a common mechanism of action. However, disruption of energy transduction and the resulting indirect activation of AMPK represent a simple mechanism requiring less molecular specificity than the activation of the insulin receptor signaling pathway or even than the inhibition of negative regulators of this pathway. Therefore, it is possible that the metabolic stress mechanism is widespread and may explain the antidiabetic activity of traditional medicines from various cultures. This mechanism is also in accordance with the defensive role of many plant secondary metabolites, evolved for fungicidal, bactericidal, and insecticidal activities, all mediated by the targeting of highly conserved energy transduction pathways ( Polya, 2003 ). Indeed, several plant compounds, including many flavonoids, are known disruptors of mitochondrial oxidative phosphorylation, acting either as uncouplers or as inhibitors ( Polya, 2003 ). Targeting mitochondrial energy transduction is inherently dangerous; increased reliance on anaerobic metabolism as a compensation for compromised mitochondrial capacity results in an increase in the rate of release of hydrogen ions, which can lead to systemic acidosis. Metabolic acidosis is indeed the primary danger associated with the use of biguanides and represents a potential complication that needs to be considered in the case of the plant products tested herein. This danger can clearly be minimized if the metabolic stress is short-lived. The results obtained in our study on the rate of acidification and on cytosolic ATP content suggest that the effects of the plant products are indeed in most cases short-lived and of low magnitude, aerobic capacity generally being restored after 30–60 min. Such effects would be expected with rapidly metabolized products and future studies, notably on active principles, will need to address this point. Our results highlight the importance of duration of AMPK activation for maximizing the enhancement of glucose uptake; the products that were notable in their sustained activation of this pathway, the extracts of Sarracenia purpurea and Sorbus decora , were also the most efficacious for increasing uptake. A less intuitive observation, however, is that there appears to be an optimal pattern of metabolic stress for enhancing muscle glucose uptake (and perhaps for maximizing other insulin-like effects of AMPK such as the inhibition of hepatic glucose output) and that sustained metabolic stress may actually be counterproductive. Indeed, the products that induced the most prolonged stress, as gauged by the depression of ATP concentration and increased flux through anaerobic glycolysis, were not the most efficacious for activating AMPK or enhancing uptake. On the other hand, the extracts of Sarracenia purpurea and of Sorbus decora produced a paradoxical combination of sustained activation of the AMPK pathway and short-lived metabolic stress. This observation therefore suggests the possibility that the potential for lactic acidosis can be uncoupled from AMPK-stimulating activity. This augers well for efforts aimed at identifying better alternatives to Metformin and the biguanides. We also observed that depression of ATP concentration was in several cases followed by a compensatory overshoot phenomenon. This provides further evidence that, for the most part, the extracts induce a sudden but short-lived metabolic stress. As an overcompensation of ATP synthesis can be explained by an AMPK-triggered stimulation of lipid and carbohydrate oxidation that is sustained several hours after the restoration of homeostasis, the concordance of these overshoots with sustained ACC phosphorylation is further support of a mismatch between the kinetics of metabolic stress and those of AMPK activation. It should be noted that ATP overcompensation and/or sustained ACC phosphorylation induced by the Rhododendron groenlandicum , Sorbus decora , and Sarracenia purpurea extracts also coincided with depressed glycolytic flux and inhibition of the insulin signaling pathway, expected effects in the face of energy surfeit. Finally, it is noteworthy that cell-type specific effects were observed in the acidification and cytosolic ATP assays. A striking example was that of the Picea mariana extract which had no effect on acidification in liver cells, concordant with its absence of effect on isolated liver mitochondria, and yet produced the highest measured rate of acidification in muscle cells. Such cell-type specificities, possibly due to molecular differences in the electron transport chain, support the hypothesis that it is possible to target AMPK-mediated effects only in some tissues while sparing others. This is highly desirable from a therapeutic point of view since in pancreatic beta cells, the activation of AMPK can decrease insulin secretion ( da Silva Xavier et al., 2003 ), while in hypothalamic cells, it can trigger hunger signaling ( Kola, 2008 ). In our previous screening study ( Spoor et al., 2006 ), it was observed that the Larix laricina and Sorbus decora extracts had no effect on glucose uptake in 3T3-L1 adipocytes, and only the Abies balsamea , Larix laricina , Rhododendron groenlandicum , and Sarracenia purpurea extracts inhibited glucose-stimulated insulin secretion in β cells. In conclusion, the medicinal plant species studied herein exert their activity through a common mechanism similar to that of Metformin, involving the activation of AMPK secondary to metabolic stress induced by the disruption of mitochondrial energy transduction. Due to its simplicity, this mechanism is likely to apply to other antidiabetic plant products used elsewhere in the world. The active products studied herein may be useful against insulin resistance and metabolic diseases. Testing for anti-hyperglycemic activity in animal models is currently underway, as is the isolation and identification of the active constituents of these products. The detailed profile of biological activity obtained in this study will help inform the choice of candidate products for integration into the diabetes care of Canadian aboriginal populations. Acknowledgments This work was supported by a Team Grant from the Canadian Institutes of Health Research to PSH, JTA, and LCM. This work was conducted with the consent and support of the Cree Nation of Mistissini and of the Cree Board of Health and Social Services of James Bay. Special thanks are given to Elizabeth Coon Come, Emma Coon Come, Rene Coon Come, Mable Gunner, Abel Mark, Kathleen Mark, Harriett Matoush, Sandy Matoush, Laurie Petawabano and Smally Petawabano and to 24 other Cree Elders who kindly agreed be interviewed. They made this article possible by allowing us to use, for the purposes of this research, their knowledge relating to medicinal plants, transmitted to them by their elders. Their trust has also enabled a useful exchange between Indigenous knowledge and Western science. 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