Diane Vallerand

Stimulation of AMP-activated protein kinase and enhancement of basal glucose uptake in muscle cells by quercetin and quercetin glycosides, active principles of the antidiabetic medicinal plant Vaccinium vitis-idaea

Several medicinal plants that stimulate glucose uptake in skeletal muscle cells were identified from among species used by the Cree of Eeyou Istchee of northern Quebec to treat symptoms of diabetes. This study aimed to elucidate the mechanism of action of one of these products, the berries of Vaccinium vitis idaea, as well as to isolate and identify its active constituents using a classical bioassay-guided fractionation approach. Western immunoblot analysis in C2C12 muscle cells revealed that the ethanol extract of the berries stimulated the insulin-independent AMP-activated protein kinase (AMPK) pathway. The extract mildly inhibited ADP-stimulated oxygen consumption in isolated mitochondria, an effect consistent with metabolic stress and the ensuing stimulation of AMPK. This mechanism is highly analogous to that of Metformin. Fractionation guided by glucose uptake activity resulted in the isolation of ten compounds. The two most active, quercetin-3-O-glycosides, enhanced glucose uptake by 38-59% (50 muM; 18 h treatment) in the absence of insulin. Quercetin aglycone, a minor constituent, stimulated uptake by 37%. The quercetin glycosides and the aglycone stimulated the AMPK pathway at concentrations of 25-100 muM, but only the aglycone inhibited ATP synthase in isolated mitochondria (by 34 and 79% at 25 and 100 muM, respectively). This discrepancy suggests that the activity of the glycosides may require hydrolysis to the aglycone form. These findings indicate that quercetin and quercetin 3-O-glycosides are responsible for the antidiabetic activity of V. vitis crude berry extract mediated by AMPK. These common plant products may thus have potential applications for the prevention and treatment of insulin resistance and other metabolic diseases.

Multiple molecular targets underlie the antidiabetic effect of Nigella sativa seed extract in skeletal muscle, adipocyte and liver cells

Aim: Nigella sativa (N. sativa) is a plant widely used in traditional medicine of North African countries. During the last decade, several studies have shown that extracts from the seeds of N. sativa have antidiabetic effects.

Antioxidant and Hepatoprotective Effects of Silibinin in a Rat Model of Nonalcoholic Steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a progressive liver disease related to the metabolic syndrome, obesity and diabetes. The rising prevalence of NASH and the lack of efficient treatments have led to the exploration of different therapeutic approaches. Milk thistle (Silibum marianum) is a medicinal plant used for its hepatoprotective properties in chronic liver disease since the 4th century BC. We explored the therapeutic effect of silibinin, the plants most biologically active extract, in an experimental rat NASH model. A control group was fed a standard liquid diet for 12 weeks. The other groups were fed a high-fat liquid diet for 12 weeks without (NASH) or with simultaneous daily supplement with silibinin–phosphatidylcholine complex (Silibinin 200 mg kg−1) for the last 5 weeks. NASH rats developed all key hallmarks of the pathology. Treatment with silibinin improved liver steatosis and inflammation and decreased NASH-induced lipid peroxidation, plasma insulin and TNF-α. Silibinin also decreased O2 ∙− release and returned the relative liver weight as well as GSH back to normal. Our results suggest that milk thistles extract, silibinin, possesses antioxidant, hypoinsulinemic and hepatoprotective properties that act against NASH-induced liver damage. This medicinal herb thus shows promising therapeutic potential for the treatment of NASH.

Hydro‐ethanolic extract of cashew tree (Anacardium occidentale) nut and its principal compound, anacardic acid, stimulate glucose uptake in C2C12 muscle cells

SCOPE Products of cashew tree (Anacardium occidentale) are used in traditional medicine for various ailments, including diabetes. METHODS AND RESULTS The anti-diabetic properties of cashew plant parts were studied using differentiated C2C12 myoblasts (myotubes) and rat liver mitochondria. Hydroethanolic extract of cashew seed (CSE) and its active component, anacardic acid (AA), stimulated glucose transport into C2C12 myotubes in a concentration-dependent manner. Extracts of other parts (leaves, bark and apple) of cashew plant were inactive. Significant synergistic effect on glucose uptake with insulin was noticed at 100 μg/mL CSE. CSE and AA caused activation of adenosine monophosphate-activated protein kinase in C2C12 myotubes after 6 h of incubation. No significant effect was noticed on Akt and insulin receptor phosphorylation. Both CSE and AA exerted significant uncoupling of succinate-stimulated respiration in rat liver mitochondria. CONCLUSION Activation of adenosine monophosphate-activated protein kinase by CSE and AA likely increases plasma membrane glucose transporters, resulting in elevated glucose uptake. In addition, the dysfunction of mitochondrial oxidative phosphorylation may enhance glycolysis and contribute to increased glucose uptake. These results collectively suggest that CSE may be a potential anti-diabetic nutraceutical.

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

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 microg/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 4mM of the AMP-mimetic AICAR, was in several cases sustained for at least 18h post-treatment. Duration of metabolic stress, however, was in most cases in the order of 1h. 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.

The Action of Antidiabetic Plants of the Canadian James Bay Cree Traditional Pharmacopeia on Key Enzymes of Hepatic Glucose Homeostasis

We determined the capacity of putative antidiabetic plants used by the Eastern James Bay Cree (Canada) to modulate key enzymes of gluconeogenesis and glycogen synthesis and key regulating kinases. Glucose-6-phosphatase (G6Pase) and glycogen synthase (GS) activities were assessed in cultured hepatocytes treated with crude extracts of seventeen plant species. Phosphorylation of AMP-dependent protein kinase (AMPK), Akt, and Glycogen synthase kinase-3 (GSK-3) were probed by Western blot. Seven of the seventeen plant extracts significantly decreased G6Pase activity, Abies balsamea and Picea glauca, exerting an effect similar to insulin. This action involved both Akt and AMPK phosphorylation. On the other hand, several plant extracts activated GS, Larix laricina and A. balsamea, far exceeding the action of insulin. We also found a significant correlation between GS stimulation and GSK-3 phosphorylation induced by plant extract treatments. In summary, three Cree plants stand out for marked effects on hepatic glucose homeostasis. P. glauca affects glucose production whereas L. laricina rather acts on glucose storage. However, A. balsamea has the most promising profile, simultaneously and powerfully reducing G6Pase and stimulating GS. Our studies thus confirm that the reduction of hepatic glucose production likely contributes to the therapeutic potential of several antidiabetic Cree traditional medicines.

Structural constraints and the importance of lipophilicity for the mitochondrial uncoupling activity of naturally occurring caffeic acid esters with potential for the treatment of insulin resistance

Caffeic acid phenethyl ester (CAPE) has recently been shown to potently stimulate glucose uptake in cultured skeletal muscle cells through the AMPK pathway and therefore to have anti-diabetic potential. We report here that CAPE increases glucose uptake in C2C12 muscle cells by 225+/-21% at 50 microM, and that activation of AMPK is a consequence of the metabolic stress resulting from an uncoupling-type disruption of mitochondrial function (complete uncoupling at 50 microM). We also observe that the therapeutic potential of CAPE is offset by its high potential for toxicity. The purpose of this study was therefore to identify other active caffeic acid derivatives, evaluate their ratio of activity to toxicity, and elucidate their structure-activity relationship. Twenty naturally occurring derivatives were tested for glucose-uptake stimulating activity in C2C12 cells following 18 h of treatment and for uncoupling activity in isolated rat liver mitochondria. Cytotoxicity was assessed in C2C12 cells by the release of lactate dehydrogenase over 18 h. In addition to CAPE, four compounds were identified to be active, both stimulating glucose uptake and uncoupling isolated mitochondria. Activity required that the caffeic acid moiety be intact and that the compound not contain a strongly ionized group. Both activity and toxicity were found to be well-correlated to predicted lipophilicity. However, two compounds exhibited little to no toxicity while still stimulating glucose uptake by 65-72%. These results support a therapeutic potential for this family of compounds and provide the framework for the design of alternatives to Metformin with an optimized balance of safety and activity.

Populus balsamifera L. (Salicaceae) mitigates the development of obesity and improves insulin sensitivity in a diet-induced obese mouse model.

ETHNOBOTANICAL RELEVANCE : In previous in vitro bioassay studies, Populus balsamifera L. (Salicaceae), a medicinal plant ethnobotanically identified from the traditional pharmacopoeia of the Cree of Eeyou Istchee (Eastern James Bay area of Canada), exhibited a strong anti-obesity potential by potently inhibiting adipogenesis in 3T3-L1 adipocytes. The aim of the study is to evaluate the effectiveness of this plant extract in mitigating the development of obesity and the metabolic syndrome in diet-induced obese (DIO) C57BL/6 mice. MATERIALS AND METHODS Mice were subjected for eight weeks to a standard diet (CHOW), a high fat diet (HFD; DIO group), or HFD to which Populus balsamifera was incorporated at 125 and 250 mg/kg. RESULTS The results showed that Populus balsamifera decreased in a dose-dependent manner the weight gain of whole body, retroperitoneal fat pad and liver as compared to DIO controls and reduced the severity of hepatic macrovesicular steatosis and triglyceride accumulation. This plant extract also decreased glycemia in the second half of the feeding period and improved insulin sensitivity by diminishing insulin levels and the leptin/adiponectin ratio, as well as augmenting adiponectin levels. These effects were associated with slightly but significantly reduced food intake with 250 mg/kg Populus balsamifera as well as with an increase in energy expenditure (increase in skin temperature and increased expression of uncoupling protein-1; UCP-1). Data also suggest other mechanisms, such as inhibition of adipocyte differentiation, decrease of hepatic inflammatory state and potential increase in hepatic fatty acid oxidation. CONCLUSION Taken together, these results confirm the potential of Populus balsamifera as a culturally adapted therapeutic approach for the care and treatment of obesity and diabetes among the Cree.

Larix laricina, an Antidiabetic Alternative Treatment from the Cree of Northern Quebec Pharmacopoeia, Decreases Glycemia and Improves Insulin Sensitivity In Vivo

Larix laricina K. Koch is a medicinal plant belonging to traditional pharmacopoeia of the Cree of Eeyou Istchee (Eastern James Bay area of Canada). In vitro screening studies revealed that, like metformin and rosiglitazone, it increases glucose uptake and adipogenesis, activates AMPK, and uncouples mitochondrial function. The objective of this study was to evaluate the antidiabetic and antiobesity potential of L. laricina in diet-induced obese (DIO) C57BL/6 mice. Mice were subjected for eight or sixteen weeks to a high fat diet (HFD) or HFD to which L. laricina was incorporated at 125 and 250 mg/kg either at onset (prevention study) or in the last 8 of the 16 weeks of administration of the HFD (treatment study). L. laricina effectively decreased glycemia levels, improved insulin resistance, and slightly decreased abdominal fat pad and body weights. This occurred in conjunction with increased energy expenditure as demonstrated by elevated skin temperature in the prevention study and improved mitochondrial function and ATP synthesis in the treatment protocol. L. laricina is thus a promising alternative and complementary therapeutic approach for the treatment and care of obesity and diabetes among the Cree.

Lingonberry (Vaccinium vitis-idaea L.) Exhibits Antidiabetic Activities in a Mouse Model of Diet-Induced Obesity

Vaccinium vitis-idaea, commonly known as lingonberry, has been identified among species used by the Cree of Eeyou Istchee of northern Quebec to treat symptoms of diabetes. In a previous study, the ethanol extract of berries of V. vitis-idaea enhanced glucose uptake in C2C12 muscle cells via stimulation of AMP-activated protein kinase (AMPK) pathway. The purpose of this study was to examine the effect of plant extract in a dietary mouse model of mild type 2 diabetes. C57BL/6 mice fed a high-fat diet (HFD, ∼35% lipids) for 8 weeks that become obese and insulin-resistant (diet-induced obesity, DIO) were used. Treatment began by adding V. vitis-idaea extract to HFD at 3 different concentrations (125, 250, and 500 mg/Kg) for a subsequent period of 8 weeks (total HFD, 16 weeks). The plant extract significantly decreased glycemia and strongly tended to decrease insulin levels in this model. This was correlated with a significant increase in GLUT4 content and activation of the AMPK and Akt pathways in skeletal muscle. V. vitis-idaea treatment also improved hepatic steatosis by decreasing hepatic triglyceride levels and significantly activated liver AMPK and Akt pathways. The results of the present study confirm that V. vitis-idaea represents a culturally relevant treatment option for Cree diabetics and pave the way to clinical studies.