Trials were performed using slight modifications to the previously reported assay. Fifty flies were aged per vial and starved in vials with 2 Kimwipes moistened with 3ml of water. A 6mm diameter circle of Whatman #1 filter paper was placed in the bottom of a 10 cm length tube to deliver the odor. A brass screen of 8/32 inch diameter was placed 5mm from the bottom of the tube to gate off the filter paper. Approximately 100 flies were inserted into the control tube and joined to the tube with test odorant. After 30minutes exposure in the dark at 25 degrees Celsius, the apparatus was photographed. Flies 5 cm from each screen were counted. Preference index was calculated. The Two-Choice Trap Assay in a plate tests less volatile odorants. Trials were performed as described. Ten female flies are placed in a Petri dish containing two traps. Traps were made with 1.5ml micro centrifuge tubes with opening cut in the bottom of the tube. Both traps contain the fly’s normal laboratory food at the base. The neck of one trap has a filter paper with test odorant, the other trap has solvent. Five microliters of hexane and five microliters of 10% DEET or test compounds in hexane were applied to the stem part of filter paper inserted into upper part of pipette tip near entrance to trap to allow flies to walk over treated surface. Traps were placed in chemical hood for 5minutes to allow hexane to volatilize before being placed in the 1% agarose treated Petri dish chamber.Assay was performed to determine24 preference for an attractive food source in the context of a repellent odor. Briefly, ten male and ten female starved flies are placed in a cylindrical chamber containing two traps: with test odorant and lure,plants in pots ideas with solvent and lure. Apple cider vinegar is the lure for all trials except for D. virilis where liquid malt was used . To create a well for separating lure from test odorant, a single cap cut from a BioRad PCR 0.2ml Tube Flat Cap strips was inserted in a snap top lid of a micro-centrifuge tube. To run the assay, 35ul of test odorant was pipetted into inner well and 90ul of lure into the outer ring. For all trials, the control trap had paraffin oil solvent in inner well and lure in outer ring. Flies were given six hours to enter traps.

Preference index was calculated.Test odorant or solvent was added to warm standard grape juice media in Petri dishes and set to solidify. Petri dishes were placed at opposite ends of a 10 gallon closed glass chamber. A 100ml beaker containing 40ml of distilled water was placed equidistant between grape plates to add moisture to the chamber. For each trial, 15 male and 25 female un-starved Canton-S flies were lightly anesthetized with carbon dioxide and released in the chamber. Assay was run for 24hours at 25 degrees Celsius on a 12hour light: 12hour dark cycle. Preference was determined by counts of eggs on grape plate containing test odorant and control. Two-Choice Blueberry Assay in a glass chamber. Fresh blueberries were obtained from a local grocer and were soaked in distilled water for 30minutes, rinsed and dried. To prepare the chamber, 31 grams of blueberries were placed in each of 2 plastic bowls. Test compound is painted on blueberries in test bowl and solvent on blueberries in control bowl. Bowls are placed at opposite ends of a 10 gallon closed glass chamber. A 100ml beaker containing 40ml of distilled water was place equidistant between fruit to add moisture to the chamber. For each trial, 15 male and 15 female un-starved flies were lightly anesthetized with carbon dioxide and released in the chamber. Assay was run for seven days at 25 degrees Celsius on a 12hour light: 12hour dark cycle. After 7 days, each bowl was covered and set aside for an additional six days for eggs and larvae to develop after which the blueberries were dissected under microscope and number of eggs, larvae, pupae and adults were recorded. Preference was determined by inferring egg-laying from a count of eggs, larvae and pupae emerging from each set of fruit.Overweight and obesity put individuals at risk of major health problems including type 2 diabetes , nonalcoholic fatty liver disease and cardiovascular disease. Consumption of Western style diets can be a major contributing factor to the increased rates of overweight and obesity in human populations, while consumption of select fruits and vegetables could attenuate these conditions. Evidence for the latter is conflicting when considering overall intakes, types of fruits and vegetables consumed, and other variables associated with population studies.

On the other hand, a large body of evidence in experimental animals suggests a benefit of select phytochemicals present in fruits and vegetables in the development of obesity and associated pathologies triggered by consumption of high fructose and/or high fat diets. Among phytochemicals, anthocyanidins are flavonoids being actively investigated for their potential to mitigate unhealthy conditions, particularly metabolic disorders. In this regard, mounting evidence supports a potential beneficial action of AC consumption on T2D and cardiovascular health. Furthermore, AC-rich food consumption is inversely correlated with overall mortality. AC are flavonoids that exist in nature as anthocyanins, the glycosylated forms of AC. They provide color to grapes, berries, blueberries, black currants, bilberries, purple corn, and black rice, among other fruits and vegetables. With the basic three-ring structure of flavonoids, AC are characterized by double bonds in the three rings and a positive charge in the B ring on the oxygen atom. Different hydroxyl substitutions in number and position define different AC, e.g. delphinidins, malvidins, and peonidins. These differences in substitutions can have a major impact on AC biological actions in animals. In this regard, we recently observed that 3-O-glucosides of cyanidin and delphinidin were more efficient than malvidin, petunidin and peonidin 3-O-glucosides at inhibiting tumor necrosis factor alpha -induced activation of transcription factor NF-κB in Caco-2 cell. Dietary energy overload can cause tissue inflammation, oxidative stress, and insulin resistance. Excess fat consumption leads to the activation of inflammatory and redox-regulated events including: i) the IκB kinase , and downstream the transcription factor NF-κB; and ii) the mitogen activated kinase c-jun N-terminal kinase . Activation of both JNK and IKK and the increased expression of the NF-κB-regulated protein tyrosine phosphatase 1B phosphatase down regulate the insulin signaling pathway leading to insulin resistance. Inflammation, oxidative stress,container size for blueberries and chronic NF-κB activation also contribute to other major adverse consequences of obesity, e.g. NAFLD and cardiovascular disease. Identifying fruits and vegetables and their active components that can provide protection against the adverse effects of consuming Western style diets has the potential to have a major impact on human health. Moreover, understanding the mechanisms by which these components act modifying cell functions is crucial to define public recommendations in terms of diets and potential supplementation.

This work investigated the capacity of a diet enriched in the AC cyanidin and delphinidin to mitigate in mice the development of obesity, dyslipidemia, steatosis, and insulin resistance promoted by the chronic consumption of a HFD. The beneficial effects of AC were mainly associated with the attenuation of liver inflammation, oxidative stress, and down regulation of the redox sensitive JNK and IKK/NF-κB. These findings stress the concept that cyanidins and delphinidins can provide benefits against excess fat consumption and its adverse health consequences.Fecal triglyceride content was measured using a modified method to that proposed by Folch et al.. Fecal samples were collected over 24 h from single cages and dried at 37 °C for 24 h. Dried feces were ground to a fine powder using a mortar and pestle. The lipid extraction was performed by homogenizing the fecal powder with 500 ml of chloroform-methanol solution. Samples were mixed for 5 min and centrifuged at 1000×g for 10 min at room temperature and the lower liquid phase containing the extracted lipids in chloroform-methanol was collected and evaporated overnight. Analysis of triglyceride content was performed by saponification using a method described by Weber et al. with minor modifications. Briefly, the lipid residue was digested by incubation with 500 µl of a KOH :ethanol solution for 30 min at 60 °C. An aliquot was combined with 215 µl of 1 M MgCl2. After centrifugation for 15 min at 2000×g at room temperature, 2 µl of the supernatant were collected and analyzed for glycerol content using the enzymatic triglyceride kit TG Color GPO/PAP AA . Analysis of liver triglyceride content was performed after extraction and saponification, basically as previously described for feces. Briefly, a 100 µl aliquot of 10% liver homogenate was mixed with 300 µl of a KOH :ethanol solution and evaporated overnight at 55 °C. The following day, 1 ml of 50% ethanol was added and samples centrifuged for 5 min at 10,000×g at room temperature. Of the resulting supernatant, 200 µl were added with 215 µl of 1 M MgCl2 and placed on ice for 10 min. After centrifugation at 10,000×g for 5 min at room temperature, 10 µl of the supernatant were analyzed for triglyceride content as described above.The liver was removed and samples fixed overnight in 4% neutralized paraformaldehyde solution. Samples were subsequently washed twice in phosphate buffer saline solution, dehydrated, and then embedded in paraffin for histological analysis. Sections were obtained from paraffin blocks and placed on glass slides. Hematoxylin and eosin staining was performed following standard procedures. Sections were examined using an Olympus BX51 microscope . Hepatic histological examination was performed using the NAFLD activity score described by Kleiner et al.. Three randomly selected fields per animal were assessed and analyzed using Pro Plus 5.1 software .Daily food intake in the groups fed the HFD was significantly lower than in those fed the control and CA diets . However, the calorie intake was similar within groups

Weekly food intake did not significantly vary within groups . Starting at week 4 and through the following weeks, the body weight gain for C, CA, and HFA40 groups was significantly lower than for the HF group . At week 14, there was a dose-dependent decrease in body weight depending on the amount of AC in the diet . At the end of the study, consumption of the HFD caused a 31% higher body weight compared to controls, while the body weight of HFA40 mice was 14% lower than in HF mice. HFD-induced obesity was associated with an increased weight of the different fat pads . HFA40 mice showed a brown fat content similar to C mice, and 72% and 49% lower visceral and retroperitoneal fat accumulation, respectively, compared to HF mice. Brown fat weight in CA mice was 43% higher than in the C group. Consumption of the HFD also caused dyslipidemia. Plasma cholesterol and triglyceride levels were 26% and 9% higher in HF than in C mice, respectively . Supplementation of HFD-fed mice with AC prevented the increase of plasma triglyceride concentrations at all the AC concentrations tested, while plasma cholesterol increase was only prevented at the highest AC supplementation level, i.e. HFA40 group. While there were no significant differences in the amount of triglycerides excreted with the feces among the control, HF and CA groups , fecal triglyceride amount in the HFA40 group was significantly higher than in the control group . Fecal cholesterol content was significantly higher in the HF and HFA40 groups than in control and CA groups .We next investigated the levels of select hormones, which are relevant to the regulation of glucose homeostasis, and that are produced by the adipose tissue, i.e. adiponectin and leptin, and by gastrointestinal enteroendocrine cells, i.e. GIP and GLP-1. While plasma adiponectin levels were similar among groups, plasma leptin was 4 times higher in the HF compared to the C group . The increase in leptin observed in HF mice was partially prevented in HFA40 mice. Plasma GLP-1 concentration was significantly higher in HF, HFA40 and CA groups compared to the C group .Consumption of the HFD caused steatosis and liver inflammation. Liver triglyceride levels were 76% higher in the HFD-fed compared to the C group. This increase in liver triglycerides was not observed in the HFA40 group. . Lipid deposition was also assessed by histological analysis after hematoxylin-eosin staining .