Recipient Organization
FORT PECK COMMUNITY COLLEGE
P. O. BOX 398
POPLAR,MT 59255
Performing Department
Biological Sciences
Non Technical Summary
The prevalence of diabetes is increasing every year across the country and is twice as high in the Native American population.Diets are far from ideal and dietary changes are necessary to mitigate diabetes. Available positive choices include decreasing the glycemic load and index of staple foods such as potatoes which also contain flavonols that have anti-obesity and anti-diabetic effects. Low glycemic potatoes will be tested in a diet induced obesity model in rats to determine if these plants can lower blood glucose levels and the weight of the animals. Plant breeding by Dr. Sands and his research group (MSU) will provide several varieties of potatoes with low glycemic properties and will be grown to maturity at the Fort Peck Community College (FPCC). Various dietary tests will be done by Dr. Teske's research group ( University of Arizona) will determine if there is an effect on the obesity and diabetic state of test animals. Since the transport mechanisms are different in obese animals some tissue samples will be sent back to FPCC for western blotting (glucose transporters) to examinehow glucose in transported in the intestine.The microbiome are bacteria necessary in the digestive tract that aid in the digestion of nutrients and when the different species are not in the correct amounts are thought to contribute to obesity and diabetes.Microbiome samples will be analyzed andsince changes from normal to obese patients and in animal models of obesity are thought to be contributors to disease.These factors will detemine if the low glycemic potatoes can lower the effects of diabetes and obesity.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Our goals for the project are to examine different cultivars (new crosses between existing plants) of potatoes and determine if they are helpful in mitigating diabetes. How these plants store their starch can be an indication of how low glycemic they are. We will examine their starch granules and also feed these potatoes to obese diabetic rats and see if there is a change in their codition. We will also measure their insulin sensitivity and how they transport glucose which is different in obese/diabetic animals as well as in humans. Finally we will also allow members of the community to taste the potatoes and even learn how to grow them themselves.
Project Methods
Potato plants seedlings from 7 cultivars and a Russet Burbank (conventional potato, control) will be stored in a cool and dry area for 6-8 weeks (i.e. cured). The first step in the project will be to plant and harvest the potatoes for testing. Plants in the greenhouse will be grown in individual pots with Miracle Grow soil and grown at 22-28 degrees C with adequate water and humidity and light.The water absorption test will be used to measure the ratio of weights for the hydrated potato flour starch gel and the dry flour samples. The capacity to absorb water is calculated by dividing the flour plus water weight by the dry flour weight.Potato samples will befreeze dried and powdered in a coffee grinder to obtain potato flour.30-40 mg of potato flour will be added to 1.5 mL of distilled water. Once the maximum amount of water has been absorbed, the supernatant will be aspirated from the tubes, carefully avoiding the gelatinous layer, and the weight calculated. the amylose concentration in a food correlates inversely with the Glycemic Index and can be calculated by using the following equation:GI= -3.0187 x+169.19, where x corresponds to the amylose percentage of the starch measured We will also use the spectrophotometric method to quantify the amylose/amylopectin ratios.20mg of freeze-dried potato from each cultivar and the control will be suspended in 2ml of 80% ethanol, and mixed.The resulting washed starch (pellet) will be mixed with 5ml of water and 5ml of 1 M KOH and vortexed. Then 1 ml of this mixture will beneutralizedand adjusted to 50ml total volume with distilled waterEach sample will be placed in a 1cm spectrophotometer cuvette and spectra will be measured and compared versus a distilled water blank.The absorbance will be recorded from 480 to 800 nm, recorded, and analyzed. Two cultivars have the highest amylose and amylose/amylopectin ratios will be used for animal studies. We expect that the all the cultivars will have significantly higher microscopy scores, amylose, and amylose/amylopectin ratios compared to the Russet Burbank.We will first identify the most effective dose of the potato that reduces blood glucose during an oral glucose test prior to conducting a formal study. Then rats will be fed a high fat diet for 14-days, which will increase weight and elevate blood glucose. Then rats will receive a daily dose of unextracted low glycemic potatoes (diced undried) by oral gavage as a supplement to their diet in a repeated measures design with 48h washout period between treatments. Sprague-Dawley rats (n = 40/sex) will be purchased and baseline endpoints for glucose homeostasis will be determined. Then the rats will be randomized by glucose levels within each sex to receive either a customized control diet or a high diet with foods similar to the human dietto establish obesity and elevate blood glucose levels. Rats will consume their respective diets for a 14-d and baseline measurements will be repeated Then rats will be further randomized to receive a daily gavage of the potato supplement or saline (control) during a 14-d treatment period to create 4 treatment groups per sex. Body weight and calorie intakewill be monitored. The morning after the final day of the treatment period, rats will be euthanized, the proximal intestine and colon will be dissected, and prepared for biotinylation, GLUT2 protein on the brush border by Western blot analysis and microbiome analysis. There are a number of tests we perform with the blood samples. The primary test will bea glucose tolerance test but we will also test for GIP and insulin levels in the blood as well. The glucose tolerance test determines how well animals respond to a glucose bolus.Then animals will receive an injection of glucose (2mg/g body weight, IP). Blood glucose levels will be measured at baseline (before the injection) and 15, 30, 60, 90, and 120 minutes after the injection.GIP and insulin levels will also be measured from the animal blood draws with ELISA assay kits.A biotinylation kit will be used to isolate proteins. A 3cm segment of intestine (jejunum) will be isolated, cleaned and secured with a dialysis clips after solutions have been placed into the tissue. After incubation , the biotin solution will bind to all the proteins and a stop solution from the kit will be used to the stop the reaction. Then the tissue will be opened and surface cells will be removed with microscope slides. The column from the kit will be used to isolate and elute the proteins that have been biotinylated. The sample containing all of the surface proteins will be measured by Western blot at FPCC . Western blotting will be performed using the primary rabbit membrane fractions will be subjected to SDS-PAGE and immunoblotting will be performed following incubation with the primary antiserausing PVDF membranescontaining the bound proteins. The membranes will be washed (3 times, 5 min)incubated with the secondary antiserum, horseradish peroxidase-conjugated goat anti-rabbit antiserum, washed again with wash buffer, soaked in chemiluminescent reagent,and photographed in a documentation system. Densitometry will then be performed to measure the brightness of the bands so they can be compared between the different animal groups.After euthanasia, microbiome samples will be taken from the proximal jejunum and the proximal colon. The microbiome in these samples will be characterized using 16S rRNA based metagenomic sequencing and an Illumina MiSeq sequencing protocol. The V3-V4 region of the 16S rRNA genes will be amplified by PCR along with a positive control, and a no template negative control (sterile dH2O).In the formal animal study, we expect saline-treated animals to have elevated insulin, GIP and GLUT2 as well as a significantly different microbiome compared to the potato supplemented animals. The Western blots will indicate a gradual if not a total disappearance of the GLUT2 glucose transporter expression on the apical membrane . We expect lower glucose levels, body weight gain, insulin, and GLUT 2in animals treated with the potato supplements and that supplementation will normalize the gut microbiota . We also expect that the ratio of Bacteroidetes to Firmicute phyla to increase as the potato supplements are added to restore the normal balance of bacteria in the gastrointestinal tract. If the potato supplements at least partially restore normal weight, the normal microbiota would also be expected to be at least partially restored as well.Our interest is in changes in the intestinal tract and the populations and shifts in the numbers of bacteria that may be different. A conventional potato (Russet Burbank) and the potato with the highest amylose/amylopectin ratio will be compared against each other in a sequential monadic presentation, which is the typical method used for product testing. The FPCC students will host an event in the community to test foods made with a conventional potato (i.e. control) and the potato with the highest amylose/amylopectin ratio. FPCC students will be instructed to sample both potatoes and rate each by completing a survey of questions after sampling each potato. Students will rate the overall taste, based on the flavor, texture, aroma, appearance and sensory characteristics (sweetness, flavor intensity, aftertaste intensity) on labeled affective magnitude (LAM) scales withlabels illustrating the absolute ratings of like/dislike, intensity or sweetness.After sampling both potatoes, respondents will answer a series of demographic questions and be able to answer open-ended questions about general likes and dislikes if they choose.