Bones, Megan

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Evaluation of enterolactone for the treatment and prevention of type-2 diabetesMegan Bones1        2       3       4        5       6        7       8        9      10      11     12      13      14 1. Centers for Disease Control and Prevention. Diabetes. Published online 2021.2. Khan MAB, Hashim MJ, King JK, Govender RD, Mustafa H, Kaabi JA.Epidemiology of Type 2 Diabetes - GlobalBurden of Disease and Forecasted Trends. J Epidemiol Glob Health. 2020;10(1):107-111.doi:10.2991/jegh.k.191028.0013. Stoffers D. The development of beta-cell mass: recent progress and potential role ofGLP-1. Horm Metab Res.2004;36(11-12):811-821. doi:10.2337/diacare.21.9.14144. Kolb H, Martin S, Kempf K. Coffee and Lower Risk of Type 2 Diabetes: Argumentsfor a Causal Relationship.Nutrients. 2021;13(4):1144. doi:10.3390/nu130411445. Zhou F, Furuhashi K, Jin Son M, Yoshizawa F, Miura Y, Yagasaki K. Antidiabeticeffect of enterolactone in culturedmuscle cells and in type 2 diabetic model db/db mice. Cytotechnology. 2017;69(3):493-502. doi:10.1007/s10616-016-9965-2I would like to thank Dr. Moore for mentoring me andsupporting me throughout this project, as well as my researchteam. I would like to thank Liberty University’s Department ofBiology and Chemistry for supporting this research.Type-2 diabetes (T2D) is an increasingly common publichealth concern that affects over 35 million Americans1 andover 462 million people worldwide2. T2D is characterized byelevated blood glucose levels caused by insulin resistance3.Despite its role in maintaining blood glucose levels, adiposetissue is often overlooked as a potential therapeutic target forT2D. Previous literature has indicated that moderate habitualcoffee consumption, both caffeinated and decaffeinated, isinversely associated with the risk of developing T2D4.Therefore, there is a current interest in this relationship,particularly the effects of small bioactive compounds found incoffee and their metabolites, and T2D. Several metabolites ofcompounds found in coffee have potential as anti-diabeticagents. One such compound is enterolactone (ENL), a gutmicrobial metabolite of matairesinol, which has also shownpromise as an anti-diabetic agent5. Previous literature hasshown that ENL promotes basal glucose uptake in L6myotubules, however its effect on adipose tissue is stillunknown5. Here, we show that low concentrations of ENLpromotes basal glucose disposal in differentiated 3T3-L1adipocytes.BackgroundHow does enterolactone effect glucose uptake in3T3-L1 adipocytes?Research Question3T3-L1 fibroblasts were grown in complete growth media(DMEM + 10% Bovine Calf Serum). Once grown toconfluency, a timeline of experiments was followed.Day 0: Ten thousand cells were seeded into each well of a 96-well plate.Day 1: The complete growth media was aspirated and 100 μLof differentiation media (complete growth media + 0.5 mMIBMX + 1 μM dexamethasone + 10 μg/mL insulin) was addedto each well.Day 4: The differentiation media was aspirated and 100 μL ofpost-differentiation media (complete growth media + 10μg/mL insulin) was added to each well.Day 7: The post-differentiation media was aspirated and 100μL of complete growth media was added to each well,supplemented with increasing concentrations of ENL (1 μM,10 μM, 20 μM, 50 μM, 100 μM) and 200 μg/mL of 2-NBDG,a fluorescently labeled glucose molecule.After treatment, the plate was incubated at 37 °C for 45minutes. The plate followed a centrifugation and washingprocedure. Then the fluorescence intensity was read, the datawas graphed (Fig. 1,3), and analyzed by a student t-test (Table1,3).Methods1. Repeat glucose uptake assays using 10 nM insulin ratherthan 100 nM insulin.2. Extend the dose-dependent range below 1 μM.3. Increase the time of ENL exposure up to 24 hours prior tomeasuring glucose uptake.4. Determine the effect of ENL on insulin-stimulated glucoseuptake.5. Characterize the mechanism of ENL-stimulated glucoseuptake.Future WorkReferences andAcknowledgmentsResults and ConclusionThough analysis of the data indicated that higherconcentrations of ENL have an inhibitory effect on glucoseuptake, (Fig. 1), we also found that a lower concentration, 1μM, significantly increased basal glucose uptake (Fig. 3).However, this data may only be described as a trend becauseof the negative and positive controls. Ideally, the negativecontrol should show the least amount of glucose uptake andthe positive control should be higher than the negativecontrol. This discrepancy may be attributed to the cell’sreceiving too much insulin (100 nM).The first set of t-test results indicate that there is nosignificant difference between the negative and positivecontrols (Table 1). The t-test results between the 20 μLnegative vehicle control and 1 μM ENL concentrationindicate that this relationship is statistically significant (Table2).CBA00.20.40.60.811.21.412-NBDG Uptake (fold induction)Negative Control1 μM10 μM20 μM50 μM100 μMPositive ControlNegative ControlPositive Control1 μM 0.5843 0.034410 μM 0.6830 0.041120 μM 0.0948 0.426150 μM 0.0718 0.3227100 μM 0.0302 0.0963Negative/Positive Control0.2329Figure 1. Normalized glucose uptake vs the different treatments given to 3T3-L1 adipocytes. Two trials of the assaywere run, with this graph showing the composite data of both trials. The negative control was a vehicle control of 100μL ethanol. The positive control was 10 nm of insulin. The ENL concentrations were 1 μM, 10 μM, 20 μM, 50 μM,and 100 μM.Table 1. T-test results the negative control, positivecontrol, and each ENL concentration from Figure 1.*00.20.40.60.811.21.41.61.82-NBDG Uptake (fold induction)Vehicle1 µMTable 2. T-test results the negative control and 1μM ENL concentration from Figure 2.Negative Control/ 1 μM ENL0.03176Figure 3. Normalized glucose uptake of the negative vehicle control and 1 μM ENL concentration. The negativevehicle control was 20 μL of ethanol.Figure 2. Chemical structure of enterolactone.

Evaluation of Enterolactone for the Treatment and Prevention of Type-2 Diabetes

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Evaluation of Enterolactone for the Treatment and Prevention of Type-2 Diabetes

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