Diabetes Volume 67, September 2018 1867 Restructuring of the Gut Microbiome by Intermittent Fasting Prevents Retinopathy and Prolongs Survival in db/db Mice Eleni Beli,1 Yuanqing Yan,2 Leni Moldovan,3 Cristiano P. Vieira,4 Ruli Gao,5 Yaqian Duan,6 Ram Prasad,4 Ashay Bhatwadekar,7 Fletcher A. White,8 Steven D. Townsend,9 Luisa Chan,10 Caitlin N. Ryan,10 Daniel Morton,10 Emil G. Moldovan,11 Fang-I Chu,12 Gavin Y. Oudit,13 Hartmut Derendorf,14 Luciano Adorini,15 Xiaoxin X. Wang,16 Carmella Evans-Molina,1,6,17 Raghavendra G. Mirmira,1,6,17 Michael E. Boulton,4 Mervin C. Yoder,1 Qiuhong Li,18 Moshe Levi,16 Julia V. Busik,19 and Maria B. Grant4 Diabetes 2018;67:1867–1879 | https://doi.org/10.2337/db18-0158 Intermittent fasting (IF) protects against the develop- (BA) metabolism, measurement of BAs demonstrated a ment of metabolic diseases and cancer, but whether significant increase of tauroursodeoxycholate (TUDCA), it can prevent diabetic microvascular complications is a neuroprotective BA, in db/db on IF but not in db/db on COMPLICATIONS not known. In db/db mice, we examined the impact of AL feeding. TGR5, the TUDCA receptor, was found in the long-term IF on diabetic retinopathy (DR). Despite no change retinal primary ganglion cells. Expression of TGR5 did not in glycated hemoglobin, db/db mice on the IF regimen change with IF or diabetes. However, IF reduced retinal displayed significantly longer survival and a reduction in DR TNF-a mRNA, which is a downstream target of TGR5 end points, including acellular capillaries and leukocyte in- activation. Pharmacological activation of TGR5 using filtration. We hypothesized that IF-mediated changes INT-767 prevented DR in a second diabetic mouse model. in the gut microbiota would produce beneficial metab- These findings support the concept that IF prevents DR olites and prevent the development of DR. Microbiome by restructuring the microbiota toward species produc- analysis revealed increased levels of Firmicutes and de- ing TUDCA and subsequent retinal protection by TGR5 creased Bacteroidetes and Verrucomicrobia. Compared activation. with db/db mice on ad libitum feeding, changes in the microbiome of the db/db mice on IF were associated with increases in gut mucin, goblet cell number, villi length, Diabetes-related blindness is a personal catastrophe for and reductions in plasma peptidoglycan. Consistent with the individual and affects more than 4.2 million Americans the known modulatory effects of Firmicutes on bile acid (1). Although diabetic retinopathy (DR) is commonly 1Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 14Department of Pharmaceutics, University of Florida, Gainesville, FL 2Department of Neurosurgery, The University of Texas Health Science Center at 15Intercept Pharmaceuticals, New York, NY Houston, Houston, TX 16Department of Biochemistry and Molecular & Cellular Biology, Georgetown 3Department of Surgery, Indiana University School of Medicine, Indianapolis, IN University, Washington, DC 4Department of Ophthalmology and Visual Sciences, University of Alabama, 17Department of Medicine, Indiana University School of Medicine, Indianapolis, IN Birmingham, AL 18Department of Ophthalmology, University of Florida, Gainesville, FL 5Department of Genetics, The University of Texas MD Anderson Cancer Center, 19Department of Physiology, Michigan State University, East Lansing, MI Houston, TX Corresponding author: Maria B. Grant, [email protected]. 6Department of Cellular and Integrative Physiology, Indiana University School of Received 7 February 2018 and accepted 18 April 2018. Medicine, Indianapolis, IN 7Department of Ophthalmology, Indiana University School of Medicine, Indian- This article contains Supplementary Data online at http://diabetes apolis, IN .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0158/-/DC1. 8Department of Anesthesia, Indiana University School of Medicine, Indianapolis, IN E.B. and Y.Y. contributed equally to the manuscript. 9Department of Chemistry, Vanderbilt University, Nashville, TN © 2018 by the American Diabetes Association. Readers may use this article as 10Second Genome, Inc., San Francisco, CA long as the work is properly cited, the use is educational and not for profit, and the 11Northeastern University, Boston, MA work is not altered. More information is available at http://www.diabetesjournals 12Department of Radiation Oncology, University of California, Los Angeles, Los .org/content/license. Angeles, CA 13Department of Medicine, University of Alberta, Edmonton, Alberta, Canada See accompanying article, p. 1745. 1868 Intermittent Fasting Affects Gut-Retina Axis Diabetes Volume 67, September 2018 classified as a microvascular complication of diabetes, it is mice were used as controls (denoted as db/db and db/m now recognized that changes in the neural cells of the hereafter). All mice were obtained from The Jackson retina occur early in the natural history of the disease and Laboratory (Bar Harbor, ME) and housed in the institu- that these changes precede alterations in the microvascula- tional animal care facilities at the University of Florida ture (2,3). Current therapies for DR, such as laser photoco- (IACUC #201106420) with strict 12 h:12 h light:dark agulation, injection of anti-VEGF antibodies, or vitreoretinal cycle; all studies were repeated at Indiana University (IACUC surgery (4), only treat late-stage disease and selectively #10604 and #11167). Blood glucose levels were tested target the microvascular component of the disease, whereas every 2 weeks in db/db mice. Animals were considered therapies for the early stages are limited to preventive strat- diabetic and used in the IF experiment if the serum glucose egies, such as lifestyle changes. level was above 250 mg/dL on two consecutive measure- Chronic calorie restriction and intermittent fasting (IF) ments. The animals were fed ad libitum (AL) before the IF represent nonpharmacological strategies for prevention was initiated at 4 months of age. The db/m and db/db mice and treatment of diseases, such as diabetes, obesity, and were each divided in two subgroups, AL feeding (control) aging. However, calorie restriction can lead to nutrient and IF, wherein the animals were fasted for 24 h every deficiencies and fatigue (5,6), low metabolic rate, and other day for 7 months, beginning at night (Fig. 1A). increased risk for subsequent obesity (7), infertility (5), Glycated hemoglobin was measured using the A1CNow+ suppression of immunity (8), and susceptibility to viral kit (Bayer HealthCare, Sunnyvale, CA) on the day prior to infections (9,10). IF prevents the adverse effects of calorie euthanasia. restriction as it restricts feeding to only a part of the day or to certain days per week. Thus, IF may be a more ben- INT-767 Treatment of Diabetic Animals eficial strategy compared with chronic calorie restriction as Eight-week-old male DBA/2J mice (stock number: 00671) it combines the beneficial effects elicited by fasting physiol- were obtained from The Jackson Laboratory and main- ogy and a recovery period during the feeding phase that tained on a 12 h:12 h light:dark cycle. Mice were injected facilitates regeneration and promotes tissue repair (11). with streptozotocin (STZ) (Sigma-Aldrich, St. Louis, MO) Accumulating evidence indicates that the gut micro- intraperitoneally (40 mg/kg made freshly in 50 mmol/L biota contribute to key physiological functions, such as sodium citrate buffer, pH 4.5 [vehicle]) for 5 consecutive energy metabolism, metabolic signaling, and regulation of days or with vehicle only. Blood glucose levels were mea- integrity of the gut barrier (12–14). Individuals with chronic sured 1 week after the last STZ injection, and mice with diseases such as diabetes exhibit altered bacterial composi- glucose levels .250 mg/dL on 2 separate days were tion (15), which likely not only influences glucose metab- considered diabetic. DBA/2J mice were fed with a Western olism but also, we reasoned, may contribute to development diet (WD) (21% milk fat, 0.2% cholesterol, TD88137) of complications. Gut microbes have a symbiotic relation- obtained from Harlan-Teklad (Madison, WI) after diabetes ship with the host: they digest exogenous, indigestible foods onset in the STZ groups and were treated for 8 weeks with generating secondary metabolites that can affect host phys- either WD or WD containing INT-767 (30 mg/kg bw/day) iology and metabolize endogenous substrates in the absence (Intercept Pharmaceuticals, New York, NY) (18). of food during prolonged fasting. Acellular Capillaries An example of endogenous metabolites being converted The eyes were fixed in 2% formalin, and trypsin digest was to beneficial secondary metabolites by the microbiome is performed for analysis of acellular capillaries as previously the generation of secondary bile acids (BAs). Primary BAs published (16). generated by the liver are highly hydrophobic and can cause colitis; however, intestinal bacteria convert them to Microbiome Analysis more hydrophilic, less toxic, secondary BAs that are potent Fecal samples (n = 180) were obtained by collection every agonists for nuclear receptors and have physiological func- 4 h for 48 h. Genomic DNA was isolated from ;0.1 g using db/db tions. Using the mouse and an IF (fasting every other the PowerSoil DNA Isolation Kit (MO Bio, Carlsbad, day) regimen for 7 months, we showed that IF prevented CA), according to manufacturer’s protocol. DNA was development of DR and increased generation of a second- sent to Second Genome, Inc. (San Francisco,