Colonic epithelial hypoxia remains constant during the progression of diabetes in male UC Davis Type-2 Diabetes Mellitus Rats

Authors: PICCOLO, BRIAN - Arkansas Children'S Nutrition Research Center (ACNC); GRAHAM, JAMES - Arkansas Children'S Nutrition Research Center (ACNC); TABOR-SIMECKA, LESLIE - Arkansas Children'S Nutrition Research Center (ACNC); RANDOLPH, CHRISTOPHER - Arkansas Children'S Nutrition Research Center (ACNC); MOODY, BECKY - Arkansas Children'S Nutrition Research Center (ACNC); ROBESON, MICHAEL - Arkansas Children'S Nutrition Research Center (ACNC); KANG, PING - Arkansas Children'S Nutrition Research Center (ACNC); FOX, RENEE - Arkansas Children'S Nutrition Research Center (ACNC); LAN, RENNY - Arkansas Children'S Nutrition Research Center (ACNC); PACK, LINDSAY - Arkansas Children'S Nutrition Research Center (ACNC); WORFORD, NOAH - Arkansas Children'S Nutrition Research Center (ACNC); Yeruva, Laxmi; LEROITH, TANYA - Arkansas Children'S Nutrition Research Center (ACNC); STANHOPE, KIMBER - Arkansas Children'S Nutrition Research Center (ACNC); HAVEL, PETER - Arkansas Children'S Nutrition Research Center (ACNC)

Submitted to: BMJ Open Diabetes Research & Care
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/12/2024
Publication Date: 3/7/2024
Citation: Piccolo, B., Graham, J.L., Tabor-Simecka, L., Randolph, C.E., Moody, B., Robeson, M.S., Kang, P., Fox, R., Lan, R., Pack, L., Worford, N., Yeruva, V., Leroith, T., Stanhope, K., Havel, P.J. 2024. Colonic epithelial hypoxia remains constant during the progression of diabetes in male UC Davis Type-2 Diabetes Mellitus Rats. BMJ Open Diabetes Research & Care. 12(2). https://doi.org/10.1136/bmjdrc-2023-003813.
DOI: https://doi.org/10.1136/bmjdrc-2023-003813

Interpretive Summary: Diabetes has been shown to change the population of bacteria that reside in the gut, and some have suggested that this change can worsen host glucose regulation. We have previously corroborated this observation using a rat model that spontaneously develops diabetes, however, the exact biological mechanism that underlies the diabetes associated change in gut bacteria populations is not known. Recent evidence has suggested that the changes in the metabolism of the gut epithelium cell layer can influence oxygen levels in the gut lumen where bacteria reside. A normal and healthy gut lumen, in which bacteria reside, has minimal oxygen; therefore, we want to determine whether the progression of diabetes in a rat model that spontaneously develops diabetes alters oxygen levels and metabolism in cells that make up their large intestine. We hypothesize that the progression of diabetes will result in higher oxygen levels in the large intestine of a rat model that has a genetic susceptibility to diabetes. Furthermore, we hypothesize that the increase in oxygen levels correlate to higher levels of bacteria known to consume oxygen. Similar to previous studies, diabetic rats in this study had different population of bacteria in their gut compared to rats that had not yet developed diabetes. Bacteria that were known to thrive in oxygen replete states (Escherichia-Shigella, and Streptococcus ) were increased in fully diabetic rats compared to rats that had not developed diabetes. However, measurements of gut tissue oxygen levels did not show any difference between diabetic and non-diabetic animals. The bacterial metabolite, butyrate, which is the main energy source of the large intestine, was greater in diabetic rats relative to non-diabetic rats. Changes in gut bacteria in this rodent model of diabetes replicated previous work in this model, suggesting that this model provided consistent results related to diabetes associated changes in gut bacteria. However, we did not find that the progression of diabetes affects gut tissue oxygen level in this model. Therefore, the biological mechanism that explains diabetes associated changes in gut bacterial populations remains to be identified

Technical Abstract: Type 2 diabetes is known to disrupt the composition of the gut microbiota; however, the exact mechanism that underlies this association has not been characterized. Colonocyte metabolism and its effect on luminal oxygen availability has recently been proposed as a mechanism by which the host can regulate commensal populations. Thus, our aim was to determine whether the progression of diabetes influences colonocyte oxygen levels in the UC Davis Type 2 Diabetes Mellitus (UCD-T2DM) Rat, which spontaneously develops diabetes while consuming a standard low-fat/low sugar rodent chow diet. Age-matched male UCD-T2M Rats (173.8 ± 3.6 days) prior to the onset of diabetes (PD, n=15), within 1-month post onset (RD, n=12), and 3-month post onset (D3M, n=12) were included in this study. Rats were given an IP injection of pimonidazole (60 mg/kg body weight) 1-hour prior to euthanization to estimate colonic oxygen levels. Colon tissue was fixed in 10% formalin, embedded in paraffin, and processed for immunohistochemical detection of pimonidazole. The colonic microbiome was assessed by 16S rRNA amplicon sequencing and content of short chain fatty acids were measured by liquid chromatography-mass spectrometry. HbA1c % increased linearly across the PD (5.9 ± 0.1), RD (7.6 ± 0.4), and D3M (11.5 ± 0.6) groups, confirming the progression of diabetes in this cohort. D3M rats had at least a 2.5% increase in sequencing reads assigned to known facultative anaerobes, Escherichia-Shigella, and Streptococcus (FDR <0.05) genera in colon contents. The intensity of pimonidazole staining of hypoxic cells did not differ across groups (P=0.37). Colon content concentrations of acetate and propionate also did not differ across UCD-T2DM groups; however, colon butyric acid levels were higher in D3M rats relative to PD rats (P <0.01). The advancement of diabetes in UCD-T2DM rats was associated with an increase in facultative anaerobes, however, this was not explained by changes in colonocyte oxygen levels. The mechanisms underlying shifts in gut microbe populations associated with the progression of diabetes in the UCD-T2DM Rat Model remain to be identified.