Reduction in Pancreatic β Cell Area is Associated with Increased Islet Macrophage Message in Female Rat Offspring following Chronic Placental Ischemia

Abstract

Placental insufficiency in a well‐nourished population can result in intrauterine growth restriction (IUGR) contributing to pathophysiology of diseases with origins early in life, including Type 2 diabetes (T2D). Certainly the risk for T2D is increased in low birth weight offspring and/or offspring of pregnancies complicated by gestational hypertension and low birth weight. Using the reduced uteroplacental perfusion pressure (RUPP) model of chronic placental ischemia in the rat, our previous studies have demonstrated β cell area is reduced and apoptosis increased in pancreatic islets of growth restricted fetus at gestation day (GD)19. Also, adult RUPP offspring have been shown to display glucose intolerance. Macrophages in pancreatic islets have been implicated as important for islet development, but whether macrophages are instrumental in T2D islet pathology and decreasing β cell area is unknown. The goal of the present study was to determine if reduction in β cell area in offspring of RUPP pregnancies persisted to postnatal day (PD)13 and was accompanied by changes in islet macrophages. On GD14, timed pregnant Sprague Dawley rats underwent either Sham surgery or RUPP surgery with clip placement on ovarian arteries and abdominal aorta to reduce uterine perfusion pressure, resulting in hypertension in the dam and IUGR. Rats were allowed to deliver pups spontaneously, and litters were culled to 8 offspring. On PD13, offspring were euthanized, and two pancreata of each sex were isolated, weighed, and fixed in neutral buffered formalin for determination of β cell area. For isolation of islets, two pancreata of each sex were inflated with collagenase via the hepatic duct. After dissection, the pancreata were further digested with collagenase at 37 C, and intact islets hand‐picked for culture overnight in media. Isolated islets were then added to RNA later for isolation of RNA and qRT PCR for the macrophage marker CD68. β cell area was determined using immunofluorescence by measuring the insulin positive area/total pancreas area in 3 sections/rat pancreas at least 100 microns apart. At PD13, β cell area was reduced in female RUPP offspring (0.012±0.001 ratio) compared to Sham (0.016±0.001 ratio) with N of 3–4 pups from at least 2 litters. Message for CD68 as an indicator of macrophages was significantly increased 2.6 ± 0.8 fold in female offspring of RUPP pregnancies compared to Sham using actin to normalize, with no significant change in CD68 in male offspring of RUPP vs Sham pregnancies. Evaluation of CD68 message in surrounding acinar tissue showed no significant change in RUPP vs Sham in either male or female offspring. Thus, our data suggest that the reduction in β cell area following chronic placental ischemia persists in female but not male offspring and is associated with increased islet macrophages. Continued studies will evaluate the potential role of macrophages in reducing β cell mass and contributing to islet cell pathology in development of T2D.Support or Funding InformationFunded in part by NIH NIDDK R03 DK114465 (EA) and American Heart Association Grant 17GRNT33650049 (JR)