Protocol for genome-scale in vivo CRISPR screening to study protection of beta cells under autoimmunity in a type 1 diabetes mouse model.

Autoimmunity-induced pancreatic beta cell failure is the main characteristic of type 1 diabetes (T1D). Here, we describe a protocol for genome-scale in vivo CRISPR-Cas9 screening for use in a mouse model of T1D. Using a non-obese-diabetic-derived mouse beta cell line, NIT-1, and a genome-wide CRISPR-Cas9 knockout library (GeCKO-v2), we describe how to identify genes that confer resistance to autoimmune killing. This protocol can be applied in other mouse models of autoimmunity. For complete details on the use and execution of this protocol, please refer to Cai et al. (2020).1.

Exposure to intrauterine diabetes and post-natal high-fat diet: Effects on the endocrine pancreas of adult rat female pups.

AIMS: To evaluate the morphological changes in the pancreatic islet cells of adult female pups born to diabetic rats and fed a high-fat diet. MAIN METHODS: Female Sprague-Dawley rats were distributed into four experimental groups (n = 10 animals/group): 1) female pups from non-diabetic dams and fed a standard diet (OC/SD), 2) female pups from non-diabetic dams and fed a high-fat (OC/HFD), 3) female pups from diabetic dams and fed a standard diet (OD/SD) and 4) female pups from diabetic dams and fed a high-fat diet (OD/HFD). In adulthood, the rats were submitted to the oral glucose tolerance test and later euthanized to collect the pancreas for the analysis of pancreatic islets. KEY FINDINGS: The OC/HFD and OD/SD groups showed an increased percentage of cells immunostained for insulin and a decreased percentage and intensity of staining for somatostatin. The OD/HFD group showed an increased percentage of cells immunostained for insulin and glucagon and a higher staining intensity for glucagon. There was a progressive increase in blood glucose in the OC/HFD, OD/SD, and OD/HFD groups. SIGNIFICANCE: The association between maternal diabetes and/or the administration of high-fat diet-induced changes in the pancreatic hormonal triad of female pups in adulthood. In turn, these changes in the pancreatic islets are not capable of causing decreased blood glucose in the offspring, contributing to the development of glucose intolerance in adulthood.

Exposure to maternal hyperglycemia and high-fat diet consumption after weaning in rats: repercussions on periovarian adipose tissue.

Clinical and epidemiological studies show that maternal hyperglycemia can change the programming of offspring leading to transgenerational effects. These changes may be related to environmental factors, such as high-fat diet (HFD) consumption, and contribute to the comorbidity onset at the adulthood of the offspring. The objective of this study was to evaluate the hyperglycemic intrauterine environment, associated or not with an HFD administered from weaning to adult life on the periovarian adipose tissue of rat offspring Maternal diabetes was chemically induced by Streptozotocin. Female offsprings were randomly distributed into four experimental groups (n = 5 animals/group): Female offspring from control or diabetic mothers and fed an HFD or standard diet. HFD was prepared with lard enrichment and given from weaning to adulthood. On day 120 of life, the rats were anesthetized and sacrificed to obtain adipose tissue samples. Then, the hyperglycemic intrauterine environment and HFD fed after weaning caused a higher body weight, total fat, and periovarian fat in adult offspring, which could compromise the future reproductive function of these females. These rats showed higher adiposity index and adipocyte area, contributing to hypertrophied adipose tissue. Therefore, maternal diabetes itself causes intergenerational changes and, in association with the HFD consumption after weaning, exacerbated the changes in the adipose tissue of adult female offspring.

Influence of Swimming Program on the Blood Pressure of Pregnant Hypertensive Rats and Their Fetuses.

The hypertension incidence and its complication on pregnant women are growing and can lead to adverse consequences on their fetuses. However, it is known that regular exercise practice can be healthful to hypertensive pregnant women but harmful to fetal growth. So, the objective of this study was to evaluate the effects of exercise beginning before pregnancy or during pregnancy on the maternal blood pressure and reproductive outcome and on the fetal development of spontaneously hypertensive rats (SHR). Pregnant SHR were randomly distributed into three experimental groups: (1) SHR-Control, non-exercised; (2) SHR-Ex0, rats submitted to physical exercise (swimming program) from day zero to 20 of pregnancy; (3) and SHR-ExPr, rats submitted to swimming program before and during pregnancy. At end of pregnancy (day 21), the rats were anesthetized, and reproductive parameters and fetal development were assessed. Blood pressure was reduced at the end of pregnancy in all the groups. Regardless of swimming exposure time, there was reduced maternal weight gain. The exercise decreased fetal weight at term pregnancy, with a higher percentage of small for gestational age (SGA) fetuses and lower number ossification sites, indicating intrauterine growth restriction (IUGR). In conclusion, our findings provide insight to support that swimming exercise in pregnant SHR impairs fetal development, causing IUGR and visceral malformations. Therefore, the indication of physical exercise must be defined very carefully, as it can compromise fetal development.

Swimming Program on Mildly Diabetic Rats in Pregnancy.

The present study aims to confirm if the moderate-intensity swimming has successful glycemic control and non-toxic oxidative stress levels and to verify the influence on pancreatic adaptations, embryo implantation, and placental efficiency. Female Wistar rats were randomly distributed to obtain mildly diabetic by streptozotocin induction at birth and the non-diabetic females given vehicle. At adulthood, pregnant rats were put at random into sedentary non-diabetic rats (ND); exercise non-diabetic rats (NDEx); sedentary diabetic rats (D); and exercise diabetic rats (DEx). The rats of the groups submitted to moderate intensity carried loads equivalent to 4% of body weight. On day 17 of gestational day, all rats were submitted to oral glucose tolerance test (OGTT). Next day (GD18), the rats were anesthetized and killed to count implantation sites and to collect placentas, blood, and muscle samples for biochemical biomarkers and pancreas for immunohistochemical analysis. The moderate exercise used was not sufficient to stimulate the aerobic pathway but presented positive results on glucose metabolism, lower embryo postimplantation loss, and pancreatic morphology compared with the sedentary diabetic group. However, the DEx group showed muscular damage, decreased antioxidant defense, and lipid peroxidation. Thus, the moderate-intensity exercise reduces glycemic levels during OGTT and causes no damage to non-diabetic rats related to other analyzed parameters in this study. The exercised diabetic rats present better glycemic metabolism in OGTT, islet pancreatic morphology, and embryofetal development. However, it is necessary an adjustment in this exercise intensity to improve the effectiveness of aerobic training for reduction of maternal muscular and lipid membrane damages.

Evaluation of neonatally-induced mild diabetes in rats: Maternal and fetal repercussions.

Many experimental studies have been performed to evaluate mild diabetes effects. However, results are divergent regarding glycemia and insulin measurement, fetal macrossomia, and placental weights. The aim was to investigate repercussions of neonatally-induced mild diabetes on the maternal organism and presence of congenital defects in their offspring in other mild diabetes model. On the day of birth, female offspring were distributed into two groups: Group streptozotocin (STZ): received 100 mg STZ/kg body weight, and Control Group: received vehicle in a similar time period. Maternal weights and glycemias were determined at days 0, 7, 14 and 21 of pregnancy. At day 21 of pregnancy, the rats were anesthetized and a laparotomy was performed to weigh and analyze living fetuses and placentas. The fetuses were classified as small (SPA), appropriate (APA) and large (LPA) for pregnancy age. Fetuses were also analyzed for the presence of external anomalies and processed for skeletal anomaly and ossification sites analysis. Statistical significance was considered as p < 0.05. In STZ group, there was increased glycemia at 0 and 14 days of pregnancy, lower weights throughout pregnancy, higher placental weight and index, an increased proportion of fetuses classified as SPA and LPA, and their fetuses presented with an increased frequency of abnormal sternebra, and absent cervical nuclei, which were not enough to cause the emergence of skeletal anomalies. Thus, this study shows that mild diabetes altered fetal development, characterized by intrauterine growth restriction. Further, the reached glycemia does not lead to any major congenital defects in the fetuses of streptozotocin-induced mild diabetic rats.