Maternal high-fat diet regulates glucose metabolism and pancreatic β cell phenotype in mouse offspring at weaning

Study approval

All experimental procedures were performed in accordance with the recommendations found in the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Peking University First Hospital (No. J201827, Beijing, China).

Animals and diets

Five-week-old C57BL/6 female mice (n = 20) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The male mice were obtained from the same facility as the female mice. All mice were maintained under controlled conditions (22 ± 2 °C; 12-h light/dark cycle) and acclimatized for one week. Then, the female mice were randomly assigned to a HFD or chow diet group for three weeks before breeding. The HFD derived 60% kcal from fat, 20% kcal from carbohydrate, and 20% kcal from protein, and the energy density was 5.24 kcal/g (D12492, Research Diets, New Brunswick). Female mice were bred with male mice. To control for potential differences in the sires, breeding was performed in harems (male: female = 1:2). Pregnancy was confirmed by the presence of a vaginal plug in the morning, which was considered to represent day 0.5 of pregnancy. After breeding, females were individually housed, and their assigned diet was continued throughout gestation and lactation. At postnatal day 1, all litters were culled to 6 pups to control for litter size effects on metabolic parameters.

One male offspring in each group was randomly selected from each litter and used for experimental studies (n = 6–8 litters in each group). At three weeks of age, mice were anesthetized by an i.p. injection of sodium pentobarbital (50 mg/kg). Blood samples were collected by piercing the right auricle. The mice were then euthanized, and their pancreas was dissected. Any surviving animals were euthanized by cervical dislocation. The female offspring were not examined in our study to prevent the influence of confounding factors related to their hormone profile and estrous cycle (Kleinert et al., 2018). In addition, our previous study showed evidence of programming effects that occurred in a sexually dimorphic manner, which was not the focus of our study (Zheng et al., 2014). Body weight was monitored weekly from birth to weaning, and food consumption by dams was measured weekly and corrected for spillage.

Glucose tolerance test

Intraperitoneal glucose tolerance tests (IPGTTs) were performed as described previously (Zheng et al., 2016). IPGTTs were performed in dams when the offspring mice were weaned. Mice were fasted for 12 h, and the baseline blood glucose (time 0) was measured in tail vein blood samples using a glucometer and glucose test strips (Contour TS, Bayer, Beijing, China). Then, mice received an intraperitoneal injection of glucose (2 mg dextrose/g body weight), and blood glucose was measured at 15, 30, 60 and 120 min following injection. The blood glucose response to the IPGTT was calculated as the area under the curve (AUC) for each mouse.

Insulin tolerance test

An intraperitoneal insulin tolerance test (ITT) was performed in animals fasted for 4 h. ITTs were performed in dams when the offspring mice were weaned. The baseline blood glucose (time 0) was measured as described above, and animals received an intraperitoneal injection of 1 U/kg body weight insulin (Humulin R, Lilly, USA). Subsequently, blood glucose levels were measured at 5, 10, 15, 30, 45 and 60 min following injection. The blood glucose response to the ITT was calculated as the AUC for each mouse.

Biochemical analyses

At 3 weeks of age, mice were intraperitoneally injected with BrdU (100 mg/kg body weight) 5 h before the animals were euthanized. After centrifugation at 3,000 rpm at 4 °C for 15 min, the serum samples were separated immediately and stored at −80 °C until further analysis. Serum insulin concentrations were measured using the Mouse Ultrasensitive Insulin ELISA kit (80-INSMSU-E01, ALPCO Diagnostics, Salem, NH, USA). All samples were measured in duplicate.

Histological analysis

The pancreas tissues were rapidly dissected, weighed, and fixed in Z-fix (Anatech Ltd. MI, USA) overnight, followed by immersion in phosphate-buffered saline (PBS). The tissue samples were dehydrated in ethanol, embedded in paraffin wax, and subsequently fixed, and serial (5 µm) paraffin-embedded tissue sections were mounted on slides for staining. The slides were stained with hematoxylin and eosin (HE). The islet size was calculated as the average of the longest and shortest diameters of each islet by ImageJ software (ImageJ, CA, USA).

Immunofluorescence staining

Adjacent slides were deparaffinized with xylene, hydrated with an ethanol gradient and rinsed with PBS. Antigen was retrieved in citric acid buffer (pH 6.0). Sections were blocked with 5% donkey serum (Sigma-Aldrich, St Louis, MO, USA) for 1 h at room temperature. Sections were incubated overnight (4 °C) with anti-insulin (1:400; Abcam, MA, USA) and anti-Ki67 (1:50; BD Pharmingen™, USA) or anti-BrdU (1:50; Dako, Glostrup, Denmark). Sections were then incubated with Alexa Fluor 594-conjugated anti-guinea pig (1:400; Jackson ImmunoResearch Laboratories, Inc.) and Alexa Fluor 488-conjugated anti-mouse (1:400; Jackson ImmunoResearch Laboratories, Inc.) fluorescent secondary antibodies for 1 h at room temperature. Nuclei were stained with 4′,6 diamino-2-phenylindole (DAPI: 1:1000, Sigma-Aldrich, St Louis, MO, USA) for 5 min at room temperature. Finally, the tissues were mounted with fluorescent mounting medium (Dako, Glostrup, Denmark). β cell apoptosis was detected by TUNEL assays using the ApopTag^® Fluorescein In Situ Apoptosis Detection Kit (Millipore, MA, USA) according to the manufacturer’s protocol. Cell counting was manually performed in a blinded fashion. BrdU (+) or Ki67 (+) β cells were assessed by immunofluorescence microscopy. Insulin (+) cells showing nuclear DAPI staining were considered β cells. Insulin (+) cells showing colocalized nuclear staining for DAPI (+) and Ki67 (+) or BrdU (+) were considered proliferating β cells. Insulin (+) cells showing colocalized nuclear staining for DAPI (+) and TUNEL (+) were considered apoptotic β cells. The double-positive staining of insulin (+)/BrdU ( +), insulin (+)/Ki67 (+) and insulin (+)/TUNEL (+) were confirmed in randomly selected cells in all experiments.

Quantification of β cell mass, proliferation and apoptosis

Photographed sections were analyzed with imaging software (ImageJ, CA, USA) to measure insulin-positive areas and to determine the number of nuclei in the insulin-positive areas. The β cell mass (mg) was calculated by multiplying the relative insulin (+) area (the percentage of the insulin-positive area over the total pancreas area) by the pancreas weight (mg). The total β cell, Ki-67 (+) β cell, BrdU (+) β cell and TUNEL (+) β cell numbers were determined from the β cell counts in each section after correction for cut nuclei. β cell proliferation was calculated as the percentage (%) of Ki-67 (+) and BrdU (+) β cells over the total number of β cells per section. β cell apoptosis was calculated as the percentage (%) of TUNEL (+) β cells over the total number of β cells per section.

Statistical analysis

Data are displayed as the mean ± standard error of the mean (SEM). GraphPad Prism software (La Jolla, CA) was utilized to analyze significant differences using Student’s t-test or analysis of variance (ANOVA), where appropriate. Statistical significance was defined as P < 0.05.

Effects of HFD on the metabolic profile in dams

There was no difference in body weight gain or pregestational body weight before breeding between HFD- and chow diet-fed dams (Figs. 1A–1B). There was no significant difference in food intake between dams fed a HFD and those fed a chow diet (Fig. 1C). To eliminate the stress of pregnancy and delivery, we did not perform glucose tolerance tests during pregnancy. IPGTT and ITT were performed in dams when offspring mice were weaned, and they revealed impaired glucose tolerance in HFD dams, with an increased AUC (Figs. 1D–1E). However, blood glucose levels indicating insulin tolerance were indistinguishable between groups (Figs. 1F–1G).

Impaired glucose metabolism in male offspring from HFD-fed dams at weaning

There was no significant difference in the birth weight of offspring between dams fed a HFD and those fed a chow diet. At weaning, male offspring of HFD-fed dams exhibited significantly higher body weight compared with the offspring of dams fed a chow diet (P < 0.05, Fig. 2A). Fasting blood glucose (FBG) (P < 0.001, Figs. 2B–2C) and insulin concentration (P < 0.05, Figs. 2B–2C) were significantly increased in male offspring of HFD-fed dams at weaning. Consistently, the blood glucose levels of the male offspring in the HFD group were increased at 15 min and 30 min after intraperitoneal glucose administration (P < 0.001, Fig. 2D), and these male offspring also had an increased AUC (P < 0.01, Fig. 2E). Furthermore, the insulin tolerance test showed higher blood glucose levels at 0 min, 5 min, 10 min and 15 min in male offspring of HFD-fed dams compared with those in male offspring in the chow group (P < 0.001, Fig. 2F), indicating decreased insulin sensitivity (P < 0.01, Fig. 2G).

Increased islet size and density in male offspring from HFD-fed dams

As shown in Fig. 3A, the representative images indicate that the male offspring from HFD-fed dams had larger islets according HE staining than the male offspring in the chow group. Increased islet sizes were found in male offspring from HFD-fed dams at weaning (P < 0.05, Fig. 3B). There was no difference in the total islet area (% pancreas areas) in male offspring at weaning between the two groups (Fig. 3C). However, we found that islet density was significantly increased in male offspring from HFD-fed dams at weaning age compared with that in male offspring in the control group (P < 0.05, Fig. 3D).

Increased β cell mass in male offspring from HFD-fed dams

Next, we aimed to determine whether a maternal HFD could regulate the β cell mass in offspring at weaning. As shown in Fig. 4A, the red area was insulin-positive and was increased in male offspring from HFD-fed dams. We found that the β cell mass was significantly increased in offspring from HFD-fed dams (P < 0.05, Fig. 4B).

Effect of a maternal HFD on β cell proliferation and apoptosis in male offspring at weaning age

Figure 5 shows representative images of proliferating cells in pancreatic sections immunostained with insulin/Ki67/DAPI and insulin/BrdU/DAPI as denoted. St least 5,000 insulin (+) cells were counted for each mouse for the calculation. The immunofluorescence costaining of Ki67 and insulin showed increased β cell proliferation in male offspring from HFD-fed dams than that in chow-fed dam offspring (P < 0.05, Fig. 6A). To confirm the difference in β cell proliferation, we used another proliferation marker, BrdU, to determine the β cell proliferation rate. BrdU can be incorporated into the newly synthesized DNA of replicating cells (during the S phase of the cell cycle, in which DNA is replicated), thus indicating cells that are actively replicating their DNA. Consistent with the results of Ki67 staining, an increased percentage of insulin (+)/BrdU (+)/DAPI (+) β cells was observed in male offspring from HFD-fed dams (P < 0.05, Fig. 6B). Because β cell apoptosis can also affect the β cell mass, we further measured the β cell apoptosis rate and found no differences in the β cell apoptosis rate in the 3-week-old male offspring (P < 0.01, Fig. 6C).