Maternal sitagliptin treatment attenuates offspring glucose metabolism and intestinal proinflammatory cytokines IL-6 and TNF-α expression in male rats

Animal treatments and diets

Ethical approval for the study was granted by the Peking Union Medical Hospital Animal Ethics Committee (Project XHDW-2015-0051, 15 Feb 2015) and conformed to the NIH Animal Care guidelines (NIH publication No. 85-23, revised 1996). Rats were placed in a 12-h light/dark cycle and temperature (22 ± 1 °C) and humidity (65%–70%) controlled housing and were given food and water ad libitum. Eight-week-old female Sprague-Dawley rats (obtained from the Institute of Laboratory Animal Sciences of the Chinese Academy of Medical Sciences and Peking Union Medical College in Beijing, China, n = 32) were randomized into one of two groups for 4 weeks: normal diet (America Institute of Nutrition-93, AIN-93, kcal %: 10% fat, 20% protein, and 70% carbohydrate; 3.85 kcal/gm, ND, n = 16) or HFD (kcal %: 45% fat, 20% protein, and 35% carbohydrate; 4.73 kcal/gm, n = 16). Then, female rats were bred with male rats. After the identification of a copulation plug, dams were housed individually. To evaluate the effect of sitagliptin on mother rats with different diet and their offspring, we divided dams fed an ND into two subgroups: the ND only group and ND-sitagliptin group (n = 8 for each group). Meanwhile, dams fed a HFD were randomized to two subgroups: the HFD only group or the HFD-sitagliptin group (n = 8 for each group). The typical human daily dose of sitagliptin is 100 mg/60 kg body weight. Thus, according to the formula: d_rat =(37 × d_human)/6 (Nair & Jacob, 2016), the corresponding dose of sitagliptin for rats is 10.28 mg/kg per day. Therefore, HFD-sitagliptin group rats were fed a HFD supplemented orally with 10 mg/kg/day sitagliptin treatment (Merck, West Point, PA, USA) as described in a previous study (Mega et al., 2011; Samaha, Said & Salem, 2019; Wojcicka et al., 2019). Dams treated with this protocol through pregnancy and lactation. The size of every litter was culled to 6 pups (3 male and 3 female rats) to ensure that there was no nutritional bias between litters. On the day of birth, one male offspring and one female offspring from each litter was randomly selected for experimental study (8 male and 8 female in each group). At weaning, offspring from all dams (8 male and 8 female for each group) were sacrificed following intraperitoneal injection of pentobarbital sodium (150 mg/kg). The intestines were immediately stored at −80 °C. Other surviving rats were kept feeding for other study. Fig. 1 shows the animal experimental protocol.

Body weight, glucose tolerance, serum insulin and inflammatory cytokines assay

Dams were weighed on the day of confirmation of pregnancy and during the pregnancy. Pups were weighed on the day after birth and weaning. At weaning, pups underwent an oral glucose tolerance test (OGTT). Briefly, after overnight food deprivation, blood was sampled from the tip of the tail in rats before and 30, 60, and 120 min after oral glucose administration via gavage (2 g/kg). Blood glucose concentrations were determined by using a blood glucose meter (Contour TS glucometer, Bayer, Hamburg, Germany). The area under the glucose tolerance curve (AUC) of the OGTT was calculated as previously described (Zhang et al., 2018). At weaning, the pups were anesthetized after 10 h of fasting. Blood samples were collected from the intraorbital retrobulbar plexus. Serum insulin was measured using an ELISA kit (EZRMI-13K, Millipore, Billerica, MA, USA). Insulin sensitivity was assessed using HOMA-IR as previously described (Zhang et al., 2018). The levels of serum proinflammatory cytokines interleukin-6 (IL-6), TNF-α and anti-inflammatory cytokine IL-10 were measured using ELISA kits (RAB0311, Merck, Darmstadt, Germany, ab46070, Abcam, Cambridge, MA, USA and RAB0246 Merck, Darmstadt, Germany).

RNA isolation, microarray processing and analysis

Previous studies showed that the programming effects of maternal high fat diet occurred in a sexually dimorphic manner (Yokomizo et al., 2014; Zheng et al., 2014), possible due to the influence of confounding factors related to female hormone profile and estrous cycle (Kleinert et al., 2018). Thus, this study mainly focused on male offspring. Total RNA was isolated from the intestines of male pups in the HFD and HFD-sitagliptin groups by using TRIzol reagent (Life Technologies Inc., Carlsbad, CA, USA). Gene expression in the intestine was detected by an Affymetrix GeneChip Rat Gene 2.0 ST whole transcript-based array (Affymetrix Technologies, Santa Clara, CA). The differential gene criteria between the two groups was 1.50-fold or higher (p < 0.05). The data obtained have been deposited in the NCBI Gene Expression Omnibus (GEO) database (accession number GSE134070).

The Gene Ontology (GO) classification system and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to assign biological meaning to the group of different genes and pathway enrichment through Database for Annotation, Visualization, and integrated Discovery (DAVID) software. STRING software (Biobyte Solution, Heidelberg, Germany) was used to analyze the connections among differentially expressed genes.

Real-time PCR

To validate the gene array results, the expression of genes (Il1b, Il6, and tumor necrosis factor (Tnf)) was analyzed using real-time PCR. Total RNA from the four groups was reverse-transcribed by Superscript II (Life Technologies, Carlsbad, CA). The primers are shown in Table 1. Real-time PCR was performed with an ABI Prism 7500 Real-Time System (Applied Biosystems, Foster City, CA) using ABI SYBR Mix (Applied Biosystems, Foster City, CA). The mRNA levels of the target gene were corrected by glyceraldehyde 3-phosphate dehydrogenase (Gapdh) using the 2^−ΔΔCt method.

Immunohistochemistry for IL-6 and TNF-α in the intestine

Intestinal sections were fixed in 10% neutral buffered formalin, cast in paraffin, sliced into 4 µm sections and placed onto microscope slides. After deparaffinization, slides were immersed in PBS. Then, sections were stained with anti-IL-6 (sc-57315, 1:100, Santa Cruz Biotechnology, Dallas, TX) and anti-TNF-α (sc-52746, 1:100, Santa Cruz Biotechnology, Dallas, TX) at 4 °C overnight. Slides were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000, Santa Cruz Biotechnology, Dallas, TX) for 1 h at room temperature. Immunolabeling was visualized with 0.05% diaminobenzidine (DAB). A Nikon 80i microscope (Nikon) was used to capture the images, and Nikon Elements (Nikon) software was used for image processing. Three slides were analyzed for each rat, and eight rats were included in each group.

Statistical analysis

Data are shown as the mean ±SD. Statistical analyses were calculated with Student’s t-test for the difference between two groups and with one-way ANOVA followed by Tukey’s post hoc test for the difference among groups. GraphPad Prism 6 (GraphPad Software Inc., CA, USA) was used for data analysis. P < 0.05 was defined as significant.

The effect of sitagliptin intervention on food intake, energy intake, body weight and blood glucose in pregnancy and lactation on dams

Food intake from HFD and HFD-sitagliptin reduced compared with ND-sitagliptin (p < 0.01, Fig. 2A) during pregnancy. However, energy intake of HFD dams was higher than that of the ND and ND-sitagliptin dams (p < 0.01, Fig. 2B). Accordingly, HFD dams gained significantly more weight than ND and ND-sitagliptin dams during pregnancy (p < 0.01, Fig. 2C). Sitagliptin intervention reduced dam food intake, energy intake and weight gain of HFD dams during pregnancy (p < 0.05or0.01, Figs. 2A, 2B, 2C). HFD dams had higher fasting blood glucose than ND and ND-sitagliptin dams at weaning time (p < 0.01, Fig. 2D); however, sitagliptin intervention reduced fasting blood glucose of HFD dams at weaning (p < 0.01, Fig. 2D).

The effect of maternal sitagliptin intervention on metabolism in offspring

No difference in birth weight was observed among pups from ND, ND-sitagliptin, HFD, and HFD-sitagliptin dams (p > 0.05, Table 2). Body weight at 3 weeks of age was higher in both of male and female offspring from HFD dams than those from ND and ND-sitagliptin dams (p < 0.05 or 0.01, Table 2). Notably, maternal sitagliptin intervention reduced offspring body weight at 3 weeks of age (p < 0.01, Table 2). Only male pups from HFD dams had higher fasting blood glucose, blood glucose after oral glucose load, and area under the curve (AUC) of blood glucose than those from ND and ND-sitagliptin dams (p < 0.01, Table 2). Maternal sitagliptin intervention reduced fasting blood glucose, blood glucose after oral glucose load and AUC of blood glucose in male offspring from HFD dams (p < 0.01, Table 2). Additionally, the serum fasting insulin concentration and HOMA-IR index in pups from HFD dams were higher than those in male pups from ND and ND-sitagliptin dams (p < 0.01, Table 2), and maternal sitagliptin intervention reduced serum fasting insulin levels and the HOMA-IR index (p < 0.01, Table 2). There was no significant difference in fasting blood glucose, AUC of blood glucose, serum insulin and HOMA-IR among ND, ND-sitagliptin and HFD groups in female offspring (p > 0.05, Table 2). Female offspring in HFD-sitagliptin group had a slight increase than those from ND group in fasting blood glucose, AUC of blood glucose and HOMA-IR (p < 0.05, Table 2).

The effect of maternal early sitagliptin intervention on gene expression in male offspring intestine from gene array results and pathways

Two hundred and one genes were differentially expressed in the intestines of pups from HFD-sitagliptin dams compared with those of pups from HFD dams (>1.50-fold, p < 0.05; 119 upregulated and 82 downregulated). To investigate the possible regulatory mechanisms of genes affected by maternal early sitagliptin intervention on offspring intestine, we analyzed biological processes, molecular functions, and cellular components through Gene Ontology and enriched KEGG pathways by DAVID. The results showed that differentially expressed genes between the HFD and HFD-sitagliptin groups were enriched in the following biological processes: inflammatory response, cellular response to lipopolysaccharide, positive regulation of calcidiol 1-monooxygenase activity, response to yeast, and positive regulation of chemokine production (p < 0.0001, Fig. 3, Table 3). They are mainly enriched in the following molecular functions: cytokine activity, interleukin-1 receptor binding, heme binding, alkane 1-monooxygenase activity, and GTPase binding (p < 0.05, Fig. 3, Table 3). Significant cellular components were extracellular space, phagocytic cup, endoplasmic reticulum membrane, actin filament, and extracellular region (p < 0.05, Fig. 3, Table 3). Enriched KEGG pathways by DAVID displayed in the KEGG pathway database showed that genes affected by maternal sitagliptin intervention on offspring intestine were significantly enriched in African trypanosomiasis, inflammatory bowel disease, retinol metabolism, malaria, tuberculosis, leishmaniasis, amoebiasis, cytokine-cytokine receptor interaction, hematopoietic cell lineage, and salmonella infection (p < 0.005, Fig. 4, Table 4). In STRING analysis, all differentially expressed genes were mapped in one network. In this network figure, Il1b, Il6, and Tnf were in the center of the differentially expressed gene network (Fig. 5, Table 5).

The effect of maternal sitagliptin intervention on gene expression in male offspring intestine from real-time PCR

To assess the reliability of the array hybridization results, three differentially expressed genes were quantified using real-time PCR. Gene array GO analysis showed that the inflammatory response was among the top biological processes. Moreover, cytokine-cytokine receptor interactions were among the top ten KEGG pathways. Proinflammatory markers Il6, Il1b and Tnf were in the center of the differentially expressed gene network. Thus, we selected these cytokines to validate the gene array results. Il6, Il1b, and Tnf expression levels were increased in the intestines of male offspring from HFD dams compared to those from ND and ND-sitagliptin dams (p < 0.01, Fig. 6). Interestingly, maternal sitagliptin intervention reduced Il6, Il1b, and Tnf expression levels in male offspring intestines form HFD dams (p < 0.01, Fig. 6).

The effect of maternal sitagliptin intervention on proinflammatory markers IL-6 and TNF-α protein expression in male offspring intestines by immunohistochemical staining

Consistent with the findings by real-time PCR, IL-6 and TNF-α expression in male offspring intestines from HFD dams was increased, compared with those from ND and ND-sitagliptin dams (p < 0.01, Fig. 7). However, maternal sitagliptin intervention reduced the immunoreactivity of IL-6 and TNF-α in male offspring from HFD dams (p < 0.01, Fig. 7).

The effect of maternal sitagliptin intervention on serum proinflammatory cytokines IL-6, TNF-α and anti-proinflammatory cytokine IL-10 in male offspring

Serum proinflammatory cytokines IL-6 and TNF-α increased significantly in male rat offspring from HFD dams (p < 0.01, Fig. 8), Maternal sitagliptin treatment reduced serum IL-6 and TNF-α levels in male offspring from HFD dams (p < 0.01, Fig. 8). However, there was no difference in serum anti-proinflammatory cytokine IL-10 levels among four groups (p > 0.05, Fig. 8).