Sci Nat (2017) 104:89 https://doi.org/10.1007/s00114-017-1510-4
ORIGINAL PAPER
Effects of prenatal caffeine exposure on glucose homeostasis of adult offspring rats Hao Kou 1,2 & Gui-hua Wang 3 & Lin-guo Pei 3,4 & Li Zhang 3 & Chai Shi 3 & Yu Guo 2,3 & Dong-fang Wu 1 & Hui Wang 2,3
Received: 10 January 2017 / Revised: 30 August 2017 / Accepted: 30 September 2017 # Springer-Verlag GmbH Germany 2017
Abstract Epidemiological evidences show that prenatal caffeine exposure (PCE) could induce intrauterine growth retardation (IUGR). The IUGR offspring also present glucose intolerance and type 2 diabetes mellitus after maturity. We have previously demonstrated that PCE induced IUGR and increased susceptibility to adult metabolic syndrome in rats. This study aimed to further investigate the effects of PCE on glucose homeostasis in adult offspring rats. Pregnant rats were administered caffeine (120 mg/kg/day, intragastrically) from gestational days 11 to 20. PCE offspring presented partial catch-up growth pattern after birth, characterizing by the increased body weight gain rates. Meanwhile, PCE had no significant influences on the basal blood glucose and insulin phenotypes of adult offspring but increased the glucose tolerance, glucose-stimulated insulin section and β cell sensitivity to glucose in female progeny. The insulin sensitivity of both male and female PCE offspring were enhanced accompanied with reduced β cell fraction and mass. Western blotting results revealed that significant augmentation in protein expression of hepatic insulin signaling elements of PCE females, including insulin receptor (INSR), insulin receptor substrate 1 (IRS-1) and the phosphorylation of Communicated by: Sven Thatje * Hui Wang [email protected] 1
Department of Pharmacy, Zhongnan Hospital, Wuhan University, Wuhan 40071, China
2
Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China
3
Hubei Provincial Key Laboratory of Developmentally Originated Diseases, Wuhan 430071, China
4
Basic Medical College of Nanyang Medical University, Nanyang 473041, China
serine–threonine protein kinase (Akt), was also potentiated. In conclusion, we demonstrated that PCE reduced the pancreatic β mass but increased the glucose tolerance in adult offspring rats, especially for females. The adaptive compensatory enhancement of β cell responsiveness to glucose and elevated insulin sensitivity mainly mediated by upregulated hepatic insulin signaling might coordinately contribute to the increased glucose tolerance. Keywords Prenatal caffeine exposure . Intrauterine growth retardation . Glucose tolerance . Insulin sensitivity . Pancreatic β cell development . Hepatic insulin signaling
Introduction Intrauterine growth retardation (IUGR) is the failure of a fetus to achieve a predicted growth potential based on the genetic constitution and environmental influences, and it primarily manifests as low birth weight. Approximately 5 to 10% of newborns worldwide are characterized by IUGR (Resnik 2002), and the incidence of IUGR in some developing countries reaches as high as 30% (Saleem et al. 2011). Epidemiological studies have shown that detrimental intrauterine environment (i.e., maternal malnutrition, protein deprivation) insults pancreatic development and permanently Bprograms^ β cell mass and function in IUGR offspring, and these irreversible alterations may greatly increase the risk of developing glucose intolerance and type 2 diabetes mellitus (T2DM) in late period of life (Bo et al. 2000; Yajnik 2000; Kahn 2001). IUGR animal models established by different methods also displayed diabetic symptoms in adult offspring (Garofano et al. 1997; Bertin et al. 1999; Garg et al. 2013).
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Caffeine is a xanthine alkaloid consumed not only in the form of coffee but also in black tea, soft beverages, energy drinks, food and cocoa (Frary et al. 2005). Upon ingestion, caffeine is rapidly absorbed and readily passes the placental barrier. The main enzyme (cytochrome P450 1A2) involved in caffeine metabolism is absent in both the placenta and the fetus which can lead to caffeine accumulation in fetal tissues (Aldridge et al. 1979). The half-life of caffeine doubles in the mother during pregnancy as the rate of caffeine metabolism decreases from the first to third trimester (Knutti et al. 1982). This delayed clearance of caffeine leads to higher exposure to caffeine for the fetus, which may in turn affect fetal growth and development. Pregnant women are commonly tried to avoid drinking coffee, but the consumption of food and beverages that contains caffeine might be ignored and thereby causing caffeine exposure during pregnancy. Epidemiological studies have revealed that prenatal caffeine consumption is associated with low birth weight and could lead to IUGR (Vik et al. 2003; Group 2008; Bakker et al. 2010; Chen et al. 2014). Reports also indicate that children who ingest caffeine-containing food or drinks are highly susceptible to metabolic syndromes such as obesity and hypertension (James et al. 2004; Cayetanot et al. 2009). Previously, we have demonstrated that 20, 60, and 180 mg/kg day (Liu et al. 2012; Xu et al. 2012b) or 30, 60 and 120 mg/kg day (Wu et al. 2015) of caffeine exposure (intragastric administration) from gestational days (GD) 11 to 20 lead to IUGR and causes fetal overexposure to high levels of maternal glucocorticoids (GC) in rats. The consequent hypothalamic-pituitary-adrenal (HPA) axis-associated neuroendocrine metabolic programming alteration induced by 120 mg/kg day of caffeine exposure from GD 11 to 20 may further increase the susceptibility to adult metabolic syndrome and relevant metabolic diseases (i.e., non-alcoholic fatty liver diseases) in adult IUGR offspring rats (Liu et al. 2012; Xu et al. 2012a; Wang et al. 2014). However, whether prenatal caffeine exposure (PCE) could alter the glucose metabolic homeostasis in IUGR adult offspring remains unknown. In the present study, we aimed to demonstrate the effects of PCE on glucose homeostasis of adult rats and explore the underlying mechanism. This study is of significance for elucidating the etiology of fetal originated diabetes and relevant diseases.
Material and methods Ethics and animal housing Animal experiments were performed in the Center for Animal Experiments of Wuhan University (Wuhan, China), which has been accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International
(AAALAC International). All animal experimental procedures were approved by and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Chinese Animal Welfare Committee. Specific pathogen-free (SPF) Wistar rats (with weights of 200–240 g for females and 260–300 g for males) were obtained from the Experimental Center of Hubei Medical Scientific Academy (No. 2009-0004, Hubei, China). Animals were housed under standard conditions (room temperature 18–22 °C; humidity 40–60%; 12:12-h light–dark cycle) and allowed free access to rat chow and distilled water. Experimental procedure The IUGR rat model was achieved as previously described (Tan et al. 2012; Xu et al. 2012a; Luo et al. 2014; Wang et al. 2014; Luo et al. 2015). To be specific, two females were placed with one male overnight. The day at which the evidence of mating was observed (i.e., vaginal plug or vaginal smear with sperm cells) was designated as gestational day (GD) 0, whereby the pregnant rats were then caged separately. From GD11 to GD20, pregnant rats were administered 120 mg/kg of caffeine (CAS #58-08-2, > 99% purity, Sigma-Aldrich, MO) once per day and the dams of control group were sham-treated with vehicle. All the animals were subjected to spontaneous delivery. On postnatal day 1, the numbers of pups were normalized to eight pups per litter to ensure adequate and standardized nutrition and the exceeding pups were euthanized by cervical dislocation. After weaning (in postnatal week 4, PW4), one male and one female pup was randomly selected from each dam and fed with lab chow (providing 22% of its energy content as protein, 63% as carbohydrates and only 5% as fat). The selected pups were divided into four groups: male control, male caffeine, female control, and female caffeine. Each group comprised eight pups. Body weights were recorded every week and the corresponding body weight gain rates were calculated as follows: Gain rate ð%Þ ¼ ½Body weight ðPWx Þ−Body weight ðPW1 Þ Body weight ðPW1 Þ 100 Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance tests (ITT) were conducted in PW20 and 21, respectively. In PW 24, the rats were anesthetized with isoflurane (Baxter Healthcare, IL) and sacrificed in a room separate from the other animals. Pancreas were resected and fixed for immunohistochemistry (IHC) and livers were frozen at − 80 °C. Intraperitoneal glucose tolerance test IPGTT was used to assess glucose tolerance in PW20. The overnight fasting can produce low, stable baseline blood
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glucose and insulin levels and obtain consistent excursions in blood glucose after glucose loading (Heikkinen et al. 2007; Muniyappa et al. 2008), so the offspring rats were prefasted overnight for 12 h and then administered glucose intraperitoneally (2 g/kg) at 8:00 a.m. Blood glucose levels at 0, 15, 30, 60, and 120 min after glucose challenge were determined by Accu-Chek® Performa glucose meter via tail clipping. The blood glucose concentrations were normalized by dividing the basal concentration of blood glucose (0 min) prior to the calculation of area under the curve (AUC) based on the trapezoidal rule. Additional blood (300 μl) was collected at 0 and 15 min from the caudal vein to prepare serum for insulin radioimmunoassay following manufacturer’s protocol (Beijing North Institute of Biological Technology, China). To estimate the ability of the β cells to respond to a glucose challenge, we calculated insulin secretion over the 15 min after the injection (ΔI15 − 0) divided by the difference between the glucose concentrations during the same time period (ΔG 15 − 0 ), that is, the ratio of ΔI 15 − 0 to ΔG 15 − 0 (I/G15 − 0) (Jha et al. 2016). Insulin tolerance test As for ITT in PW21, we prefasted the animals for 6 h since 8:00 a.m. in order to get a sufficient depletion of endogenous blood insulin and reduce the possibility of hypoglycemia induced by overnight fasting. An intraperitoneal injection of 0.75 units/kg human insulin (Eli Lilly, IN) were performed at 14:00 p.m. The measurement of blood glucose levels at 0, 15, 30, 60, and 120 min after insulin challenge and the corresponding AUC calculation were the same as described in IPGTT. Morphometric analysis of pancreas Pancreas were weighed and then fixed in 10% formaldehyde and embedded in paraffin. Six complete longitudinal sections (5 μm) with at least 200-μm intervals from each embedded pancreas were obtained through their maximal width and subsequently subjected to insulin IHC. The sections were deparaffinized, rehydrated, and heated in 10 mmol/l citrate buffer (pH 6.0) at 92 °C for 10 min in a microwave oven for antigen retrieval. After cooling at room temperature, the sections were washed three times with PBS, and endogenous peroxidase was blocked by 30-min incubation with 3% H2O2 at room temperature. Then, the sections were incubated overnight at 4 °C with mouse anti-insulin (1:1000, Sigma). Detection was with a streptavidin–biotin–peroxidase complex developed with aminoethylcarbazol (Zymed, San Francisco, CA). As a negative control, pancreatic sections underwent similar treatment, and no positive reactions to the antibody against insulin were observed (not shown).
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The tissue sections were viewed and captured at a magnification of × 20 or × 400 using a Nikon Eclipse Ci-S upright microscope coupled with a DS-U3 digital camera control unit (Nikon, Tokyo, Japan). Morphometric analyses were performed with NIS-Elements Br software (Nikon, Tokyo, Japan). The size of the insulin-positive cell area as well as the pancreatic tissue area of the entire section was measured on six sections per animal, with a total of five animals being analyzed per group. The β fractions (%) were determined as the ratio of the insulin-positive cell area to the total pancreatic tissue area on the entire section. The β cell masses (mg) were obtained through multiplying the β cell fractions (%) by the entire pancreas mass. Western blotting The hepatic protein expressions of insulin receptor (INSR), insulin receptor substrate-1 (IRS-1), serine–threonine protein kinase (Akt), and phosphorylated Akt (pAkt) were determined by western blotting. The liver homogenate tissues were rinsed with ice-cold PBS and then lysed for 30 min at 4 °C in RIPA lysis buffer containing Protease Inhibitor Cocktail (Sigma, St. Louis, MO) and Phosphatase Inhibitor Cocktail (Sigma, St. Louis, MO). The supernatant was harvested, and protein concentrations were determined using a BCA protein assay kit (Beyotime, Shanghai, China) following the manufacturer’s protocol. Aliquots of protein lysates (50 mg/lane) were isolated by SDS-PAGE (10% gels) and blotted onto PVDF membranes (Millipore, Billerica, MA). Membranes were blocked in 5% non-fat milk for 1 h and incubated overnight at 4 °C with the primary antibodies (CST, Danvers, MA), including anti-INSR (1:1000), anti-IRS-1 (1:1000), anti-Akt (1:1000), anti-pAkt (1:1000), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000). After washing in TBST, they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 45 min and visualized using ECL HRP substrate (PerkinElmer, Inc., Boston, MA). Signals of antibody binding were detected by Chemi-doc Image Analyzer (Bio-Rad, Hercules, CA). The immunoblotting experiments were performed three times using different samples. Protein band intensities were analyzed by ImageJ (NIH, Bethesda, MA), and relative protein levels of target genes were normalized by GAPDH protein level. Data analysis and statistics Q u a n t it a t i v e p a r a m e t r i c d a t a w e r e e x p r e s s e d a s mean ± S.E.M., and non-parametric data were indicated as median with interquartile (Battiston et al. 2017). Shapiro– Wilk test was employed to examine the normality of data. The homogeneity of variances was evaluated by Levene’s test. For the repeated measures data (i.e., postnatal bodyweight and the corresponding gain rates, relative glycemic values in
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IPGTT and ITT, serum insulin in IPGTT), two-way repeated measures ANOVA was used to analyze overall effects of PCE over time, followed by multivariate ANOVA for the comparison at every individual time point. The other data (i.e., basal blood glucose-insulin phenotype, AUC, I/G15 − 0, β cell fraction and mass, protein expression of hepatic insulin signaling pathway) were split by gender, and the differences between groups within gender were compared by unpaired Student’s t test assuming normal distribution and equal variance. If normal distribution and equal variance not assumed, log transformation was performed to normalize the data distribution prior to t test evaluation or unpaired Mann–Whitney U test (equivalent to Wilcoxon W) was applied when necessary. Statistical significance was set at P < 0.05. All the statistics were performed with SPSS for Windows version 17 (SPSS Science, Inc., Chicago, IL).
Results
offspring were also greater than those of the control group (P < 0.01). All of the above reveals that PCE increased the glucose tolerance in female adult offspring, accompanied with an enhancement of glucose-stimulated insulin secretion and β cell responsiveness to glucose. However, no such effects were observed in males. Insulin sensitivity ITT was employed to determine whole body insulin sensitivity of adult offspring in PW21. As shown in Fig. 3, the linear glucose decay of both male and female PCE offspring in the first 30 min tended to be greater than those of their controls, and the AUCs in PCE male and female offspring were all significantly decreased when compared with the controls (P < 0.01). In addition, the relative blood glucose levels at 60 and 120 min were all reduced in the PCE group compared with the controls (P < 0.05). These results indicated that the whole body insulin sensitivity of PCE offspring was partly increased.
Postnatal bodyweights Pancreatic β cell fraction and mass As shown in Fig. 1a, the body weights of both male and female offspring from PCE group were lower than those of control animals in PW1 (P < 0.01; Fig. 1a). Although the absolute body weights of PCE offspring fell behind controls from PW4 to PW24, the corresponding bodyweight gain rates in the whole stage or each time point were greater than those of control animals (P < 0.05, P < 0.01; Fig. 1b–e). These results indicate that PCE offspring presented a partial Bcatchup^ growth pattern of body weights without any gender difference.
To determine the effects of PCE on pancreatic morphological development, we measured the fraction and mass of pancreatic β cells. The total pancreatic masses were not altered in the PCE offspring compared with control animals (Fig. 4b, e). Morphometric analysis further revealed that the pancreatic β cell fraction and mass in both male and female offspring from PCE group were lower than those in control counterparts (P < 0.05, P < 0.01; Fig. 4c, d, f, g). Hepatic insulin signaling protein expression
Basal glucose-insulin phenotype, glucose tolerance, and β cell responsiveness to glucose In PW20, glucose tolerance was evaluated by IPGTT after 12 h of fasting. The basal levels of blood glucose (Fig. 2a, e) and insulin (Fig. 2c, g) did not differ between the control and PCE groups. For male offspring, glucose tolerance of the PCE group was not changed when compared to the controls (Fig. 2b). Meanwhile, injection of the glucose caused a pronounced increase in blood concentrations of insulin at 15 min only in the control animals (P < 0.05) but not in the PCE progeny (Fig. 2c). The calculated I/G15 − 0 of the PCE group tended to be lower than those of control counterparts. For female offspring, glucose tolerance of the PCE animals was significantly increased when compared with the controls (P < 0.05; Fig. 2f), as evidenced by the lowered relative blood glucose levels and the corresponding reduced AUC after glucose injection. In addition, PCE also induced higher insulin secretion at 15 min after glucose load than the controls (P < 0.05; Fig. 2g). The calculated I/G15 − 0 of the PCE
The western blotting results showed that PCE significantly enhanced the protein expression levels of hepatic INSR, IRS-1, and total Akt only in the females (P < 0.01, P = 0.068; Fig. 5b, d), but not males. Moreover, the phosphorylation of Akt was also increased in females, as observed by the augmented ratios of pAkt to total Akt proteins (P < 0.01; Fig. 5e).
Discussion Abnormal homeostasis and perturbed metabolism of blood glucose in IUGR adult offspring have been demonstrated by numerous animal studies (Varvarigou 2010). However, we found the interesting phenomenon that PCE offspring rats presented normal glucose homeostasis (i.e., unchanged basal blood glucose and insulin levels), accompanied with the unchanged or increased glucose tolerance. Previous review proposed that IUGR offspring may undergo an age-dependent
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Fig. 1 Effects of prenatal caffeine exposure on postnatal growth and development in offspring rats. Pregnant rats were administered of 120 mg/kg of caffeine once per day from gestational days (GD) 11 to 20. a Body weights at postnatal week (PW) 1. b, c Male body weights and corresponding gain rates from PW4 to 24. d, e Female body mass and corresponding gain rates from PW 4 to 24. Mean ± S.E.M., n = 8 animals/
group per gender. *P < 0.05, **P < 0.01 vs control. Differences of body weights in PW1 were compared by using unpaired Student’s t test. Effects of prenatal caffeine exposure on postnatal body weights, and corresponding gain rates over time were determined by two-way repeated measures ANOVA followed by multivariate ANOVA for the comparison at every individual time point
loss of glucose tolerance even if a better glucose tolerance than control was found in young adult life (Ozanne 2001).
In addition, IUGR offspring from different modeling methods are shown to be glucose-intolerant at various postnatal time
Fig. 2 Effects of prenatal caffeine exposure on basal glucose-insulin phenotype, glucose tolerance and β cell responsiveness to glucose in adult offspring rats. Pregnant rats were administered of 120 mg/kg of caffeine once per day from gestational days (GD) 11 to 20. In postnatal week 20, intraperitoneal glucose tolerance test (IPGTT) was performed to evaluate the glucose tolerance in offspring rats via an intraperitoneal injection of glucose (2 mg/kg) following 12 h prefasting. The β cells responsiveness to glucose was measured as the insulin secretion over the 15 min after the injection (ΔI15 − 0) divided by the difference between the glucose concentrations during the same time period (ΔG15 − 0). a, e Blood glucose levels at 0 min during IPGTT. b, f Normalized blood
glucose levels during IPGTT and area under the curve (AUC). c, g Blood insulin levels at 0 and 15 min during IPGTT. d, h Ratio of ΔI15 − 0 to ΔG15 − 0 (I/G15 − 0). Mean ± S.E.M., n = 8 animals/group per gender. *P < 0.05, **P < 0.01, vs control; #P < 0.05, ##P < 0.01, vs 0 min. Effects of prenatal caffeine exposure on normalized blood glucose over time during IPGTT were determined by two-way repeated measures ANOVA followed by multivariate ANOVA for the comparison at every individual time point. Differences of blood glucose at 0 min, AUC, and I/G15 − 0 between groups were compared by using unpaired Student’s t test
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Fig. 3 Effects of prenatal caffeine exposure on insulin sensitivity in adult offspring rats. Pregnant rats were administered of 120 mg/kg of caffeine once per day from gestational days (GD) 11 to 20. In postnatal week 21, insulin tolerance test (ITT) was performed to evaluate the whole body insulin sensitivity in offspring rats via an intraperitoneal injection of insulin (2 mg/kg) following 6 h prefasting from the morning. a Normalized blood glucose levels during ITT and area under the curve (AUC) in male
offspring. b Normalized blood glucose levels during ITT and AUC in female offspring. Mean ± S.E.M., n = 8 animals/group per gender. *P < 0.05, **P < 0.01, vs control. Effects of prenatal caffeine exposure on normalized blood glucose over time during ITT were determined by two-way repeated measures ANOVA followed by multivariate ANOVA for the comparison at every individual time point. Differences of AUC between groups were compared by using unpaired Student’s t test
points (Simmons et al. 2001; Inoue et al. 2009; Yuan et al. 2011). Therefore, we deduced that the unchanged and increased glucose tolerance in PCE male and female offspring might be associated with our experimental method which induces IUGR by PCE during second and third trimesters. Additionally, we conducted IPGTT in PW20 that is just within the young adult stage of rats, which may also be attributed to the enhancement of glucose tolerance in PCE adult offspring. Glucose tolerance relies on several important aspects, including β cell mass and function-associated insulin secretion as well as insulin availability of targeting organs (Kahn et al. 1993). In physiological condition, both pancreatic insulin secretion and peripheral insulin sensitivity coordinately regulate and maintain the dynamic balance of glucose metabolism. In IPGTT, although the blood insulin levels of PCE male offspring at 15 min after glucose load and the calculated I/G15 − 0 tended to be lower than their controls, the glucose tolerance of PCE male offspring remains unaltered, as observed by the unchanged AUC. These results indicate that there might be no significant reduction of β cell function in PCE male offspring rats, even if the decreased β cell mass could be found. In addition, the increased insulin sensitivity may enhance the glucose disposal so that the in vivo secretion of insulin remains enough to metabolize blood glucose from glucose injection and maintain the normal glucose tolerance in PCE male offspring. With regard to female offspring, the basal blood glucose and insulin levels (0 min in IPGTT) of PCE females were not changed when compared with the control, even though a significant decrease of β cell mass could be observed. Meanwhile, we also found that the β cell sensitization to glucose and whole body insulin sensitivity of PCE females were all potentiated to some extent, as evidenced by the elevated I/G15 − 0 in IPGTT and decreased AUCs of relative glucose curves in ITT. Basing on a series of animal studies, Rafacho
et al. have demonstrated that one of the major adaptive compensatory responses for pancreatic β cell to face the new metabolic demand for insulin includes an increase in responsiveness to glucose (Rafacho et al. 2008; Rafacho et al. 2010a, b). Meanwhile, the IUGR infants who display catch-up growth pattern are often characterized by enhanced metabolic demand to insulin and other growth factor (i.e., insulin growth-like factor 1, IGF1) (Luo et al. 2010). Therefore, we speculated that the increase in β cell sensitization to glucose might be one of the adaptive compensatory responses to the diminished β cell mass in PCE females with a postnatal catch-up growth pattern. The enhanced β cell sensitivity to glucose may trigger much more insulin secretion, which partly recovers the relative deficiency of total insulin biosynthesis due to the decreased β cell population and hence maintains the constant basal blood insulin levels as well as normal glucose homeostasis. In addition, as another compensatory response to the reduced β cell mass, the increased insulin sensitivity was not only observed in the PCE female adult offspring but also has been confirmed in prenatal protein deprived IUGR offspring (Gosby et al. 2010; Lim et al. 2011). Accordingly, when facing the transient glucose challenge in IPGTT, the insulinsensitive PCE females could secret much more insulin than the controls at 15 min due to the increased β cell sensitivity to glucose, which might be contributed to the increased glucose tolerance. Interestingly, Camacho et al. also found the enhanced insulin secretion and insulin sensitivity in young lambs born with IUGR caused by placental insufficiency (Camacho et al. 2017), which is consistent with our observation. Early environmental insults may alter the timing or amplitude of developmental changes leaving the individual with a β cell population poorly suited both quantitatively and qualitatively for postnatal life and ultimately leading to altered glucose metabolism (Hill and Duvillie 2000). Maternal calorie
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Fig. 4 Effects of prenatal caffeine exposure on pancreatic β cell fraction and mass in adult offspring rats. Pregnant rats were administered of 120 mg/kg of caffeine once per day from gestational days (GD) 11 to 20. In postnatal week 24, the offspring rats were sacrificed to collect pancreas. The morphometric analysis of pancreatic β cells was performed based on the insulin immunohistochemical staining. a Representative insulin-staining images (with a magnification × 40). b, e Pancreatic mass. c, f Pancreatic β cell fraction. d, g Pancreatic β cell mass. Mean ± S.E.M., n = 5 animals/ group per gender. *P < 0.05, **P < 0.01 vs control. Differences between groups were compared by using unpaired Student’s t test
restriction during late gestation in mice showed that β cell mass was not only reduced in the newborn but also lower than control after maturity (Inoue et al. 2009). We previously found PCE reduced the population of β cells in fetal rats (unpublished data). In this study, the pancreatic β cell mass of both male and female PCE adult offspring remained lower than those of control animals, indicating that PCE may perturb fetal β cell development and result in postnatal insufficient β cell mass. Insulin/INSR and IGF1/IGF1 receptor (IGF1R) signaling not only participate in the regulation of hepatic glucose metabolism (Cherrington et al. 1998) but also play an important role in the postnatal catch-up growth in offspring with IUGR
(Shen et al. 2014; Wang et al. 2014). In this study, the increased insulin sensitivity of PCE female offspring may be partially explained by the upregulation of hepatic insulin signaling, as supported by the increased protein expressions of INSR and IRS-1 as well as incremental phosphorylation of Akt. However, there were no profound alterations in hepatic insulin signaling in PCE males. Both clinical and experimental evidences have revealed that IUGR male offspring presented increased insulin sensitivity that is associated with the increased insulin signaling in muscle (Ozanne et al. 1996; Jensen et al. 2005), which might be one of the potential reasons for the enhanced insulin sensitivity in PCE male offspring.
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programming the insulin/IGF1 signaling pathway in the liver and muscle (Liu et al. 2012; Xu et al. 2012b; Kou et al. 2014; Wang et al. 2014). Furthermore, we proposed a GC–IGF1 axis programming as the possible underlying mechanism for the development of caffeine-induced IUGR and enhanced susceptibility to adult metabolic syndrome (Wang et al. 2014). Therefore, the GC–IGF1 axis programming may underlie the increased expression of hepatic insulin signaling in PCE female offspring and partly contribute to the observed glucose metabolic phenotype. Both epidemiological survey and animal experiments identify that chronic adult-onset diseases with fetal origin are characterized by gender differences (Laguna-Barraza et al. 2012). Interestingly, here we also found significant gender differences of multiple parameters existed in PCE adult offspring, such as glucose tolerance, glucose-stimulated insulin secretion, and hepatic insulin signaling. An ovarian estrogen, such as estradiol, could ameliorate pancreatic insulin biosynthesis to some extent in a diabetic state (Godsland 2005). On diabetic rodent models, males are much more susceptible to suffering insulin deficiency (Louet et al. 2004). In addition, as a crucial endocrine axis regulating postnatal growth and glucose metabolism, the growth hormone (GH)–IGF1 axis has been verified to be interacted with sex hormones during puberty. Therefore, we assume that gender differences in PCE adult offspring may partially be due to the sensitivity variation of effects of androgens and estrogens on the GH–IGF1 axis, which requires further investigation.
Conclusions Fig. 5 Effects of prenatal caffeine exposure on the protein expressions of key hepatic insulin signaling elements in adult offspring rats. Pregnant rats were administered of 120 mg/kg of caffeine once per day from gestational days (GD) 11 to 20. In postnatal week 24, the offspring rats were sacrificed to collect liver. The relative protein expressions of insulin receptor (INSR), insulin receptor substrate 1 (IRS-1), and serine–threonine protein kinase (Akt) were determined by western blotting and then normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The phosphorylation of Akt was measured as the ratio of phosphorylated Akt (pAkt) to total Akt. a Representative western blotting image. b, d Relative protein expression levels of INSR, IRS-1 and Akt. c, e Ratio of pAkt to Akt. Mean ± S.E.M., n = 3 animals/group per gender. **P < 0.01 vs control. Differences between groups were compared by using unpaired Student’s t test
In recent decades, findings from retrospective human epidemiological studies and experimental animal models have strongly implicated environmental stimulus or insult during fetal and immediate postnatal periods as an important factor contributing to the development of metabolic disturbance via the phenomenon of metabolic programming (Gluckman et al. 2007). Our previous studies have demonstrated that PCE alters the peripheral glucose metabolic programming via inducing fetal overexposure to the elevated maternal GC and further
In conclusion, we demonstrated that PCE reduced the pancreatic β mass but increased the glucose tolerance in adult offspring rats, especially for females. The adaptive compensatory enhancement of β cell responsiveness to glucose and elevated insulin sensitivity mainly mediated by upregulated hepatic insulin signaling might coordinately contribute to the increased glucose tolerance. Our study provided experimental basis and proposed potential mechanism for interpreting the increased susceptibility to diabetes in PCE-induced IUGR adult offspring. Funding informationThis work was granted by the National Science & Technology Pillar Program of China (No. 2013BAI12B01-3), National Natural Science Foundation of China (Nos. 81220108026, 81430089, 81473290, 81703631) and Hubei Province Health and Family Planning Scientific Research Project (Nos. WJ2017C0003, WJ2017M028). Compliance with ethical standards Animal experiments were performed in the Center for Animal Experiments of Wuhan University (Wuhan, China), which has been accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). All animal experimental procedures were approved by and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Chinese Animal Welfare Committee.
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Page 9 of 10 89 James J, Thomas P et al (2004) Preventing childhood obesity by reducing consumption of carbonated drinks: cluster randomised controlled trial. Br Med J 328(7450):1237–1239. https://doi.org/10.1136/bmj. 38077.458438.EE Jensen CB, Storgaard H et al (2005) Young, low-birth-weight men are not more susceptible to the diabetogenic effects of a prolonged free fatty acid exposure than matched controls. Metab Clin Exp 54(10):1398– 1406. https://doi.org/10.1016/j.metabol.2005.05.005 Jha PK, Foppen E et al (2016) Sleep restriction acutely impairs glucose tolerance in rats. Physiol Rep 4(12). 10.14814/phy2.12839 Kahn HS (2001) Glucose tolerance in adults after prenatal exposure to famine. Lancet 357(9270):1798–1799. https://doi.org/10.1016/ S0140-6736(00)04911-4 Kahn SE, Prigeon RL et al (1993) Quantification of the relationship between insulin sensitivity and beta-cell function in human-subjects—evidence for a hyperbolic function. Diabetes 42(11):1663– 1672. https://doi.org/10.2337/diabetes.42.11.1663 Knutti R, Rothweiler H et al (1982) The effect of pregnancy on the pharmacokinetics of caffeine. Arch Toxicol Suppl = Archiv fur Toxikologie Supplement 5:187–192 Kou H, Liu Y et al (2014) Maternal glucocorticoid elevation and associated blood metabonome changes might be involved in metabolic programming of intrauterine growth retardation in rats exposed to caffeine prenatally. Toxicol Appl Pharmacol. https://doi.org/10. 1016/j.taap.2014.01.007 Laguna-Barraza R, Bermejo-Alvarez P et al (2012) Sex-specific embryonic origin of postnatal phenotypic variability. Reprod Fertil Dev 25(1):38–47. https://doi.org/10.1071/RD12262 Lim K, Armitage JA et al (2011) IUGR in the absence of postnatal Bcatchup^ growth leads to improved whole body insulin sensitivity in rat offspring. Pediatr Res 70(4):339–344 Liu Y, Xu D et al (2012) Fetal rat metabonome alteration by prenatal caffeine ingestion probably due to the increased circulatory glucocorticoid level and altered peripheral glucose and lipid metabolic pathways. Toxicol Appl Pharmacol 262(2):205–216. https://doi. org/10.1016/j.taap.2012.05.002 Louet JF, LeMay C et al (2004) Antidiabetic actions of estrogen: insight from human and genetic mouse models. Curr Atheroscler Rep 6(3): 180–185 Luo ZC, Xiao L et al (2010) Mechanisms of developmental programming of the metabolic syndrome and related disorders. World J Diabetes 1(3):89–98. https://doi.org/10.4239/wjd.v1.i3.89 Luo H, Deng Z et al (2014) Prenatal caffeine ingestion induces transgenerational neuroendocrine metabolic programming alteration in second generation rats. Toxicol Appl Pharmacol 274(3):383–392. https://doi.org/10.1016/j.taap.2013.11.020 Luo H, Li J et al (2015) Prenatal caffeine exposure induces a poor quality of articular cartilage in male adult offspring rats via cholesterol accumulation in cartilage. Sci Rep 5:17746. https://doi.org/10.1038/ srep17746 Muniyappa R, Lee S et al (2008) Current approaches for assessing insulin sensitivity and resistance in vivo: advantages, limitations, and appropriate usage. Am J Phys Endocrinol Metab 294(1):E15–E26. https://doi.org/10.1152/ajpendo.00645.2007 Ozanne SE (2001) Metabolic programming in animals. Br Med Bull 60: 143–152. https://doi.org/10.1093/Bmb/60.1.143 Ozanne SE, Wang CL et al (1996) Altered muscle insulin sensitivity in the male offspring of protein-malnourished rats. Am J Phys 271(6 Pt 1): E1128–E1134 Rafacho A, Giozzet VA et al (2008) Functional alterations in endocrine pancreas of rats with different degrees of dexamethasone-induced insulin resistance. Pancreas 36(3):284–293. https://doi.org/10.1097/ MPA.0b013e31815ba826 Rafacho A, Marroqui L et al (2010a) Glucocorticoids in vivo induce both insulin hypersecretion and enhanced glucose sensitivity of stimulus-
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secretion coupling in isolated rat islets. Endocrinology 151(1):85– 95. https://doi.org/10.1210/en.2009-0704 Rafacho A, Quallio S et al (2010b) The adaptive compensations in endocrine pancreas from glucocorticoid-treated rats are reversible after the interruption of treatment. Acta Physiol (Oxf) 200(3):223–235. https://doi.org/10.1111/j.1748-1716.2010.02146.x Resnik R (2002) Intrauterine growth restriction. Obstet Gynecol 99(3): 490–496. https://doi.org/10.1016/S0029-7844(01)01780-X Saleem T, Sajjad N et al (2011) Intrauterine growth retardation—small events, big consequences. Ital J Pediatr 37:41. https://doi.org/10. 1186/1824-7288-37-41 Shen L, Liu Z et al (2014) Prenatal ethanol exposure programs an increased susceptibility of non-alcoholic fatty liver disease in female adult offspring rats. Toxicol Appl Pharmacol 274(2):263–273. https://doi.org/10.1016/j.taap.2013.11.009 Simmons RA, Templeton LJ et al (2001) Intrauterine growth retardation leads to the development of type 2 diabetes in the rat. Diabetes 50(10):2279–2286. https://doi.org/10.2337/diabetes.50.10.2279 Tan Y, Liu J et al (2012) Caffeine-induced fetal rat over-exposure to maternal glucocorticoid and histone methylation of liver IGF-1 might cause skeletal growth retardation. Toxicol Lett 214(3):279– 287. https://doi.org/10.1016/j.toxlet.2012.09.007 Varvarigou AA (2010) Intrauterine growth restriction as a potential risk factor for disease onset in adulthood. J Pediatr Endocr Met 23(3): 215–224 Vik T, Bakketeig LS et al (2003) High caffeine consumption in the third trimester of pregnancy: gender-specific effects on fetal growth. Paediatr Perinat Epidemiol 17(4):324–331
Sci Nat (2017) 104:89 Wang LL, Shen L et al (2014) Intrauterine metabolic programming alteration increased susceptibility to non-alcoholic adult fatty liver disease in prenatal caffeine-exposed rat offspring. Toxicol Lett 224(3): 311–318. https://doi.org/10.1016/j.toxlet.2013.11.006 Wu YM, Luo HW et al (2015) Prenatal caffeine exposure induced a lower level of fetal blood leptin mainly via placental mechanism. Toxicol Appl Pharmacol 289(1):109–116. https://doi.org/10.1016/j.taap. 2015.09.007 Xu D, Wu Y et al (2012a) A hypothalamic-pituitary-adrenal axisassociated neuroendocrine metabolic programmed alteration in offspring rats of IUGR induced by prenatal caffeine ingestion. Toxicol Appl Pharmacol 264(3):395–403. https://doi.org/10.1016/j.taap. 2012.08.016 Xu D, Zhang BJ et al (2012b) Caffeine-induced activated glucocorticoid metabolism in the hippocampus causes hypothalamic-pituitaryadrenal axis inhibition in fetal rats. PLoS One 7(9). https://doi.org/ 10.1371/journal.pone.0044497 Yajnik C (2000) Interactions of perturbations in intrauterine growth and growth during childhood on the risk of adult-onset disease. Proc Nutr Soc 59(2):257–265 Yuan QX, Chen L et al (2011) Postnatal pancreatic islet beta cell function and insulin sensitivity at different stages of lifetime in rats born with intrauterine growth retardation. PloS one 6(10):e25167. https://doi. org/10.1371/journal.pone.0025167
ORIGINAL PAPER
Effects of prenatal caffeine exposure on glucose homeostasis of adult offspring rats Hao Kou 1,2 & Gui-hua Wang 3 & Lin-guo Pei 3,4 & Li Zhang 3 & Chai Shi 3 & Yu Guo 2,3 & Dong-fang Wu 1 & Hui Wang 2,3
Received: 10 January 2017 / Revised: 30 August 2017 / Accepted: 30 September 2017 # Springer-Verlag GmbH Germany 2017
Abstract Epidemiological evidences show that prenatal caffeine exposure (PCE) could induce intrauterine growth retardation (IUGR). The IUGR offspring also present glucose intolerance and type 2 diabetes mellitus after maturity. We have previously demonstrated that PCE induced IUGR and increased susceptibility to adult metabolic syndrome in rats. This study aimed to further investigate the effects of PCE on glucose homeostasis in adult offspring rats. Pregnant rats were administered caffeine (120 mg/kg/day, intragastrically) from gestational days 11 to 20. PCE offspring presented partial catch-up growth pattern after birth, characterizing by the increased body weight gain rates. Meanwhile, PCE had no significant influences on the basal blood glucose and insulin phenotypes of adult offspring but increased the glucose tolerance, glucose-stimulated insulin section and β cell sensitivity to glucose in female progeny. The insulin sensitivity of both male and female PCE offspring were enhanced accompanied with reduced β cell fraction and mass. Western blotting results revealed that significant augmentation in protein expression of hepatic insulin signaling elements of PCE females, including insulin receptor (INSR), insulin receptor substrate 1 (IRS-1) and the phosphorylation of Communicated by: Sven Thatje * Hui Wang [email protected] 1
Department of Pharmacy, Zhongnan Hospital, Wuhan University, Wuhan 40071, China
2
Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China
3
Hubei Provincial Key Laboratory of Developmentally Originated Diseases, Wuhan 430071, China
4
Basic Medical College of Nanyang Medical University, Nanyang 473041, China
serine–threonine protein kinase (Akt), was also potentiated. In conclusion, we demonstrated that PCE reduced the pancreatic β mass but increased the glucose tolerance in adult offspring rats, especially for females. The adaptive compensatory enhancement of β cell responsiveness to glucose and elevated insulin sensitivity mainly mediated by upregulated hepatic insulin signaling might coordinately contribute to the increased glucose tolerance. Keywords Prenatal caffeine exposure . Intrauterine growth retardation . Glucose tolerance . Insulin sensitivity . Pancreatic β cell development . Hepatic insulin signaling
Introduction Intrauterine growth retardation (IUGR) is the failure of a fetus to achieve a predicted growth potential based on the genetic constitution and environmental influences, and it primarily manifests as low birth weight. Approximately 5 to 10% of newborns worldwide are characterized by IUGR (Resnik 2002), and the incidence of IUGR in some developing countries reaches as high as 30% (Saleem et al. 2011). Epidemiological studies have shown that detrimental intrauterine environment (i.e., maternal malnutrition, protein deprivation) insults pancreatic development and permanently Bprograms^ β cell mass and function in IUGR offspring, and these irreversible alterations may greatly increase the risk of developing glucose intolerance and type 2 diabetes mellitus (T2DM) in late period of life (Bo et al. 2000; Yajnik 2000; Kahn 2001). IUGR animal models established by different methods also displayed diabetic symptoms in adult offspring (Garofano et al. 1997; Bertin et al. 1999; Garg et al. 2013).
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Caffeine is a xanthine alkaloid consumed not only in the form of coffee but also in black tea, soft beverages, energy drinks, food and cocoa (Frary et al. 2005). Upon ingestion, caffeine is rapidly absorbed and readily passes the placental barrier. The main enzyme (cytochrome P450 1A2) involved in caffeine metabolism is absent in both the placenta and the fetus which can lead to caffeine accumulation in fetal tissues (Aldridge et al. 1979). The half-life of caffeine doubles in the mother during pregnancy as the rate of caffeine metabolism decreases from the first to third trimester (Knutti et al. 1982). This delayed clearance of caffeine leads to higher exposure to caffeine for the fetus, which may in turn affect fetal growth and development. Pregnant women are commonly tried to avoid drinking coffee, but the consumption of food and beverages that contains caffeine might be ignored and thereby causing caffeine exposure during pregnancy. Epidemiological studies have revealed that prenatal caffeine consumption is associated with low birth weight and could lead to IUGR (Vik et al. 2003; Group 2008; Bakker et al. 2010; Chen et al. 2014). Reports also indicate that children who ingest caffeine-containing food or drinks are highly susceptible to metabolic syndromes such as obesity and hypertension (James et al. 2004; Cayetanot et al. 2009). Previously, we have demonstrated that 20, 60, and 180 mg/kg day (Liu et al. 2012; Xu et al. 2012b) or 30, 60 and 120 mg/kg day (Wu et al. 2015) of caffeine exposure (intragastric administration) from gestational days (GD) 11 to 20 lead to IUGR and causes fetal overexposure to high levels of maternal glucocorticoids (GC) in rats. The consequent hypothalamic-pituitary-adrenal (HPA) axis-associated neuroendocrine metabolic programming alteration induced by 120 mg/kg day of caffeine exposure from GD 11 to 20 may further increase the susceptibility to adult metabolic syndrome and relevant metabolic diseases (i.e., non-alcoholic fatty liver diseases) in adult IUGR offspring rats (Liu et al. 2012; Xu et al. 2012a; Wang et al. 2014). However, whether prenatal caffeine exposure (PCE) could alter the glucose metabolic homeostasis in IUGR adult offspring remains unknown. In the present study, we aimed to demonstrate the effects of PCE on glucose homeostasis of adult rats and explore the underlying mechanism. This study is of significance for elucidating the etiology of fetal originated diabetes and relevant diseases.
Material and methods Ethics and animal housing Animal experiments were performed in the Center for Animal Experiments of Wuhan University (Wuhan, China), which has been accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International
(AAALAC International). All animal experimental procedures were approved by and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Chinese Animal Welfare Committee. Specific pathogen-free (SPF) Wistar rats (with weights of 200–240 g for females and 260–300 g for males) were obtained from the Experimental Center of Hubei Medical Scientific Academy (No. 2009-0004, Hubei, China). Animals were housed under standard conditions (room temperature 18–22 °C; humidity 40–60%; 12:12-h light–dark cycle) and allowed free access to rat chow and distilled water. Experimental procedure The IUGR rat model was achieved as previously described (Tan et al. 2012; Xu et al. 2012a; Luo et al. 2014; Wang et al. 2014; Luo et al. 2015). To be specific, two females were placed with one male overnight. The day at which the evidence of mating was observed (i.e., vaginal plug or vaginal smear with sperm cells) was designated as gestational day (GD) 0, whereby the pregnant rats were then caged separately. From GD11 to GD20, pregnant rats were administered 120 mg/kg of caffeine (CAS #58-08-2, > 99% purity, Sigma-Aldrich, MO) once per day and the dams of control group were sham-treated with vehicle. All the animals were subjected to spontaneous delivery. On postnatal day 1, the numbers of pups were normalized to eight pups per litter to ensure adequate and standardized nutrition and the exceeding pups were euthanized by cervical dislocation. After weaning (in postnatal week 4, PW4), one male and one female pup was randomly selected from each dam and fed with lab chow (providing 22% of its energy content as protein, 63% as carbohydrates and only 5% as fat). The selected pups were divided into four groups: male control, male caffeine, female control, and female caffeine. Each group comprised eight pups. Body weights were recorded every week and the corresponding body weight gain rates were calculated as follows: Gain rate ð%Þ ¼ ½Body weight ðPWx Þ−Body weight ðPW1 Þ Body weight ðPW1 Þ 100 Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance tests (ITT) were conducted in PW20 and 21, respectively. In PW 24, the rats were anesthetized with isoflurane (Baxter Healthcare, IL) and sacrificed in a room separate from the other animals. Pancreas were resected and fixed for immunohistochemistry (IHC) and livers were frozen at − 80 °C. Intraperitoneal glucose tolerance test IPGTT was used to assess glucose tolerance in PW20. The overnight fasting can produce low, stable baseline blood
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glucose and insulin levels and obtain consistent excursions in blood glucose after glucose loading (Heikkinen et al. 2007; Muniyappa et al. 2008), so the offspring rats were prefasted overnight for 12 h and then administered glucose intraperitoneally (2 g/kg) at 8:00 a.m. Blood glucose levels at 0, 15, 30, 60, and 120 min after glucose challenge were determined by Accu-Chek® Performa glucose meter via tail clipping. The blood glucose concentrations were normalized by dividing the basal concentration of blood glucose (0 min) prior to the calculation of area under the curve (AUC) based on the trapezoidal rule. Additional blood (300 μl) was collected at 0 and 15 min from the caudal vein to prepare serum for insulin radioimmunoassay following manufacturer’s protocol (Beijing North Institute of Biological Technology, China). To estimate the ability of the β cells to respond to a glucose challenge, we calculated insulin secretion over the 15 min after the injection (ΔI15 − 0) divided by the difference between the glucose concentrations during the same time period (ΔG 15 − 0 ), that is, the ratio of ΔI 15 − 0 to ΔG 15 − 0 (I/G15 − 0) (Jha et al. 2016). Insulin tolerance test As for ITT in PW21, we prefasted the animals for 6 h since 8:00 a.m. in order to get a sufficient depletion of endogenous blood insulin and reduce the possibility of hypoglycemia induced by overnight fasting. An intraperitoneal injection of 0.75 units/kg human insulin (Eli Lilly, IN) were performed at 14:00 p.m. The measurement of blood glucose levels at 0, 15, 30, 60, and 120 min after insulin challenge and the corresponding AUC calculation were the same as described in IPGTT. Morphometric analysis of pancreas Pancreas were weighed and then fixed in 10% formaldehyde and embedded in paraffin. Six complete longitudinal sections (5 μm) with at least 200-μm intervals from each embedded pancreas were obtained through their maximal width and subsequently subjected to insulin IHC. The sections were deparaffinized, rehydrated, and heated in 10 mmol/l citrate buffer (pH 6.0) at 92 °C for 10 min in a microwave oven for antigen retrieval. After cooling at room temperature, the sections were washed three times with PBS, and endogenous peroxidase was blocked by 30-min incubation with 3% H2O2 at room temperature. Then, the sections were incubated overnight at 4 °C with mouse anti-insulin (1:1000, Sigma). Detection was with a streptavidin–biotin–peroxidase complex developed with aminoethylcarbazol (Zymed, San Francisco, CA). As a negative control, pancreatic sections underwent similar treatment, and no positive reactions to the antibody against insulin were observed (not shown).
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The tissue sections were viewed and captured at a magnification of × 20 or × 400 using a Nikon Eclipse Ci-S upright microscope coupled with a DS-U3 digital camera control unit (Nikon, Tokyo, Japan). Morphometric analyses were performed with NIS-Elements Br software (Nikon, Tokyo, Japan). The size of the insulin-positive cell area as well as the pancreatic tissue area of the entire section was measured on six sections per animal, with a total of five animals being analyzed per group. The β fractions (%) were determined as the ratio of the insulin-positive cell area to the total pancreatic tissue area on the entire section. The β cell masses (mg) were obtained through multiplying the β cell fractions (%) by the entire pancreas mass. Western blotting The hepatic protein expressions of insulin receptor (INSR), insulin receptor substrate-1 (IRS-1), serine–threonine protein kinase (Akt), and phosphorylated Akt (pAkt) were determined by western blotting. The liver homogenate tissues were rinsed with ice-cold PBS and then lysed for 30 min at 4 °C in RIPA lysis buffer containing Protease Inhibitor Cocktail (Sigma, St. Louis, MO) and Phosphatase Inhibitor Cocktail (Sigma, St. Louis, MO). The supernatant was harvested, and protein concentrations were determined using a BCA protein assay kit (Beyotime, Shanghai, China) following the manufacturer’s protocol. Aliquots of protein lysates (50 mg/lane) were isolated by SDS-PAGE (10% gels) and blotted onto PVDF membranes (Millipore, Billerica, MA). Membranes were blocked in 5% non-fat milk for 1 h and incubated overnight at 4 °C with the primary antibodies (CST, Danvers, MA), including anti-INSR (1:1000), anti-IRS-1 (1:1000), anti-Akt (1:1000), anti-pAkt (1:1000), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000). After washing in TBST, they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 45 min and visualized using ECL HRP substrate (PerkinElmer, Inc., Boston, MA). Signals of antibody binding were detected by Chemi-doc Image Analyzer (Bio-Rad, Hercules, CA). The immunoblotting experiments were performed three times using different samples. Protein band intensities were analyzed by ImageJ (NIH, Bethesda, MA), and relative protein levels of target genes were normalized by GAPDH protein level. Data analysis and statistics Q u a n t it a t i v e p a r a m e t r i c d a t a w e r e e x p r e s s e d a s mean ± S.E.M., and non-parametric data were indicated as median with interquartile (Battiston et al. 2017). Shapiro– Wilk test was employed to examine the normality of data. The homogeneity of variances was evaluated by Levene’s test. For the repeated measures data (i.e., postnatal bodyweight and the corresponding gain rates, relative glycemic values in
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IPGTT and ITT, serum insulin in IPGTT), two-way repeated measures ANOVA was used to analyze overall effects of PCE over time, followed by multivariate ANOVA for the comparison at every individual time point. The other data (i.e., basal blood glucose-insulin phenotype, AUC, I/G15 − 0, β cell fraction and mass, protein expression of hepatic insulin signaling pathway) were split by gender, and the differences between groups within gender were compared by unpaired Student’s t test assuming normal distribution and equal variance. If normal distribution and equal variance not assumed, log transformation was performed to normalize the data distribution prior to t test evaluation or unpaired Mann–Whitney U test (equivalent to Wilcoxon W) was applied when necessary. Statistical significance was set at P < 0.05. All the statistics were performed with SPSS for Windows version 17 (SPSS Science, Inc., Chicago, IL).
Results
offspring were also greater than those of the control group (P < 0.01). All of the above reveals that PCE increased the glucose tolerance in female adult offspring, accompanied with an enhancement of glucose-stimulated insulin secretion and β cell responsiveness to glucose. However, no such effects were observed in males. Insulin sensitivity ITT was employed to determine whole body insulin sensitivity of adult offspring in PW21. As shown in Fig. 3, the linear glucose decay of both male and female PCE offspring in the first 30 min tended to be greater than those of their controls, and the AUCs in PCE male and female offspring were all significantly decreased when compared with the controls (P < 0.01). In addition, the relative blood glucose levels at 60 and 120 min were all reduced in the PCE group compared with the controls (P < 0.05). These results indicated that the whole body insulin sensitivity of PCE offspring was partly increased.
Postnatal bodyweights Pancreatic β cell fraction and mass As shown in Fig. 1a, the body weights of both male and female offspring from PCE group were lower than those of control animals in PW1 (P < 0.01; Fig. 1a). Although the absolute body weights of PCE offspring fell behind controls from PW4 to PW24, the corresponding bodyweight gain rates in the whole stage or each time point were greater than those of control animals (P < 0.05, P < 0.01; Fig. 1b–e). These results indicate that PCE offspring presented a partial Bcatchup^ growth pattern of body weights without any gender difference.
To determine the effects of PCE on pancreatic morphological development, we measured the fraction and mass of pancreatic β cells. The total pancreatic masses were not altered in the PCE offspring compared with control animals (Fig. 4b, e). Morphometric analysis further revealed that the pancreatic β cell fraction and mass in both male and female offspring from PCE group were lower than those in control counterparts (P < 0.05, P < 0.01; Fig. 4c, d, f, g). Hepatic insulin signaling protein expression
Basal glucose-insulin phenotype, glucose tolerance, and β cell responsiveness to glucose In PW20, glucose tolerance was evaluated by IPGTT after 12 h of fasting. The basal levels of blood glucose (Fig. 2a, e) and insulin (Fig. 2c, g) did not differ between the control and PCE groups. For male offspring, glucose tolerance of the PCE group was not changed when compared to the controls (Fig. 2b). Meanwhile, injection of the glucose caused a pronounced increase in blood concentrations of insulin at 15 min only in the control animals (P < 0.05) but not in the PCE progeny (Fig. 2c). The calculated I/G15 − 0 of the PCE group tended to be lower than those of control counterparts. For female offspring, glucose tolerance of the PCE animals was significantly increased when compared with the controls (P < 0.05; Fig. 2f), as evidenced by the lowered relative blood glucose levels and the corresponding reduced AUC after glucose injection. In addition, PCE also induced higher insulin secretion at 15 min after glucose load than the controls (P < 0.05; Fig. 2g). The calculated I/G15 − 0 of the PCE
The western blotting results showed that PCE significantly enhanced the protein expression levels of hepatic INSR, IRS-1, and total Akt only in the females (P < 0.01, P = 0.068; Fig. 5b, d), but not males. Moreover, the phosphorylation of Akt was also increased in females, as observed by the augmented ratios of pAkt to total Akt proteins (P < 0.01; Fig. 5e).
Discussion Abnormal homeostasis and perturbed metabolism of blood glucose in IUGR adult offspring have been demonstrated by numerous animal studies (Varvarigou 2010). However, we found the interesting phenomenon that PCE offspring rats presented normal glucose homeostasis (i.e., unchanged basal blood glucose and insulin levels), accompanied with the unchanged or increased glucose tolerance. Previous review proposed that IUGR offspring may undergo an age-dependent
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Fig. 1 Effects of prenatal caffeine exposure on postnatal growth and development in offspring rats. Pregnant rats were administered of 120 mg/kg of caffeine once per day from gestational days (GD) 11 to 20. a Body weights at postnatal week (PW) 1. b, c Male body weights and corresponding gain rates from PW4 to 24. d, e Female body mass and corresponding gain rates from PW 4 to 24. Mean ± S.E.M., n = 8 animals/
group per gender. *P < 0.05, **P < 0.01 vs control. Differences of body weights in PW1 were compared by using unpaired Student’s t test. Effects of prenatal caffeine exposure on postnatal body weights, and corresponding gain rates over time were determined by two-way repeated measures ANOVA followed by multivariate ANOVA for the comparison at every individual time point
loss of glucose tolerance even if a better glucose tolerance than control was found in young adult life (Ozanne 2001).
In addition, IUGR offspring from different modeling methods are shown to be glucose-intolerant at various postnatal time
Fig. 2 Effects of prenatal caffeine exposure on basal glucose-insulin phenotype, glucose tolerance and β cell responsiveness to glucose in adult offspring rats. Pregnant rats were administered of 120 mg/kg of caffeine once per day from gestational days (GD) 11 to 20. In postnatal week 20, intraperitoneal glucose tolerance test (IPGTT) was performed to evaluate the glucose tolerance in offspring rats via an intraperitoneal injection of glucose (2 mg/kg) following 12 h prefasting. The β cells responsiveness to glucose was measured as the insulin secretion over the 15 min after the injection (ΔI15 − 0) divided by the difference between the glucose concentrations during the same time period (ΔG15 − 0). a, e Blood glucose levels at 0 min during IPGTT. b, f Normalized blood
glucose levels during IPGTT and area under the curve (AUC). c, g Blood insulin levels at 0 and 15 min during IPGTT. d, h Ratio of ΔI15 − 0 to ΔG15 − 0 (I/G15 − 0). Mean ± S.E.M., n = 8 animals/group per gender. *P < 0.05, **P < 0.01, vs control; #P < 0.05, ##P < 0.01, vs 0 min. Effects of prenatal caffeine exposure on normalized blood glucose over time during IPGTT were determined by two-way repeated measures ANOVA followed by multivariate ANOVA for the comparison at every individual time point. Differences of blood glucose at 0 min, AUC, and I/G15 − 0 between groups were compared by using unpaired Student’s t test
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Fig. 3 Effects of prenatal caffeine exposure on insulin sensitivity in adult offspring rats. Pregnant rats were administered of 120 mg/kg of caffeine once per day from gestational days (GD) 11 to 20. In postnatal week 21, insulin tolerance test (ITT) was performed to evaluate the whole body insulin sensitivity in offspring rats via an intraperitoneal injection of insulin (2 mg/kg) following 6 h prefasting from the morning. a Normalized blood glucose levels during ITT and area under the curve (AUC) in male
offspring. b Normalized blood glucose levels during ITT and AUC in female offspring. Mean ± S.E.M., n = 8 animals/group per gender. *P < 0.05, **P < 0.01, vs control. Effects of prenatal caffeine exposure on normalized blood glucose over time during ITT were determined by two-way repeated measures ANOVA followed by multivariate ANOVA for the comparison at every individual time point. Differences of AUC between groups were compared by using unpaired Student’s t test
points (Simmons et al. 2001; Inoue et al. 2009; Yuan et al. 2011). Therefore, we deduced that the unchanged and increased glucose tolerance in PCE male and female offspring might be associated with our experimental method which induces IUGR by PCE during second and third trimesters. Additionally, we conducted IPGTT in PW20 that is just within the young adult stage of rats, which may also be attributed to the enhancement of glucose tolerance in PCE adult offspring. Glucose tolerance relies on several important aspects, including β cell mass and function-associated insulin secretion as well as insulin availability of targeting organs (Kahn et al. 1993). In physiological condition, both pancreatic insulin secretion and peripheral insulin sensitivity coordinately regulate and maintain the dynamic balance of glucose metabolism. In IPGTT, although the blood insulin levels of PCE male offspring at 15 min after glucose load and the calculated I/G15 − 0 tended to be lower than their controls, the glucose tolerance of PCE male offspring remains unaltered, as observed by the unchanged AUC. These results indicate that there might be no significant reduction of β cell function in PCE male offspring rats, even if the decreased β cell mass could be found. In addition, the increased insulin sensitivity may enhance the glucose disposal so that the in vivo secretion of insulin remains enough to metabolize blood glucose from glucose injection and maintain the normal glucose tolerance in PCE male offspring. With regard to female offspring, the basal blood glucose and insulin levels (0 min in IPGTT) of PCE females were not changed when compared with the control, even though a significant decrease of β cell mass could be observed. Meanwhile, we also found that the β cell sensitization to glucose and whole body insulin sensitivity of PCE females were all potentiated to some extent, as evidenced by the elevated I/G15 − 0 in IPGTT and decreased AUCs of relative glucose curves in ITT. Basing on a series of animal studies, Rafacho
et al. have demonstrated that one of the major adaptive compensatory responses for pancreatic β cell to face the new metabolic demand for insulin includes an increase in responsiveness to glucose (Rafacho et al. 2008; Rafacho et al. 2010a, b). Meanwhile, the IUGR infants who display catch-up growth pattern are often characterized by enhanced metabolic demand to insulin and other growth factor (i.e., insulin growth-like factor 1, IGF1) (Luo et al. 2010). Therefore, we speculated that the increase in β cell sensitization to glucose might be one of the adaptive compensatory responses to the diminished β cell mass in PCE females with a postnatal catch-up growth pattern. The enhanced β cell sensitivity to glucose may trigger much more insulin secretion, which partly recovers the relative deficiency of total insulin biosynthesis due to the decreased β cell population and hence maintains the constant basal blood insulin levels as well as normal glucose homeostasis. In addition, as another compensatory response to the reduced β cell mass, the increased insulin sensitivity was not only observed in the PCE female adult offspring but also has been confirmed in prenatal protein deprived IUGR offspring (Gosby et al. 2010; Lim et al. 2011). Accordingly, when facing the transient glucose challenge in IPGTT, the insulinsensitive PCE females could secret much more insulin than the controls at 15 min due to the increased β cell sensitivity to glucose, which might be contributed to the increased glucose tolerance. Interestingly, Camacho et al. also found the enhanced insulin secretion and insulin sensitivity in young lambs born with IUGR caused by placental insufficiency (Camacho et al. 2017), which is consistent with our observation. Early environmental insults may alter the timing or amplitude of developmental changes leaving the individual with a β cell population poorly suited both quantitatively and qualitatively for postnatal life and ultimately leading to altered glucose metabolism (Hill and Duvillie 2000). Maternal calorie
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Fig. 4 Effects of prenatal caffeine exposure on pancreatic β cell fraction and mass in adult offspring rats. Pregnant rats were administered of 120 mg/kg of caffeine once per day from gestational days (GD) 11 to 20. In postnatal week 24, the offspring rats were sacrificed to collect pancreas. The morphometric analysis of pancreatic β cells was performed based on the insulin immunohistochemical staining. a Representative insulin-staining images (with a magnification × 40). b, e Pancreatic mass. c, f Pancreatic β cell fraction. d, g Pancreatic β cell mass. Mean ± S.E.M., n = 5 animals/ group per gender. *P < 0.05, **P < 0.01 vs control. Differences between groups were compared by using unpaired Student’s t test
restriction during late gestation in mice showed that β cell mass was not only reduced in the newborn but also lower than control after maturity (Inoue et al. 2009). We previously found PCE reduced the population of β cells in fetal rats (unpublished data). In this study, the pancreatic β cell mass of both male and female PCE adult offspring remained lower than those of control animals, indicating that PCE may perturb fetal β cell development and result in postnatal insufficient β cell mass. Insulin/INSR and IGF1/IGF1 receptor (IGF1R) signaling not only participate in the regulation of hepatic glucose metabolism (Cherrington et al. 1998) but also play an important role in the postnatal catch-up growth in offspring with IUGR
(Shen et al. 2014; Wang et al. 2014). In this study, the increased insulin sensitivity of PCE female offspring may be partially explained by the upregulation of hepatic insulin signaling, as supported by the increased protein expressions of INSR and IRS-1 as well as incremental phosphorylation of Akt. However, there were no profound alterations in hepatic insulin signaling in PCE males. Both clinical and experimental evidences have revealed that IUGR male offspring presented increased insulin sensitivity that is associated with the increased insulin signaling in muscle (Ozanne et al. 1996; Jensen et al. 2005), which might be one of the potential reasons for the enhanced insulin sensitivity in PCE male offspring.
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programming the insulin/IGF1 signaling pathway in the liver and muscle (Liu et al. 2012; Xu et al. 2012b; Kou et al. 2014; Wang et al. 2014). Furthermore, we proposed a GC–IGF1 axis programming as the possible underlying mechanism for the development of caffeine-induced IUGR and enhanced susceptibility to adult metabolic syndrome (Wang et al. 2014). Therefore, the GC–IGF1 axis programming may underlie the increased expression of hepatic insulin signaling in PCE female offspring and partly contribute to the observed glucose metabolic phenotype. Both epidemiological survey and animal experiments identify that chronic adult-onset diseases with fetal origin are characterized by gender differences (Laguna-Barraza et al. 2012). Interestingly, here we also found significant gender differences of multiple parameters existed in PCE adult offspring, such as glucose tolerance, glucose-stimulated insulin secretion, and hepatic insulin signaling. An ovarian estrogen, such as estradiol, could ameliorate pancreatic insulin biosynthesis to some extent in a diabetic state (Godsland 2005). On diabetic rodent models, males are much more susceptible to suffering insulin deficiency (Louet et al. 2004). In addition, as a crucial endocrine axis regulating postnatal growth and glucose metabolism, the growth hormone (GH)–IGF1 axis has been verified to be interacted with sex hormones during puberty. Therefore, we assume that gender differences in PCE adult offspring may partially be due to the sensitivity variation of effects of androgens and estrogens on the GH–IGF1 axis, which requires further investigation.
Conclusions Fig. 5 Effects of prenatal caffeine exposure on the protein expressions of key hepatic insulin signaling elements in adult offspring rats. Pregnant rats were administered of 120 mg/kg of caffeine once per day from gestational days (GD) 11 to 20. In postnatal week 24, the offspring rats were sacrificed to collect liver. The relative protein expressions of insulin receptor (INSR), insulin receptor substrate 1 (IRS-1), and serine–threonine protein kinase (Akt) were determined by western blotting and then normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The phosphorylation of Akt was measured as the ratio of phosphorylated Akt (pAkt) to total Akt. a Representative western blotting image. b, d Relative protein expression levels of INSR, IRS-1 and Akt. c, e Ratio of pAkt to Akt. Mean ± S.E.M., n = 3 animals/group per gender. **P < 0.01 vs control. Differences between groups were compared by using unpaired Student’s t test
In recent decades, findings from retrospective human epidemiological studies and experimental animal models have strongly implicated environmental stimulus or insult during fetal and immediate postnatal periods as an important factor contributing to the development of metabolic disturbance via the phenomenon of metabolic programming (Gluckman et al. 2007). Our previous studies have demonstrated that PCE alters the peripheral glucose metabolic programming via inducing fetal overexposure to the elevated maternal GC and further
In conclusion, we demonstrated that PCE reduced the pancreatic β mass but increased the glucose tolerance in adult offspring rats, especially for females. The adaptive compensatory enhancement of β cell responsiveness to glucose and elevated insulin sensitivity mainly mediated by upregulated hepatic insulin signaling might coordinately contribute to the increased glucose tolerance. Our study provided experimental basis and proposed potential mechanism for interpreting the increased susceptibility to diabetes in PCE-induced IUGR adult offspring. Funding informationThis work was granted by the National Science & Technology Pillar Program of China (No. 2013BAI12B01-3), National Natural Science Foundation of China (Nos. 81220108026, 81430089, 81473290, 81703631) and Hubei Province Health and Family Planning Scientific Research Project (Nos. WJ2017C0003, WJ2017M028). Compliance with ethical standards Animal experiments were performed in the Center for Animal Experiments of Wuhan University (Wuhan, China), which has been accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). All animal experimental procedures were approved by and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Chinese Animal Welfare Committee.
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