Objective
A maternal high-fat diet creates an increased risk of offspring obesity and systemic hypertension. Although the renal renin-angiotensin system (RAS) is known to regulate blood pressure, it is now recognized that the RAS is also activated in adipose tissue during obesity. We hypothesized that programmed offspring hypertension is associated with the activation of the adipose tissue RAS in the offspring of obese rat dams.
Study Design
At 3 weeks of age, female rats were weaned to a high-fat diet (60% k/cal; n = 6) or control diet (10% k/cal; n = 6). At 11 weeks of age, these rats were mated and continued on their respective diets during pregnancy. After birth, at 1 day of age, subcutaneous adipose tissue was collected; litter size was standardized, and pups were cross-fostered to either control or high-fat diet dams, which created 4 study groups. At 21 days of age, offspring were weaned to control or high-fat diet. At 6 months of age, body fat and blood pressure were measured. Thereafter, subcutaneous and retroperitoneal adipose tissue was harvested from male offspring. Protein expression of adipose tissue RAS components were determined by Western blotting.
Results
The maternal high-fat diet induced early and persistent alterations in offspring adipose RAS components. These changes were dependent on the period of exposure to the maternal high-fat diet, were adipose tissue specific (subcutaneous and retroperitoneal), and were exacerbated by a postnatal high-fat diet. Maternal high-fat diet increased adiposity and blood pressure in offspring, regardless of the period of exposure.
Conclusion
These findings suggest that programmed adiposity and the activation of the adipose tissue RAS are associated with hypertension in offspring of obese dams.
Obesity and its associated health problems represent a worldwide epidemic. Maternal obesity has been shown to increase the risk of offspring obesity and its related diseases, such as heart disease, stroke, and hypertension. Hence, as the prevalence of obesity among pregnant women continues to rise, an increasing number of children are exposed to an “obese intrauterine environment” during development. It is clear that there is a strong correlation between the incidence of hypertension and obesity, more specifically visceral and abdominal obesity. According to data from the Framingham Cohort, obesity by itself accounts for 78% and 65% of essential hypertension in men and women, respectively. Although the mechanisms underlying these associations are not elucidated fully, adipose tissue clearly is a critical factor in the development of obesity-hypertension.
The renin-angiotensin system (RAS) is a classic endocrine system that is involved in blood pressure homeostasis. Angiotensinogen, the precursor of the bioactive peptide angiotensin II (Ang II), is synthesized mainly in the liver and cleaved enzymatically in the circulation by renin to angiotensin I and subsequently by the angiotensin-converting enzyme (ACE) to Ang II. The major vasopressor effects of Ang II are mediated through its receptor type 1 (AT1) whereas its interaction with receptor type 2 (AT2) modulates cell proliferation and renal sodium excretion. In addition to the classic pathway of Ang II synthesis, adipose tissue has the ability to synthesize Ang II independently of the circulating RAS. Recent observations indicate that all components of the RAS are expressed in white adipose tissue from rodents and humans, which suggests that the adipogenic RAS may be involved in the pathogenesis of obesity-related hypertension. Accordingly, studies have shown that adipose-derived angiotensinogen can contribute to approximately 20% of plasma angiotensinogen concentrations and can modulate blood pressure. Angiotensinogen-knockout mice are hypotensive with hypotrophic adipocytes and adipose tissue–specific angiotensinogen gene expression in angiotensinogen-knockout mice limits angiotensinogen expression to the adipose tissue but results in systemic concentrations of angiotensinogen levels that are 20% of wild-type levels, which shows that angiotensinogen that is produced in adipocytes can enter the circulation. Furthermore, over-expression of adipose angiotensinogen in mice induces hypertension with increased body fat and plasma angiotensinogen levels, which indicates that an increased adipose tissue mass may result in higher circulating angiotensinogen levels, a finding that has been confirmed in obese individuals.
Increasing evidence suggests that adult cardiovascular and metabolic disorders can be “programmed” in utero and in the early postnatal period. Notably, both maternal under- or over-nutrition results in offspring obesity and hypertension. Despite this convincing evidence, there are no studies that have investigated the regulation of the adipogenic RAS in programmed, obesity-mediated hypertension. We hypothesized that the adipose tissue RAS is activated in the obese, hypertensive offspring that were exposed to maternal high-fat diet during pregnancy and/or lactation. We determined the protein expression of adipose RAS components in newborn rats and adult offspring that were exposed to maternal high-fat diet during pregnancy and/or lactation. We further investigated the changes in visceral and nonvisceral adipose tissue and the additive impact of a high-fat, postweaning diet on the adipose RAS.
Materials and Methods
A rat model of maternal obesity was created with a high-fat diet before mating and throughout pregnancy and lactation. Studies were approved by the Animal Care and Use Committee of the Los Angeles Biomedical Research Institute at Harbor–University of California, Los Angeles, and were in accordance with the American Association for Accreditation of Laboratory Care and National Institutes of Health guidelines. Sprague Dawley rats (Charles River Laboratories, Hollister, CA) were housed in a facility with constant temperature and humidity and a controlled 12-/12-hour light/dark cycle. Weanling female rats were fed a high-fat diet (60% k/cal fat, 20% k/cal protein, 20% k/cal carbohydrate; D12492 ; Research Diets Inc, New Brunswick, NJ; n = 6) or normal-fat control diet (10% k/cal fat, 20% k/cal protein, 70% k/cal carbohydrate; n = 6). At 11 weeks of age, rats were mated and continued on their respective diets during pregnancy and lactation.
Offspring
At birth, pups were culled to 8 per litter (4 male and 4 female) to normalize rearing and were cross-fostered, thereby generating 4 paradigms of maternal diets during pregnancy/lactation ( Figure 1 ). To control for the cross-fostering effects, the control diet and high-fat diet pups similarly were cross-fostered among dams of the same group ( Figure 1 ). At 3 weeks of age, offspring in each of the 4 groups were housed individually and weaned. To examine the effects of a high-fat diet, 1 male from each litter was weaned randomly to a normal-fat diet, and 1 male was weaned to a high-fat diet. Thus, there were 4 maternal feeding paradigms during pregnancy/lactation (control diet or high-fat diet) and 2 offspring feeding paradigms (control diet or high-fat diet) that were examined for male offspring ( Figure 1 ). We elected to study males because females would have required estrus assessment, and estrogen is known to affect adiposity and lipid metabolism.
Body weight and composition
Male offspring were weighed at 1 day and at 6 months of age. In addition, at 6 months of age, a noninvasive dual-energy X-ray absorptiometry scan was performed with the DXA system with a software program for small animals (6 males from 6 litters per group and postweaning diet; QDR 4500A; Hologic, Bedford, MA). An in vivo scan of whole body composition was obtained that allowed determination of the percentage of body fat.
Blood pressure
Measurements were undertaken in conscious animals with the use of noninvasive tail-cuff sphygmomanometry (ML125 NIPB System; AD Instruments, Colorado Springs, CO) method. Several cuff sizes were used depending on the weight of the animal. To circumvent the potential problem of restrain-induced stress, the animals were acclimatized for at least 1 week with placement in the restraint. Because adult offspring that were weaned to the high-fat diet had markedly increased bodyweights, we were unable to measure blood pressure because of unavailability of tail-cuff size in that range.
Tissue collection, protein extraction and Western blotting
At 1 day of age, subcutaneous adipose tissue was collected, and tissue was pooled from 4 males per litter. In each group, 6 litters were studied. At 6 months of age, both subcutaneous and retroperitoneal (visceral) intraabdominal fat depot adipose tissue were collected; 6 males from 6 litters per group per postnatal weaning diet were studied, Adipose tissue samples were frozen in liquid nitrogen and stored at –80°C until protein analysis. Protein was extracted in radioimmunoprecipitation assay buffer that contained protease inhibitors (HALT cocktail; Pierce, Rockford, IL). Supernatant protein concentration was determined by bicinchoninic acid solution (Pierce). Protein expression was determined as previously described. The primary and secondary antibodies were angiotensinogen (Santa Cruz SC-7419; primary 1:500, secondary 1:2000); ACE (Santa Cruz SC-23909; primary 1:500, secondary 1:2000); AT1 (Santa Cruz SC-1573; primary 1:500, secondary 1:2000); and AT2 (Santa Cruz SC-9040; primary 1:500, secondary 1:2000). All commercial antibodies were optimized for binding specificity, and bands that are depicted have the expected molecular weights.
Statistical analysis
At 1 day of age, differences between high-fat diet and control diet were compared with the use of the unpaired Student t test. At 6 months of age, differences between the 4 groups were compared by 1-way or 2-way analysis of variance, as appropriate, with the experimental group (eg, control diet/control diet) and postweaning diet (control diet or high-fat diet) as factors and with the use of Dunnett’s post-hoc test. Probability values of ≤ .05 were considered significant.
Results
Bodyweight and body fat
Consumption of a high-fat diet resulted in maternal obesity both at term (control diet dams [378 ± 4 g] vs high-fat diet dams [430 ± 7 g]; P < .05) and at the end of the lactation period (control diet dams [309 ± 5 g] vs high-fat diet dams [345 ± 6 g]; P < .05).
At 1 day of age, high-fat diet male newborn rats had similar body weights to the control diet male newborn rats (7.4 ± 0.2 vs 7.3 ± 0.1 g). However, the maternal diet during lactation had a significant impact on body weights of the adult offspring. High-fat diet newborns that were nursed by high-fat diet dams (high-fat diet/high-fat diet) and weaned to a normal-fat diet exhibited significantly higher body weight at 6 months of age, whereas high-fat diet newborns that were nursed by control dams (high-fat diet/control diet) continued to exhibit similar body weights as the control diet newborns. In addition, control diet newborns that were nursed by high-fat diet dams (control diet/high-fat diet) showed significantly increased body weight. This pattern was maintained when offspring were weaned to a postweaning high-fat diet; all groups showed increased body weights compared with the respective offspring that were weaned to a normal-fat diet ( Figure 1 ).
All offspring that were exposed to maternal high-fat diet had significantly increased percentages of body fat when compared with the control diet, regardless of the timing of high-fat diet exposure (pregnancy and/or lactation; Figure 1 ).
Blood pressure
At 6 months of age irrespective of body weight and regardless of the timing of exposure to maternal high-fat diet, all high-fat diet groups that had been weaned to normal-fat diet showed significantly increased systolic and diastolic blood pressure compared with the control diet groups ( Figure 2 , A and B). As stated in the Materials and Methods section of this article, we were unable to measure blood pressure of offspring that were weaned to a high-fat diet.
Adipose protein expression of angiotensinogen
At 1 day of age, high-fat diet newborns had significantly decreased protein expression of subcutaneous adipose angiotensinogen ( Figure 3 , A). At 6 months of age, high-fat diet offspring showed differential protein expression of angiotensinogen, depending on the timing of exposure to the maternal high-fat diet and the composition of the postweaning diet. When high-fat diet/high-fat diet offspring were weaned to a control diet, angiotensinogen protein expression was comparable with the control diet/control diet animals in subcutaneous adipose tissue, but with a decrease in retroperitoneal adipose tissue ( Figure 4 ). In contrast, high-fat diet/control diet and control diet/high-fat diet offspring that were weaned to a control diet had markedly increased angiotensinogen level in both subcutaneous and retroperitoneal adipose tissue. When weaned to a high-fat diet, angiotensinogen protein expression was increased significantly in subcutaneous and decreased in retroperitoneal adipose in all high-fat diet groups compared with the control diet/control diet group ( Figure 4 ).
Adipose protein expression of ACE
At 1 day of age, high-fat diet newborns had significantly increased protein expression of subcutaneous adipose ACE ( Figure 3 , B). At 6 months of age, when high-fat diet/high-fat diet offspring were weaned to a control diet, the changes in the protein expression of ACE were similar to the changes in angiotensinogen; ACE expression was comparable with control diet/control diet in subcutaneous adipose and decreased relative to control diet/control diet in retroperitoneal adipose tissue ( Figure 4 ). In contrast, when high-fat diet/control diet and control diet/high-fat diet male offspring were weaned to a control diet, the subcutaneous adipose ACE protein expression was unchanged, despite elevated subcutaneous adipose tissue angiotensinogen in these animals. Retroperitoneal adipose ACE expression was increased significantly in high-fat diet/control diet rats that were weaned to a control diet. In contrast, subcutaneous and retroperitoneal adipose ACE expression was decreased significantly in all high-fat diet groups that were weaned to a high-fat diet, except for control diet/high-fat diet animals in which the retroperitoneal adipose tissue ACE expression levels were not different to the control diet/control diet group ( Figure 4 ).
Adipose protein expression of the AT1 and AT2 receptors
At 1 day of age, high-fat diet newborns had significantly increased protein expression of subcutaneous adipose vasoreceptor AT1 and proliferative receptor AT2 ( Figure 3 , C and D). At 6 months of age, subcutaneous adipose tissue AT1 receptor expression was decreased (high-fat diet/high-fat diet), unchanged (high-fat diet/control diet), or increased (control diet/high-fat diet); AT2 receptor expression was increased significantly in all high-fat diet groups when offspring were weaned to a control diet ( Figure 5 , A). In retroperitoneal adipose tissue, AT1 receptor expression was increased in all high-fat diet groups that were weaned to a control diet ( Figure 5 , B). Conversely, although AT2 receptor expression was increased in high-fat diet/control diet and control diet/high-fat diet groups, it was decreased in high-fat diet/high-fat diet offspring that were weaned to a control diet ( Figure 5 , B). When offspring were weaned to a high-fat diet, the protein expression of the AT1 receptor in subcutaneous adipose tissue was increased significantly in the control diet/high-fat diet and the high-fat diet/control diet groups; AT2 receptor expression was increased significantly in all high-fat diet groups ( Figure 5 , A). In retroperitoneal adipose tissue, the protein expression of the AT1 receptor was increased significantly in the control diet/high-fat diet group; AT2 receptor expression was increased in high-fat diet/high-fat diet and control diet/high-fat diet, but not high-fat diet/control diet animals that had been weaned to a high-fat diet ( Figure 5 , B).
