Long-term Metabolic Dysfunction Programming in Female Mice by Serial Moderate Restriction of a High-fat High-sucrose Diet.

BACKGROUND: While intermittent fasting leads to weight loss and improved glucose metabolism, food insecurity, the insufficient access to food for a healthy life, is associated with obesity and adverse cardiometabolic health, especially in women. We aimed to characterize the effects of intermittently restricted feeding on energy balance and glucose tolerance in female mice. METHODS: Female C57BL/6J mice were fed a high-fat, high-sucrose diet and intermittently food restricted to 60% of control littermates' ad libitum intake, starting at weaning and until week 19. Restricted mice were subsequently allowed ad libitum access to the same diet. Body composition and energy balance were measured at weeks 18.5, 19, 30, and 40. At week 42, mice underwent an intraperitoneal glucose tolerance test and plasma appetitive hormones measurements after nutrient gavage. RESULTS: During the food restriction phase, restricted mice accrued lower weight and fat mass than controls despite periodic ad libitum food access. Reintroduction of continuous ad libitum food caused increased food intake during the light phase and increased body mass in restricted mice. Minor differences in body composition-adjusted energy expenditure between groups were observed at week 40. At week 42, glucose tolerance was impaired in restricted mice compared to controls, and trends toward lower levels of postprandial anorexigenic hormones glucagon-like peptide-1 and pancreatic polypeptide were observed. CONCLUSION: Our findings suggest that repeated intermittent food restriction leads to changes in eating behavior that predispose to glucose intolerance when food is freely available. Future studies are needed to elucidate the specific mechanisms underlying these changes.

Modulatory effects of estrous cycle on ingestive behaviors and energy balance in young adult C57BL/6J mice maintained on a phytoestrogen-free diet.

The estrous cycle is known to modify food, fluid, and electrolyte intake behaviors and energy homeostasis in various species, in part through fluctuations in estrogen levels. Simultaneously, commonly commercially available rodent dietary formulations greatly vary in soy protein content, and thereby the delivery of biologically active phytoestrogens. To explore the interactions among the estrous cycle, sodium, fluid, and caloric seeking behaviors, and energy homeostasis, young adult C57BL/6J female mice were maintained on a soy protein-free 2920x diet and provided water, or a choice between water and 0.15 mol/L NaCl drink solution. Comprehensive metabolic phenotyping was performed using a multiplexed Promethion (Sable Systems International) system, and estrous stages were determined via daily vaginal cytology. When provided food and water, estrous cycling had no major modulatory effects on intake behaviors or energy balance. When provided a saline solution drink choice, significant modulatory effects of the transition from diestrus to proestrus were observed upon fluid intake patterning, locomotion, and total energy expenditure. Access to saline increased total daily sodium consumption and aspects of energy expenditure, but these effects were not modified by the estrous stage. Collectively, these results indicate that when supplied a phytoestrogen-free diet, the estrous cycle has minor modulatory effects on ingestive behaviors and energy balance in C57BL/6J mice that are sensitive to sodium supply.NEW & NOTEWORTHY When provided a phytoestrogen-free diet, the estrous cycle had very little effect on food and water intake, physical activity, or energy expenditure in C57BL/6J mice. In contrast, when provided an NaCl drink in addition to food and water, the estrous cycle was associated with changes in intake behaviors and energy expenditure. These findings highlight the complex interactions among estrous cycling, dietary formulation, and nutrient presentation upon ingestive behaviors and energy homeostasis in mice.

Differences in Fluid, Electrolyte, and Energy Balance in C57BL/6J Mice (Mus musculus) in Metabolic Caging at Thermoneutral or Standard Room Temperatures.

The Guide for the Care and Use of Laboratory Animals recommends mice be pair or group housed and provided with nesting materials. These provisions support social interactions and are also critical for thermoregulatory behaviors such as huddling and burrowing. However, studies of fluid and electrolyte balance and digestive function may involve use of metabolic caging (MC) systems in which mice are housed individually on wire-mesh floors that permit quantitative collection of urine and feces. MC housing prevents mice from performing their typical huddling and burrowing behaviors. Housing in MC can cause weight loss and behavioral changes in rodents. Here, we tested the hypothesis that MC housing of mice at standard room temperature (SRT, 22 to 23 °C) exposes them to cold stress, which causes metabolic changes in the mice as compared with standard housing. We hypothesized that performing MC studies at a thermoneutral temperature (TNT, 30 °C) would minimize these changes. Fluid, electrolyte, and energy balance and body composition were assessed in male and female C57BL/6J mice housed at SRT or TNT in MC, static microisolation cages, or a multiplexed metabolic phenotyping system designed to mimic static microisolation cages (Promethion, Sable Systems International). In brief, as compared with MC housing at SRT, MC housing at TNT was associated with lower food intake and energy expenditure, absence of weight loss, and lower urine and fecal corticosterone levels. These results indicate that housing in MC at SRT causes cold stress that can be mitigated if MC studies are performed at TNT.

Early-life sodium deprivation programs long-term changes in ingestive behaviors and energy expenditure in C57BL/6J mice.

Postnatal growth failure remains a significant problem for infants born prematurely, despite aggressive efforts to improve perinatal nutrition. Though often dysregulated in early life when children are born preterm, sodium (Na) homeostasis is vital to achieve optimal growth. We hypothesize that insufficient Na supply in this critical period contributes to growth restriction and programmed risks for cardiometabolic disease in later adulthood. Thus, we sought to ascertain the effects of prolonged versus early-life Na depletion on weight gain, body composition, food and water intake behaviors, and energy expenditure in C57BL/6J mice. In one study, mice were provided a low (0.04%)- or normal/high (0.30%)-Na diet between 3 and 18 wk of age. Na-restricted mice demonstrated delayed growth and elevated basal metabolic rate. In a second study, mice were provided 0.04% or 0.30% Na diet between 3 and 6 wk of age and then returned to standard (0.15%)-Na diet through the end of the study. Na-restricted mice exhibited growth delays that quickly caught up on return to standard diet. Between 6 and 18 wk of age, previously restricted mice exhibited sustained, programmed changes in feeding behaviors, reductions in total food intake, and increases in water intake and aerobic energy expenditure while maintaining normal body composition. Although having no effect in control mice, administration of the ganglionic blocker hexamethonium abolished the programmed increase in basal metabolic rate in previously restricted mice. Together these data indicate that early-life Na restriction can cause programmed changes in ingestive behaviors, autonomic function, and energy expenditure that persist well into adulthood.

Genetic Ablation of Prorenin Receptor in the Rostral Ventrolateral Medulla Influences Blood Pressure and Hydromineral Balance in Deoxycorticosterone-Salt Hypertension.

Non-enzymatic activation of renin via its interaction with prorenin receptor (PRR) has been proposed as a key mechanism of local renin-angiotensin system (RAS) activation. The presence of renin and angiotensinogen has been reported in the rostral ventrolateral medulla (RVLM). Overactivation of bulbospinal neurons in the RVLM is linked to hypertension (HTN). Previous studies have shown that the brain RAS plays a role in the pathogenesis of the deoxycorticosterone (DOCA)-salt HTN model. Thus, we hypothesized that PRR in the RVLM is involved in the local activation of the RAS, facilitating the development of DOCA-salt HTN. Selective PRR ablation targeting the RVLM (PRRRVLM-Null mice) resulted in an unexpected sex-dependent and biphasic phenotype in DOCA-salt HTN. That is, PRRRVLM-Null females (but not males) exhibited a significant delay in achieving maximal pressor responses during the initial stage of DOCA-salt HTN. Female PRRRVLM-Null subsequently showed exacerbated DOCA-salt-induced pressor responses during the "maintenance" phase with a maximal peak at 13 d on DOCA-salt. This exacerbated response was associated with an increased sympathetic drive to the resistance arterioles and the kidney, exacerbated fluid and sodium intake and output in response to DOCA-salt, and induced mobilization of fluids from the intracellular to extracellular space concomitant with elevated vasopressin. Ablation of PRR suppressed genes involved in RAS activation and catecholamine synthesis in the RVLM but also induced expression of genes involved in inflammatory responses. This study illustrates complex and sex-dependent roles of PRR in the neural control of BP and hydromineral balance through autonomic and neuroendocrine systems. Graphical abstract.

Sodium and Growth in Preterm Infants: A Review.

Aim: This article is intended to review the relationship between sodium homeostasis and growth, outline reasons why preterm infants may become sodium deficient, and share data from our group and others regarding the potential benefits of dietary sodium supplementation. Background: Despite tremendous efforts over the past 20 years to optimize neonatal nutrition, postnatal growth failure in preterm infants remains a significant problem. Compelling associations have been identified between in-hospital growth failure and cardiometabolic and neurodevelopmental disorders, heightening the need to further identify the optimal nutritional needs of preterm infants. Results: The impact of sodium deficiency may have on somatic growth is poorly studied and reported upon within the human literature. In contrast, animal studies dating back almost 100 years highlight the nutritional importance of dietary sodium. Sodium homeostasis during early postnatal life is understudied and underappreciated by neonatologists. Conclusion: Insufficient sodium intake during early life is likely a critical yet underappreciated contributor to growth failure. Total body sodium depletion may be an important risk factor driving complications of premature birth. Clinical significance: Increased awareness of sodium homeostasis in preterm infants may improve outcomes in this population. Sodium intake recommendations are provided based on the interpretation of currently available literature.

Cardiometabolic Effects of DOCA-Salt in Mice Depend on Ambient Temperature.

BACKGROUND: Mice prefer warmer environments than humans. For this reason, behavioral and physiological thermoregulatory responses are engaged by mice in response to a standard room temperature of 22 to 24 °C. Autonomic mechanisms mediating thermoregulatory responses overlap with mechanisms activated in hypertension, and, therefore, we hypothesized that housing at thermoneutral temperatures (TNs; 30 °C) would modify the cardiometabolic effects of deoxycorticosterone acetate (DOCA)-salt in mice. METHODS: The effects of DOCA-salt treatment upon ingestive behaviors, energy expenditure, blood pressure, heart rate (HR), and core temperature were assessed in C57BL/6J mice housed at room temperature or TN. RESULTS: Housing at TN reduced food intake, energy expenditure, blood pressure, and HR and attenuated HR responses to acute autonomic blockade by chlorisondamine. At room temperature, DOCA-salt caused expected increases in fluid intake, sodium retention in osmotically inactive pools, blood pressure, core temperature, and also caused expected decreases in fat-free mass, total body water, and HR. At TN, the effects of DOCA-salt upon fluid intake, fat gains, hydration, and core temperature were exaggerated, but effects on energy expenditure and HR were blunted. Effects of DOCA-salt upon blood pressure were similar for 3 weeks and exaggerated by TN housing in the fourth week. CONCLUSIONS: Ambient temperature robustly influences behavioral and physiological functions in mice, including metabolic and cardiovascular phenotype development in response to DOCA-salt treatment. Studying cardiometabolic responses of mice at optimal ambient temperatures promises to improve the translational relevance of rodent models.

ARRB2 (ß-Arrestin-2) Deficiency Alters Fluid Homeostasis and Blood Pressure Regulation.

BACKGROUND: GPCRs (G protein-coupled receptors) are implicated in blood pressure (BP) and fluid intake regulation. There is a developing concept that these effects are mediated by both canonical G protein signaling and noncanonical ß-arrestin mediated signaling, but the contributions of each remain largely unexplored. Here, we hypothesized that ß-arrestin contributes to fluid homeostasis and blood pressure (BP) regulation in deoxycorticosterone acetate (DOCA) salt hypertension, a prototypical model of salt-sensitive hypertension. METHODS: Global ß-arrestin1 (Arrb1) and ß-arrestin2 (Arrb2) knockout mice were employed to evaluate drinking behavior, and BP was evaluated in Arrb2-knockout mice. Age- and sex-matched C57BL/6 mice served as controls. We measured intake of water and different sodium chloride solutions and BP employing a 2-bottle choice paradigm with and without DOCA. RESULTS: Without DOCA (baseline), Arrb2-knockout mice exhibited a significant elevation in saline intake with no change in water intake. With DOCA treatment, Arrb2-knockout mice exhibited a significant increase in both saline and water intake. Although Arrb2-knockout mice exhibited hypernatremia at baseline conditions, we did not find significant changes in total body sodium stores or sodium palatability. In a separate cohort, BP was measured via telemetry in Arrb2-knockout and C57BL/6 mice with and without DOCA. Arrb2-knockout did not exhibit significant differences in BP before DOCA treatment when provided water alone, or when provided a choice of water and saline. However, Arrb2-knockout exhibited an increased pressor response to DOCA-salt. CONCLUSIONS: These findings suggest that in salt-sensitive hypertension, ARRB2, but not ARRB1 (ß-arrestin 1), might counterbalance the canonical signaling of GPCRs.

Chronic intracerebroventricular infusion of angiotensin II causes dose- and sex-dependent effects on intake behaviors and energy homeostasis in C57BL/6J mice.

The renin-angiotensin system (RAS) within the brain is implicated in the control of fluid and electrolyte balance, autonomic functions, blood pressure, and energy expenditure. Mouse models are increasingly used to explore these mechanisms; however, sex and dose dependencies of effects elicited by chronic intracerebroventricular (ICV) angiotensin II (ANG II) infusion have not been carefully established in this species. To examine the interactions among sex, body mass, and ICV ANG II on ingestive behaviors and energy balance, young adult C57BL/6J mice of both sexes were studied in a multiplexed metabolic phenotyping system (Promethion) during chronic infusion of ANG II (0, 5, 20, or 50 ng/h). At these infusion rates, ANG II caused accelerating dose-dependent increases in drinking and total energy expenditure in male mice, but female mice exhibited a complex biphasic response with maximum responses at 5 ng/h. Body mass differences did not account for sex-dependent differences in drinking behavior or total energy expenditure. In contrast, resting metabolic rate was similarly increased by ICV ANG II in a dose-dependent manner in both sexes after correction for body mass. We conclude that chronic ICV ANG II stimulates water intake, resting, and total energy expenditure in male C57BL/6J mice following straightforward accelerating dose-dependent kinetics, but female C57BL/6J mice exhibit complex biphasic responses to ICV ANG II. Furthermore, control of resting metabolic rate by ANG II is dissociable from mechanisms controlling fluid intake and total energy expenditure. Future studies of the sex dependency of ANG II within the brain of mice must be designed to carefully consider the biphasic responses that occur in females.

Cardiometabolic effects of DOCA-salt in male C57BL/6J mice are variably dependent on sodium and nonsodium components of diet.

Hypertension characterized by low circulating renin activity accounts for roughly 25%-30% of primary hypertension in humans and can be modeled experimentally via deoxycorticosterone acetate (DOCA)-salt treatment. In this model, phenotypes develop in progressive phases, although the timelines and relative contributions of various mechanisms to phenotype development can be distinct between laboratories. To explore interactions among environmental influences such as diet formulation and dietary sodium (Na) content on phenotype development in the DOCA-salt paradigm, we examined an array of cardiometabolic endpoints in young adult male C57BL/6J mice during sham or DOCA-salt treatments when mice were maintained on several common, commercially available laboratory rodent "chow" diets including PicoLab 5L0D (0.39% Na), Envigo 7913 (0.31% Na), Envigo 2920x (0.15% Na), or a customized version of Envigo 2920x (0.4% Na). Energy balance (weight gain, food intake, digestive efficiency, and energy efficiency), fluid and electrolyte homeostasis (fluid intake, Na intake, fecal Na content, hydration, and fluid compartmentalization), renal functions (urine production rate, glomerular filtration rate, urine Na excretion, renal expression of renin, vasopressin receptors, aquaporin-2 and relationships among markers of vasopressin release, aquaporin-2 shedding, and urine osmolality), and blood pressure, all exhibited changes that were subject to interactions between diet and DOCA-salt. Interestingly, some of these phenotypes, including blood pressure and hydration, were dependent on nonsodium dietary components, as Na-matched diets resulted in distinct phenotype development. These findings provide a broad and robust illustration of an environment × treatment interaction that impacts the use and interpretation of a common rodent model of low-renin hypertension.

Methods for the Comprehensive in vivo Analysis of Energy Flux, Fluid Homeostasis, Blood Pressure, and Ventilatory Function in Rodents.

Cardiovascular disease represents the leading cause of death in the United States, and metabolic diseases such as obesity represent the primary impediment to improving cardiovascular health. Rodent (mouse and rat) models are widely used to model cardiometabolic disease, and as a result, there is increasing interest in the development of accurate and precise methodologies with sufficiently high resolution to dissect mechanisms controlling cardiometabolic physiology in these small organisms. Further, there is great utility in the development of centralized core facilities furnished with high-throughput equipment configurations and staffed with professional content experts to guide investigators and ensure the rigor and reproducibility of experimental endeavors. Here, we outline the array of specialized equipment and approaches that are employed within the Comprehensive Rodent Metabolic Phenotyping Core (CRMPC) and our collaborating laboratories within the Departments of Physiology, Pediatrics, Microbiology & Immunology, and Biomedical Engineering at the Medical College of Wisconsin (MCW), for the detailed mechanistic dissection of cardiometabolic function in mice and rats. We highlight selected methods for the analysis of body composition and fluid compartmentalization, electrolyte accumulation and flux, energy accumulation and flux, physical activity, ingestive behaviors, ventilatory function, blood pressure, heart rate, autonomic function, and assessment and manipulation of the gut microbiota. Further, we include discussion of the advantages and disadvantages of these approaches for their use with rodent models, and considerations for experimental designs using these methods.

Short-term Housing in Metabolic Caging on Measures of Energy and Fluid Balance in Male C57BL/6J Mice ( Mus musculus).

Metabolic caging is an important tool for quantitative urine and feces collection in rodents, although significant limitations and problems accompany its use. Despite strong opinions among investigators regarding the effects of metabolic caging on energy and fluid homeostasis, careful quantitative analysis of the impact of this caging type-particularly when used for mice-is lacking. The current study assessed the effects of metabolic caging, with or without modifications such as plastic platform inserts, on ingestive behaviors, energy expenditure, accuracy of urine and fecal collection, and ambulatory activities in male C57BL/6J mice. Housing mice in metabolic cages, regardless of platform inclusion, increased energy expenditure without modifying food intake, presumably due to the inability of mice to perform normal thermoregulatory behaviors (burrowing and huddling). Surprisingly, mice in metabolic cages actively avoided platforms, and the inclusion of platforms modified the behavior of the mice and had position-dependent effects that reduced the accuracy of urine collection. Moving mice from cohousing to individual housing in home cages also increased ingestive behaviors and energy expenditure. We conclude that single housing of male C57BL/6J mice increases energy expenditure, that this increase is potentiated in metabolic caging conditions, and that platforms in metabolic cages alter mouse behavior and urine collection. Additional future work is needed to determine the potential benefits of using higher ambient temperature for studies of mice in metabolic caging and whether the above effects occur in females and other strains of mice and other rodent species.

Maturational changes in sodium metabolism in periviable infants.

BACKGROUND: Sodium depletion results in impaired somatic growth. The sodium requirements of extremely preterm (periviable) infants early in life are not known. We therefore investigated sodium homeostasis in this population over the first 10 weeks following birth. METHODS: This was a longitudinal, observational study of sodium intake and urine sodium excretion in a convenience cohort of 23 infants born at 22 0/7-23 6/7-week gestation. RESULTS: Sodium intake ranged from 5.2 ± 0.4 to a maximum of 7.9 ± 0.5 mEq/kg/day at 2 and 8 weeks of postnatal age, respectively, while urinary sodium loss was 7.7 ± 1.0 mEq/kg/day and 6.9 ± 0.7 mEq/kg/day at these time points. Sodium balance (sodium intake - urine sodium output) was first positive at 6 weeks of age, though a positive sodium balance exceeding 1.4 mEq/kg/day (i.e., a balance associated with weight gain of 30 g/day) was not observed until 10 weeks. CONCLUSIONS: Infants born at 22-23-week gestational age have a prolonged period of high urinary losses of sodium and negative sodium balance. Sodium intakes greater than those currently recommended by the American Academy of Pediatrics are needed to achieve a significant positive sodium balance in this population.

Dissociable effects of dietary sodium in early life upon somatic growth, fluid homeostasis, and spatial memory in mice of both sexes.

Postnatal growth failure is a common morbidity for preterm infants and is associated with adverse neurodevelopmental outcomes. Although sodium (Na) deficiency early in life impairs somatic growth, its impact on neurocognitive functions has not been extensively studied. We hypothesized that Na deficiency during early life is sufficient to cause growth failure and program neurobehavioral impairments in later life. C57BL/6J mice were placed on low- (0.4), normal- (1.5), or high- (3 g/kg) Na chow at weaning (PD22) and continued on the diet for 3 wk (to PD40). Body composition and fluid distribution were determined serially by time-domain NMR and bioimpedance spectroscopy, and anxiety, learning, and memory were assessed using the elevated plus maze and Morris water maze paradigms in later adulthood (PD63-PD69). During the diet intervention, body mass gains were suppressed in the low- compared with normal- and high-Na groups despite similar caloric uptake rates across groups. Fat mass was reduced in males but not in females fed low-Na diet. Fat-free mass and hydration were significantly reduced in both males and females fed the low-Na diet, although rapidly corrected after return to normal diet. Measures of anxiety-like behavior and learning in adulthood were not affected by diet in either sex, yet memory performance was modified by a complex interaction between sex and early life Na intake. These data support the concepts that Na deficiency impairs growth and that the amount of Na intake which supports optimal somatic growth during early life may be insufficient to fully support neurocognitive development.

Quantification of body fluid compartmentalization by combined time-domain nuclear magnetic resonance and bioimpedance spectroscopy.

The measurement of fluid compartmentalization, or the distribution of fluid volume between extracellular (ECF) and intracellular (ICF) spaces, historically requires complicated, burdensome, and often terminal methodologies that do not permit repeated or longitudinal experiments. New technologies including time-domain nuclear magnetic resonance (TD-NMR)-based methods allow for highly accurate measurements of total body water (TBW) within minutes in a noninvasive manner, but do not permit dissection of ECF versus ICF reservoirs. In contrast, methods such as bioimpedance spectroscopy (BIS) allow dissection of ECF versus ICF reservoirs but are hampered by dependence on many nuanced details in data collection that undermine confidence in experimental results. Here, we present a novel combinatorial use of these two technologies (NMR/BIS) to improve the accuracy of BIS-based assessments of ECF and ICF, while maintaining the advantages of these minimally invasive methods. Briefly, mice undergo TD-NMR and BIS-based measures, and then fat masses as derived by TD-NMR are used to correct BIS outputs. Mice of the C57BL/6J background were studied using NMR/BIS methods to assess the effects of acute furosemide injection and diet-induced obesity on fluid compartmentalization, and to examine the influence of sex, body mass and composition, and diet on TBW, ECF, and ICF. We discovered that in mice, sex and body size/composition have substantial and interactive effects on fluid compartmentalization. We propose that the combinatorial use of NMR/BIS methods will enable a revisioning of the types of longitudinal, kinetic studies that can be performed to understand the impact of various interventions on body fluid homeostasis.

Fetal storage of osmotically inactive sodium.

Work in adult humans and animals suggest sodium (Na) is stored in tissue reservoirs without commensurate water retention. These stores may protect from water loss, regulate immune function, and participate in blood pressure regulation. A role for such stores early in life, during which total body Na sufficiency is vital for optimal growth, has not been explored. Using data from previously published literature, we calculated total body stores of Na, potassium (K), and chloride (Cl) during fetal development (24-40 wk gestation) using two methods 1) based on the distribution of body water mass within extracellular and intracellular compartments, and 2) reported total mineral content. Based on differences between the models, we argue that Na, and to a lesser extent Cl, but not K, are stored in osmotically inactive pools within the fetus that increase with advancing gestational age. Because human breastmilk is relatively Na deficient, we speculate the fetal osmotically inactive Na pool is vital for providing a sufficient total body Na content that supports optimal postnatal growth.