Krüppel-like factor 4 in transcriptional control of the three unique isoforms of Agouti-related peptide in mice.

Agouti-related peptide (AgRP/Agrp) within the hypothalamic arcuate nucleus (ARC) contributes to the control of energy balance, and dysregulated Agrp may contribute to metabolic adaptation during prolonged obesity. In mice, three isoforms of Agrp are encoded via distinct first exons. Agrp-A (ENSMUST00000005849.11) contributed 95% of total Agrp in mouse ARC, whereas Agrp-B (ENSMUST00000194654.2) dominated in placenta (73%). Conditional deletion of Klf4 from Agrp-expressing cells (Klf4Agrp-KO mice) reduced Agrp mRNA and increased energy expenditure but had no effects on food intake or the relative abundance of Agrp isoforms in the ARC. Chronic high-fat diet feeding masked these effects of Klf4 deletion, highlighting the context-dependent contribution of KLF4 to Agrp control. In the GT1-7 mouse hypothalamic cell culture model, which expresses all three isoforms of Agrp (including Agrp-C, ENSMUST00000194091.6), inhibition of extracellular signal-regulated kinase (ERK) simultaneously increased KLF4 binding to the Agrp promoter and stimulated Agrp expression. In addition, siRNA-mediated knockdown of Klf4 reduced expression of Agrp. We conclude that the expression of individual isoforms of Agrp in the mouse is dependent upon cell type and that KLF4 directly promotes the transcription of Agrp via a mechanism that is superseded during obesity.NEW & NOTEWORTHY In mice, three distinct isoforms of Agouti-related peptide are encoded via distinct first exons. In the arcuate nucleus of the hypothalamus, Krüppel-like factor 4 stimulates transcription of the dominant isoform in lean mice, but this mechanism is altered during diet-induced obesity.

Mitochondrial-targeted antioxidant attenuates preeclampsia-like phenotypes induced by syncytiotrophoblast-specific Gαq signaling.

Syncytiotrophoblast stress is theorized to drive development of preeclampsia, but its molecular causes and consequences remain largely undefined. Multiple hormones implicated in preeclampsia signal via the Gαq cascade, leading to the hypothesis that excess Gαq signaling within the syncytiotrophoblast may contribute. First, we present data supporting increased Gαq signaling and antioxidant responses within villous and syncytiotrophoblast samples of human preeclamptic placenta. Second, Gαq was activated in mouse placenta using Cre-lox and DREADD methodologies. Syncytiotrophoblast-restricted Gαq activation caused hypertension, kidney damage, proteinuria, elevated circulating proinflammatory factors, decreased placental vascularization, diminished spiral artery diameter, and augmented responses to mitochondrial-derived superoxide. Administration of the mitochondrial-targeted antioxidant Mitoquinone attenuated maternal proteinuria, lowered circulating inflammatory and anti-angiogenic mediators, and maintained placental vascularization. These data demonstrate a causal relationship between syncytiotrophoblast stress and the development of preeclampsia and identify elevated Gαq signaling and mitochondrial reactive oxygen species as a cause of this stress.

Angiotensin AT1A receptor signal switching in Agouti-related peptide neurons mediates metabolic rate adaptation during obesity.

Resting metabolic rate (RMR) adaptation occurs during obesity and is hypothesized to contribute to failed weight management. Angiotensin II (Ang-II) type 1 (AT1A) receptors in Agouti-related peptide (AgRP) neurons contribute to the integrative control of RMR, and deletion of AT1A from AgRP neurons causes RMR adaptation. Extracellular patch-clamp recordings identify distinct cellular responses of individual AgRP neurons from lean mice to Ang-II: no response, inhibition via AT1A and Gαi, or stimulation via Ang-II type 2 (AT2) receptors and Gαq. Following diet-induced obesity, a subset of Ang-II/AT1A-inhibited AgRP neurons undergo a spontaneous G-protein "signal switch," whereby AT1A stop inhibiting the cell via Gαi and instead begin stimulating the cell via Gαq. DREADD-mediated activation of Gαi, but not Gαq, in AT1A-expressing AgRP cells stimulates RMR in lean and obese mice. Thus, loss of AT1A-Gαi coupling within the AT1A-expressing AgRP neuron subtype represents a molecular mechanism contributing to RMR adaptation.

Cardiometabolic Consequences of Deleting the Regulator of G protein Signaling-2 (Rgs2) From Cells Expressing Agouti-Related Peptide or the ANG (Angiotensin) II Type 1A Receptor in Mice.

BACKGROUND: RGS (regulator of G protein signaling) family members catalyze the termination of G protein signaling cascades. Single nucleotide polymorphisms in the RGS2 gene in humans have been linked to hypertension, preeclampsia, and anxiety disorders. Mice deficient for Rgs2 (Rgs2Null) exhibit hypertension, anxiety, and altered adipose development and function. METHODS: To study cell-specific functions of RGS2, a novel gene-targeted mouse harboring a conditional allele for the Rgs2 gene (Rgs2Flox) was developed. These mice were bred with mice expressing Cre-recombinase via the Agouti-related peptide locus (Agrp-Cre) to cause deletion of Rgs2 from all cells expressing Agrp (Rgs2Agrp-KO), or a novel transgenic mouse expressing Cre-recombinase via the ANG (angiotensin) type 1A receptor (Agtr1a/ AT1A) promoter encoded in a bacterial artificial chromosome (BAC-AT1A-Cre) to delete Rgs2 in all Agtr1a-expressing cells (Rgs2AT1A-KO). RESULTS: Whereas Rgs2Flox, Rgs2Agrp-KO, and BAC-AT1A-Cre mice exhibited normal growth and survival, Rgs2AT1A-KO exhibited pre-weaning lethality. Relative to littermates, Rgs2Agrp-KO exhibited reduced fat gains when maintained on a high fat diet, associated with increased energy expenditure. Similarly, surviving adult Rgs2AT1A-KO mice also exhibited increased energy expenditure. Surprisingly, given the hypertensive phenotype previously reported for Rgs2Null mice and evidence supporting a role for RGS2 in terminating AT1A signaling in various cell types, Rgs2AT1A-KO mice exhibited normal blood pressure, ingestive behaviors, and renal functions, both before and after chronic infusion of ANG (490 ng/kg/min, sc). CONCLUSIONS: These results demonstrate the development of a novel mouse with conditional expression of Rgs2 and illustrate the role of Rgs2 within selected cell types for cardiometabolic control.

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.

Vascular Dysfunction in Preeclampsia.

Preeclampsia is a life-threatening pregnancy-associated cardiovascular disorder characterized by hypertension and proteinuria at 20 weeks of gestation. Though its exact underlying cause is not precisely defined and likely heterogenous, a plethora of research indicates that in some women with preeclampsia, both maternal and placental vascular dysfunction plays a role in the pathogenesis and can persist into the postpartum period. Potential abnormalities include impaired placentation, incomplete spiral artery remodeling, and endothelial damage, which are further propagated by immune factors, mitochondrial stress, and an imbalance of pro- and antiangiogenic substances. While the field has progressed, current gaps in knowledge include detailed initial molecular mechanisms and effective treatment options. Newfound evidence indicates that vasopressin is an early mediator and biomarker of the disorder, and promising future therapeutic avenues include mitigating mitochondrial dysfunction, excess oxidative stress, and the resulting inflammatory state. In this review, we provide a detailed overview of vascular defects present during preeclampsia and connect well-established notions to newer discoveries at the molecular, cellular, and whole-organism levels.

Exercise in the heat blunts improvements in aerobic power.

INTRODUCTION: PGC-1a has been termed the master regulator of mitochondrial biogenesis. The exercise-induced rise in PGC-1a transcription is blunted when acute exercise takes place in the heat. However, it is unknown if this alteration has functional implications after heat acclimation and exercise training. PURPOSE: To determine the impact of 3 weeks of aerobic exercise training in the heat (33 °C) compared to training in room temperature (20 °C) on thermoregulation, PGC-1a mRNA response, and aerobic power. METHODS: Twenty-one untrained college aged males (age, 24 ± 4 years; height, 178 ± 6 cm) were randomly assigned to 3 weeks of aerobic exercise training in either 33 °C (n = 12) or 20 °C (n = 11) environmental temperatures. RESULTS: The 20 °C training group increased 20 °C [Formula: see text]̇O2peak from 3.21 ± 0.77 to 3.66 ± 0.78 L·min-1 (p < 0.001) while the 33 °C training group did not improve (pre, 3.16 ± 0.48 L·min-1; post, 3.28 ± 0.63 L·min-1; p = 0.283). PGC-1a increased in response to acute aerobic exercise more in 20 °C (6.6 ± 0.7 fold) than 33 °C (4.6 ± 0.7 fold, p = 0.031) before training, but was no different after training in 20 °C (2.4 ± 0.3 fold) or 33 °C (2.4 ± 0.5 fold, p = 0.999). No quantitative alterations in mitochondrial DNA were detected with training or between temperatures (p > 0.05). CONCLUSIONS: This research indicates that exercise in the heat may limit the effectiveness of aerobic exercise at increasing aerobic power. Furthermore, this study demonstrates that heat induced blunting of the normal exercise induced PGC-1a response is eliminated after 3 weeks of heat acclimation.

Exercise- and Cold-Induced Human PGC-1α mRNA Isoform Specific Responses.

Cold exposure in conjunction with aerobic exercise stimulates gene expression of PGC-1α, the master regulator of mitochondrial biogenesis. PGC-1α can be expressed as multiple isoforms due to alternative splicing mechanisms. Among these isoforms is NT-PGC-1α, which produces a truncated form of the PGC-1α protein, as well as isoforms derived from the first exon of the transcript, PGC-1α-a, PGC-1α-b, and PGC-1α-c. Relatively little is known about the individual responses of these isoforms to exercise and environmental temperature. Therefore, we determined the expression of PGC-1α isoforms following an acute bout of cycling in cold (C) and room temperature (RT) conditions. Nine male participants cycled for 1h at 65% Wmax at -2 °C and 20 °C. A muscle biopsy was taken from the vastus lateralis before and 3h post-exercise. RT-qPCR was used to analyze gene expression of PGC-1α isoforms. Gene expression of all PGC-1α isoforms increased due to the exercise intervention (p < 0.05). Exercise and cold exposure induced a greater increase in gene expression for total PGC-1α (p = 0.028) and its truncated isoform, NT-PGC-1α (p = 0.034), but there was no temperature-dependent response in the other PGC-1α isoforms measured. It appears that NT-PGC-1α may have a significant contribution to the reported alterations in the exercise- and temperature-induced PGC-1α response.

Effects of 7°C environmental temperature acclimation during a 3-week training period.

Cold environmental temperatures during exercise and recovery alter the acute response to cellular signaling and training adaptations. Approximately 3 wk is required for cold temperature acclimation to occur. To determine the impact of cold environmental temperature on training adaptations, fitness measurements, and aerobic performance, two groups of 12 untrained male subjects completed 1 h of cycling in 16 temperature acclimation sessions in either a 7°C or 20°C environmental temperature. Fitness assessments before and after acclimation occurred at standard room temperature. Muscle biopsies were taken from the vastus lateralis muscle before and after training to assess molecular markers related to mitochondrial development. Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) mRNA was higher in 7°C than in 20°C in response to acute exercise before training (P = 0.012) but not after training (P = 0.813). PGC-1α mRNA was lower after training (P < 0.001). BNIP3 was lower after training in the 7°C than in the 20°C group (P = 0.017) but not before training (P = 0.549). No other differences occurred between temperature groups in VEGF, ERRα, NRF1, NRF2, TFAM, PINK1, Parkin, or BNIP3L mRNAs (P > 0.05). PGC-1α protein and mtDNA were not different before training, after training, or between temperatures (P > 0.05). Cycling power increased during the daily training (P < 0.001) but was not different between temperatures (P = 0.169). V̇o2peak increased with training (P < 0.001) but was not different between temperature groups (P = 0.460). These data indicate that a 3-wk period of acclimation/training in cold environmental temperatures alters PGC-1α gene expression acutely but this difference is not manifested in a greater increase in V̇o2peak and is dissipated as acclimation takes place.NEW & NOTEWORTHY This study examines the adaptive response of cellular signaling during exercise in cold environmental temperatures. We demonstrate that peroxisome proliferator-activated receptor-γ coactivator 1α mRNA is different between cold and room temperature environments before training but after training this difference no longer exists. This initial difference in transcriptional response between temperatures does not lead to differences in performance measures or increases in protein or mitochondria.

Effects of exercise in a cold environment on gene expression for mitochondrial biogenesis and mitophagy.

Cold exposure during cycling and recovery enhances PGC-1α transcription, but aspects of mitophagy and a more intense cold exposure without recovery occurring in the cold have not been explored. PURPOSE: Determine the expression of genes related to mitochondrial biogenesis and mitophagy following an acute cycling bout at a temperature below freezing compared to that of room temperature. METHODS: Eleven male participants cycled at 65% Wmax for 1 h at -2 °C and 20 °C and then recovered at room temperature for 6 h. A muscle biopsy was taken from the vastus lateralis before exercise, 3 h, and 6 h post-exercise for gene expression analysis. RESULTS: Exercising heart rate and skin temperature were lower in the cold (p < 0.001; p = 0.004), while core temperature was higher (p = 0.016). Temperature had no effect on gene expression (p > 0.05). BNIP3 and BNIP3L mRNA were not influenced by exercise (p = 0.329; p 0.233). PGC-1α and VEGF were higher after cycling (p < 0.001), but the extent of PGC-1α upregulation was reduced 6 h post-exercise (p 0.006). TFAM increased 6 h post-exercise (p = 0.001). NRF2, ERRα, PINK1, and PARK2 decreased 3 h post-exercise (p 0.035; p = 0.005; p = 0.002; p = 0.001), but this downregulation was diminished after 6 h of recovery (p = 0.017; p 0.006; p = 0.043; p = 0.047). NRF1 was marginally attenuated with exercise (p = 0.001). CONCLUSIONS: Exercise induced alterations in gene expression for mitochondrial biogenesis and mitophagy, but these effects were independent of temperature.