Chronic Phase Shifts Are Associated With Higher Systolic Blood Pressure and Impaired Glucose Tolerance Independently of Estrogen Deficiency in Female Spontaneously Hypertensive Rats

Abstract

Postmenopausal shift workers face increased cardiometabolic risk due to estrogen decline and shift work–induced circadian misalignment. Yet, their combined effects remain poorly understood, especially in hypertensive individuals. This study investigated whether circadian misalignment worsens cardiometabolic parameters in a hypertensive ovariectomized rat model. Female spontaneously hypertensive rats (SHR) were ovariectomized or sham-operated (7-week-old), and then exposed to a chronic phase shift (CPS) protocol or a control light schedule for 10 weeks (n = 9 per group). Measurements included body mass, food and water intake, blood pressure (BP), fasting glucose, glucose tolerance, organ masses, and low-density lipoprotein (LDL) concentration. Ovariectomized rats were heavier and had greater food intake and organ masses than sham-operated rats. However, food intake and organ masses were reduced relative to body mass. CPS rats had greater water intake and reduced liver mass than control light rats. In addition, ovariectomized rats showed lower glucose concentration than sham-operated rats, whereas CPS rats showed higher glucose concentration than control light rats during the oral glucose tolerance test. Moreover, the CPS rats had higher systolic BP. The LDL and fasting glucose concentrations were similar. No interaction between ovariectomy and CPS was observed. These findings suggest that estrogen deficiency increases body mass, but does not worsen cardiometabolic parameters in female SHR. CPS-induced circadian misalignment altered water intake, liver mass, systolic BP and glucose tolerance in the CPS condition in female SHR. This study was unable to monitor physiological or behavioral indicators to confirm circadian misalignment by the CPS protocol. However, the findings provide novel insights into how CPSs independently impair cardiometabolic outcomes in female SHR, with implications for understanding risk in postmenopausal shift workers.

Keywords

hypertension metabolic syndrome glucose control circadian disruption circadian rhythm women’s health

Endogenous circadian rhythms, generated by the master circadian pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus, ensure optimal alignment of behavioral cycles (i.e., sleep/wake, feeding/fasting, rest/activity, etc.) with internal physiological processes (digestion, glucose metabolism, blood pressure [BP] control, etc.) (Green et al., 2008; Silver and Rainbow, 2013; Meléndez-Fernández et al., 2023). Light exposure influences SCN activity, with varying effects depending on the timing of exposure, as represented by phase response curves (PRCs) (Rusak and Groos, 1982; Benloucif and Dubocovich, 1996; Vitaterna et al., 2001; Khalsa et al., 2003; Kripke et al., 2007; Youngstedt et al., 2019). Irregular light exposure such as that experienced by shift workers disrupts circadian rhythms, leading to misalignment of behavioral and physiological processes (Touitou et al., 2017; Chellappa et al., 2020; Price et al., 2022; Meléndez-Fernández et al., 2023).

Circadian misalignment is associated with negative cardiometabolic outcomes in both humans (Scheer et al., 2009; Morris et al., 2015; Dicom et al., 2023) and rodent models (Penev et al., 1998; Martino et al., 2007; Casiraghi et al., 2016; Zitting et al., 2022; Trebucq et al., 2023). Given that postmenopausal women and ovariectomized rodent models already exhibit heightened cardiometabolic susceptibility, it is plausible that circadian misalignment may further compound this risk (Rüger and Scheer, 2009). Recent evidence from murine models suggests sex-specific metabolic adaptations in response to circadian misalignment (In Het Panhuis et al., 2023; Ma et al., 2024). Moreover, estrogen deficiency and circadian misalignment may synergistically worsen cardiometabolic outcomes (Zhu et al., 2015; Anderson et al., 2023). Thus, sex hormones may differentially influence cardiometabolic responses under circadian misalignment.

Postmenopausal women are at an increased risk of developing cardiometabolic disorders (Zuo et al., 2009; Figueiredo Neto et al., 2010; de Marchi et al., 2017). In contrast, premenopausal women exhibit a protective advantage against cardiometabolic risk compared with age-matched men (Chikwati et al., 2023; Meloni et al., 2023). However, this advantage diminishes following menopause, leading to a risk profile equivalent to that of men (Zhernakova et al., 2022; Chikwati et al., 2023; Meloni et al., 2023). This shift coincides with the significant decline in estrogen levels during the menopausal transition (Cignarella et al., 2010; Roa-Díaz et al., 2021).

Estrogen deficiency due to ovariectomy (surgical menopause) has been linked with negative cardiometabolic outcomes in both human studies (Colditz et al., 1987; Dørum et al., 2008; Dam et al., 2019) and rodent models, including normotensive (Van Lenten et al., 1983; McElroy and Wade, 1987; Fahmy et al., 2018) and hypertensive rats (Hinojosa-Laborde et al., 2000; Santos et al., 2004; da Silva et al., 2009). Estrogen loss has also been implicated in the onset of hypertension, further exacerbating cardiometabolic risk (Staessen et al., 1989; Zanchetti et al., 2005; Lima et al., 2012). Thus, the association between estrogen loss and heightened cardiometabolic risk underscores postmenopausal women’s susceptibility to developing cardiometabolic disorders.

The impact of this interaction between sex hormones, circadian misalignment, and cardiometabolic processes, especially in the context of hypertension, remains unclear. This highlights the necessity to understand the potential interactions between circadian misalignment and estrogen loss. This study aimed to address this gap by investigating whether circadian misalignment worsens estrogen deficiency–induced cardiometabolic outcomes. We used ovariectomized female spontaneously hypertensive rats (SHR), a rodent model of human essential hypertension (Kren et al., 1997; Pravenec et al., 2014; Jama et al., 2022), in combination with a chronic phase shift (CPS) protocol similar to that used by Tran et al. (2021).

Materials and Methods

Experimental Design

All experimental procedures were approved by the Animal Research Ethics Committee of the University of the Witwatersrand (Approval reference: 2017/11/76/C) and were conducted according to the National Institutes of Health Guide for the care and use of laboratory animals. We also used the ARRIVE checklist when writing our report (Percie du Sert et al., 2020). Five-week-old female SHR (n = 36) were obtained from the Wits Research Animal Facility in batches at 6 separate occasions and acclimated to the laboratory environment for 2 weeks. To minimize batch effects, each cohort included animals from all intervention groups (i.e., surgery and lighting schedule changes) conducted under consistent environmental and procedural conditions. The rats were housed individually in clear plastic cages under controlled conditions of constant temperature (26 ± 2 °C) and relative humidity (28 ± 7%), with a 12:12 light-dark cycle. Sawdust and shredded filter paper were used as bedding, and colored play balls were provided as environmental enrichment.

Environmental conditions were monitored and recorded using a data logger (HOBO MX1104 Analog/Temp/RH/Light; Onset Computer Corporation, Bourne, MA, USA). The rats’ general health was monitored twice weekly using the rat grimace scale. The assessment included indicators such as fur condition, body posture, orbital tightening, nose/cheek flattening, ear changes, and whisker changes. All pre-euthanasia procedures, such as body mass, BP and glucose measurements, were conducted in the animal facility where the rats were housed. The rats were briefly removed from their cages as needed. Post-euthanasia procedures were conducted in a dedicated laboratory.

At 7 weeks old, rats were randomly assigned to undergo either bilateral ovariectomy (ovx, n = 18) or a sham operation (sham-operated, n = 18), as previously described (Maluleke et al., 2022). Briefly, the rats were anesthetized by intraperitoneal injections of 100 mg/kg ketamine and 5 mg/kg xylazine cocktail, in addition to 100 mg/kg meloxicam, followed by inhaled isoflurane maintenance (2%-4%) (0900-1200 h). The ovaries, arteries, and fallopian tubes were ligated, and then ovaries were excised. The sham operation involved the same procedure without removing the ovaries. Postoperative care included meloxicam and atipamezole (100 mg/kg each), as well as daily health monitoring, with particular attention to the wound site.

Following recovery, rats were acclimated to weekly procedures and the 12:12 light-dark (0700-1900 h light and 1900-0700 h dark) control light schedule for 3 weeks, as previously described (Martino et al., 2007). The light intensity was maintained at approximately 200 lux during the light phase (day-time) and 0 lux during the dark phase (night-time). At 12 weeks old, rats were randomly assigned to remain on the control light schedule (ovx, n = 9 and sham-operated, n = 9) or exposed to the CPS protocol (ovx, n = 9 and sham-operated, n = 9). The random allocation was done in a non-systematic manner to ensure unbiased distribution across groups. The sample size has been justified in most ovariectomized SHR studies, utilizing 4 to 10 rats per group (da Silva et al., 2017; Pereira et al., 2022; Pizzato et al., 2023; Wang and Yu, 2023; Neves et al., 2024).

The CPS protocol involved a weekly reversal of the 12:12 light-dark cycle, as previously described (Tran et al., 2021)—the CPS protocol: 0700-1900 h light and 1900-0700 h dark schedule during the odd-numbered weeks and the opposite 0700-1900 h dark and 1900-0700 h light schedule during even-numbered weeks. The light-dark cycle shifts in CPS protocol occurred on the first day of every week (Mondays); thus, there were 24 h of light or darkness on the day of the light-dark cycle reversal. During the dark phase, measurements were conducted under dim, indirect lighting to allow visibility while minimizing unintended circadian disruption.

The investigators were aware of the group allocations throughout the study period prior to euthanasia. However, from the point of anesthetizing the rats through to the post-euthanasia assays, the investigators were blinded to group allocations. This blinding was maintained throughout the outcome assessment and data analysis to reduce potential bias. Figure 1 illustrates the light intensity data obtained weekly in both the control light and CPS protocol schedules. All the rats were euthanized after 10 weeks of the 12:12 light-dark cycle intervention.

Figure 1.

The 10-week light-dark intervention study design. The rats were assigned to either the control light (left) or the CPS (right) schedules. The control light-exposed rats were maintained on a 12:12 light-dark cycle with lights-on at 0700 h and lights-off at 1900 h every day. In contrast, the CPS-exposed rats were maintained on a 12:12 light-dark cycle reversed every Mondays weekly (lights-on at 0700 h and lights-off at 1900 h during odd-numbered weeks; lights-on at 1900 h and lights-off at 0700 h during the even-numbered weeks). The white and black bars indicate the duration of periods of light (light phase) and darkness (dark phase), respectively. Light exposure data recorded by the HOBO are represented as a heat map and expressed as mean light intensity (in lux) in the white bars, with the light phase ~200 lux and dark phase = 0 lux in the black bars. CPS = chronic phase shift.

Body Mass, Food, and Water Intake

Body mass, food, and water intake were measured weekly (1000-1200 h Monday, a week after the shift in the light-dark cycle) using a digital scale (LBK6 weighing scale, Adam Equipment S.A. (Pty), Johannesburg, South Africa). The rats had ad libitum access to standard rat chow and plain drinking water throughout the study duration regardless of the appointed light schedule. Specifically, every Monday, food and water supplies were restocked in excess for the following week. This prevented circadian food entrainment caused by daily feeding. To calculate food and water intake weekly the following formula was used: Intake (per week) = initial amount–remaining amount. Food and water spillage within the cage were not measured; thus, intake values may slightly overestimate actual consumption. To account for differences in body mass, the following formula was used: Relative to body mass value = (measured value (g)/body mass (g)) × 100.

Noninvasive BP Measurements

BP was measured once weekly for each rat on a specific day between Tuesday and Thursday (1000-1200 h) using a noninvasive tail-cuff technique that utilizes the NIBP250 BP monitor system (Biopac Systems Inc., Goleta, CA, USA), as previously described (Mogane et al., 2019; Maluleke et al., 2022; Mkhize et al., 2025). Briefly, a conscious rat was restrained and placed on an electric heating pad (Almohadilla Super Plus 13133, San-UP S.A, San Martín, Argentina) to maintain a temperature of ~32 °C. When the rat was relaxed and inactive, systolic and diastolic BP readings were recorded with a 200 mm Hg cuff pressure.

Fasting Blood Glucose Concentration and Oral Glucose Tolerance Test

Weekly Fasting Blood Glucose Measurements

Rats were fasted overnight with free access to plain drinking water. Blood glucose concentration was measured weekly (1000-1200 h Friday) using a calibrated glucose meter (Contour plus, Bayer (Pty) Ltd, Isando-Johannesburg, South Africa) after a tail prick with a 25G × 15 mm needle (Axiology Labs (Pty) Ltd, Meyerton, Vereeniging, South Africa).

Oral Glucose Tolerance Test

Three days before euthanasia, rats were fasted again overnight with free access to plain drinking water. After measuring baseline (0 min) fasting glucose concentration, rats were gavaged with glucose solution (D-(+)-Glucose, Merck Chemicals (Pty) Ltd, Johannesburg, South Africa) at a dose of 2 g/kg body mass. Then, blood glucose concentration was measured at 15, 30, 60, 120, and 180 min post-gavage as described above for the weekly glucose measurements.

Organ and Blood Sampling

At euthanasia, blood samples were collected via cardiac puncture under anesthesia (1000-1300 h), which euthanized the rats by cardiac puncture–induced exsanguination (blood loss). The organs were harvested (heart, left ventricle [LV], left kidney, and liver). Blood was centrifuged (3000 rpm for 10 min), and then sera were collected and stored in a –80 °C freezer.

LDL Concentration Measurement

Rat sera were centrifuged (1000×g at 2-8 °C for 15 min) after reaching room temperature (18-25 °C). LDL concentration was measured using a Rat LDL Enzyme-Linked Immunosorbent Assay (ELISA) kit (Elabscience Biotechnology Inc., Houston, TX, USA) following the manufacturer’s instructions. The kit had a sensitivity of 0.10 μg/mL, a detection range of 0.16-10 μg/mL, and <10% repeatability coefficient of variation.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism version 10.2 for Windows (GraphPad Software Inc., San Diego, CA, USA) and SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA). All continuous variables were assessed for normality using QQ plots in GraphPad Prism, and all met the criteria for normal distribution. Data are presented as mean ± SD.

For variables collected over time (weekly body mass, food, water intake, systolic BP, diastolic BP, fasting blood glucose, and the oral glucose tolerance test [OGTT] at the end of the light intervention), we performed a mixed-model analysis (REML) with a random intercept, assuming a linear effect of time for the weekly variables and a categorical effect of time for OGTT, with fixed effects being estrogen status (ovariectomized vs sham-operated; sham-operated being set as the reference group) and light schedule status (CPS vs Ctr light, Ctr light being set as the reference group). For single time-point variables (organ masses and LDL concentration), a 2-way analysis of variance (ANOVA) was performed, with the main effects of estrogen status and light schedule status. Statistical significance was set at p < 0.05.

Results

The Characteristics of Ovariectomized (Ovx) and Sham-operated SHR Under CPS or Control Light Schedules

Figure 2 illustrates changes in weekly body mass. All groups exhibited increased body mass over time (β = 6.012, p < 0.0001). Ovx rats had higher body mass than sham-operated rats (β = 41.99, p < 0.0001). No interaction between time and intervention group, or estrogen status and light schedule status was observed.

Figure 2.

Changes in weekly body mass in female SHR. Body mass measurements were taken weekly, starting from the week of surgery (Sx) and continuing through the 10-week light-dark intervention period (week 0-10). The dotted line at week 0 separates the pre-intervention period (baseline) from the 10-week light-dark intervention period. A significant increase in body mass was observed in all groups over time (β = 6.012, p < 0.0001). There was a significant effect of estrogen status on body mass, but not of light schedule status. Ovx rats had higher body mass than sham-operated rats (β = 41.99, p < 0.0001), and no interaction between time and intervention group, or estrogen status and light schedule status was observed. Data expressed as mean ± SD, n = 9 per group. SHR = spontaneously hypertensive rat; Ctr Light = control light; CPS = chronic phase shift; Ovx = ovariectomy; Sx = pre-surgery body mass.

Figure 3a illustrates changes in weekly food intake. All groups exhibited a decrease in food consumption over time (β = –0.240, p < 0.0001). Ovx rats consumed more food than sham-operated rats (β = 1.150, p < 0.0001). Relative to body mass shown in Figure 3b, all groups exhibited a decrease in food consumption over time (β = –0.397, p < 0.0001). Ovx rats consumed less food than sham-operated rats (β = –0.769, p < 0.0001). No significant interaction between time and intervention group, or estrogen status and light schedule status was observed for absolute food intake or food intake relative to body mass.

Figure 3.

Changes in weekly (a) food intake and (b) relative (to body mass) food intake in female SHR. Food intake was measured weekly from the week of surgery (Sx) and continuing through the 10-week light-dark intervention period (week 0-10). The dotted line at week 0 separates the pre-intervention period (baseline) from the 10-week light-dark intervention period. Both food intake (β = –0.240, p < 0.0001) and relative food intake (β = –0.397, p < 0.0001) significantly decreased in all groups over time. Ovariectomized rats had an increased food intake compared with sham-operated rats (β = 1.150, p < 0.0001). However, relative to body mass, ovariectomized rats had decreased food intake compared with sham-operated rats (β = –0.769, p < 0.0001). No significant interaction between time and intervention group, or estrogen status and light schedule status was observed for absolute food intake or food intake relative to body mass. Data expressed as mean ± SD, n = 9 per group. SHR = spontaneously hypertensive rat; Ctr Light = control light; CPS = chronic phase shift; Ovx = ovariectomy; Sx = pre-surgery food intake.

Figure 4a illustrates changes in weekly water intake. No significant effect of time or light schedule status was observed. However, ovx rats exhibited an increased water intake than sham-operated rats (β = 3.41, p = 0.027). While no significant interaction between time and estrogen status, or estrogen status and light schedule status was found, a significant interaction between time (as a linear effect) and light schedule status was observed (β = 0.34, p = 0.017). This indicates that there is a progressive increase in water intake over time in CPS rats than in control light rats.

Figure 4.

Changes in weekly (a) water intake and (b) relative (to body mass) water intake in female SHR. Water intake was measured weekly from the week of surgery (Sx) and continued through the 10-week light-dark intervention period (week 0-10). The dotted line at week 0 separates the pre-intervention period (baseline) from the 10-week CPS intervention period. No significant effect of time or light schedule observed. All groups show a decrease in water intake over time (β = –0.619, p < 0.0001). However, there is a Time × Light schedule status interaction, for both absolute (β = 0.34, p = 0.017) and relative water intake (β = 0.180, p = 0.011), indicating a progressive rise in water intake over time in CPS rats than control light rats. The ovx rats exhibited an increased water intake than sham-operated rats (β = 3.41, p = 0.027). However, there is a Time × Estrogen status interaction, indicating that the ovx rats exhibit a steeper decline in water intake over time than sham-operated rats (β = –0.242, p = 0.001. Data expressed as mean ± SD, n = 9 per group. SHR = spontaneously hypertensive rat; Ctr Light = control light; CPS = chronic phase shift; Ovx = ovariectomy, Sx = pre-surgery water intake.

All groups exhibited a decrease in water intake relative to body mass over time (β = –0.619, p < 0.0001), as shown in Figure 4b. A significant interaction between time and estrogen status was observed, with ovx rats exhibiting a steeper decline in water intake over time than sham-operated rats (β = –0.242, p = 0.001). Furthermore, there was a significant interaction between time and light schedule status (β = 0.180, p = 0.011), indicating a progressive increase in water intake over time in the CPS rats than in control light rats.

All groups exhibited an increase in weekly systolic BP over time (β = 0.433, p < 0.0001), as shown in Figure 5a. The CPS rats had higher systolic BP than the control light rats (β = 0.688, p = 0.012), irrespective of time and estrogen status. No significant effect of estrogen status was observed. Weekly diastolic BP measurements (Figure 5b) and fasting blood glucose concentration (Figure 6a) were similar across all groups. There was no significant interaction between time and intervention group, or estrogen status and light schedule status for systolic BP or diastolic BP.

Figure 5.

Changes in weekly (a) systolic and (b) diastolic BP in female SHR during the 10-week light-dark intervention. The BP was measured noninvasively using an NIBP250 monitor system. All groups show an increase in weekly systolic BP over time (β = 0.433, p < 0.0001), with CPS rats showing higher systolic BP than the control light rats (β = 0.688, p = 0.012). The diastolic BP remained similar across all groups, with no significant interaction of time and intervention group, or estrogen status and light schedule status. Data expressed as mean ± SD, n = 9 per group. BP = blood pressure; SHR = spontaneously hypertensive rat; CPS = chronic phase shift; Ctr Light = control light; Ovx = ovariectomy.

Figure 6.

Changes in (a) weekly fasting blood glucose concentrations and (b) 3-h oral OGTT results in female SHR. The fasting blood glucose concentration was measured weekly during the 10-week light-dark intervention. The 3-h OGTT was performed 3 days before the end of the 10-week light-dark intervention. No significant differences were observed in fasting blood glucose between all the groups. OGTT: A significant effect of time (entered as a categorical variable) was observed (p < 0.0001). Glucose peaked at 15-30 min post-administration and gradually returned to baseline levels by 120-180 min. Ovx rats had lower glucose concentration than sham-operated rats irrespective of time or light schedule status (β = –0.360, p = 0.049). CPS rats had higher glucose concentration than control light rats irrespective of time or estrogen status (β = 0.394, p = 0.032). There was no significant interaction between time and intervention group, or estrogen status and light schedule status. Data expressed as mean ± SD, n = 9 per group. OGTT = oral glucose tolerance test; SHR = spontaneously hypertensive rat; CPS = chronic phase shift; Ctr Light = control lighting; Ovx = ovariectomy.

During the OGTT (Figure 6b), a significant effect of time (entered as a categorical variable) was observed (p < 0.0001). Glucose levels peaked at 15-30 min post-administration (all post hoc tests with Tukey adjustment comparing 15- and 30-min glucose with the other time points (0, 60, 120, and 180) showing higher glucose at 15 and 30 min; with all adjusted p values < 0.0001) and gradually returned to baseline by 120-180 min. Ovx rats exhibited lower glucose concentration than sham-operated rats (fixed effect of estrogen status; β = –0.360, p = 0.049), when controlling for time and light schedule status. CPS rats had higher glucose concentration than control light rats (fixed effect of light schedule status; β = 0.394, p = 0.032), when controlling for time and light schedule status. There was no significant interaction between time and intervention group, or between estrogen status and light schedule status.

Table 1 also presents post-euthanasia characteristics. Ovx rats had larger absolute heart (p = 0.0006), LV (p < 0.0001), kidney (p = 0.016), and liver (p < 0.0001) masses than sham-operated rats. Ovx rats had smaller heart (p < 0.0001), LV (p < 0.0001), kidney (p = 0.001), and liver (p < 0.0001) masses than sham-operated rats relative to body mass. In addition, CPS rats exhibited smaller liver mass than control light rats, both in absolute (p = 0.031) and relative mass (p = 0.012). LDL concentrations were similar across all groups, and no significant interaction was observed between estrogen status and light schedule status for any of these parameters.

Table 1.

Characteristics of ovx and sham-operated female SHR after 10-week light-dark intervention.

Characteristics	Sham control lightn = 9	Sham CPSn = 9	Ovx control lightn = 9	Ovx CPSn = 9
Heart mass, g	0.86 ± 0.06	0.88 ± 0.06	0.95 ± 0.07***	0.95 ± 0.06***
LV mass, g	0.64 ± 0.04	0.65 ± 0.05	0.72 ± 0.04***	0.71 ± 0.05***
Liver mass, g	7.1 ± 0.5	6.8 ± 0.4^†	8.1 ± 0.8***	7.6 ± 0.5***^†
Kidney mass, g	0.66 ± 0.07	0.64 ± 0.09	0.71 ± 0.05*	0.69 ± 0.06*
Normalized heart mass, g/100 g	0.45 ± 0.03	0.46 ± 0.04	0.38 ± 0.02***	0.38 ± 0.02***
Normalized LV mass, g/100 g	0.34 ± 0.02	0.34 ± 0.02	0.29 ± 0.01***	0.29 ± 0.02***
Normalized liver mass, g/100 g	3.7 ± 0.3	3.5 ± 0.2^†	3.2 ± 0.2***	3.1 ± 0.2***^†
Normalized kidney mass, g/100 g	0.34 ± 0.03	0.33 ± 0.04	0.29 ± 0.02**	0.28 ± 0.02**
LDL, ug/mL	9.1 ± 6.5	13.9 ± 10.0	9.7 ± 6.6	15.8 ± 12.1

Abbreviations: SHR = spontaneously hypertensive rats; CPS = chronic phase shift; Ovx = ovariectomy; LV = left ventricle; LDL = low-density lipoprotein. Data are expressed as mean ± SD.

p = 0.05 ovx vs sham-operated. **p = 0.01 ovx vs sham-operated. ***p = 0.001 ovx vs sham-operated. ^†p = 0.05 CPS vs control light.

Discussion

The key findings of this study indicate that a CPS protocol designed to induce circadian misalignment was associated with increased systolic BP and water intake, reduced liver mass, and impaired glucose tolerance in female SHR, regardless of ovariectomy or sham operation status. These findings suggest that CPS-induced circadian misalignment exacerbates cardiometabolic disturbances in female SHR. In addition, estrogen deficiency independently increased body mass, food intake, and absolute organ masses. However, food intake and organ masses were decreased relative to body mass, suggesting a primary alteration in body composition. Notably, the absence of interaction between CPS-induced circadian misalignment and estrogen deficiency indicates that these conditions independently affect cardiometabolic parameters in female SHR. SHR exhibit a genetic predisposition to circadian disruptions, including spontaneous phase advance, a shorter period and lower amplitude of circadian rhythms, and higher desynchrony with peripheral clocks compared with normotensive controls (Cui et al., 2011; Sládek et al., 2012; Polidarová et al., 2013; Olejníková et al., 2015). While these inherent circadian disruptions could potentially lessen the effects of additional misalignment, this study highlights a sex- and strain-specific response of CPS on cardiometabolic parameters (specifically, higher systolic BP and lower glucose tolerance) in female SHR.

Previous findings report that circadian misalignment in male rodents—SHR, Wistar, F344 rats, and C57BL/6 mice—often results in cardiometabolic disruptions, including increased body mass, glucose intolerance, and elevated BP (Tsai et al., 2005; Salgado-Delgado et al., 2008, Casiraghi et al., 2016; Rumanova et al., 2019; Wang et al., 2022). However, fewer studies have examined circadian misalignment effects in female rodents, particularly hypertensive models. Chronic circadian misalignment had no effect on body mass or BP in adult female Wistar rats (Zeman et al., 2016) but induced less pronounced BP increases in female Bmal1-KO rats than in males (Rhoads et al., 2021). In addition, recent studies report glucose intolerance in males but not in female rodents—C57BL/6 mice and fat sand rats in response to circadian misalignment/circadian disruption–inducing protocols (chronic jet lag and short photoperiod) (Ma et al., 2024; Tan et al., 2024). However, findings from other studies demonstrate impaired glucose metabolism in female C57BL/6 mice under chronic circadian disruption (Zhu et al., 2015; Zhong et al., 2019). The severity of impairment varied depending on the duration and extent of misalignment. These findings collectively suggest that the effects of circadian misalignment on females are more variable and less consistent than in males.

In this study, CPS had no effect on body mass or food intake in female SHR, consistent with Zeman et al. (2016). Studies show that circadian misalignment may not consistently affect body mass or food intake (Dauchy et al., 2010; Wang et al., 2022), and its effects on BP remain inconsistent. Increased BP is frequently observed in male rodents (Zhang et al., 2000; Rhoads et al., 2021; Wang et al., 2021, 2022) while females exhibit subtle or no changes (Zeman et al., 2016; Wang et al., 2021). In our study, the CPS protocol led to subtle yet significant increases in systolic BP in the female SHR, independent of ovariectomy or sham operation status. Male sex hormones are known to exacerbate hypertension (Reckelhoff et al., 1998; Huisman et al., 2006), which may explain the more pronounced effects in males in other studies.

The CPS protocol also led to increased water intake, consistent with findings observed in male F344 rats (Tsai et al., 2005), although some studies reported decreased water intake (Spiteri, 1982; Rosenwasser et al., 2010; Guerrero- et al., 2017). Variations in the circadian protocols used may account for these discrepancies. Nevertheless, increased water intake may reflect disruptions in fluid regulation (George et al., 1975; Duffy et al., 2016; Scheuermaier et al., 2024), leading to compensatory thirst (Mutter et al., 2021; Perrier et al., 2021). While Tsai et al. (2005) reported no liver mass changes—with 12-h light-dark cycle shifts twice weekly and a 10-day recovery period, this study observed reduced liver mass in the rats exposed to CPS. Since the liver plays a crucial role in glucose metabolism (Zhang et al., 2010; Lamia et al., 2008), its dysfunction can impair glucose regulation (Salgado-Delgado et al., 2013). The impaired glucose tolerance observed in this study is consistent with previous findings in rat models with circadian misalignment induced by either manipulation of the light-dark cycle or restricted feeding (Salgado-Delgado et al., 2010; Varcoe et al., 2011; Zhong et al., 2019; Tan et al., 2024). The reduced liver mass observed in this study may have contributed to impaired glucose clearance and reduced insulin sensitivity (Qian et al., 2018; Zhong et al., 2019; Her et al., 2024), exacerbating glucose intolerance in female SHR.

Beyond the CPS effects described above, estrogen deficiency significantly increased body mass gain in female SHR, consistent with previous studies (Santos et al., 2004; Lin et al., 2016; da Silva et al., 2017). Despite this increase, food intake was reduced relative to body mass, likely reflecting decreased physical activity and altered metabolic processes (Lovejoy et al., 2008; Rogers et al., 2009). Although absolute organ masses increased, the proportional gain was smaller than the overall body mass. This indicates a shift toward greater fat accumulation, consistent with estrogen decline–related increased adiposity and metabolic dysregulation (Chen and Heiman, 2001; Sowers et al., 2007; Abildgaard et al., 2021). Although metabolic abnormalities such as elevated fasting glucose, glucose intolerance, and insulin resistance are often reported in estrogen-deficient SHR (Sanches et al., 2012; Conti et al., 2015; Brito-Monzani et al., 2018), findings remain inconsistent.

Variability in the age of ovariectomy and environmental factors may account for these inconsistencies. Indeed, fasting blood glucose increased following fructose-induced insulin resistance and ovariectomy at 3 months old (Sanches et al., 2012; Conti et al., 2015; Brito-Monzani et al., 2018), whereas no changes were observed in female SHR ovariectomized at a younger age (3-12 weeks old) (Iwase et al., 1996; Swislocki et al., 2002; das Graças Abeles et al., 2012). In this study, estrogen deficiency did not significantly affect fasting blood glucose but was associated with lower glucose concentration during the OGTT. Lower OGTT blood glucose levels have been observed in female rats (non-ovariectomized) showing higher insulin sensitivity and enhanced muscle glucose uptake (Yki-Järvinen, 1984; Bilu et al., 2022). While ovariectomy is associated with glucose intolerance (Ben-Shmuel et al., 2015; Lei et al., 2021), the decrease in glucose during OGTT in ovariectomized female SHR could reflect a reduction in overall glucose availability due to lower food intake relative to body mass as observed in this study.

Environmental factors, such as temperature, may contribute to inconsistent findings regarding LDL concentration in estrogen-deficient SHR. Increased LDL was observed at cooler room temperatures (22 °C) (Packer et al., 2002; Kim et al., 2011), but not at higher temperatures (24 °C) (Delgado et al., 2017), consistent with this study. Prolonged exposure to cooler temperatures has been associated with elevated cholesterol levels (Umishio et al., 2022), with LDL concentration rising as temperature decreases (Hong et al., 2012; Shiue, 2016; Sartini et al., 2017). Thus, housing conditions, alongside diet and age, may influence metabolic outcomes more than estrogen deficiency alone. Furthermore, the effect of estrogen deficiency on BP in female SHR remains inconclusive. Some studies report increased BP (Dantas et al., 1999; Peng et al., 2003; Lin et al., 2016; Shimojo et al., 2018), while others report no effect (Giménez et al., 2006; Jazbutyte et al., 2008; Martin et al., 2008; Tatchum-Talom et al., 2011; Costa et al., 2018; Lin et al., 2020; Chen et al., 2021). Consistent with the latter findings, this study found no differences in BP. The discrepancies may be attributed to variations in experimental methodologies, such as telemetry versus noninvasive tail-cuff measurements. Notably, male SHR consistently exhibit higher BP, likely due to androgen-driven hypertensive effects (Reckelhoff et al., 1999). Thus, possible protective effects of female hormones that persist despite ovariectomy may explain the absence of BP changes in this study.

Finally, no interaction between CPS-induced circadian misalignment and estrogen deficiency was observed in this study. Although both these conditions induce cardiometabolic disturbances, such as insulin resistance, hypertension, elevated cholesterol, and glucose intolerance (Penev et al., 1998; Morris et al., 2016; Chikwati et al., 2023), the lack of interaction suggests they may operate independently. Unlike menopause that involves age-related metabolic changes and hormonal shifts, the early onset of estrogen deficiency may result in fewer compounded effects. In addition, the genetic profile of SHR may also mitigate the impact of CPS on other cardiometabolic parameters. Therefore, the strain-specific characteristics of SHR combined with early estrogen deficiency may explain the lack of additive effects in this study. A recent study by Ma et al. (2024) demonstrated a sex-dependent effect on metabolic disturbances under circadian misalignment, whereby males developed glucose intolerance during chronic jet lag induced by 6-h daily phase shifts, but females did not. Notably, testosterone played a key role as castration reduced circadian misalignment–induced metabolic disturbances (no evidence of glucose intolerance in castrated mice compared with non-castrated mice during the chronic jet lag protocol), while testosterone replacement restored them. When taken together, Ma et al.’s (2024) findings (castrated mice do not develop glucose intolerance during chronic jet lag) and our study’s findings (ovariectomy is associated with lower glucose levels during OGTT, when controlling for light schedule) suggest that the absence of ovarian hormones alone does not exacerbate circadian misalignment–induced metabolic disturbances, but rather testosterone may be a primary driver of these effects. Taken together, these findings reinforce the notion that circadian misalignment and estrogen deficiency independently influence cardiometabolic outcomes. Their interaction may be limited, and potentially due to sex-and species-specific responses or genetic factors.

Limitations

This study was unable to monitor physiological or behavioral indicators (e.g., core body temperature, locomotor activity, fasting/feeding activity, or hormonal rhythms) commonly used to confirm circadian rhythms and re-entrainment in rodents (Reid, 2019). Therefore, we cannot establish with certainty whether circadian rhythms were misaligned by the CPS protocol, nor can we ascertain whether the changes observed during the CPS protocol (i.e., increased water intake, lower glucose tolerance during OGTT, and higher systolic BP) were driven by changes in the timing of rest/activity, fasting/feeding and/or hormonal/molecular rhythms, or a combination thereof, during the CPS protocol.

While data collection was consistent within each group across weekly cycles, parameters were collected on different days relative to each CPS phase shift. This may introduce variability due to ongoing re-entrainment in the CPS group. Thus, future work should consider standardizing the timing of all measurements relative to the phase shift cycles (i.e., measure at consistent intervals each phase shift) to ensure that all rats are in comparable physiological states during assessment. In addition, BP was measured using the noninvasive tail-cuff technique, which may have underestimated the effect size of the systolic BP increase compared with direct methods like telemetry (Wilde et al., 2017; Harrison et al., 2024). It is possible that the timing of our experimental intervention interacted with the maturation of hypertension, potentially influencing the BP outcomes observed.

Conclusion

This study demonstrates that CPS-induced chronic circadian misalignment and estrogen deficiency independently contribute to cardiometabolic disturbances in female SHR. The 10 weeks of CPS of the photoperiod increased systolic BP, water intake, and impaired glucose tolerance. However, the CPS did not worsen the overall cardiometabolic profile in estrogen-deficient SHR. SHR may show limited effects of CPS-induced circadian misalignment during a shift work paradigm study due to inherent circadian disruptions, yet we were able to observe the expected effects of CPS-induced circadian misalignment on glucose tolerance and BP. This abnormal circadian regulation could be valuable in unraveling molecular pathways contributing to cardiometabolic risk under chronic circadian misalignment, such as in shift work. In addition, sex differences play a role in the response to circadian misalignment, as male rodents typically exhibit more pronounced cardiometabolic disturbances than females. On the contrary, estrogen deficiency led to increased body mass but did not worsen other cardiometabolic parameters in female SHR. While early estrogen deprivation may not disrupt the metabolic function in young female SHR, these findings warrant further investigation into possible compensatory mechanisms mitigating broader metabolic dysfunction. Furthermore, the lack of combined effects between circadian misalignment and estrogen deficiency may reflect species differences. These findings underscore the complexity of circadian and hormonal influences on cardiometabolic health, particularly in female hypertensive models.

Footnotes

Acknowledgements

We would like to thank the Wits Research Animal Facility’s staff for their assistance with the care and welfare of the animals. We also thank Dr. Monica Gomes for her technical assistance. This work was supported by grants from the Faculty Research Committee (FRC) of the Wits Faculty of Health Sciences (Grant number: 001.401.8521101. 0000000.000000 PHSMFR0). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions

Refentshe Amandu’s Nthlane: Data Acquisition, Data interpretation, Investigation, Methodology, Writing—original draft, Writing—revision & editing. Karine Scheuermaier: Conceptualization, Data interpretation, Supervision, Writing revision & editing. Siluleko Advice Mkhize: Investigation, Writing—revision & editing. Frédéric Sébastien Michel: Conceptualization, Formal analysis, Investigation, Methodology, Supervision, Visualization, Writing—original draft, Writing—review & editing.

Conflict of Interest Statement

The authors have no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability

The data presented in this manuscript are available upon reasonable request to the corresponding author.

Ethical Approval

All experimental procedures were approved by the Animal Research Ethics Committee (AREC) of the University of the Witwatersrand (Approval reference: 2017/11/76/C), in compliance with the National Institutes of Health guidelines for the care and use of laboratory animals.

ORCID iD

Refentshe Amandu’s Nthlane

Protocol Registration

A study protocol was prepared prior to the study and approved by the Faculty of Health Sciences at the University of the Witwatersrand. This research was conducted as part of a postgraduate project, and a letter of protocol approval is available upon request to the corresponding author.

References

Abildgaard

Ploug

Al-Saoudi

Wagner

Thomsen

Ewertsen

Bzorek

Pedersen

Lindegaard

(2021) Changes in abdominal subcutaneous adipose tissue phenotype following menopause is associated with increased visceral fat mass. Sci Rep 11:14750.

Anderson

Meng

Brooks

Tang

Lordan

Sengupta

Nayak

Mřela

Sarantopoulou

Lahens

, et al. (2023) Sexual dimorphism in the response to chronic circadian misalignment on a high-fat diet. Sci Transl Med 15:eabo2022.

Benloucif

Dubocovich

(1996) Melatonin and light induce phase shifts of circadian activity rhythms in the C3H/HeN mouse. J Biol Rhythms 11:113-125.

Ben-Shmuel

Scheinman

Rashed

Orr

Gallagher

LeRoith

Rostoker

(2015) Ovariectomy is associated with metabolic impairments and enhanced mammary tumor growth in MKR mice. J Endocrinol 227:143-151.

Bilu

Kronfeld-Schor

Zimmet

Einat

(2022) Sex differences in the response to circadian disruption in diurnal sand rats. Chronobiol Int 39:169-185.

Brito-Monzani

Sanches

Bernardes

Ponciano

Moraes-Silva

Irigoyen

Llesuy

De Angelis

(2018) Hypertension induces additional cardiometabolic impairments and attenuates aerobic exercise training adaptations in fructose-fed ovariectomized rats. Hypertens Res 41:88-95.

Casiraghi

Alzamendi

Giovambattista

Chiesa

Golombek

(2016) Effects of chronic forced circadian desynchronization on body weight and metabolism in male mice. Physiol Rep 4:e12743.

Chellappa

Morris

Scheer

(2020) Circadian misalignment increases mood vulnerability in simulated shift work. Sci Rep 10:8614.

Chen

Yuan

Zhang

(2021) mTOR inhibitor improves testosterone-induced myocardial hypertrophy in hypertensive rats. J Endocrinol 252:179-193.

10.

Chen

Heiman

(2001) Increased weight gain after ovariectomy is not a consequence of leptin resistance. Am J Physiol Endocrinol Metab 280:e315-e322.

11.

Chikwati

Mahyoodeen

Jaff

Ramsay

Micklesfield

Wade

Agongo

Asiki

Choma

SSR

Boua

, et al. (2023) Cardiometabolic disease risk factors in pre- and postmenopausal women from four sub-Saharan African countries: a cross-sectional study. Maturitas 172:60-68.

12.

Cignarella

Kratz

Bolego

(2010) Emerging role of estrogen in the control of cardiometabolic disease. Trends Pharmacol Sci 31:183-189.

13.

Colditz

Willett

Stampfer

Rosner

Speizer

Hennekens

(1987) Menopause and the risk of coronary heart disease in women. New Eng J Med 316:1105-1110.

14.

Conti

Brito

Bernardes

Dias

Malfitano

Morris

Llesuy

Irigoyen

, et al. (2015) Positive effect of combined exercise training in a model of metabolic syndrome and menopause: autonomic, inflammatory, and oxidative stress evaluations. Am J Physiol Regul Integr Comp Physiol 309:R1532-R1539.

15.

Costa

Ceravolo

Echem

Hashimoto

Costa

Santos-Eichler

Oliveira

Jiménez-Altayó

Akamine

Dantas

, et al. (2018) Detrimental effects of testosterone addition to estrogen therapy involve cytochrome P-450-induced 20-HETE synthesis in aorta of Ovariectomized Spontaneously Hypertensive Rat (SHR), a Model of postmenopausal hypertension. Front Physiol 9:490.

16.

Cui

Kohsaka

Waki

Bhuiyan

MER

Gouraud

Maeda

(2011) Metabolic cycles are linked to the cardiovascular diurnal rhythm in rats with essential hypertension. PLoS ONE 6:e17339.

17.

Dam

van der Schouw

Onland-Moret

Groenwold

RHH

Peters

SAE

Burgess

Wood

Chirlaque

Moons

KGM

Oliver-Williams

, et al. (2019) Association of menopausal characteristics and risk of coronary heart disease: a pan-European case–cohort analysis. Int J Epidemiol 48:1275-1285.

18.

Dantas

Scivoletto

Fortes

Nigro

Carvalho

(1999) Influence of female sex hormones on endothelium-derived vasoconstrictor prostanoid generation in microvessels of spontaneously hypertensive rats. Hypertens 34:914-919.

19.

das Graças Abeles

de Souza Cordeiro

de Sousa Martins

Pesquero

dos Reis

Andrade

Botion

(2012) Estrogen therapy attenuates adiposity markers in spontaneously hypertensive rats. Metab 61:1100-1107.

20.

da Silva

Gabriel-Costa

Wang

Ahmad

Sun

Varagic

Sudo

Ferrario

Dell Italia

Sudo

, et al. (2017) Blunting of cardioprotective actions of estrogen in female rodent heart linked to altered expression of cardiac tissue chymase and ACE2. J Renin Angiotensin Aldosterone Syst 18:1470320317722270.

21.

da Silva

VJD

Miranda

Oliveira

Rodrigues Alves

CHF

Van Gils

GHF

Porta

Montano

(2009) Heart rate and arterial pressure variability and baroreflex sensitivity in ovariectomized spontaneously hypertensive rats. Life Sci 84:719-724.

22.

Dauchy

Tirrell

Hill

Davidson

Greene

Tirrell

Sauer

Blask

(2010) Dark-phase light contamination disrupts circadian rhythms in plasma measures of endocrine physiology and metabolism in rats. Comp Med 60:348-356.

23.

Delgado

NTB

Rouver

Freitas-Lima

de Paula

TDC

Duarte

Silva

Lemos

Santos

AMC

Mauad

, et al. (2017) Pomegranate extract enhances endothelium-dependent coronary relaxation in isolated perfused hearts from spontaneously hypertensive ovariectomized rats. Front Pharmacol 7:522.

24.

de Marchi

Dell’Agnolo

Lopes

TCR

Gravena

AAF

Demitto

Brischiliari

SCR

Borghesan

DHP

Carvalho

, et al. (2017) Prevalence of metabolic syndrome in pre- and postmenopausal women. Arch Endocrinol Metab 61:160-166.

25.

Dicom

Huang

Hilal

(2023) Association between shift work schedules and cardiovascular events in a multi-ethnic cohort. Int J Environ Res Public Health 20:2047.

26.

Dørum

Tonstad

Liavaag

Michelsen

Hildrum

Dahl

(2008) Bilateral oophorectomy before 50 years of age is significantly associated with the metabolic syndrome and Framingham risk score: a controlled, population-based study (HUNT-2). Gynecol Oncol 109:377-383.

27.

Duffy

Scheuermaier

Loughlin

(2016) Age-related sleep disruption and reduction in the circadian rhythm of urine output: contribution to nocturia? Curr Aging Sci 9:34-43.

28.

Fahmy

Sayyed

Elrahim

EAA

Farag

RTA

(2018) Superimposed effect of ovariectomy on type 2 diabetes mellitus in Wistar rats. Alexandria Med J 54:129-137.

29.

Figueiredo Neto

de Figuerêdo

Barbosa

Costa

GRC

Nina

(2010) Metabolic syndrome and menopause: cross-sectional study in gynecology clinic. Arq Bras Cardiol 95:339-345.

30.

George

Messerli

Genest

Nowaczynski

Boucher

Kuchel Orofo-Oftega

(1975) Diurnal variation of plasma vasopressin in man. J Clin Endocrinol Metab 41:332-338.

31.

Giménez

García

Serna

Bonacasa

Carbonell

Quesada

Hernández

(2006) 17Beta-oestradiol enhances the acute hypotensive effect of captopril in female ovariectomized spontaneously hypertensive rats. Exp Physiol 91:715-722.

32.

Green

Takahashi

Bass

(2008) The meter of metabolism. Cell 134:728-742.

33.

Guerrero-Vargas

Navarro-Espíndola

Guzmán-Ruíz

Basualdo

del

Espitia-Bautista

López-Bago

Lascurain

Córdoba-Manilla

Buijs

, et al. (2017) Circadian disruption promotes tumor growth by anabolic host metabolism; experimental evidence in a rat model. BMC Cancer 17:625.

34.

Harrison

Bader

Lerman

Fink

Karumanchi

Reckelhoff

Sequeira-Lopez

MlS

Touyz

(2024) Tail-cuff versus radiotelemetry to measure blood pressure in mice and rats. Hypertens 81:3-5.

35.

Her

Lin

Liu

Root

Regal

Alejandro

Cao

(2024) Circadian disruption across lifespan impairs glucose homeostasis and insulin sensitivity in adult mice. Metabolites 14:126.

36.

Hinojosa-Laborde

Lange

Haywood

(2000) Role of female sex hormones in the development and reversal of Dahl hypertension. Hypertens 35:484-489.

37.

Hong

Kim

Lim

Kim

Yoon

Park

(2012) Association of cold ambient temperature and cardiovascular markers. Sci Total Environ 435:74-79.

38.

Huisman

Schutte

Van Rooyen

Malan

Schutte

Kruger

(2006) The influence of testosterone on blood pressure and risk factors for cardiovascular disease in a black South African population. Ethn Dis 16:693-698.

39.

In het Panhuis

Schönke

Siebeler

Banen

Pronk

Streefland

Afkir

Sips

Kroon

Rensen

, et al. (2023) Circadian disruption impairs glucose homeostasis in male but not in female mice and is dependent on gonadal sex hormones. FASEB J 37:e22772.

40.

Iwase

Wakisaka

Yoshinari

Sato

Yoshizumi

Nunoi

Fujishima

(1996) Effect of gonadectomy on the development of diabetes mellitus, hypertension, and albuminuria in the rat model. Metab 45:155-161.

41.

Jama

Muralitharan

O’Donnell

Bertagnolli

Broughton

BRS

Head

Marques

(2022) Rodent models of hypertension. Br J Pharmacol 179:918-937.

42.

Jazbutyte

Arias-Loza

Widder

Govindaraj

von Poser-Klein

Bauersachs

Fritzemeier

Hegele-Hartung

Neyses

, et al. (2008) Ligand-dependent activation of ER{beta} lowers blood pressure and attenuates cardiac hypertrophy in ovariectomized spontaneously hypertensive rats. Cardiovasc Res 77:774-781.

43.

Khalsa

SBS

Jewett

Cajochen

Czeisler

(2003) A phase response curve to single bright light pulses in human subjects. J Physiol 549:945-952.

44.

Kim

Baek

Rhyu

(2011) Cardioprotective effects of aqueous Schizandra chinensis fruit extract on ovariectomized and balloon-induced carotid artery injury rat models: effects on serum lipid profiles and blood pressure. J Ethnopharmacol 134:668-675.

45.

Kren

Pravenec

Krenova

Wang

Merriouns

Wong

St Lezin

Lau

, et al. (1997) Genetic isolation of a region of chromosome 8 that exerts major effects on blood pressure and cardiac mass in the spontaneously hypertensive rat. J Clin Invest 99:577-581.

46.

Kripke

Elliott

Youngstedt

Rex

(2007) Circadian phase response curves to light in older and young women and men. J Circadian Rhythms 5:1-13.

47.

Lamia

Storch

Weitz

(2008) Physiological significance of a peripheral tissue circadian clock. Proc Natl Acad Sci U S A 105:15172-15177.

48.

Lei

Yang

Lei

Liu

Nie

Yang

Zhang

Yang

, et al. (2021) Ovariectomy impaired hepatic glucose and lipid homeostasis and altered the gut microbiota in mice with different diets. Front Endocrinol (Lausanne) 12:708838.

49.

Lima

Wofford

Reckelhoff

(2012) Hypertension in postmenopausal women. Curr Hypertens Rep 14:254-260.

50.

Lin

Cheng

Chu

Shyu

Kao

Lin

Kuo

Yang

Lee

(2016) The coexistence of hypertension and ovariectomy additively increases cardiac apoptosis. Int J Mol Sci 17:2036.

51.

Lin

Hong

Zhou

Huang

Shyu

Chen

Ting

Cheng

Yang

Lee

(2020) Exercise training attenuates cardiac inflammation and fibrosis in hypertensive ovariectomized rats. J Appl Physiol 128:1033-1043.

52.

Lovejoy

Champagne

de Jonge

Xie

Smith

(2008) Increased visceral fat and decreased energy expenditure during the menopausal transition. Int J Obes (Lond) 32:949-958.

53.

Matsuo

Kurogi

Miyamoto

Morita

Shinozuka

Taniguchi

Ikegami

Yasuo

(2024) Sex-dependent effects of chronic jet lag on circadian rhythm and metabolism in mice. Biol Sex Differ 15:1-18.

54.

Maluleke

Millen

AME

Michel

(2022) The effects of estrogen deficiency and aging on myocardial deformation and motion in normotensive female rats. Menopause 29:89-95.

55.

Martin

Redetzke

Vogel

Mark

Eyster

(2008) Effect of ovariectomy on blood pressure and venous tone in female spontaneously hypertensive rats. Am J Hypertens 21:983-988.

56.

Martino

Tata

Belsham

Chalmers

Straume

Lee

Pribiag

Khaper

Liu

Dawood

, et al. (2007) Disturbed diurnal rhythm alters gene expression and exacerbates cardiovascular disease with rescue by resynchronization. Hypertens 49:1104-1113.

57.

McElroy

Wade

(1987) Short- and long-term effects of ovariectomy on food intake, body weight, carcass composition, and brown adipose tissue in rats. Physiol Behav 39:361-365.

58.

Meléndez-Fernández

Liu

Nelson

(2023) Circadian rhythms disrupted by light at night and mistimed food intake alter hormonal rhythms and metabolism. Int J Mol Sci 24:3392.

59.

Meloni

Cadeddu

Cugusi

Donataccio

Deidda

Sciomer

Gallina

Vassalle

Moscucci

Mercuro

, et al. (2023) Gender differences and cardiometabolic risk: the importance of the risk factors. Int J Mol Sci 24:1588.

60.

Mkhize

Nthlane

Xhakaza

Verhaert

Baijnath

Millen

Michel

(2025) Decreased blood pressure with acute administration of quercetin in L-NAME-induced hypertensive rats. Basic Clin Pharmacol Toxicol 136:e14113.

61.

Mogane

Mokotedi

Millen

AME

Michel

(2019) Increased systolic blood pressure associated with hypertriglyceridemia in female Sprague-Dawley rats. Can J Physiol Pharmacol 97:971-979.

62.

Morris

Purvis

Scheer

FAJL

(2016) Circadian misalignment increases cardiovascular disease risk factors in humans. Proc Natl Acad Sci U S A 113:e1402-e1411.

63.

Morris

Yang

Garcia

Myers

Bozzi

Wang

Buxton

Shea

Scheer

(2015) Endogenous circadian system and circadian misalignment impact glucose tolerance via separate mechanisms in humans. Proc Natl Acad Sci 112:E2225-E2234.

64.

Mutter

Smith

Menze

Zakharia

Nguyen

(2021) Diabetes insipidus: pathogenesis, diagnosis, and clinical management. Cureus 13: e13523.

65.

Neves

Ferreira

Shecaira

Rascio Henriques Dutra

Kimura

Gomes

Irigoyen

De Angelis

(2024) P099 exercise training further attenuates blood pressure variability and renal damage in hydrochlorothiazide treated-hypertensive and postmenopausal rats. J Hypertens 42:e92.

66.

Olejníková

Polidarová

Paušlyová

Sládek

Sumová

(2015) Diverse development and higher sensitivity of the circadian clocks to changes in maternal-feeding regime in a rat model of cardio-metabolic disease. Chronobiol Int 32:531-547.

67.

Packer

Pelaez

Johnson

(2002) Gender dichotomy in reactivity to the vasoactive oxidant hydrogen peroxide in spontaneously hypertensive rats. J Gend Specif Med 5:17-23.

68.

Penev

Kolker

Zee

Turek

(1998) Chronic circadian desynchronization decreases the survival of animals with cardiomyopathic heart disease. Am J Physiol 275:H2334-H2337.

69.

Peng

Clark

Wei

Wyss

(2003) Estrogen depletion increases blood pressure and hypothalamic norepinephrine in middle-aged spontaneously hypertensive rats. Hypertens 41:1164-1167.

70.

Percie du Sert

Hurst

Ahluwalia

Alam

Avey

Baker

Browne

Clark

Cuthill

Dirnagl

, et al. (2020) The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. J Cereb Blood Flow Metab 40:1769-1777.

71.

Pereira

Oliveira

Coletto-Nunes

Souza

PPC

Menani

De Luca

Andrade

CAF

(2022) Central angiotensinergic mechanisms in female spontaneously hypertensive rats treated with estradiol Appetite 174:106012.

72.

Perrier

Armstrong

Bottin

Clark

Dolci

Guelinckx

Iroz

Kavouras

Lang

Lieberman

, et al. (2021) Hydration for health hypothesis: a narrative review of supporting evidence. Eur J Nutr 60:1167-1180.

73.

Pizzato

Terraciano

Zanon

Kuhl

Alves Garcez

Passos

Tirloni

Berger

(2023) Estrogen depletion modulates aortic prothrombotic signaling in normotensive and spontaneously hypertensive female rats. Mol Cell Endocrinol 561:111827.

74.

Polidarová

Sládek

Nováková

Parkanová

Sumová

(2013) Increased Sensitivity of the circadian system to temporal changes in the feeding regime of spontaneously hypertensive rats—a potential role for Bmal2 in the liver. PLoS ONE 8:e75690.

75.

Pravenec

Křen

Landa

Mlejnek

Musilová

Šilhavý

Šimáková

Zídek

(2014) Recent progress in the genetics of spontaneously hypertensive rats. Physiol Res 63:S1-S8.

76.

Price

Khazova

Udovičić

(2022) Assessment of the light exposures of shift-working nurses in London and Dortmund in relation to recommendations for sleep and circadian health. Ann Work Expo Health 66:447-458.

77.

Qian

Dalla Man

Morris

Cobelli

Scheer

FAJL

(2018) Differential effects of the circadian system and circadian misalignment on insulin sensitivity and insulin secretion in humans. Diabetes Obes Metab 20:2481-2485.

78.

Reckelhoff

Zhang

Granger

(1998) Testosterone exacerbates hypertension and reduces pressure-natriuresis in male spontaneously hypertensive rats. Hypertens 31:435-439.

79.

Reckelhoff

Zhang

Srivastava

Granger

(1999) Gender differences in hypertension in spontaneously hypertensive rats: role of androgens and androgen receptor. Hypertens 34:920-923.

80.

Reid

(2019) Assessment of circadian rhythms. Neurol Clin 37:505-526.

81.

Rhoads

Meek

Jin

Pollock

(2021) Chronic circadian misalignment increases blood pressure in male and female Bmal1-KO rats. FASEB J 35:02903.

82.

Roa-Díaz

Raguindin

Bano

Laine

Muka

Glisic

(2021) Menopause and cardiometabolic diseases: what we (don’t) know and why it matters. Maturitas 152:48-56.

83.

Rogers

James

Perfield

Strissel

Obin

Greenberg

(2009) Reduced energy expenditure and increased inflammation are early events in the development of ovariectomy-induced obesity. Endocrinol 150:2161-2168.

84.

Rosenwasser

Clark

Fixaris

Belanger

Foster

(2010) Effects of repeated light–dark phase shifts on voluntary ethanol and water intake in male and female Fischer and Lewis rats. Alcohol 44:229-237.

85.

Rüger

Scheer

FAJL

(2009) Effects of circadian disruption on cardiometabolic system. Rev Endocr Metab Disord 10:245-260.

86.

Rumanova

Okuliarova

Molcan

Sutovska

Zeman

(2019) Consequences of low-intensity light at night on cardiovascular and metabolic parameters in spontaneously hypertensive rats 1. Can J Physiol Pharmacol 97:863-871.

87.

Rusak

Groos

(1982) Suprachiasmatic stimulation phase shifts rodent circadian rhythms. Science 215:1407-1409.

88.

Salgado-Delgado

Angeles-Castellanos

Buijs

Escobar

(2008) Internal desynchronization in a model of night-work by forced activity in rats. Neurosci 154:922-931.

89.

Salgado-Delgado

Angeles-Castellanos

Saderi

Buijs

Escobar

(2010) Food intake during the normal activity phase prevents obesity and circadian desynchrony in a rat model of night work. Endocrinol 151:1019-1029.

90.

Salgado-Delgado

Saderi

Basualdo

del

Guerrero-Vargas

Escobar

Buijs

(2013) Shift work or food intake during the rest phase promotes metabolic disruption and desynchrony of liver genes in male rats. PLoS ONE 8:e60052.

91.

Sanches

de Oliveira Brito

Candido

da Silva Dias

Jorge

Irigoyen

De Angelis

(2012) Cardiometabolic benefits of exercise training in an experimental model of metabolic syndrome and menopause. Menopause 19:562-568.

92.

Santos

Pereira

LMM

Mandarim-de-Lacerda

(2004) The effect of enalapril on the cardiac remodelling in ovariectomized spontaneously hypertensive rats. Int J Exp Pathol 85:287-294.

93.

Sartini

Barry

Whincup

Wannamethee

Lowe

Jefferis

Lennon

Welsh

Ford

Sattar

, et al. (2017) Relationship between outdoor temperature and cardiovascular disease risk factors in older people. Eur J Prev Cardiol 24:349-356.

94.

Scheer

FAJL

Hilton

Mantzoros

Shea

(2009) Adverse metabolic and cardiovascular consequences of circadian misalignment. Proc Natl Acad Sci U S A 106:4453-4458.

95.

Scheuermaier

Chang

Duffy

(2024) Sleep-independent circadian rhythm of aldosterone secretion in healthy young adults. Sleep Health 10:S103-S107.

96.

Shimojo

da Silva Dias

Malfitano

Sanches

Llesuy

Ulloa

Irigoyen

De Angelis

(2018) Combined aerobic and resistance exercise training improve hypertension associated with menopause. Front Physiol 9:1471.

97.

Shiue

(2016) Cold homes are associated with poor biomarkers and less blood pressure check-up: English Longitudinal Study of Ageing, 2012–2013. Environ Sci Pollut Res Int 23:7055-7059.

98.

Silver

Rainbow

(2013) The suprachiasmatic nucleus and the circadian timekeeping system of the body. In: Pfaff

Rubenstein

Volkow

, editors. Neuroscience in the 21st century: from basic to clinical. Cham: Springer. p. 1847-1888.

99.

Sládek

Polidarová

Nováková

Parkanová

Sumová

(2012) Early chronotype and tissue-specific alterations of circadian clock function in spontaneously hypertensive rats. PLoS ONE 7:e46951.

100.

Sowers

Zheng

Tomey

Karvonen-Gutierrez

Jannausch

Yosef

Symons

(2007) Changes in body composition in women over six years at midlife: ovarian and chronological aging. J Clin Endocrinol Metab 92:895-901.

101.

Spiteri

(1982) Circadian patterning of feeding, drinking and activity during diurnal food access in rats. Physio Behav 28:139-147.

102.

Staessen

Bulpitt

Fagard

Lijnen

Amery

(1989) The influence of menopause on blood pressure. J Hum Hypertens 3:427-433.

103.

Swislocki

Burgie

Rodnick

(2002) Effects of ovariectomy on indices of insulin resistance, hypertension, and cardiac energy metabolism in middle-aged spontaneously hypertensive rats (SHR). Horm Metab Res 34:516-522.

104.

Tan

JTM

Cheney

Bamhare

NES

Hossin

Bilu

Sandeman

Nankivell

Solly

Kronfeld-Schor

Bursill

(2024) Female Psammomys obesus are protected from circadian disruption-induced glucose intolerance, cardiac fibrosis and adipocyte dysfunction. Int J Mol Sci 25:7265.

105.

Tatchum-Talom

Eyster

Kost

Martin

(2011) Blood pressure and mesenteric vascular reactivity in SHR seven months after gonadectomy. J Cardiovasc Pharmacol 57:357-364.

106.

Touitou

Reinberg

Touitou

(2017) Association between light at night, melatonin secretion, sleep deprivation, and the internal clock: health impacts and mechanisms of circadian disruption. Life Sci 173:94-106.

107.

Tran

Jochum

Shaikh

Wilber

Zhang

Hayden

Forsyth

Voigt

Bishehsari

Keshavarzian

, et al. (2021) Circadian misalignment by environmental light/dark shifting causes circadian disruption in colon. PLoS ONE 16:e0251604.

108.

Trebucq

Lamberti

Rota

Aiello

Borio

Bilen

Golombek

Plano

Chiesa

(2023) Chronic circadian desynchronization of feeding-fasting rhythm generates alterations in daily glycemia, LDL cholesterolemia and microbiota composition in mice. Front Nutr 10:1154647.

109.

Tsai

Hwang

Huang

Tzeng

(2005) Repeated light-dark shifts speed up body weight gain in male F344 rats. Am J Physiol Endocrinol Metab 289:e212-e217.

110.

Umishio

Ikaga

Kario

Fujino

Suzuki

Hoshi

Ando

Yoshimura

Yoshino

Murakami

(2022) Association between indoor temperature in winter and serum cholesterol: a cross-sectional analysis of the smart wellness housing survey in Japan. J Atheroscler Thromb 29:1791-1807.

111.

Van Lenten

Melchior

Roheim

(1983) Lipoprotein metabolism in the ovariectomized rat. J Lipid Res 24:1475-1484.

112.

Varcoe

Wight

Voultsios

Salkeld

Kennaway

(2011) Chronic phase shifts of the photoperiod throughout pregnancy programs glucose intolerance and insulin resistance in the rat. PLoS ONE 6:e18504.

113.

Vitaterna

Takahashi

Turek

(2001) Overview of circadian rhythms. Alcohol Res Health 25:85-93.

114.

Wang

(2023) Study on the mechanism of cardiac hypertrophy induced by testosterone through mtorc2 in ovariectomized female spontaneously hypertensive rats. J Hypertens 41:e42-e43.

115.

Wang

Hou

Song

Zuo

Chi

Zhang

(2022) Harm of circadian misalignment to the hearts of the adolescent Wistar rats. J Transl Med 20:352.

116.

Wang

Zhang

Bai

Zhang

Chi

(2021) Effects of inverted photoperiods on the blood pressure and carotid artery of spontaneously hypertensive rats and Wistar–Kyoto rats. J Hypertens 39:871-879.

117.

Wilde

Aubdool

Thakore

Baldissera

Alawi

Keeble

Nandi

Brain

(2017) Tail-cuff technique and its influence on central blood pressure in the mouse. J Am Heart Assoc 6:e005204.

118.

Yki-Järvinen

(1984) Sex and insulin sensitivity. Metab 33:1011-1015.

119.

Youngstedt

Elliott

Kripke

(2019) Human circadian phase–response curves for exercise. J Physiol 597:2253-2268.

120.

Zanchetti

Facchetti

Cesana

Modena

Pirrelli

Sega

; SIMONA Participants (2005) Menopause-related blood pressure increase and its relationship to age and body mass index: the SIMONA epidemiological study. J Hypertens 23:2269-2276.

121.

Zeman

Molcan

Herichova

Okuliarova

(2016) Endocrine and cardiovascular rhythms differentially adapt to chronic phase-delay shifts in rats. Chronobiol Int 33:1148-1160.

122.

Zhang

Zannou

Sannajust

(2000) Effects of photoperiod reduction on rat circadian rhythms of BP, heart rate, and locomotor activity. Am J Physiol Regul Integr Comp Physiol 279:R169-R178.

123.

Zhang

Liu

Dentin

Pongsawakul

Liu

Hirota

Nusinow

Sun

Landais

Kodama

, et al. (2010) Cryptochrome mediates circadian regulation of cAMP signaling and hepatic gluconeogenesis. Nat Med 16:1152-1156.

124.

Zhernakova

Sinha

Andreu-Sánchez

Prins

Kurilshikov

Balder

Sanna

Franke

Kuivenhoven

Zhernakova

, et al. (2022) Age-dependent sex differences in cardiometabolic risk factors. Nat Cardiovasc Res 1:844-854.

125.

Zhong

Yang

Zhang

Pan

Zhang

Zhou

Wang

, et al. (2019) Circadian misalignment alters insulin sensitivity during the light phase and shifts glucose tolerance rhythms in female mice. PLoS ONE 14:e0225813.

126.

Zhu

Zou

Yang

Saito

Othrell Hinton

Jr Yan

Ding

Fukuda

, et al. (2015) Estrogens prevent metabolic dysfunctions induced by circadian disruptions in female mice. Endocrinol 156:2114-2123.

127.

Zitting

Vetrivelan

Yuan

Vujovic

Wang

Bandaru

Quan

Klerman

Scheer

FAJL

Buxton

Williams

, et al. (2022) Chronic circadian disruption on a high-fat diet impairs glucose tolerance. Metab 130:155158.

128.

Zuo

Shi

Guo

Hussain

(2009) Prevalence of metabolic syndrome and factors associated with its components in Chinese adults. Metab 58:1102-1108.