Male wild-type (WT) and ghrelin gene-deficient (ghrl^-/- ) mice were generated by homologous recombination as previously reported (1). Mice, 10 weeks old, were included in this study and housed five per cage. The mice were maintained on a 12-h light-dark cycle (lights on from 7:00 to 19:00) with access to food and water ad libitum. The room temperature was maintained at 23 ± 1°C. All animal studies were handled in accordance with the Guide for the Care and Use of Laboratory Animals, as adopted by the U.S. National Institutes of Health, and the specific protocols were approved by the Institutional Animal Care and Use Committee of Kurume University School of Medicine (approval no. 2010-026, 2011-062, 2020-005, 2021-006-1, 2022-125, 2023-097, 2024-022-1).

Body weight, tissue wet weight, and blood pressure were measured in 12-week-old intact WT and ghrl^-/- mice. Blood pressure was measured from 13:00 to 15:00 using a tail-cuff plethysmograph with a blood pressure-measuring system (MODEL MK-2000ST; Muromachi Kikai, Tokyo, Japan). The blood pressure was measured following 15 min warming at 37°C in an animal holder made of dark brown acryl, allowing blood pressure measurement under relatively stress-free conditions. Five measurements were taken for each animal and the data represented the average of three values, excluding the maximum and minimum values.

The hearts and kidneys from 12-week-old mice were quickly immersed in cold 4% paraformaldehyde/PBS. After dehydration and permeabilization, the tissue was paraffin-embedded and incised at a thickness of 5 μm. The sections were stained with a hematoxylin and eosin staining kit (Scy Tek Laboratories, UT, USA) and viewed under an optical microscope (Evident, Tokyo, Japan).

Intact 12-week-old WT and ghrl^-/- mice were used. Whole blood was collected by decapitation at 14:00, and serum was obtained after centrifugation. The serum aldosterone levels were measured using an ELISA kit (Cayman, Chemical Co., Ann Arbor, MI).

The mice were anesthetized by intraperitoneal injection of a mixture of ketamine and xylazine (5 mg/kg; Dainippon Pharmaceutical, Osaka, Japan). At the age of 10 weeks, mice were separated into individual cages and implanted with the telemetry device (PA-C10; Data Sciences International, St. Paul, MN, USA) to the abdominal aorta, according to the manufacturer’s outlines. Briefly, after an incision of abdominal skin and musculature, the abdominal aorta was exposed, and the catheter of the transmitter was introduced upstream into the vessel. The transmitter body was placed subcutaneously in the left flank. After implantation, the mice were rapidly recovered with atipamezole. A minimum of 1 week was allowed for recovery and for the mice to regain their pre-surgical weight. The diurnal rhythm of blood pressure and heart rate in mice was measured 4 weeks after surgery. During the experiment, a PhysiolTel-Receiver (model RPC-1; Data Sciences International) was placed under each animal’s cage to record the blood pressure. For the diurnal rhythm of blood pressure, data were obtained every 5 min and graphed.

The rat ghrelin was provided by Dr. Hiroshi Hosoda (National Cerebral and Cardiovascular Center). To investigate the changes in blood pressure after ghrelin administration, the 12-week-old mice with implanted telemetric devices were administered 100 μg/kg ghrelin intraperitoneally at 7:00 or 19:00.

To determine the role of ghrelin in blood pressure and heart rate regulation during torpor induction, the mice implanted with telemetric devices had their blood pressure and heart rate measured while receiving GHRP-6 (Sigma, St. Louis, MO, USA), a ghrelin receptor agonist. Mini-osmotic pumps filled with GHRP-6 (Model 2002; Durect Co., Cupertino, CA, USA) were implanted subcutaneously 10 days before reaching 14 weeks of age. The mini-osmotic pumps delivered GHRP-6 at 42 ng/0.5 μl/h/g body weight for 14 days.

WT and ghrl^-/- mice, and ghrl^-/- mice receiving continuous administration of GHRP-6 with osmotic mini-pumps were all initiated on a 48-h fast at the end of the light phase (19:00) when they reached 14 weeks of age. During this period, they were free to drink, and after 48 h, they were fed again. To better understand the changes in blood pressure and heart rate during fasting, we calculated the difference in mean blood pressure (ΔBP) and mean heart rate (ΔHR). First, the average of 12 data obtained during the first hour of fasting (18:00-18:55) was determined and set as the reference value, or zero. Next, the data acquired during all fasting and refeeding periods were used to calculate the mean value of the 12 data obtained every hour. These values were subtracted from the reference value to obtain the difference (ΔBP and ΔHR, respectively).

The heart rate variability (HRV) was measured to assess the autonomic function (25). The data were recorded by electrocardiography using a PowerLab system and analyzed using the LabChart software (version 6.0; AD Instruments Pty Ltd., Australia). The frequency-domain variables included: low (LF, 0.04–0.15 Hz) and high (HF, 0.15–0.4 Hz) frequencies. Subsequently, the LF/HF ratio was calculated.

The data are displayed as mean ± S.E.M. The significance of the differences within each group was determined using one-way ANOVA, followed by Bonferroni’s post-hoc test. When two groups were compared, the Student’s t-test or two-way ANOVA followed by Bonferroni’s post-hoc test was used. The analyses were performed using the Prism software (version 5.0; GraphPad Software, San Diego, CA, USA); p < 0.05 was considered to be significant.

To determine the effect of ghrelin deficiency on blood pressure, we measured the blood pressure in ghrl^-/- mice using tail-cuff plethysmography and found that blood pressure was higher in ghrl^-/- mice than in WT mice ( Table 1 ). Consistent with previous reports (26, 27), ghrelin deficiency did not cause any changes in body weight, and there were no differences in heart and kidney tissue wet weight, body weight ratio, or histology ( Table 1 , Figure 1A ). Since blood pressure is known to be regulated by humoral and neural factors, we next investigated whether ghrelin deficiency affects the basal state of humoral factors. The renin-angiotensin-aldosterone system, vasopressin, and atrial natriuretic peptide are known as humoral factors that regulate blood pressure, and the renin-angiotensin-aldosterone system has been implicated in blood pressure regulation by ghrelin (28). Therefore, we measured serum aldosterone levels and found no differences between the two groups ( Figure 1B ), suggesting that the kidney function was normal despite the ghrelin gene deficiency. In other words, the humoral factors that regulate blood pressure were shown to be normal despite ghrelin deficiency.

To determine whether blood pressure in ghrl^-/- mice was always high, the blood pressure was continuously measured using an automated telemetry system. A clear diurnal rhythm was observed in the blood pressure of WT mice, which was higher during the dark phase, the active phase of nocturnal mice, and lower during the light phase, the resting phase of nocturnal mice ( Figure 2A ). In contrast, the blood pressure of ghrl^-/- mice was indistinct and was generally higher than that of WT mice ( Figure 2A ). Such diurnal variation in blood pressure was also consistent with diurnal variation in heart rate ( Supplementary Figure 1 ). To quantitatively evaluate the blood pressure data of WT and ghrl^-/- mice, histograms were generated from the data of light (8:00-18:00) and dark (20:00-6:00) phases ( Figure 2B ), and mean and median values were calculated ( Table 2 ). The blood pressure in both WT and ghrl^-/- mice was higher during the dark phase of activity than during the light phase of rest, indicating that the central clock was normal. Both mean and median blood pressure were higher in ghrl^-/- mice only during the light phase ( Table 2 ). Thus, the ghrelin gene deficiency prevented blood pressure reduction during the light phase, which is the resting phase in nocturnal mice.

Subsequently, we analyzed the effects of ghrelin on blood pressure reduction in WT and ghrl^-/- mice during the light and dark phases. Ghrelin induced a transient decrease in blood pressure in all the groups ( Figure 3 ). The ghrelin-induced reduction in blood pressure was greater in ghrl^-/- mice than in WT mice ( Figure 3 ). In particular, the blood pressure was markedly reduced in ghrl^-/- mice treated with ghrelin during the light phase ( Figure 3 ). These results suggest that even ghrl^-/- mice with high blood pressure can have a lower blood pressure.

Ghrelin secretion in mice is maximal during fasting, when torpor is induced (1). Therefore, we measured the blood pressure and heart rate in ghrl^-/- mice during torpor induction. In WT mice, a decrease in blood pressure was observed with the passage of fasting time while maintaining the light-dark cycle, and the decrease in blood pressure was cancelled by re-feeding ( Figure 4A ). In ghrl^-/- mice, the individual differences in blood pressure fluctuation patterns were large, and large blood pressure fluctuations were observed in short cycles. However, the overall trend was that the blood pressure remained high during the fasting period ( Figure 4A ). To determine whether the blood pressure abnormalities observed in ghrl^-/- mice could be improved by activation of the ghrelin receptor system, we performed fasting experiment in ghrl^-/- mice implanted with an osmotic mini-pump filled with the ghrelin receptor agonist GHRP-6 and found that the blood pressure in ghrl^-/- mice was improved to the level of WT mice ( Figure 4A ). To quantitatively evaluate these observations, we calculated the mean blood pressure 24-48 hours after the onset of fasting and found that it was higher only in ghrl^-/- mice, with no difference between WT mice and ghrl^-/- mice pretreated with GHRP-6: WT mice: 98.7 ± 3.7 mmHg, ghrl^-/- mice: 108.2 ± 2.2 mmHg, ghrl^-/- +GHRP-6 mice: 89.9 ± 4.8 mmHg. In addition, we calculated hourly average data and shown as blood pressure changes from the start of fasting ( Figure 4B ). WT mice and ghrl^-/- mice pretreated with GHRP-6 showed similar blood pressure trends, with a low value at approximately 36 h after the start of fasting during torpor induction ( Figure 4B ). In contrast, fasting did not produce a sufficient decrease in blood pressure in ghrl^-/- mice ( Figure 4B ). In all groups, the blood pressure increased quickly after re-feeding; however, this increase was greater in ghrl^-/- mice ( Figure 4B ). These results showed that the decrease in blood pressure seen during torpor does not occur in ghrl^-/- mice. In addition, it was shown that preadministration of GHRP-6 using an osmotic mini-pump can reduce the mean blood pressure of ghrl^-/- mice to the same level as that of WT mice. The change in heart rate during fasting was also similar to the change in blood pressure, but differences between the ghrl^-/- mice group and the other two groups were observed from an earlier time from the start of fasting ( Supplementary Figure 2 ). These results suggest that activation of the ghrelin-ghrelin receptor system is required for active blood pressure reduction during torpor induction.

As there were no morphological or endocrinological abnormalities in the heart or kidneys of ghrl^-/- mice ( Figure 1 ), we analyzed the autonomic nervous system activity to understand the mechanism by which ghrelin lowered blood pressure during torpor induction. Analysis of HRV is widely used as a standard method for assessing autonomic nervous functions (25). Particularly, LF and HF spectral components of HRV are used as the separate metrics of sympathetic and parasympathetic functions (25). The LF/HF ratio, a measure of sympathetic activity, was higher and normalized HF% (nHF%), a measure of parasympathetic activity, was lower in ghrl^-/- mice after 36 h of fasting ( Figure 5 ). Furthermore, these changes were cancelled in ghrl^-/- mice pretreated with GHRP-6 using an osmotic mini-pump and were comparable to those in WT mice ( Figure 5 ). Taken together, these results indicated that blood pressure is higher in ghrl^-/- mice because of significant sympathetic activity due to ghrelin deficiency and that this effect is suppressed by the activation of the ghrelin-ghrelin receptor system. Thus, it was suggested that the suppression of the sympathetic nerve activity by activation of the ghrelin-ghrelin receptor system is necessary during torpor induction.