Effect of PACAP on Heat Exposure

Abstract

Heat stroke is a life-threatening illness caused by exposure to high ambient temperatures and relative humidity. The incidence of heat stroke is expected to increase due to climate change. Although pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in thermoregulation, the role of PACAP on heat stress remains unclear. PACAP knockout (KO) and wild-type ICR mice were subjected to heat exposure at an ambient temperature of 36 °C and relative humidity of 99% for 30-150 min. After heat exposure, the PACAP KO mice had a greater survival rate and maintained a lower body temperature than the wild-type mice. Moreover, the gene expression and immunoreaction of c-Fos in the ventromedially preoptic area of the hypothalamus, which is known to harbor temperature-sensitive neurons, were significantly lower in PACAP KO mice than those in wild-type mice. In addition, differences were observed in the brown adipose tissue, the primary site of heat production, between PACAP KO and wild-type mice. These results suggest that PACAP KO mice are resistant to heat exposure. The heat production mechanism differs between PACAP KO and wild-type mice.

Keywords: PACAP; core body temperature; heat stroke; hypothalamus.

Figure 1

Conditions of heat stroke in ICR mice and the survival rate during heat exposure. (a) During heat exposure, WBGT (°C), ambient temperatures (ATs) (°C), and relative humidity (RH) (%) were measured with a digital thermo-hygrometer. The measurements were repeated four times and expressed as the mean ± SD. The WBGT (AT and RH) before heat exposure was 37.3 ± 0.0 °C (34.3 ± 0.0 °C and 99.9 ± 0.0%). It reached 42.0 ± 0.1 °C (38.0 ± 0.0 °C and 99.9 ± 0.0%) at 150 min. (b) The survival rate (%) of the wild-type (WT) and PACAP KO (KO) mice under heat exposure. WT (red line) and PACAP KO (blue line) mice (n = 10 each) were exposed to heat exposure for 150 min, and the survival time was observed. The survival rates at 150 min were 20.0% and 80.0% in the WT and PACAP KO groups, respectively (p < 0.05, by Kaplan–Meier survival analysis).

Figure 2

Decrease in body weight (BW; %) of the wild-type and PACAP KO mice. After 3 h water restriction, BW decreased by approximately 3% in each group. The decrease in BW after 60 min of heat exposure was significantly higher in the wild-type mice than in the PACAP KO mice (Student’s t-test, * p < 0.05).

Figure 3

Morphological observations (HE staining) of livers (a–d) and kidneys (e,f) of the wild-type (a,c,e) and PACAP KO (b,d,f) mice after 60 min of heat exposure. (a) The hepatic sinuses surrounding the area of the central vein (V) were uncertain. (c) The higher-magnification image demonstrated that the vacuolations in the hepatocytes (arrowhead) and leukocytes (arrow) were present in the wild-type mice and relatively suppressed in the PACAP KO mice (b,d). The renal tissues did not show obvious pathological changes or differences between the groups (e,f).

Figure 4

Changes in core body temperature throughout the circadian rhythm and heat exposure. (a) The core body temperature in the WT (n = 5) and PACAP KO (KO; n = 5) mice during the circadian rhythm. The core body temperature changed between 35 and 38 °C in a day. However, no significant differences were observed between the groups. (b) The core body temperature in the wild-type and PACAP KO mice during 180 min of dehydration and 60 min of heat exposure. The core body temperature slightly increased after measuring BW (−180 min) and gradually returned to physiological levels. The core body temperature drastically increased in the heat stroke chamber and exceeded 42 °C. The PACAP KO mice exhibited a similar pattern to that of the wild-type mice. However, the PACAP KO mice exhibited a significantly slower decrease during dehydration and increase during heat exposure. Data are expressed as the mean ± SE. * p < 0.05 (Student’s t-test).

Figure 5

c-Fos immunoreaction and gene expression in the VMPO of the hypothalamus in the wild-type and PACAP KO mice after heat exposure (HE). (a) Immunostaining of c-Fos in the VMPO in the wild-type and PACAP KO mice. Schematic drawing from Paxinos and Flanklin’s atlas and representative photomicrographs of coronal sections from the VMPO at 0.3–0.5 mm from the bregma [32]. (b) Quantitative analysis of c-Fos+ cell count in the VMPO (n = 5 of each group). The PACAP KO mice exhibited significantly fewer c-Fos+ cells than the wild-type mice pre-HE (p < 0.05). The number significantly increased in both groups after HE, with no significant difference. (c) c-Fos expression before and after HE. c-Fos expression was significantly lower in PACAP KO mice than in wild-type mice before HE. (d) Adcyap1 was expressed in the VMPO of the wild-type mice and increased after HE. Data are expressed as the mean ± SE. * p < 0.05, ** p < 0.01 (Student’s t-test).

Figure 6

Morphohistological comparison of brown adipose tissues (BATs) in the wild-type and PACAP KO mice. (a) Gross comparison of BAT between the wild-type (n = 5) and PACAP KO (n = 5) mice. No significant difference was observed between the two groups. (b) Wet weight of BAT also demonstrated no significant differences between the two groups. Data are shown as the mean ± standard error (SE).

Figure 7

Thermogenesis-related gene expression of BAT in the wild-type and PACAP KO mice before and after heat exposure. The expression of thermogenesis-related genes, such as Ucp1 (a), Adrb3 (b), and Lipe (c), was determined with SYBR green-based real-time PCR before and 30 and 60 min after heat exposure in the wild-type (n = 8–12) and PACAP KO (n = 7–12) mice. The gene expression was normalized with a housekeeping gene, Gapdh, and expressed in arbitrary units (AUs). All data are shown as the mean ± standard error (SE). * p < 0.05 (Student’s t-test).

Figure 8

Schematic pathway of non-shivering heat thermogenesis in BAT and putative thermoregulation of PACAP under normal and heat conditions. (a) Schematic nerve pathway for non-shivering thermoregulation with BAT. Non-shivering thermogenesis is regulated by the sympathetic nerve innervation. Preganglionic sympathetic nerves from the DMH and RPa project to postganglionic nerves, which innervate into the BAT. The nerves release noradrenaline (NA) and enhance a key thermogenetic molecule, UCP1, mediated by β3-adrenaline receptor (b3AR) and hormone-sensitive lipase (HSL) in the BAT. (b) Putative thermoregulation of the wild-type and PACAP KO mice under normal and heat conditions. The NA contents in the BAT were lower in PACAP KO mice than wild-type mice [24,45]. To compensate for their lower NA contents, the PACAP KO mice exhibited increased expression of b3AR and HSL, therefore, maintaining the UCP1 level and core body temperature. Simultaneously, heat exposure increased PACAP in the POA to inhibit the DMH and RPa nerve signals and decreased the expression of b3AR and HSL to suppress thermogenesis. On the other hand, because of the POA signaling, and because downregulation of b3AR and HSL was involved in the same pathway, the thermoregulation was less effective. However, in PACAP KO mice, thermogenesis depends highly on b3AR and HSL in normal conditions. Therefore, strong suppression of b3AR and HSL expression might result in the suppression of UCP1 and thermogenesis.

Figure 9

Representative images of the PACAP KO and wild-type mice. Higher bands indicate homozygous and lower bands indicate wild type.

Figure 10

Heat stroke chamber: Ten mice were placed in an acrylic box from the top input port. Humidity was controlled by an ultrasonic humidifier and recorded by a digital thermo-hygrometer.