Heat stroke-induced hepatic lipid dysregulation: histological and lipidomic insights
Takahiro Deguchi, Hiroki Tanaka, Kie Horioka, Chihiro Matsuhisa, Akira Hayakawa, Shuhei Takauji, Shimpei Watanabe, Masanori Goto, Yumiko Fujii, Kumi Takasawa, Akira Takasawa
TL;DR
This study explores how heat stroke causes liver damage in mice, revealing lipid accumulation and changes in cholesterol levels that could help diagnose and treat heat stroke.
Contribution
The study identifies heat stroke-induced hepatic lipid dysregulation and elevated 27-hydroxycholesterol as novel insights into liver injury mechanisms.
Findings
Heat stroke causes lipid accumulation in hepatocytes surrounding the central vein.
27-hydroxycholesterol levels significantly increase 24 hours post-heat exposure.
Lipid accumulation may mediate inflammation and act as a protective response.
Abstract
Global warming has increased summer temperatures, leading to a rise in heat stroke-related deaths in Japan. Heat stroke disrupts the body's adaptation to high temperatures, often resulting in severe complications, including liver damage and even death. However, despite the increasing incidence, pathological autopsies remain rare, and the histological changes associated with heat stroke are poorly understood. In this study, we investigated the pathogenesis of heat stroke using a mouse model. Mice were exposed to 45 °C for 30 min and dissected immediately or 24, 48, and 72 h post-exposure. Histological analysis revealed significant lipid accumulation in hepatocytes surrounding the central vein at 24, 48, and 72 h. At 24 h, hepatocytes also exhibited features of early degeneration, including cytoplasmic lysis and chromatin condensation. Lipidomics analysis of liver tissue collected 24 h…
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Taxonomy
TopicsThermoregulation and physiological responses · Climate Change and Health Impacts · Adipose Tissue and Metabolism
Introduction
Climate change is one of the most critical issues of the twenty-first century, with global and annual average temperatures having increased by approximately 0.8 °C since the nineteenth century. Among the physiological impacts of rising temperatures on the human body is an elevated risk of heat stroke, a life-threatening condition that places a significant strain on healthcare resources [1, 2]. Epidemiological data indicate that both the incidence and mortality rates of heat stroke and other heat-related health conditions are projected to rise further [3]. Heat stroke is generally categorized into exertional and non-exertional types. Exertional heat stroke primarily affects younger populations, such as athletes and laborers engaged in strenuous physical activity, while non-exertional heat stroke mainly impacts older adults with comorbid conditions like diabetes mellitus, hypertension, cardiovascular disease, and chronic kidney disease [4].
Heat stroke can lead to multiple organ failures, including those of the central nervous system (CNS), liver, and kidneys. The CNS, being especially vulnerable to elevated body temperatures, can exhibit symptoms such as delirium, dizziness, weakness, agitation, nausea, and vomiting. Severe heat stroke may result in life-threatening complications, including disseminated intravascular coagulation (DIC), acute respiratory distress syndrome (ARDS), acute kidney injury (AKI), cardiac dysfunction, and hepatic dysfunction [4–6]. Acute liver injury, in particular, is a crucial clinical finding that can lead to fatal outcomes [7]. Studies have shown that acute liver failure may be associated with widespread hepatocellular necrosis, affecting cells in both zone 3 and zone 2 of the liver lobule [8, 9]. However, these histological observations were made several days post-heat stress and do not shed light on early-phase changes. Additionally, few reports exist on the histopathology of heat stroke fatalities; while hemorrhagic changes in organs other than the liver have been documented, liver-specific findings remain unreported [10, 11].
Consequently, the early histological changes and molecular mechanisms in the liver during heat stroke remain poorly understood. To address this gap, we conducted a histological and molecular biological analysis using a mouse model of heat stroke, aiming to provide insights into the early changes associated with this condition.
Materials and methods
Animal models
Eight-week-old male C57BL/6 mice were used to establish the heat stroke model. To induce a rapid increase in body temperature, preliminary testing was conducted to determine conditions under which rectal temperature would rise within 30 min, referring to previous report from Miyamoto et al*.* [12]. Based on these trials, an environmental temperature of 45 °C was deemed appropriate. Mice were subsequently placed in an incubator set at 45 °C for 30 min, followed by a recovery period. Heat exposure was terminated when rectal temperatures increased from an average of 35.3 °C to 39.2 °C, at which point the mice exhibited reduced activity. Following heat stress exposure, organs were collected immediately (0 h), and then at 24, 48, and 72 h post-stress. Samples were taken from 5 mice at each time point, with an additional group of 5 non-stressed mice serving as controls. Rectal temperatures measured immediately before dissection of these mice are shown in Fig. 1A. Organs such as the liver, kidney, intestine, and lungs were then harvested. All animal procedures were approved by the Bioethics Committee of Asahikawa Medical University.Fig. 1. Morphological changes in a mouse model of heat stroke. A Rectal temperatures measured immediately before dissection (n = 5 each). B Gross image of the liver 24 h after heat stress treatment. C HE staining of liver tissue. D HE staining of the small intestine, kidney, lung, and skeletal muscle. Scale bars: 100 μm. Cont: control, 24 h: 24 h after heat stress treatment. *P < 0.05, ns: no significance
Histopathological analysis
Organ samples were fixed in 3.7% paraformaldehyde for 24 h, dehydrated, paraffin-embedded, sectioned into 2-μm slices, and stained with hematoxylin–eosin (HE). After staining, sections were examined under an optical microscope. For Oil Red O staining, liver tissues were fixed in 3.7% paraformaldehyde, embedded in OCT compound (Sakura Finetek Japan Co., Ltd., Tokyo, Japan), and frozen. Cryosections were prepared at 5 μm thickness and subsequently stained. For immunohistochemical staining, slices of paraffin-embedded specimens were stained with M30 cytodeath antibody (Roche Diagnostic, Mannheim, Germany). Deparaffinized and rehydrated sections were placed in an alkaline buffer (Agilent Technologies, Santa Clara, CA, USA) and subjected to antigen retrieval using a pressure cooker. For immunohistochemical staining, sections were also treated with 3% hydrogen peroxide to block endogenous peroxidase activity. The sections were then incubated overnight with M30 cytodeath antibody diluted 1:200, followed by washing and incubation with the appropriate secondary antibody. For immunohistochemistry, an HRP-conjugated horse anti-mouse IgG antibody (Vector Labs, Burlingame, CA, USA) was used, while an Alexa Fluor™ 488-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA) was used for immunofluorescence staining. Immunohistochemical sections were visualized using DAB chromogen and counterstained with hematoxylin. For immunofluorescence, sections were counterstained with DAPI and examined under a fluorescence microscope. The number of lipid droplets and M30 cytodeath-positive cells were quantitatively analyzed using QuPath [13]. For these quantifications, one liver lobe was sampled from each animal, and five randomly selected images were captured per sample at × 200 magnification. Data from five animals per experimental group were collected for comparative analysis.
Serum alanine aminotransferase analysis
Mouse serum alanine aminotransferase (ALT) activities were measured by a dry chemistry analyzer FUJI DRI-CHEM (Fujifilm, Tokyo, Japan).
Lipidomics
Mouse liver tissue was dissolved in an acetonitrile/ethanol extraction buffer (9:1, containing 0.075% formic acid), vortexed for 10 min, and centrifuged at 1000 × g for 10 min. The supernatant was collected and analyzed using an Orbitrap LC–MS system (Thermo Fisher Scientific, Waltham, MA, USA). Metabolite separation was performed on a reversed-phase C18 column with non-target analysis at 40 °C and a flow rate of 0.5 mL/min. Both negative and positive polarity spectrometric data were acquired in the 100–600 m/z range. Raw data were processed, and metabolites were quantified using MS-DIAL (RIKEN CSRS/IMS, Yokohama, Japan). Samples were n = 3 control and 24 h post heat stress, respectively.
Statistics
GraphPad Prism 10 (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses. The differences in the experimental values were analyzed via one-way ANOVA followed by the Kruskal–Wallis test for multiple comparison, unpaired t-test. P-values < 0.05 were considered statistically significant.
Results
Accumulation of lipid droplets in the liver of heat-stressed mice
Liver samples taken 24 h after heat stressing the mice were grossly white in color compared to controls (Fig. 1B). H&E staining showed numerous lipid droplets in the cytoplasm of hepatocytes (Fig. 1C), but no significant changes were observed in other organs (Fig. 1D).
Time course change of lipid droplets in the liver
To more clearly visualize lipid droplet accumulation in hepatic tissue, we performed Oil Red O staining and quantified lipid droplets exceeding a defined size threshold in the micrographs. Immediately after heat stress, lipid droplets were only sparsely and locally observed in liver sections (Fig. 2A). However, 24 h post-exposure, lipid accumulation became more widespread, extending throughout hepatic zones 1 to 3 (Fig. 2A, B). Over time, lipid droplets decreased, and their distribution became more localized to zone 3, surrounding the central vein (Fig. 2A).Fig. 2. Time-course analysis of lipid droplet accumulation in the liver. A HE staining (Left) and Oil Red O staining (Right) in the liver. Scale bars: 100 μm. B Number of lipid droplets in photographs taken at × 200 magnification (n = 5 each). ****P < 0.0001, ns no significance. PV portal vein, CV central vein
Cell death in the liver of heat-stressed mice
In liver samples collected 24 h after heat stress, necrosis-like features were observed in some hepatocytes, including loss of nuclear structure, chromatin condensation, and indistinct cell margins (Fig. 3A). HE staining revealed these degenerative changes without evidence of significant inflammatory cell infiltration, indicating that hepatocyte injury occurred in the absence of a marked inflammatory response. This finding is particularly notable given that inflammation is commonly implicated in heat stress-induced liver damage. M30 immunostaining, an established marker of apoptotic and necrotic cell death, further confirmed the presence of degenerating hepatocytes (Fig. 3B and Supplementary Fig. 1). These morphological changes were transient and diminished over time (Fig. 3B, C). Additionally, serum ALT levels increased and peaked between 24 and 48 h post-heat stress, then declined by 72 h (Fig. 3C), consistent with the histological evidence of reversible hepatocellular injury.Fig. 3. Histological evidence of cell death. A HE staining showing necrosis-like changes. Scale bars: 50 μm. Cont: control, 24 h: 24 h after heat stress treatment. Arrows indicate cells that have undergone necrosis. B Immunohistochemical staining for M30. Scale bars: 100 μm. C Left: Graph of average number of M30-positive cells in micrographs (× 200 field of view, n = 5 each)., Right: Graph of serum ALT levels (n = 5 each). *P < 0.05, **P < 0.01, ****P < 0.0001, ns: no significance
Lipidomics analysis in the liver of heat-stressed mice
Histological observations revealed that lipid accumulation peaked in the liver 24 h after heat stress. Based on this finding, we conducted a lipidomics analysis to characterize the specific types of lipids that accumulated in hepatic tissue. Using LC–MS, we compared liver samples from heat-stressed mice and controls, focusing on metabolites with high identification confidence. Lipid species showing statistically significant differences at 24 h post-stress are summarized in the accompanying table. Among these, 27-HC exhibited the most marked increase in abundance relative to control liver tissue (Fig. 4 and Table 1).Fig. 4. Comparison of 27-hydroxycholesterol levels in liver tissue. Graph of the results of the determination of 27-hydroxycholesterol levels shown in Table 1 (n = 3 each). P < 0.05Table 1Lipid species showing statistically significant differences in liver samples 24 h after heat stress treatment compared to controlsNameFormulaFC (HS/cont)p-value27-hydroxycholesterolC27H46O1.4430.022CarnitineC7H15NO31.2740.006N-acyl ethanolaminesC15H31NO21.1380.025N-acyl glycineC18H29NO31.1220.024MonoacylglycerolC19H32O41.0700.024SulfonolipidC26H47NO6S1.0440.045Ceramide hydroxy fatty acid-dihydrosphingosineC23H47NO40.9360.006Brassicasterol esterC31H50O20.8330.042N-acyl taurineC20H39NO4S0.2870.034FC* fold change, HS 24 h after heat stress treatment, cont control
Discussion
There have been many cases in which patients who were rushed to the hospital for heat stroke developed fulminant hepatitis in their later years. However, the pathogenesis of these cases remained unclear. In the present study, we used a mouse model to investigate the early hepatic responses to heat stress and found that abnormal lipid metabolism occurs at an early stage following exposure.
By 24 h post-heat stress, fatty liver formation was clearly evident both macroscopically and histologically, with numerous lipid droplets observed in hepatocytes. Unlike metabolic dysfunction-associated steatotic liver disease (MASLD), where large, triglyceride-rich lipid droplets are typically seen [14–17], our heat stress model revealed smaller lipid droplets and a significant increase in 27-HC, as shown by lipidomics analysis. MASLD is generally driven by chronic factors such as excess caloric intake, elevated endotoxins, and insulin resistance, and murine models typically require several weeks of high-fat or choline-deficient diets to induce visible steatosis [16, 17]. In contrast, our model demonstrated striking hepatic lipid accumulation within just 24 h of a single 30-min heat exposure, regardless of nutritional status. This rapid onset underscores the profound and immediate impact of heat stress on hepatic lipid metabolism.
Additionally, hepatocyte death was observed at 24 h post-heat stress, as evidenced by positive M30 immunostaining and necrosis-like morphological changes. While previous studies have suggested that inflammation underlies heat stress-induced liver damage [8, 9], our histological analysis revealed no significant inflammatory cell infiltration. This raises the possibility that alternative mechanisms, such as lipid-derived signaling, may mediate liver injury and repair in this context.
Among the lipids altered by heat stress, 27-HC stands out as a major oxysterol and intermediate in hepatic bile acid synthesis [18]. Beyond its metabolic role, 27-HC has been shown to modulate immune responses and inflammation, with studies reporting both pro-inflammatory [18–21] and anti-inflammatory [22] effects depending on the context. Notably, 27-HC also functions as a ligand for nuclear receptors such as liver X receptor (LXR) and estrogen receptor (ER), which are involved in cellular survival and lipid homeostasis.
In our study, cell death and elevated 27-HC levels were observed at 24 h post-heat stress. However, these cell death and lipid accumulation subsided by 48–72 h, as evidenced by decreased ALT levels. The absence of inflammation alongside hepatocyte death raises the intriguing possibility that 27-HC may activate protective pathways via ER and LXR signaling, promoting hepatocyte survival and recovery. Further studies will be necessary to determine whether these survival pathways are indeed upregulated in liver tissue following heat stress.
Additionally, among the lipid species analyzed, N-acyl taurine was significantly reduced in the liver post-heat stress. N-acyl taurine, a fatty acid-derived metabolite, has been reported to function as a second messenger regulating the secretion of hormones involved in glucose metabolism [23]. Although this study did not directly assess glucose dynamics, the observed reduction in N-acyl taurine warrants future investigation into metabolic disturbances during heat stroke.
In conclusion, our findings demonstrate that acute heat stress rapidly induces hepatic fat accumulation, hepatocyte death, and a marked increase in 27-HC. This response may play a protective role in mitigating liver injury, highlighting a previously underappreciated link between heat stress and hepatic lipid signaling.
Supplementary Information
Below is the link to the electronic supplementary material.Supplementary file 1. Immunofluorescent staining for M30. Scale bars: 100 μm. Arrows indicate M30-positive cells