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Journal of Diabetes Investigation logoLink to Journal of Diabetes Investigation
. 2026 Feb 5;17(4):608–621. doi: 10.1111/jdi.70256

Heat stress alters metabolic pathways and nitric oxide signaling in keratinocytes under hyperglycemia

Syeda Tayyiba Rahat 1, Ilkka Miinalainen 2, Anni I Nieminen 3, Seppo Vainio 1,4,5,6,7, Nsrein Ali 1,7,✉
PMCID: PMC13042437  PMID: 41640368

ABSTRACT

Background

Diabetic patients are particularly vulnerable to heat exposure due to impaired thermoregulation and reduced sweating ability. The impact of heat on skin cell function, particularly keratinocytes, is poorly understood. Recent studies highlight the critical role of nitric oxide (NO) in thermoregulation and heat stress responses, but its specific involvement in keratinocyte responses and metabolic profiles remains unexplored.

Objective

This proof‐of‐concept study investigates the metabolic profiles of HaCat keratinocytes under normal and high‐glucose conditions during varied heat exposures.

Methods

We conducted experiments using a metabolomics approach, NO levels assessments, western blot analysis, and evaluations of mitochondrial morphology.

Results

Our findings indicate that acute heat exposure over 90 minutes significantly alters metabolic pathways, particularly amino acid metabolism (including arginine, valine, leucine, and serine), the pyrimidine metabolite uracil, and glycolysis, notably lactate production. Arginine metabolism was uniquely affected by high glucose combined with heat, aligning with previous clinical observations.

Furthermore, we discovered that changes in NO production correlated with heat exposure duration, and that NO levels in extracellular vesicles (EVs) from HaCat cells were inversely related to intracellular NO levels. Additionally, we observed alterations in HSP‐70 protein expression and mitochondrial morphology, supporting cellular adaptation to thermal stress.

Conclusion

This study is the first to demonstrate heat‐induced metabolic changes in keratinocytes involving arginine and NO, highlighting their potential as clinical biomarkers for thermal stress adaptation, with implications for both healthy individuals and diabetic patients.

Keywords: Glucose, Keratinocytes, Metabolites

This illustration depicts the effects of thermal stress on human keratinocytes maintained in both normal and high‐glucose conditions. Under normal glucose conditions, heat exposure enhances arginine metabolism and increases nitric oxide (NO) production, which promotes alterations in mitochondrial morphology and elevates heat shock protein 70 (Hsp70) levels. In contrast, under high‐glucose conditions, heat exposure suppresses arginine metabolism, leading to a significant decline in NO production. This reduction is accompanied by a decrease in Hsp70 expression, mitochondrial resistance, and an increased release of NO‐enriched extracellular vesicles (EVs). Overall, NO acts as a crucial signaling molecule, highlighting the detrimental effects of combined heat and high glucose stress on keratinocyte health and function. Created in BioRender. https://BioRender.com/p70wb2p.

graphic file with name JDI-17-608-g001.jpg

INTRODUCTION

Diabetes is a metabolic disorder characterized by elevated blood glucose levels, insulin resistance, and damage to multiple organs 1 , 2 . Emerging evidence suggests that diabetic individuals are more vulnerable to heat exposure than non‐diabetics, with studies reporting higher mortality rates in this population during heat events 3 , 4 .

The skin, through its sweat glands, plays a crucial role in thermoregulation by facilitating heat dissipation via sweat production 5 , 6 . In individuals with diabetes, this thermoregulatory function is impaired due to several complications. These include endothelial and non‐endothelial signaling dysfunction linked to cardiovascular issues; metabolic abnormalities such as chronic hyperglycemia and irregular insulin distribution, altered autonomic nervous function, and reduced skin blood flow. Collectively, these factors contribute to a diminished ability to sweat 7 , 8 .

Interestingly, clinical studies have reported therapeutic benefits of heat treatments in diabetes, including improved blood glucose control and reduced insulin resistance 9 , 10 . One hypothesis is that heat exposure influences both the rate and composition of sweat. Furthermore, the interaction between nitric oxide (NO) and heat shock protein (HSP) pathways has been identified as a potential molecular mechanism promoting sweat production under heat stress 11 , 12 , 13 .

NO, a free radical generated by the oxidation of L‐arginine 14 , 15 , 16 has gained increasing attention for its diverse biological roles, particularly in the skin 17 , 18 , 19 . It plays a crucial role in regulating oxidative stress and mitochondrial activity due to its biphasic nature. At low concentrations, NO exerts protective effects on mitochondria by promoting mitochondrial biogenesis, enhancing efficiency, and modestly regulating reactive oxygen species (ROS). However, at elevated concentrations, NO can lead to oxidative stress by inhibiting the electron transport chain, increasing ROS and reactive nitrogen species. This can trigger mitochondrial fragmentation and dysfunction, resulting in cell injury and death 20 , 21 , 22 . Additionally, NO is vital for thermoregulation, contributing to both central and peripheral mechanisms of body temperature control. It is involved in the body's responses to heat stress, fever, and immune‐related temperature changes, with concentrations of NO rising during heat exposure. This increase is believed to influence neuronal activation in the hypothalamus, further affecting the body's thermal response 16 , 23 , 24 .

HSPs, particularly HSP‐70, are critical molecular chaperones involved in protein homeostasis and cytoprotection. Their expression is dynamically induced under stress conditions, including heat 14 , 25 , 26 . Altered HSP levels have been associated with diabetic complications 11 , 27 . HSP‐70, specifically, has been linked to keratinocyte responses to heat, promoting anti‐inflammatory mechanisms and protection against photodamage 28 .

In our recent work, we identified metabolites in sweat extracellular vesicles (EVs) using a novel extraction and collection technique. These metabolites were influenced by heat and correlated with glucose levels in healthy individuals, suggesting a role for sweat in systemic metabolic signaling 29 .

In the present study, we aim to establish a causal relationship between heat exposure and skin responses in the context of diabetes. Using an in vitro model of human immortalized keratinocytes (HaCaT), cultured under normal (5 mM) and high (25 mM) glucose conditions to simulate healthy and diabetic environments, respectively, cells were exposed to heat for varying durations to investigate metabolic profiles and further elucidate the underlying molecular pathways involved.

Our findings demonstrate that heat exposure induces distinct metabolic alterations in keratinocytes, particularly within the arginine‐NO axis. Notably, in high‐glucose conditions, arginine metabolism and downstream NO production were significantly modulated. Interestingly, the NO levels in EVs displayed an inverse pattern compared to intracellular levels. Repeated heat exposure further amplified NO production, which was associated with upregulated HSP‐70 expression and enhanced mitochondrial integrity.

This study provides novel insights into the role of skin in thermoregulation, particularly under diabetic conditions. It identifies arginine and NO as potential clinical biomarkers for monitoring skin response to heat stress and suggests that targeting their pathway through HSPs may offer therapeutic strategies for improving heat management in patients with diabetes.

MATERIALS AND METHOD

Cell culture and glucose treatment

HaCat cell lines were maintained in normal glucose (5 mM D‐Glucose) and high‐glucose (25 mM D‐Glucose) DMEM (Thermo Fisher) to mimic normoglycemic and diabetic conditions, respectively. DMEM media was supplemented with 1% Penicillin/Streptomycin (Sigma), 10% FBS (Gibco) and maintained at 37°C in a humidified incubator under 5% CO2. The media was replaced every 48 h after the seeding of the cells (seeding density 2.2 × 106 cells). When needed the cells were maintained in different passages after detachment with Trypsin–EDTA (Gibco Life Technology).

A cell viability assay was performed following heat exposure treatment using trypan blue dye (Cat# T10282 Invitrogen™, CA, USA). Cell counting was conducted using the TC20™ automated cell counter (Bio‐Rad Laboratories, CA, USA), which provides an accurate count of both viable and nonviable cells. A representative figure relevant to this assay is presented in Figure S1B, showing the results 48 h after heat exposure.

Heat exposure

Acute and chronic heat exposures

On the day of treatment, HaCat cell cultures were rinsed with growth media to eliminate dead cells. Following this, fresh growth media were added to all control and treatment plates. Control cell plates were incubated for 90 min and 48 h at 37°C in a humidified incubator with 5% CO2, while treatment plates were incubated at 40°C under the same conditions. To ensure precise control of temperature and humidity during heat exposure, a temperature data logger with an integrated USB interface (LOG32TH, Carl Roth) was employed.

After heat exposure, cells were rinsed with 1× PBS and subsequently collected for protein, metabolomics, and electron microscopy analyses, in which each was conducted according to its respective protocols. This procedure was repeated twice using two independent cell passages, with three biological replicates conducted for each passage.

Repetitive heat exposure

On the day of the treatment, HaCat cells maintained in 5 and 25 mM glucose levels were subjected to repeated heat exposure. In this experimental setup, the cells were exposed to 40°C in a humidified incubator under 5% CO2 for 90 min every 24 h, repeated three times. Following each heat exposure, the cells were returned to an incubator at 37°C under the same incubation conditions. Cells for analysis were collected immediately after the third heat exposure and used for further analyses as described in the section above.

Isolation of extracellular vesicles from keratinocytes

Following 48 h of heat exposure, EVs were isolated using our previously published protocol with some modifications 29 , 30 . Briefly, the culture medium was collected and filtered through 40 μm Sterile Cell Strainers (CLS431750, Corning™) to remove dead cells. The supernatant was cleared from cellular components by centrifugation twice at 2,500 × g for 15 min at 4°C. The filtrate was then refiltered through 0.8 μm vacuum filter units (Thermo Fisher Scientific, Waltham, MA, USA). The filtrate was centrifuged in a Sorvall AH‐629 rotor at 100,000× g for 12 h, in a Sorvall Ultracentrifuge Machine WX ultra 90 (VWR, Thermo Electron Corporation, Radnor, PA, USA). The supernatant was collected; the pellets were washed using 1× PBS followed by centrifugation at 100,000× g for 2 h and then were resuspended in 1× PBS. The characterization of EVs was performed using nano tracking analysis and electron microscopy (negative staining), as described in our previous studies 29 , 30 and detailed in the Data S1, as previously described.

Electron microscopy–negative staining of HaCat cells

HaCat cells were fixed in a 1% glutaraldehyde and 4% formaldehyde mixture in 0.1 M phosphate buffer (pH 7.2) for 10 min. Cells were detached and fixation continued for 1 h and stored at +4°C in the fixative. After fixation, cells were centrifuged to form a pellet, immersed in 2% agarose in distilled water, and postfixed in 1% osmium tetroxide, dehydrated in acetone, and embedded in Epon LX 112 (Ladd Research Industries, Vermont, USA). Thin sections (70 nm) were cut with a Leica Ultracut UCT ultramicrotome, stained using uranyl acetate and lead citrate, and examined in a Tecnai G2 Spirit transmission electron microscope (FEI Europe, Eindhoven, The Netherlands) operated at 100 kV. Images were captured with a Quemesa bottom‐mounted CCD camera (Olympus Soft Imaging Solutions GMBH, Münster Germany) using Radius software (EMSIS GmbH, Münster, Germany).

The number of mitochondria was counted in ten cells from both normal and high‐glucose conditions. Mitochondria were classified into two groups: (1) “normal mitochondria,” which can exhibit various shapes (elongated or round) with organized cristae and an electron‐dense matrix, and (2) “abnormal mitochondria,” which are swollen, enlarged, and round, characterized by disarrangement of their cristae and a partially or completely electron‐lucent matrix, as previously reported in our publication 31 .

Nitrite concentration determination

HaCat cells were exposed to heat as explained earlier in the experimental design. Cell lysate was prepared using RIPA buffer (Cell Signaling Technology). Nitrite concentration was determined using a Griess reagent kit (G7921, Thermo Scientific). The standard nitrite concentration solutions were prepared up to 100 mg. Cell lysate samples and standards were combined with Griess reagent following the protocol provided with the kit, and incubated for 30 min. After incubation, the absorbance at 544 nm was measured with a spectroscope (Victor V3, Perkin Elmers, Massachusetts, USA). The standard curve was plotted using nitrite standards and their respective absorbance at 544 nm. Nitrite concentration in cell lysate was calculated by the corresponding standard curve equation.

Statistical analysis

All analyses were performed with GraphPad Prism software, version (9.3.1), which was used for the statistical analyses. The two‐tailed Student t‐test was employed and *P‐values less than 0.05 were considered significant.

RESULTS

Thermal stress induces metabolic remodeling in skin keratinocytes

Recently, we demonstrated that metabolite levels in sweat‐derived EVs are modulated by heat exposure, highlighting their potential as noninvasive indicators of metabolic changes in both healthy and diabetic individuals 29 , 30 . A key limitation of our clinical study, however, was the lack of access to baseline metabolite profiles under conditions of high glucose alone or heat exposure alone under normal glucose conditions. To overcome this, and building on evidence that eccrine sweat glands play a central role in maintaining skin homeostasis 6 , 32 , 33 , and given their ectodermal origin with keratinocytes, we sought to further explore these metabolic responses in a controlled in vitro setting.

We used HaCaT keratinocyte cells as a model system to investigate the individual and combined effects of heat and glucose stress on cellular metabolism. To mimic normoglycemic and diabetic environments, cells were cultured under normal (5 mM) and high (25 mM) glucose conditions, as previously reported 34 , 35 , 36 , 37 , 38 . Afterward, the cells were exposed to heat stress at 40°C for 90 min. We assessed the impact of temperature changes on cell numbers and viability using trypan blue staining to distinguish living from dead cells. Our results indicated no significant difference in cell survival between cells cultured in normal‐ or high‐glucose conditions when exposed to heat compared with those maintained at room temperature of 37°C (Figure S1A,B). These findings were further supported by ultrastructural electron microscopy analyses presented in a later section of this article.

Subsequently, metabolite profiling was conducted using a targeted metabolomics approach as described by Rahat et al. 29 (Figure 1a). Our analysis revealed that heat exposure significantly decreased six metabolites under high‐glucose conditions compared to normal glucose. Notably, arginine and valine (amino acids), along with uracil (a pyrimidine metabolite), were markedly downregulated (Figure 1b–d). Conversely, levels of leucine and serine (amino acids), as well as lactate (a glycolysis product), were significantly elevated (Figure 1e–g). Interestingly, these intracellular shifts contrast with our previous observations in sweat EVs from individuals with type 2 diabetes 29 .

Figure 1.

Figure 1

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Metabolic composition of keratinocytes upon acute heat exposure. (a) Schematic overview of the experimental design. (b–g) Relative levels of arginine (b), valine (c), uracil (d), leucine (e), serine (f), and lactate (g) in keratinocytes maintained under high‐ versus normal glucose conditions following 90 min of heat exposure. Data represent fold changes in metabolite peak areas ± SEM, normalization to the cell number and expressed relative to non‐heated control cells. Two‐tailed Student's t‐test was used. *P < 0.05.

To determine whether the observed metabolic changes were specifically induced by the combined effect of high glucose and heat, we examined cells exposed to acute heat under normal glucose (5 mM) conditions. In this setting, none of the previously identified metabolites, including arginine, uracil, leucine, serine, or lactate, were significantly altered, although arginine displayed an opposite trend compared to the combined condition (Figure S2A–F). Similarly, exposing cells to high glucose at 37°C did not significantly affect the levels of arginine, leucine, serine, or lactate. However, the latter three metabolites exhibited trends consistent with those observed under the combined stress condition, albeit to a lesser extent (Figure S2G,J–L). Notably, valine and uracil were significantly decreased in high‐glucose compared to normal glucose conditions (Figure S2H,I).

Collectively, these findings suggest that arginine is uniquely responsive to the combined effects of heat and high‐glucose exposure, highlighting its potential as a specific marker of this dual stress interaction in clinical settings. This also confirms our previous clinical observation reported in Rahat et al. 29 .

Thermal regulation of nitric oxide in skin keratinocytes

Building on our observation that arginine levels significantly decreased following heat exposure under high‐glucose conditions, we sought to investigate the underlying molecular mechanism. Arginine serves as a precursor for nitric oxide (NO), which is synthesized via its oxidation by nitric oxide synthase (NOS) 13 , 14 , 15 , 16 . Given the reduction in arginine upon acute heat exposure, we hypothesized a concomitant decrease in NO production. To test this, NO levels were quantified using the Griess assay as previously described 39 (Figure 2a). Following 90 min of heat exposure, NO concentrations were substantially lower in keratinocytes cultured under high‐glucose conditions compared to their normal glucose counterparts (Figure 2b), suggesting a link between decreased arginine availability and reduced NO production in heat‐stressed hyperglycemic keratinocytes.

Figure 2.

Figure 2

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Nitric oxide is a downstream target of both acute and chronic heat exposure in high glucose‐maintained keratinocytes and their EVs. (a) Schematic representation of the experimental workflow for assessing NO levels under acute heat exposure. (b) NO concentration in HaCat cells maintained in normal‐ and high‐ glucose conditions after 90 min of acute heat exposure. (c) Schematic representation of the experimental workflow for assessing NO levels under chronic heat exposure. (d) NO concentration in HaCat cells after 48 h of chronic heat exposure under normal‐ and high‐ glucose conditions. (e) NO concentration in EVs isolated from HaCat cells maintained in normal‐ and high‐ glucose conditions after 48 h of heat exposure. Data are presented as mean ± SEM from five independent experiments in (b) and three in (d) and (e), normalized to either total cellular protein (μg) or EV particle concentration. Two‐tailed Student's t‐test was used. *P < 0.05.

Previous studies have suggested that glucose concentration may influence NO production 35 , 40 , 41 . To investigate this in our model and determine whether the observed NO reduction was specific to the combined effects of heat and glucose, HaCat cells were incubated at 37°C in media containing 5, 12, or 25 mM glucose, and NO levels were measured (Figure S3A). While a moderate, nonsignificant increase in NO production was observed at higher glucose concentrations, the differences were not statistically significant (Figure S3B). These results suggest that NO production is not solely driven by glucose concentration but rather by a synergistic interaction between hyperglycemia and heat stress.

Building on the growing evidence that the molecular composition of EVs does not necessarily mirror the molecular profile of their parent cells or tissues 42 , 43 , and our current findings showing that keratinocytes' metabolic profiles under heat exposure contrast with those observed in sweat EVs collected from healthy and type 2 diabetic patients in clinical settings 29 , we aimed to assess NO levels in keratinocytes' EVs following heat exposure and compare their patterns with those observed in keratinocytes themselves. Due to insufficient EV yield after 90 min in our experimental setup, which differs from clinical conditions, we extended the culture duration to 48 h based on evidence indicating this timeframe optimally enriches EVs 31 , 44 , 45 , 46 . Keratinocytes were cultured under normal‐ and high‐glucose conditions with heat treatment. Following exposure, both cells and culture media were collected: cells were used for intracellular NO analysis, while the media were processed for EV isolation and subsequent NO quantification (Figure 2c). A significant reduction in NO production was observed in high‐glucose cells compared to normal glucose controls (Figure 2d), confirming the sustained effect of thermal stress on NO synthesis. In contrast, EVs isolated from culture supernatants after 48 h, characterized using nano tracking analysis (NTA) and electron microscopy (Figure S4A,B), revealed that high‐glucose conditions induced a greater release of EVs containing significantly elevated NO levels (Figure 2e). These results support the role of EVs as a compensatory mechanism regulating cellular stress responses in hyperglycemic keratinocytes.

Altogether, our data reveal NO as a potential biomarker in keratinocytes and their EVs for monitoring the cellular effects of heat exposure in diabetic conditions, with contrasting levels observed in the two matrices.

Thermal stress alerts HSP‐70 signaling and mitochondrial morphology in human keratinocytes

Heat Shock Protein 70 (HSP‐70) plays a protective role in response to heat stress by functioning as a molecular chaperone to maintain cellular integrity 26 , 47 . Recent studies have also proposed that HSP‐70 may modulate the NO pathway 14 . To investigate the impact of acute and chronic heat exposure on HSP‐70 expression, we conducted western blot analysis (Figure 3a).

Figure 3.

Figure 3

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HSP‐70 protein levels are altered in response to acute and chronic heat exposure and are associated with changes in mitochondrial morphology. (a) Schematic representation of the experimental methodology. (b) Western blotting analysis of HSP‐70 in HaCat cells after 90 min of acute heat exposure. (c) Quantification of HSP‐70 protein expression levels following acute heat exposure during 90 min. (d) Western blotting analysis of HSP‐70 expression in HaCat cells following 48 h of chronic heat exposure. (e) Quantification of HSP‐70 protein expression levels after 48 h of heat exposure. Data are presented as averages, normalized to control ± SEM from three independent experiments. β‐Actin was used as a loading control. Two‐tailed Student's t‐test was used. *P < 0.05. (f) Acute heat exposure induces abnormal mitochondrial phenotype in keratinocytes, characterized by swelling, enlarged round shapes, disorganized cristae, and a partially or completely electron‐lucent matrix (black dotted rectangle). In contrast, mitochondria in cells maintained in 25 mM glucose retained a normal appearance, displaying various shapes (elongated or round), well‐organized cristae, and an electron‐dense matrix (black dotted rectangle). Scale bar = 500 nm.

Following 90 min of heat exposure, western blot images and subsequent band intensity quantification revealed a significant decrease in HSP‐70 levels in high glucose‐maintained cells compared to those in normal glucose conditions (Figures 3b,c and S5). Similarly, after 48 h of heat exposure, HSP‐70 expression was substantially downregulated, indicating a progressive decline with increasing heat (Figure 3d,e and S6). To confirm that high glucose alone affects HSP‐70 expression, we examined its levels under basal conditions (37°C). The results showed no significant difference, suggesting that glucose concentration alone does not modulate HSP‐70 expression (Figure S7A). These findings align with our ongoing study, which indicates that high glucose induces metabolic reprogramming in keratinocytes through specific molecular modulators, as identified using a 2D gel‐based proteomics approach (Ali et al., In Preparation). Collectively, the data suggest that the observed reductions in HSP‐70 expression under heat stress are temporally correlated with changes in arginine and NO levels in both acute and chronic heat exposure scenarios.

Considering the established role of HSP‐70 in maintaining mitochondrial homeostasis by supporting protein folding and preserving aggregation 25 , 26 , 48 , 49 , we investigated whether the heat‐induced decline in HSP‐70 expression might trigger mitochondrial phenotype alterations under high‐glucose conditions. Electron microscopy images revealed that the majority of mitochondria in cells maintained in 25 mM glucose appeared structurally normal after acute heat exposure, exhibiting various shapes (elongated or round), well‐organized cristae, and an electron‐dense matrix. In contrast, the majority of mitochondria in cells maintained in 5 mM glucose appeared abnormal, displaying swelling, enlargement, and rounded shapes, along with irregularly organized cristae and a partially or completely electron‐lucent matrix (Figure 3f, dashed rectangle). These observations were supported by the low magnification of electron microscopy images showing the ultrastructural details of 15–20 cells and their total mitochondria and indicating a healthy cellular state (Figure S8A,B). These findings align with our viability data, showing that heat did not induce cytotoxicity, as presented in Figure S1A,B. Subsequently, the number of normal and abnormal mitochondria under both glucose conditions after 90 min of heat exposure was quantified using ten cells per condition, based on our previously published data 31 . The results demonstrated an increase in abnormal mitochondria in cells under normal glucose conditions, whereas cells maintained under high‐glucose conditions showed a significant decrease in abnormal mitochondria (Figure 4a). Building on our observations and the reported evidence linking HSP‐70 to the maintenance of mitochondrial homeostasis 25 , 26 , 48 , 49 , we propose that mitochondria in high‐glucose conditions are more resilient than those in normal glucose conditions, reflecting a protective mechanism.

Figure 4.

Figure 4

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Acute and repetitive heat exposure influence keratinocytes responses under high‐glucose conditions. (a) Quantification of normal and abnormal mitochondria in ten cells from both normal‐ and high‐glucose conditions after 90 min of heat exposure. Results are shown as the averages ± SEM using ten cells from each glucose condition. Two‐tailed Student's t‐test was used. *P < 0.05. (b) Schematic representation of the experimental methodology. (c) Nitrate concentration in normal‐ and high glucose‐maintained HaCat upon repetitive heat exposure. Data are presented as the average ± SEM from three independent experiments, normalized to protein concentration and control levels. (d) Western blotting analysis of HSP‐70 expression in HaCat cells following three consecutive 90‐min acute heat exposures. (e) Quantification of HSP‐70 protein levels based on band intensity. Results are shown as the averages ± SEM from three independent experiments, normalized control levels. β‐Actin was used as a loading control. Two‐tailed Student's t‐test was used. *P < 0.05.

Supporting this hypothesis, further analysis of metabolites significantly influenced by high glucose revealed a marked decrease in hypoxanthine, an established marker of oxidative stress 44 , 45 , 46 , 50 , alongside a trend toward increased levels of γ‐Aminobutyric acid (GABA)‐related metabolites, which are associated with antioxidant mechanisms 51 , 52 , in high‐glucose cells under basal conditions (37°C; Figure S7B,C). These observations align with our ongoing data indicating that high glucose induces metabolic reprogramming in keratinocytes through specific target modulators (Ali N. et al., in preparation).

Taken together, these findings reveal that heat exposure influences HSP‐70 expression levels in a similar pattern to NO, suggesting a potential link between these two factors and the modulation of mitochondrial morphology in keratinocytes under heat stress.

Repetitive heat exposure enhances NO and HSP‐70 levels and modulates mitochondrial phenotype in keratinocytes

Recent clinical studies suggest that the duration and frequency of heat exposure are critical factors for achieving therapeutic benefits, such as improved insulin sensitivity and glycemic control in diabetic patients 12 , 13 , 14 , 15 . Building on this, we investigated whether repeated heat exposure could enhance NO production and HSP‐70 expression in high glucose‐maintained keratinocytes. Cells underwent three rounds of 90 min heat exposure, as illustrated in Figure 4b. NO levels and HSP‐70 protein expression were assessed using the Griess assay and western blotting, respectively.

Our findings demonstrate a significant increase in NO production in keratinocytes cultured under high‐glucose conditions compared to those maintained in normal glucose environments (Figure 4c). Consistently, Western blot images revealed markedly higher expression levels of HSP‐70 in high‐glucose cells, which was further confirmed by densitometric quantification of band intensity (Figure 4d,e and S9).

Electron microscopy revealed altered ultrastructural mitochondrial morphology in both normal‐ and high‐glucose‐maintained cells following repeated heat exposure (Figure 5a). Disorganized cristae networks were observed, suggesting that repetitive heat exposure may enhance mitochondrial remodeling, similar to what we observed after acute heat exposure in cells maintained under normal glucose conditions (Figure 3f). Quantification of normal and abnormal mitochondria in ten cells from each condition (normal and high glucose) supports these histological observations, showing an increase in abnormal mitochondria compared to normal in both glucose conditions. This increase was significant in cells maintained in 25 mM glucose. Importantly, these structural changes may reflect an adaptive mechanism by which mitochondria engage in coping with repeated heat exposure, consistent with previous studies suggesting beneficial effects mediated through NO and HSP‐70 11 , 12 , 13 .

Figure 5.

Figure 5

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Repeated heat exposure alters mitochondrial morphology in keratinocytes under high‐glucose conditions. (a) Morphological examination of mitochondria in both keratinocytes incubated with normal and high glucose following repeated heat exposure. Scale bar = 500 nm. (b) Quantification of normal and abnormal mitochondria in ten cells from each condition (normal and high glucose). Results are shown as the averages ± SEM using ten cells from each glucose condition. Two‐tailed Student's t‐test was used. *P < 0.05.

Collectively, these findings demonstrate, for the first time, the beneficial effects of repeated heat exposure on mitochondrial function in keratinocytes, mediated by increased NO production and HSP‐70 expression.

DISCUSSION

In the present study, we focused specifically on keratinocytes, as a representative model for sweat gland cells to investigate their intrinsic response under diabetic‐like conditions and in the absence of neuronal input. Using HaCat cells, our goal was to examine how different durations and frequences of heat exposure influence their metabolic patterns. We employed the same experimental design as in our recent clinical study 29 . Through targeted metabolomics, we were able to identify keratinocyte‐autonomous thermoregulatory metabolic profile responses, distinguishing them from impairments typically attributed to neuropathic factors 7 , 8 .

Sweat glands are extensively innervated, and proper neuronal signaling is essential for initiating sweat secretion 5 , 6 , 8 , 53 . In diabetes, this neuro–glandular interaction is disrupted, and numerous clinical reports have shown that neuropathic dysfunction is closely linked to impaired sweat gland function and, consequently, compromised thermoregulation 54 , 55 , 56 , 57 , 58 , 59 . Additionally, many clinical studies have demonstrated that heat may affect the production of sweat and its molecular composition in both healthy individuals and diabetic patients 56 , 60 , 61 , 62 , 63 , 64 . In agreement, our recently published article supports this statement, showing that heat after exercise may influence the metabolic composition of sweat EVs 30 .

In a previous clinical report, arginine was identified as the highest metabolite present in the sweat of healthy individuals following heat exposure. Additionally, the concentration of arginine in sweat was found to be independent of its variations in blood 65 . In line with this, our current study demonstrates that arginine is the unique metabolite associated with the dual effects of heat and high glucose, accompanied by a similar pattern of NO production. This finding aligns with previous clinical reports indicating that NO levels are altered during heat‐induced exercise, which enhances sweat production 60 , 61 , 64 , 66 , 67 . One explanation for this phenomenon is linked to the activity of nitric oxide synthase (NOS), an enzyme expressed in sweat glands that produces NO through the oxidation of arginine. This process ultimately influences sweat production and composition 21 , 68 , 69 , supporting a regulatory role for NO in thermal adaptation.

Emerging clinical studies have demonstrated the therapeutic potential of heat exposure in improving insulin sensitivity, glycemic control, and inflammation in type 2 diabetes (T2D) patients, with frequent sessions being recommended 10 , 11 , 14 , 27 , 70 . In alignment with this, our findings show that repeated heat exposure restores NO levels and is accompanied by enhanced mitochondrial integrity in keratinocytes maintained under high‐glucose conditions. However, the differing NO levels in sweat EVs from keratinocytes indicate that the EV molecular components may not mirror those of their parental cells or tissues, as previously reported 42 , 43 . Building on our results and the existing literature claiming heat as a potential therapeutic solution for diabetes management, our study proposes that arginine and NO may serve as potential clinical biomarkers, particularly in sweat EVs, offering a noninvasive approach for diabetes management and monitoring the physiological benefits of heat exposure.

While diabetes is associated with decreased levels of HSPs in insulin‐sensitive tissues 71 , 72 , 73 , 74 , repetitive heat exposure has been shown to induce HSP‐70 expression in healthy keratinocytes 28 . Our study confirms this finding in keratinocytes maintained in high‐glucose conditions. We observed that increased HSP‐70 levels correlate with elevated NO production, suggesting a functional interplay between the HSP‐70 and NO pathways as a molecular mechanism through which heat promotes cellular adaptation, as previously reported 11 , 12 , 13 , 14 , 27 .

This interaction appears to support mitochondrial resilience, as evidenced by improved mitochondrial integrity in heat‐exposed, high‐glucose cells. Complementing these findings, we also detected shifts in redox‐related metabolites such as hypoxanthine and GABA, molecules known to be involved in oxidative stress responses, indicating a broader rebalancing of the cellular redox state under thermal stress. These redox adaptations are particularly relevant in high‐glucose environments, where excess reactive oxygen species (ROS) generation disrupts cellular homeostasis 75 , 76 , 77 . NO, acting as a redox‐sensitive signaling molecule, is known to play a pivotal role in modulating oxidative stress and maintaining redox equilibrium 22 . Thus, the coordinated induction of HSP‐70 and NO may represent a compensatory mechanism to counteract glucose‐induced oxidative imbalance, with implications for diabetes management.

A more direct mechanism may link HSP‐70 to mitochondrial protection. Previous studies have shown that repetitive heat exposure can protect keratinocytes from UVB‐induced apoptosis by enhancing mitochondrial integrity through HSP‐70 induction 78 . Similarly, in other cell types, HSP‐70 overexpression has been found to inhibit cytochrome c release and prevent mitochondrial membrane permeabilization during stress, thereby reducing apoptosis 79 , 80 .

These findings suggest that HSP‐70 may support mitochondrial stability through both direct and indirect mechanisms, particularly by modulating nitric oxide (NO) signaling and preserving redox homeostasis. This aligns with the dual‐pathway hypothesis outlined in our graphical abstract. This adapted mechanism is crucial for sustaining keratinocyte survival in the context of diabetes and the skin features associated with this metabolic disorder.

CONCLUSION

Our study identifies potential clinical biomarkers in keratinocytes in response to heat stress under diabetic conditions, which are dependent on the presence of neural connections, supporting our previous clinical observations. Our findings demonstrate an association between amino acid metabolism, specifically arginine and its downstream product nitric oxide (NO), as central players in the molecular mechanism by which keratinocytes respond to heat stress. The dynamic responses of NO to repeated heat exposure may also hold clinical value in managing diabetes. Elucidating the molecular mechanisms involving HSP‐70 and mitochondrial activity supports an adaptive pathway utilized by keratinocytes to cope with heat stress conditions.

In conclusion, considering previous clinical observations that identify both arginine and NO as key players in beneficial heat therapies, our study provides proof‐of‐concept evidence for the use of these molecules as therapeutic markers and potential therapeutic routes involving HSP‐70 and mitochondria.

Limitations and future directions

Despite these insights, several limitations should be acknowledged. First, although HaCaT cells are a widely accepted in vitro model for keratinocytes, they do not fully replicate the complexity of native sweat gland physiology. Second, our results are based on acute and repetitive heat exposure protocols under controlled conditions, which may not fully capture real‐life thermal stress or long‐term clinical outcomes, especially in diabetic individuals undergoing insulin therapy or other treatments. Third, while we identified changes in nitric oxide levels and associated metabolic shifts, the underlying signaling mechanisms, especially those involving calcium dynamics, mitochondrial respiration, and membrane potential, remain to be elucidated. Additionally, the direct link between HSP‐70 and mitochondrial function, as proposed in the graphical abstract, warrants further investigation.

Future studies involving primary human sweat gland cells, in vivo validation, and broader metabolic profiling are necessary to strengthen the translational potential of our findings. Additionally, exploring the interplay between Trisk95, calcium dynamics, and NO signaling may help uncover new regulatory mechanisms relevant to thermal adaptation and metabolic health.

AUTHOR CONTRIBUTION

NA and SJ‐V created the idea. NA and ST‐R designed the experiments. ST‐R performed the experiments related to NO concentration and HSP‐70 expression. A‐IN performed the metabolomics analysis and provided the data. I.M. assessed mitochondrial morphology via electron microscopy. NA prepared the figures and wrote the paper with the help of ST‐R. SV edited the paper. All authors reviewed and edited the paper.

FUNDING

This work was supported by the Finnish Research Impact Foundation Tandem Industry Academia grant (NA), the European Regional Development Fund project PrintoDise (SV, ST‐R), the Infotech Oulu Institute projects (NA, SV), NETSKINMODELS COST Action CA21108 and the Academy of Finland Flagship GeneCellNano (ST‐R, NA, SV). The FIMM Metabolomics Unit was supported by HiLIFE and Biocenter Finland (A‐IN).

DISCLOSURE

The authors declare no conflict of interest.

Approval of the research protocol: N/A.

Informed consent: N/A.

Approval date of registry and registration no: N/A.

Animal study: N/A.

Supporting information

Data S1. Supplementary Materials.

Figure S1. Morphological and viability assay of HaCat cells under heat exposure.

Figure S2. Metabolic profile of keratinocytes under acute heat exposure and high‐glucose conditions.

Figure S3. Nitric oxide production remains unchanged across varying glucose concentrations.

Figure S4. Characterization of HaCat‐derived EVs.

Figure S5. Uncropped western blot showing HSP70 expression in HaCat cells 90 min after heat exposure.

Figure S6. Uncropped western blot showing HSP70 expression in HaCat cells 48 h after heat exposure.

Figure S7. HSP‐70 expression and metabolic profile under basal conditions (37°C) in cells maintained in normal‐ or high‐ glucose environments.

Figure S8. Acute heat exposure induces alterations in mitochondrial morphology under high‐glucose conditions.

Figure S9. Uncropped western blot showing HSP70 expression in HaCat cells following repeated heat exposure.

JDI-17-608-s001.docx (3.4MB, docx)

ACKNOWLEDGMENT

We thank J. Kekolahti‐Liias and P. Haipus for their excellent technical assistance in the laboratory. We also appreciate Mira Mäkelä for her contributions to the project and intellectual support. Additionally, we acknowledge Keerthanaa Balasubramanian Shanthi and Naveed Ahmad for providing the initial protocols for the isolation of EVs in cells, which we subsequently modified for our study. We would like to thank the facilities and expertise of Helsinki Metabolomics Center, supported by HiLIFE and Biocenter Finland.

DATA AVAILABILITY STATEMENT

The data sets generated and/or analyzed during the current study are openly available from the University of Oulu data repository at https://oulurepo.oulu.fi/ or from the corresponding author on request.

REFERENCES

  • 1. Galicia‐Garcia U, Benito‐Vicente A, Jebari S, et al. Pathophysiology of type 2 diabetes mellitus. Int J Mol Sci 2020; 21: 6275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. IDF Diabetes Atlas 7th.pdf [online]. https://www.diabetesatlas.org/upload/resources/previous/files/7/IDF%20Diabetes%20Atlas%207th.pdf. Accessed October 30, 2022.
  • 3. Cuschieri S, Calleja Agius J. The interaction between diabetes and climate change – A review on the dual global phenomena. Early Hum Dev 2021; 155: 105220. [DOI] [PubMed] [Google Scholar]
  • 4. Xu Z, Tong S, Ho HC, et al. Associations of heat and cold with hospitalizations and post‐discharge deaths due to acute myocardial infarction: What is the role of pre‐existing diabetes? Int J Epidemiol 2022; 51: 134–143. [DOI] [PubMed] [Google Scholar]
  • 5. Murota H, Matsui S, Ono E, et al. Sweat, the driving force behind normal skin: An emerging perspective on functional biology and regulatory mechanisms. J Dermatol Sci 2015; 77: 3–10. [DOI] [PubMed] [Google Scholar]
  • 6. Baker LB. Physiology of sweat gland function: The roles of sweating and sweat composition in human health. Temperature (Austin) 2019; 6: 211–259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Fealey RD, Low PA, Thomas JE. Thermoregulatory sweating abnormalities in diabetes mellitus. Mayo Clin Proc 1989; 64: 617–628. [DOI] [PubMed] [Google Scholar]
  • 8. Greaney JL, Kenney WL, Alexander LM. Sympathetic regulation during thermal stress in human aging and disease. Auton Neurosci 2016; 196: 81–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Sebők J, Édel Z, Dembrovszky F, et al. Effect of HEAT therapy in patiEnts with type 2 diabetes mellitus (HEATED): Protocol for a randomised controlled trial. BMJ Open 2022; 12: e062122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Sebők J, Édel Z, Váncsa S, et al. Heat therapy shows benefit in patients with type 2 diabetes mellitus: A systematic review and meta‐analysis. Int J Hyperthermia 2021; 38: 1650–1659. [DOI] [PubMed] [Google Scholar]
  • 11. Bathaie SZ, Jafarnejad A, Hosseinkhani S, et al. The effect of hot‐tub therapy on serum Hsp70 level and its benefit on diabetic rats: A preliminary report. Int J Hyperthermia 2010; 26: 577–585. [DOI] [PubMed] [Google Scholar]
  • 12. Henstridge DC, Bruce CR, Drew BG, et al. Activating HSP72 in rodent skeletal muscle increases mitochondrial number and oxidative capacity and decreases insulin resistance. Diabetes 2014; 63: 1881–1894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Molina MN, Ferder L, Manucha W. Emerging role of nitric oxide and heat shock proteins in insulin resistance. Curr Hypertens Rep 2015; 18: 1. [DOI] [PubMed] [Google Scholar]
  • 14. Hooper PL. Diabetes, nitric oxide, and heat shock proteins. Diabetes Care 2003; 26: 951–952. [DOI] [PubMed] [Google Scholar]
  • 15. Lundberg JO, Weitzberg E. Nitric oxide signaling in health and disease. Cell 2022; 185: 2853–2878. [DOI] [PubMed] [Google Scholar]
  • 16. Jobgen WS, Fried SK, Fu WJ, et al. Regulatory role for the arginine–nitric oxide pathway in metabolism of energy substrates. J Nutr Biochem 2006; 17: 571–588. [DOI] [PubMed] [Google Scholar]
  • 17. Daniela B‐G, Thomas R, Kolb‐Bachofen V. Nitric oxide in human skin: Current status and future prospects. J Invest Dermatol 1998; 110: 1–7. [DOI] [PubMed] [Google Scholar]
  • 18. Cals‐Grierson M‐M, Ormerod AD. Nitric oxide function in the skin. Nitric Oxide 2004; 10: 179–193. [DOI] [PubMed] [Google Scholar]
  • 19. Arany I, Brysk MM, Brysk H, et al. Regulation of inducible nitric oxide synthase mRNA levels by differentiation and cytokines in human keratinocytes. Biochem Biophys Res Commun 1996; 220: 618–622. [DOI] [PubMed] [Google Scholar]
  • 20. Andrabi SM, Sharma NS, Karan A, et al. Nitric oxide: Physiological functions, delivery, and biomedical applications. Adv Sci 2023; 10: 2303259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Cinelli MA, Do HT, Miley GP, et al. Inducible nitric oxide synthase: Regulation, structure, and inhibition. Med Res Rev 2020; 40: 158–189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Joshi MS, Ponthier JL, Lancaster JR. Cellular antioxidant and pro‐oxidant actions of nitric oxide. Free Radic Biol Med 1999; 27: 1357–1366. [DOI] [PubMed] [Google Scholar]
  • 23. Chachlaki K, Garthwaite J, Prevot V. The gentle art of saying NO: How nitric oxide gets things done in the hypothalamus. Nat Rev Endocrinol 2017; 13: 521–535. [DOI] [PubMed] [Google Scholar]
  • 24. Leshan RL, Greenwald‐Yarnell M, Patterson CM, et al. Leptin action through hypothalamic nitric oxide synthase‐1–expressing neurons controls energy balance. Nat Med 2012; 18: 820–823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Kim JY, Han Y, Lee JE, et al. The 70‐kDa heat shock protein (Hsp70) as a therapeutic target for stroke. Expert Opin Ther Targets 2018; 22: 191–199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Havalová H, Ondrovičová G, Keresztesová B, et al. Mitochondrial HSP70 chaperone system—The influence of post‐translational modifications and involvement in human diseases. Int J Mol Sci 2021; 22: 8077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Hooper PL, Balogh G, Rivas E, et al. The importance of the cellular stress response in the pathogenesis and treatment of type 2 diabetes. Cell Stress Chaperones 2014; 19: 447–464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Scieglinska D, Krawczyk Z, Sojka DR, et al. Heat shock proteins in the physiology and pathophysiology of epidermal keratinocytes. Cell Stress Chaperones 2019; 24: 1027–1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Rahat ST, Mäkelä M, Nasserinejad M, et al. Clinical‐grade patches as a medium for enrichment of sweat‐extracellular vesicles and facilitating their metabolic analysis. Int J Mol Sci 2023; 24: 7507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Ali N, Rahat ST, Mäkelä M, et al. Metabolic patterns of sweat‐extracellular vesicles during exercise and recovery states using clinical grade patches. Front Physiol 2023; 14: 1295852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Ali N, Rezvani HR, Motei D, et al. Trisk 95 as a novel skin mirror for normal and diabetic systemic glucose level. Sci Rep 2020; 10: 12246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Lin Y, Chen L, Zhang M, et al. Eccrine sweat gland and its regeneration: Current status and future directions. Front Cell Dev Biol 2021; 9: 667765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Chen R, Zhu Z, Ji S, et al. Sweat gland regeneration: Current strategies and future opportunities. Biomaterials 2020; 255: 120201. [DOI] [PubMed] [Google Scholar]
  • 34. Cheng T‐L, Lai C‐H, Chen P‐K, et al. Thrombomodulin promotes diabetic wound healing by regulating toll‐like receptor 4 expression. J Invest Dermatol 2015; 135: 1668–1675. [DOI] [PubMed] [Google Scholar]
  • 35. Nakai K, Fujii S, Yamamoto A, et al. Effects of high glucose on NO synthesis in human keratinocyte cell line (HaCaT). J Dermatol Sci 2003; 31: 211–218. [DOI] [PubMed] [Google Scholar]
  • 36. Chen Y‐Y, Huang S‐M, Cheng Y‐W, et al. Investigation of the keratinocyte transcriptome altered in high‐glucose environment: An in‐vitro model system for precision medicine. J Dermatol Sci 2023; 109: 37–46. [DOI] [PubMed] [Google Scholar]
  • 37. Wang M, Huang H, Xu Y, et al. Metabolic rewiring in keratinocytes by miR‐31‐5p identifies therapeutic intervention for psoriasis. EMBO Mol Med 2023; 15: e15674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Wang J, Pothana K, Chen S, et al. Ultraviolet B irradiation alters the level and miR contents of exosomes released by keratinocytes in diabetic condition. Photochem Photobiol 2022; 98: 1122–1130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Weller R, Pattullo S, Smith L, et al. Nitric oxide is generated on the skin surface by reduction of sweat nitrate. J Invest Dermatol 1996; 107: 327–331. [DOI] [PubMed] [Google Scholar]
  • 40. Nakai K, Kubota Y, Kosaka H. Inhibition of nuclear factor kappa B activation and inducible nitric oxide synthase transcription by prolonged exposure to high glucose in the human keratinocyte cell line HaCaT. Br J Dermatol 2004; 150: 640–646. [DOI] [PubMed] [Google Scholar]
  • 41. Rizwan H, Kumar S, Kumari G, et al. High glucose‐induced increasing reactive nitrogen species accumulation triggered mitochondrial dysfunction, inflammation, and apoptosis in keratinocytes. Life Sci 2023; 312: 121208. [DOI] [PubMed] [Google Scholar]
  • 42. Hayasaka R, Tabata S, Hasebe M, et al. Metabolomics of small extracellular vesicles derived from isocitrate dehydrogenase 1‐mutant HCT116 cells collected by semi‐automated size exclusion chromatography. Front Mol Biosci 2023; 9: 1049402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Eirin A, Zhu X‐Y, Puranik AS, et al. Comparative proteomic analysis of extracellular vesicles isolated from porcine adipose tissue‐derived mesenchymal stem/stromal cells. Sci Rep 2016; 6: 36120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Johnson TA, Jinnah HA, Kamatani N. Shortage of cellular ATP as a cause of diseases and strategies to enhance ATP. Front Pharmacol 2019; 10: 98. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. Farthing DE, Farthing CA, Xi L. Inosine and hypoxanthine as novel biomarkers for cardiac ischemia: From bench to point‐of‐care. Exp Biol Med (Maywood) 2015; 240: 821–831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Fujiwara M, Sato N, Okamoto K. Hypoxanthine reduces radiation damage in vascular endothelial cells and mouse skin by enhancing ATP production via the salvage pathway. Radiat Res 2022; 197: 583–593. [DOI] [PubMed] [Google Scholar]
  • 47. Saibil H. Chaperone machines for protein folding, unfolding and disaggregation. Nat Rev Mol Cell Biol 2013; 14: 630–642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48. Hartl FU, Bracher A, Hayer‐Hartl M. Molecular chaperones in protein folding and proteostasis. Nature 2011; 475: 324–332. [DOI] [PubMed] [Google Scholar]
  • 49. Kampinga HH, Craig EA. The Hsp70 chaperone machinery: J‐proteins as drivers of functional specificity. Nat Rev Mol Cell Biol 2010; 11: 579–592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50. Wu W, Song Y, Sun M, et al. Corneal metabolic biomarkers for moderate and high myopia in human. Exp Eye Res 2023; 237: 109689. [DOI] [PubMed] [Google Scholar]
  • 51. Tritsch NX, Granger AJ, Sabatini BL. Mechanisms and functions of GABA co‐release. Nat Rev Neurosci 2016; 17: 139–145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52. Zhang Q, Zhu L, Li H, et al. Insights and progress on the biosynthesis, metabolism, and physiological functions of gamma‐aminobutyric acid (GABA): A review. PeerJ 2024; 12: e18712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53. Hu Y, Converse C, Lyons MC, et al. Neural control of sweat secretion: A review. Br J Dermatol 2018; 178: 1246–1256. [DOI] [PubMed] [Google Scholar]
  • 54. Levy DM, Rowley DA, Abraham RR. Changes in cholinergic sweat gland activation in diabetic neuropathy identified by computerised sweatspot analysis. Diabetologia 1991; 34: 807–812. [DOI] [PubMed] [Google Scholar]
  • 55. Liu Y, Billiet J, Ebenezer GJ, et al. Factors influencing sweat gland innervation in diabetes. Neurology 2015; 84: 1652–1659. [DOI] [PubMed] [Google Scholar]
  • 56. Petrofsky JS, Lee S, Patterson C, et al. Sweat production during global heating and during isometric exercise in people with diabetes. Med Sci Monit 2005; 11: CR515–CR521. [PubMed] [Google Scholar]
  • 57. Berghoff M, Kilo S, Hilz MJ, et al. Differential impairment of the sudomotor and nociceptor axon‐reflex in diabetic peripheral neuropathy. Muscle Nerve 2006; 33: 494–499. [DOI] [PubMed] [Google Scholar]
  • 58. Chahal S, Vohra K, Syngle A. Association of sudomotor function with peripheral artery disease in type 2 diabetes. Neurol Sci 2017; 38: 151–156. [DOI] [PubMed] [Google Scholar]
  • 59. Liu B, Cheng X, Chen M, et al. Changes of sweat gland function in type 2 diabetes mellitus patients with peripheral neuropathy. Int J Diabetes Dev Ctries 2024; 44: 84–90. [Google Scholar]
  • 60. Stapleton JM, Fujii N, Carter M, et al. Diminished nitric oxide‐dependent sweating in older males during intermittent exercise in the heat. Exp Physiol 2014; 99: 921–932. [DOI] [PubMed] [Google Scholar]
  • 61. Schmidt MD, McGarr GW, Muia CM, et al. Regional influence of nitric oxide on cutaneous vasodilatation and sweating during exercise‐heat stress in young men. Exp Physiol 2020; 105: 773–782. [DOI] [PubMed] [Google Scholar]
  • 62. Kennedy WR, Sakuta M, Sutherland D, et al. The sweating deficiency in diabetes mellitus: Methods of quantitation and clinical correlation. Neurology 1984; 34: 758–763. [DOI] [PubMed] [Google Scholar]
  • 63. Avila S, Buono MJ. Priming of the sweat glands explains reflex sweati.pdf [online]. 2012. https://www.tandfonline.com/doi/pdf/10.3109/02656736.2011.613891?needAccess=true. [DOI] [PubMed]
  • 64. Welch G, Foote KM, Hansen C, et al. Nonselective NOS inhibition blunts the sweat response to exercise in a warm environment. J Appl Physiol 2009; 106: 796–803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65. Hier SW, Cornbleet T, Bergeim O. The amino acids of human sweat. J Biol Chem 1946; 166: 327–333. [PubMed] [Google Scholar]
  • 66. McNamara TC, Keen JT, Simmons GH, et al. Endothelial nitric oxide synthase mediates the nitric oxide component of reflex cutaneous vasodilatation during dynamic exercise in humans. J Physiol 2014; 592: 5317–5326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67. Lee K, Mack GW. Role of nitric oxide in methacholine‐induced sweating and vasodilation in human skin. J Appl Physiol 2006; 100: 1355–1360. [DOI] [PubMed] [Google Scholar]
  • 68. Abu‐Soud HM, Gachhui R, Raushel FM, et al. The ferrous‐dioxy complex of neuronal nitric oxide synthase: Divergent effects of l‐arginine and tetrahydrobiopterin on its stability*. J Biol Chem 1997; 272: 17349–17353. [DOI] [PubMed] [Google Scholar]
  • 69. Shimizu Y, Sakai M, Umemura Y, et al. Immunohistochemical localization of nitric oxide synthase in Normal human skin: Expression of endothelial‐type and inducible‐type nitric oxide synthase in keratinocytes. J Dermatol 1997; 24: 80–87. [DOI] [PubMed] [Google Scholar]
  • 70. Pallubinsky H, Phielix E, Dautzenberg B, et al. Passive exposure to heat improves glucose metabolism in overweight humans. Acta Physiol (Oxf) 2020; 229: e13488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71. Gupte AA, Bomhoff GL, Swerdlow RH, et al. Heat treatment improves glucose tolerance and prevents skeletal muscle insulin resistance in rats fed a high‐fat diet. Diabetes 2009; 58: 567–578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 72. Hooper PL, Hooper PL. Inflammation, heat shock proteins, and type 2 diabetes. Cell Stress Chaperones 2009; 14: 113–115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73. Kurucz I, Morva Á, Vaag A, et al. Decreased expression of heat shock protein 72 in skeletal muscle of patients with type 2 diabetes correlates with insulin resistance. Diabetes 2002; 51: 1102–1109. [DOI] [PubMed] [Google Scholar]
  • 74. Oster H. Getting hot about diabetes—Repeated heat exposure improves glucose regulation and insulin sensitivity. Acta Physiol 2020; 229: e13524. [DOI] [PubMed] [Google Scholar]
  • 75. D'Autréaux B, Toledano MB. ROS as signalling molecules: Mechanisms that generate specificity in ROS homeostasis. Nat Rev Mol Cell Biol 2007; 8: 813–824. [DOI] [PubMed] [Google Scholar]
  • 76. Bandeira S d M, da Fonseca LJS, Guedes G d S, et al. Oxidative stress as an underlying contributor in the development of chronic complications in diabetes mellitus. Int J Mol Sci 2013; 14: 3265–3284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 77. Giacco F, Brownlee M. Oxidative stress and diabetic complications. Circ Res 2010; 107: 1058–1070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 78. Maytin EV, Wimberly JM, Kane KS. Heat shock modulates UVB‐induced cell death in human epidermal keratinocytes: Evidence for a hyperthermia‐inducible protective response. J Invest Dermatol 1994; 103: 547–553. [DOI] [PubMed] [Google Scholar]
  • 79. Stankiewicz AR, Lachapelle G, Foo CPZ, et al. Hsp70 inhibits heat‐induced apoptosis upstream of mitochondria by preventing Bax translocation. J Biol Chem 2005; 280: 38729–38739. [DOI] [PubMed] [Google Scholar]
  • 80. Gu ZT, Li L, Wu F, et al. Heat stress induced apoptosis is triggered by transcription‐independent p53, Ca2+ dyshomeostasis and the subsequent Bax mitochondrial translocation. Sci Rep 2015; 5: 11497. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1. Supplementary Materials.

Figure S1. Morphological and viability assay of HaCat cells under heat exposure.

Figure S2. Metabolic profile of keratinocytes under acute heat exposure and high‐glucose conditions.

Figure S3. Nitric oxide production remains unchanged across varying glucose concentrations.

Figure S4. Characterization of HaCat‐derived EVs.

Figure S5. Uncropped western blot showing HSP70 expression in HaCat cells 90 min after heat exposure.

Figure S6. Uncropped western blot showing HSP70 expression in HaCat cells 48 h after heat exposure.

Figure S7. HSP‐70 expression and metabolic profile under basal conditions (37°C) in cells maintained in normal‐ or high‐ glucose environments.

Figure S8. Acute heat exposure induces alterations in mitochondrial morphology under high‐glucose conditions.

Figure S9. Uncropped western blot showing HSP70 expression in HaCat cells following repeated heat exposure.

JDI-17-608-s001.docx (3.4MB, docx)

Data Availability Statement

The data sets generated and/or analyzed during the current study are openly available from the University of Oulu data repository at https://oulurepo.oulu.fi/ or from the corresponding author on request.

Articles from Journal of Diabetes Investigation are provided here courtesy of Wiley

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