Prevention by Heat Stimulation of Metabolic Syndrome Progression Based upon the Underlying Molecular Mechanism

Abstract

Metabolic syndrome (MS) is a pathological condition that causes high blood pressure, abnormal glucose metabolism, and lipid metabolism based on visceral fat accumulation. Insulin resistance and atherosclerosis caused by chronic inflammation of visceral adipose tissue are fundamental pathologies of lifestyle-related diseases. It is well known that diet and exercise are important in preventing these diseases. However, exercise is limited in people with various locomotive disorders. In recent years, the use of heat therapy to treat insulin resistance has attracted attention. Many researchers are interested in strengthening the skeletal muscle functions as a metabolic organ. We are verifying the thermal effect of skeletal muscles on underlying mechanism of MS progression such as chronic inflammation, cell death and heat shock protein 70 family (HSP70). This chapter reviews recent reports on whether hyperthermia may safely contribute to the prevention of MS and its progression to type 2 diabetes and atherosclerosis. It was thought that the chaperone function of HSP70 could be used to influence inflammatory cytokines and contribute to the prevention of insulin resistance and atherosclerosis. Thermal effects may be useful, especially when physical activity is limited. Safe and effective interventions to prevent MS and its progression require further research.

1. Introduction

Metabolic syndrome (MS) is a metabolic condition defined by specific clinical and paraclinical criteria [1]. MS has an accumulation of metabolic abnormalities based on visceral fat accumulation, which is a risk factor for lifestyle-related diseases such as hypertension, dyslipidemia, glucose intolerance, and atherosclerosis.

Lifestyles that cause these include unhealthy eating, lack of exercise, and poor-quality sleep. Particular attention should be paid to high-calorie meals and sedentary lifestyles. High-calorie meals lead to postprandial hypertriglyceridemia. There is a strong relationship between postprandial hyperlipidemia and atherosclerotic cardiovascular disease (ASCVD). Serum triglycerides levels are a surrogate marker of triglyceride-rich lipoproteins (TRLs) such as chylomicrons (CMs), very low-density lipoproteins (VLDLs), and their remnants [2]. High triglycerides due to hyperlipidemia after a high-calorie meal are a factor in reducing the size of low-density lipoproteins (LDL). VLDL has a poor affinity for LDL receptors compared to LDL, and is more easily metabolized in the periphery than in the liver. The remaining lipoproteins can then penetrate the artery wall. Residual lipoproteins, even more than LDL, increase monocyte rolling and adhesion on endothelial cells and translocation between endothelial cells. Residual lipoproteins are involved in inflammation, platelet activation, and endothelial dysfunction by activating transcription factors such as nuclear factor-κB (NF-κB) in endothelial cells and monocytes [3]. In pathological conditions that develop postprandial hyperlipidemia, decreased lipoprotein lipase (LPL) activity lowers high-density lipoprotein (HDL) cholesterol and reduces cholesterol efflux from atherosclerotic plaques [3]. Postprandial hyperlipidemia is likely to be complicated by postprandial hyperglycemia, and postprandial hyperglycemia also induces endothelial dysfunction [4].

It is well known that prolonged sitting time in daily life increases the risk of MS. The longer you spend sitting, the less energy you use [5]. The metabolic effects of a sedentary lifestyle include energy imbalances and muscle inactivity that lead to weight gain and obesity [6]. Even in the absence of obesity, young patients with sarcopenia due to muscle atrophy exhibit early-onset insulin resistance due to muscle atrophy [7]. Even in healthy individuals, limited physical activity was associated with a significant increase in total cholesterol and triglycerides [8]. Physical inactivity can rapidly induce insulin resistance and microvascular dysfunction in healthy individuals, preceding the development of obesity [8]. However, the biological mechanisms linking sedentary lifestyles and metabolic abnormalities are not completely understood [9].

Visceral fat accumulation and insulin resistance are hallmarks of MS, including cardiovascular risk factors such as ASCVD. In visceral adipose tissue, an inflammatory state occurs with adipocyte hypertrophy, angiogenesis, and macrophage infiltration. Chemokine monocyte chemoattractant protein1(MCP1)/C-C motif chemokine 2 (CCL2) is produced from adipocytes, and monocytes migrate from blood vessels into adipose tissue, leading to macrophage infiltration [10]. Infiltrated macrophages highly express Toll-like receptor 4 (TLR4), produce tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-12(IL-12), etc., and progress to a chronic inflammatory state.

Furthermore, free fat acid (FFA) released from adipocytes causes dysfunction in glucose metabolism [11]. Adipose tissue influences insulin action through the secretion of pro-inflammatory cytokines and other factors [1].

C-reactive protein (CRP), the inflammatory marker, is associated with MS, and is also associated with type 2 diabetes and cardiovascular disease. It has been established that CRP levels are elevated in MS, and high-sensitivity CRP (hsCRP) has also been proposed as an additional clinical indicator for the development of MS and hsCRP-modified chronic heart disease risk [11]. Increased CRP in patients with MS increases the likelihood of cardiovascular events due to its effects on the activation of vascular cells, especially monocytes, and the formation of endothelial cell dysfunction [12]. The rate of CRP production is highly correlated with MS characteristics such as waist circumference, increased triglycerides levels, and decreased HDL-cholesterol levels, as well as increased IL-6 levels and decreased adiponectin levels [13]. CRP is also produced in adipocytes and can be overexpressed in individuals with central obesity, ultimately leading to insulin resistance and type 2 diabetes [13].

We have previously reported the relationship between dietary [14] as well as exercise habits [15] and indicators of lifestyle-related diseases, although it is difficult to continue. Furthermore, there are handicapped people who are unable to take daily exercise. We have repeatedly verified the effect of thermal stimulation on skeletal muscles as a preventive method of ASCVD for such people [15, 16, 17]. Skeletal muscle plays an important role in in vivo metabolism, and exercise and thermal stimulation of skeletal muscle are thought to further enhance the ASCVD prevention effect.

We described the effects and possibilities of thermal stimulation of skeletal muscle on glucose metabolism, apoptosis, and anti-inflammatory effects [18]. Heat shock protein 70 (HSP70), one of the heat shock protein (HSP) family, is induced by thermal stimulation, and is known to be involved in improving underlying pathological conditions such as ASCVD and type 2 diabetes.

We have previously reported that thermal stimuli-induced HSP70 might be useful to prevent ASCVD [19]. We believe that HSP70-targeted interventions may control atherosclerosis, insulin resistance, inflammatory conditions, and other underlying pathologies of ASCVD.

Since HSP70 is a heat-responsive protein, its expression is induced by heat stimulation. Furthermore, since HSP70 has a chaperone function, its expression fluctuates depending on various biological conditions. HSP expression is associated with oxidative stress [20], heat stress [21], ischemia [22], exercise [23] and metabolic stress [24].

The factors that control HSP70 activity are the heat shock transcription factor 1 (HSF1) binding to the heat shock element (HSE) [25, 26], protein binding protein 1 (HspBP1) [27], and HSP70-ATPase [28], etc. The carboxy terminus of HSP70 binding protein (CHIP) has been reported as a factor regulating HSP70 induction [26]. CHIP is a cytoplasmic protein (ubiquitin ligase), and its amino acid sequence differs among species, but it is highly conserved across the globe and is most abundantly expressed in cardiac muscle, skeletal muscle, and brain [28].

Bruxe1 et al. [29] showed that chronic heat treatment of adult mice with atherosclerosis exerted effects in regulating the anti-inflammatory, anti-aging molecular axis (SIRT1-HSF1-HSP). They also reported that heat treatment dramatically reduced plasma triglycerides, total cholesterol, LDL cholesterol, oxidative stress, fasting blood glucose levels, and insulin resistance, and increased HDL cholesterol levels [29].

Diet and exercise are important for preventing MS. When motor function is impaired, treatment by thermal stimulation could be used on behalf of exercise to enhance skeletal muscle function and prevent the progression of type 2 diabetes and atherosclerosis. However, there are few reports on how to utilize thermal effects to treat atherosclerosis and chronic inflammation in daily life.

In this chapter, we will introduce the molecular mechanism of the thermal effect, including what we have been working on so far. The aim is to provide a perspective for future practical use.

2. Effect of heat stimulation on MS progression

2.1 Insulin resistance/type 2 diabetes

It is well known that in type 2 diabetes, visceral fat is the main cause of chronic inflammation that smolders throughout the body. It has been shown that the inflammatory cytokine TNF-α increases in adipose tissue of obesity, that TNF-α phosphorylates insulin receptors and causes insulin resistance, and that macrophages in adipose tissue develop chronic inflammation [30]. Insulin resistance precedes important medical conditions such as cardiovascular atherosclerosis.

The HSP72 inhibits the activation of stress kinases such as c-Jun-NH2 terminal kinase (JNK), which interfere with the insulin signaling pathway. JNK is an important regulator of intracellular signal transduction. JNK promotes insulin resistance by inhibiting the phosphorylation of insulin receptor substrate 1 (IRS-1), a key protein in the insulin signaling cascade [31]. JNK activation, especially in skeletal muscle, is one of the factors that causes insulin resistance. One study demonstrated that high-fat diet (HFD)-induced JNK activity was suppressed in HSP72-overexpressing mice [32]. As diabetes progresses, JNK becomes more activated, while HSP72 expression decreases [33]. The HSP72 has also been shown to protect against age-related insulin resistance [33] and HFD-induced obesity and insulin resistance [32]. This mechanism may be due to many factors, including blockade of JNK signaling by HSP72 in skeletal muscle, activation of AMP-activated protein kinase (AMPK) and prevention of intramuscular lipid accumulation, and promotion of mitochondrial production and oxidative metabolism by increasing sirtuin activity [32].

It has also been reported that insulin resistance is induced when eHSP72 increases [34, 35]. The mechanism leading to insulin resistance may involve extracellular HSP70 (eHSP70)-mediated stimulation of TLR2/4. Thereafter, TLR2/4-dependent JNK activation is linked to Ser 312 of insulin receptor substrate 1 (Irs1) in humans. Akt activation is inhibited [35], leading to decreased glucose uptake and a state of resistance to the action of insulin. Furthermore, chronically elevated eHSP72 status has been shown to have direct effects such as decreased pancreatic β cell and islet viability, insulin secretion, and mitochondrial function in humans and rodents [34]. As mentioned in the previous section, intracellular HSP70 (iHSP70) and eHSP70 should be explained separately, and it is important to utilize their roles.

2.2 Atherosclerosis

Atherosclerosis is a disease that affects everyone as we age, and its complications, primarily coronary heart disease and stroke, are the leading cause of death in developed countries [36, 37]. Clinical manifestations of atherosclerosis are mainly observed in older adults and are associated with the metabolome, endotheliosis, immune dysfunction, telomere shortening, and other systemic age-related changes [38].

2.2.1 Chronic inflammation

Atherosclerosis is based on chronic inflammation. Chronic inflammation is a low-level inflammation that continues to smolder throughout the body, and although there are few clinical signs, it is a reaction system that can lead to fatal damage when noticed [39]. Atherosclerosis is a chronic inflammatory disease of elastic and myoelastic arteries with the formation of atherosclerotic plaques (cholesterol) that lead to narrowing or thrombosis (if the plaque is unstable) [40]. In addition to atherosclerosis, diseases based on chronic inflammation have been reported to include type 2 diabetes, chronic lung disease, colorectal cancer, and prostate cancer [39]. Chronic inflammation, which is the underlying pathology of these diseases, is non-classical inflammation. Atherosclerosis is thought to be the result of low-grade chronic inflammation [40, 41].

The acute inflammatory response occurs within 48 hours and is a response to protect organisms from pathogens and repair tissue [29]. In acute inflammation, HSP suppresses inflammation by blocking NF-κB and other downstream proinflammatory signals through an anti-inflammatory program centered on HSF1-dependent expression of HSP and other anti-aggregation protein chaperones [29]. In other words, inflammation is suppressed by the HS reaction. The HSP72 mRNA increases during the acute phase response, and its expression level correlates with the amount of tissue inflammation, and these changes occur regardless of the underlying disease [42].

However, when the damaging stimulus becomes chronic, HSF1 expression is markedly blunted and cells stop producing cardioprotective HSPs (HSP27, HSP72, etc.) with anti-inflammatory properties [29]. Chronic inflammation is caused by innate immune system cells such as macrophages, dendritic cells, and neutrophils. These cells contain nucleotide-binding multimer domain (Nod)-like receptors that become activated when they detect pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). When the inflammasome is formed by attracting apoptosis-as-associated speck-like protein and procaspase-1, caspase-1 is activated. Caspase-1 converts all IL-1β and IL-18 into mature forms and removes dangerous PAMPs and DAMPs. Once the treatment is complete, the tissue will return to normal, but if the removal process does not proceed, inflammatory cytokines such as IL-6 and TNF-α will increase one after another, leading to tissue destruction [39, 43]. High-calorie diets, inappropriate eating habits, and low physical activity are chronic harmful stimuli that trigger inflammatory signals on vascular endothelial cells and vascular smooth muscle cells [29]. The stimulation then becomes an inflammatory signal to endothelial cells, which causes vascular smooth muscle cells (VSMCs) to homing monocytes/macrophages and transform foam cells [29]. It is known that when chronic inflammation such as atherosclerosis continues, the pathological condition of atherosclerosis progresses and the blood concentration of HSP70 increases. In advanced human atherosclerosis, increased expression of HSP70 is induced by activation of signal transducers and activators of transcription (STAT) and NF-κB and inflammatory cytokines [44]. The HSP72 also has anti-inflammatory effects within cells caused by inhibition of TNF-α, IL-1β, and IL-6. Reduced HSP72 expression is associated with worsened vascular inflammation.

In the case of atherosclerosis, when LDL cholesterol particles accumulate excessively in the intima of blood vessels and undergo oxidation and glycation modification, monocytes are stimulated, and chemokines gather [39]. Monocytes eventually mature into macrophages, phagocytose LDL cholesterol, accumulate cholesterol, become foam cells, and form precursors to plaque on blood vessel walls. It is thought that chronic inflammation targets plaque precursors, and additional inflammatory cytokines such as TNF-α, IL-6, and interferon-gamma (IFN-γ) are mobilized, resulting in long-term chronic inflammation and hardening of the arteries [39, 40]. In addition, immune responses to HSPs are expressed in chronic diseases such as atherosclerosis, and HSPs induce the receptors of the innate immune system, followed by the expression of regulatory adaptive immune responses through the production of regulatory cytokines [45].

Thus, there are a number of factors that act as inducers of the inflammatory process in atherosclerosis, including aging of the vascular endothelium, metabolic dysfunction, autoimmunity, and in some cases also infectious damaging factors [40].

In our current research, we aim to utilize IL-6, hs-CRP, and HSP70 as indicators of intervention using warm compresses. IL-6 is an inflammatory cytokine, and its induction also affects chronic inflammation, which is the underlying pathology of atherosclerosis. The hs-CRP is a marker of systemic low-grade inflammation, and elevated plasma levels are widely known to be associated with cardiovascular events in various populations. Furthermore, it has been pointed out that inflammation “upstream” of hs-CRP itself is a risk factor for cardiovascular events [46]. Sharif et al. [41] found that low-grade inflammation measured by hs-CRP was an independent risk factor for vascular-related and all-cause mortality in a prospective cohort study of patients with type 2 diabetes. The authors described the possibility of this as a therapeutic target to reduce residual cardiovascular risk.

The HSP70 is expected to be applied to monitoring and treatment due to its anti-inflammatory effect on chronic inflammation [18, 42]. The expression level of HSP72 correlates with the amount of tissue inflammation, which is induced by inflammatory cytokines [42]. The HSP72 is known to have anti-inflammatory effects. It has been suggested that HSP72 may elicit additional anti-inflammatory effects by localizing to the extracellular space and macrophages. In mammals, HSP72 binds with high affinity to macrophages and peripheral blood mononuclear cells and is internalized by endocytosis and localized intracellularly [47]. In the intracellular and extracellular space, HSP72 inhibits the release of inflammatory cytokines [48]. The HSP72 may play an important role in reducing the risk of developing insulin resistance by locally reducing inflammation [49]. Inflammation is a phenomenon induced by JNK activity and may be mediated by HSP70. The role of iHSP70 is known to inhibit JNK activation through several mechanisms, including direct contact of the protein to JNK [50]. Chronic inflammation is the underlying pathology of ASCVD, and HSP72 may be effective in preventing and treating ASCVD.

Meanwhile, eHSP70 has been reported to activate NF-κB and activator protein 1, stimulating the release of inflammatory cytokines and the generation of reactive oxygen species (ROS) [51]. Higher levels of eHSP72 are also present in conditions where chronic inflammation and oxidative stress occur, in such patients with type 1 diabetes [52] and type 2 diabetes [53]. In fact, serum eHSP72 concentrations are positively correlated with human inflammatory markers such as CRP, monocyte count, and TNF-α [54, 55]. Therefore, eHSPs play an important role in inducing cellular immune responses. However, the mechanisms underlying the association between HSP70 and inflammatory diseases are not completely understood.

Furthermore, there is a recent report on the ratio of iHSP70 to eHSP70 and inflammation [56]. When cells undergo stress, iHSP70 present in cells is transferred to the circulation system through mechanisms such as cell necrosis and ATP-binding cassette (ABC) transporters [57, 58]. This HSP was named eHSP70. The iHSP70 exerts anti-inflammatory effects through heat shock response. In contrast, eHSP70 mediates pro-inflammatory pathways [51] and may be associated with insulin resistance in type 2 diabetes. In this way, iHSP70 and eHSP70 play antagonistic roles, and it has been proposed to use the ratio of eHSP70 to iHSP70 as a biomarker [51, 56]. And HSP70 has a variety of functions as mentioned above. The effect also affects the whole body.

In particular, by utilizing skeletal muscles distributed throughout the whole body, we believe that we can expect to be able to utilize not only their role as biomarkers but also their effects themselves.

2.2.2 Cell death

The concept that age-related inflammation (inflammaging) is deeply involved in the pathological progression of age-related diseases such as atherosclerosis has been proposed. It is known that blood inflammatory markers such as CRP and IL-6 are elevated in the elderly [59]. Increased blood concentrations of inflammatory cytokines such as IL-1, LI-6, and TNF-α have been observed even in healthy long-lived individuals. The main cause of weak chronic inflammation associated with aging is the activation of macrophages. It is a defensive response/adaptive phenomenon of the immune system against oxidative stress that accumulates in the body throughout life and antigenic loads such as cancer, viral, and bacterial infections [60]. Furthermore, it has been reported that inflammatory indicators are associated with malnutrition and hypohydremia in healthy long-lived individuals [61].

In response to various stressors, vascular cells produce high levels of HSPs to block inflammation and maintain homeostasis against stress. The HSP70 protein levels change with age in adult and aged animals, and HSP expression levels increase with age [62]. However, protein level homeostasis has been shown to be lost during vascular aging, potentially resulting in decreased expression of chaperone proteins including HSP70 [63]. The HSP70 is involved in the regulation of oxidative stress and inflammation, and may also regulate the rate of aging [62].

Various factors contribute to why inflammation becomes more likely to occur with age, including changes in the endocrine system and metabolism, accelerated cell death, and the accumulation of senescent cells that release senescence-associated secretory phenotype (SASP) [59]. The SASP is a general term for physiologically active molecules secreted from senescent cells. SASP induces inflammation through autocrine and paracrine pathways, transmits senescence signals to neighboring cells, exacerbates telomere dysfunction, and promotes cellular senescence through ROS-mediated pathways in the tissue microenvironment [64]. The following theories exist as mechanisms by which disorders accumulate and bodily functions decline with age: flu radicals, mutations, epigenetics, cell destruction due to abnormal protein accumulation, membrane senescence, cell senescence due to telomere-independent oncogenes, senescence via p38 activation due to chronic stress and oxidative stress accumulation, and cells due to endoplasmic reticulum (ER) stress aging. Cellular senescence is a state of persistent cell cycle arrest in dysfunctional cells that acquire SASP [65].

Thus, cellular senescence caused by various stresses forms the basis of chronic inflammation, and suppression of tissue regeneration further progresses aging. As cells age, the transcription factor NF-κB is activated, leading to the induction of the production of the inflammatory cytokine SASP. As we age, these inflammatory signals and processes are no longer properly controlled, resulting in pathological inflammation. As a result, the risk of developing cancer, arteriosclerotic disease, obesity metabolic disease, etc., increases [66]. Cellular senescence is a biological defense mechanism that suppresses canceration, but it also promotes chronic inflammation through SASP. It causes cancer and further accelerates aging [10].

In addition, DAMPs derived from dead cells that cannot be properly disposed of due to phagocytosis or from cells damaged by oxidative stress activate the innate immune response. It has been pointed out that this may promote inflammaging by inducing inflammatory cytokines and dieters [67]. DAMPs are self-molecules released from dead cells caused by oxidative stress due to aging mitochondria in diseased tissues [59]. This released self-molecule is also recognized by innate immune receptors such as TLRs and activates an immune response. In atherosclerosis, DAMPs may be released from dead macrophage cells that turn into foam [68]. HSP is also a DAMP derived from the cytoplasm and has TLR2 and TLR4 as receptors [59]. DAMPs are said to be involved in the chronicity of inflammation, but they have various effects in vivo that go beyond inducing inflammation. Elucidation of the detailed mechanism is expected [59]. Moiseeva et al. [69] found that senescent cells, one of the factors of chronic inflammation, are almost absent in intact skeletal muscle tissue even in old age. However, it has been stated that upon injury, the accumulation of active oxygen and DNA damage increases the appearance of senescent cells and induces inflammation [69]. Furthermore, in young individuals, induction of inflammation due to injury to muscle tissue has important implications for tissue regeneration, but senescent cells cannot be removed due to weakened immune function, and it has been pointed out that they are harmful [69].

Aging promotes inflammation and poses a risk of developing age-related diseases, but this risk can be reduced, for example, by suppressing proteins related to angiopoietin-like protein 2 (ANGPTL2) [70]. Oike and Manabe [70] also analyzed the study results reported by Tyshkovskiy et al. [71]. In normal aging, inflammatory pathways are activated, but in long-lived organisms with natural history, activation of inflammatory pathways is attenuated. It has also been pointed out that activation of inflammatory pathways was significantly suppressed in mice whose lifespans were extended by the intervention [70]. Calorie restriction causes a decrease in fasting blood sugar levels and suppression of the PI3K-AKT and mTOR-S6K pathways due to decreases in insulin and IGF-1 [10]. In addition, increased antioxidant enzymes, increased HSP/autophagy, and decreased ER stress lead to delayed aging and extended lifespan [10].

For these reasons, some type of intervention against inflammation and aging is necessary for healthy longevity [70].

Cell death often occurs at sites where an inflammatory response occurs. There are three types of cell death: Necrosis, autophagy, and apoptosis. Necrosis and apoptosis induce inflammation [10].

As mentioned above, chronic inflammation and cell death are linked, especially under the influence of aging. In our previous study [19], we confirmed that thermal stimulation of skeletal muscle-derived cells (SMDC) significantly increased the expression of HSP70 and changed the expression of factors related to cell death such as apoptosis. No morphological changes or cell detachment were observed 20 hours after the start of heat stimulation of SMDCs. The number of cells seeded at the beginning of the subculture was comparable. It is well known that HSPs play an important role in the apoptotic pathway. The HSPs interact with a variety of important apoptotic proteins. The HSP70 inhibits the apoptotic pathway by acting as a chaperone, and overexpression of HSP70 was shown to suppress apoptosis [72]. The HSPs can essentially block all apoptotic pathways, most of which involve the activation of cysteine proteases called caspases [49]. JNK induces apoptosis by inhibiting mitochondrial respiration and increasing reactive oxygen species (ROS) production [31]. JNK activation is an important step in the cellular response to stress, but prolonged JNK activation can lead to cell damage and cell death [73]. It is well known that when HSP72 expression increases, JNK expression is suppressed. Therefore, as in the previous study [19], if the expression of HSP72 was significantly increased by thermal stimulation of SMDCs, it is unlikely that JNK activity would promote apoptosis. Furthermore, since there was no morphological change while affecting gene expression [19], heat stress may activate chaperone function through HSP72 activity. However, thermal stimulation of SMDCs may affect some mechanisms related to apoptosis. This may be one of the factors involved in chronic inflammation when utilizing whole body thermal stimulation. The HSPs are also involved in the unfolded protein response (UPR). We demonstrated that the expression of UPR stress markers, including activation of transcription factor 6 (ATF6) and HSP70 (HSPA5), was reduced [19]. This indicates that heat stimulation enhances the chaperone function of HSPs and leads to the activation of enzymes involved in protein folding. Therefore, thermal stimulation of SMDCs may have reduced intracellular endoplasmic ER stress.

Demyanenko et al. [74] reported that eHSP70 reduces the level of p53 protein and inhibits the pro-apoptotic effect of WR1065 in neurons. This indicates that eHSP70 is involved in regulating p53 activity through the JNK-dependent signaling pathway [74]. The p53 is an important regulator of apoptosis. Additionally, Demyanenko et al. [75] also reported that eHSP70 reduces glial cell apoptosis. The iHSP70 has also been reported to inhibit apoptosis by activating NF-κB, producing nitric oxide, and inhibiting superoxide dismutase activity [51].

As mentioned above, the pathway by which iHSP70 and eHSP70 inhibit apoptosis has also been elucidated. Intrinsic effects are the main factor when exploiting thermal stimulation effects in living organisms. However, if the effects of HSP70 are to be utilized against chronic inflammation and atherosclerosis, it is necessary to verify the effects of iHSP70 and eHSP70 separately.

In addition, cell death, which is strongly related to inflammation, induces inflammation, and is also induced by inflammatory cytokines, which are thought to form positive feedback. Therefore, it is difficult to separate cause and effect regarding inflammation and cell death [10]. It is thought that the same thing can be said about aging. Therefore, we believe it is important to respect the overall outcome of interventions.

3. Lifestyle and MS risk markers

It is well known that dietary recommendations for maintaining good health are important in preventing ASCVD. Diet and exercise are known to increase HSP, reduce inflammation, improve insulin signaling, and reverse the cycle that leads to type 2 diabetes [76]. Heat stimulation and exercise have been used to increase HSP70 and improve insulin resistance in type 2 diabetes [77].

3.1 Diet

Vahid et al. [78] reported that dietary patterns and healthy eating index (HEI) were inversely correlated with plasma hs-CRP, plasma IL-1β, and plasma IL-4 levels. Angelini et al. [79] report that a high-calorie diet secretes HSP70 and glucose-regulated protein 78 kDa (GRP78) from the human jejunum, increasing circulating levels of HSP70 and insulin resistance. Plasma HSP70 concentration is negatively correlated with insulin sensitivity [79]. In vitro validation in the same study also showed that HSP inhibits Akt phosphorylation and glucose uptake in HepG2 [79]. Thus, secretion of stress response proteins HSP and GRP from the small intestine is thought to be associated with insulin resistance as important mediators of intercellular signaling in response to nutrients [79].

Dietary components play an important role in the progression of inflammation. Intake of vegetables and fruits, or macronutrients and micronutrients, n-3 polyunsaturated fatty acids (PUFA), monounsaturated fatty acids (MUFA), flavonoids, and vitamins C and E has been shown to reduce systemic inflammation. However, saturated fatty acids (SFA), high glycemic index carbohydrates, etc., increase serum levels of pro-inflammatory cytokines [80]. Healthy dietary patterns, such as Mediterranean and vegetarian diets, may improve inflammatory processes and reduce levels of circulating inflammatory biomarkers, thereby reducing the risk of age-related diseases [81, 82]. A high-protein meal replacement program lowers hs-CRP and also supports weight loss [83, 84]. In this way, the content and style of meals have a major impact on underlying MS conditions such as chronic inflammation and insulin resistance.

On the other hand, it has also been reported that continuing to eat a healthy diet tended to gradually reduce hs-CRP plasma concentrations over the long term, but this was not statistically significant [85]. In our previous study [14], we also reported that the food formula has more influence on the parameters of lifestyle-related diseases than the dietary content. Additionally, whether diet results in changes in chronic inflammation-related proteins is sensitive to weight loss, making interpretation of the results difficult. It is very likely that weight loss, rather than the specific diet chosen, is most important for achieving lower circulating hs-CRP levels [86]. There is a lack of evidence for the effects of dietary changes alone on systemic inflammation.

3.2 Exercise

Systemic exercise is well known to reduce chronic inflammation by improving skeletal muscle function. Regular physical activity, such as aerobic and resistance exercise, mainly leads to decreased levels of inflammatory mediators such as CRP, IL-6, and TNFα, and improved anti-inflammatory capacity [87, 88]. However, a meta-analysis evaluating aerobic exercise in healthy subjects found a significant reduction in hs-CRP associated with weight loss and body fat composition, but not with independent exercise [89]. Data on the association between exercise and systemic inflammation are less clear [86]. In a review of several studies, Maierean et al. [86] found that the evidence for a decrease in hs-CRP in exercising subjects was inconclusive, or that improvements were seen in the presence of weight loss. Furthermore, we have not found sufficient evidence that exercise itself in any category (aerobic exercise, strength training, vigorous exercise capacity, etc.) is associated with blood hs-CRP.

Regarding HSP70, research on changes in expression in skeletal muscle due to exercise is widely known. The reduction of exercise-induced inflammation and oxidative stress is also exerted in the recovery process of injured tissues after exercise through the chaperone function of HSP70. There was a change in serum HSP70 concentration immediately after 60 minutes of exercise [90]. Serum-sensitive HSP70 is influenced by several pathophysiological processes, such as recovery from exercise, the process of repairing damaged tissues, and the reduction of oxidative stress due to inflammation [55]. Furthermore, chronic inflammation may be the molecular basis of frailty and impede healthy longevity. Therefore, HSP70 and inflammatory cytokines are thought to be useful biomarkers to verify the preventive effects of exercise on various diseases [55].

3.3 Thermal stimulation

We previously reported the effects of thermal stimulation on human skeletal muscle in vivo and SMDC in vitro [17]. Since the HSP70 family is overexpressed by heat stimulation, there have been reports utilizing heat responsiveness. The HSP70 family is also a chaperone protein and is involved in many physiological mechanisms. It is attracting attention as a potential disease prevention target and disease biomarker [25, 91]. Inducible proteins such as HSPs may be important in reducing lifestyle risk factors and managing disease [77]. The HSPs have been shown to reduce oxidative stress, inhibit inflammatory pathways, and enhance the metabolic properties of skeletal muscle [92].

Systemic and local heat stimuli activate the HSP72 heat shock pathway and induce the expression of associated genes. This results in weight loss and improved glucose metabolism. Expression of HSPA1A/HSPA1B (HSP70/HSP 72) decreases with obesity, insulin resistance, and aging. Thermal stimulation and the subsequent increase in chaperone function increase the levels of HSP70 and slow the vascular aging process. The HSP70 family has protective effects against HFD-induced obesity, and insulin resistance [77, 93]. Weekly in vivo hyperthermia improved glucose tolerance, increased muscle strength and hepatic HSP72 protein content, and decreased muscle triglycerides accumulation [77]. A review article [31] also agrees that HSP72 levels are reduced in metabolic syndrome with insulin resistance and type 2 diabetes, and HSP72 expression levels are closely associated with obesity.

Skeletal muscle plays an important role in insulin-stimulated glucose uptake [94]. We previously investigated gene expression changes after heat stimulation in SMDCs [16, 19]. We reported that thermal stimulation of SMDCs enhances glucose uptake through various insulin-dependent and independent mechanisms and changes the expression of genes involved in improving insulin resistance. A significant increase in HSP72 (HSPA1A) mRNA expression levels was observed. Increased HSP72 expression in skeletal muscle is associated with increased expression of LPL, indicating that increased mitochondria in skeletal muscle leads to suppressed insulin resistance [32]. Thermal stimulation of skeletal muscle may be a way to promote glucose metabolism through HSP activity [95]. Conversely, decreased HSP70 induces insulin resistance through JNK activation. The HSP72 induction has been shown to directly inhibit JNK activation, which improves insulin sensitivity and glucose tolerance at both the skeletal muscle and systemic levels. In particular, induction of HSP72 by chronic heat treatment protects skeletal muscle from insulin resistance.

On the other hand, HSP70 promotes insulin resistance in tissues other than skeletal muscle [79]. The improved insulin response without dietary restriction in HSP72-overexpressing mice further resulted in increased insulin-stimulated glucose uptake in the anterior tibial muscle. This effect was also observed in white and brown adipose tissues that do not overexpress HSP72, suggesting that increased HSP72 expression in skeletal muscle also indirectly affects adipocytes [32].

Thermal stimulation from bathing can enhance the chaperone function of HSP70. Changes in serum HSP70 concentration were observed 15 minutes to 2 days after bathing [54]. Immediately after bathing in a chloride hot spring, immunity such as NK cell activity improves, and protein repair function by HSP70 increases [54]. When skeletal muscle is exposed to heat stress, HSP70 increases and exerts a chaperone function to suppress muscle atrophy [52]. Accumulation of misfolded proteins is involved in skeletal muscle atrophy. Hyperthermia significantly decreases muscle atrophy markers such as CD68, KLF, and MAFbx, and significantly increases muscle hypertrophy markers such as AKT, mTOR, and HSP70 [53]. These thermal stimulation therapies are thought to be effective in maintaining muscle in diabetic patients [53].

In adult mice with atherosclerosis, systemic heat therapy reverses the inhibition of the SIRT1-HSF1-HSP molecular axis and exerts anti-inflammatory effects, and chronic heat treatment improves chronic inflammation in atherosclerosis [29]. The HSF1-dependent HSP expression due to the heat shock response may be suppressed by the progression of atherosclerosis. Bruxel et al. [29] showed that expression of SIRT1, HSF1, HSP27, HSP72, and HSP73 was suppressed in parallel with increased expression of NF-κB-dependent VCAM1 adhesion molecule in the aorta of adult mice with atherosclerosis. However, the group that received whole-body heat treatment completely reversed the suppression of these heat shock responsive proteins and significantly inhibited both VCAM1 expression and NF-κB DNA-binding activity. Uchiyama et al. [96] found that in vascular endothelial cells, increased expression of human HSF1 was associated with elevated steady-state levels of eNOS and thrombomodulin, endothelin-1 (ET-1) and plasminogen activator inhibitor-1 (PAI-1) level has decreased. In particular, HSP70 and HSP90 strongly induced the expression of eNOS and decreased the expression of PAI-1 [96]. These results suggested that HSF1 activity could be used for cardiovascular treatment. Thus, HSP70 expression improves endothelial function in advanced human atherosclerosis [96].

In addition, mice receiving whole-body hyperthermia improved blood sugar levels, reduced atherosclerotic lesions and plasma levels of triacylglycerol, total cholesterol, and LDL cholesterol, and increased plasma HDL cholesterol, even while consuming a high-cholesterol diet [29]. Regarding this observation, Bruxel et al. [29] suggested that the heat-induced hyperadrenergic state may be involved in the reduction of plasma lipids involved in atherogenesis.

Elevated serum HSP72 concentrations are associated with an increased number of vascular lesions [20]. In myocardial infarction, HSP72 expression has been shown to increase due to an anti-apoptotic effect to protect cardiomyocytes from apoptosis caused by ischemic injury [97]. It was also demonstrated that there was a positive correlation between serum HSP70 levels and systolic blood pressure in patients with newly diagnosed hypertension, and that HSP70 levels were elevated in vascular endothelial cells of diabetic patients who had progressed to the inflammatory phase [98]. Hooper [76] state that increasing HSP reduces inflammation and improves insulin action. Chronic inflammation, the underlying pathology of ASCVD, has been shown to be influenced by a vicious metabolic cycle. Obesity, a sedentary lifestyle, and a diet high in fat calories accelerate this cycle by decreasing insulin signaling, increasing inflammatory cytokines, and inducing a low HSP state [76]. Similarly, prolonged stress increases stress hormones (cortisol, catecholamines, and glucagon), which ultimately impair insulin signaling and reduce HSP levels. Therefore, reduced HSP can lead to insulin resistance, and it is associated with inflammation [76]. In our previous study [16], thermal stimulation of skeletal muscle cells significantly increased the expression of HSPA1A encoded by HSP70. In the gene profile that showed simultaneous expression changes of many factors related to improved insulin resistance, atherosclerosis, and inflammation were identified. Comprehensive analysis using multiple analysis methods suggested that thermal stress on skeletal muscle cells is involved in ASCVD in a preventive manner.

In addition to chronic thermal stimulation, acute thermal treatment may also be effective in improving insulin resistance. Gupte et al. [99] exposed the lower body of older rats to short-term thermal stimulation using a thermal blanket and demonstrated that heat exposure improved insulin-stimulated glucose uptake in skeletal muscle. The mechanism underlying this effect has been suggested to be HSP72-mediated inhibition of JNK. Therefore, both chronic and acute thermal stimulation may be effective in increasing HSP72 expression. These reports also indicate that thermal stimulation of skeletal muscle, a central regulator of metabolism, can be effectively used to influence systemic levels. Due to numerous reports regarding insulin resistance, type 2 diabetes, and HSP70, there is hope in recent years for type 2 diabetes management using HSP70 as a target factor. The target factor HSP70 may be modulated by interventions such as long-term physical exercise and hyperthermia.

Healthy people can expect systemic effects and improvements in skeletal muscle function through exercise and daily bathing. However, when physical activity is limited, it is more important to effectively utilize skeletal muscle function. We believe that it may be particularly effective in treating neurological and muscular diseases in young people and in their prime age.

Heat-stimulated HSP70 overexpression is effective in controlling blood sugar levels and suppressing chronic low-grade inflammation in individuals with limited physical activity levels, such as those with severe obesity, muscular dystrophy, people in wheelchairs, and the elderly [62]. Demyanenko et al. [74] reported that eHSP70 attenuates apoptosis and necrosis in glial cells, but not in neurons, indicating that eHsp70 also has a neuroprotective effect. Increased eHSP70 expression appears to have little risk of promoting muscle and nerve atrophy [18]. On the other hand, there are also reports that HSP70 expression promotes muscle atrophy. The eHSP70 induces muscle wasting in cancer patients [100]. In zebrafish, HSP70 is one of the factors that causes muscle atrophy due to excessive exercise [101]. Furthermore, both pro- and anti-inflammatory effects of HSP70 in rheumatoid arthritis have been reported and require further validation [102]. Therefore, thermal stimulation treatment may be a hopeful candidate to prevent MS and its progression for people who cannot exercise because of locomotive syndromes such as neurological, muscle, bone, and joint disease.

As mentioned above, it may be possible to influence MS prevention by leveraging the chaperone function of HSP70 to contribute to chronic inflammation. As mentioned above, there is accumulating evidence suggesting a role for thermal stimulation in ASCVD prevention. Especially when physical activity is restricted, it is important to create a habit of incorporating heat into your life, along with diet.

Serum HSP72 concentration levels do not fluctuate in response to apparent endogenous circadian rhythms and climate change [103]. Therefore, changes in serum HSP70 levels may reflect an in vivo response. A study by Lubkowska et al. [90] suggests that the extracellular concentration of HSP70 may be an important indicator of impaired glucose homeostasis. The HSP72 is involved in a variety of diseases, including those that cause ASCVD, such as diabetes and atherosclerosis, and HSP72 could potentially be used as a disease biomarker.

However, as reported by Koelman et al. [85], it is difficult to observe a significant decrease in hs-CRP based on dietary content alone. Furthermore, regarding hs-CRP, a cardiovascular events prediction index, there is insufficient evidence to show that treatments that reduce hs-CRP improve CV outcomes in subjects with normal hs-CRP baseline [86]. In our survey of young people, there were no significant differences in bathing habits, arterial stiffness, HSP72, IL-6, or hs-CRP. Furthermore, although ASCVD is based on chronic inflammation, there are not many reports that utilize inflammatory markers or HSP70 as an effect of lifestyle interventions, so future research is expected.

4. Conclusion

Many factors are involved in MS prevention based on chronic inflammation caused by visceral fat accumulation. One of these, HSP70, uses its chaperone function to influence inflammatory cytokines and may contribute to the prevention of insulin resistance and atherosclerosis. Furthermore, the utilization of HSP70 is highly safe in vivo. It is necessary to verify that inflammatory markers and HSP70 can be used as a comprehensive intervention outcome while respecting diet, exercise, thermal habits, etc., in the lives of individuals. In particular, when physical activity is limited before reaching old age, this may lead to improve the function of skeletal muscle, which is the largest metabolic organ in vivo. When using thermal stimulation, it is important to ensure safety. Further investigation is required for safe and effective interventions to prevent MS and its progression.

Acknowledgments

This study was supported by Grant-in-Aid for Scientific-Research from JSPS KAKENHI Grant Number JP22K17448.

Conflict of interest

The authors declare no conflict of interest.