**3. Crisis of the mitochondrial free radical theory of aging (MFRTA)**

From the described above different free radicals and environmental pollutants, we see that all of them have the potential ability to cause damages to cellular and mitochondrial functions, although by different mechanisms. Some of the biologically active molecules and radicals show clear tissue and organ specificities. For example, singlet oxygen damages predominantly skin and eyes; ozone and nitrogen dioxide - lung epithelium, nitric oxide and peroxynitrite - vascular endothelium and neuronal cells. However, none of the above ROS is directly related, with the exception of the superoxide radical, to the inevitable aging mechanism [55].

After decades of research a vast amount of accumulated knowledge revealed serious inconsistencies between the data obtained and the MFRTA, which call into question the correctness of the free radical theory in its current paradigms. We refer the reader to excellent reviews on this topic [16, 18, 56–58]. Taking into consideration the controversies regarding various species, in this Chapter we will focus only on those inconsistencies that directly relate to the topic of humans and mammalians aging mechanism. Animals are often used for modeling of aging mechanisms [59–62]. We shall discuss the following most important for MFRTA facts, which undermine this theory.

#### **3.1 MtDNA are protected from the direct effects of reactive oxygen species**

Recently, it has been established that neither of the above listed ROS and biologically active molecules are capable to cause directly mutations of mtDNA, which was and still is for many researchers the main hallmark of the aging process and it is considered as the main pathogenic mechanism of many diseases [30–33]. From the beginning of free radical theory of aging, mutations of mtDNA were the only reliable markers of oxidative stress. As a matter of fact, the MFRTA itself arose on the basis that changes in the production of ROS were always accompanied by parallel changes in the number of mtDNA mutations [13, 14, 25]. Recently, however, it was concluded that there was no reliable evidence for the direct involvement of ROS in mtDNA mutations [31, 57]. There are two main reasons for this conclusion. First, the commonly studied radicals are not active enough to cause mutations [55]. Secondly, mtDNA are encased into a protein coating of nucleoid, which prevents direct contact of mtDNA and radicals [31].

#### **3.2 Antioxidant supplementation interventions do not increase longevity**

Antioxidant supplementation interventions do not increase longevity, as would be predicted by the MFRTA. This Antioxidant Paradox is considered as the strongest evidence against the MFRTA; it comes from studies that manipulate antioxidant levels. Many studies have shown that administration of low-molecular weight antioxidants failed to extend longevity [reviewed in 16, 56, 57, 63]. Barja (2014) suggested that the lack of antioxidants to exert effects on longevity could be explained by the spatial separation: a free radical, which causes aging, acts in the lipid phase of membranes, while antioxidants exerts their effects mostly in the water phase of cells [16]. Barja (2014) also summarized the numerous studies of life longevities on various species: "Only two parameters currently correlate with species longevity in the right sense: the mitochondrial rate of reactive oxygen species (mitoROS) production and the degree of fatty acid unsaturation of tissue membranes" [16]. As we will see, these are very correct suggestions.

*Metabolic Syndrome as the First Stage of Eldership; the Beginning of Real Aging DOI: http://dx.doi.org/10.5772/intechopen.95464*

#### **3.3 The rate of O2 • production controls lifespan independently of SOD**

Muller (2000) has stressed that SOD activity interspecies variation is not correlated with the maximal life span (MLSP) in mammals and the activity of other antioxidant enzymes negatively correlates with MLSP. However, there is a strong negative correlation between longevity and the O2 • and H2O2 production rates by isolated mitochondria from diverse mammalian species. The longevity depends not on the amount of superoxide in a cell, but on the rate of its production [18]. To explain this unexpected observation, Muller suggested that a significant fraction (between 10% and 50%) of O2 • is not produced as aqueous O2 • but instead is produced as lipid-phase HO2 • in the inner mitochondrial membrane. In other words, Muller has proposed that it is not the O2 • in the water phase, but its protonated form - hydroperoxyl radical (HO2 • ) in the membrane lipid phase exerts damaging effects on longevity, where the radical cannot be affected by a superoxide dismutase. Muller suggested that hydroperoxyl radical may initiate lipid peroxidation and the formation of peroxynitrous acid [18]. Unfortunately, the proposal that the hydroperoxyl radical is the aging main cause remained unnoticed for the next two decades, as well as the earlier similar attempts of other researchers before Muller [48, 63].

#### **4. Hydroperoxyl radical HO2 • as the systemic aging cause**

Here we present in brief our current views on the mechanisms of the hydroperoxyl radical formation and its damaging effects, which we consider as the systemic aging main mechanism. The details of the mechanism are presented in recent publications [55, 64–67].

#### **5. Properties of the hydroperoxyl radical or perhydroxyl radical (• HО2)**

Lipid phase of the mitochondrial membrane has 4–5 fold higher concentration of oxygen than the cells' water in the cytosol. When O2 acquires an electron from the respiratory chain and becomes O2 • , it must quickly leave the lipid phase of the membrane. However, before O2 • riches the bulk of the matrix or cytosol, it crosses the thing layer of the structured water near the charged surfaces of the membrane. The inner leaflet of the inner mitochondrial membrane contains approximately 80–90% of total cardiolipin (CL), which, together with phosphatidylethanolamine (PEA), accommodate respiratory complexes and ATP-synthase into the mitochondrial cristae sharp curves [67]. Since CL bears strong negative charge, at some arears of the inner membrane aggregates of CL form areas with strong negative charge, called antennae, which attract protons [reviewed in 66, 68]. For this reason, the thin layer of structured water near the charged surfaces of the inner mitochondrial membrane has up to three units more acid pH than the bulk of a compartment. This is a very important issue, because 1000 times higher concentration of H<sup>+</sup> increases the probability of the hydroperoxyl radical formation in the O2 • + H<sup>+</sup> ↔ • HО2 reversible reaction (pKα of the reaction is 4.8) [68]. Highly hydrophobic • HО2 returns back into the membrane's lipid phase. The described mechanism explains why the aging process depends only on the rate of superoxide formation, but not on the concentration of O2 • and the activities of SODs. Hydroperoxyl radical is a much stronger oxidizing agent than superoxide radical, and has a specific propensity to abstract H atoms from α-tocopherol and,

particularly, from the polyunsaturated fatty acids, such as arachidonic acid (C20:4 n6) and docosahexaenoic acid (C22:6 n3) [69].

For unknown reason, the perhydroxyl radical for a long time was almost completely excluded from the oxidative stress literature [61, 70]. Mitchell (2000) proposed perhydroxyl radical damaging mechanism through formation of peroxynitrite radical in the membrane [18]. This possible situation has been discussed by Gebicki and Bielski [69]. These authors indicated that although both • NO and • HО<sup>2</sup> аre hydrophobic radicals, the radicals are spatially separated since • NO is normally present in the blood vessel endothelium, and thus the mitochondrial hydroperoxyl radicals have little chance to meet nitric oxide, and even if this might happen, the negatively charged ONOO<sup>−</sup> hardly could be a systemic damaging factor because it is immediately excluded from the membrane's lipid phase [69]. Bielski et al. (1983) studied reactions of • HО2 with linoleic (C18:2), linolenic (C18:3) and arachidonic acids (C20:4) in water/ethanol solutions [68]. The obtained kinetic parameters of the reactions indicate that • HО2 reacts with the double allyl hydrogens of polyunsaturated fatty acids, and the more double bonds was present in a PUFA, the more active was the reaction. The abstraction of H atoms by • HО2 was exothermic, which indicates that it is irreversible and highly probable, when • HО2 encounters a PUFA. Since the reactions were performed in the water/ethanol solution, H2O2 formed cleaved heterolytically (2H2O2 → H2O + O2), and the final products of the fatty acids with • HО2 reactions were stable hydroperoxides without much variation in their structure [68].

## **6. The isoprostane pathway of lipid peroxidation (IPLP)**

When • HО2 reacts inside the lipid phase of the membrane with a PUFA, which is still part of a phospholipid, the products are a racemic mixture of a very large number of various stereo- and positional isomers [71]. Many of these products are similar to enzymatically produced prostaglandins, and were named Isoprostanes (IsoPs). For this reason, this type of PUFA autoxidation was named the Isoprostane Pathway of Lipid Peroxidation (IPLP) [71–73]. IsoPs possess potent biological activity and thus may convey abnormal cellular signaling and inflammation [71, 74]. Furthermore, many products of IPLP are very toxic, such as γ-ketoaldehydes. They form adducts with primary amines of the lysine-containing proteins and phosphatidylethanolamine (PEA). The most active among γ-ketoaldehydes are isolevuglandins (IsoLG) produced from arachidonic acid can be only detected as adducts with proteins or ethanolamine of PEA [74]. In addition to arachidonic acid the most common PUFA among phospholipids, other PUFA such as eicosapentaenoic acid (20:5 n3) and docosahexaenoic acid (22:6 n3), have been found as substrates for the IPLP [75]. Because docosahexaenoic acid (DHA) is present in a larger quantity in neurons, the products of IPLP were correspondingly named neuroprostanes and neuroketals. From arachidonic acid, which has four double bonds, the racemic mixture may contain up to eight hundreds of different products, whereas the products number from the containing 6 double bonds docosahexaenoic acid may be more than one thousand [71–73].

IsoPs and the cyclooxygenase derived prostaglandins (PGs) have a number of distinctions in their origin and properties, which have been discussed in a number of publications [65, 71, 76, 77]. Here we briefly list the most important distinctions: 1) The side chains of normal PGs are almost always oriented *trans* to the prostane ring whereas the products of IPLP have mostly the side chains with *cis* orientation [71, 76]; 2) The IsoPs are formed *in situ* from PUFA, which

*Metabolic Syndrome as the First Stage of Eldership; the Beginning of Real Aging DOI: http://dx.doi.org/10.5772/intechopen.95464*

are esterified to phospholipids, while PGs are generated exclusively from the free AA and DHA [77]. 3) The products of IPLP are the racemic mixture of molecules with a very large number of possible stereo- and positional isomers, whereas the products of the enzymatically produced prostaglandins have mainly one optical isomer [71, 76].

**Figure 1A** very schematically presents the pathways of arachidonic acid oxidation, and **Figure 1B** illustrates the suggested mechanism of AA oxidation by • HО<sup>2</sup> [65]. The key event after abstraction of the first H atom is that H2O2 formed under hydrophobic condition undergoes homolytic cleavage with formation of two molecules of the hydroxyl radicals H2O2 → 2 • OH, which instantly subtract another two H atoms from the same PUFA with formation of two molecules of H2O. The remaining molecule of the AA has completely disarranged double bonds, becomes extremely unstable and quickly attaches randomly two O2 molecules, and undergoes intramolecular transitions with formation of one out of many possible positional and stereoisomers. Thus • HO2 converts into 2 H2O and AA loses two out of 4 double bonds, and becomes one of hundreds isoprostanes. The differences between IPLP and the "classical" lipid peroxidation have been discussed in [65, 79].

#### **Figure 1.**

*Autoxidation of arachidonic acid with transformation of the molecule into various ring structures. (A) this part of the figure was adapted from the article [78] and shows different intermediary metabolites during autoxidation of arachidonic acid (AA). HO2 • is the only candidate for initiation of the isoprostane autoxidation of PUFA [65]. (B) the suggested sequence of transformations of HO2 • and AA in the course of isoprostane lipid peroxidation (IPLP) [65]. After abstraction of the first H atom from a molecule of AA, HO2 • turns into H2O2, which in the hydrophobic milieu undergoes homolytic cleavage into two molecules of • OH radicals, which instantly subtract the next two H atoms from the AA with formation of two molecules of H2O and the remains of the AA has completely disarranged double bonds. A very fast abstraction of three H atoms from any of the four double bonds leads to the fact that the residue of the AA becomes extremely unstable and quickly attaches two O2 molecules in a random way and undergoes intramolecular transitions with formation of one out of many possible positional and stereoisomers. The more PUFA has double bonds, the more possible isomers for the end product of the reaction. Abbreviations: F2-IsoP, E2-IsoP, D2-IsoP – Isoprostanes with, correspondingly, prostane rings F2, E2, D2 or A2 and J2; IsoTxA2 and IsoTxB2 – Isothromboxanes with rings A2* и *B2, correspondingly, which were formed from prostaglandins H2 (PGH2); E2-IsoK* и *D2-IsoK – Isoketals with rings E2* и *D2.*

## **7. IPLP as the mechanism of aging**

It has been demonstrated by researchers from the Vanderbilt University that IsoPs are the most early and reliable markers of lipid peroxidation *in vivo*, and recent studies provided valuable information about participation of IPLP in pathogenesis of numerous human diseases [80–82]. According to our model of IPLP initiation by HO2 • , the hydroperoxyl radical upon encounter with a PUFA produces one of many variants of isoPG, γ-ketoaldehyde or isolevuglandins. When reacting with fatty acids with two unsaturated bonds, such as linoleic acids of cardiolipin, HO2 • produces corresponding hydroperoxides.

The different toxic products of the IPLP evidently cause numerous and different lesions to mitochondria gradually causing wear and tear of mitochondrial and cellular functions. We distinguish two types of direct lesions to mitochondria: one dysfunctions type is caused by oxidation of CL and PEA, which result in structural changes of respirosomes and ATP-synthase complexes. The second dysfunctions type is caused by direct damages by toxic products, like isolevuglandins, which directly form adducts with PEA and lysine of proteins. This type of damages may explain mtDNA replication damages [33, 34, 83]. Anderson et al. [83] have shown that mtDNA replicase *exo* domain, Pol gamma, is far more sensitive to oxidation than *pol* domain. The authors suggested that under oxidative conditions, exonuclease activity therefore declines more rapidly than polymerase. The oxidized Pol gamma becomes editing-deficient, displaying a 20-fold elevated mutations than the unoxidized enzyme [83]. PEA may be damaged by both pathways: via PUFA at C2 autoxidation, and via formation of adducts of ethanolamine with IsoLG produced upon activation if IPLP by HO2 • . Of note, most of the ROS have very short lifetime (seconds) but IsoLG produce rather stable adducts (lifetime days), which can accumulate with age and, therefore, contribute to the development of age-associated conditions.

## **8. The importance of fatty acids oxidation for increased rate of ROS production**

Mammalian tissues mitochondria generate superoxide and hydrogen peroxide (ROS) from 11 different sites depending on substrates used and the redox state of the electron transport chain [21]. All mitochondrial ROS production sites have distinct properties [21, 84]. They can be divided into two groups: six sites operate at the redox potential of the NADH/NAD<sup>+</sup> isopotential pool, about −280 mV, and five sites operate at the redox potential of the ubiquinol/ubiquinone (QH2/Q ) isopotential pool, about +20 mV [21, 84].

Much of the published literature on contribution of separate respiratory complexes in generation of ROS have potential problems for several reasons: first, the authors often used inhibitors of the respiratory chain, which is far from situation *in situ*, secondly, most authors used a single substrate, whereas *in situ* mitochondria metabolize several substrates simultaneously [85–88]. Most importantly and, with few exceptions, authors never used fatty acids as substrates for mitochondrial respiration. Brandt and colleagues provided evidence that the highest rates of ROS production (O2 • + H2O2) are observed during β-oxidation of fatty acids [21, 84, 89]. During β-oxidation of fatty acids the membrane's pool of CoQ becomes fully reduced to CoQH2 and this may reverse the transport of reducing equivalents at the level of succinate dehydrogenase (SDH), also known as Complex II, and thus activate the reverse electron transport and involve the sites of respiratory Complexes I and III, and SDH (Complex II) in production of ROS [21, 89].

*Metabolic Syndrome as the First Stage of Eldership; the Beginning of Real Aging DOI: http://dx.doi.org/10.5772/intechopen.95464*

**Figure 2.**

*Production of superoxide radicals by rat heart mitochondria oxidizing palmitoyl-carnitine. Designations: 1. Supporting substrate only; 2. Palmitoyl-carnitine only, and 3. Palmitoyl-carnitine + supporting substrate. Substrates: Figure 2A - pyruvate 2.5 mM + malate 2 mM, Figure 2B - glutamate 5 mM + malate 2 mM, and Figure 2C. – Succinate 5 mM. Experimental conditions are described in [88]. The incubation medium contained: Amplex red 2 μM, horse radish peroxidase 2 units, substrates as indicated above, volume 1 ml. The reaction was initiated by addition of 50 μg of mitochondria. Initial rates were measured for 3 minutes. Numbers at the traces are the rates of H2O2 production in picomol H2O2/min/mg protein RHM. The rates were corrected for the time control rate with RHM incubated without added substrates. The figure was taken from [88].*

It has been recently shown that active oxidation of palmitoyl-carnitine by mitochondria in all metabolic states absolutely requires the simultaneous presence of any of the other mitochondrial metabolites such as: pyruvate, succinate, malate or glutamate, which were designated as "fatty acid oxidation supporting substrates" [87, 88]. In the presence of supporting substrates, fatty acids are actively oxidized not only by the isolated heart mitochondria [88], but also by the brain synaptic mitochondria [87], which breaks the old myth that the brain's energy metabolism is supported almost exclusively by glucose [reviewed in 87].

Most importantly, active oxidation of fatty acids in the presence of supporting substrates results in a manifold increase in ROS production in resting mitochondria (**Figure 2**). Earlier, we have proposed that oxidative damages and thus accelerated aging, are more common for organs, which have a wide range of physiological activities, such as heart, skeletal muscles and brain [65, 88]. When these organs are at low workloads or at rest, the very efficient oxidation of fatty acids may redirect excessive electrons to generation of ROS.

### **9. OK, MRFTA is valid, but what is aging?**

In the sections above, we presented evidence that in spite of complications of the MFRTA based on old paradigms, the latest discoveries clearly support the essence of this theory, and the introduction of the perhydroxyl radical as a new mediator of oxidative stress resolve most of the contradictions. However, MFRTA still requires further consideration and we have to find answers to simple questions: what is aging and when the aging begins?

From the beginning, researchers working on MFRTA held the point of view that aging is a pathological process caused by oxidative stress that affects our health, and over time, we succumb to a myriad of age-related pathologies and eventually die [12, 58]. But is aging simply the process of errors accumulation? The Dictionary of Science and Technology designates aging as "the process of growing older or changing over time" [90]. In other words, a person after birth goes through a series of genetically controlled transitions, which are called postembryonic ontogenesis [91]. From the point of view of a human ontogeny, we can roughly divide a person's individual life into five periods: infancy, childhood, adolescence, reproductive period and post-reproductive, or aging period. For several reasons, we hardly expect that during infancy, childhood and adolescence "a myriad of age-related pathologies"

might be accumulated [58]. We will consider the last two periods: "reproductive", which comprises the ages from 20 to 50, and the "aging period", which begins after the age of 50 [92].

Among recent definitions of aging there were few in more general terms: "Aging is characterized by a gradual decline in various health parameters across multiple biochemical, physiological and behavioral systems" [93]. Correspondingly, some researchers started looking for a subset of aging individuals with lack of resilience within these general physiological systems, a condition termed frailty. Frailty has been defined in broad terms as an age-associated syndrome characterized by increased vulnerability to external influences, a diminished capacity to respond correctly to stressors and as an overall loss of fitness. In general, frail individuals are at a greater risk of falls, dependency, disability, institutionalization, hospitalization and mortality [94]. Frailty can be measured in relation to the accumulation of deficits using a frailty index. A frailty index can be developed from most aging databases [95, 96].

In another parallel world of Science, a large group of researchers study the metabolic syndrome, a nosological entity established at the end of 80s of the last century. The metabolic syndrome (MetS) was defined as a condition of simultaneous existence of several risk factors, including obesity, insulin resistance, atherogenic dyslipidemia and hypertension, which are interrelated, age-dependent, and share underlying mediators and metabolic pathways [97]. Undoubtedly, both concepts of frailty and metabolic syndrome are important from a medical perspective, but apparently did little for understanding the mechanisms of aging. Due to intensive research on both concepts, however, there has been made a great discovery: the rates of aging and energy metabolism are sex-specific. In our opinion, these discoveries are crucial for understanding the mechanisms of aging as the process of transition of an individual from the reproduction period to the last stage of ontogeny – aging.

#### **10. Sex-specific differences in the rates of aging and longevity**

Most animals and plants are sexual, in spite of the reproductive advantages experienced by asexual variants. Evidently there where selective forces that gave an advantage to sexuality and genetic recombination at either the population or individual level. The effect of sex and recombination increases the efficiency of natural selection, which is a major factor favoring evolution [98, 99]. It has been experimentally shown that sex increases the rate of adaptation to a new harsh environment, but has no measurable effect on fitness in a new benign environment where there is little selection [100]. Nonetheless, we are still far from a definitive answer to the question of why sexual reproduction is so common [99]. Recently, the hypothesis has been put forward that the internal production of ROS 2 billion years ago started the eukaryotic sex (re)evolution [101]. It has also been stressed that earlier theoretical works on sexual reproduction ignored important complexities that face natural populations, such as genetic drift and the spatial structure of populations [102].

The data accumulated show that in many species, including humans, females have slower rate of aging and longer life span than males [99, 103–105]. We suggest that this observation has important general biological goals for a female: bearing and raising a new generation despite any external difficulties and metabolic restrictions. These goals demand that females have to be efficient enough, but not superefficient from the metabolic (thermodynamic) point of view, resilient to harsh environmental conditions, and, in accord with the MFRTA, have lower rate of oxidative stress. Numerous studies of various species showed that in general females *Metabolic Syndrome as the First Stage of Eldership; the Beginning of Real Aging DOI: http://dx.doi.org/10.5772/intechopen.95464*

have slower production of ROS than males [99]. As always, there are some exclusions from the general rule, but again they hold the same conclusion: the longer living gender produces less ROS [106].

In the next sections we will discuss possible mechanisms underlying the slower aging and slower rates of ROS production in the longer living females.

## **11. Sex-specific differences in the rates of fat utilization for the sake of energy metabolism**

Laboratory animals are indispensable part of biomedical research and widely used for modeling physiological and pathological situations in humans [107]. For ethical and technical restrictions, it is impossible to study many biomedical problems on humans, whereas animal research provides a degree of experimental control and precision not usually feasible in studies using human subjects [108]. Meanwhile, the animals used in most experiments were males, because researchers usually avoid using females for the reason of their reproductive cycles and hormone fluctuations that may affect the results of their studies [109]. For these reasons research on sex differences has begun relatively recently, but today the related literature is enormous. Human studies on metabolic differences between men and women were stimulated largely due to the progress of the sport medicine. Here, we will discuss only those works, which have direct relation to our subject under discussion: what sex metabolic differences underlie the fact that females live longer than males in many species, including humans [108].

Evidently, the sex differences in the body structure and metabolism depend on the stage of a person's ontogeny. Vijay et al. (2015) studied sexual differences in the expression of mitochondria-related genes in rat heart at different ages that correspond to different stages of the rat's reproductive capacities [110]. The authors studied the whole genome expression profiling in the hearts of young (8-week), adult (21-week), and old (78-week) male and female Fischer 344 rats, and the expression of 670 unique genes related to various mitochondrial functions was analyzed. A significant (p < 0.05) sexual dimorphism in expression was observed in young animals for 46, adult for 114 and old rats for 41 genes, respectively [110]. Importantly, in young and adult hearts, sexual dimorphism was not noted in genes encoding oxidative phosphorylation**.** Adult males showed higher expression of genes associated with the pyruvate dehydrogenase complex as compared to females. In old rats a majority of genes involved in oxidative phosphorylation had higher expression in females. This clearly shows that sexual dimorphism largely depends on the stage of ontogeny. Other studies demonstrated better preservation of myocardial mass and a greater cardiac contractility in women than men during aging [111]. The better heart health in aged women might be the result of either genetically predetermined factors or less oxidative damages and slower aging as compared to men.

It is a well-known observation that women generally have a higher amount of body fat than men. Distribution of fat is also different: women store more fat in the gluteal-femoral region, whereas men have more body fat in the abdominal (visceral) region [112, 113]. Importantly, that visceral fat accumulation is accompanied with multiple endocrine perturbations, including elevated cortisol and androgens in women, as well as low growth hormone and, in men, testosterone secretion. The consequences of the hormones effects will be more expressed in visceral than subcutaneous adipose tissues, because omental fat has higher cellularity, innervation and blood flow. Furthermore, the density of cortisol and androgen receptors seems to be higher in visceral fat than in other regions of adipose tissue [114]. In addition, there are epidemiological and metabolic associations between centralized (visceral) fat accumulation and disease [114]. This is an important fact because visceral obesity is a common symptom for men and women with metabolic syndrome.

Physiological experiments with oral administration of triglycerides, labeled with a small amount of oleic acid, revealed the following regional differences in the order of lipid uptake: omental = retroperitoneal > subcutaneous abdominal > subcutaneous femoral adipose tissues in men, with a similar rank order for half-life of the triglyceride, indicating also a turn-over of triglycerides in that order. Testosterone amplifies these differences in men. In premenopausal women, the visceral fat accumulation is smaller than in men, and subcutaneous abdominal has a higher turnover than femoral adipose tissue [114]. Among regional gender differences of fat metabolism, there is an interesting evidence that *in vivo*, catecholamine mediated leg free fatty acid release is lower in women than in men, whereas free fatty acid release from the upper body depots is comparable [112].

These experiments *in vivo* indicate that sex variations in fat metabolism are controlled by sex-specific hormones. This presumes that in the post-reproductive stage of ontogenesis, the sexual dimorphism in fat metabolism should be weaker or absent. Results of studies *in vitro* also indicate that this difference is diminished at the menopause, and may be restored by estrogen therapy. This suggest that the functional effects of estrogens in women are similar to those of testosterone in men. As we will show in the next section, the effects of both hormones are targeted on substrate oxidations by mitochondria, and, thus, on the rates of ROS production. However, the mechanism of estrogen on fat metabolism might be indirect because human adipose tissue does not possess specific estrogen and progesterone receptors [114].

As regards protein metabolism, no gender differences in the basal level net muscle protein balance have been found [115]. In general, testosterone increases muscle protein synthesis and net muscle protein balance, resulting in increased muscle mass. At young age, boys and girls have similar amounts of testosterone. At puberty testosterone levels increase much more dramatically in males, as does muscle mass. Furthermore, although no evidence exists in humans, the in-vitro and rat data suggest that ovarian hormones inhibit muscle protein synthesis [115].

## **12. Sex differences in substrate utilization during physical activities**

The indirect effect of the sex hormones on fat metabolism is supported by the data on sexual dimorphism in utilization of fatty acids during physical activities, which demonstrate that the proportion of energy derived from fat during exercise is higher in women than in men [112, 116]. Carter et al. (2001) investigated the effect of endurance training on whole body substrate, glucose, and glycerol utilization during 90 min of exercise in males and females [116]. First, during submaximal physical loads females show a lower respiratory exchange ratio (RER) than males, which indicates on a proportionately lower carbohydrate and higher fat oxidation [116, 117]. In comparison with females, exercising males had a greater increase in leucine oxidation but not lysine levels, which indicated that during intensive physical activity males increase their need for amino acids to fuel energy needs. Under the same conditions, females responded by increased mobilization of fat, thereby requiring less alternative fuels such as carbohydrate and amino acids [116–118]. The overall conclusion of these experiments was that females oxidize a greater proportion of fat and less carbohydrates and amino acids as compared with males. Thus physiologists support our finding that fatty acids oxidation requires simultaneous presence of other mitochondrial metabolites derived from carbohydrates or

*Metabolic Syndrome as the First Stage of Eldership; the Beginning of Real Aging DOI: http://dx.doi.org/10.5772/intechopen.95464*

proteins [88]. Because women have lesser consumption of supporting substrates, the rate of fatty acids oxidation should be also diminished. This might explain why, in general, women demonstrate lower levels of physical performance during endurance sports and produce less ROS [119–121].

Knowing the fact that mitochondria oxidize fatty acids only in the presence of supporting substrates [87, 88], the data presented above suggest that females require less or different supporting substrates for effective oxidation of fatty acids. We can, therefore, predict that the isolated skeletal muscle or heart mitochondria from females must be different from males in terms of the type and requirement of supporting substrates, and also produce ROS at a slower rate. Unfortunately, until now we have been the only ones who have studied the oxidation of fatty acids in the presence of various supporting substrates, but we used male rats only in our experiments [87, 88]. **Figure 3** illustrates that the rates of ROS production strongly depend on the type of supporting substrates, namely pyruvate, glutamate and succinate, and their various mixtures [55, 87, 88]. Thus the *in vitro* experiments with the isolated mitochondria from different organs require further investigation in order to elucidate molecular mechanisms of the sex diversity at the mitochondrial level. Since males showed higher expression of genes associated with the pyruvate dehydrogenase complex, as compared to females [111], and consume more carbohydrates and amino acids during endurance training [116–118] we can suggest that women's higher longevity and slower rate of aging is associated with the less efficient oxidation of fatty acids and thus slower rates of oxidative stress. However, this conclusion evidently regards only women at the reproductive stage, when, according to Ventura-Clapier et al. (2020), women are "protected" [122]. These authors also stated that "apart from comparisons between males and females, there is a crucial need for studying the female physiology and woman pathology. In particular the biological step that constitutes menopause in women appears to be the border between "female protection" and "female susceptibility" to cardiovascular diseases, which needs to be deciphered further".

#### **Figure 3.**

*Effect of substrate mixtures on production of ROS by isolated rat heart mitochondria oxidizing palmitoyl-carnitine. The incubation medium contained: Amplex red 2 μM, horse radish peroxidase 2 units, volume 1 ml. The reaction was initiated by addition of 50* μ*g of mitochondria. Initial rates were measured for 3 minutes. Substrates: 1. Succinate 5 mM; 2. Succinate + pyruvate 2.5 mM + glutamate 5 mM + malate 2 mM; 3. Palmitoyl-carnitine 50 μM + succinate; 4. Palmitoyl-carnitine + succinate + pyruvate + glutamate + malate. Numbers at the traces are the rates of H2O2 production in picomol H2O2/min/mg protein RHM. The rates were corrected for the time control rate with RHM incubated without added substrates. The figure was taken from [55].*
