The Impact of H2S on Obesity-Associated Metabolic Disturbances

Over the last several decades, hydrogen sulfide (H2S) has gained attention as a new signaling molecule, with extensive physiological and pathophysiological roles in human disorders affecting vascular biology, immune functions, cellular survival, metabolism, longevity, development, and stress resistance. Apart from its known functions in oxidative stress and inflammation, new evidence has emerged revealing that H2S carries out physiological functions by targeting proteins, enzymes, and transcription factors through a post-translational modification known as persulfidation. This review article provides a critical overview of the current state of the literature addressing the role of H2S in obesity-associated metabolic disturbances, with particular emphasis on its mechanisms of action in obesity, diabetes, non-alcoholic fatty liver disease (NAFLD), and cardiovascular diseases.


Introduction
Gasotransmitters are small molecules of endogenous gas that have the noticeable ability to diffuse into cells to interact with their targets, inducing an array of intracellular signaling and pathophysiological responses [1,2]. Hydrogen sulfide is the most recent addition to the gasotransmitters family, the first two being nitric oxide (NO) and carbon monoxide (CO) [3].
Instead of binding to plasma membrane receptors, the high solubility of gasotransmitters in lipids allows them to penetrate cell membranes without requiring a specific transporter or receptor. Gasotransmitters are generated endogenously by specific enzymes and can generate various functions at physiologically relevant concentrations by targeting specific cellular and molecular targets [4]. A gaseous substance is not readily stored in vesicular structures and so must be resynthesized as needed. This implies that the biosynthetic enzymes must be subject to tightly regulated mechanisms [5]. Abnormal generation and metabolism of these gasotransmitters have been extensively demonstrated to affect diverse biological processes, such as vascular biology, immune functions, cellular survival, metabolism, longevity, development, and stress resistance [4].
Perhaps the most remarkably unique feature of gasotransmitters relates to the molecular mechanisms whereby they signal to their targets. Gasotransmitters chemically modify intracellular proteins, thus affecting cellular metabolism in a more immediate fashion than other signal transduction mechanisms [5].
H 2 S has been traditionally considered only as a toxic agent for living organisms [6]. Nowadays, it is considered a gaseous mediator that plays important regulatory roles in innate immunity and inflammatory responses, impacting the development of cardiovascular and metabolic disorders [7][8][9][10][11]. Dysfunctional tissue H 2 S metabolism is increasingly implicated in different pathologies, from cardiovascular [12,13] and neurodegenerative diseases [14][15][16] to cancer [17,18]. There is emerging evidence supporting the importance of H 2 S in the pathophysiology of obesity, type 2 diabetes, NAFLD, and cardiovascular diseases. Understanding the precise mechanisms that control H 2 S homeostasis and their Figure 1. Overview of the transsulfuration pathway. There are four enzymatic pathways for the biosynthesis of H 2 S, including CBS, CSE, MPST coupled with CAT, and MPST coupled with DAO. Non-enzymatic H 2 S generation occurs in the presence of reducing equivalents such as NADPH and NADH, reactive sulfur species in persulfides, thiosulfate, and polysulfides that are reduced into H 2 S and other metabolites. The transsulfuration pathway intersects with the transmethylation pathway at homocysteine. Methionine is an indispensable amino acid and is transmethylated intracellularly via S-adenosylmethionine (SAM), an important methyl donor for most biological methylation reactions, producing S-adenosylhomocysteine (SAH) in this process, and is then hydrolyzed to homocysteine. Homocysteine can be remethylated back to methionine by N5,N10-methylenetetrahydrofolate reductase (MTHFR). The cysteine generated by the pathway can be conducted into GSH synthesis by the action of the enzymes γ-glutamyl cysteine synthetase (γ-GCS) and glutathione synthetase (GS) or converted to other sulfur-containing molecules such as taurine. Taurine is generated by the action of three enzymes, CDO, cysteine sulfinic acid decarboxylase (CSAD), and hypotaurine dehydrogenase (HTAU-DH).
Recently, an alternative enzymatic pathway to the transsulfuration pathway has been identified for the enzymatic generation of H 2 S within mitochondria, known as the 3mercaptopyruvate pathway. The pathway requires two enzymes, 3-mercaptopyruvate sulfurtransferase and the PLP-dependent enzyme cysteine aminotransferase (CAT). 3mercaptopyruvate (3MP) is produced by CAT from L-cysteine and α-ketoglutarate [21]. Thereafter, MPST transfers a sulfur atom from 3MP onto itself, which leads to the formation of a persulfide, MPST-SS. H 2 S and MPST are then released from the persulfideof MPST-SS in the presence of reductants such as thioredoxin (TRX) and dihydrolipoic acid (DHLA) [33]. Recently, another source of 3MP was found in mammals by Shibuya et al.: D-cysteine [34]. Specifically, D-cysteine is transformed into 3MP by peroxisome-located d-amino acid oxidase (DAO). Metabolite exchanges between peroxisome and mitochondria can import 3MP into mitochondria, where it is further catalyzed into H 2 S by MPST. Because of the exclusive location of DAO in the brain and kidney, this H 2 S-generating pathway is currently believed to exist only in these two organs [17].

Non-Enzymatic Synthesis of H 2 S
Several cases of the non-enzymatic production of H 2 S have been reported, and these are suspected to represent a small proportion of all generated endogenous H 2 S. It is postulated that coordinated activities of PLP and iron (Fe 3+ ) catalyze the generation of H 2 S using cysteine as a substrate, in a non-enzymatic manner in specific circumstances. Regulation of H 2 S production via this pathway may contribute to the pathophysiology of conditions with iron dysregulation such as hemolysis, iron overload, and hemorrhagic disorders [35].
H 2 S can also be generated from sulfane sulfur via non-enzymatical reduction in the presence of an endogenous reductant, such as nicotinamide adenine dinucleotide phosphate (NADPH) and nicotinamide adenine dinucleotide (NADH), which are supplied by oxidation of glucose via glycolysis or from phosphogluconate via NADPH oxidase [36].
In the presence of such reductants, reactive sulfur species in persulfides, thiosulfate, and polysulfides can be reduced into H 2 S and other metabolites [37]. Essentially, all the components of this non-enzymatic route are available in mammals, including reducible sulfur, suggesting the necessity of this pathway in mammalian systems. In accordance with this, hyperglycemia is demonstrated to promote H 2 S generation by enhancing this pathway [38].

The Relationship between H 2 S and Obesity
Obesity is characterized by the excessive accumulation and storage of fat in the body (regionally, globally, or both) that may be harmful to health and is defined by a body mass index (BMI) of 30 kg/m 2 or greater, being considered morbidly obesity when BMI is over 35 kg/m 2 .
The epidemic of obesity presents a serious threat to human health around the world. The worldwide prevalence of obesity has increased dramatically over the past 30 years, fueled by economic growth, industrialization, mechanized transport, urbanization, an increasingly sedentary lifestyle, and a nutritional transition to processed foods and highcalorie diets [39]. According to the World Health Organization (WHO), 30% of Americans and 10%-20% of Europeans are obese, and on estimate more than 1.9 billion adults worldwide are overweight. High body mass carries with it an increased risk of the development of a number of serious cardiovascular and metabolic diseases, such as type 2 diabetes, hypertension, dyslipidemia, stroke, osteoarthritis, as well as several different forms of cancer [40]. There is emerging evidence supporting the importance of H 2 S in the pathophysiology of obesity.

The Importance of H 2 S in Obesity
Few studies have evaluated circulating sulfide in humans, with discrepant results. Whiteman et al. were the first ones to demonstrate the involvement of H 2 S in obesity [41]. These authors found that plasma H 2 S levels were significantly decreased in non-obese individuals with type-2 diabetes and in overweight participants with altered glucose [41]. However, the mechanisms that mediate the loss of H 2 S were not elucidated in that early study.
Additionally, other animal studies have demonstrated that exogenous H 2 S administration led to increased insulin sensitivity and improved glucose tolerance after a high-fat diet was fed to mice in parallel to weight gain [42,43]. Supplementation with H 2 S donors or increasing endogenous H 2 S biosynthesis was sufficient to stimulate fat mass accumulation in mice and fruit flies, whereas the depletion of endogenous H 2 S biosynthesis prevented high fat diet-induced fat mass (HFD-induced fat mass) [43,44]. Later, Alkhouri et al. reported that the H 2 S concentration in exhaled air was higher by 1/3 in obese children compared to lean children [45].
Recently, Comas and colleagues demonstrated that serum sulfide concentrations were increased in subjects with morbid obesity in proportion to fat mass [46]. Longitudinally, weight gain resulted in increased serum sulfide concentration, whereas weight loss had opposite effects, being the percent change in serum sulfide positively correlated with the percent change in BMI and waist circumference [46]. Ren and colleagues demonstrated that a milk protein concentrate diet prevents obesity induced by HFD in Sprague Dawley rats. This protection against obesity was associated with increased transsulfuration pathway and plasma H 2 S levels, which in turn maintained redox homeostasis and reduced lipid disorders induced by HFD [47].
Due to the lack of standardized methodology and variation in models and study cohorts, we see contradicting reports about H 2 S in obesity (Table 1). Depending on the technique used to measure sulfide, reported circulating levels of sulfide vary widely in obese and lean individuals. Furthermore, more human and animal studies are needed to fully comprehend the physiological and pathophysiological roles of H 2 S in obesity.

Persulfidation Might Prevent the Negative Effects of Obesity-Associated Oxidative Stress
Signaling by H 2 S is proposed to occur via persulfidation, a posttranslational modification of cysteine residues (RSH) to persulfides (RSSH) [48], thought to be one of its main beneficial mechanisms of action [28]. Like other posttranslational modifications, persulfidation potentially alters a protein's structure, function, stability, and/or macromolecular interactions [48][49][50]. Nonetheless, due to their enhanced nucleophilicity, persulfides react readily with reactive oxygen species (ROS), whereas H 2 S itself is a poor ROS scavenger. When exposed to ROS, proteins undergo oxidation to form sulfenic acids (P-SOH), sulfinic acids (P-SO 2 H), and sulphonic acids (P-SO 3 H), which cause the irreversible inactivation of the protein [51].
The detrimental actions of ROS are well-known. ROS are physiologically important, acting as second messengers in cell signaling, and they also play a pivotal role in cellular homeostasis [52,53] and are associated with cellular damage by oxidizing cellular constituents such as proteins, lipids, and DNA [54]. These physiological effects are mainly mediated by changes in the redox state of crucial intracellular and/or surface thiols. In the last few years, several studies have shown a strong association between obesity and altered redox state, consistent with the idea that an increased caloric intake and/or obesity are associated with a pro-oxidant environment and increased oxidative damage [55][56][57][58][59]. A lot of evidence has shown that obesity is a state of chronic oxidative stress, although it is not completely understood if the alteration in redox balance is a trigger rather than a result of obesity [57,[60][61][62][63].
Given the fact that adipose tissue expansion during the progression of obesity can result in the excess production of toxic radical species that can cause the generation of oxidative stress through ROS, it is tempting to speculate that increased ROS in obesity leads to reduced persulfidation, resulting in cysteine thiol overoxidation, altering the integrity and activity of relevant adipose-tissue-related proteins ( Figure 2). Antioxidants 2021, 10, x FOR PEER REVIEW 6 of 24 oxidative stress through ROS, it is tempting to speculate that increased ROS in obesity leads to reduced persulfidation, resulting in cysteine thiol overoxidation, altering the integrity and activity of relevant adipose-tissue-related proteins ( Figure 2).

Figure 2.
Proposed mechanism that occurs in thiols in obesogenic conditions. In obesity, adipose tissue hypertrophy is associated with increased ROS levels, which oxidizes protein thiols to sulfenic acids. The decreased expression of H2Ssynthesizing enzymes and H2S production in adipose tissue avoids the formation of persulfides by sulfenic acid. The oxidizing environment of obesity mediates the conversion of sulfenic acids to sulfonate, altering the integrity and activity of relevant adipose-tissue-related proteins.
Aside from decreased persulfidation in obesity due to ROS, alternative mechanisms have also been proposed. Dietary restriction (DR) regimens are known to reduce adiposity. Hine et al. reported that dietary restriction of sulfur amino acids (SAAs) methionine (Met) and cysteine (Cys) and decreased mTORC1 activation led to the increased expression of CTH, resulting in increased H2S production in the liver, and an increased lifespan [9]. Methionine and cysteine are considered to be the principal SAAs in the diet because they are incorporated into proteins. Elevation of plasma SAAs is generally associated with an unfavorable lipid profile, and plasma total cysteine is independently associated with body mass index [64]. Another study reported that higher plasma total cysteine was associated with an increased risk of obesity and IR in Hispanic children and adolescents [65]. These studies suggest that a pro-oxidant environment and increased oxidative damage, paired with increased SAAs in obesity, lead to decreased transsulfuration pathway (TSP) activity and decreased H2S, which could result in decreased persulfidation in obesity.
Aging is known to be associated with decreased persulfidated protective pools, associated with the loss of protein expression of the three H2S-producing enzymes [66]. Hine et al. showed that hepatic-produced H2S was elevated in long-lived hypopituitary mouse models, and that the thyroid hormone (TH) and growth hormone (GH) negatively regulate hepatic H2S via the repression of CTH [67]. In addition to the onset of age-related diseases in senior adults, there are some conserved aging phenotypes in human and animals-redox imbalance, mitochondrial dysfunction, increased apoptosis, cellular senescence, insufficient autophagy, and increased inflammation [68]. Similar to aging, obesity and excess calorie intake appear to perpetuate the onset of age-related diseases through similar mechanisms [69]. These findings indicate that obesity accelerates aging, allowing for cysteine overoxidation and reduced persulfidation. Proposed mechanism that occurs in thiols in obesogenic conditions. In obesity, adipose tissue hypertrophy is associated with increased ROS levels, which oxidizes protein thiols to sulfenic acids. The decreased expression of H 2 S-synthesizing enzymes and H 2 S production in adipose tissue avoids the formation of persulfides by sulfenic acid. The oxidizing environment of obesity mediates the conversion of sulfenic acids to sulfonate, altering the integrity and activity of relevant adipose-tissue-related proteins.
Aside from decreased persulfidation in obesity due to ROS, alternative mechanisms have also been proposed. Dietary restriction (DR) regimens are known to reduce adiposity. Hine et al. reported that dietary restriction of sulfur amino acids (SAAs) methionine (Met) and cysteine (Cys) and decreased mTORC1 activation led to the increased expression of CTH, resulting in increased H 2 S production in the liver, and an increased lifespan [9]. Methionine and cysteine are considered to be the principal SAAs in the diet because they are incorporated into proteins. Elevation of plasma SAAs is generally associated with an unfavorable lipid profile, and plasma total cysteine is independently associated with body mass index [64]. Another study reported that higher plasma total cysteine was associated with an increased risk of obesity and IR in Hispanic children and adolescents [65]. These studies suggest that a pro-oxidant environment and increased oxidative damage, paired with increased SAAs in obesity, lead to decreased transsulfuration pathway (TSP) activity and decreased H 2 S, which could result in decreased persulfidation in obesity.
Aging is known to be associated with decreased persulfidated protective pools, associated with the loss of protein expression of the three H 2 S-producing enzymes [66]. Hine et al. showed that hepatic-produced H 2 S was elevated in long-lived hypopituitary mouse models, and that the thyroid hormone (TH) and growth hormone (GH) negatively regulate hepatic H 2 S via the repression of CTH [67]. In addition to the onset of age-related diseases in senior adults, there are some conserved aging phenotypes in human and animals-redox imbalance, mitochondrial dysfunction, increased apoptosis, cellular senescence, insufficient autophagy, and increased inflammation [68]. Similar to aging, obesity and excess calorie intake appear to perpetuate the onset of age-related diseases through similar mechanisms [69]. These findings indicate that obesity accelerates aging, allowing for cysteine overoxidation and reduced persulfidation.
Supporting the importance of H 2 S and persulfidation in the physiology of adipose tissue (Figure 2), Comas et al. recently demonstrated that proteins required for an appro-priate adipogenesis and adipose tissue functionality present higher persulfidation levels in adipocytes in comparison to preadipocytes, sustaining the idea that persulfidation preserves the function of adipogenesis-related proteins [70].
Altogether, the evidence collectively suggests that obesity might be associated with reduced protein persulfidation, paving the way to cysteine overoxidation and impairing the activity and function of adipogenesis-related proteins.

H 2 S in Adipose Tissue
Adipose tissue is one of the most abundant organs in the human body, but for a long time it has been given little importance and it was considered only as a passive energy storage site [71]. The worldwide epidemic of obesity and type 2 diabetes has greatly increased interest in the biology and physiology of adipose tissues. It is now known that apart from the energy reservoir function, adipose tissue functions as a thermal insulator, mechanical shock absorber, and more importantly as an endocrine organ [72].
Feng et al. first identified the endogenous CTH and CBS gene expression pathways in adipose tissue, and suggested CTH as the primary pathway of H 2 S generation in adipose tissue [73]. The same year, Fang et al. also demonstrated that CTH protein expression and endogenous H 2 S production in rat perivascular adipose tissue were detectable and that the endogenous H 2 S generated was predominantly CTH-catalyzed [74]. Subsequent studies confirmed that CBS, CTH, and MPST genes were expressed in adipose tissue depots [75,76], and suggested that H 2 S affects diverse metabolisms that take place in the adipose tissue, such as lipid, glucose, and mitochondrial metabolism. H 2 S is also involved in the regulation of inflammatory and oxidative stress-associated responses in adipose tissue through adipokine and antioxidant control [77]. Studies of an in vitro 3T3-L1 mouse cell line model pointed to a possible role of H 2 S in adipocyte differentiation through the modulation of peroxisome proliferator-activated receptor gamma (PPARγ) activity [43,44,78]. The overexpression of the H 2 S generation enzyme CTH and the administration of the H 2 S donor sodium hydrosulfide (NaHS) to 3T3-L1 cells in an environment of high glucose restored adiponectin secretion and decreased the secretion of proinflammatory cytokines [79]. Recently, Comas et al. first reported the relevance of H 2 S and human adipose tissue physiology in the context of obesity [70]. Experiments in human adipose tissue explants and in isolated preadipocytes demonstrated that exogenous H 2 S or the activation of endogenous H 2 S biosynthesis resulted in increased adipogenesis, insulin action, sirtuin deacetylase, and PPARγ transcriptional activity, whereas inhibition through chemical compound or gene knockdown of one of the H 2 S-generating enzymes (CTH, CBS, MPST) led to altered adipocyte differentiation, cellular senescence, and increased inflammation. In morbidly obese subjects, reduced gene expression of H 2 S-synthesising enzymes in visceral and subcutaneous adipose tissue depots was reported, whereas weight loss interventions improved the expression of these enzymes. In human preadipocytes, the expression of CTH, CBS, and MPST genes and hydrogen sulfide production were dramatically increased during adipocyte differentiation [70]. In addition, a recent study demonstrated the relevance of selenium-binding-protein 1 (SELENBP1) in 3T3-L1 adipocyte differentiation through the modulation of cellular H 2 S levels and the expression of H 2 S-producing enzymes [80].

H 2 S and Lipid Metabolism
The effect of H 2 S on the regulation of lipid metabolism in adipose tissue is controversial. In primary adipocytes, inhibition of the CTH/H 2 S system was achieved with stimulated adipocyte lipolysis by means of increased activation of the PKA-perilipin1/hormone sensitive lipase (HSL) pathway, whereas GYY4137 or L-cysteine reversed this effect. In adipose tissue of normal chow and HFD mice, the CTH inhibitor DL-propargylglycine (PAG or PPG) increased lipolysis, evidenced by elevated serum glycerol, whereas H 2 S donors lowered lipolysis only in HFD mice [42]. Different studies have reported the use of slow hydrogen sulfide-releasing agents derived from garlic with effects on lipid metabolism. In isolated human adipocytes and Wistar rats, diallyl sulfide (DAS) downregulated the mRNA and protein expression of lipolytic genes like hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), whereas it upregulated the expression of lipogenic gens like PPARγ [81]. Aged black garlic (AGB) extract suppressed lipogenesis by reducing the expression of PPARγ, whereas it enhanced lipolysis by upregulating HSL phosphorylation at Ser563 and downregulating perilipin in mature 3T3-L1 adipocytes [82].
On the other hand, infusing Na 2 S in adipose tissue of rats resulted in increased glycerol and cAMP release in control and obese rats, whereas PAG partially reduced glycerol levels in obese rats, suggesting an H 2 S-cAMP-PKA lipolysis mechanism [77].
A mechanistic way by which hydrogen sulfide could regulate lipid metabolism is persulfidation. Ding et al. showed that H 2 S post-translationally modified perilipin 1 (Plin-1) via cysteine persulfidation, resulting in increased Plin1 activity, which blocks HSL translocation to lipid droplets, resulting in decreased lipolysis and promoting adipocyte lipid accumulation [83].

H 2 S and Adipose Tissue Inflammation
Obesity predisposes patients to a pro-inflammatory state via increased inflammatory mediators interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), as well as reduced levels of adiponectin, which has a totally anti-inflammatory function [84]. In recent years, substantial basic scientific research has led to a reasonably clear understanding of the role of H 2 S as an inflammatory mediator implicated in different inflammatory conditions, such as acute pancreatitis, sepsis, joint inflammation, and chronic obstructive pulmonary disease (COPD) [85]. Nevertheless, our understanding of the molecular mechanisms by which adipose tissue H 2 S contributes to inflammation in the obesity context is still scarce.
Adipose tissue macrophages (ATMs) are able to adopt either a proinflammatory (M1) or an anti-inflammatory (M2) phenotype. During obesity, the proinflammatory M1 phenotype is predominant. Store-operated calcium entry (SOCE), causing an influx of Ca 2+ , occurs in macrophage polarization to the M1 phenotype [86]. Increased amounts of CSE mRNA and protein and reduced H 2 S concentrations were found in ATMs isolated from obese mice and RAW264.7 macrophages under inflammatory conditions. Nonetheless, the H 2 S production capacity was found to be markedly increased in the previous conditions, leading to the increased consumption of H 2 S and reducing its bioavailability in proinflammatory conditions. This decrease in the concentration of H 2 S was associated with increased Ca 2+ entry through the amplification of SOCE activity, facilitating the proinflammatory M1 phenotype in obese adipose tissue [87].
Recently, we examined the role of H 2 S in the regulation of inflammation during adipogenesis. Treatment with slow-releasing H 2 S donor GYY4137 attenuated the negative effect of inflammation on adipogenesis in 3T3-L1 during differentiation [88].
Active research on the role of H 2 S in inflammation will unravel the pathophysiology of its actions in adipose tissue inflammatory conditions and may help to develop novel therapeutic approaches.

The Possible Role of H 2 S on Glucose Metabolism
Most human cells utilize glucose as the primary substrate, with cellular uptake requiring insulin. Insulin signaling is therefore critical for these tissues. However, a decrease in insulin sensitivity due to the disruption of various molecular pathways causes insulin resistance (IR) [89]. Insulin resistance is one of the main characteristics of pathological manifestations associated with type 2 diabetes mellitus. In general, several intrinsic and extrinsic cellular mechanisms have been identified, which display a cause-effect relationship between weight gain and peripheral insulin resistance [90]. Intrinsic cell-signaling pathways include mitochondrial dysfunction, oxidative stress, and endoplasmic reticulum (ER) stress, whereas alterations in adipokines and fatty acids levels and the presence of inflammation in metabolic tissue are the dominant extrinsic mechanisms that modulate insulin's peripheral actions [90].
H 2 S has emerged as a regulator of glucose metabolism and energy homeostasis, through different mechanisms targeting the pancreas, liver, adipose tissue, and skeletal muscle (Figure 3). extrinsic cellular mechanisms have been identified, which display a cause-effect relationship between weight gain and peripheral insulin resistance [90]. Intrinsic cell-signaling pathways include mitochondrial dysfunction, oxidative stress, and endoplasmic reticulum (ER) stress, whereas alterations in adipokines and fatty acids levels and the presence of inflammation in metabolic tissue are the dominant extrinsic mechanisms that modulate insulin's peripheral actions [90].
H2S has emerged as a regulator of glucose metabolism and energy homeostasis, through different mechanisms targeting the pancreas, liver, adipose tissue, and skeletal muscle (Figure 3).

H2S and the Pancreas
The pancreas is a complex gland, active in digestion and metabolism through the secretion of digestive enzymes, including the secretion of the blood sugar-lowering hormone insulin by β-cells and its opponent glucagon via the secretion of α-cells [91].
Insulin release from pancreatic β-cells is tightly regulated and allows the sensitive response of insulin levels to calorigenic nutrients in the body. Glucose, free fatty acids, and amino acids serve as fuel stimuli for insulin release, promoting insulin granule exocytosis. Additional hormonal factors influence the regulation pathway [92]. Insulin therapy is the most effective method of lowering blood glucose, thus pharmacological agents that augment insulin release are a key part of the treatment of diabetes.
At the start of the century, the pathophysiological implications of the CTH/H2S system in diabetes were reported. Yusuf et al. first demonstrated that CBS mRNA and H2S synthesis were increased in the pancreas from streptozotocin-induced diabetic rats [96]. Later, CSE and CBS gene expression were reported in mouse pancreatic acini [97]. In HIT-

H 2 S and the Pancreas
The pancreas is a complex gland, active in digestion and metabolism through the secretion of digestive enzymes, including the secretion of the blood sugar-lowering hormone insulin by β-cells and its opponent glucagon via the secretion of α-cells [91].
Insulin release from pancreatic β-cells is tightly regulated and allows the sensitive response of insulin levels to calorigenic nutrients in the body. Glucose, free fatty acids, and amino acids serve as fuel stimuli for insulin release, promoting insulin granule exocytosis. Additional hormonal factors influence the regulation pathway [92]. Insulin therapy is the most effective method of lowering blood glucose, thus pharmacological agents that augment insulin release are a key part of the treatment of diabetes.
At the start of the century, the pathophysiological implications of the CTH/H 2 S system in diabetes were reported. Yusuf et al. first demonstrated that CBS mRNA and H 2 S synthesis were increased in the pancreas from streptozotocin-induced diabetic rats [96]. Later, CSE and CBS gene expression were reported in mouse pancreatic acini [97]. In HIT-T15, a hamster insulin-secreting cell line, low levels of CBS were reported and no CTH mRNA transcripts were found [94]. In Zucker diabetic fatty (ZDF) rats, both CSE and CBS transcripts were detected in pancreatic islet tissues, with the expression level of CSE being significantly higher than that of CBS [98]. The expression of CBS protein in mouse pancreatic islets and a mouse β-cell line MIN6 was reported by Kaneko et al. [99,100]. [93]. No MPST protein expression was detected by Western blot pancreases from C57BL/6J; however, later, strong levels of MPST protein were detected in the pancreas of C57/BL mice via immunohistochemistry using anti-rabbit IgG conjugated with HRP polymer and diaminobenzidine-hydrogen peroxide. Specifically, islets were strongly stained, whereas acinar wells were not stained [101]. Interestingly, Bronowicka-Adamska et al. reported for the first time the expression and activity of all enzymes involved in H 2 S production in the pancreas and suggested the most important role of MPST in Wistar Kyoto rats [102]. More human and animal studies are needed to elucidate the role of the interplay between H 2 S synthesizing enzymes, which seems to be a species-specific system.

H 2 S and the Liver
The human liver possesses the remarkable ability to produce glucose that is released to the systemic circulation and used by other tissues, particularly during periods of fasting. Hepatic glucose production derives from glycogen breakdown (glycogenolysis) and de novo synthesis of glucose (gluconeogenesis). In the fed state, plasma glucose is derived from the ingestion of nutrients, and the liver maintains normal plasma glucose levels by promoting glycogen synthesis and inhibiting gluconeogenesis [103]. All H 2 S-producing enzymes are expressed in the liver, which plays an important role in glucose and lipid homeostasis, xenobiotic metabolism, and antioxidant defense [104][105][106][107]. The published literature regarding the role of H 2 S in regulating glucose metabolism is controversial. Compared with non-diabetic rats, H 2 S production and CSE and CBS mRNA levels in the liver were increased in STZ diabetic rats, whereas insulin treatment reversed these effects [96]. Alternatively, H 2 S formation and CSE activity and protein expression in the liver were suppressed in STZ-induced type 1 diabetic rats [108]. Livers taken from insulinsensitizer metformin-treated SJL mice (100 mg/kg b.w. per day) exhibited increased H 2 S concentrations [109].
In HepG2 hepatocytes, H 2 S has been shown to downregulate glucose uptake and glycogen storage, mediated through decreased AMP-activated protein kinase (AMPK) activation, resulting in increased activity of the gluconeogenic enzyme known as phosphoenolpyruvate carboxykinase (PEPCK), leading to hyperglycemia [110]. The same group also reported a stimulatory effect of H 2 S on liver glucose production under physiologic conditions [111]. On the other hand, Kundu et al. showed that H 2 S mitigates hyperglycemia in the liver by remodeling kinase B1-adenosine monophosphate-activated protein kinase signaling [112]. Another mechanism suggested by Guo et al. implies that CSE deficiency promoted liver gluconeogenesis in HepG2 via forkhead Box O1 (FoxO1) accumulation [113]. Other hepatoprotective effects of H 2 S donors were correlated with Nrf2 translocation and increased expression of antioxidant genes, which is regulated by kelch-like ECH-associated protein 1 (Keap1) persulfidation [114,115].
Further study will be needed to explore the regulatory mechanism of diabetes mellitus and its related conditions on the production of H 2 S in the liver.

H 2 S and Adipose Tissue
Adipose tissue plays a central role in regulating whole-body energy and glucose homeostasis through its subtle functions at both organ and systemic levels. On one hand, adipose tissue stores energy in the form of lipids and controls the lipid mobilization and distribution in the body. On the other hand, adipose tissue acts as an endocrine organ and produces numerous bioactive factors such as adipokines that communicate with other organs and modulate a range of metabolic pathways [116]. As explained, previous studies indicate that H 2 S plays an important role in adipose tissue, and all three enzymes are expressed in human adipose tissue [70].
Insulin exerts a critical control on anabolic responses in adipose tissue (AT) by stimulating glucose and free fatty acid uptake, inhibiting lipolysis and stimulating de novo fatty acid synthesis in adipocytes. In addition, insulin regulates AT growth and differentiation by enhancing the gene expression of various fat-specific transcription factors, including sterol regulatory element-binding protein 1 (SREBP-1c) and PPARγ [117]. Insulin increases glucose uptake in adipocytes by regulating the intracellular localization of glucose transporter 4 (GLUT4), the main glucose transporter involved in the insulin-regulated glucose transport from the cytosol compartment to the plasma membrane [118].
Various studies have shown that H 2 S regulates insulin sensitivity in adipocytes. In the adipose tissue of fructose induced-diabetic rats, the CTH/H 2 S system was upregulated and negatively associated with glucose uptake in AT, suggesting a pathological role of H 2 S in insulin resistance [73]. Huang et al. reported that H 2 S mediated TNF-α-stimulated insulin resistance, as the treatment of 3T3-L1 adipocytes with TNF-α lead to a deficiency in insulin-stimulated glucose consumption and uptake and an increase in endogenous H 2 S generation [119]. Chemical inhibition of CTH with PPG and β-cyano-L-alanine attenuated TNF-α-induced insulin resistance in 3T3-L1 adipocytes [119].
Other findings have suggested a beneficial role of H 2 S in glucose metabolism. Manna et al. showed a molecular mechanism in 3T3-L1 adipocytes by which H 2 S acts on 1,25-dihydroxyvitamin D3, upregulating GLUT4 protein levels and the translocation of GLUT4, essential for normal glucose metabolism [120]. Through persulfidation of PPARγ (Cys139) in 3T3-L1 cells and in the adipose tissue of HFD-mice, Cai et al. demonstrated that the CSE/H 2 S system attenuates insulin resistance and promotes the conversion of glucose into triglyceride storage through persulfidation [43]. Manna and Jain found that the administration of exogenous H 2 S using Na 2 S donors or stimulating the synthesis of H 2 S with L-cysteine increased phosphatidylinositol 3,4,5-trisphosphate (PIP3), which phosphorylated Akt and promoted glucose uptake and utilization in 3T3-L1 cells [121]. They also showed that exogenous H 2 S or L-cysteine supplementation increased insulin receptor substrate 1 (IRS1) phosphorylation and GLUT4 activation, resulting in upregulation of the metabolic actions of insulin and an improvement in glucose metabolism [121]. In another in vivo study, administration of exogenous H 2 S through NaHS or H 2 S gas solution promoted adipocytes to uptake glucose, thus reducing fasting blood glucose levels and increasing glucose tolerance [122]. H 2 S can protect adipocytes against high concentrations of glucose-induced adipocyte dysfunction, evidenced by the restored monocyte chemotactic protein (MCP)-1 and adiponectin secretion [79].
Recently Comas et al., showed that in ex vivo adipose tissue explants the stimulation of H 2 S synthesis via L-cysteine and pyridoxal 5 -phosphate (PLP) enhanced the expression of adipogenic genes in association with the insulin sensitivity, indicating a beneficial role of adipose tissue H 2 S in insulin pathway. In different human cohorts, insulin sensitivity was positively associated mainly with subcutaneous CTH and CBS, and weight loss resulted in increased CTH, CBS, and MPST mRNA, in parallel with improved insulin sensitivity [70]. In line with this, it is known that subcutaneous adipose tissue, VAT, is associated with insulin resistance, whereas SAT is associated with a decreased risk of insulin resistance [123]. In human adipocytes, exogenous administration of H 2 S using GYY4137 increased insulin action, whereas chemical inhibition with PPG attenuated insulin-induced Ser 473 Akt phosphorylation. Altogether, these findings suggest a mechanism by which H 2 S increases insulin action through the activation of PPARγ transcriptional activity in differentiated adipocytes [70]. Further study will be needed to explore the regulatory mechanism of adipose tissue H 2 S and glucose metabolism.

H 2 S and Skeletal Muscle
Skeletal muscle differentiation follows an organized sequence of events, including commitment, cell cycle withdrawal, and cell fusion to form multinucleated myotubes [124]. The skeletal muscle is the largest organ in the body by mass. It is also the regulator of glucose homeostasis, responsible for 80% of postprandial glucose uptake from the circulation. Skeletal muscle is essential for metabolism, both for its role in glucose uptake and its importance in exercise and metabolic disease [125]. Human skeletal muscles express significant amounts of CBS and CSE, whereas mouse skeletal muscles completely lack these enzymes [126]. A recent report suggested that all the three enzymes (CBS, CSE, MPST) were present in detectable levels in rat skeletal muscles [127]. Nonetheless, their expression is very low when compared to that of the liver and kidney. Only human skeletal muscles express CBS and CSE enzymes that are comparable to the expression levels in the liver in relative abundance [128].
Xue et al. provided evidence of the insulin-sensitizing effect of exogenous H 2 S administration (NaHS) in an in vitro model of myotubes (L6) and in vivo in Wistar Rats [122]. Using C2C12 mouse myotubes, exogenous H 2 S administration (NaHS) upregulated CSE expression and genes involved in GSH biosynthesis in parallel to increased H 2 S and glucose uptake and decreased ROS, whereas CSE knockdown had the opposite effects [129]. Chronic NaHS treatment (30 µmol·kg−1·day−1) in Goto-Kakizaki diabetic rats decreased fasting blood glucose, increased insulin sensitivity, and increased glucose tolerance with increased phosphorylation of PI3K and Akt in muscles [122]. Expression of CSE was declined at 20 weeks in skeletal muscle of db/db mice, compared to the control group, whereas administration of NaHS restores the expression of CSE at 20 weeks. In C2C12 myoblasts high glucose and palmitate and oleate reduced H 2 S and expression of CSE significantly [130]. It seems that H 2 S plays an important role in skeletal muscle, improving glucose homeostasis and increasing glucose uptake.

Implication of H 2 S in NAFLD
Non-alcoholic fatty liver disease (NAFLD) has become the principal cause of chronic liver disease worldwide, involving a spectrum of disturbances mainly characterized by fatty acid infiltration and fat deposition in the liver parenchyma, which can further progress to fibrosis, inflammation, and eventually develop into cirrhosis and hepatocellular carcinoma [131]. Identifying altered molecular pathways that trigger the onset and progression of the disease is a key point in ensuring early diagnoses and developing treatments.
In the liver, although the three H 2 S-generating enzymes are detectable, their roles in endogenous H 2 S generation are differently described [108,132,133]. It was found that CTH expression is about 60-fold greater than that of CBS in the murine liver [133]. Apart from endogenous hepatic synthesis, the liver is likely exposed to high levels of H 2 S exogenous sources as a consequence of its location, making the liver a key regulator of H 2 S levels by maintaining a high capacity for H 2 S clearance from the circulation [134].
Pathological processes involved with NAFLD are linked with hepatic H 2 S pathways and include lipid metabolism dysfunction, oxidative stress, insulin resistance, inflammation, and mitochondrial dysfunction. Malfunction of hepatic H 2 S metabolism is involved in the pathogenesis of many liver diseases, such as hepatic fibrosis and cirrhosis [135] (Figure 4).

Mitochondrial and Lipid Metabolism Dysfunction
Since the first studies on NAFLD, many researchers have pointed out that it is primarily characterized by the presence of mitochondrial dysfunction [136]. Mitochondria play a pivotal role in hepatocyte metabolism, being the primary site for the oxidation of fatty acids and oxidative phosphorylation [137].
The CBS/CSE system, which may be regulated by several fatty acids, has been actively investigated in the pathogenesis of NAFLD and has been proposed as a potential therapeutic target for NAFLD [138].
Impaired endogenous H2S synthesis was reported to be associated with fatty liver induced by HFD feeding [106,139] or methionine and choline-deficient (MCD) diet feed-

Mitochondrial and Lipid Metabolism Dysfunction
Since the first studies on NAFLD, many researchers have pointed out that it is primarily characterized by the presence of mitochondrial dysfunction [136]. Mitochondria play a pivotal role in hepatocyte metabolism, being the primary site for the oxidation of fatty acids and oxidative phosphorylation [137].
The CBS/CSE system, which may be regulated by several fatty acids, has been actively investigated in the pathogenesis of NAFLD and has been proposed as a potential therapeutic target for NAFLD [138].
Impaired endogenous H 2 S synthesis was reported to be associated with fatty liver induced by HFD feeding [106,139] or methionine and choline-deficient (MCD) diet feeding [104]. Treatment with H 2 S donor sodium hydrosulfide (NaHS), prevented nonalcoholic steatohepatitis (NASH) by reducing hepatic triglyceride and cholesterol levels in rodents fed with the MCD diet through increasing peroxisome proliferator-activated receptor alpha (PPARα) and reducing SREBP-1c gene expression in the liver, suggesting an antisteatogenic effect of H 2 S through the prevention of oxidative stress and inflammation [104]. In fatty livers of mice induced by HFD, administration of NaHS significantly reduced hypertriglyceridemia and improved NAFLD by activating liver autophagy flux and the AMPK-mTOR signaling pathway [139]. However, a recent study suggested that hepatic MPST promoted hepatic steatosis in HFD fed mice and the knockdown of MPST-stimulated H 2 S production, whereas overexpression of MPST markedly reduced the formation of H 2 S via inhibition of CSE/H 2 S and subsequent upregulation of SREBP-1c, c-Jun N-terminal kinase phosphorylation, and oxidative stress [138]. In addition, that study also demonstrated that the inhibition of MPST in L02 cells reduced free fatty acids (FFAs) and increased the expression of CTH and H 2 S [138].

Oxidative Stress
Oxidative stress is an important pathophysiological mechanism in NAFLD pathogenesis. Growing evidence supports a key role for oxidative stress caused by the generation of ROS in the progression of NAFLD [141]. Disturbances in lipid metabolism lead to hepatic lipid accumulation, which affects various reactive oxygen species (ROS) generators, including mitochondria, the endoplasmic reticulum, and NADPH oxidase [142].
Oxidative stress involves molecular or cellular damage, resulting from deficiency of antioxidants and/or antioxidant enzyme systems, and disrupting the cellular reductionoxidation balance [143]. The human body is equipped with a variety of antioxidants that serve to counterbalance the effect of oxidants, with H 2 S being one of the most important. In fact, H 2 S protects cells in various diseases by acting as an antioxidant that reduces excessive amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS) [144]. ROS are highly reactive molecules and can damage cell structures such as carbohydrates, nucleic acids, lipids, and proteins, and alter their functions [145].
Several studies have highlighted the role of H 2 S in cellular redox homeostasis, which can be summarized in two main mechanisms: (i) modulating levels and activity of classic cellular antioxidants, such as glutathione (GSH) and thioredoxin (TRX) [146], and (ii) increasing the activity or expression of the transcription nuclear factor (erythroid-derived 2)-like 2 (NRF2) and the histone deacetylase protein family of sirtuins (SIRTs), which in turn increase the expression of antioxidant enzymes (AOE) [114,115].

H 2 S on Cardiovascular Disease
The epidemiological tendency in the 20th century was accompanied by an increase in noncommunicable diseases, of which cardiovascular diseases (CVDs) are now the leading cause of mortality and morbidity worldwide [147]. CVDs are a cluster of diseases and injuries that affect the cardiovascular system, including the heart and blood vessels. Multiple factors are contributing to the epidemic of CVDs, including the rapid aging of the popu-lation, improved survival rate from other illnesses, progressive urbanization, increased calorie consumption, decreased physical activity, mental stress, and air pollution [148][149][150].
The main condition underlying CVD is atherosclerosis, a chronic inflammatory condition that involves different cell types, and several cytokines and adhesion molecules.
Atherosclerosis is a chronic and slowly progressive cardiovascular disease that affects arterial blood vessels by thickening and hardening as consequences of high plasma cholesterol concentrations, especially cholesterol in the form of low-density lipoprotein (LDL) [151]. In these processes, reactive oxygen species play a pivotal role, as they can cause the oxidation of lipids such as low-density lipoprotein (LDL) and polyunsaturated fatty acids that are deposited in the vascular wall, allowing plaque calcification, directly damaging cellular components, and further promoting inflammation by activating several pro-atherogenic transcriptional factors [152]. The accumulation of plaques consequentially narrows the arterial lumen and restricts the blood supply, causing atherosclerotic lesions, which through the action of several cytokines can rupture and lead to occlusion of the vascular lumen. Depending on the area of rupture, these can manifest as acute myocardial infractions or stroke or acute ischemia of any nearby organ [153].
Over the last few years, our understanding of atherosclerotic processes has vastly improved; however, there are still many mechanisms that have not been fully elucidated. One of the first reported physiological roles of H 2 S was its capacity to induce vasorelaxation, acting as a K(ATP) channel opener, and displaying important antihypertensive effects and cardioprotection properties [11,[154][155][156]. In line with this, there is a growing body of evidence linking H 2 S with cardioprotection, including decreasing heart rate, exerting inotropic and proangiogenic effects, decreasing blood pressure, and causing vasodilation [157]. Reduced levels of H 2 S have been found in patients with acute or stable coronary artery disease [158], hypertension [159], and heart failure [160]. The impact of H 2 S on cardiovascular disease has been extensively updated and reviewed in recent studies [161][162][163][164][165].

Hypertension
In 2003, Zhang et al. demonstrated that H 2 S could exert beneficial effects on the pathogenesis of hypoxic pulmonary hypertension in rats [166]. Later, other groups using hypertensive experimental animal models showed that in rats which had spontaneous hypertension (SHR), the level of hydrogen sulfide and the gene expression and activity of CSE were reduced [167]. In SHR rats, reduced arterial pressure after systemic treatment with NaHS or after intracerebroventricular NaHS treatment were also reported [168].
CSE levels are reduced in the vessel wall of spontaneously hypertensive rats and both CSE and CBS are reduced in the resistance vessels of rats rendered hypertensive after dexamethasone treatment [167,169,170]. In addition, animals with salt-sensitive hypertension have lower levels of CBS [171]. A causal link between low CSE levels and high blood pressure was established following the observation that CSE KO mice exhibit hypertension [172].
Moreover, administration of sulfide salts or the slowly releasing H 2 S donor GYY4137 reduced blood pressure in a genetic model of hypertension, as well as in rats rendered hypertensive by angiotensin-II or L-NAME administration [173][174][175].
Other naturally occurring H 2 S donors such as garlic-derived compounds or isothiocyanates present in vegetables also demonstrated cardioprotective effects [176]. Allicin, a garlic-derived H 2 S donor, is known for lowering arterial blood pressure, among other cardiovascular protection systems [177]. Isothiocyanates, which are abundant in cruciferae like mustard and broccoli, are also considered important in the cardiovascular system. In the rat aortic ring, H 2 S derived from isothiocyanates was responsible for observed vasorelaxant effects [178]. Other synthetic H 2 S donors like thioamino acids, including thioglycine and thiovaline, were found to induce significant relaxation of the pre-contracted mouse aortic ring [179].
A great number of studies have been carried on the investigation of the modulation of blood pressure by means of exogenous and endogenous H 2 S. However, in humans, few studies have measured plasma H 2 S levels in association with hypertension. In a small cohort of patients with type 2 diabetes, lower plasma levels of H 2 S were associated with higher systolic blood pressure (SBP) and diastolic blood pressure (DBP) [41]. Reduced H 2 S plasma levels have been confirmed in a human cohort of hypertensive patients [180,181].

Atherosclerosis
There are strong indications that the loss of H 2 S contributes to the establishment and progression of the disease of atherosclerosis. Hydrogen sulfide shows an anti-atherosclerotic action by attenuating oxidative stress, reducing blood platelet activation, reducing the inflammation process, and preventing the proliferation of vascular smooth muscle cells [182][183][184].
Atherosclerosis is a lipoprotein-driven disease that leads to plaque formation at specific sites of the arterial tree through intimal inflammation, necrosis, fibrosis, and calcification [185]. H 2 S is known to reduce endothelial dysfunction by preventing plaque formation by counteracting the main aspects of atherosclerosis, such as oxidation, adhesion, proliferation, and calcification [165].
Increased oxidative stress can accelerate the development of atherosclerosis by means of endothelial cell dysfunction, adhesion of molecules, vascular smooth muscle proliferation and migration, platelet activation, lipid oxidation, matrix metalloproteinase activation, and alterations in vasomotor activity [186]. Several studies have highlighted the role of H 2 S in cellular redox homeostasis, which can be summarized in two main mechanisms: (i) modulating the levels and activity of classic cellular antioxidants, such as glutathione (GSH) and thioredoxin (TRX), and (ii) increasing the activity or expression of transcription nuclear factor (erythroid-derived 2)-like 2 (NRF2) and the histone deacetylase protein family of sirtuins (SIRTs), which in turn increase the expression of antioxidant enzymes. H 2 S can ameliorate vascular calcification by decreasing the activation of alkaline phosphatase and reducing the gene expression of osteopontin [187].
Plasma H 2 S and aortic H 2 S levels were decreased in apolipoprotein E knockout (ApoE(−/−)) mice. CSE expression was reduced in oxidized LDL (Ox-LDL)-stimulated human aortic endothelial cells (HAEC) and in the aorta of high-fat-diet-induced ApoE(−/−) mice [188]. The expression of CSE is mainly upregulated in macrophages, foam cells, and myofibroblasts from the atherosclerotic lesions of human patients with carotid specimens. In mouse and human atherosclerosis, CSE expression is upregulated, but circulating and plasma levels of H 2 S are reduced, a phenomenon that can be attributed to the inhibition of CSE enzyme activity [189].
The main therapeutic strategy to lower or reverse atherosclerosis is statins, but the clinical benefits of statins are somewhat limited [190,191]. Surprisingly, recent reports have mentioned that treatment using statins can improve H 2 S production, but it is the lipophilic atorvastatin, rather than the hydrophilic pravastatin, that increases the net H 2 S production [192][193][194][195][196].

Conclusions
This review summarizes and discusses the literature concerning the roles and mechanisms of H 2 S on obesity-associated metabolic disturbances, including insulin resistance, NAFLD, and cardiovascular diseases. Impaired H 2 S metabolism is involved in obesity and adipose tissue disturbances; however, scientific understandings of the role of H 2 S and the mechanisms by which it is altered in obesity remain somewhat contradictory. Significant progress has been made in this field during recent years, but more research is warranted to address this discrepancy in the future. The majority of studies suggest that the deficiency of endogenous H 2 S synthesis is associated with NAFLD, due to altered pathways that include lipid metabolism dysfunction, oxidative stress, insulin resistance, inflammation, and mitochondrial dysfunction. However, the mechanisms involved are complex and are still incompletely understood. Based on the evidence, it is clear that H 2 S plays a protective role in cardiovascular diseases, including atherosclerosis. However, there are still many controversies surrounding the signaling pathways, beneficial roles, and harmful effects of H 2 S in cardiovascular diseases. H 2 S therapy has only entered a preliminary stage in terms of basic medical research or preclinical research. Nevertheless, based on the existing knowledge about the beneficial effects of H 2 S, the future of H 2 S as a potential therapy against obesity-associated metabolic disturbances is expected to be promising and exciting. Funding: This work was partially supported by research grants PI19/01712 from the Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (FEDER) from Spain and VII Spanish Diabetes Association grants to Basic Diabetes Research Projects led by young researchers. CIBEROBN Fisiopatología de la Obesidad y Nutrición is an initiative from the Instituto de Salud Carlos III.

Conflicts of Interest:
The authors declare no conflict of interest.