Regulation of carbohydrate metabolism by nitric oxide and hydrogen sulfide: Implications in diabetes

Abstract

Nitric oxide (NO) and hydrogen sulfide (H₂S) are two gasotransmitters that are produced in the human body and have a key role in many of the physiological activities of the various organ systems. Decreased NO bioavailability and deficiency of H₂S are involved in the pathophysiology of type 2 diabetes and its complications. Restoration of NO levels have favorable metabolic effects in diabetes. The role of H₂S in pathophysiology of diabetes is however controversial; H₂S production is decreased during development of obesity, diabetes, and 8 its complications, suggesting the potential therapeutic effects of H₂S. On the other hand, 9 increased H₂S levels disturb the pancreatic β-cell function and decrease insulin secretion. In addition, there appear to be important interactions between NO and H₂S at the levels of both biosynthesis and signaling pathways, yet clear an insight into this relationship is lacking. H₂S potentiates the effects of NO in the cardiovascular system as well as NO release from its storage pools. Likewise, NO increases the activity and the expression of H₂S-generating enzymes. Inhibition of NO production leads to elimination/attenuation of the cardioprotective effects of H₂S. Regarding the increasing interest in the therapeutic applications of NO or H₂S-releasing molecules in a variety of diseases, particularly in the cardiovascular disorders, much is to be learned about their function in glucose/insulin metabolism, especially in diabetes. The aim of this 18 review is to provide a better understanding of the individual and the interactive roles of NO and 19 H₂S in carbohydrate metabolism.

Graphical Abstract

1. Introduction

Nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H₂S) collectively are known as gasotransmitters [1]. Although all three are considered to be toxic gases, they are nevertheless synthesized in the human body and play a key role in many of the physiological activities of the various organ systems [1–7]. Although the origins of NO go back to the late eighteenth century, its biological role was recognized in 1980 [8] and its vasorelaxatory effects were established a few years later in 1987 [9]. In comparison, H₂S is relatively a new player on the scene and the gradual discovery of H₂S-producing enzymes has shed more light on its physiological functions 15 and cellular signaling.

Decreased NO bioavailability and deficiency of H₂S are considered to be involved in pathophysiology of many disease such as type 2 diabetes [10–12]. Restoration of NO levels has been associated with many favorable metabolic effects in type 2 diabetes [10, 13, 14]. The role of H₂S in pathophysiology of diabetes is however controversial, as both inhibition and stimulation of the H₂S system have been suggested to be potential therapeutic approaches [15, 16].

There are many similarities between the biological characteristics of NO and H₂S in terms of their biosynthesis, biological targets, effects, metabolism, and elimination. In addition, these molecules regulate many physiological functions with some cross talk between the enzymes that are involved in their generation and also the pathways that these two gasotransmitters affect (reviewed in [17, 18]). For example, both NO and H₂S are well known antioxidants and recently it was demonstrated that H₂S at low doses potentiates the anti-oxidative effects of NO in diabetic rats [19]. Cross talk between H₂S and NO was initially reported in 1997 by Hosoki et al. who showed that H₂S at a concentration that did not produce any appreciable vascular relaxation, it potentiated the vasorelaxatory effects of NO [20]. While the individual physiological functions of H₂S and NO as well as their potential relationship in many organ systems are extensively studied, our understanding about their potential roles in regulation of carbohydrate metabolism in particular the role of H₂S is woefully incomplete. This review discusses the effects of NO and 10 H₂S and also their interactions in carbohydrate metabolism.

2. Nitric oxide synthesis

NO is produced in all tissues [21–24] by NO synthase (NOS)-dependent and independent pathways (Figure 1) [25, 26]. In mice, total NO formation has been reported to be about 0.2 mmol/kg/day of which approximately 70% is derived from endothelial NOS (eNOS, NOS-3) [10]. In Wistar rats and humans, the rate of NO production is about 0.33–0.85μmol/kg/h and 0.9 μmol/kg/h, respectively [27–30].

Figure 1. Hydrogen sulfide and nitric oxide biosynthetic pathways.

Hydrogen sulfide (H₂S) and nitric oxide (NO) are produced by enzymatic and non-enzymatic pathways. Non-enzymatic production of H₂S is mediated through reducing elemental sulfur or organic polysulfides. Enzymatic production of H₂S is mediated by cystathionine γ-lyase (CSE), cystathionine-beta synthase (CBS), and 3-mercaptopyruvate sulfuretransferase (3-MST). NO is produced by nitrate/nitrite pathway which can be enzymatic or non-enzymatic. NO is also produced from L8 arginine by neuronal NO synthase (nNOS), inducible NO synthase (iNOS), and endothelial NO 9 synthase (eNOS).

2.1. NOS-dependent NO synthesis

In NOS-dependent pathway, NO is produced from L-arginine (the L-arginine-NO pathway) by the three isoforms of NOS namely, neuronal (nNOS/NOS-1), inducible (iNOS/NOS-2), and eNOS/NOS-3 which are heme-containing dioxygenases enzymes [31–34]. These isoforms are active as homodimers [35–38] and proper dimerization is critical for their activity [39]; L-arginine, tetrahydrobiopterin (BH₄), and heme are essential for stabilizing the active dimeric form of all NOS isoforms [38]. NOS monomers cannot catalyze NO formation and have a limited capacity to produce superoxide anions [37]. These isoforms vary in amino acid sequence, cellular location, function, and post-translational modifications [35, 40]. eNOS which produces relatively low quantities of NO, is mostly expressed in the vascular endothelium, but has also been found in epithelial cells, neurons, and cardiomyocytes as well as in hepatocytes and adipocytes [41, 42]. nNOS is expressed to highest relative abundance in neurons, skeletal muscle, and epithelial cells [43, 44]. eNOS and nNOS are firmly regulated through phosphorylation, compartmentalization in caveolae, calcium/calmodulin, and interact with plasma membrane ionotropic receptors [45]. iNOS is primarily identified in macrophages but its expression can be stimulated in virtually any cells or tissues and can produce large amounts of NO for long periods of time provided the necessary substrate is available [45, 46]. Some studies have also suggested a mitochondria-localized NOS isoform [47]; however, the specific contribution of this isoform remains unclear.

All isoforms of NOS bind calmodulin as a prosthetic group; increased in intracellular Ca²⁺ levels are necessary for binding of calmodulin to eNOS and nNOS (half-maximal activity between 200–400 nM) while due to a different amino acid structure of the calmodulin-binding site in iNOS, in this isoform calmodulin is constitutively active at extremely low intracellular Ca²⁺ concentrations (<40 nM) [35, 37]. Overproduction of NO by iNOS and also exogenous NO could lead to inhibition of eNOS and nNOS [32, 48]. Of note, the NO concentration necessary for the inhibition of eNOS and nNOS is considerably lower than that required for iNOS inhibition [48]. NO could be protective or toxic depending on its concentration, source, location, and environment [49].

For NO production, NOS isoforms catalyze oxidation of L-arginine to NO and L-citrulline [23, 50, 51]. Reduced nicotinamide-adenine-dinucleotide phosphate (NADPH) and molecular oxygen are co-substrates [36, 52] and flavin mononucleotide, flavin adenine dinucleotide, and BH₄ are cofactors of NOS [36, 52], of which, BH₄ is critical and rate-limiting [23]. Exposure to oxidative stress such as seen in diabetes, results in the conversion of BH₄ to 7,8- dihydrobiopterin (BH₂) and thus leads to a dysfunctional eNOS, as BH₂ is inactive as a cofactor and competes with BH₄ for BH₄ binding [53]. Anti-oxidants like vitamin C and folate increase BH₄ bioavailability and could affect NO formation [23].

2.2. NOS-independent NO synthesis

NOS-independent NO production from nitrate and nitrite was initially reported in 1994 in the stomach following protonation of swallowed salivary nitrite [54, 55]. Decreased NOS-derived NO production in tissues makes the nitrate-nitrite-NO pathway important [22]. Oxidation of NOS-derived NO as well as direct exposure through the diet are two major sources of nitrate in mammals [56]. Total body pool of nitrate is about 0.53 and 0.47 mM in men and women, respectively [57]; the relative contribution of the dietary source and NOS-derived NO to the total body pool of nitrate varies, but with a moderately high intake of green leafy vegetables (~200 g per day), the dietary source clearly dominates [58].

About 25% (20–28%) of circulating nitrate (from diet or endogenous NO) is actively taken up by the salivary glands and concentrated by a factor of 10 in about 5 hours [59–63]; nitrate is reduced to the more reactive nitrite anion (NO₂⁻) by the oral commensal bacteria with potent nitrate reductase enzymes [64]. For this reason, the salivary nitrate and nitrite concentrations are normally 10–100 and 1000-fold higher than their plasma levels, respectively [65, 66]. After oral loading, nitrate/nitrite is rapidly absorbed in the duodenum and jejunum [10, 67]. In the stomach, part of this nitrite is reduced to NO but most of it is absorbed to the circulation [10, 60, 68]. Nitrite reduction to NO in blood and tissues could be enzymatic or non-enzymatic [64, 69–71], which is generally enhanced during hypoxic, ischemic, and acidic conditions [32, 46, 50, 64, 68–71].

3. Nitric oxide signaling pathways

Actions of NO could be cyclic guanosine monophosphate (cGMP)-dependent and/or cGMP independent (mostly reactive nitrogen species-mediated) [72]. cGMP-dependent signaling pathway is the most important physiologic signaling pathway activated by NO [26, 65, 73–75]. In this pathway, only low concentrations of NO (5–10 nM) are required to activate guanylyl cyclase (GC) [36, 74, 76], which converts guanosine triphosphate to cGMP [36]. GC has two isoforms: soluble (sGC is cytosolic) and membrane (particulate), of which, sGC is the receptor for NO [36]. sGC is a heterodimer of α and β subunits which contains a ferrous heme prosthetic group on histidine 105 residue of the β subunit [36]. When NO binds to the ferrous heme iron, it causes disruption of histidine 105 and inhibition of the catalytic activity of sGC by the heme is overcomed. The catalytic domain near the C-terminal of the α and β subunits is then activated resulting in an increase in V_max and a decrease in K_m of the enzyme [36]. Elevated cGMP levels activate protein kinase G (PKG), which is a serine/threonine kinase and exists as a homodimer [35]. PKG has two isoforms: PKGI and PKGII [77]; PKGI is the common isoform involved in NO/cGMP/PKG signaling pathway [78], which exerts its vasodilatory effects in the vascular smooth muscle cells through decreased [Ca²⁺]_i [79] and Ca²⁺ sensitivity [80].

In addition to NO/cGMP/PKG signaling pathway, nitrosative post-translational modifications such as S-nitrosylation, which is a reversible covalent attachment of NO to cysteine residues of proteins, is a key mechanism for NO signaling [35, 73, 81]. S-nitrosylation activates or inhibits protein function and therefore can be beneficial or detrimental [82, 83]. Despite the presence of cysteine residues on almost all proteins and production of NO by most cells, only some proteins are nitrosylated [84]. Specificity in S-nitrosylation depends on the presence of metal ions (Mg²⁺ or Ca²⁺), local pH, and the acid-base motifs [85].

4. Hydrogen sulfide synthesis

H₂S is a colorless gas produced by enzymatic and non-enzymatic pathways in the body (Figure 1) [86]. Non-enzymatic production of H₂S is responsible for a limited amount of H₂S in mammalian cells [87] and is mediated through reducing elemental sulfur or organic polysulfides via glucose-supported and thiol-dependent as well as glutathione-dependent cellular reactions [88–90]. Enzymatic production of H₂S is organ-specific and has been attributed to two pyridoxal 5′-phosphate (PLP) dependent enzymes, cystathionine β synthase (CBS, EC 4.2.1.22) and cystathionine γ lyase (CSE, EC 4.4.1.1), as well as a non-PLP dependent enzyme, 3-mercaptopyruvate sulfurtransferase (3-MST, EC 2.8.1.2) along with cysteine aminotransferase (CAT, EC 2.6.1.3) [2, 86, 91]. CBS and CSE are heme-proteins being in the cytosol, while 3-MST is a zinc-dependent protein, localized in the cytosol and in particular in the mitochondrial matrix, with an optimal pH of ~8 [86, 92].

L-cysteine, which is the major source of H₂S production, is desulfhydrated during the transsulfuration pathway by all H₂S-producing enzymes [2, 91]. H₂S can also be produced from homocysteine; in normal conditions, about 70% of H₂S is produced from L-cysteine and 30% from homocysteine [93]. An additional biosynthetic pathway for H₂S production has also been reported from D-cysteine involving 3-MST and D-amino acid oxidase [94]. This pathway operates predominantly in the cerebellum and the kidneys. In the cerebellum it protects the cerebellar neurons from oxidative stress and in the kidneys, it attenuates ischemia-reperfusion injury more effectively than L-cysteine [94].

Tissue production of H₂S and in particular the liver contributes to plasma level of H₂S [95]. Using a polarographic H₂S sensor, rate of H₂S production in rat liver, brain, aorta, and heart is 12.3 ± 4.6, 10.6 ± 3.2, 5.8 ± 1.7, and 1.1 ± 0.3 pmol/s/mg protein, respectively [96]. Concentration of H₂S in the plasma and tissue is under 1 μM, however using the methylene blue method and S²⁻ ion selective electrodes, which are the most commonly employed methods, artificially inflated H₂S concentrations and values from 20 to 300 μM are still being reported as “physiological” [97]. H₂S at high concentrations (> 700 ppm or ~ 20,000 μM) is lethal to both 10 humans and animals [98].

H₂S is a weak acid and equilibrates with hydrosulfide anions (HS⁻, pKa 7.04 and S²⁻, pKa 11.96) in aqueous solution; with a pKa1 value of 6.76 and pKa2 >12 at 37 °C [17, 99], 18.5% of H₂S remains undissociated at physiologic pH=7.40 [99]. H₂S dissolves well in lipids and its solubility is 5-fold higher than in water and therefore can freely penetrate cells [99]; while HS⁻ does not pass. Much of the inactivation of H₂S occurs through mitochondrial oxidation during three consecutive reactions; two membrane-bound sulfide: quinone oxidoreductases and a sulfur dioxygenase are involved in oxidation of sulfide to thiosulfate [100].

5. H₂S signaling pathways

It is believed that most of H₂S signaling is done through S-sulfhydration of target proteins, a reaction which transfers a sulfhydryl group (-SH) to a cysteine residue of a protein to form a hydropersulfid moiety (-SSH) [101, 102] or persulfide group [103]. S-sulfhydration or more correctly sulfuration of a protein modifies its functions, stability, and localization within the cells [104]. S-sulfhydration also contributes to modification of inflammation, endoplasmic reticulum (ER) stress signaling and vascular tone [104]. Regulation of ion channels via S-sulfhydration has been reported in several studies; S-sulfhydration of adenosine triphosphate (ATP)-dependent K⁺ channels (K_ATP) activates it by decreasing ATP binding and increasing phosphatidylinositol 4, 5-bisphosphate (PIP₂) binding; S-sulfhydration also activates small and intermediate calcium-activated potassium channels (SK_Ca and IK_Ca) in vascular endothelial cells [95]. In addition, S-sulfhydration of inositol-3-phosphate receptor (IP₃R) inhibits Ca²⁺ release from the ER [103].

6. Role of NO in carbohydrate metabolism

6.1. Insulin secretion and NO

Glucose enters the pancreatic β-cells through a low affinity glucose transporter (GLUT-2). Glucose is phosphorylated by glucokinase and pyruvate is generated through glycolysis in the cytoplasm; pyruvate is then metabolized by pyruvate carboxylase and pyruvate dehydrogenase and passes into the mitochondria where it increases the cytoplasmic ATP/adenosine diphosphate (ADP) ratio; increased ATP/ADP ratio causes closure of K_ATP channels [105, 106]. In the β-cells, K_ATP channels are the primary determinant of the membrane potential, so closure of these channels causes membrane depolarization and the subsequent activation of L-type voltage-dependent Ca²⁺ channels (VDCC). Elevation of cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) is followed by insulin vesicle exocytosis [105], which is mediated by SNAp REceptors (SNARE), located in both vesicle (v-SNAREs) and target (t-SNAREs) membrane. Syntaxin and synaptosome associated protein-25 (SNAP-25) families are known as t-SNARE and v-SNAREs including the vesicle-associated membrane proteins (VAMPs) [107].

Expression of all three isoforms of NOS has been reported in the pancreatic β-cells [56, 106, 108–110]. The role of NO in insulin secretion is controversial; iNOS-derived NO decreases, while eNOS-derived NO increases insulin secretion [32, 111]. In addition, NO stimulates the activity of the insulin gene promoter, with comparable increases in endogenous insulin mRNA levels in both Min6 β-cells (a pancreatic β-cell line derived from transgenic mouse expressing the large T-antigen of SV40 [112]) and isolated rat islets of Langerhans [113]. Both glucose and insulin increase NO production in β-cells [114]; glucose-induced NO production at physiological concentrations increases insulin secretion, however, higher NO levels inhibit insulin secretion [114]. By contrast, it has been reported that both eNOS- and iNOS-activity are greatly increased by high glucose concentrations (20 mM) in intact islets from freely fed mice; fasting induces islet iNOS activity at both physiological (7 mM) and high (20 mM) glucose concentrations; inhibition of NOS by L-NAME (L-N^G-Nitroarginine methyl ester) increases insulin secretion both during freely fed conditions and after fasting [115].

Possible mechanisms for increased insulin secretion by NO include: a) mitochondrial depolarization, which induces calcium release from the mitochondria and therefore increases insulin secretion [32], b) increase in islet blood flow, which supplies oxygen and nutrients to the islets [111], c) increased formation of mitochondrial reactive oxygen species (ROS), which is an obligatory signal for glucose-induced insulin secretion [111], and d) S-nitrosylation of glucokinase (at cysteine-371) or syntaxin 4 (at cysteine-141), which facilitates glucose stimulated insulin secretion (Figure 2) [116]. Besides effects on insulin secretion, NO also inhibits an insulin-degrading enzyme (IDE) [117], a ubiquitously expressed cytosolic protease, by S-nitrosylation [116]. Inhibition of NOS causes glucose intolerance by doubling degradation of the secreted insulin and a 40% drop in β-cell glucose sensitivity in non-diabetic subjects [114, 116], suggesting that NO reduces the burden of β-cells via inhibiting circulating insulin clearance [114]. Interleukin 1 beta (IL-1β) has been shown to be a contributing factor in β-cell dysfunction and decreased insulin secretion [118], nitrite-mediated NO decreased elevated IL-1β levels and increased insulin secretion in diabetic rats [13], indicating the anti-inflammatory effects of NO in increased insulin secretion.

Figure 2. Mechanisms of nitric oxide-stimulated insulin secretion in pancreatic β-cell.

Glucose enters the pancreatic β-cells through glucose transporter type 2 (GLUT-2) (1). Glucose is phosphorylated by glucokinase (2) and increases cytoplasmic adenosine triphosphate (ATP)/adenosine diphosphate (ADP) ratio (3); increased ATP/ADP ratio closes (ATP)15 dependent K⁺ (K_ATP) channels (4) and causes membrane depolarization (5) and the subsequent activation of L-type voltage-dependent Ca²⁺ channels (VDCC) (6). Elevation of cytosolic free Ca²⁺ concentration is followed by activation of synaptotagmin as a calcium sensor (7) and then exocytosis of insulin granules into the circulation (8).

Nitric oxide (NO) causes mitochondrial depolarization, which induces calcium release from mitochondria. NO also facilitates glucose-stimulated insulin secretion by S-nitrosylation of glucokinase or syntaxin 4.

6.2. Insulin signaling and NO

Insulin activates the insulin receptor (IR), which belongs to a tyrosine kinase family of transmembrane signaling proteins containing two extracellular α-subunits and two transmembrane β-subunits [118, 119]. When insulin binds to the α-subunits, this results results in tyrosine autophosphorylation of the β subunits [119]. Transphosphorylation among β-subunits leads to further kinase activity and recruitment and phosphorylation of receptor substrates including insulin receptor substrates (IRSs), SH2 (Src-homology2)-containing proteins (Shc), and Grb2 (Growth factor receptor binding protein 2)-associated binder (GAB) [119]. Phosphorylated IRSs provide docking sites for intracellular molecules containing SH2 domains including type 1A phosphatydylinositol (3, 4, 5)-triphosphate kinase (PI3K), which phosphorylates PIP₂ to generate PI (3, 4, 5)-triphosphate (PIP3) [120]. PIP3 is an allosteric regulator of phosphoinositide-dependent kinase (PDK), which in turn leads to phosphorylation and activation of Akt (PKB), a serine/threonine protein kinase [120, 121]. Mitogen-activated protein kinase (MAPK) pathway and PI3K-Akt pathway are two major signaling pathways for insulin actions [122]. Most of insulin’s metabolic actions are mediated through the PI3K-Akt 19 pathway [119].

In endothelial cells, insulin increases eNOS activity through the PI3K-Akt pathway (PI3K-Akt-eNOS pathway) [37, 38, 121, 123] and provides an important step in regulating eNOS activity and glucose uptake [39]. Insulin by enhancing the synthesis and therefore availability of NADPH and BH₄ in endothelial cells increases NO production [38]. In the PI3K-Akt-eNOS pathway, eNOS is activated by phosphorylation at serine 1177 [39, 123], which is not phosphorylated in the resting endothelial cells [37].

6.3. NO and glucose metabolism in skeletal muscle

nNOS is the main isoform of NOS in skeletal muscle and is called nNOSμ [43, 44]; eNOS is expressed at low levels and is mainly associated with the vascular endothelium [44]; although there is essentially no expression of iNOS in healthy skeletal muscle [43], it can however be induced in response to inflammatory cytokines [124]. All isoforms may transcriptionally be regulated by hypoxia; expressions of both vascular and skeletal muscle eNOS are increased by chronic exercise [125, 126]; eNOS in blood vessels is also increased by sheer stress [127]. Expression of nNOS is upregulated following muscle activity [125], crush injury [128], and 11 ageing [129], while it is downregulated following denervation [130].

NOS activity is regulated developmentally; total activity in diaphragm homogenates has been reported to be 40–45 pmol/min/mg protein from embryonic day 18 to postnatal day 1; the activity is decreased to ~25 pmol/min/mg protein by day 7 and then to the adult rate by day 30 [131]. Rat diaphragm produces ~3–5 pmol NO/min/mg muscle during passive incubation and is increased approximately six-fold in actively contracting muscle in a tissue bath [132, 133]. Resting cordofemoralis and quadratus femoralis muscles produces ~1 pmol/min/mg muscle and this is increased two-three-fold by electrical stimulation in vitro [134]. Resting mouse diaphragm and soleus muscles produce ~13 pmol NO/min/mg and there is no difference between muscle types or between wild-type and eNOS-deficient mice [135].

Effect of NO on glucose uptake was first recognized when NOS inhibition was shown to attenuate 2-deoxyglucose uptake by rat limb muscle [134] both under basal conditions and during repetitive contractions in vitro [125]. These studies further demonstrated that exogenous NO was associated with an increase in 2-deoxyglucose uptake which was additive with the increase in uptake stimulated by insulin. Local infusion of the NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), into the femoral artery attenuates elevated skeletal muscle glucose uptake during moderate cycling exercise in healthy and type 2 diabetes participants and this effect occurs independently of blood flow [136]. In addition, NO stimulates rate of lactate release and glucose oxidation in isolated rat skeletal muscle [137], which is responsible for approximately 80% of insulin-stimulated glucose uptake [138].

The mechanism(s) by which NO stimulate glucose metabolism in skeletal muscle are not well characterized, however it has been demonstrated that NO decreases insulin resistance by increasing mRNA expression, protein levels, and translocation of GLUT4 in skeletal muscle of type 2 diabetic rats [13, 14, 121]. sGC/cGMP/PKG pathway as well as various post-translational protein modifications are involved in the NO-mediated intramuscular GLUT4 translocation [137]. In resting muscle, using various NO donors, the cGMP analogue (8-bromo-cGMP), and phosphodiesterase 5 (PDE5) inhibitor (zaprinast), which prevents degradation of cGMP, increases cGMP level with a parallel increase in muscle glucose uptake [137, 139], whereas inhibiting sGC, decreases cGMP level and NO-induced glucose uptake [137]. In contrast to basal muscle glucose uptake, inhibition of sGC and PKG during ex vivo contraction have no effect on muscle glucose uptake [140], indicating that a cGMP/PKG-independent mechanism may be involved in NO-induced glucose uptake during contraction.

6.4. NO and glucose metabolism in adipose tissue

The expression of iNOS and eNOS have been reported in white adipose tissue [41]. eNOS is mostly membrane bound, while iNOS is found in the cytoplasm of adipocytes and macrophages. Although protein expression of nNOS has been reported in the cytoplasm and the mitochondria of adipocytes [141, 142], its expression does not appear to be present in significant amounts [143]. In addition, nitrate-mediated NO production is increased in response to hypoxia in rats and primary adipocytes [69].

In adipose tissue, NO stimulates insulin-dependent [144] and insulin-independent [41, 142] uptake and oxidation of glucose. NO-released from S-Nitrosoglutathione and S-Nitroso-N acetylpenicillamine (GSNO and SNAP) at low doses (<1 mM), increases while at high doses (10 and 20 mM) inhibits insulin-stimulated glucose uptake in isolated adipocytes of normoglycemic and streptozotocin (STZ)-induced diabetic rats [145]. Increased NO production also up-regulates brown adipocyte-associated genes [69], which can be a physiological adaptation of adipocytes undergoing hypertrophy in obesity.

In adipose tissue, the reaction between eNOS-mediated NO and sGC occurs at nM concentrations of NO [146, 147]. In addition, nitrite-mediated NO production by the nitrite reductase activity of xanthine oxidase (XO) also acts through the sGC/cGMP-signaling pathway [69]. NO scavenger (CPTIO) and GC inhibitor (LY83583) reduce SNP-stimulated glucose uptake to the basal level, suggesting that SNP-stimulated glucose uptake is mediated by NO and GC [41]. sGC/cGMP-dependent stimulation of AMP-activated protein kinase (AMPK) increases gene expression and PKG-dependent AMPK phosphorylation and therefor increases glucose uptake in adipose tissue [142]. The anti-inflammatory effects of NO may have an important role in decreasing insulin resistance, since in adipose tissue of diabetic rats; nitrite-derived NO in a dose-dependent manner reduced both mRNA levels of TNF-α and adipocyte size [149].

6.5. NO and glucose metabolism in hepatocytes

Glucose uptake by the liver is not affected by insulin directly and is accomplished passively via glucose transporters of which GLUT2 is the main hepatic glucose transporter [148]. The physiological control of glucose uptake across the plasma membrane of a hepatocyte depends on the intracellular glucose phosphorylation/dephosphorylation balance; however, insulin by stimulating the activity of glucokinase, it indirectly promotes hepatic glucose uptake (reviewed in [149]). Within the cells, hexokinase isoenzymes phosphorylate free glucose to glucose 6-phosphate which may follow three metabolic pathways: a) isomerization into glucose 1-phosphate, that is transformed into UDP–glucose (precursor of glycogen), UDP–glucuronate and UDP–galactose; b) isomerization into fructose 6-phosphate, which may either start the hexosamine pathway by combining with glutamine or continue into the glycolytic pathway to form pyruvate and then acetyl-CoA; c) oxidation into gluconolactone and start the pentose phosphate pathway [150].

In the liver, iNOS is expressed primarily in the cytoplasm of periportal hepatocytes and eNOS is expressed in hepatocytes, hepatic sinusoidal, endothelium of hepatic arteries, terminal hepatic venules, and epithelium of biliary ducts [42]. In the liver, NO induces mitochondrial biogenesis through an increase in cGMP levels and activation of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) [151], decreases gluconeogenesis through decreasing mRNA expression of phosphoenolpyruvate carboxykinase (PEPCK) [53], inhibits glycogen synthesis by decreasing the activity of glycogen synthase [152], and reduces glycolysis. Besides hepatic glucose metabolism, NO is also involved in the regulation of lipid metabolism at the level of lipogenesis and lipolysis. Whether NO stimulates or inhibits lipid oxidation or synthesis appears to be dependent on the isoforms on NOS stimulated, tissue site and the intracellular redox state [142].

BH₄ suppresses gluconeogenesis and increases AMPKα phosphorylation in hepatocytes from wild-type mice but not in hepatocytes isolated from eNOS^−/− mice or in the presence of NOS inhibitors, suggesting that eNOS acts upstream of AMPK activation in suppression of hepatic gluconeogenesis by BH₄ [53]. AMPK is a central regulator of glucose metabolism as liver-specific AMPK^−/− mice exhibit hyperglycemia, glucose intolerance, and increased hepatic glucose production [153]. BH₄ is mainly produced in the liver [154] and this is impaired by oxidative stress, such as seen in liver cirrhosis and diabetes [155, 156]. Furthermore, nitrate/nitrite-derived NO restores decreased phospho-AMPK (p-AMPK)/AMPK ratios in the liver of high fat feeding mice and this effect is abolished in the presence of sGC inhibitors [157]. NO decreases glucose production from lactate and also by inhibiting the conversion of glycogen synthase b into synthase a, it inhibits glycogen synthesis as observed in isolated rat hepatocytes [152].

6.6. NO and insulin resistance

Insulin resistance can be defined as the reduced metabolic actions of insulin in target tissues namely liver, skeletal muscle, and adipose tissue [158]. Insulin responsiveness is defined as the maximal effect of insulin (V_max or concentration of insulin exerting maximal biological response), and insulin sensitivity is defined as the insulin concentration that is required for 50% of its maximal response (EC₅₀/ED₅₀) [158]. Defects in insulin receptor decreases insulin sensitivity while defects that are post-receptor, reduce its responsiveness [118].

Insulin resistance is associated with decreased NO bioavailability as eNOS^−/− animals show a number of features of insulin resistance; in addition, polymorphisms in the eNOS gene are associated with insulin resistance susceptibility and metabolic syndrome in humans; inhibition of iNOS prevents while inhibition of eNOS and nNOS promotes insulin resistance [159, 160]. It is interesting to note that absence of eNOS promotes insulin resistance in both skeletal muscle and liver, while absence of nNOS impairs insulin sensitivity only in the liver of eNOS/nNOS doubleknockout mice [160]. Deletion of all isoforms of NOS (eNOS/nNOS/iNOS triple knockout) in mice, causes visceral obesity, hypertension, hypertriglyceridemia, and impaired glucose tolerance [161]. Insulin resistance caused by eNOS dysfunction is thought to be induced by endothelial dysfunction, which decreases skeletal muscle blood flow and glucose uptake [162]. eNOS^−/− mice have lower glucose transport in skeletal muscle, indicating that eNOS also regulates glucose uptake in skeletal muscle [162].

Absence of iNOS improves glucose tolerance, normalizes insulin sensitivity, and prevents disorders in PI3K/Akt signaling in high fat-fed mice [163]. Increased iNOS expression is associated with increased S-nitrosation of the insulin receptor, IRS-1, and Akt in skeletal muscle of obese mice, suggesting that S-nitrosation of proteins in insulin signaling pathway is responsible for iNOS-induced insulin resistance [164]. Furthermore, free fatty acid (FFA) induced loss of pancreatic β-cells is due to NO overproduction, which leads to interleukin 1 beta (IL-1β)-mediated β-cell dysfunction and death [108]. Selective overexpression of iNOS in liver causes hepatic insulin resistance, hyperglycemia and hyperinsulinemia [165]; the iNOS-specific inhibitor (L-NIL), reverses hyperglycemia, hyperinsulinemia, and insulin resistance in ob/ob mice [166].

These studies suggest that NOS isoforms play a central role in the regulation of glucose metabolism and insulin resistance and represent several therapeutic targets for management of type 2 diabetes.

6.7. Obesity and diabetes: The NO connection

NO bioavailability has been shown to be decreased in obesity and type 2 diabetes in animal models of obesity and diabetes as well as in obese and diabetic humans [32, 38, 121, 167]. Diminished NO bioavailability is an independent predictor of type 2 diabetes [116], hypertension [168], and atherosclerosis [168]. The role of glucose levels in NO production is controversial; it has been reported that NOS activity and subsequently NO production gradually increases due to an elevation in glucose concentrations within the pancreatic islets [169] and cultured human aortic endothelial cells (HAECs) [170]. In contrast, other groups have demonstrated that hyperglycemia contributes to endothelial dysfunction and leads to a decrease in NO bioavailability by inhibiting basal levels of eNOS expression/activity or increased NO quenching (increased NO oxidation) [32, 38, 39]. Moreover, it has been shown that uncoupling of NOS, led to decreased availability/transport of L-arginine, and an increased in arginase activity resulted in reduced NO production [57, 171, 172].

Diminished expression of NOS isoforms in particular, expression and activity of eNOS are found in both adipose tissue and skeletal muscle of obese humans and rodents [173–175]. Increased mRNA expression of caveolin-1 in adipose tissues is another mechanism for reduced eNOS-derived NO in obesity and type 2 diabetes [176]. Caveolae are a specialized type of lipid raft that appear in the plasma membrane, especially in adipocytes [176]. Caveolin-1, an essential protein for caveolae formation [177], directly binds to eNOS [168] and inhibits its activity [37] resulting in decreased NO production [168, 177]. Obesity and high-fat diets (HFD) by impairing eNOS phosphorylation at serine 1177, reduce eNOS activity [39, 46]. This site of phosphorylation is critical for NO production and can be regulated by Akt [178], which is activated by insulin.

The reason for decreased eNOS phosphorylation in obesity and diabetes could be due to FFA-induced insulin resistance [179]. Elevated FFA which is observed in obesity and type 2 diabetes, by stimulation of Toll-like receptor 4 (TLR4) or TLR2 and NFκB decreases PI3K-Akt-mediated phosphorylation of eNOS at serine 1177 and therefore reduces eNOS activity [32]. Increases in tumor necrosis factor-α (TNF-α) that are seen in obesity and diabetes, by decreasing the stability of eNOS mRNA [180] result in downregulation of its expression and abundance [175, 181]. Increased TNF-α acutely increases eNOS activity most likely through activation of PI3K-Akt and sphingomyelinase/sphingosine-1-phosphate pathways; high levels of NO however through a negative feedback loop, leads to downregulation of eNOS [32]. Elevated ROS levels in obesity and type 2 diabetes [36, 37, 182] also cause PI3K-Akt-eNOS pathway inhibition, which also 6 decreases NO bioavailability [39].

Expression of iNOS is increased in pancreatic β-cells [108], skeletal muscle [183], liver [166], and adipose tissue [184] in obesity and diabetes. Elevated TNF-α levels also increase iNOS in adipocytes which downregulates uncoupling protein 2 (UCP-2) [185] and decreases energy expenditure in adipose tissue.

In addition to decreases in eNOS-mediated NO production, uncoupling of NOS and NO quenching contribute to decreased NO bioavailability in obesity and diabetes. Obesity and diabetes are associated with decreased BH₄ and increased BH₂ levels; indeed, BH₄ to BH₂ ratio is very critical in preventing glucose-induced eNOS uncoupling [186]. Uncoupling of nNOS has also been reported in penile arteries of obese rats and leads to nitrergic dysfunction, which is corrected by elevating BH₄ levels [187].

Increased oxidative stress and inflammation are the major causes of increased NO quenching in obesity and diabetes. Uncoupled eNOS produces superoxide anion instead of NO [23, 32], which rapidly combines with NO to produce peroxynitrite [36, 39]. On the other hand, excessive iNOS-derived NO increases the formation of peroxynitrite, which enhances eNOS uncoupling [32]. Elevated peroxynitrite levels in obesity and diabetes leads to oxidation, nitration, and S-nitrosylation of proteins, lipids, and DNA [37, 116]. Obesity and diabetes are associated with chronic inflammation [72, 188]. Inflammatory cytokines by decreasing the stability of eNOS mRNA reduce its expression [39]. Moreover, inflammatory cytokines upregulate cationic amino acid transporter-2 and downregulate cationic amino acid transporter-1, the arginine transporters that enhance L-arginine availability for iNOS and decrease it for eNOS [23].

6.8. NO and diabetes complications

NO exerts several protective roles in the prevention and treatment of diabetes complications [189, 190]; NO has cardioprotective effect in ischemia-reperfusion injury [190] and it improves endothelial dysfunction, cardiomyopathy, and nephropathy in animal models of diabetes [189–191].

6.8.1. NO and diabetic cardiovascular complications

It is well known that cardiovascular disease is a common complication of diabetes that can lead to a significant number of mortalities in diabetic patients; diabetic men and women being two times and five times more likely to suffer from congestive heart failure than non-diabetic individuals, respectively [192]. High glucose levels in diabetes cause multiple biochemical modifications in endothelial cells and myocytes; glucose enters the cells by GLUT-1, whose activity is insulin independent and is predominantly regulated by extracellular glucose concentrations [193][194]. Therefore, endothelial cells are more sensitive to hyperglycemia induced injury than other cell types. During diabetes, hyperglycemia causes activation of NAD(P)H oxidase (NOX), which by using NADPH converts oxygen into superoxide anions, which in turn reacts with NO and forms peroxynitrite (ONOO-); peroxynitrite then reacts with BH₄, and that this loss of BH₄ leads eNOS uncoupling in endothelial cells [192].

Blocking NO production with L-NAME in diabetic rats increase mean arterial pressure (MAP); suggesting that in the onset of diabetes, NO is important in prevention of hypertension, most likely through actions to maintain glomerular filtration and suppress renin secretion [189]. Nitrate-mediated NO in STZ-nicotinamide-induced diabetic rats was shown to provide cardioprotection against ischemia-reperfusion injury through regulating eNOS and iNOS expression and inhibiting lipid peroxidation in an ex vivo heart preparation [190]. Nitrite4 mediated NO in db/db mice subjected to permanent unilateral femoral artery ligation, restored ischemic hind limb blood flow in a vascular endothelial growth factor (VEGF)-dependent and NO-mediated manner [195]. L-arginine treatment improved hypertension and vascular responsiveness in STZ-induced diabetic rats [191]. SNP infusions in diabetic rats decreased MAP and increased vascular conductance (flow/MAP) in a dose-dependent manner [196]. These findings indicate that impaired metabolic pathways in diabetes by decreasing NO synthesis and bioavailability lead to the impaired of endothelium-dependent vasodilatation, which could be improved by exogenous NO donors.

6.8.2. NO and renal complications of diabetes

Approximately 30% of patients with type 2 diabetes develop nephropathy; thus, it appears that hyperglycemia is necessary but not sufficient to result renal failure [197]. In the kidney, NO controls glomerular and renal hemodynamics and promotes natriuresis and diuresis [198], along with renal adaptation to dietary salt intake [199]. nNOS is mostly expressed in macula densa [200] and in small degree in specialized neurons within renal arteries of the hilus, arcuate and interlobular arteries [201]. eNOS is mostly found in renal vascular endothelium, although tubular expressions of eNOS have also been reported [201]. Although iNOS is weakly expressed in the kidney, its expression is dramatically increased by pro-inflammatory stimuli such as ischemia– reperfusion and lipopolysaccharide [202, 203]. Expression and activity of nNOS [204], iNOS [205], and eNOS [205] as well as NO metabolites [204, 206, 207] were found to be decreased in the kidney of diabetic animals.

Sodium nitrite, L-arginine, and daidzein (caveolin inhibitor) administration in HFD-STZ-induced diabetic rats have been shown to reduce blood urea nitrogen (BUN), serum creatinine, proteinuria, urinary output, kidney weight/ body weight, and renal cortical collagen content [207]; however, treatment with L-NAME, decreased the L-arginine-and daidzein-induced ameliorative effects in diabetic nephropathy [207]. In addition, nitrite treatment improved some parameters of glomerular injury, including urinary protein and albumin excretion, histopathological glomerular hypertrophy, and mesangial matrix accumulation in STZ-induced diabetic rats [206]. Improvement in renal function following L-arginine treatment and deterioration following NOS inhibitor (Nω-Nitro-L-arginine) have been reported in the ischemic acute renal failure of diabetic rats [208]. L-arginine prevents reduction in protein and mRNA expression of aquaporin-2, a water and sodium transporter in the renal outer medulla of diabetic rats [209].

7. Role of H₂S in carbohydrate metabolism

The potential role of H₂S in carbohydrate metabolism was first reported in 1990 when Hayden and colleagues demonstrated that H₂S inhalation (2.2 mM) increased circulating glucose levels in postpartum rats [210]. Later on, a growing body of evidence showed that H₂S is generated in pancreatic β-cells as well as in insulin target tissues including the liver, adipose tissue and skeletal muscles where it may control insulin secretion and insulin resistance.

7.1. H₂S and the pancreas

Expressions of CSE [211–213], CBS [212–214], and 3-MST [215] have been documented in the pancreas; further analyses in the islets showed that expression of CSE, but not CBS, was dramatically increased following glucose stimulation, which suggests that CSE may act as an inducible H₂S-generating enzyme, while CBS is constitutive [216]. CSE appears to be the major H₂S-synthesisng enzyme in the pancreas, as most of the H₂S produced from cultured insulinoma INS-1E cells was abolished after inhibition of CSE by propargylglycine (PPG) [211], and also following partially knockdown of the CSE gene, using an CSE-siRNA technique [211]. H₂S production rate at basal glucose concentration is about 12 nmole/g/min in INS-1E cells [211], about 30 nmole/g/30 min in fresh rat pancreas [212], and 8 nmole/g/min in isolated rat islets [213].

H₂S protects pancreatic β-cells against apoptosis induced oxidative stress or glucotoxicity in mice treated with glucose (5 and 20 mM); in this study, both low and high-glucose concentrations increased CSE expression but CBS expression remained relatively constant, suggesting that CSE-mediated H₂S may act as an “intrinsic brake” against glucose-induced apoptotic death in β-cells [216]. Exogenous H₂S has also been shown to protect β-cells from apoptosis induced by hydrogen peroxide, fatty acids, and cytokines; and through phosphorylation and activation of Akt signaling, promote cell proliferation and survival [217]. H₂S by reducing elevated thioredoxin binding protein-2 (TBP-2) expression in isolated islets of HFD-fed CSE knockout (CSE-KO) mice, protected β-cells from glucotoxicity-induced apoptosis [218]; considering the role of TBP-2 in β-cell apoptosis, it can be speculated that H₂S may protect β-cells from glucotoxicity-induced apoptosis by suppressing TBP-2 expression levels.

Conversely, exogenous H₂S or overexpression of CSE in INS-1E cells, reduced cell viability and induced apoptosis; in this study, H₂S increased expression of ER stress indices such as binding of immunoglobulin protein (BiP), CCAAT/enhancer-binding protein homologous protein (CHOP), and sterol regulatory element-binding transcription factor 1 (SREBF1) [219], which play a key role in pancreatic β-cell apoptosis and development of diabetes [220]. These contradictory effects of H₂S on β-cell apoptosis may be due to the different types of insulin secreting cells used in different studies and their sensitivity to H₂S [218], differences between exogenous and endogenous H₂S [221], and differences in the dose and duration of exposure [222].

7.2. Insulin secretion and H₂S

H₂S can influence insulin secretion [211, 214, 223] and modulate circulating glucose levels [224]. Inhibitory effects of sodium hydrosulfide (NaSH, 10 μM-1 mM) and L-cysteine (0.1–10 7 mM) on glucose (10 mM)-induced insulin secretion have been observed in both isolated mouse islets and pancreatic beta cell line, MIN6 cells, an effect that was not observed at a low glucose concentration (3 mM) [214]. In addition, NaSH treatment (100 μM) inhibited insulin secretion by about 70% from HIT-T15 cells [225]. Overexpression of CSE in INS-1E cells virtually abolished high glucose (16 mM)-stimulated insulin secretion; however, basal insulin secretion was not altered [211]. NaSH at concentrations of 100 and 300 μM decreased glucose-stimulated insulin secretion by about 26% and 45%, respectively from isolated mouse β-cells; moreover, glucose induced insulin release in CSE-KO mice was three times higher than wild-type mice [223]. Indeed, increased extracellular glucose levels have been shown to decrease intracellular H₂S production followed by increased insulin secretion [211]. With an increase in glucose level from 5 mM to 16 mM, the insulin secretion became 3-fold greater in INS-1E cells [211]. Taken together, these studies showan inhibitory effect of H₂S on glucose stimulated insulin secretion.

One of the mechanisms through which H₂S inhibits insulin secretion is through opening of K_ATP channels, as the inhibitory effects of NaSH and L-cysteine on insulin secretion were reproduced after using tolbutamide (a K_ATP blocker), α-ketoisocaproate (a mitochondrial fuel), and high K⁺ condition (30 mmol/L) [214]. Similar conclusions were drawn by an independent group of investigators, who showed that endogenous H₂S levels may be a switch for turning K_ATP channels on/off at different glucose concentrations in INS-1E cells [211]. Interaction of H₂S with K_ATP channels seems to be mediated through functional manipulation, probably by decreasing selective cysteine residues of K_ATP channel protein, independent of cytosolic second messengers [211]. It has been suggested that S-sulfhydration of K_ATP channels is an underlying mechanism by which H₂S could influence insulin secretion (Figure 3) [222].

Figure 3. Mechanisms of hydrogen sulfide-inhibited insulin secretion from pancreatic β-cell.

Hydrogen sulfide (H₂S) inhibits insulin secretion by opening of K_ATP channels via Ssulfhydration. Opening of K_ATP channels causes membrane hyperpolarization and therefore closing of VDCC. H₂S also inhibits VDCC directly via S-sulfhydration. H₂S inhibits glucose-induced mitochondrial membrane hyperpolarization and ATP production. G6P, Glucose 6-phosphate; SNAP 23/25, Synaptosome Associated Protein 23/25; VAMP, Vesicle Associated Membrane Protein.

In a study by Kaneko et al., L-cysteine and NaSH inhibited glucose-induced [Ca²⁺]_i oscillation without obvious changes in the mean [Ca²⁺]_i value in mouse pancreatic β-cells [214]. L-cysteine and NaSH also inhibited Ca²⁺-stimulated insulin secretion from permeabilized islets (treated with 9 streptolysin-O; SLO); interestingly, NaSH and L-cysteine also inhibited insulin release induced by the co-presence of guanosine 5′−0–3-thiotriphosphate (GTPγS) and Ca²⁺ from SLO-treated islets, under this condition, [Ca²⁺]_i is not altered by either Ca²⁺ influx or mobilization, because of its chelation by ethylene glycol-bis (β-aminoethyl ether)-N,N,N’,N’-tetra acetic acid (EGTA) [214], indicating that the inhibitory effect of H₂S on insulin release at least in part is independent of [Ca²⁺]_i. This study also revealed that modulation of glucose metabolism in the pancreatic β-cells may be another mechanism which H₂S inhibits insulin secretion, as NaSH and L-cysteine inhibited glucose-induced mitochondrial membrane hyperpolarization and ATP production [214].

Inhibition of L-type VDCC is another mechanism through which H₂S inhibits insulin secretion in β-cells. Tang and colleagues demonstrated that NaSH and ACS67, another H₂S donor, decreased insulin secretion from pancreatic β-cell as well as L-type VDCC current density by 45% and 18%, respectively [223]. Baseline VDCC current was higher in β-cells of CSE-KO mice and PPG increased baseline Ca²⁺ current in β-cells of wild-type mice; in addition, Bay K-8644, the specific agonist of VDCC, increased glucose-induced insulin secretion, an effect which was abolished by NaSH [223].

Despite the reported inhibitory effects of H₂S on insulin secretion from the pancreatic β-cells, lower plasma H₂S levels in type 2 diabetic patients may be a compensatory response [222], and contribute to the development of hyper-insulinemia to maintain normal glucose concentrations [222].

By contrast to these studies, the stimulatory effect of H₂S on insulin secretion has been reported by Takahashi et al., who showed that inhibition of CBS by β-cyano-L-alanine, reduces cysteine hydropersulfide (Cys-SSH) levels, and 2-methylthio modification, as well as decreases in glucose-induced insulin release in two different β-cell lines were abolished by Cys-SSH precursor, Cys-S2-Cys, but not by NaSH [226]. Silencing of CBS or CSE by the respective siRNAs resulted in a decrease in 2-methylthio modification in HeLa cells concomitantly with reducing intracellular cysteine persulfide (Cys-SSH), one of the possible products of CBS and CSE-catalyzed reactions [226].

7.3. Glucose metabolism in skeletal muscle and H₂S

Both CSE and CBS are expressed in human skeletal muscle cells [227]; although all H₂S producing enzymes (CBS, CSE, 3-MST) are expressed in rat skeletal muscle, these enzymes surprisingly are absent in mice skeletal muscle [228]. The rate of H₂S production in the skeletal muscles of Sprague-Dawley rats has been found to be 0.17 nmol/min/mg with an H₂S content of 2.06 nmol/mg [228].

There is limited evidence demonstrating the potential role of H₂S on carbohydrate metabolism in skeletal muscle. CSE gene knockdown resulted in a decrease in glucose uptake by cultured rat L6 myotubes and NaSH treatment at 25, 50, and 100 μM for 24h potentiated insulin-induced glucose uptake by 1.54, 1.72 and 2.06-fold, respectively, an effect which was mediated through increased phosphorylation of insulin receptors (IRs), PI3K, and Akt [229]. Moreover, NaSH (30 μmol/kg/day) increased phosphorylation of PI3K and Akt in skeletal muscles of Goto-Kakizaki rats, an experimental model of type 2 diabetes [229]. Inhalation of 5 mg/L H₂S inhibited aerobic metabolism, resulting in a significant accumulation of circulating lactate during exercise in healthy men [230]. This metabolic shift was probably due to a decrease in citrate synthase levels, which is a rate limiting enzyme in the tricarboxylic acid (TCA) cycle in skeletal muscle [230].

H₂S-mediated activation of Wnt/β-catenin signaling pathway [231] may partly elucidate the H₂S effect on insulin sensitivity in skeletal muscle cells. Activation of skeletal muscle Wnt/β-catenin signaling increases insulin sensitivity through decreasing intra-myocellular lipid deposition and down-regulation of SREBP-1c, inhibiting MAPK pathway, and also trough increasing activation of the Akt/PKB and AMPK pathways [232].

7.4. Glucose metabolism in adipose tissue and H₂S

H₂S is produced in epidydimal, perirenal, and brown adipose tissue with the rate of 4.76, 2.93, and 4.65 nmol/min/protein, respectively. In addition, H₂S-producing rates in cultured adipocytes and preadipocytes were 2.89 and 2.17 nmol/min/mg protein, respectively [233]. Although both CBS and CSE are expressed in adipose tissue, CSE appears to be the main H₂S-producing enzyme [233], as CSE inhibitors decreased H₂S production by more than 80% in adipose tissue.

Aging is associated with upregulation of CSE expression and H₂S production in adipocytes while hyperglycemia through increases in ROS down-regulates CSE expression [233]. Moreover, CSE expression was higher in adipose tissue macrophages of obese animals; however, endogenous H₂S level was lower, which indicates reduced H₂S bioavailability in obesity [234]. Decreased H₂S bioavailability in obesity activates store-operated Ca²⁺ entry pathway in adipose tissue macrophages and increases cytokine production [234]. CSE protein expression has been shown to be increased in lipopolysaccharides (LPS)-induced inflammation in a mouse macrophage cell line (RAW264.7 cells), whereas H₂S level is decreased due to enhanced cellular demand and/or consumption of H₂S [234]. Indeed, decreased H₂S bioavailability following hyperglycemia and 5 obesity plays a key role in the development of metabolic syndrome [235].

H₂S plays a regulatory role in adipogenesis [236] and adipose tissue metabolism [235]. Adipogenesis and adipose tissue maturation are promoted by endogenous and exogenous H₂S [236]. Differentiation of 3T3L1 cells is associated with upregulation of all H₂S-producing enzymes (CBS, CSE, 3-MST) and incubation of preadipocytes with GYY4137 (a slow H2S releasing compound) or NaSH, increase adipocyte differentiation factors, such as proliferator11 activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (CEBPα) [236].

The role of H₂S on glucose uptake in adipose tissue and therefore development of insulin resistance is controversial as both inhibitory and stimulatory effects of H₂S have been reported [233, 237–239]. H₂S (10–1000 μM) as well as L-cysteine decreased basal and insulin-stimulated glucose uptake in the mature adipocytes, an effect which was reversed by CSE inhibitors (PPG or BCA) [233]. CSE expression and H₂S production increased in adipose tissue of high fructose fed rats, which is a commonly used model of insulin resistance and hyperlipidemia [233]. Increased adipose tissue CSE-H₂S system in insulin-resistant rats correlated with impaired insulin-induced glucose uptake [233]. In this study, H₂S-inhibited glucose uptake of adipocytes was mediated through PI3K but not K_ATP channel pathway, which may inhibit GLUT4 translocation [233]. These findings suggest that adipose-released H₂S may contribute to the pathogenesis of insulin resistance and diabetes [233]. In addition, TNF-α inhibits insulin-stimulated glucose uptake of 3T3-L1 adipocytes, this effect is accompanied by an increase in CSE activity, expression, and H₂S generation [240]. A CSE inhibitor attenuated the detrimental effect of TNF-α on insulin-stimulated glucose uptake of 3T3-L1 adipocytes; however, a CBS inhibitor had no effect [240]. The authors accordingly suggested that the detrimental effects of TNF-α on insulin sensitivity may be partially mediated by H₂S and modulation of CSE-H₂S 5 system can be a potential therapeutic approach for insulin resistance [240].

By contrast, Manna et al. examined the effects of L-cysteine (100, 500, and 1000 μM) and Na₂S (10 and 100 μM) on phosphatase and tensin homolog (PTEN), PI3K, PIP₃, phospho-AKT and glucose uptake in high glucose (25 mM)-treated 3T3L1 adipocyte cells. H₂S decreased PTEN (a negative regulator of glucose utilization) and increased PI3K and PIP3 levels, as well as restored phosphorylation of IRS1, phospho-AKT, and GLUT4 translocation in adipocytes, and eventually increased glucose utilization [237]. The protective role of L-cysteine was blocked by PAG, in addition, H₂S and L-cysteine had no effect on glucose utilization by cells cultured at normoglycemic conditions [237]. The same group also reported that hyperglycemia decreases CSE expression and H₂S production by 3T3-L1 cells and suggested that hyperglycemia-induced insulin resistance at least in part is mediated through downregulation of the CSE-H₂S system [16].

Similarly, Cai et al. demonstrated that H₂S gas and GYY4137 stimulate glucose uptake by 3T3-L1 adipocytes which was associated with persulfidation of PPAR-γ at Cys¹³⁹ [238]. H₂S-stimulated glucose uptake was abolished when Cys¹³⁹ was replaced by serine producing a mutant PPAR-γ; this mutant could not be persulfidated, indicating that PPARγ C¹³⁹ site is a major sulhydration site [238]. In addition, CSE‒H₂S inhibited adipose-tissue PDE activity whereas it increased PPARγ activity and adipocyte numbers in mice fed a HFD; H₂S induced PPARγ sulfhydration and reduced insulin resistance but did not continuously increase obesity [238], which may explain why some obese patients (CSE‒H₂S system not downregulated) do not have diabetes [238]. H₂S also inhibited lipolysis through the protein kinase A (PKA)perilipin/hormone-sensitive lipase pathway, which promotes and sensitizes insulin response in adipocytes [239].

Vitamin D has a favorable effect on insulin sensitivity and it is considered to be of potential value in management of type 2 diabetes. It has been shown that, 1, 25-dihydroxycholecalciferol (1,25-(OH)2-D3), the active metabolite of vitamin D3, up-regulates CSE expression and H₂S production in 3T3-L1 adipocytes cultured in high glucose medium [16]. In addition, 1,25-(OH)2 D3 through increased IRS-1 phosphorylation, PI3K activity and Akt phosphorylation, increased insulin-stimulated glucose uptake, these effects were abolished by CSE inhibitor or CSE gene knockdown [16].

NaSH (25 and 50 μM) promoted insulin-stimulated glucose uptake in 3T3-L1 adipocytes; H₂S increased phosphorylation of the insulin receptor β-subunit, PI3K activity and Akt phosphorylation in cells cultured at both normo- or hyperglycemic conditions [229]. NaSH also increased insulin receptor tyrosine phosphorylation and its kinase activity in a cell free system, indicating that insulin receptor may be the direct target for the stimulatory effects of H₂S [229].

7.5. Effects of H₂S on hepatic glucose metabolism

All three H₂S-producing enzymes are expressed in the liver but H₂S production seems to be primarily catalyzed by CSE [241, 242]. H₂S plays an important role in glucose metabolism and insulin signaling in hepatocytes; H₂S stimulates hepatic glucose production and activates gluconeogenesis and glycogenolysis [243]. CSE protein was shown to be downregulated upon starvation in murine liver extracts [244], and fasting-induced downregulation of CSE can prevent hepatic glucose production and release into the circulation. Hepatic CBS expression is up-regulated in both prediabetic insulin-resistance and frank diabetic stages of Zucker diabetic fatty (ZDF) rats [245].

NaSH (100 μM) decreased glucose uptake and glycogen content in HepG₂ cells; in addition, primary hepatocytes from CSE-KO mice showed a 2-fold increase in glucose consumption rate [246]. CSE-KO mice had higher glucose consumption and glycogen content in their liver tissues; however, lower glucose was produced by hepatocytes via gluconeogenesis and glycogenolysis pathways in these mice [246]. Overexpression of CSE in HepG₂ cells, increased endogenous H₂S production and decreased glycogen content [246]. Decreased AMPK activation and suppression of glucokinase activity were responsible for H₂S-downregulated glucose uptake and glycogen storage; furthermore, H₂S-increased glucose production was mediated by inhibition of AKT signaling which is followed by activation of PEPCK [246]. H₂S-increased PEPCK activity has also been reported to be induced by increased glucocorticoid receptor activity and decreased AMPK phosphorylation [221]. Similarly, activity of glucose 6-phosphatase and fructose-1,6-bisphosphatase, the rate-limiting gluconeogenic enzymes, is promoted by H₂S through S- sulfhydration [247]. H₂S also increases the expression of theses enzymes indirectly through S- sulfhydration of the peroxisome proliferator-activated receptor gamma coactivator 1-ɑ (PGC-1ɑ) [221]. H₂S-induced glucose production is also mediated by S-sulfhydration and increased activity of pyruvate carboxylase, a key enzyme providing fuel for gluconeogenesis [248]. Taken together, these studies suggest that H₂S may play a pivotal role in hepatic insulin resistance and is further involved in the pathogenesis of type 2 diabetes.

Mitochondrial dysfunction is associated with the pathogenesis of insulin resistance. CSE-KO hepatocytes were shown to have less mitochondrial content and DNA which were restored by NaSH [249]; CSE-KO hepatocytes exhibited lower levels of transcription factors involved in mitochondrial biogenesis including nuclear respiratory factors-1 and −2 (NRF-1, NRF-2), PGC-1α, PGC-1β, and PGC-related protein (PPRC) [249]. NaSH treatment (30 μM) upregulated PPRC by S-sulfhydration, yet downregulated PGC-1β protein levels [249]. Knockdown of either PGC-1α or PPRC reduced NaSH-induced mitochondrial biogenesis in hepatocytes, while knockdown of both genes completely abolished effects of NaSH on mitochondrial biogenesis [249].

7.6. Pathophysiology of diabetes: Role of H₂S

H₂S deficiency is related to the pathophysiology of diabetes; however, the potential role of H₂S in diabetes seems to be complex. Contribution of H₂S in the onset and progression of diabetes has been reported in several studies. Expressions of CSE and CBS mRNAs, as well as endogenous H₂S production are higher in both the liver and pancreas of STZ-induced diabetic rats [212]. Similarly, activities of the hepatic CBS and CSE increased in STZ-induced diabetic rats, this effect was normalized by insulin treatment [250]. Pancreatic CSE expression and H₂S production are higher in ZDF rats than in Zucker fatty and Zucker lean rats [213]; PPG treatment of ZDF rats resulted in an increased serum insulin concentration and decreased hyperglycemia [213]. We recently showed that NaSH at high doses (1.6–5.6 mg/kg) aggravated carbohydrate metabolism while at low doses (0.28 and 0.56 mg/kg) it had no effect [251]. A positive 18 correlation has been found between H₂S concentration and disrupted insulin secretion in pancreatic β-cells [252], suggesting that inhibition of pancreatic H₂S may be a new therapeutic approach for the management of diabetes [213, 252]. However, it has been suggested that hyperglycemia-induced pancreatic CSE overexpression in the early stages of diabetes, may have as a protective mechanism, as H₂S neutralizes oxidative/nitrosative stress [15].

Conversely, endogenous H₂S production and plasma H₂S levels decreased in both non-obese diabetic mice [253], STZ-treated diabetic rats [12, 251] and patients with type 2 diabetes [11, 12] which was parallel with the severity of diabetes [11, 253], in particular in those with a history of cardiovascular disease [11]. Moreover, metformin administration in diabetic rats was associated with elevated H₂S levels in the kidney, heart, liver, and brain [254]. Hyperglycemia causes H₂S deficiency in endothelial cells and administration of H₂S could be a potential therapeutic approach in hyperglycemia [255]. Indeed, high glucose levels have been shown to inhibit H₂S production via specificity protein 1 (SP1) and p38 MAPK phosphorylation in INS-1E cells and freshly isolated rat pancreatic islets [256]. In addition, hyperglycemia and increased ROS levels decrease CSE expression [257] and increase H₂S consumption [255], leading to lower H₂S levels in diabetes.

To summarize, whether H₂S has a beneficial or deleterious effects on glucose metabolism in type 2 diabetes is inconclusive and further studies are needed to clear the potential role of H₂S manipulation in treatment of diabetes. Regarding the association between diabetes and H₂S deficiency [12, 258], H₂S modulation may have potential therapeutic effects in diabetes.

7.7. Diabetes complications and H₂S

H₂S plays multiple protective roles in the prevention and improvement of complications associated with diabetes [259]. Favorable effects of H₂S have been reported in endothelial dysfunction, retinopathy, cardiomyopathy, and nephropathy [255, 260–262].

7.7.1. Diabetic cardiovascular complications and H₂S

Increasing evidence indicates that H₂S has multiple beneficial roles in diabetic cardiovascular complications. H₂S attenuates the development of diabetic cardiomyopathy as exemplified by Barr and colleagues who examined the role of H₂S in the pathogenesis of diabetic cardiomyopathy in mice that were fed a HFD [263]. These mice had reduced circulating and cardiac H₂S levels, hallmark features of type-2 diabetes, and also marked cardiac dysfunction. H₂S treatment restored sulfide levels and attenuated HFD-induced cardiac dysfunction; the protective effects of H₂S were associated with the activation of adiponectin-AMPK signaling and suppression of HFD-induced ER stress [263]. Adiponectin plays an important role in maintaining cardiovascular health and there is a correlation between decreased adiponectin levels and increase cardiovascular risk [264]. Adiponectin delivers much of its metabolic-regulatory effects through the induction of AMPK [265]. AMPK stimulates glucose transport by increasing the expression of GLUT4 and also modulates cardiac fatty acid oxidation and subsequent lipid accumulation through the phosphorylation and inhibition of acetyl-coenzyme A carboxylase [266].

NaSH administration was found to improve ventricular function and attenuate cardiac hypertrophy and myocardial fibrosis in STZ-induced diabetic rats [262]; NaSH also reduced hyperglycemia-induce inflammation, oxidative stress and apoptosis in the cardiac tissue; favorable effects of NaSH were associated with increased activation of Nrf2 and protein expression of its downstream targets, increased activation of PI3K/Akt pathway and also blockade of c-Jun N-terminal kinase (JNK) and p38 MAPK pathways [262]. Similarly, using diabetic mice, Ye et al. demonstrated that the protective effects of NaSH on cardiac structure and function as well as reduction in apoptosis are associated with increased FoxO1 phosphorylation and the prevention of FoxO1 nuclear translocation in the injured tissue [267]. FoxO1 is critical for cellular processes that are involved in reducing oxidative stress, cell resistance to apoptosis, cell survival, energy metabolism, and cell death [268]. Furthermore, favorable effects of H₂S in diabetic cardiomyopathy were attributed to considerable reductions in the up-regulated matrix metalloproteinase 2 and transforming growth factor (TGF)-β1 in the hearts of diabetic animals [269].

Xie et al. showed that H₂S reduces aortic atherosclerotic plaque formation with reductions in superoxide generation and the adhesion molecules in STZ-induced LDLr(−/−) mice [270], a protective effect attributed to inhibition of oxidative stress via kelch ECH associating protein 1 (Keap1) sulfhydrylation at Cys151 to activate Nrf2 signaling [270]. H₂S also inhibits development of atherosclerosis via blocking diabetes-induced oxidative and inflammatory stress in endothelial cells, decreases in ROS levels and prevents foam cell formation by macrophages [271]. H₂S supplementation in diabetic rats normalized plasma H₂S levels and improved the endothelium-dependent relaxant responses of the thoracic aorta ex vivo, without affecting the degree of hyperglycemia [255].

7.7.2. Renal complications of diabetes and H₂S

Yamamoto and colleagues examined the role of NaSH in diabetic nephropathy, using pancreatic β-cell specific calmodulin-overexpressing transgenic mice as a model of diabetes [272]. In this study, diabetic mice had higher BUN and albuminuria as well as decreased CSE expression in the kidneys, however CBS expression was not altered. Renal peritubular capillary (PTC) blood flow velocity, diameter and blood flow were decreased in the kidneys and the hematocrit was on the low side. NaSH treatment dilated PTC diameter and increased blood flow in diabetic mice but had no effect on PTC blood flow velocity [272]. These findings suggest that the CSE-H₂S system in the proximal tubules may regulate the interstitial microcirculation and that H₂S releasing compounds may offer a useful strategy for the treatment of diabetic nephropathy.

NaSH improved renal tissue fibrosis in STZ-treated diabetic rats by inhibition of autophagy, up regulation of superoxide dismutase, and down-regulation of serine/threonine kinase, TGF-β1 and NF-κB protein, as key mediators of diabetic nephropathy [273]. Similarly, NaSH administration to diabetic rats attenuated high BUN levels, and reduced renal collagen and TGF-β1 expression, without affecting hyperglycemia levels [274]. NaSH also improved renal function and decreased mesangial matrix deposition, glomerular basement membrane thickening, and renal interstitial fibrosis in diabetic rats [260]; furthermore, NaSH by activation of the Nrf2 antioxidant pathway and inhibiting NF-κB signaling decreased high glucose-induced oxidative stress [260]. H₂S inhibited the renin-angiotensin system in the diabetic kidney and attenuated high glucose induced mesangial cell proliferation by suppression of the MAPK signaling pathway [260].

8. NO and H₂S interactions in carbohydrate metabolism

In the past few years, much attention has been given to the interactions between NO and H₂S. There is a growing body of evidence showing that these two gasotransmitters interact with each other at the levels of both biosynthesis and biological responses. Although the individual roles of these two gasotransmitters in both physiological and pathophysiological function are well appreciated, consequences of their interactions need further research. Understanding the interactions between these two molecules will prove fruitful in managing several pathophysiological conditions.

8.1. Biosynthesis of H₂S and NO and their interaction

We recently examined the combination effects of sodium nitrite and NaSH on carbohydrate metabolism in type 2 diabetic rats [19]. In this study, nitrite supplementation increased serum total sulfide levels and NaSH administration increased serum NO_x (nitrate+nitrite), however, the combined treatments further increased NO_x but not sulfide levels. In addition, nitrite supplementation increased mRNA expression levels of CSE and CBS in the soleus muscle and CBS in the liver and epididymal adipose tissue of diabetic rats [19]. NaSH administration was also shown to decrease total NOS and iNOS activities as well as NO content in diabetic rats [275].

Effects of H₂S on activity and expression of NOS enzymes are controversial. H₂S was shown to increase eNOS activity [17, 101, 276], have no effect on iNOS and nNOS activities [99], or inhibit all NOS isoforms [4]. These controversies may at least in part be due to the time of NOS activity measurement after H₂S administration, as the stimulatory effects of H₂S on eNOS activity are transient [277]. Moreover, low concentrations of L-cysteine (0.1–3 mM and 0.01–1 mM, respectively) promote the activities of nNOS and iNOS but not that of eNOS, and high concentrations inhibit the activity of all NOS enzymes (10 mM for nNOS and 3–10 mM for iNOS or eNOS) [4]. This effect of L-cysteine is considered to be independent of its property as a reductant and zinc-chelator, because DTT (threo-1, 4-dimercapto-2, 3-butanediol), a reducing agent and also NaSH that possess similar properties did not mimic the facilitating effects of L-cysteine at low concentrations [4], thus the underlying mechanisms for these paradoxical effects of L-cysteine on the NOS activity have yet to be clarified.

The stimulatory effects of H₂S on eNOS activity is partly due to an elevated [Ca²⁺]_i levels [1, 17, 95, 101, 278–280], resulting in phosphorylation of eNOS at S1177 [103, 280, 281], and inhibition of eNOS S-nitrosylation [85]. Furthermore, H₂S prevents eNOS degradation [281] and stabilizes the dimeric active form of eNOS [103] through sulfhydration of Cys443 [17, 85]. H₂S-increased [Ca²⁺]_i is mediated through increasing IP₃-dependent intracellular Ca²⁺ mobilization, activating K_ATP channels, and favoring the reverse mode of Na⁺-Ca²⁺ exchanger [1, 17, 101]. Inhibitory effect of NaSH on eNOS is concentration dependent with an IC₅₀ of 170 μM [4]; which is due to the inhibition of BH₄ function [282] and decreasing the phospho-eNOS (serine 1177) [283].

The interaction between NO and H₂S is also influences the diabetic cardiomyopathy; Yang and colleagues showed that iNOS expression in STZ-induced diabetic rats is positively correlated with the severity of myocardial injury [275]. In this study, the cardioprotective effects of H₂S (56 μmol/kg/day NaSH) were related to the decrease in activity and expression of iNOS; inhibition of H₂S exacerbated myocardial injury [275].

Effect of NO on H₂S synthesis is also not straightforward. It has been reported that NO increases the activity and expression of CSE [93, 102, 282, 284, 285], has no effect on H₂S-producing enzymes [286], or even directly inhibits CSE activity in vitro with an IC₅₀ of about 100 nM [95]. NO-increased H₂S synthesis is mediated by cGMP as it is reduced by inhibition of sGC [287]. CSE also is a target of S-nitrosylation at its multiple reactive cysteine residues [288]. Heme-containing proteins are targets of NO, thus, CBS activity might be affected by NO [289].

8.2. Interaction between NO and H₂S in biological responses

The interaction between thiol molecules and NO is the bases of NO-induced S-nitrosylation. As H₂S is a thiol molecule, it is possible that H₂S interacts with NO to form nitrothiols (RSNO). Incubation of NaSH with SNP in vitro resulted in a time dependent release of nitrite, indicating formation of nitrosothiol [290]. In addition, incubation of liver homogenates from LPS-treated rats with NaSH or L-cysteine and pyridoxal phosphate to increase endogenous production of H₂S, increased nitrite formation as an outcome of the interaction between exogenous NaSH and endogenous NO [290]. In addition to nitrosothiol, interaction of NO and H₂S and their respective metabolites can generate different species that have significantly different physiological functions as compared to either NO and/or H₂S [291–294]. These products include nitrosopersulfide (SSNO⁻) [294–298], thionitrous acid (HSNO) [293, 295, 297, 299], and nitroxyl (HNO) [294, 296, 297].

In our previous study, NaSH at a low dose, which had no effect on carbohydrate metabolism, potentiated the favorable metabolic effects of sodium nitrite in type 2 diabetic rats [19]. These favorable effects were associated with improvement of liver function, reduced oxidative stress, and increased mRNA expression and protein levels of GLUT4 in insulin-sensitive tissues [19]. H₂S stimulates NO release from nitrite via increasing the activity of XO [300] and also enhances production of sulfinyl nitrite (an NO donor) [301], suggesting that nitrite in the presence of H₂S becomes more biologically active. In addition, H₂S maintains sGC in an NO-activatable form [103] and increases cGMP levels by inhibiting PDE5 [17].

The interaction between NO and H₂S has also been reported in diabetes-induced nephropathy; NaSH administration decreased fasting blood glucose, insulin, lipid profile, urea and creatinine, as well as insulin resistance in HFD-STZ-induced type 2 diabetic rats [302]. NaSH also decreased TNF-α, NF-κB, TGF-β, caspase 3, malondialdehyde (MDA), and H₂O₂ production, whereas it increased catalase and superoxide dismutase activities in renal tissue of diabetic rats. However, chronic administration of L-NAME in combination with NaSH diminished the favorable effects of NaSH on diabetes-induced nephropathy, serum insulin, urea, and creatinine, as well as tissue levels of TNF-α, NF-κB, TGF-β, caspase 3, MDA, and H₂O₂ production, and activity of antioxidant enzymes [302]. Collectively, these data strongly suggest that NO has a significant role in the protective effects of H₂S in diabetes-induced nephropathy.

Expression of NOX4, the major source of ROS in the kidney, is increased in diabetes. Genetic deletion or chemical inhibitors of NOX4 ameliorate kidney injury that is diabetes induced [303]. Reduced activity of AMPK is also associated with high glucose-induced increases in NOX4 expression; AMPK activation prevents the deleterious effects of glucose in the kidney via inhibition of high glucose-induced NOX4 protein expression and subsequent ROS generation [304]. NaSH inhibited high glucose-induced NOX4 expression via activation of AMPK, an effect which was reversed by L-NAME, suggesting a role for NO in mediating the H₂S effect; in this study, NaSH increased mRNA and protein expression of iNOS but not that of eNOS in the mouse kidney proximal tubular epithelial cells [305]. In addition, compound C, a selective AMPK inhibitor, blocked the NaSH-induced increases in iNOS expression [305]. These data demonstrate that H₂S stimulates iNOS expression in an AMPK-dependent manner to inhibit the high glucose-induced increase in NOX4.

9. Conclusion and perspectives

All H₂S-producing enzymes are found in the pancreas and insulin sensitive tissues; however, CSE seems to be the major enzyme in these tissues. All NO-producing enzymes are also expressed in the pancreas and insulin sensitive tissues; although nNOS expression does not appear to be present in any significant amounts in the adipose tissue, it is the main isoform of NOS in skeletal muscle. Both NO and H₂S have roles in pathophysiology of diabetes and regulation of blood glucose levels. Deficiencies in H₂S and NO systems have been reported in diabetic animal models as well as in human studies. The role of H₂S in carbohydrate metabolism is however controversial, H₂S causes inhibition of insulin secretion by activation of K_ATP channels and inhibition of VDCC in pancreatic β-cells. In addition, H₂S through inhibition of GLUT4, inhibits insulin-stimulated glucose uptake in insulin sensitive tissues. By contrast, H₂S protects β-cells against glucotoxicity-induced apoptosis and up-regulates insulin signaling pathways essential for glucose utilization, suggesting a potential therapeutic approach in diabetes. Regarding H₂S administration in animal models, its multiple functions against diabetes-induced cardiovascular diseases, nephropathy, and other complications have been documented.

In addition, iNOS-derived NO decreases while eNOS-derived NO increases insulin secretion. eNOS^−/− mice have lower glucose transport in skeletal muscle, indicating regulation of glucose uptake by NO. Regarding effects of these gasotranmitters on carbohydrate metabolism, translational work in this area requires a deep understanding of the biology and pharmacology of H₂S and NO, as well as an ability to integrate this scientific knowledge with the principles of drug development. In addition, it should be noted that in compare to NO, H₂S is the youngest member of the gasotransmitter family and certainly needs further work to fully elucidate its effects on carbohydrate metabolism. Deficiencies in both H₂S and NO contribute to the development of diabetes, and in this context, combination treatment modalities may prove fruitful in managing diabetes. Another area that warrants attention is the possible roles of these gasotransmitters in the diabetes-cancer-axis; since undesirable changes in the H₂S and NO systems contribute to the pathophysiological conditions of both, this topic needs further research.

Abbreviations

3-MST

3-Mercaptopyruvate sulfurtransferase

AMPK

AMP-activated protein kinase

ATP

Adenosine triphosphate

BH₂

7,8- Dihydrobiopterin

BH₄

Tetrahydrobiopterin

CAT

Cysteine aminotransferase

CBS

Cystathionine β synthase

cGMP

Cyclic guanosine monophosphate

CSE

Cystathionine γ lyase

EGTA

Ethylene glycol-bis (β-aminoethyl ether)-N,N,N’,N’-tetra acetic acid

eNOS

Endothelial nitric oxide synthase

ER

Endoplasmic reticulum

FFA

Free fatty acid

G6P

Glucose 6-phosphate

GSNO

S-Nitrosoglutathione

H₂S

Hydrogen sulfide

HFD

High-fat diet

IL-1β

Interleukin 1 beta

iNOS

Inducible nitric oxide synthase

IP₃R

Inositol-3-phosphate receptor

K_ATP

K⁺ channels

L-NAME

L-N^G-Nitroarginine methyl ester

L-NMMA

NG-monomethyl-L-arginine

MAPK

Mitogen-activated protein kinase

NADPH

Nicotinamide-adenine-dinucleotide phosphate

NF-κB

Nuclear factor kappa-light-chain-enhancer of activated B cells

nNOS

Neuronal nitric oxide synthase

NO

Nitric oxide

NO₂⁻

Nitrite

NO₃⁻

Nitrate

NOS

Nitric oxide synthase

Nrf2

Nuclear factor-like 2

PDE5

Phosphodiesterase 5

PEPCK

Phosphoenolpyruvate carboxykinase

PIP₂

Phosphatidylinositol 4, 5-bisphosphate

PIP3

Phosphatidylinositol (3, 4, 5)-triphosphate

PKG

Protein kinase G

PLP

Pyridoxal-5′-phosphate

PPG

Propargylglycine

ROS

Reactive oxygen species

sGC

Soluble guanylyl cyclase

SH

Sulfhydryl group

SNAP

S-Nitroso-N-acetylpenicillamine

SNAP23/25

Synaptosome associated protein 23/25

SNP

Sodium nitroprusside

SREBF1

Sterol regulatory element-binding transcription factor 1

SSH

Hydropersulfid moiety

STZ

Streptozotocin

TLR4

Toll-like receptor 4

TNF-α

Tumor necrosis factor-α

UCP-2

Uncoupling protein 2

VAMP

Vesicle associated membrane protein

VDCC

L-type voltage-dependent Ca²⁺ channels

XO

Xanthine oxidase