*2.2. Inositol Incorporation in Organisms*

Despite eight out of the nine inositol isomers occurring naturally, only myo-inositol (MI; *cis*-1,2,3,5-*trans*-4,6-cyclohexanehexol), scyllo-inositol (SI; *cis*-1,3,5-*trans*-2,4,6-cyclohexanehexol), and d-chiro-inositol (DCI; *cis*-1,2,4*-trans*-3,5,6-cyclohexanehexol) have been detected as major inositols present in mammalian tissue. Some studies have reported the possible presence of neo-inositol (NI; *cis*-1,2,3-*trans*-4-5-6-cyclohexanehexol) in bovine brains [12] and also epi-inositol (EI; *cis*-1-*trans*-2,3,4,5,6-cyclohexanehexol) and muco-inositol (*cis*-1,4-*trans*-2,3,5,6-cyclohexanehexol) in the liver, muscle, blood, brain, and several other rat tissues with a rate of isomerization from MI of 0.6% [13]. MI interconverts to other inositols via an specific epimerase [13,14] that differs from the epimerase catalyzing the conversion from MI to DCI [15,16]. MI and SI are the most abundant forms of inositols in organisms, representing over 90% of the total inositol content of mammalian cells. SI has been reported to be incorporated into lipids in plants [17]. However, SI is not incorporated into PI lipids at detectable levels, even under SI administration in mammalian tissue [18]. The other inositol found in great levels in mammals is DCI, which acts as a second messenger for insulin signaling, presumably as IG, as will be depicted later. A summary of the current knowledge on inositol stereoisomers sources, distribution, and pharmacological properties can be found in Table 1.


**Table 1.** Biological sources of inositol stereoisomers, distribution in mammalian tissue, and current pharmacological applications for the treatment of human diseases. PIs: Phosphatidylinositols; IP3: Inositol 1,4,5-trisphosphate; GPIs: Glycosylphosphatidylinositols.


**Table 1.** *Cont.*

Inositols can be obtained from the diet as they are abundant components of the cell membrane in plants and animals. The most abundant form of MI incorporation is either free, or in the form of (myo)-inositol hexaphosphate or IP6 (1,2,3,4,5,6-hexayl hexakis (dihydrogen (phosphate))), also known as phytic acid. Major sources of IP6 are plants, as IP6 is a major reservoir of phosphorus, energy, and a source of cations and MI in the cell wall. However, IP6 cannot be obtained from dietetic sources because its bioavailability is very limited. This is due to its negative charge density, being necessary to be dephosphorylated via bacterial phytases and phosphatases, providing free MI or other IPs before entering bloodstream [29,30]. The only animals that can carry out this transformation belong to the ruminant mammal group. Western diets can provide around 0.5–1 g/day of MI [31,32]. DCI is mainly obtained as the methylated form d-pinitol (DPIN; 3-o-methyl-d-chiro-inositol), which is demethylated to DCI under acidic conditions in the gastrointestinal tract. DPIN acts as an osmolyte in plants, allowing tolerance to heat, high salinity, and drought stress [33]. The *Leguminosae* family is a major source of DPIN, especially carob pods, which provide 10–80 g/kg of DPIN [34]. Many herbal extracts contain methyl-inositol derivatives like sequoyitol (5-o-methyl-myo-inositol) or l-quebrachitol (2-o-methyl-l-chiro-inositol) (Figure 1B) [35,36]. Dietary supply of other inositols seems to be scarce, as evidenced by their low presence in organisms.

Although inositols can be obtained from diet, the main inositol isomer, MI, is also synthesized in the body in great enough quantities for the whole supply required, as kidneys produce 2 g/day each of MI (4 g total) and other tissues contribute to a small extent to MI synthesis, like the brain and testis [37]. De novo MI synthesis occurs primarily from glucose into cytosol. The process follows glucose phosphorylation into glucose-6-phosphate (G6P) via hexokinase. D-3-*myo*-inositol-phosphate synthase (MIPS) catalyzes the conversion from G6P into myo-inositol 3-phosphate (MIP), and dephosphorylation occurs with inositol monophosphatase (IMPase), rendering free MI. MIPS is a phosphoprotein whose activity is regulated by the glycogen synthase kinase 3 (GSK3) homolog MCK1 in yeast [38,39]. Although this may suggest GSK3 regulation of MIPS activity in humans, there are no data of this proposed mechanism in mammalian cells. Expression of inositol-3-phosphate synthase 1 (Isyna1), a gene encoder for IMPase transcription, is tightly regulated by the disposal of phosphatidic acid (PA) and inositol hexakisphosphate synthase 1 (IP6K1). PA is considered a "metabolic sensor", as its synthesis is upregulated by high levels of glucose or the presence of growth factors and promotes cell growth via a mammalian target of rapamycin (mTOR) [40,41]. Interaction of PA with IP6K1 leads to its translocation into the nucleus and synthesis of IP7, which negatively regulates *Isyna1* via DNA methylation [42].

The transport of inositol isomers into cells is regulated by the sodium-myo-inositol co-transporters (SMIT1 and SMIT2) with a 2 Na<sup>+</sup> to 1 myo-inositol stoichiometric ratio, similar to that of the sodium-glucose co-transporter SGLT1 [43–45]. SMIT1 and SMIT2 both have a different affinity for inositol isomers, which are competitors for inositol transport, and are differentially regulated by monosaccharides. DCI and MI are both effectively transported into cells via SMIT2 with a similar affinity, whereas DPIN, a methyl derivative of DCI, binds to SMIT2 with lower affinity and is a competitor for MI/DCI transport [46]. SI has also a high affinity for the SMIT1 and SMIT2 transporters, whereas other inositol derivatives like sequoyitol, D-ononitol (4-o-methyl-d-chiro-inositol), or viburnitol (1-deoxy-l-chiro-inositol) are also transported with lower affinity [47]. Inositol transport is inhibited in the presence of l-fructose and d-glucose, as they are also competitors of SMIT1 and SMIT2 [46,47]. SMIT2 is highly expressed in mammalian kidneys and is responsible for the reabsorption of inositols into the bloodstream [48,49]. DCI uptake via SMIT2 is highly upregulated in the presence of insulin in human L6 myoblasts, which could explain the lower concentration of DCI in insulin-sensitive tissues and lower DCI re-uptake in the renal tubes in cases of hyperglycemia and insulin resistance [46,50,51], along with decreased epimerase activity [15,16]. The fact that inositol transport is highly dependent on glucose concentrations and insulin signaling limits its potential use in association with food (i.e., as an ingredient), where it would be necessary to adjust the timing, dose, and number of doses to the given metabolic profile of the user.

Inositols may also be transported by a described H+/myo-inositol transporter (HMIT) in a 1 H<sup>+</sup> to 1 myo-inositol stoichiometric ratio [43,52]. Inositol HMIT transport is pH-dependent and phlorizin-sensitive [52]. HMIT is highly expressed in the brain, but its transcript is also detected in white, brown, and epididymal adipose tissues, and also in the kidney in rats [52]. HMIT is reported to transport MI, SI, DCI, and muco-inositol, but not allo-inositol, and is blocked by phloretin and phlorizin, which are well-described inhibitors of the Na+/glucose transporters SGLT1 and SGLT2. [52]. There are not many reports about the physiological role of HMIT in inositol transport in the peripheral tissues. HMIT differently controls inositol transport and signaling in the neurons and astrocytes along with SMIT1 and SMIT2, which may stand for a specific role of inositols in osmoregulation, insulin signaling, Ca2<sup>+</sup> mobilization, and membrane composition in the brain [52–55]. A more detailed mechanism of inositol transport in the brain will be discussed in the other sections of this review.

MI, when available in the cell, is incorporated into PI. All MI-containing phospholipids are derived from PI, which is the most abundant form throughout the cell, constituting 10–15% of mammalian membrane phospholipids [56]. PI synthesis is performed next to the endoplasmic reticulum via PI synthase (PIS). This process requires cytidine diphosphate diacylglycerol (CDP-DAG) and MI. PIS has a low affinity for MI, hence MI availability is the rate-limiting factor for PI synthesis (ref). Some PI is channeled to the luminal face of the endoplasmic reticulum to later derive glycan PI or GPIs, which are acylated and transported to plasma membrane, serving as "anchors" for proteins in the external

surface of the plasma membrane [57]. PIs may also be substrate for PI kinases, deriving PI-3, -4, and -5 monophosphate (PI(3)P, PI(4)P, and PI(5)P), which may suffer posterior phosphorylation and be converted into PI-3,5, -3,4, and -4,5 biphosphate (PI(3,5)P2, PI(3,4)P2, and PI(4,5)P2), the latter also rendering the triphosphate form PI(3,4,5)P3, as reviewed in [57,58]. Phospholipase C may cleave PI(4,5)P2 and form inositol (3,4,5) phosphate or IP3, which is subsequently derived into other IPs or recycled back to MI [58].
