54.2.1

Metabolic Role and Effect of Deficiency

Thiamin ( Fig. 54.1 ) was the first member of the family of water-soluble vitamins to be described, with reference to the effects of its deficiency (beriberi) being suggested some 4000 years ago in old Chinese medical literatures. Thiamin (mainly in its pyrophosphate forms) acts as an enzymatic cofactor for transketolase, pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and branched-chain ketoacid dehydrogenase, enzymes involved in many critical metabolic reactions that relate to energy metabolism. Because thiamin bridges the glycolytic and the pentose phosphate metabolic pathways (which generate chemical-reducing power in cells), it also plays an important role in reducing cellular oxidative stress. Thus, low intracellular levels of thiamin will lead to impairment in energy metabolism (acute energy failure) and to a propensity for oxidative stress. While most of thiamin’s biological activity is attributed to its pyrophosphate form (i.e., thiamin pyrophosphate, TPP, which is also the most abundant form of this vitamin in cells and accumulates mainly in mitochondria), recent studies have shown that other forms of the vitamin, namely, thiamin triphosphate, also possess biological activity in nerve cells where it regulates the function of membrane chloride channels and acts as a phosphate group donor to other membrane proteins (Ref. and references therein). Thiamin deficiency (and suboptimal levels) represents a significant nutritional problem worldwide and leads to several clinical abnormalities including neurological (neuropathy and/or Wernicke-Korsakoff syndrome) and cardiovascular (peripheral vasodilatation, biventricular myocardial failure, edema, and potentially acute fulminant cardiovascular collapse) disorders. On the other hand, optimization of thiamin levels appears to have the potential of preventing diabetic retinopathy and tissue damage caused by the hyperglycemia of diabetes and alleviating fatigue in patients with inflammatory bowel diseases (IBDs). Systemic thiamin deficiency and suboptimal levels occur in a high percentage of chronic alcoholics, diabetics, patients with celiac sprue and renal diseases, post bariatric surgery, patients with sepsis, and in subjects on long-term furosemide diuretic therapy. In addition, thiamin deficiency and suboptimal levels occur in the elderly population despite an average daily intake of the vitamin that exceeds their recommended requirement. Also recognized is the occurrence of thiamin deficiency disorders that occur despite normal plasma level of this vitamin. Examples of the latter are thiamin-responsive megaloblastic anemia (TRMA) and thiamin-responsive/Wernicke’s-like encephalopathy. TRMA is an autosomal recessive disorder characterized by megaloblastic anemia, sensorineural deafness, and non-type 1 diabetes mellitus, which is caused by mutations in the human thiamin transporter-1 (hTHTR-1; product of the SLC19A2 gene). Such mutations in hTHTR-1 gene led to an impairment in thiamin uptake (by the affected tissues) and the development of localized thiamin deficiency. Thiamin-responsive/Wernicke’s-like encephalopathy is characterized by seizures, ophthalmoplegia, nystagmus, and ataxia and is believed to be due to mutations in the human thiamin-transporter-2 (hTHTR-2; product of the SLC19A3 gene). In both of the latter disorders, oral administration of pharmacological doses of thiamin brings about significant improvements in many of the clinical symptoms of the affected individuals.

54.2.2

Physiology of the Intestinal Thiamin Absorption Process

The human intestine encounters thiamin from two sources: a dietary source and from microbiota. Dietary thiamin exists mainly in the phosphorylated form, which must be hydrolyzed to free thiamin prior to absorption (the small intestine does not have a transport system for intact TPP, that is, it is unlike the large intestine that has such a system; see below), a process that is achieved by the action of the abundant intestinal phosphatases. Absorption of the liberated free thiamin (which exists in the monocationic form at gut luminal pH) then takes place mainly in the proximal part of the small intestine. The mechanism of thiamin absorption in the small intestine has been investigated using a variety of intestinal preparations from different species including human. Collectively, these studies have shown the involvement of a specific carrier-mediated mechanism that is inhibited by thiamin structural analogues, but not by unrelated organic cations. Studies using purified brush-border membrane vesicles (BBMV) isolated from human and animal small intestine have shown the involvement of a pH (but not Na ⁺ )-dependent, electroneutral, and amiloride-sensitive carrier-mediated mechanism for thiamin uptake. The mechanism by which thiamin exits out of enterocytes, that is, transport across the basolateral membrane (BLM), has also been studied using purified human and animal intestinal basolateral membrane vesicles (BLMV) and has been found to involve a specific, pH-dependent, electroneutral, carrier-mediated process. With regard to potential metabolism during absorption, studies have shown that some of the absorbed free thiamin is rephosphorylated (mainly to TPP) in intestinal epithelial cells; however, only free thiamin exits the intestinal absorptive cells into the serosal side. There is recent emerging evidence that indicates that the intracellularly generated TPP accumulates in mitochondria via a carrier-mediated process and this important process has major relevance to intestinal energy metabolism in health and disease.

As to the microflora-generated thiamin, this source provides considerable amounts of free and phosphorylated (TPP) forms of the vitamin. While gut microbiota of all human generate thiamin, recent studies that classified the human gut microbiota into three functionally different enterotypes (enterotypes 1, 2, and 3) have reported that enterotype 2 (which is enriched in Prevotella and Desulfovibrio ) is especially overrepresented in enzymes that are involved in TPP biosynthesis, implying a larger level of thiamin generation in the group that harbors this enterotype. As to absorbability of the microbiota-generated forms of thiamin in the large intestine, recent investigations have shown that the human colonocytes do indeed possess an efficient and specific carrier-mediated mechanism for uptake of free thiamin and a separate and specific carrier-mediated mechanism for uptake of intact TPP. The latter system was shown to have high affinity (apparent K _m of 0.157 μM) and specificity for TPP (it does not transport free thiamin or thiamin monophosphate), is pH- and Na ⁺ -independent, and energy-dependent. It is also interesting to note that the gene that encodes the colonic TPP transporter (TPPT) (i.e., SLC44A4; see below) has recently been identified as an ulcerative colitis susceptibility gene. Based on the mentioned evidence, it is clear that the microbiota-generated thiamin is absorbable and contributes to overall thiamin nutrition, especially when it come to cellular nutrition and health of the local colonocytes.

54.2.3

Molecular Biology of the Intestinal Thiamin Uptake Systems

Mammalian cells appear to utilize two members of the solute carrier SLC19A gene family (i.e., THTR-1 and THTR-2) to transport free thiamin and one to transport TPP. The SLC19A2 encodes a protein of 497 amino acids (THTR-1), while the SLC19A3 encodes a protein of 496 amino acids (THTR-2). Both of the human THTR-1 and -2 are predicted to have 12 putative transmembrane domains (TMDs) with their N- and C-terminal tails extending into cell interior. The hTHTR-1 and hTHTR-2 share 48% identity and 64% similarity with one another. They also share 40% and 39% identity at the amino acid level, respectively, with the human reduced folate carrier (hRFC). However, neither hTHTR-1 nor hTHTR-2 transport folate and the hRFCs do not transport free thiamin.

The hTHTR-1 appears to function in the micromolar range, while the hTHTR-2 appears to function in the nanomolar range. Both the hTHTR-1 and hTHTR-2 are expressed in the human small and large intestine, with expression of the former being markedly higher than that of the latter. The membrane domain of the polarized human enterocyte at which hTHTR-1 and hTHTR-2 proteins are expressed have also been investigated using immunological and confocal imaging approaches. Expression of the hTHTR-1 protein was shown to be at both the apical and BLM domains of the polarized enterocytes, with a slightly higher expression at the latter compared to the apical membrane domain. On the other hand, expression of the hTHTR-2 protein was shown to be restricted to only the apical membrane domain of the polarized absorptive epithelia.

As to the molecular identity of the colonic TPPT, this has been recently delineated in studies by Nabokina et al., which showed that the uptake system is the product of the SLC44A4 gene. Expression of the human TPPT along the intestinal tract was found to be restricted to the colon ( Fig. 54.4 ). Also, cell surface biotinylation assay and live-cell confocal imaging studies have shown that the human TPPT is exclusively expressed at the apical membrane domain of polarized epithelial cells ( Fig. 54.5 ). The tissue-specific expression of the TPPT in the colon appears to be determined by epigenetics as well as microribonucleic acid (miRNA)-mediated mechanisms. The latter was suggested by findings that in the colon, histone H3 in the 5′-regulatory region of Slc44a4 is trimethylated at lysine 4 and acetylated at lysine 9, whereas the trimethylation at lysine 27 was negligible. In the jejunum, on the other hand, histone H3 was found to be hypertrimethylated at lysine 27 (repressor mark). Involvement of miRNA(s) in the tissue-specific expression of TPPT was suggested by findings showing the 3′-UTR of SLC44A4 to be the target of specific miRNAs/RNA-binding proteins in noncolonic, but not in colonic, epithelial cells.

Expression of the hTPPT ( SLC44A4 ) in different human tissues and cell lines. Expression in the GI tract is highest the colon; high expression was also observed in the prostate, trachea, and lung.

(From Nabokina SM, Inoue K, Subramanian VS, Valle JE, Yuasa H, Said HM. Molecular identification and functional characterization of the human colonic thiamine pyrophosphate transporter. J Biol Chem 2014; 289 :4405–16.)

Apical membrane targeting of hTPPT-1-YFP in polarized epithelial cells. Targeting of YFP (vector alone), hTPPT-YFP, hSMVT-GFP (a known apical membrane marker), and hSVCT2-YFP (a known basolateral membrane marker) in epithelial cells in lateral ( XY , top row ) and axial ( XZ , bottom row ).

(From Nabokina SM, Inoue K, Subramanian VS, Valle JE, Yuasa H, Said HM. Molecular identification and functional characterization of the human colonic thiamine pyrophosphate transporter. J Biol Chem 2014; 289 :4405–16.)

54.2.4

Cell Biology of the Intestinal Thiamin Absorption Process

Insight into the mechanisms involved in intracellular trafficking and membrane targeting of the human thiamin transporters in epithelial cells have also been emerging from studies utilizing the live-cell confocal imaging approach. Using a series of truncated hTHTR-1 constructs fused with the enhanced green fluorescent protein (EGFP) (i.e., hTHTR1-EGFP), expression of the full-length fusion protein was shown to be at both the apical and the BLM domains of intestinal epithelial cells. Analysis of the localization pattern of truncated mutants of the hTHTR-1 has shown that while the C-terminus region of the polypeptide does not appear to have a role in membrane targeting, whereas an essential role for the N-terminal and the backbone was found. Also, truncation of the hTHTR-1 polypeptide within a region where several TMRA truncations are clustered results in intracellular retention of the mutant protein. As to intracellular trafficking, the hTHTR-1 protein was found to reside inside numerous trafficking vesicles whose movement is temperature dependent and requires an intact microtubule network for their mobility (Ref. ; real-time movies can be viewed at website: http://www.jcb.org/cgi/content/full/278/6/3976/DC1 ).

With regard to the hTHTR-2 protein, this carrier is expressed exclusively at the apical membrane domain of the polarized intestinal epithelial cells. Live-cell imaging investigations utilizing a series of hTHTR-2 truncations have shown that while the cytoplasmic C-terminal is not essential for membrane targeting of the protein, an essential role for the transmembrane backbone is evident. In addition, trafficking vesicles were found to be involved in the intracellular movement of hTHTR-2 to the cell surface, and that mobility of these vesicles depends on an intact microtubule network that was disrupted by overexpression of the dynactin subunit dynamitin (p50) (dynactin is involved in trafficking of vesicles via conventional cytoplasmic dynein, a minus end-directed motor protein).

Other studies have identified accessory proteins that influence the functionality and cell biology of the hTHTR-1 and -2 in intestinal epithelial cells ( Fig. 54.6 ). Using a bacterial two-hybrid system to screen a human intestinal cDNA library with the complete coding sequence of hTHTR-1 as bait, studies have identified a member of the tetraspanin family of proteins, Tspan-1, as an interacting partner with hTHTR-1. Coimmunoprecipitation assay, glutathione S -transferase (GST)-pulldown assay, and live-cell confocal imaging have confirmed the existence of such an interaction between hTspan-1 and hTHTR-1 in human intestinal epithelial Caco-2 cells. This interaction appears to have biological and functional consequences as it decreased the rate of degradation of the hTHTR-1 protein, and thus, influenced its functionality. More recent investigations have utilized a yeast split-ubiquitin two-hybrid approach to identify the human transmembrane 4 superfamily 4 (hTM4SF4) as an interacting partner of hTHTR-2 in human intestinal epithelial cells ( Fig. 54.6 ). This interaction was again confirmed by GST-pull-down assay in vitro , by live-cell confocal imaging in intestinal epithelial cells, and shown to have functional consequences on the hTHTR-2 activity.

Diagram showing the accessory proteins that interact with human intestinal thiamin transporters hTHTR-1 and -2, the human biotin transporter hSMVT, and human folate membrane transporter hRFC, and the human ascorbic acid transporter in intestinal epithelial cells.

54.2.5

Structure-Activity Aspects of the Thiamin Transporters

Knowledge about the structure-function activity of the hTHTR-1 and hTHTR-2 proteins has been emerging in recent years from clinical and experimental findings. With regard to the hTHTR-1, over 18 different clinical mutations have been identified in patients with TRMA. These mutations are either mis-sense or nonsense in nature with the latter type leading to an early truncation of the carrier protein. The effect of a number of these mutations (e.g., P51L and T158R, W358X, and Delta383fs) on functionality of the hTHTR-1 has been investigated experimentally using mutants generated by site-directed mutagenesis followed by expression in intestinal and other appropriate cell types. The results revealed a spectrum of mutant phenotypes with all exhibiting impaired thiamin uptake function due to either a change in hTHTR-1 level of expression/stability, mis-targeting, and/or altered transport activity. Other studies have examined the role of the only conserved anionic amino acid residue (located at position 138) in the predicted TMDs of hTHTR-1 in the transport of the cationic thiamin, as well as the role of the two putative N-glycosylation sites (predicted to be at positions 63 and 314). The result showed a critical role for the residue at position 138 in the function of hTHTR-1. On the other hand, neither of the putative glycosylation sites was found to play a role in the function or membrane targeting of the hTHTR-1 protein.

As to the hTHTR-2 protein, again data from clinical and experimental studies have shed light into important aspects of its structure-activity relationship. Two clinical mutations (K44E and E320Q) in hTHTR-2 have been described in patients with thiamin-responsive Wernicke’s-like encephalopathy. Experimental testing of these two mutants showed impaired functionality of hTHTR-2 in both cases. The K44E mutant caused thiamin transport dysfunction by impairing hTHTR-2 trafficking to the cell membrane. Another two mutations in the hTHTR-2 protein (G23V and T422A) have been reported in patients with biotin-responsive basal ganglia disease (although it is unclear at present as to why mutations in a specific thiamin transporter precipitates this condition despite the fact that hTHTR-2 does not transport biotin; Ref. ). Both of these mutations in hTHTR-2 were found to cause functional impairment, with no defect in the expression of this transporter at cell membrane. It is interesting to mention here that both of the affected residues (i.e., at positions 23 and 422) are conserved in the hTHTR-2 sequences of all species cloned so far, as well as in other members of the SLC19A gene family, that is, the hTHTR-1 and the (hRFC; product of the SLC19A1 gene), thus, supporting an essential role of these residues in transporter functionality. Hydropathy analyses suggested that these residues (G23V in TM1, T422A in TM11) lie within the TMDs of the hTHTR-2 protein. Crystallographic data from other major facilitator superfamily transporters showed that both TM1 and TM11 contribute to the central hydrophilic cavity of the transporters, underscoring the likelihood that mutation of residues within these regions could impair transporter function (reviewed in Ref. ). Other studies have examined the role of the three negatively charged conserved glutamic acid residues within the TMDs of hTHTR-1 and hTHTR-2 (i.e., E120, E320, and E346) as well as the role of the two putative N-glycosylation sites in the hTHTR-2 polypeptide (i.e., N45, N166) in the function and cell biology of the hTHTR-2 protein. Results of these investigations showed that mutating any of the conserved glutamic acid residues lead to a significant impairment in transport of the positively charged thiamin. Furthermore, while mutating E120 and E320 led to a decrease in the level of expression of hTHTR-2 at the apical membrane domain of intestinal epithelial cells, mutating E346 did not affect this parameter. Finally, the two putative glycosylation sites of the hTHTR-2 polypeptide (i.e., N45Q, N166Q) do not appear to affect the functionality or level of expression of the hTHTR-2 in intestinal epithelial cells.

With regard to the colonic TPPT, recent studies have shown that the protein is glycosylated at positions N69, N155, N197, N393, and N416. However, only glycosylation at N69, N155, and N393 appears to be important for function, most likely through an effect on protein conformation and/or interaction with the substrate.

54.2.6

Regulation of the Intestinal Thiamin Uptake Process

The intestinal thiamin uptake process appears to be under the regulation of a variety of extracellular/environmental conditions and intracellular factors. Since a number of these conditions/factors exert their regulatory effect(s) at the transcription level, it is relevant to first describe the basal transcriptional regulation of the SLC19A2, SLC19A3 , and SLC44A4 genes.

54.2.7

Conditions/Factors That Negatively Impact the Intestinal Thiamin Uptake Process

54.3.1

Metabolic Role and Effect of Deficiency

Riboflavin (RF; Fig. 54.1 ) is required for normal cellular metabolism, proliferation, and growth. In its biologically active forms [flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD)], the vitamin plays a key metabolic role in the transfer of electrons in biological oxidation-reduction reactions involving carbohydrate, lipid, amino acid, and certain water-soluble vitamins (pyridoxine and folate). Studies have also shown a role for riboflavin in protein folding in the endoplasmic reticulum and in the regulation of cellular energy expenditure. More recent investigations have demonstrated antioxidant and antiinflammatory properties for riboflavin, as well as a role for this vitamin in normal immune function. Riboflavin deficiency leads to a variety of clinical abnormalities, including degenerative changes of the nervous system, anemia, skin lesions, cataract, and growth retardation. It also leads to an increase in the susceptibility to cancer. Deficiency/suboptimal levels of riboflavin occur in chronic alcoholism, diabetes mellitus, IBD, Brown-Vialetto Van Laere, and Fazio Londe syndromes [the latter are rare neurological disorders caused by mutations in riboflavin transporters-2 and -3 (RFVT-2 and RFVT-3)]. In contrast to the deleterious effect of riboflavin deficiency, optimizing riboflavin status has been reported to reduce the risk of esophageal squamous cell carcinoma (Ref. and references therein).

54.3.2

Physiology of the Intestinal Riboflavin Absorption Process

The human intestine encounters two sources of RF: a dietary source and a microbiota source. Dietary riboflavin exists mainly in the form of FMN and FAD and is bound (noncovalently) to proteins. These coenzymes are first released from proteins via the combined action of gastric acid and hydrolases and then hydrolyzed further (prior to absorption) by intestinal phosphatases to free riboflavin. The mechanism of absorption of free riboflavin has been studied using a variety of human and animal intestinal preparations. Collectively, these studies have shown that transport of free riboflavin occurs mainly in the proximal part of the small intestine and involves an efficient and specific Na ⁺ -independent, carrier-mediated mechanism. This mechanism is inhibited by riboflavin structural analogues and by the Na ⁺ /H ⁺ exchange inhibitor amiloride ( K _i for amiloride ≈ 0.48 mM). Other studies have shown that the intestinal riboflavin uptake process is sensitive to the effect of the tricyclic phenothiazine drug chlorpromazine, a compound that shares structural similarities with riboflavin. Once internalized, some of the riboflavin is phosphorylated and used by enterocytes, and the rest is transported out of the cell across the BLM via a specific carrier-mediated process.

With regard to the microbiota-generated riboflavin in the large intestine, the amount of the vitamin produced depends on the type of the ingested diet and is higher following ingestion of a vegetable-based diet compared to a meat-based diet. Also, a considerable amount of the microbiota produced riboflavin was found to exist in the large intestinal lumen in the form of free riboflavin, and thus, available for absorption. Other investigations have shown that the large intestine is capable of absorbing luminally introduced free riboflavin. Insight into the mechanism involved in colonic uptake of riboflavin came from studies using the human colonic epithelial NCM460 cells, which showed the involvement of an efficient and specific carrier-mediated mechanism that is similar to the one described in the small intestine. Subsequent studies in rats have confirmed the existence of a specialized carrier-mediated mechanism for riboflavin uptake in the colon. With the demonstration of existence of an efficient carrier-mediated system for riboflavin uptake in the large intestine, the belief that this source of riboflavin contributes to the overall host nutrition of the vitamin, and especially toward the cellular nutrition of the localized colonocytes, is further strengthened.

54.3.3

Molecular Biology of the Intestinal Riboflavin Uptake Process

Insight into the molecular identity of the transport systems involved in intestinal riboflavin uptake has recently emerged with the identification of three putative human riboflavin transporters-1, -2, and -3 (RFVT-1, RFVT-2, and RFVT-3) that are encoded by three different but homologous genes: the SLC52A1 , SLC52A2 , and SLC52A3 genes, respectively. These transporters exhibit no similarity with the yeast or bacterial riboflavin transporters, nor do they show similarity with any other mammalian transporter. The hRFVT-1 and hRFVT-2 share > 87% sequence similarity at the protein level, while hRFVT-3 shares around 43% similarity with hRFVT-1 and hRFVT-2. The hRFVT-1, -2, and -3 are all expressed in the gut, with expression of the hRFVT-3 being significantly higher than that of hRFVT-1 and hRFVT-2 ; hRFVT-3 is also more efficient in transporting riboflavin than the other hRFVTs. Furthermore, live-cell confocal imaging of polarized intestinal (and renal) epithelial cells showed the hRFVT-3 to be predominantly expressed at the apical membrane domain, while expression of hRFVT-1 is mostly at the BLM domain of these cells; expression of hRFVT-2 was localized inside intracellular vesicles (with some expression at the BLM domain).

The relative contribution of the predominantly expressed riboflavin transporter in the intestine (i.e., RFVT-3) toward total carrier-mediated riboflavin uptake has also been recently addressed using an in vitro SLC52A3 gene-specific silencing siRNA approach and human intestinal epithelial Caco-2 cells with the results showing a major role for these transporters in intestinal riboflavin uptake. This was confirmed in vivo in studies utilizing an intestinal-specific (conditional) RFVT-3-knockout (cKO) mouse model developed by the Cre/Lox approach ( Fig. 54.7 ). In the latter study, all the RFVT-3 cKO mice developed systemic riboflavin deficiency indicating that RFVT-3 is involved in determining the overall riboflavin body homeostasis. Also observed was a significant increase in the level of expression of oxidative stress-responsive genes in the intestine of the cKO mice, which suggest a role for riboflavin in the maintenance of normal intestinal physiology.

Effect of conditional (intestinal-specific) knockout of the RFVT-3 on intestinal riboflavin absorption. Severe inhibition in carrier-mediated riboflavin absorption, but not in absorption of the unrelated biotin (B), in jejunal loops of cKO mice in vivo was evident compared to absorption in sex-matched litter-mates. WT , wild type; KO , knock out.

(From Subramanian VS, Lambrecht N, Lytle C, Said HM. Conditional (intestinal-specific) knockout of the riboflavin transporter-3 (RFVT-3) impairs riboflavin absorption. Am J Physiol 2016; 310 :G285–92.)

54.3.4

Cell biology of the Intestinal Riboflavin Absorption Process

As mentioned earlier, the RFVT-3 is expressed exclusively at the apical membrane domain of intestinal epithelial cells. What dictates cell surface expression of the protein appears to be a sequence in the C-terminal portion of the polypeptide that includes two conserved cysteine residues (one at position 463 and the other at position 467). Mutating these cysteine residues was shown to lead to severe impairment in riboflavin uptake due to retention of the protein in the endoplasmic reticulum. A potential disulfide bridge between C463 and C386 (also predicted by modeling analysis and mutating the C386 residue) was found to lead to intracellular retention of RFVT-3. Intracellular trafficking of RFVT-3 was shown to involve distinct vesicular structures whose mobility depends on existence of an intact microtubule network.

54.3.5

Structure-Activity Aspects of the Riboflavin Transporters

Early studies using Caco-2 cells exposed to specific sulfhydryl group reagents have reported a role for sulfhydryl groups in the function of the intestinal riboflavin uptake carriers. Since the sulfhydryl group reagents used in these studies were membrane impermeable, it was assumed that the affected sulfhydryl group(s) were located at the exofacial surface of the cell membrane. More recent investigations examined the effect of clinical mutations in SLC52A3 found in patients with the Brown-Vialetto-Van Laere syndrome (BVVLS; a rare neurological disease characterized by ponto-bulbar palsy, bilateral sensorineural deafness, and respiratory insufficiency) on cell biology of RFVT-3. Mutants P28T, E36K, E71K, and R132W were all found to be functionally impaired and that the impairment was due to retention of the mutated RFVT-3 within the endoplasmic reticulum. These findings suggest a role for the affected residues in normal cell biology of RFVT-3.

54.3.6

Regulation of the Intestinal Riboflavin Uptake Process

The intestinal riboflavin uptake process is under the regulation of extracellular/environmental conditions and intracellular factors. Since a number of these conditions exert their effect(s) at the transcription level, it is relevant to first describe the basal transcriptional regulation of the intestinally relevant SLC52A1 and SLC52A3 genes.

54.3.7

Factors and Conditions That Negatively Impact the Intestinal Riboflavin Uptake Process

Chronic alcohol consumption in humans is associated with a higher prevalence of riboflavin suboptimal/deficient levels. This appears to be mediated, at least in part, via interference with intestinal riboflavin absorption process as shown in studies utilizing rats chronically fed an alcohol liquid diet. These studies showed that chronic alcohol feeding leads to a significant inhibition in carrier-mediated riboflavin uptake across both the jejunal brush-border and BLM. This inhibition was found to be associated with a parallel reduction in the expression of RFVT-1 and RFVT-3 at the protein, mRNA, and hnRNA levels. The latter suggest that the inhibitory effect of chronic alcohol feeding is mediated, at least in part, at the level of transcription of the slc52a1 and slc52a3 genes. Chronic alcohol feeding was also shown to inhibit colonic uptake of riboflavin. Thus, absorption of both dietary- and microbiota-generated riboflavin appears to be negatively impacted by chronic alcohol exposure.

Other studies have shown that the Na ⁺ /H ⁺ exchanger amiloride and the tricyclic phenothiazine agent, chlorpromazine (which shares structural similarities with RF), inhibit intestinal riboflavin uptake.

54.4.1

Metabolic Role and Effect of Deficiency

The main function of niacin ( Fig. 54.1 ) is as a precursor for the synthesis of the coenzymes nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). Most of the NAD- and NADP-linked enzymes are involved in reactions that maintain the redox state of cells such as glycolysis and the pentose phosphate shunt. Niacin also appears to have lipid-lowering effects and is in use clinically for that purpose. Severe niacin deficiency in humans leads to pellagra, a disease characterized by inflammation of mucous membranes, skin lesions, and diarrhea. Deficiency and suboptimal levels of niacin occurin chronic alcoholism and in patients with Hartnup’s disease. Patients with the latter disorder have mutations in the gene that encodes the transporter of the amino acid tryptophan, which is a precursor for the endogenous production of niacin.

54.4.2

Physiology of the Intestinal Niacin Absorption Process

Humans have access to niacin from endogenous and exogenous sources. The endogenous source of niacin is provided via the metabolic conversion of tryptophan to niacin via the so called “kunurenine pathway.” The exogenous source is provided through the diet and via production of the vitamin by the large intestinal microbiota. Our knowledge of the mechanism of intestinal absorption of dietary niacin has expanded in recent years. Early animal studies have concluded that niacin is taken up by intestinal absorptive cells either via simple diffusion of the undissociated form of nicotinic acid (i.e., according to the pH partition hypothesis and is assisted by acidic microclimate at the luminal surface of the intestine; p K _a of nicotinic acid is 4.9), or via a carrier-mediated mechanism involving a monocarboxylate transporter(s). The latter studies, however, have reported an apparent K _m of 3.52–17.0 mM. Such a high apparent K _m raises a concern with regard to the physiological relevance of the described system, because intestinal luminal concentration of niacin under physiological conditions is estimated to be in the micromolar range. Recent studies, however, using human intestinal epithelial Caco-2 cells and purified intestinal BBMV isolated from human organ donors have shown the involvement of an acidic pH-dependent (but Na ⁺ -independent) high-affinity carrier-mediated mechanism for niacin uptake (apparent K _m of 0.53 μM). This system is highly specific for niacin and is not affected by monocarboxylic acids. In addition, the system was highly sensitive to the effect of membrane-impermeable sulfhydryl group reagents and appears to be under the regulation of an intracellular protein tyrosine kinase (PTK)-mediated signaling pathway. A similar high affinity carrier-mediated system (apparent K _m of 0.73 ± 0.16 μM) was subsequently described for niacin uptake in human liver cells. Currently, there is nothing known about the molecular identity of the intestinal niacin uptake system or about how the vitamin exits the polarized enterocytes across the BLM.

As to the microbiota-generated niacin in the large intestine, recent studies utilizing human colonic epithelial NCM460 cells, native human colonic apical membrane vesicles (AMV) isolated from organ donors, and mouse colonic loops in vivo as models have shown the existence of an acid pH (but not Na ⁺ dependent) carrier-mediated (apparent K _m of 2.5 μM) process for niacin uptake. The process was also shown to be probenecid sensitive, but was not affected by other bacterially produced monocarboxylates. Furthermore, the colonic niacin uptake process was found to be under the regulation of an intracellular PTK- and a Ca ^{2 +} -calmodulin-mediated regulatory pathways; it was also adaptively regulated by the prevailing substrate level in the extracellular environment.

54.5.1

Metabolic Role and Effect of Deficiency

Pantothenic acid ( Fig. 54.1 ) is required for the biosynthesis of coenzyme A and acyl carrier proteins in mammalian tissues, and thus is involved in the metabolism of carbohydrate, fat, and to a lesser extent protein. Spontaneous pantothenic acid deficiency has not been reported in humans, most likely because of the ubiquitous distribution of the vitamin.

54.5.2

Physiology of the Intestinal Pantothenic Acid Absorption Process

The intestinal tract is exposed to two sources of pantothenic acid, one being dietary and the other is bacterial (i.e., microbiota-generated vitamin; Ref. ). Dietary pantothenate exists mainly in the form of coenzyme A, which is hydrolyzed to free pantothenic acid in the intestinal lumen prior to uptake. Free pantothenic acid is then transported into the absorptive cells via the sodium-dependent multivitamin transporter, SMVT, which also transports biotin and lipoate (Refs. ; see also Section 54.7 ). Absorption of the microbiota-generated pantothenic acid in the large intestine also appears to utilize the SMVT system. There is little known about regulation of the intestinal pantothenic uptake process, but it may be similar to the regulation of intestinal biotin uptake process since the two vitamins share the same intestinal uptake system. There is nothing known at present regarding the mechanism of exit of pantothenic acid out of the intestinal absorptive cells.

54.6.1

Metabolic Role and Effect of Deficiency

Pyridoxine ( Fig. 54.1 ) and other members of the B ₆ family (pyridoxal and pyridoxamine) exist in varying proportions in the diet in both the phosphorylated and nonphosphorylated forms. Pyridoxal 5′-phosphate represents the most biologically active form of the vitamin and is a cofactor for enzymes that catalyze transaminase, decarboxylase, and synthetase reactions in pathways that include carbohydrate, lipid, protein (including metabolism of the amino acid homocysteine), and in production of neurotransmitters. Vitamin B ₆ deficiency in humans leads to a variety of clinical abnormalities that includes microcytic anemia, dermatitis/glossitis, and neurological disorders. Deficiency/suboptimal levels of vitamin B ₆ occur in conditions such as chronic alcoholism and in diabetes, in patients with celiac and renal diseases, and following long-term use of hydrazines (e.g., isoniazid, which is used for treatment of tuberculosis) as well as penicillamine. Patients with vitamin B ₆ -dependent seizure, an autosomal-recessive disorder (believed to be due to an abnormality in pyridoxine transport into cells), also shows low blood levels of vitamin B _6.

54.6.2

Physiology of Intestinal Vitamin B ₆ Transport

Vitamin B ₆ is synthesized by plant cells and by most unicellular microorganisms. However, humans and other mammals cannot synthesize the vitamin, and thus must obtain the micronutrient from exogenous sources via intestinal absorption. As with a number of other water-soluble vitamins, two sources of vitamin B ₆ are available to the intestine: a dietary source and a bacterial source (the latter is in references to the vitamin generated by the microbiota of the large intestine; Ref. ). Dietary phosphorylated forms of vitamin B ₆ are hydrolyzed to free forms prior to absorption. A variety of intestinal preparations have been used to study the mechanisms(s) of intestinal absorption of vitamin B ₆ . Some studies have reported inability of the intestine to accumulate vitamin B _6, while others have reported lack of a saturable uptake process. This led to the conclusion that intestinal vitamin B ₆ uptake is via simple diffusion process. This conclusion, however, has been challenged by Said et al. who used human intestinal epithelial Caco-2 cells to show the existence of an efficient and specific carrier-mediated mechanism for pyridoxine uptake. The latter studies have also shown that the intestinal uptake process of pyridoxine is highly dependent on acidic extracellular pH, but is Na ⁺ -independent. In addition, amiloride was found to inhibit (in a competitive manner) pyridoxine uptake by intestinal epithelial cells (apparent K _i of 0.39 mM). These findings on the involvement of carrier-mediated pyridoxine uptake process in intestinal epithelial cells are similar to those reported for the vitamin uptake by renal epithelial cells. Other recent investigations have reported an important role for the intestine in vitamin B ₆ metabolism.

With regard to the microbiota-generated vitamin B ₆ , a significant amount of this source of vitamin exists in the free form in the surrounding medium (i.e., not trapped inside bacterial cells), and thus is available for absorption. Evidence suggesting that this source of vitamin B ₆ is indeed bioavailable to the host comes from studies showing that the amount of vitamin B ₆ excreted in the urine is significantly higher than the total amount of the vitamin consumed orally. Direct evidence for the ability of the colonic epithelial cells to absorb vitamin B ₆ came from studies using mouse and human colonic preparations, which showed the involvement of an efficient and specific carrier-mediated mechanism (apparent K _m of 2.1 μM) for pyridoxine uptake.

Currently, there is little known about the molecular identity of the intestinal pyridoxine carrier in any species. However, the structure of the first vitamin B ₆ transporter in any living organism ( Saccharomyces cerevisiae ) has been reported. This system was predicted to have 12 TMDs and appears to be regulated by substrate availability in the extracellular environment.

54.6.3

Regulation of the Intestinal Vitamin B ₆ Uptake Process

The intestinal vitamin B ₆ uptake process appears to be under the regulation of intracellular and extracellular factors. A role for a PKA-mediated signaling pathway in regulating intestinal pyridoxine uptake has been reported. In the latter study, pretreatment of Caco-2 cells with compounds that increase in intracellular cAMP levels, and thus activate PKA, caused a significant inhibition in pyridoxine uptake. This inhibition was mediated via a decrease in the activity (and/or the number), but not affinity, of the pyridoxine uptake carriers. The molecular mechanism through which cAMP exerts its effect on intestinal pyridoxine uptake process, however, is not clear and requires further investigations.

The intestinal pyridoxine uptake process is also adaptively regulated by the prevailing extracellular pyridoxine levels, with a significant induction in the uptake occurring in cells maintained in the presence of low pyridoxine level compared to those maintained in the presence of high level. The mechanism involved in this adaptive regulation appears to be, at least in part, mediated via transcriptional mechanism(s).

54.7.1

Metabolic Role and Effect of Deficiency

Biotin ( Fig. 54.1 ) is an indispensable micronutrient for normal human health due to its essential nature for cellular metabolism, proliferation, and survival. Biotin acts as a cofactor for five carboxylases that are critical for fatty acid, glucose, and amino acid metabolism. Important roles for biotin in cellular energy metabolism (i.e., ATP production) and in regulation of cellular oxidative stress, as well as in gene expression (where expression of over 2000 human genes appears to be affected by biotin status ) have also been reported. Emerging evidence has also been accumulating documenting an important role for biotin in the function of the immune system. In reference to the latter, biotin was shown to be important for activity of the human dendritic cells and the human natural killer (NK) lymphocytes, for the generation of cytotoxic T lymphocytes, and for the maturation and responsiveness of immune cells. An increase in the levels of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1b, and interferon-γ (IFN-γ) has been observed in biotin deficiency. Biotin also appears to play an important role in the maintenance of normal intestinal integrity and homeostasis as its deficiency leads to an increase in intestinal permeability and changes in level of expression of tight junction (TJ) proteins, as well as to spontaneous inflammation in the large intestine. Biotin also appears to influence the colonization and invasiveness of certain enteropathogenic bacteria, and it mediates the effect of probiotic bacteria on microbial community in the gut. Finally, animal studies have shown that biotin deficiency during pregnancy could lead to embryonic growth retardation, congenital malformation, and death. Thus, it is not surprising that deficiency/suboptimal levels of this vitamin lead to disturbances in the normal function of many organ systems and ultimately to clinical manifestations of disease. The latter includes dermatological abnormalities, neurological disorders, and growth retardation (reviewed in Refs. ). Deficiency/suboptimal levels of biotin occur in conditions including IBD, chronic alcoholism, in patients on long-term therapy with anticonvulsant drugs, and those receiving long-term parenteral nutrition. In addition, inborn errors in biotin metabolism (multiple carboxylase deficiency ) and patients with biotin-responsive basal ganglia disease (an autosomal recessive disease) appear to benefit from biotin supplementation implying suboptimal cellular levels of the vitamin.

54.7.2

Physiology of the Intestinal Biotin Absorption Processes

The human intestine is exposed to two sources of biotin: a dietary source and a bacterial source (the latter is in reference to the biotin that is generated by the gut microbiota). Dietary biotin exists both in free and protein-bound forms. The protein-bound form is first digested to biocytin (biotinyl- l -lysine) and biotin-short peptides via the action of gastrointestinal (GI) proteases and peptidases. Biocytin and biotin-short peptides cannot be absorbed as such (cannot be recognized by the intestinal biotin uptake system) until they are further digested into free biotin, a process performed by biotinidase, an enzyme that is believed to be of pancreatic origin. Biotinidase has been cloned, and a number of clinical mutations have been identified in its sequence in patients with the autosomal recessive disorder “biotinidase deficiency.” Patients with the latter disorder display neurological and cutaneous abnormalities. These patients cannot convert biocytine or biotin-short peptides to free biotin in the intestinal lumen nor can they recycle endogenous biocytin (and biotin-short peptides) that is generated from catabolism of biotin-bound enzymes/proteins to free biotin in different tissues. This leads to a reduction in the fraction of dietary biotin that is bioavailable/absorbed, and to a reduction in ability of different cells to take in sufficient amounts of this vitamin. Supplementation with high pharmacological doses of “free” biotin brings about a favorable clinical outcome in these patients as; such a treatment could prevent the development of the abnormal clinical symptoms if initiated early.

Absorption of free biotin (a substrate that is negatively charged at physiological pH) in the small intestine occurs via an efficient Na ⁺ -dependent carrier-mediated mechanism; absorption is higher in the proximal compared to the distal part of the small intestine. Functional, immunological, and live-cell confocal imaging studies have localized the Na ⁺ -dependent, carrier-mediated, biotin uptake system at the apical BBM domain of the polarized enterocytes. The exit of biotin from the polarized absorptive cells across the BLM is also carrier-mediated, but is Na ⁺ -independent in nature. One of the interesting features of the intestinal Na ⁺ -dependent biotin absorption process is its ability to transport not only free biotin but also two other functionally unrelated nutrients, namely, the water-soluble vitamin pantothenic acid (which is important for energy, fat, and protein metabolism) and the metabolically important substrate, lipoate (a potent intracellular and extracellular antioxidant). It is for the latter reason that the uptake system involved in the uptake of biotin is referred to as the “sodium-dependent multivitamin transporter,” or “SMVT.”

With regard to the biotin generated by the gut microbiota, a substantial portion of this biotin exists in the free form, and thus available for absorption. While gut microbiota of all humans generates biotin, recent studies that classified the human gut microbiome into three functional enterotypes of which enterotype 1 is especially overrepresented in enzymes involved in the biotin biosynthetic pathway, therefore, implying a larger level of biotin generation in the group that harbors this enterotype. As to absorption of the microbiota-generated biotin, both human and animal colonocytes are capable of absorbing the vitamin via the same SMVT-dependent process that operates in the small intestine. The demonstration of the existence of a carrier-mediated mechanism for absorption of the microbiota-generated biotin in the large intestine clearly suggests that this source of biotin is bioavailable and contributes toward overall host biotin nutrition, and especially toward the cellular nutrition of the local colonocytes. The latter may have consequences in relation to the maintenance of normal colonic health.

54.7.3

Molecular Biology of the Intestinal Biotin Uptake Process

The SMVT system (product of the SLC5A6 gene) has been cloned from the intestine of a number of species including human. Functional identity of the cloned SMVT was established by expression in a number of heterologous cellular systems, where the protein was found to transport both biotin and pantothenic acid in a Na ⁺ -dependent manner and with similar kinetic parameters as those determined in native intestinal preparations (reviewed in Ref. ). Functional, immunological, and confocal imaging studies have shown that the protein is expressed exclusively at the apical membrane domain of the polarized intestinal epithelial cells ( Fig. 54.8 ). The SMVT polypeptide is predicted to have 12 TMD and is N-glycosylated at N138 and N489. This glycosylation appears to be important for function of the SMVT system. In addition, putative phosphorylation sites for several intracellular protein kinases are predicted in the SMVT polypeptide; of these putative sites, the T286 site has been experimentally shown to be important for the protein kinase-C (PKC)-mediated regulation of biotin uptake.

Cellular distribution of hSMVT (fused to GFP) in human intestinal epithelial Caco-2 cells and expression of the hSMVT protein in apical membrane of native human colonocytes. (A) XY and Z confocal image showing Caco-2 cell expressing hSMVT-EGFP and dsRed (a cytoplasmic dye) 48-h posttransfection. Lower panels show distribution of GFP alone. (B) Western blot analysis showing expression of hSMVT protein at native human colonic apical (but not basolateral) membrane (colonic tissue was obtained from organ donors).

(From Subramanian VS, Marchant JS, Boulware MJ, Ma TY, Said HM. Membrane targeting and intracellular trafficking of the human sodium-dependent multivitamin transporter in polarized epithelial cells. Am J Physiol 2009; 296 :C663–71.)

While another potential biotin transport system has been suggested in other tissues, the SMVT system appears to be the only system that operates in the mammalian intestinal tract. The latter conclusion is based on recent findings from in vitro studies utilizing gene-specific silencing (siRNA) approach, and in vivo investigations utilizing conditional (intestinal-specific) SMVT-knockout (cKO) mouse model, which is generated by utilizing Cre/lox technology. In the latter studies, a complete inhibition of gut biotin (and pantothenic acid) uptake in the SMVT-cKO mice was observed. Also, all the cKO animals developed biotin deficiency, severe growth retardation, and decreased bone density and length. Furthermore, two-third of the cKO animals died prematurely prior to reaching the age of 10 weeks. An unexpected observation found in all the SMVT-cKO animals was the development of a spontaneous intestinal chronic active inflammation, especially in the cecum ( Fig. 54.9 ). This inflammation is reminiscent to that seen in IBD. The latter finding suggests that the SMVT system plays a role in the maintenance of normal intestinal mucosal integrity and immunity. The suggestion was confirmed by subsequent investigations showing a significant increase in gut permeability and marked changes in level of expression of TJ proteins in the intestine of the SMVT-cKO mice ( Fig. 54.10 ). These changes were found to be related to the state of biotin deficiency that develops in the SMVT-cKO mice, as shown in studies utilizing wild-type mice made biotin deficient by dietary means. In the latter studies, chronic active inflammation also developed in the cecum of the biotin-deficient mice compared to their pair-fed controls ( Fig. 54.11 ); and was associated with a significant increase in the level of expression of proinflammatory cytokines in the cecal mucosa, an increase in intestinal permeability, and changes in level of expression of TJ proteins ( Fig. 54.12 ). Since no changes in permeability and expression of TJ proteins occur in confluent intestinal epithelial Caco-2 monolayers maintained under chronic biotin-deficient conditions, it was suggested that the role of intestinal SMVT in the maintenance of normal mucosal integrity is most likely mediated via its function in providing biotin to immune cells of the gut mucosa. The latter is likely given the roles played by biotin in maintaining normal innate and adaptive immune functions.

Histology of the small intestine (A–C) and cecum (D–F) of conditional (intestinal-specific) SMVT KO mice and their sex-matched littermates. Small intestine: (A) normal small intestinal morphology of the wild-type littermates, (B) shortening of the villi and focal dysplastic changes (B insert). (C) Small intestinal villi length in mm (left y -axis) and total area of dysplasia as percentage (right y -axis). Cecum: A representative section of wild-type cecum (D) and that of KO mouse showing significant submucosal edema (open arrow) and acute inflammation involving surface (closed arrows) and crypts (D insert). (F) Number of neutrophils in 10 high power fields (400 ×, left y -axis), and total area of dysplasia and submucosal edema as percentage (right y -axis).

(From Ghosal A, Lambrecht N, Subramanya SB, Kapadia R, Said HM. Conditional knockout of the Slc5a6 gene in mouse intestine impairs biotin absorption. Am J Physiol 2013; 304 :G64–71.)

Effect of conditional (intestinal-specific) SMVT KO on level of expression of TJ proteins in the cecum. Cecal tissue lysates were used for Western blot and the blots were probed with anticlaudins-1 and -2, ZO-1, and MLCK antibodies. The expression of TJ proteins was normalized relative to β-actin. The graphs show relative protein expression of (A) claudin-1, (B) claudin-2, (C) ZO-1, and (D) MLCK in the cecum of cKO mice and their sex-matched littermates.

(From Sabui S, Bohl J, Kapadia R, Cogburn K, Ghosal A, Lambrecht N, et al. Role of sodium-dependent multivitamin tranporter (SMVT) in the maintenance of intestinal mucosal integrity. Am J Physiol Gastrointest Liver Physiol 2016 [in press].)

Histology of the cecum (A–D) of biotin-deficient mice and their pair-fed controls. A representative section of cecum of a biotin deficient mouse (A, B) and its pair-fed control (C, D); hematoxylin and eosin (H&E), 40 × (A, C), 200 × (B, D). (C, D) Normal morphology of the pair-fed control cecum. (A, B) Significant cryptitis and neutrophils within epithelial crypt (arrows) .

(From Sabui S, Bohl J, Kapadia R, Cogburn K, Ghosal A, Lambrecht N, et al. Role of sodium-dependent multivitamin tranporter (SMVT) in the maintenance of intestinal mucosal integrity. Am J Physiol Gastrointest Liver Physiol 2016 [in press].)

Effect of dietary-induced biotin deficiency on level of expression of TJ proteins in mouse cecum. The graphs show relative expression of Western blot analysis of claudin-1 (A), claudin-2 (B), ZO-1 (C), and MLCK (D) proteins in the cecum of biotin-deficient mice and their pair-fed controls. Data are mean ± SE of at least three separate pairs of biotin-deficient mice and their pair-fed controls (* P < 0.05; ** P < 0.01).

54.7.4

Cell Biology of the Intestinal Biotin Absorption Process

As mentioned earlier, the SMVT protein is expressed exclusively at the apical membrane domain of intestinal epithelial cells ( Fig. 54.8 ). What determines targeting of the hSMVT protein to the BBM of these cells has been investigated using live-cell confocal imaging and shown to be dictated by a sequence(s) in the C-terminal tail of the polypeptide. Furthermore, distinct trafficking vesicles were found to be involved in the intracellular movement of SMVT, and that mobility of these vesicles appears to be dependent on the existence of intact microtubule network.

The SMVT protein also appears to have an interacting partner, the PDZ domain containing 11 (PDZD11) in intestinal epithelial cells that affects its function ( Fig. 54.4 ). The interaction between SMVT and its partner was shown in studies utilizing yeast two-hybrid screening approach, and was confirmed by in vitro (by GST-pull-down assay), in vivo (by two-hybrid luciferase and coimmunoprecipitation assays), and confocal imaging of living cells. Functional consequence of this interaction was shown in studies involving coexpression of the SMVT with PDZD11, which led to an increase in biotin uptake; on the other hand, knocking down PDZD11 was found to lead to an inhibition in the vitamin uptake. Finally, the interaction of PDZD11 with the SMVT polypeptide was found to occur at the PDZ-binding domain of the transporter located at its cytoplasmic tail.

54.7.5

Structure-Activity Aspects of the hSMVT

Knowledge about the structure – function relationship of the hSMVT has also been forthcoming in recent years. In one study, the role for each of the 10 conserved (among species) cysteine residues in the function of hSMVT has been examined following their individual mutation by mean of site-directed mutagenesis. Only C294 was found to be essential for functionality, and its role appears to be mediated via insuring a proper expression of the hSMVT protein at the cell surface as shown by biotinylation assay.

Other studies have shown that the hSMVT polypeptide is glycosylated at positions N138 and N489 and that this glycosylation is important for its function.

54.7.6

Regulation of Intestinal Biotin Absorption Process

A variety of extracellular/environmental conditions and intracellular factors appear to regulate the intestinal biotin uptake process. Since a number of these conditions/factors work via transcriptional mechanism(s), it is relevant to first describe the basic transcriptional regulation of the SLC5A6 gene (which encodes SMVT) in human intestinal epithelial cells. In this regard, the 5′-regulatory region (promoter) of the human SLC5A6 genes was found to have two promoters (promoters 1 and 2). Activity of these promoters was confirmed in vitro using the Firefly luciferase reporter gene assay following transient transfection into human intestinal epithelial cells, and in vivo in transgenic mice carrying the hSMVT promoter-luciferase construct. A role for the nuclear factors Kruppel-like factor 4 (KLF-4) and activator protein (AP-2) in regulating the activity of the SLC5A6 promoter has also been reported.

54.7.7

Factors That Affect the Intestinal Biotin Uptake Process

A significant reduction in plasma biotin levels has been observed in chronic alcoholics. Studies utilizing animal models that were fed alcohol chronically have shown that this, at least in part, is mediated via inhibition in intestinal biotin uptake. This inhibition was associated with a significant reduction in expression of SMVT at the protein, mRNA, and hnRNA levels as well as a decrease in the activity of the SLC5A6 promoter in transgenic animals carrying this human construct. Chronic alcohol feeding also caused a significant inhibition in colonic uptake of biotin suggesting that uptake of the bacterially generated biotin is also negatively impacted by the chronic alcohol exposure. Similarly, findings were observed in studies utilizing human intestinal epithelial cells chronically exposed to alcohol. It is worthwhile mentioning that chronic alcohol exposure also inhibits biotin uptake by other cell types of the digestive system, namely, pancreatic acinar cells, as well as renal reabsorption process of the vitamin. The alcohol-mediated inhibition in biotin uptake in the case of pancreatic acinar cells was found to be mediated at the transcriptional level of the Slc5a6 gene and involves changes in the methylation status of the CPG islands predicted in the promoter of this gene and a decrease in the level of expression of the transcription factor KLF4,which plays an important role in regulating Slc5a6 transcription.

Other studies have shown that intestinal infection with Salmonella enterica serovar Typhimurium ( S. typhimurium ) inhibits gut biotin uptake, and that the inhibition is mediated via the action of proinflammatory cytokines. In the latter investigation, infecting mice with wild-type S. typhimurium (but not with its nonpathogenic isogenic invA spiB mutant) led to a significant inhibition in jejunal/colonic biotin uptake and in the level of expression of SMVT. No such inhibition in biotin uptake, however, was seen when cultured intestinal cells were infected with S. typhimurium suggesting that the effect of S. typhimurium infection in vivo is indirect and is likely mediated by proinflammatory cytokines; indeed, levels of these cytokines were found to be markedly induced in the intestine of the S. typhimurium -infected mice. Consistent with this suggestion was the observations that exposure of cultured colonic epithelial cells to the proinflammatory cytokines TNF-α and IFN-γ led to a significant inhibition in biotin uptake as well as in level of expression SMVT and activity of the SLC5A6 promoter. The inhibitory effect of proinflammatory cytokines on biotin uptake and on activity of the SLC5A6 promoter appeared to be mediated, at least in part, via the nuclear factor kappa-B signaling pathway.

Exposure of gut epithelia to the endotoxin lipopolysaccharide (a major component of the outer membrane of gram-negative bacteria that is released from bacterial cell walls by shedding or through bacterial lysis) has also been shown to lead to inhibition in colonic biotin uptake. This effect appears to be mediated via interference with the level of expression of the SMVT protein at the cell surface and is mediated via protein CK-2 (Lakhan R and Said HM; manuscript in preparation).

Other factors that interfere with the intestinal biotin uptake process are anticonvulsant drugs like carbamazepine and primidone.

54.8.1

Metabolic Role and Effect of Deficiency

Folates refer to a group of one-carbon derivatives of the folic acid ( Fig. 54.1 ) that are required for the synthesis of pyrimidine and purine nucleotides, precursors of DNA and RNA, respectively. Folate is also involved in the metabolism of several amino acids including homocysteine. At the cellular level, folate deficiency leads to derangement of one-carbon metabolism, defect(s) in DNA synthesis and methylation, mis-incorporation of uracil into DNA, and disturbance in the metabolism of several amino acids. At the clinical level, folate deficiency and suboptimal levels are associated with a variety of abnormalities that include megaloblastic anemia, growth retardation, and neural tube defects in the developing embryo (the latter represents a relatively common birth defect in humans). In contrast, optimizing folate body homeostasis is associated with a marked reduction in the incidence of neural tube defects and may also provide protection against cardiovascular diseases and certain types of cancers. Folate deficiency/suboptimal levels are highly prevalent throughout the world and occur due to a variety of causes—including the impairment in intestinal absorption of the vitamin. A variety of conditions have been reported to affect/interfere with the normal intestinal folate absorption process such as the autosomal-recessive disorder “hereditary folate malabsorption syndrome” (HFMS), intestinal diseases (e.g., celiac disease, tropical sprue), drug interactions (e.g., sulfasalazine, trimethoprim, pyrimethamine, diphenylhydantoin), and chronic alcohol consumption.

54.8.2

Physiology of the Intestinal Folate Absorption Process

Two sources of folate are available to the gut: a dietary source (which is mainly processed and absorbed in the small intestine) and a gut microbiota-generated source. Dietary folates exist mainly in the free (i.e., folate monoglutamate) and polyglutamate forms, with the latter being the predominant form in the diet. The conjugated folate polyglutamates cannot be absorbed due to their size, multiple negative charges, and high hydrophilicity, and thus must be hydrolyzed to folate monoglutamate forms prior to uptake. This hydrolysis step is performed by the action of the enzyme folate hydrolase (also known as folylpoly-γ-glutamate carboxypeptidase). Two forms of the enzyme exist in intestinal epithelial cells. The first form is expressed at the apical BBM domain, and the second form is expressed intracellularly (most probably in the lysosomes). The BBM form of the enzyme is expressed mainly in the proximal part of the small intestine, while the intracellular form is expressed uniformly along the entire length of the small intestine. The intestinal BBM form of the folate hydrolase has been cloned and shown to be adaptively upregulated in folate deficiency ; this form is also developmentally regulated.

In describing the mechanism of folate uptake across biological membranes, it is important to mention here that the folate monoglutamate molecule is hydrophilic in nature and is negatively charged at physiological pH (p K _a values for its α and γ carboxyl groups are 3.5 and 4.8, respectively). The mechanism of dietary folate monoglutamates uptake, by the small intestine (which also applies to the folate that enters the small intestine via the enterohepatic cycling of the vitamin ), has been extensively studied using a variety of human and animal intestinal preparations (reviewed in Ref. ). Collectively, these studies have shown that absorption of dietary folate monoglutamates occurs mainly in the proximal part of the small intestine and involves a specific and Na ⁺ -independent carrier-mediated mechanism. This mechanism, however, is highly dependent on acidic extracellular pH with a markedly higher uptake at acidic (pH 5–6) compared to neutral/alkaline (pH 7–7.4) pH. Studies with purified intestinal BBMV have also shown that the folate uptake system at the apical BBM domain is concentrative and is sensitive to the inhibitory effect of the anion transport inhibitors 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS), and acetamidoisothiocyanostilbene-2,2′-disulfonic acid. The effect of extracellular acidic pH on intestinal folate uptake is in part mediated via providing a trans-membrane pH gradient (suggesting the involvement of a folate ⁻ :H ⁺ cotransport mechanism and possibly a folate ⁻ :OH ⁻ exchange mechanism) and in part via a direct effect of the acidic extracellular pH on the activity of the carrier(s) involved. Other studies have shown that the intestinal folate uptake process has similar affinity for reduced (e.g., 5-methyltetrahydrofolate, 5-formyltetrahydrofolate), oxidized (e.g., folic acid), and substituted (e.g., methotrexate) folate derivatives. The mechanism of folate exit out of the intestinal absorptive cells, that is, transport across the BLM, has also been characterized using purified intestinal BLMV and again shown to involve a specific and DIDS sensitive carrier-mediated mechanism.

As to the microbiota-generated folate in the large intestine, previous studies have shown that a substantial portion of this folate exists in the absorbable monoglutamate forms and that the large intestine is indeed capable of absorbing some of this vitamin. The mechanism of folate absorption in the large intestine has been investigated using purified colonic AMV isolated from the colon of human organ donors and human-derived nontransformed colonic epithelial NCM460 cells. The results showed the involvement of an efficient and specific carrier-mediated mechanism. The identification of such an efficient uptake mechanism, for the microbiota-generated folate in the human large intestine, supports the belief that this source of folate may contribute to the overall nutritional requirement host for the vitamin,and especially to the cellular nutrition of the local colonocytes. Interferences with this mechanism may contribute to the development of localized folate deficiency and premalignant changes in the colonic mucosa.

54.8.3

Molecular Biology of the Intestinal Folate Uptake Process

Mammalian cells utilize three distinct and specific systems to transport folates across their plasma membrane. These systems are the ubiquitously expressed reduced folate carrier (RFC; the product of the SLC19A1 gene), which operates optimally at neutral/alkaline pH of 7.0–7.4 ; the transmembrane proton-coupled folate transporter (PCFT; the product of the SLC46A1 gene), which operates optimally at acidic pH 5.5–6.0 (with minimal activity at pH 7 and above) ; and the membrane anchored (via a glycosyl phosphatidyl-inositol linkage) folate receptors (FRs), which operates optimally at neutral pHs. While both the RFC and PCFT systems are expressed and functional in the intestinal tract, the FRs are not expressed in the normal intestine.

The human RFC (hRFC) polypeptide consists of 591 amino acids and is predicted to have 12 putative TMDs with both of the C- and N-terminals facing the cell interior. In addition, it has a large central intracellular loop that connects TM domains (TMDs) 1–6 and 7–12, three putative PKC phosphorylation sites, and one potential N-glycosylation site. The RFC protein is predicted to carry a net positive charge at physiological pH, which may be important for its interaction with the negatively charged substrate; also, the protein is N-glycosylated at position N58. The hRFC shares high degree of sequence homology with RFC of other mammals (e.g., mouse, rat, hamster, and chimpanzee). Functional identity of the cloned RFC has been verified by expression in different mammalian cells as well as Xenopus oocytes. RFC functions as an anion exchanger (apparent K _m of 1–5 μM) moving the negatively charged folate molecule against its concentration gradient in exchange for downhill movement of an anion. Affinity of RFC is much higher for reduced folate derivatives (like the naturally occurring 5-methyltetrahydrofolate) than for oxidized folates (like the synthetic folic acid). The RFC protein is expressed at the BBM domain of the polarized enterocytes and at the apical membrane of colonocytes. Considering the optimal pH for function of this carrier and its distribution pattern along the intestinal tract, it is suggested that this transporter plays a role in folate uptake in the lower half of the small intestine and in the large intestine (where the pH at the intestinal surface is near the neutral range; Ref. ).

As to the hPCFT, this protein consists of 459 amino acids with 12 putative TMDs, two potential N-glycosylation sites, and with both the N- and C-terminals facing the cell interior. A critical role for PCFT in intestinal absorption of dietary folate has been established by the identification of loss-of-function mutations in the hPCFT gene in patients with HFMS, and in studies utilizing PCFT-knockout mouse model (Refs. and references therein). Folate transport mediated by PCFT is electrogenic and involves the net movement of a positive charge (proton), that is, it functions as a folate ⁻ :H ⁺ symport (Refs. and references therein). Folate is transported by this system against a concentration gradient utilizing the energy generated by the downhill movement of protons. The hPCFT mRNA is expressed mainly in the proximal part of the human small intestine with less expression in the distal small intestine and the large intestine. Furthermore, expression of the hPCFT is restricted to the apical membrane domain of the polarized enterocytes.

Since the PCFT and RFC systems are both expressed in the intestine (although at different levels in different regions of the gut) and both are localized at the apical membrane domain of the polarized intestinal epithelial cells, it is reasonable to conclude that both transporters contribute to overall gut folate absorption, but that the relative contribution is determined by the prevailing pH at the mucosal surface. Thus, PFCT is believed to play a predominant role in the absorption of dietary folate in the proximal half of the small intestine (where the pH at the luminal surface is around pH 5.8–6.0 as established by direct measurements with pH microelectrodes; 268), while RFC is believed to play a major role in folate absorption in the large intestine where the pH is in the neutral/slightly alkaline range.

With regard to the molecular identity of the folate transport system at the BLM domain of the intestinal absorptive epithelial cells, this is believed to be a member(s) of the multidrug-resistance-associated proteins (Ref. and references therein) although the apparent K _m of these carriers is markedly higher than the levels of folate encountered by the intestinal BLM.

54.8.4

Cell Biology of the Intestinal Folate Uptake Process

Studies on the mechanisms involved in membrane targeting and intracellular trafficking of the hRFC and hPCFT proteins to the cell membrane have also been forthcoming. Using confocal imaging of live human intestinal epithelial cells and a series of truncated fusion proteins of hRFC with GFP (i.e., hRFC-GFP), the molecular determinants that dictate the targeting of the hRFC protein to the cell membrane were found to reside within the hydrophobic backbone of the polypeptide and not within its N- or C-terminal domains. Also, the integrity of the hRFC backbone was found to be critical for exporting the polypeptide from the endoplasmic reticulum to the cell surface. Numerous trafficking vesicles that contain the hRFC-EGFP fusion protein have also been identified and appear to be involved in the intracellular movement of the protein (real-time movies can be viewed at website: http://www.jbc.org.easyaccess2.lib.cuhk.edu.hk/cgi/content/full/277/36/33325/DC1 ). This intracellular trafficking process is dependent on the existence of an intact microtubule network; their disruption was shown to lead to a severe inhibition in the motility of the hRFC-containing trafficking vesicles as well as in the final expression of the hRFC protein at the cell membrane.

A recent study has identified a novel accessory protein that interacts with the hRFC in intestinal epithelial cells and influences its function ( Fig. 54.4 ). Using a bacterial two-hybrid system to screen a BacterioMatch II human intestinal cDNA library using the large intracellular loop between TMDs 6 and 7 of the hRFC as the bait, the dynein light-chain road block-1 (DYNLRB1) protein was identified as an interacting partner with hRFC. Existence of a direct protein-protein interaction between hRFC and DYNLRB1 was confirmed by in vitro pull-down assay, in vivo mammalian two-hybrid luciferase assay, and coimmunoprecipitation analysis. Moreover, confocal imaging of live human intestinal epithelial cells demonstrated colocalization of DYNLRB1 with hRFC. Coexpression of DYNLRB1 with hRFC led to a marked increase in folate uptake, while inhibiting the endogenous DYNLRB1 (with gene-specific siRNA or with vanadate, a specific pharmacological inhibitor) led to a marked decrease in the vitamin uptake.

With regard to cell biology of the hPCFT, live-cell confocal imaging studies have shown that the protein is expressed in a functional form at the apical membrane domain of epithelial cells and that its C-terminal sequence is not essential for membrane targeting or function. The latter investigations also showed that mutations that ablate a consensus beta-turn sequence separating predicted TM2 and TM3 abolishes apical folate uptake as a consequence of endoplasmic reticulum retention of mutant proteins. Furthermore, cell surface delivery of the hPCFT polypeptide was disrupted by microtubule depolymerization or by overexpression of the dynactin complex dynamitin (p50).

54.8.5

Structure-Activity Aspects of PCFT and RFC

Knowledge about structure – function activity of the hPCFT and hRFC systems has been emerging in recent years from clinical and experimental findings. Initial studies utilizing group-specific reagents have suggested possible involvement of positively charged histidine residues in intestinal uptake of the negatively charged folate. These histidine residues were believed to be located at or near the folate-binding site(s) of the involved folate uptake system(s). Subsequent molecule (site-directed mutagenesis) studies have confirmed a critical role for the conserved histidine residues located at positions 247 and 281 of the hPCFT polypeptide. In addition, clinical mutations identified in PCFT in patients with HFMS have implied a role for the amino acid residues located at positions 65, 66, 113, 147, 318, 376, and 425 of the polypeptide (Ref. and references therein). These mutations were shown to lead to spectrum of consequences that include an early stop codon and a frame shift (both of which lead to absence of hPCFT protein), or to a defective intracellular trafficking/membrane targeting of the protein and/or protein instability (Ref. and references therein). Other studies have reported a role for the residues located at positons 161, 232, 299, 304 in hPCFT function. Also, recent investigations have shown that the hPCFT forms homo-oligomers, which have functional significance, and that TMDs 2, 3, 4, and 6 play a role in the formation of these oligomers. Finally, experimental evidence was obtained to show that TMDs 1, 2, 7, and 11 forms an extracellular gate in PCFT in the inward-open confirmation.

With regard to RFC, this protein exists as homo-oligomer, but the monomer form appears to function independently (i.e., oligomerization is not a prerequisite for function but appears to be important for trafficking from the ER to the plasma membrane; Ref. and references therein). Many individual amino acid residues in the hRFC protein have been reported as being important for function including residues located at positions 29, 44, 45, 46, 48, 106, 107, 132, 133, 313, and 373 of the polypeptide (Ref. and references therein). Furthermore, studies have reported a functional role for the intracellular loop between TMDs 6 and 7, while no role was found for the N- or C-terminal tails of the RFC protein. Moreover, the membrane translocation pathway of the hRFC protein appears to be formed from TMDs 1, 2, 4, 5, 7, 8, 10, and 11 (reviewed in Ref. ).

54.8.6

Regulation of the Intestinal Folate Uptake Process

The intestinal folate uptake process is under the regulation of a variety of extracellular/environmental conditions and intracellular factors. Since a number of these conditions act via transcriptional mechanisms, it is important to first discuss what is known about the transcriptional regulation of the folate transport systems.

54.8.7

Factors/Conditions That Negatively Impact the Intestinal Folate Uptake Process

It is well known that chronic alcoholism is associated with folate deficiency. While a variety of factors contribute to the development of this deficiency, an inhibition in intestinal absorption of folate is one of the significant contributors. Chronic alcohol use has been shown to affect both the initial hydrolysis phase of dietary folate polyglutamates and the subsequent absorption of the generated folate monoglutamates. The latter was associated with a significant reduction in folate uptake across the BBM and BLM domains of the absorptive epithelial cells as well as with a marked reduction in level of expression of RFC (level of PCFT was not investigated in these studies). It is interesting to mention here that a recent study examining the effect of chronic alcohol feeding on folate uptake by pancreatic acinar cells has shown significant reduction in the level of expression of both RFC and PCFT.

Other studies have reported an impairment in the activity of the intestinal folylpoly-γ-glutamate carboxypeptidase (folate hydrolase) in disease conditions that affect the intestinal mucosa (e.g., celiac disease and tropical sprue ), and as a result of chronic alcohol consumption and long-term use of the drug sulfasalazine.