Loss of Sucrase-Isomaltase Function Increases Acetate Levels and Improves Metabolic Health in Greenlandic Cohorts.

BACKGROUND & AIMS: The sucrase-isomaltase (SI) c.273_274delAG loss-of-function variant is common in Arctic populations and causes congenital sucrase-isomaltase deficiency, which is an inability to break down and absorb sucrose and isomaltose. Children with this condition experience gastrointestinal symptoms when dietary sucrose is introduced. We aimed to describe the health of adults with sucrase-isomaltase deficiency. METHODS: The association between c.273_274delAG and phenotypes related to metabolic health was assessed in 2 cohorts of Greenlandic adults (n = 4922 and n = 1629). A sucrase-isomaltase knockout (Sis-KO) mouse model was used to further elucidate the findings. RESULTS: Homozygous carriers of the variant had a markedly healthier metabolic profile than the remaining population, including lower body mass index (ß [standard error], -2.0 [0.5] kg/m2; P = 3.1 × 10-5), body weight (-4.8 [1.4] kg; P = 5.1 × 10-4), fat percentage (-3.3% [1.0%]; P = 3.7 × 10-4), fasting triglyceride (-0.27 [0.07] mmol/L; P = 2.3 × 10-6), and remnant cholesterol (-0.11 [0.03] mmol/L; P = 4.2 × 10-5). Further analyses suggested that this was likely mediated partly by higher circulating levels of acetate observed in homozygous carriers (ß [standard error], 0.056 [0.002] mmol/L; P = 2.1 × 10-26), and partly by reduced sucrose uptake, but not lower caloric intake. These findings were verified in Sis-KO mice, which, compared with wild-type mice, were leaner on a sucrose-containing diet, despite similar caloric intake, had significantly higher plasma acetate levels in response to a sucrose gavage, and had lower plasma glucose level in response to a sucrose-tolerance test. CONCLUSIONS: These results suggest that sucrase-isomaltase constitutes a promising drug target for improvement of metabolic health, and that the health benefits are mediated by reduced dietary sucrose uptake and possibly also by higher levels of circulating acetate.

Dietary adaptation to high starch involves increased relative abundance of sucrase-isomaltase and its mRNA in nestling house sparrows.

Dietary flexibility in digestive enzyme activity is widespread in vertebrates but mechanisms are poorly understood. When laboratory rats are switched to a higher carbohydrate diet, the activities of the apical intestinal α-glucosidases (AGs) increase within 6-12 h, mainly by rapid increase in enzyme transcription, followed by rapid translation and translocation to the intestine's apical, brush-border membrane (BBM). We performed the first unified study of the overall process in birds, relying on activity, proteomic, and transcriptomic data from the same animals. Our avian model was nestling house sparrows (Passer domesticus), which switch naturally from a low-starch insect diet to a higher starch seed diet and in whom the protein sucrase-isomaltase (SI) is responsible for all maltase and sucrase intestinal activities. Twenty-four hours after the switch to a high-starch diet, SI activity was increased but not at 12 h post diet switch. SI was the only hydrolase increased in the BBM, and its relative abundance and activity were positively correlated. Twenty-four hours after a reverse switch back to the lower starch diet, SI activity was decreased but not at 12 h post diet switch. Parallel changes in SI mRNA relative abundance were associated with the changes in SI activity in both diet-switch experiments, but our data also revealed an apparent diurnal rhythm in SI mRNA. This is the first demonstration that birds may rely on rapid increase in abundance of SI and its mRNA when adjusting to high-starch diet. Although the mechanisms underlying dietary induction of intestinal enzymes seem similar in nestling house sparrows and laboratory rodents, the time course for modulation in nestlings seemed half as fast compared with laboratory rodents. Before undertaking modulation, an opportunistic forager facing limited resources might rely on more extensive or prolonged environmental sampling, because the redesign of the intestine's hydrolytic capacity shortly after just one or a few meals of a new substrate might be a costly mistake.

Digestive enzyme expression in the large intestine of children with short bowel syndrome in a late stage of adaptation.

BACKGROUND AND AIMS: Intestinal adaptation in short bowel syndrome (SBS) includes morphologic processes and functional mechanisms. This study investigated whether digestive enzyme expression in the duodenum and colon is upregulated in SBS patients. METHOD: Sucrase-isomaltase (SI), lactase-phlorizin hydrolase (LPH), and neutral Aminopeptidase N (ApN) were analyzed in duodenal and colonic biopsies from nine SBS patients in a late stage of adaptation as well as healthy and disease controls by immunoelectron microscopy (IEM), Western blots, and enzyme activities. Furthermore, proliferation rates and intestinal microbiota were analyzed in the mucosal specimen. RESULTS: We found significantly increased amounts of SI, LPH, and ApN in colonocytes in most SBS patients with large variation and strongest effect for SI and ApN. Digestive enzyme expression was only partially elevated in duodenal enterocytes due to a low proliferation level measured by Ki-67 staining. Microbiome analysis revealed high amounts of Lactobacillus resp. low amounts of Proteobacteria in SBS patients with preservation of colon and ileocecal valve. Colonic expression was associated with a better clinical course in single cases. CONCLUSION: In SBS patients disaccharidases and peptidases can be upregulated in the colon. Stimulation of this colonic intestinalization process by drugs, nutrients, and pre- or probiotics might offer better therapeutic approaches.

Adaptation of intestinal epithelial hydrolysis and absorption of dietary carbohydrate and protein in mammals and birds.

The small intestine of mammals and birds exhibits fascinating variation across taxa, body size, and life history features such as locomotion and diet. In the intestine's brush border membrane (BBM), hydrolases are more abundant than transporters in both mammals and birds, but there are differences among the groups in abundance of certain hydrolases and possibly in transporters. For example, mammals express two α-glucosidases, sucrase-isomaltase (SI) and maltase glucoamylase (MGAM), whereas songbirds we studied have only SI, and the chicken expresses SI plus another α-glucosidase that functions similarly to MGAM but is not a true ortholog. For intestinal absorption of sugars and amino acids, small fliers rely on a paracellular pathway to a greater extent than do nonflying mammals, which rely more on transporters. Possibly having evolved in fliers as compensation for lower intestinal nominal surface area (NSA), the fliers' reliance on paracellular absorption is supported by their greater villous surface enlargement that leads to more (per cm2 NSA) tight junctions and greater clearance of passively absorbed compounds. To match digestive capacity to nutrient load, a positive relationship is often observed between dietary intake of macronutrients and intestinal activity of the enzymes and transporters of their respective constituents. In enterocytes, rapid, fine-tuned adjustment to high dietary carbohydrate and protein involves rapid, specific correlated increase in activity and abundance of hydrolases and transporters in the BBM and increases in their mRNA.

Dextran Sodium Sulfate-Induced Impairment of Protein Trafficking and Alterations in Membrane Composition in Intestinal Caco-2 Cell Line.

A key morphological feature of inflammatory bowel disease (IBD) is the loss of the barrier function of intestinal epithelial cells. The present study investigates endoplasmic reticulum (ER) stress in addition to alterations in protein and membrane trafficking in a dextran sulfate sodium (DSS)-induced IBD-like phenotype of intestinal Caco-2 cells in culture. DSS treatment significantly reduced the transepithelial electric resistance (TEER) and increased the epithelial permeability of Caco-2 cells, without affecting their viability. This was associated with an alteration in the expression levels of inflammatory factors in addition to an increase in the expression of the ER stress protein markers, namely immunoglobulin-binding protein (BiP), C/EBP homologous protein (CHOP), activation transcription factor 4 (ATF4), and X-box binding protein (XBP1). The DSS-induced ER-stress resulted in impaired intracellular trafficking and polarized sorting of sucrase-isomaltase (SI) and dipeptidyl peptidase-4 (DPPIV), which are normally sorted to the apical membrane via association with lipid rafts. The observed impaired sorting was caused by reduced cholesterol levels and subsequent distortion of the lipid rafts. The data presented confirm perturbation of ER homeostasis in DSS-treated Caco-2 cells, accompanied by impairment of membrane and protein trafficking resulting in altered membrane integrity, cellular polarity, and hence disrupted barrier function.

Loss of MYO5B Leads to Reductions in Na+ Absorption With Maintenance of CFTR-Dependent Cl- Secretion in Enterocytes.

BACKGROUND & AIMS: Inactivating mutations in MYO5B cause microvillus inclusion disease (MVID), but the physiological cause of the diarrhea associated with this disease is unclear. We investigated whether loss of MYO5B results in aberrant expression of apical enterocyte transporters. METHODS: We studied alterations in apical membrane transporters in MYO5B-knockout mice, as well as mice with tamoxifen-inducible, intestine-specific disruption of Myo5b (VilCreERT2;Myo5bflox/flox mice) or those not given tamoxifen (controls). Intestinal tissues were collected from mice and analyzed by immunostaining, immunoelectron microscopy, or cultured enteroids were derived. Functions of brush border transporters in intestinal mucosa were measured in Ussing chambers. We obtained duodenal biopsy specimens from individuals with MVID and individuals without MVID (controls) and compared transporter distribution by immunocytochemistry. RESULTS: Compared to intestinal tissues from littermate controls, intestinal tissues from MYO5B-knockout mice had decreased apical localization of SLC9A3 (also called NHE3), SLC5A1 (also called SGLT1), aquaporin (AQP) 7, and sucrase isomaltase, and subapical localization of intestinal alkaline phosphatase and CDC42. However, CFTR was present on apical membranes of enterocytes from MYO5B knockout and control mice. Intestinal biopsies from patients with MVID had subapical localization of NHE3, SGLT1, and AQP7, but maintained apical CFTR. After tamoxifen administration, VilCreERT2;Myo5bflox/flox mice lost apical NHE3, SGLT1, DRA, and AQP7, similar to germline MYO5B knockout mice. Intestinal tissues from VilCreERT2;Myo5bflox/flox mice had increased CFTR in crypts and CFTR localized to the apical membranes of enterocytes. Intestinal mucosa from VilCreERT2;Myo5bflox/flox mice given tamoxifen did not have an intestinal barrier defect, based on Ussing chamber analysis, but did have decreased SGLT1 activity and increased CFTR activity. CONCLUSIONS: Although trafficking of many apical transporters is regulated by MYO5B, trafficking of CFTR is largely independent of MYO5B. Decreased apical localization of NHE3, SGLT1, DRA, and AQP7 might be responsible for dysfunctional water absorption in enterocytes of patients with MVID. Maintenance of apical CFTR might exacerbate water loss by active secretion of chloride into the intestinal lumen.

Functional variants in the sucrase-isomaltase gene associate with increased risk of irritable bowel syndrome.

OBJECTIVE: IBS is a common gut disorder of uncertain pathogenesis. Among other factors, genetics and certain foods are proposed to contribute. Congenital sucrase-isomaltase deficiency (CSID) is a rare genetic form of disaccharide malabsorption characterised by diarrhoea, abdominal pain and bloating, which are features common to IBS. We tested sucrase-isomaltase (SI) gene variants for their potential relevance in IBS. DESIGN: We sequenced SI exons in seven familial cases, and screened four CSID mutations (p.Val557Gly, p.Gly1073Asp, p.Arg1124Ter and p.Phe1745Cys) and a common SI coding polymorphism (p.Val15Phe) in a multicentre cohort of 1887 cases and controls. We studied the effect of the 15Val to 15Phe substitution on SI function in vitro. We analysed p.Val15Phe genotype in relation to IBS status, stool frequency and faecal microbiota composition in 250 individuals from the general population. RESULTS: CSID mutations were more common in patients than asymptomatic controls (p=0.074; OR=1.84) and Exome Aggregation Consortium reference sequenced individuals (p=0.020; OR=1.57). 15Phe was detected in 6/7 sequenced familial cases, and increased IBS risk in case-control and population-based cohorts, with best evidence for diarrhoea phenotypes (combined p=0.00012; OR=1.36). In the population-based sample, 15Phe allele dosage correlated with stool frequency (p=0.026) and Parabacteroides faecal microbiota abundance (p=0.0024). The SI protein with 15Phe exhibited 35% reduced enzymatic activity in vitro compared with 15Val (p<0.05). CONCLUSIONS: SI gene variants coding for disaccharidases with defective or reduced enzymatic activity predispose to IBS. This may help the identification of individuals at risk, and contribute to personalising treatment options in a subset of patients.

Structure-function analysis of human sucrase-isomaltase identifies key residues required for catalytic activity.

Sucrase-isomaltase (SI) is an intestinal membrane-associated α-glucosidase that breaks down di- and oligosaccharides to absorbable monosaccharides. SI has two homologous functional subunits (sucrase and isomaltase) that both belong to the glycoside hydrolase family 31 (GH31) and differ in substrate specificity. All GH31 enzymes share a consensus sequence harboring an aspartic acid residue as a catalytic nucleophile. Moreover, crystallographic structural analysis of isomaltase predicts that another aspartic acid residue functions as a proton donor in hydrolysis. Here, we mutagenized the predicted proton donor residues and the nucleophilic catalyst residues in each SI subunit. We expressed these SI variants in COS-1 cells and analyzed their structural, transport, and functional characteristics. All of the mutants revealed expression levels and maturation rates comparable with those of the wild-type species and the corresponding nonmutated subunits were functionally active. Thereby we determined rate and substrate specificity for each single subunit without influence from the other subunit. This approach provides a model for functional analysis of the single subunits within a multidomain protein, achieved without the necessity to express the individual subunits separately. Of note, we also found that glucose product inhibition regulates the activities of both SI subunits. We experimentally confirmed the catalytic function of the predicted proton donor residues, and sequence analysis suggested that these residues are located in a consensus region in many GH31 family members. In summary, these findings reveal the kinetic features specific for each human SI subunit and demonstrate that the activities of these subunits are regulated via product inhibition.

Posttranslational Processing and Function of Mucosal Maltases.

The final step of carbohydrate digestion in the intestine is performed by 2 major α-glucosidases of the intestinal mucosa, sucrase-isomaltase (SI) and maltase-glucoamylase. Both of these enzymes are type II membrane glycoproteins, which share a significant level of homology in gene and protein structures and yet have differences in the posttranslational processing, substrate specificity and functional capacity. Insufficient activity of these disaccharidases particularly SI as a result of genetic mutations or secondary intestinal pathologies is associated with carbohydrate maldigestion and gastrointestinal intolerances. This review will discuss the maturation profiles of SI and maltase-glucoamylase relative to their functional capacities and deficiencies.

Molecular pathogenicity of novel sucrase-isomaltase mutations found in congenital sucrase-isomaltase deficiency patients.

BACKGROUND & AIMS: Congenital sucrase-isomaltase deficiency (CSID) is a genetic disorder associated with mutations in the sucrase-isomaltase (SI) gene. The diagnosis of congenital diarrheal disorders like CSID is difficult due to unspecific symptoms and usually requires invasive biopsy sampling of the intestine. Sequencing of the SI gene and molecular analysis of the resulting potentially pathogenic SI protein variants may facilitate a diagnosis in the future. This study aimed to categorize SI mutations based on their functional consequences. METHODS: cDNAs encoding 13 SI mutants were expressed in COS-1 cells. The molecular pathogenicity of the resulting SI mutants was defined by analyzing their biosynthesis, cellular localization, structure and enzymatic functions. RESULTS: Three biosynthetic phenotypes for the novel SI mutations were identified. The first biosynthetic phenotype was defined by mutants that are intracellularly transported in a fashion similar to wild type SI and with normal, but varying, levels of enzymatic activity. The second biosynthetic phenotype was defined by mutants with delayed maturation and trafficking kinetics and reduced activity. The third group of mutants is entirely transport incompetent and functionally inactive. CONCLUSIONS: The current study unraveled CSID as a multifaceted malabsorption disorder that comprises three major classes of functional and trafficking mutants of SI and established a gradient of mild to severe functional deficits in the enzymatic functions of the enzyme. GENERAL SIGNIFICANCE: This novel concept and the existence of mild consequences in a number of SI mutants strongly propose that CSID is an underdiagnosed and a more common intestinal disease than currently known.

Improved Starch Digestion of Sucrase-deficient Shrews Treated With Oral Glucoamylase Enzyme Supplements.

BACKGROUND AND OBJECTIVE: Although named because of its sucrose hydrolytic activity, this mucosal enzyme plays a leading role in starch digestion because of its maltase and glucoamylase activities. Sucrase-deficient mutant shrews, Suncus murinus, were used as a model to investigate starch digestion in patients with congenital sucrase-isomaltase deficiency.Starch digestion is much more complex than sucrose digestion. Six enzyme activities, 2 α-amylases (Amy), and 4 mucosal α-glucosidases (maltases), including maltase-glucoamylase (Mgam) and sucrase-isomaltase (Si) subunit activities, are needed to digest starch to absorbable free glucose. Amy breaks down insoluble starch to soluble dextrins; mucosal Mgam and Si can either directly digest starch to glucose or convert the post-α-amylolytic dextrins to glucose. Starch digestion is reduced because of sucrase deficiency and oral glucoamylase enzyme supplement can correct the starch maldigestion. The aim of the present study was to measure glucogenesis in suc/suc shrews after feeding of starch and improvement of glucogenesis by oral glucoamylase supplements. METHODS: Sucrase mutant (suc/suc) and heterozygous (+/suc) shrews were fed with C-enriched starch diets. Glucogenesis derived from starch was measured as blood C-glucose enrichment and oral recombinant C-terminal Mgam glucoamylase (M20) was supplemented to improve starch digestion. RESULTS: After feedings, suc/suc and +/suc shrews had different starch digestions as shown by blood glucose enrichment and the suc/suc had lower total glucose concentrations. Oral supplements of glucoamylase increased suc/suc total blood glucose and quantitative starch digestion to glucose. CONCLUSIONS: Sucrase deficiency, in this model of congenital sucrase-isomaltase deficiency, reduces blood glucose response to starch feeding. Supplementing the diet with oral recombinant glucoamylase significantly improved starch digestion in the sucrase-deficient shrew.

Histone deacetylase inhibition impairs normal intestinal cell proliferation and promotes specific gene expression.

Mechanisms that maintain proliferation and delay cell differentiation in the intestinal crypt are not yet fully understood. We have previously shown the implication of histone methylation in the regulation of enterocytic differentiation. In this study, we investigated the role of histone deacetylation as an important epigenetic mechanism that controls proliferation and differentiation of intestinal cells using the histone deacetylase inhibitor suberanilohydroxamic acid (SAHA) on the proliferation and differentiation of human and mouse intestinal cells. Treatment of newly confluent Caco-2/15 cells with SAHA resulted in growth arrest, increased histone acetylation and up-regulation of the expression of intestine-specific genes such as those encoding sucrase-isomaltase, villin and the ion exchanger SLC26A3. Although SAHA has been recently used in clinical trials for cancer treatment, its effect on normal intestinal cells has not been documented. Analyses of small and large intestines of mice treated with SAHA revealed a repression of crypt cell proliferation and a higher expression of sucrase-isomaltase in both segments compared to control mice. Expression of SLC26A3 was also significantly up-regulated in the colons of mice after SAHA administration. Finally, SAHA was also found to strongly inhibit normal human intestinal crypt cell proliferation in vitro. These results demonstrate the important implication of epigenetic mechanisms such as histone acetylation/deacetylation in the regulation of normal intestinal cell fate and proliferation.

Functional analysis of sucrase-isomaltase mutations from chronic lymphocytic leukemia patients.

Next-generation sequencing techniques have emerged as powerful tools for the understanding of cancer genomes. In recent years, whole-exome and whole-genome sequencing strategies have enabled the annotation of a comprehensive mutation landscape of chronic lymphocytic leukemia (CLL), the most frequent leukemia in western countries. Several recurrently mutated genes have been identified, with a subset being validated as neoplastic drivers. Still, a main challenge remains for the differentiation between driver and passenger mutations among candidates as well as for the functional description of the newly discovered leukemogenic genes that could be utilized for personalized anti-tumor strategies. In this scenario, we have identified the metabolic enzyme sucrase-isomaltase (SI) as one of the most frequently mutated genes in a cohort of 105 CLL patients. Here, we demonstrate that these SI mutations result in loss of enzyme function by preventing the biosynthesis of catalytically competent SI at the cell surface. Transcriptome analyses of RNA from CLL patients with SI loss-of-function mutations have uncovered gene expression patterns that depict ample metabolic reprogramming, pinpointing SI as a putative player in the cancer-associated metabolic switch. These results highlight SI as a relevant target for clinical evaluation in future CLL studies.

Effects of lactoferrin on intestinal epithelial cell growth and differentiation: an in vivo and in vitro study.

This study was designed to analyse the effects of human (h) and bovine lactoferrin (bLF) on the growth and differentiation of intestinal cells using the mice model supplemented with Lactoferrin (LF) and the enterocyte-like model of Caco-2 cells which spontaneously differentiate after confluency. In mice, bLF supplementation increased jejunal villus height and the expression of several intestinal brush border membrane enzymes activities. Addition of bLF or hLF to undifferentiated Caco-2 cells was able to increase cell proliferation with confluency being reached more rapidly. Moreover, when Caco-2 cells were grown in the presence of LF for 3 weeks, brush-border membrane-associated enzyme activities i.e. sucrase, alkaline phosphatase and neutral aminopeptidase, as well as the L-glutamate transporter expression were all increased indicating an increased Caco-2 cell differentiation. Accordingly, cDNA Atlas array and Western blot analysis of cell cycle proteins shown a decreased expression of Cdck2 and an increased TAF1 expression; these proteins being implicated in the regulation of numerous genes related to cellular proliferation and differentiation. These modifications were associated with an inhibition of Caco-2 cell spontaneous apoptosis. Altogether, our results indicate that LF increase in vivo and in vitro enterocyte differentiation. In addition, LF was found to increase in vitro enterocyte proliferation resulting in higher cell density in cell flasks, an effect that was likely partly due to a reduction of the cellular apoptosis. The different stimulation patterns observed for the different parameters associated with cell differentiation in relationship with specific gene regulation is discussed.

Mammalian maltase-glucoamylase and sucrase-isomaltase inhibitory effects of Artocarpus heterophyllus: An in vitro and in silico approach.

Alpha-glucosidase (maltase, sucrase, isomaltase and glucoamylase) activities which are involved in carbohydrate metabolism are present in human intestinal maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI). Hence, these proteins are important targets to identify drugs against postprandial hyperglycemia thereby for diabetes. To find natural-based drugs against MGAM and SI, Artocarpus heterophyllus leaf was explored for MGAM and SI inhibition in in vitro and in silico. A. heterophyllus leaf aqueous active fraction (AHL-AAF) was prepared using Soxhlet extraction followed by silica column chromatography. The phytoconstituents of AHL-AAF were determined using LC-ESI-MS/MS. AHL-AAF showed dose-dependent and mixed inhibition against maltase (IC50 = 460 µg/ml; Ki = 300 µg/ml), glucoamylase (IC50 = 780 µg/ml; Ki = 480 µg/ml), sucrase (IC50 = 900 µg/ml, Ki = 504 µg/ml) and isomaltase (IC50 = 860 µg/ml, Ki = 400 µg/ml). AHL-AAF phytoconstituents interaction with N-terminal (Nt) and C-terminal (Ct) subunits of human MGAM and SI was analyzed using induced-fit docking, molecular dynamics (MD), and binding free energy calculation. In docking studies, rhamnosyl hexosyl methyl quercetin (RHMQ), P-coumaryl-O-16-hydroxy palmitic acid (PCHP), and spirostanol interacted with active site amino acids of human MGAM and SI. Among these RHMQ stably interacted with all the subunits (Nt-MGAM, Ct-MGAM, Nt-SI and Ct-SI) whereas PCHP with Ct-MGAM and Nt-SI during MD analysis. In molecular docking, the docking score of RHMQ with NtMGAM, CtMGAM, NtSI and CtSI was -8.48, -12.88, -11.98 and -11.37 kcal/mol. The docking score of PCHP for CtMGAM and NtSI was -8.59 and -8.4 kcal/mol, respectively. After MD simulation, the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values further confirmed the stable protein-ligand interaction. The RMSD value of all the complexes were around 2.5 Šand the corresponding RMSF values were also quite low. In MM/GBSA analysis, the involvement of Van der Waals and lipophilic energy in the protein/ligand interactions are understood. Further binding free energy for Nt-MGAM-PCHP, Nt-MGAM-RHMQ, Nt-SI-PCHP, Nt-SI-RHMQ, Ct-MGAM-PCHP, Ct-MGAM-RHMQ and Ct-SI-RHMQ complexes was found to be -24.94, -46.60, -46.56, -44.48, -40.3, -41.86 and -19.39 kcal/mol, respectively. Altogether, AHL-AAF showed inhibition of α-glucosidase activities of MGAM and SI. AHL-AAF could be further studied for its effect on diabetes in in vivo.

Inhibition of human starch digesting enzymes and intestinal glucose transport by walnut polyphenols.

One approach to controlling type 2 diabetes (T2D) is to lower postprandialglucose spikesby slowing down the digestion of carbohydrates and the absorption of glucose in the small intestine. The consumption of walnuts is associated with a reduced risk of chronic diseases such as T2D, suggested to be partly due to the high content of (poly)phenols. This study evaluated, for the first time, the inhibitory effect of a (poly)phenol-rich walnut extract on human carbohydrate digesting enzymes (salivary and pancreatic α-amylases, brush border sucrase-isomaltase) and on glucose transport across fully differentiated human intestinal Caco-2/TC7 monolayers. The walnut extract was rich in multiple (poly)phenols (70 % w/w) as analysed by Folin-Ciocalteau and by LCMS. It exhibited potent inhibition of both human salivary (IC50: 32.2 ± 2.5 µg walnut (poly)phenols (WP)/mL) and pancreatic (IC50: 56.7 ± 1.7 µg WP/mL) α-amylases, with weaker effects on human sucrase (IC50: 990 ± 20 µg WP/mL), maltase (IC50: 1300 ± 80 µg WP/mL), and isomaltase (IC25: 830 ± 60 µg WP/mL) activities. Selected individual walnut (poly)phenols inhibited human salivary α-amylase in the order: 1,3,4,6-tetragalloylglucose > ellagic acid pentoside > 1,2,6-tri-O-galloyl-ß-D-glucopyranose, with no inhibition by ellagic acid, gallic acid and 4-O-methylgallic acid. The (poly)phenol-rich walnut extract also attenuated (up to 59 %) the transfer of 2-deoxy-D-glucose across differentiated Caco-2/TC7 cell monolayers. This is the first report on the effect of (poly)phenol-rich extracts from any commonly-consumed nut kernel on any human starch-digesting enzyme, and suggests a mechanism through which walnut consumption may lower postprandial glucose spikes and contribute to their proposed health benefits.

Glucocorticoids and microbiota regulate ontogeny of intestinal fucosyltransferase 2 requisite for gut homeostasis.

At weaning, the intestinal mucosa surface glycans change from predominantly sialylated to fucosylated. Intestinal adaptation from milk to solid food is regulated by intrinsic and extrinsic factors. The contribution by glucocorticoid, an intrinsic factor, and colonization by microbiota, an extrinsic factor, was measured as the induction of α1,2/3-fucosyltransferase and sucrase-isomaltase (SI) activity and gene expression in conventionally raised, germ-free, and bacteria-depleted mice. In conventionally raised mice, cortisone acetate (CA) precociously accelerated SI gene expression up to 3 weeks and fut2 to 4 weeks of age. In germ-free mice, CA treatment induces only SI expression but not fucosyltransferase. In post-weaning bacteria-deficient (germ-free and bacteria-depleted) mice, fut2 expression remains at low suckling levels. In microbiota deficient mice, intestinal fut2 (but not fut1, fut4 or fut7) was induced only by adult microbiota, but not immature microbiota or CA. Fut2 induction could also be restored by colonization by Bacteroides fragilis, but not by a B. fragilis mutant unable to utilize fucose. Restoration of fut2 expression (by either microbiota or B. fragilis) in bacteria-depleted mice is necessary for recovery from dextran sulfate sodium-induced mucosal injury. Thus, glucocorticoids and microbes regulate distinct aspects of gut ontogeny: CA precociously accelerates SI expression and, only in colonized mice, fut2 early expression. The adult microbiota is required for the fut2 induction responsible for the highly fucosylated adult gut phenotype and is necessary for recovery from intestinal injury. Fut2-dependent recovery from inflammation may explain the high incidence of inflammatory disease (Crohn's and necrotizing enterocolitis) in populations with mutant FUT2 polymorphic alleles.

Maltase-glucoamylase modulates gluconeogenesis and sucrase-isomaltase dominates starch digestion glucogenesis.

OBJECTIVES: Six enzyme activities are needed to digest starch to absorbable free glucose; 2 luminal α-amylases (AMY) and 4 mucosal maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) subunit activities are involved in the digestion. The AMY activities break down starch to soluble oligomeric dextrins; mucosal MGAM and SI can either directly digest starch to glucose or convert the post-α-amylolytic dextrins to glucose. We hypothesized that MGAM, with higher maltase than SI, drives digestion on ad limitum intakes and SI, with lower activity but more abundant amount, constrains ad libitum starch digestion. METHODS: Mgam null and wild-type (WT) mice were fed with starch diets ad libitum and ad limitum. Fractional glucogenesis (fGG) derived from starch was measured and fractional gluconeogenesis and glycogenolysis were calculated. Carbohydrates in small intestine were determined. RESULTS: After ad libitum meals, null and WT had similar increases of blood glucose concentration. At low intakes, null mice had less (f)GG (P = 0.02) than WT mice, demonstrating the role of Mgam activity in ad limitum feeding; null mice did not reduce fGG responses to ad libitum intakes demonstrating the dominant role of SI activity during full feeding. Although fGG was rising after feeding, fractional gluconeogenesis fell, especially for null mice. CONCLUSIONS: The fGNG (endogenous glucogenesis) in null mice complemented the fGG (exogenous glucogenesis) to conserve prandial blood glucose concentrations. The hypotheses that Mgam contributes a high-efficiency activity on ad limitum intakes and SI dominates on ad libitum starch digestion were confirmed.

Prevalence of congenital sucrase-isomaltase deficiency in Turkey may be much higher than the estimates.

Congenital sucrase-isomaltase deficiency (CSID) is a rare autosomal carbohydrate malabsorption disorder caused by mutations in the sucrase-isomaltase gene. While the prevalence of CSID is high in the indigenous populations of Alaska and Greenland, it is imprecise and ambiguous in the Turkish pediatric population. In this cross-sectional case-control study, which is retrospective in nature, next-generation sequencing (NGS) results obtained from records of 94 pediatric patients with chronic nonspecific diarrhea were reviewed. Demographic characteristics, clinical symptoms and treatment responses of those diagnosed with CSID were evaluated. We identified one new, homozygous frame-shift mutation and 10 other heterozygous mutations. Two cases were from the same family and nine were from different families. While the median age at onset of symptoms was 6 months (0-12), median age at diagnosis was 60 months (18-192) with a median delay of 5 years and 5 months (10 months -15 years and 5 months) in diagnosis. Clinical symptoms included diarrhea (100%), abdominal pain (54.5%), vomiting after consuming sucrose (27.2%), diaper dermatitis (36.3%) and growth retardation (81%). Our clinical study revealed that sucrase-isomaltase deficiency may have been underdiagnosed in patients with chronic diarrhea in Turkey. In addition, the frequency of heterozygous mutation carriers was significantly higher than that of homozygous mutation carriers and those with a heterozygous mutations responded well to the treatment.