Conditioning with slowly digestible starch diets in mice reduces jejunal α-glucosidase activity and glucogenesis from a digestible starch feeding.

OBJECTIVES: Maltase-glucoamylase (Mgam) and sucrase-isomaltase (Si) are mucosal α-glucosidases required for the digestion of starch to glucose. We hypothesized that a dietary approach to reduce Mgam and Si activities can reduce glucose generation and absorption, and improve glucose control. METHODS: Rice starch was entrapped in alginate microspheres to moderate in vitro digestion properties. Three groups of 8-wk old mice (n = 8) were conditioned for 7 d with low 13C-starch-based materials differing in digestion rates (fast, slow, and slower), and then given a digestible 13C-labeled cornstarch test feeding to determine its digestion to glucose. RESULTS: Conditioning of the small intestine with the slowly digestible starches for 7 d reduced jejunal α-glucosidase and sucrase activities, as well as glucose absorption for the slowly digestible starch slower group (P < 0.01). A correlative relationship was found between glucose absorption from a cornstarch test feeding given at d 7 and jejunal α-glucosidase and sucrase activities (R2 = 0.64; 0.67). However, total prandial glucose levels during the 2-h feeding period did not differ. CONCLUSIONS: Decreased glucogenesis from a digestible starch feeding was found in mice conditioned on slowly digestible starch diets, suggesting that a dietary approach incorporating slowly digestible starches may change α-glucosidase activities to moderate glucose absorption rate.

Metabolic Impacts of Maltase Deficiencies.

The mucosal maltase enzymes are characterized by an activity that produces glucose from linear glucose polymers, assayed with the disaccharide maltose. The related enzyme isomaltase produces glucose from branched glucose polymers, assayed with palatinose. Maltase and isomaltase activities are part of the 4 disaccharidases assayed from clinical duodenal biopsy homogenates. The reported maltase activities are more difficult to interpret than lactase or sucrase activities because both the sucrase-isomaltase and maltase-glucoamylase proteins have overlapping maltase activities. The early work of Dahlqvist identified 4 maltase activities from human small intestinal mucosa. On one peptide, sucrase (maltase Ib) and isomaltase (maltase Ia) activities shared maltase activities but identified the enzymes as sucrase-isomaltase. On the other peptide, no distinguishing characteristics of the 2 maltase activities (maltases II and III) were detected and the activities identified as maltase-glucoamylase. The nutritional/clinical importance of small intestinal maltase and isomaltase activities are due to their crucial role in the digestion of food starches to absorbable free glucose. This review focuses on the interpretation of biopsy maltase activities in the context of reported lactase, sucrase, maltase, and palatinase biopsy assay activity patterns. We present a classification of mucosal maltase deficiencies and novel primary maltase deficiency (Ib, II, III) and provide a clarification of the role of maltase activity assayed from clinically obtained duodenal biopsies, as a path toward future clinical and molecular genomic investigations.

Phenolic compounds increase the transcription of mouse intestinal maltase-glucoamylase and sucrase-isomaltase.

Diverse natural phenolic compounds show inhibition activity of intestinal α-glucosidases, which may constitute the molecular basis for their ability to control systemic glycemia. Additionally, phenolics can modify mRNA expression for proteins involved in nutritional, metabolic or immune processes. To explore the possibility that phenolics can regulate the mRNA expression, enzymatic activity, and protein synthesis/processing of intestinal Maltase-Glucoamylase (MGAM) and Sucrase-Isomaltase (SI), small intestinal explants from Balb/c mice were cultured for 24 h in the presence or absence of gallic acid, caffeic acid, and (+)-catechin at 0.1, 0.5, and 1 mM. We measured the levels of MGAM and SI mRNA expression by qRT-PCR, maltase and sucrase activities by a standard colorimetric method and the molecular size distribution of MGAM and SI proteins by western blotting. mRNA expression for MGAM was induced by the three phenolic compounds at 0.1 mM. mRNA expression for SI was induced by caffeic and gallic acids, but not by (+)-catechin. Caffeic acid was the most effective inducer of mRNA expression of these enzymes. Total maltase and sucrase activities were not affected by treatment with phenolics. The proportion of high molecular size forms of MGAM was significantly increased by two of the three phenolic compounds, but little effect was observed on SI proteins. Thus, changes in the protein synthesis/processing, affecting the proportions of the different molecular forms of MGAM, may account for the lack of correlation between mRNA expression and enzymatic activity.

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.

Contribution of the Individual Small Intestinal α-Glucosidases to Digestion of Unusual α-Linked Glycemic Disaccharides.

The mammalian mucosal α-glucosidase complexes, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI), have two catalytic subunits (N- and C-termini). Concurrent with the desire to modulate glycemic response, there has been a focus on di-/oligosaccharides with unusual α-linkages that are digested to glucose slowly by these enzymes. Here, we look at disaccharides with various possible α-linkages and their hydrolysis. Hydrolytic properties of the maltose and sucrose isomers were determined using rat intestinal and individual recombinant α-glucosidases. The individual α-glucosidases had moderate to low hydrolytic activities on all α-linked disaccharides, except trehalose. Maltase (N-terminal MGAM) showed a higher ability to digest α-1,2 and α-1,3 disaccharides, as well as α-1,4, making it the most versatile in α-hydrolytic activity. These findings apply to the development of new glycemic oligosaccharides based on unusual α-linkages for extended glycemic response. It also emphasizes that mammalian mucosal α-glucosidases must be used in in vitro assessment of digestion of such carbohydrates.

Taste cell-expressed α-glucosidase enzymes contribute to gustatory responses to disaccharides.

The primary sweet sensor in mammalian taste cells for sugars and noncaloric sweeteners is the heteromeric combination of type 1 taste receptors 2 and 3 (T1R2+T1R3, encoded by Tas1r2 and Tas1r3 genes). However, in the absence of T1R2+T1R3 (e.g., in Tas1r3 KO mice), animals still respond to sugars, arguing for the presence of T1R-independent detection mechanism(s). Our previous findings that several glucose transporters (GLUTs), sodium glucose cotransporter 1 (SGLT1), and the ATP-gated K(+) (KATP) metabolic sensor are preferentially expressed in the same taste cells with T1R3 provides a potential explanation for the T1R-independent detection of sugars: sweet-responsive taste cells that respond to sugars and sweeteners may contain a T1R-dependent (T1R2+T1R3) sweet-sensing pathway for detecting sugars and noncaloric sweeteners, as well as a T1R-independent (GLUTs, SGLT1, KATP) pathway for detecting monosaccharides. However, the T1R-independent pathway would not explain responses to disaccharide and oligomeric sugars, such as sucrose, maltose, and maltotriose, which are not substrates for GLUTs or SGLT1. Using RT-PCR, quantitative PCR, in situ hybridization, and immunohistochemistry, we found that taste cells express multiple α-glycosidases (e.g., amylase and neutral α glucosidase C) and so-called intestinal "brush border" disaccharide-hydrolyzing enzymes (e.g., maltase-glucoamylase and sucrase-isomaltase). Treating the tongue with inhibitors of disaccharidases specifically decreased gustatory nerve responses to disaccharides, but not to monosaccharides or noncaloric sweeteners, indicating that lingual disaccharidases are functional. These taste cell-expressed enzymes may locally break down dietary disaccharides and starch hydrolysis products into monosaccharides that could serve as substrates for the T1R-independent sugar sensing pathways.

Dietary phenolic compounds selectively inhibit the individual subunits of maltase-glucoamylase and sucrase-isomaltase with the potential of modulating glucose release.

In this study, it was hypothesized that dietary phenolic compounds selectively inhibit the individual C- and N-terminal (Ct, Nt) subunits of the two small intestinal α-glucosidases, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI), for a modulated glycemic carbohydrate digestion. The inhibition by chlorogenic acid, caffeic acid, gallic acid, (+)-catechin, and (-)-epigallocatechin gallate (EGCG) on individual recombinant human Nt-MGAM and Nt-SI and on mouse Ct-MGAM and Ct-SI was assayed using maltose as the substrate. Inhibition constants, inhibition mechanisms, and IC50 values for each combination of phenolic compound and enzymatic subunit were determined. EGCG and chlorogenic acid were found to be more potent inhibitors for selectively inhibiting the two subunits with highest activity, Ct-MGAM and Ct-SI. All compounds displayed noncompetitive type inhibition. Inhibition of fast-digesting Ct-MGAM and Ct-SI by EGCG and chlorogenic acid could lead to a slow, but complete, digestion of starch for improved glycemic response of starchy foods with potential health benefit.

Branch pattern of starch internal structure influences the glucogenesis by mucosal Nt-maltase-glucoamylase.

To produce sufficient amounts of glucose from food starch, both α-amylase and mucosal α-glucosidases are required. We found previously that the digestion rate of starch is influenced by its susceptibility to mucosal α-glucosidases. In the present study, six starches and one glycogen were pre-hydrolyzed by α-amylase for various time periods, and then further hydrolyzed with the mucosal α-glucosidase, the N-terminal subunit of maltase-glucoamylase (Nt-MGAM), to generate free glucose. Results showed that α-amylase amplified the Nt-MGAM glucogenesis, and that the amplifications differed in various substrates. The amount of branches within α-amylase hydrolysate substrates was highly related to the rate of Nt-MGAM glucogenesis. After de-branching, the hydrolysates showed three fractions, Fraction 1, 2, and 3, in size exclusion chromatographs. We found that the α-amylase hydrolysates with higher quantity of the Fraction 3 (molecules with relatively short chain-length) and shorter average chain-length of this fraction had lower rates of Nt-MGAM glucogenesis. This study revealed that the branch pattern of α-amylase hydrolysates modulates glucose release by Nt-MGAM. It further supported the hypothesis that the internal structure of starch affects its digestibility at the mucosal α-glucosidase level.

Different sucrose-isomaltase response of Caco-2 cells to glucose and maltose suggests dietary maltose sensing.

Using the small intestine enterocyte Caco-2 cell model, sucrase-isomaltase (SI, the mucosal α-glucosidase complex) expression and modification were examined relative to exposure to different mono- and disaccharide glycemic carbohydrates. Caco-2/TC7 cells were grown on porous supports to post-confluence for complete differentiation, and dietary carbohydrate molecules of glucose, sucrose (disaccharide of glucose and fructose), maltose (disaccharide of two glucoses α-1,4 linked), and isomaltose (disaccharide of two glucoses α-1,6 linked) were used to treat the cells. qRT-PCR results showed that all the carbohydrate molecules induced the expression of the SI gene, though maltose (and isomaltose) showed an incremental increase in mRNA levels over time that glucose did not. Western blot analysis of the SI protein revealed that only maltose treatment induced a higher molecular weight band (Mw ~245 kDa), also at higher expression level, suggesting post-translational processing of SI, and more importantly a sensing of maltose. Further work is warranted regarding this putative sensing response as a potential control point for starch digestion and glucose generation in the small intestine.

Mucosal C-terminal maltase-glucoamylase hydrolyzes large size starch digestion products that may contribute to rapid postprandial glucose generation.

SCOPE: The four mucosal α-glucosidases, which differ in their digestive roles, generate glucose from glycemic carbohydrates and accordingly can be viewed as a control point for rate of glucose delivery to the body. In this study, individual recombinant enzymes were used to understand how α-glucan oligomers are digested by each enzyme, and how intermediate α-amylolyzed starches are hydrolyzed, to elucidate a strategy for moderating the glycemic spike of rapidly digestible starchy foods. METHODS AND RESULTS: The C-terminal maltase-glucoamylase (ctMGAM, commonly termed "glucoamylase") was able to rapidly hydrolyze longer maltooligosaccharides, such as maltotetraose and maltopentaose, to glucose. Moreover, it was found to convert larger size maltodextrins, as would be produced early in α-amylase digestion of starch, efficiently to glucose. It is postulated that ctMGAM has the additional capacity to hydrolyze large α-amylase products that are produced immediately on starch digestion in the duodenum and contribute to the rapid generation of glucose from starch-based meals. CONCLUSION: The findings suggest that partial inhibition of ctMGAM, such as by natural inhibitors found in foods, might be used to moderate the early stage of high glycemic response, as well as to extend digestion distally; thereby having relevance in regulating glucose delivery to the body.

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.

Enzyme-synthesized highly branched maltodextrins have slow glucose generation at the mucosal α-glucosidase level and are slowly digestible in vivo.

For digestion of starch in humans, α-amylase first hydrolyzes starch molecules to produce α-limit dextrins, followed by complete hydrolysis to glucose by the mucosal α-glucosidases in the small intestine. It is known that α-1,6 linkages in starch are hydrolyzed at a lower rate than are α-1,4 linkages. Here, to create designed slowly digestible carbohydrates, the structure of waxy corn starch (WCS) was modified using a known branching enzyme alone (BE) and an in combination with ß-amylase (BA) to increase further the α-1,6 branching ratio. The digestibility of the enzymatically synthesized products was investigated using α-amylase and four recombinant mammalian mucosal α-glucosidases. Enzyme-modified products (BE-WCS and BEBA-WCS) had increased percentage of α-1,6 linkages (WCS: 5.3%, BE-WCS: 7.1%, and BEBA-WCS: 12.9%), decreased weight-average molecular weight (WCS: 1.73×10(8) Da, BE-WCS: 2.76×10(5) Da, and BEBA-WCS 1.62×10(5) Da), and changes in linear chain distributions (WCS: 21.6, BE-WCS: 16.9, BEBA-WCS: 12.2 DPw). Hydrolysis by human pancreatic α-amylase resulted in an increase in the amount of branched α-limit dextrin from 26.8% (WCS) to 56.8% (BEBA-WCS). The α-amylolyzed samples were hydrolyzed by the individual α-glucosidases (100 U) and glucogenesis decreased with all as the branching ratio increased. This is the first report showing that hydrolysis rate of the mammalian mucosal α-glucosidases is limited by the amount of branched α-limit dextrin. When enzyme-treated materials were gavaged to rats, the level of postprandial blood glucose at 60 min from BEBA-WCS was significantly higher than for WCS or BE-WCS. Thus, highly branched glucan structures modified by BE and BA had a comparably slow digesting property both in vitro and in vivo. Such highly branched α-glucans show promise as a food ingredient to control postprandial glucose levels and to attain extended glucose release.

Enterocyte loss of polarity and gut wound healing rely upon the F-actin-severing function of villin.

Efficient wound healing is required to maintain the integrity of the intestinal epithelial barrier because of its constant exposure to a large variety of environmental stresses. This process implies a partial cell depolarization and the acquisition of a motile phenotype that involves rearrangements of the actin cytoskeleton. Here we address how polarized enterocytes harboring actin-rich apical microvilli undergo extensive cell remodeling to drive injury repair. Using live imaging technologies, we demonstrate that enterocytes in vitro and in vivo rapidly depolarize their microvilli at the wound edge. Through its F-actin-severing activity, the microvillar actin-binding protein villin drives both apical microvilli disassembly in vitro and in vivo and promotes lamellipodial extension. Photoactivation experiments indicate that microvillar actin is mobilized at the lamellipodium, allowing optimal migration. Finally, efficient repair of colonic mechanical injuries requires villin severing of F-actin, emphasizing the importance of villin function in intestinal homeostasis. Thus, villin severs F-actin to ensure microvillus depolarization and enterocyte remodeling upon injury. This work highlights the importance of specialized apical pole disassembly for the repolarization of epithelial cells initiating migration.

DDR2 plays a role in fibroblast migration independent of adhesion ligand and collagen activated DDR2 tyrosine kinase.

Discoidin domain receptor-2 (DDR2) is a cell surface tyrosine kinase receptor that can be activated by soluble collagen and has been implicated in diverse physiological functions including organism growth and wound repair. In the current studies, we used fibronectin and collagen-coated 2D surfaces and collagen matrices in combination with siRNA technology to investigate the role of DDR2 in a range of fibroblast motile activities. Silencing DDR2 with siRNA inhibited cell spreading and migration, and similar inhibition occurred regardless whether cells were interacting with fibronectin or collagen surfaces. Under the assay conditions used, DDR2 tyrosine kinase activation was not observed unless soluble collagen was added to the incubation medium. Finally silencing DDR2 also inhibited human fibroblast migration in 3D collagen matrices but had no effect on 3D collagen matrix remodeling and contraction. Taken together, our findings suggest that DDR2 is required for normal fibroblast spreading and migration independent of adhesion ligand and collagen activation of DDR2 tyrosine kinase.

Starch source influences dietary glucose generation at the mucosal α-glucosidase level.

The quality of starch digestion, related to the rate and extent of release of dietary glucose, is associated with glycemia-related problems such as diabetes and other metabolic syndrome conditions. Here, we found that the rate of glucose generation from starch is unexpectedly associated with mucosal α-glucosidases and not just α-amylase. This understanding could lead to a new approach to regulate the glycemic response and glucose-related physiologic responses in the human body. There are six digestive enzymes for starch: salivary and pancreatic α-amylases and four mucosal α-glucosidases, including N- and C-terminal subunits of both maltase-glucoamylase and sucrase-isomaltase. Only the mucosal α-glucosidases provide the final hydrolytic activities to produce substantial free glucose. We report here the unique and shared roles of the individual α-glucosidases for α-glucans persisting after starch is extensively hydrolyzed by α-amylase (to produce α-limit dextrins (α-LDx)). All four α-glucosidases share digestion of linear regions of α-LDx, and three can hydrolyze branched fractions. The α-LDx, which were derived from different maize cultivars, were not all equally digested, revealing that the starch source influences glucose generation at the mucosal α-glucosidase level. We further discovered a fraction of α-LDx that was resistant to the extensive digestion by the mucosal α-glucosidases. Our study further challenges the conventional view that α-amylase is the only rate-determining enzyme involved in starch digestion and better defines the roles of individual and collective mucosal α-glucosidases. Strategies to control the rate of glucogenesis at the mucosal level could lead to regulation of the glycemic response and improved glucose management in the human body.

Modulation of starch digestion for slow glucose release through "toggling" of activities of mucosal α-glucosidases.

Starch digestion involves the breakdown by α-amylase to small linear and branched malto-oligosaccharides, which are in turn hydrolyzed to glucose by the mucosal α-glucosidases, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI). MGAM and SI are anchored to the small intestinal brush-border epithelial cells, and each contains a catalytic N- and C-terminal subunit. All four subunits have α-1,4-exohydrolytic glucosidase activity, and the SI N-terminal subunit has an additional exo-debranching activity on the α-1,6-linkage. Inhibition of α-amylase and/or α-glucosidases is a strategy for treatment of type 2 diabetes. We illustrate here the concept of "toggling": differential inhibition of subunits to examine more refined control of glucogenesis of the α-amylolyzed starch malto-oligosaccharides with the aim of slow glucose delivery. Recombinant MGAM and SI subunits were individually assayed with α-amylolyzed waxy corn starch, consisting mainly of maltose, maltotriose, and branched α-limit dextrins, as substrate in the presence of four different inhibitors: acarbose and three sulfonium ion compounds. The IC(50) values show that the four α-glucosidase subunits could be differentially inhibited. The results support the prospect of controlling starch digestion rates to induce slow glucose release through the toggling of activities of the mucosal α-glucosidases by selective enzyme inhibition. This approach could also be used to probe associated metabolic diseases.