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Superior metabolic flexibility, or the ability to efficiently switch between oxidation of carbohydrate and fat, is inversely associated with obesity and type 2 diabetes. This study examined the impact of dietary carbohydrate digestion rate on metabolic substrate utilization and metabolic flexibility. We employed percent relative cumulative frequency (PRCF) analyses coupled with a new application of modeling using the Mixed Weibull Cumulative Distribution function to examine respiratory exchange ratio (RER) data from wild-type mice and mice lacking the mucosal maltase-glucoamylase enzyme (Mgam, null) under different dietary carbohydrate conditions. We further devised a Metabolic Flexibility Factor (MFF) to quantitate metabolic flexibility, with higher MFF indicating higher metabolic flexibility. The collective results indicated that a diet high in slowly digestible starch exhibited higher metabolic flexibility (MFF) than diets high in resistant starch, sucrose, or fat. These findings show a new-found benefit of consuming slowly digestible carbohydrates for improved metabolic health.

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.

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.

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 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.

OBJECTIVES: To analyze the role of each subunit from Sucrase-Isomaltase (SI) and Maltase-Glucoamylase (MG) on the glucose production from corn starch (NC), Waxy starch (WX) and Hylon V (HLV) starch predigested with pancreatic amylase (Amy). METHODS: The starches were selected based on their ratios of α-1,4 and α-1,6 linkages. Starches were predigested with Amy for 0, 10, 20, 30, 60 and 120 min. After predigestion starches were hydrolyzed with different combinations of recombinant C terminal (C) and N terminal (N) subunits of Sucrase-Isomaltase (SI) and Maltase-Glucoamylase (MG), each at the relative maltase activity of: 40% C-SI, 20% N-SI, 32% C-MG and 8% N-MG, resembling the observed in the human intestinal mucosa. Glucose release was quantified by a real-time glucose oxidase-peroxidase method at 37°C. RESULTS: C-MG subunit hydrolyzed the three starches without predigestion by Amy at rates of 2.9 ± 0.02, 2.5 ± 0.02 and 2.4 ± 0.05 ng/min of glucose for WX, HLV and NC, respectively. C-SI subunit produced only 0.9 ± 0.02 ng/min glucose from the starches not predigested by Amy. After 30 min of Amy predigestion, the rates of glucose release by C-MG were modified to 13.3 ± 0.09, 12.3 ± 0.08 and 11.6 ± 0.08 ng/min for HLV, NC and WX, respectively. The same predigestion modified glucose release by C-SI subunit to 3.9 ± 0.02, 4.7 ± 0.08 and 5.8 ± 0.03 for WX, NC and HLV, respectively. In starches predigested 60 or 120 min, C-MG subunit alone or together with N-MG showed lower activity than expected, suggesting the occurrence of substrate inhibition. Together the four MG and SI subunits showed clear differences on the rates of glucose production from the starches predigested with Amy for 120 min (22.8 ± 0.133 for WX, 26.4 ± 0.2 for NC and 26.7 ± 0.8 for HLV; all ng/min) which correlated with the glucose production by the N and C SI subunits together (15.5 ± 0.9 for WX, 19.4 ± 0.09 for NC and 23.85 ± 0.12 for HLV; all ng/min). CONCLUSIONS: We found complex interactions among the starch structure, extent of amylase digestion and the rate of glucose release by each C and N subunits of SI and MG. Thus, the rate of intestinal starch digestion depends on the structure of the starches, the predigestion by amylase and, importantly, the relative abundance and catalytic properties of the individual subunits of MG and SI. FUNDING SOURCES: Consejo Nacional de Ciencias y Tecnología (CONACyT), Mexico.

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.

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.

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.

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.

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.

Objectives: Six enzyme activities are needed to digest starch to absorbable free glucose; 2 luminal a-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-a-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:Mgamnulland 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 fGG (P ¼0.02) 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.

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.

Inhibition of intestinal α-glucosidases and pancreatic α-amylases is an approach to controlling blood glucose and serum insulin levels in individuals with Type II diabetes. The two human intestinal glucosidases are maltase-glucoamylase and sucrase-isomaltase. Each incorporates two family 31 glycoside hydrolases responsible for the final step of starch hydrolysis. Here we compare the inhibition profiles of the individual N- and C-terminal catalytic subunits of both glucosidases by clinical glucosidase inhibitors, acarbose and miglitol, and newly discovered glucosidase inhibitors from an Ayurvedic remedy used for the treatment of Type II diabetes. We show that features of the compounds introduce selectivity towards the subunits. Together with structural data, the results enhance the understanding of the role of each catalytic subunit in starch digestion, helping to guide the development of new compounds with subunit specific antidiabetic activity. The results may also have relevance to other metabolic diseases such as obesity and cardiovascular disease.

The detailed mechanistic aspects for the final starch digestion process leading to effective alpha-glucogenesis by the 2 mucosal alpha-glucosidases, human sucrase-isomaltase complex (SI) and human maltase-glucoamylase (MGAM), are poorly understood. This is due to the structural complexity and vast variety of starches and their intermediate digestion products, the poorly understood enzyme-substrate interactions occurring during the digestive process, and the limited knowledge of the structure-function properties of SI and MGAM. Here we analyzed the basic catalytic properties of the N-terminal subunit of MGAM (ntMGAM) on the hydrolysis of glucan substrates and compared it with those of human native MGAM isolated by immunochemical methods. In relation to native MGAM, ntMGAM displayed slower activity against maltose to maltopentose (G5) series glucose oligomers, as well as maltodextrins and alpha-limit dextrins, and failed to show the strong substrate inhibitory "brake" effect caused by maltotriose, maltotetrose, and G5 on the native enzyme. In addition, the inhibitory constant for acarbose was 2 orders of magnitude higher for ntMGAM than for native MGAM, suggesting lower affinity and/or fewer binding configurations of the active site in the recombinant enzyme. The results strongly suggested that the C-terminal subunit of MGAM has a greater catalytic efficiency due to a higher affinity for glucan substrates and larger number of binding configurations to its active site. Our results show for the first time, to our knowledge, that the C-terminal subunit of MGAM is responsible for the MGAM peptide's "glucoamylase" activity and is the location of the substrate inhibitory brake. In contrast, the membrane-bound ntMGAM subunit contains the poorly inhibitable "maltase" activity of the internally duplicated enzyme.

BACKGROUND: Starches are the major source of dietary glucose in weaned children and adults. However, small intestine alpha-glucogenesis by starch digestion is poorly understood due to substrate structural and chemical complexity, as well as the multiplicity of participating enzymes. Our objective was dissection of luminal and mucosal alpha-glucosidase activities participating in digestion of the soluble starch product maltodextrin (MDx). PATIENTS AND METHODS: Immunoprecipitated assays were performed on biopsy specimens and isolated enterocytes with MDx substrate. RESULTS: Mucosal sucrase-isomaltase (SI) and maltase-glucoamylase (MGAM) contributed 85% of total in vitro alpha-glucogenesis. Recombinant human pancreatic alpha-amylase alone contributed

Digestion of starch requires activities provided by 6 interactive small intestinal enzymes. Two of these are luminal endo-glucosidases named alpha-amylases. Four are exo-glucosidases bound to the luminal surface of enterocytes. These mucosal activities were identified as 4 different maltases. Two maltase activities were associated with sucrase-isomaltase. Two remaining maltases, lacking other identifying activities, were named maltase-glucoamylase. These 4 activities are better described as alpha-glucosidases because they digest all linear starch oligosaccharides to glucose. Because confusion persists about the relative roles of these 6 enzymes, we ablated maltase-glucoamylase gene expression by homologous recombination in Sv/129 mice. We assayed the alpha-glucogenic activities of the jejunal mucosa with and without added recombinant pancreatic alpha-amylase, using a range of food starch substrates. Compared with wild-type mucosa, null mucosa or alpha-amylase alone had little alpha-glucogenic activity. alpha-Amylase amplified wild-type and null mucosal alpha-glucogenesis. alpha-Amylase amplification was most potent against amylose and model resistant starches but was inactive against its final product limit-dextrin and its constituent glucosides. Both sucrase-isomaltase and maltase-glucoamylase were active with limit-dextrin substrate. These mucosal assays were corroborated by a 13C-limit-dextrin breath test. In conclusion, the global effect of maltase-glucoamylase ablation was a slowing of rates of mucosal alpha-glucogenesis. Maltase-glucoamylase determined rates of digestion of starch in normal mice and alpha-amylase served as an amplifier for mucosal starch digestion. Acarbose inhibition was most potent against maltase-glucoamylase activities of the wild-type mouse. The consortium of 6 interactive enzymes appears to be a mechanism for adaptation of alpha-glucogenesis to a wide range of food starches.

Maltase-Glucoamylase (MGAM) and Sucrase-Isomaltase (SI) are α-glucosidases of the membrane of small intestinal enterocytes responsible for the digestion of dietary carbohydrates into glucose and other monosaccharides. MGAM and SI mRNA expression, protein synthesis and catalytic activity of MGAM and SI are found predominantly in enterocytes; however, they are also found in lower amounts in other non-intestinal tissues, notably blood leukocytes and kidney cells. Little information exists on the functions, the mRNA expression, the nature of the respective proteins and catalytic properties of MGAM and SI in non-intestinal tissues. To correlate the transcription, synthesis and activity of these enzymes, we measured the mRNA expression of MGAM and SI; the catalytic activities of maltase, sucrase, and isomaltase; and the presence of the respective proteins in small intestine and other tissues such as spleen, kidney and pancreas of 8 weeks old mice. Small intestine showed the highest level of mRNA expression of M...

SXT-related integrating conjugative elements (ICEs) became prevalent in Asian Vibrio cholerae populations after V. cholerae O139 emerged. Here, we describe an SXT-related ICE, ICE Vch Mex1, in a Mexican environmental V. cholerae isolate. Identification of ICE Vch Mex1 represents the first description of an SXT-related ICE in the Western Hemisphere. The significant differences between the SXT and ICE Vch Mex1 genomes suggest that these ICEs have evolved independently.

Sugars normally are absorbed in the small intestine. When carbohydrates are malabsorbed, the osmotic load produced by the high amount of low molecular weight sugars and partially digested starches in the small intestine can cause symptoms of intestinal distention, rapid peristalsis, and diarrhea. Colonic bacteria normally metabolize proximally malabsorbed dietary carbohydrate through fermentation to small fatty acids and gases (ie, hydrogen, methane, and carbon dioxide). When present in large amounts, the malabsorbed sugars and starches can be excreted in the stool. Sugar intolerance is the presence of abdominal symptoms related to the proximal or distal malabsorption of dietary carbohydrates. The symptoms consist of meal-related abdominal cramps and distention, increased flatulence, borborygmus, and diarrhea. Infants and young children with carbohydrate malabsorption show more intense symptoms than adults; the passage of undigested carbohydrates through the colon is more rapid and is associated with detectable carbohydrates in copious watery acid stools. Dehydration often follows feeding of the offending sugar. In this review we present the clinical and current molecular aspects of disaccharidase digestion.

Using papain digestion together with molecular sieving and ion-exchange HPLC, maltase–glucoamylase (MGA) was purified from small intestinal mucosa of CBA/J mice. The purified enzyme displayed an apparent M.W. of 500–600 kDa by SDS–PAGE analysis and under fully denaturing conditions was found to comprise at least three different glycoproteins with apparent M.W. of 410, 275, and 260 kDa, respectively. Thus, murine MGA displayed structural homology to the enzymes obtained from rat and rabbit intestines and differed substantially from the structures reported for the human, pig, and chicken counterparts. The enzyme showed spontaneous degradation during storage at −20 °C with accumulation particularly of the 275 and 260 kDa proteins. In addition, IgG obtained from sera of MGA-deficient CBA/Ca mice previously immunized with murine MGA reacted with the native enzyme, as well as with the 410, 275, and 260 kDa components. These results indicated that the 410 kDa component might constitute a precursor of the components with lower apparent M.W.

While luminal α-Amylase (AMY) is recognized as a starch digesting enzyme, the products require that glucose be hydrolyzed from the non-reducing ends of the oligosaccharide produced. Two mucosal enzyme complexes are required for glucogenesis: Maltase-glucoamylase (Mgam) is a fast and Sucrase-isomaltase (Si) is a slow α-glucosidase. Mgam was knocked-out in mice with the objective of determining role of Si on in vivo starch digestion to glucose. 8 mice were fed 0.5 gm. of chow containing normal maize starch. 2 h after feeding they were euthanized and 4 segments of small bowel (SB) snap-frozen. The segments were thawed and homogenized in water. Protein assays were by Bradford method. The homogenate was denatured in 80% ethanol at 80°C. Supernatant was collected by centrifugation. Free glucose of concentrated supernatant was determined by glucose oxidase/peroxidase. Soluble oligosaccharides were determined by diluting supernatant with buffer, pH 4.5, in 10% ethanol and 2.5 units of amyloglucosidase (AMG) added...

Some polyphenols inhibit the activities of the four subunits of intestinal α-glucosidases, maltase-glucoamylase (Mgam) and sucrase-isomaltase (Si). The use of polyphenols as modulators of the rate ...

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.

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.

MicroRNAs (miRNAs) are involved in gene regulation of several physiological processes. Alterations in the concentrations of miRNAs may result in cancer and autoimmune diseases. In cells of the immune system, miRNA expression is regulated by several cytokines and this expression is related to the inflammatory process. In the present work we evaluated miR-155 and miR-146a levels in peripheral blood mononuclear cells (PBMC) from patients with type 2 diabetes (T2D). We analysed the expression of miRNAs in PBMC from T2D patients (n=20) and control subjects (n=20) using real-time PCR. The quantity of IL-1β and IL-6 in culture supernatants was measured by ELISA. The basal expression of miR-155 and miR-146a in patients with T2D was decreased compared to control subjects and associated with age, gender and metabolic control but not with the therapeutic treatment used. We found significant correlations between the basal expression of miR-155 and miR-146a with HbA1c, Glucose and BMI, as well as of miR-155 expression stimulated by LPS with the values of TG, HbA1c, Glucose and BMI. Additionally, we detected an altered distribution of miR-155 and miR-146a expression related with HbA1c, glucose and BMI using the analysis of a three dimensional association of variables in the group of T2D patients. Downregulated levels of miR-155 could play an important role in the pathogenesis of T2D due to their relationship with metabolic control.

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 β-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.

Some polyphenolic compounds inhibit the intestinal alpha-glucosidases. However, it is not clear how those compounds affect the enzyme activity of the individual C or N terminals subunits of maltase-glucoamylase (Mgam) and sucrase-isomaltase (Si). ObjectiveTo modulate the enzymatic activity of the most active C terminal-Mgam (Ct-Mgam) and C terminal Si (Ct-Si) by selected polyphenols in order to control the glucose release from the starch digestion. MethodsMaltase activities of recombinant Ct-Mgam and Ct-Si subunits were measured by Tris Glucose- Oxidase (TGO) method. The reactions were stopped by TGO and the optical density was measured at 450 nm. ResultsMaltase activity of both enzymes was inhibited. IC50 values were 0.58 and 0.571 mM for caffeic acid, 0.039 and 0.014 mM chlorogenic acid, 0.139 and 0.213 mM for (+)-catechin hydrate, 0.383 and 0.177 mM for gallic acid, 0.041 and 0.014 mM for (−) epigallocatechin gallate for Ct-Si and Ct-Mgam, respectively. ConclusionsThe results indicate that the potencie...

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.

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.

For starch digestion to glucose, two luminal α-amylases and four gut mucosal α-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal α-glucosidases on cooked (gelatinized) starch. Gelatinized normal maize starch was digested with N- and C-terminal subunits of recombinant mammalian maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) of varying amounts and digestion periods. Without the aid of α-amylase, Ct-MGAM demonstrated an unexpected rapid and high digestion degree near 80%, while other subunits showed 20 to 30% digestion. These findings suggest that Ct-MGAM assists α-amylase in digesting starch molecules and potentially may compensate for developmental or pathological amylase deficiencies.

Chronic inflammation is an important contributor to the insulin resistance observed in type 2 diabetes (T2D). We evaluated the expression and function of the P2X(7) receptor and CD39/Entpd1, molecules involved in the cellular regulation of inflammation, in peripheral blood mononuclear cells from T2D patients, and their correlation with the concentration of HbA1c in blood. T2D patients with deficient metabolic control (DC) showed increased proportion of P2X(7)(+) cells compared with healthy individuals; T2D-DC subjects also displayed higher proportion of CD14(+), CD4(+) and CD19(+) subpopulations of P2X(7)(+) cells when compared with T2D patients with acceptable metabolic control. A significant association was observed between the proportion of P2X(7)(+)CD14(+) cells and blood concentration of LDL-c. In addition, the percentages of CD39(+) cells and CD39(+)CD19(+) cells were significantly associated with HbA1c and fasting plasma glucose levels. No changes were observed in the function of P2X(7)(+) cells from T2D patients; however, enhanced CD39/Entpd1 enzyme activity and low serum levels of IL-17 were detected. Therefore, CD39(+) cells could have a balancing regulatory role in the inflammatory process observed in patients with T2D.

Starch digestion requires two luminal enzymes, salivary and pancreatic alpha-amylase (AMY), and four small intestinal mucosal enzyme activities from the N- and C-terminals of maltase-glucoamylase (...

Congenital sucrase-isomaltase deficiency (CSID) is characterized by absence or deficiency of the mucosal sucrase-isomaltase enzyme. Specific diagnosis requires upper gastrointestinal biopsy with evidence of low to absent sucrase enzyme activity and normal histology. The hydrogen breath test (BT) is useful, but is not specific for confirmation of CSID. We investigated a more specific 13C-sucrose labeled BT.Determine whether CSID can be detected with the 13C-sucrose BT without duodenal biopsy sucrase assay, and if the 13C-sucrose BT can document restoration of sucrose digestion by CSID patients after oral supplementation with sacrosidase (Sucraid).Ten CSID patients were diagnosed by low biopsy sucrase activity. Ten controls were children who underwent endoscopy and biopsy because of dyspepsia or chronic diarrhea with normal mucosal enzymes activity and histology. Uniformly labeled 13C-glucose and 13C-sucrose loads were orally administered. 13CO2 breath enrichments were assayed using an infrared spectrophotometer. In CSID patients, the 13C-sucrose load was repeated adding Sucraid. Sucrose digestion and oxidation were calculated as a mean percent coefficient of glucose oxidation averaged between 30 and 90 minutes.Classification of patients by 13C-sucrose BT percent coefficient of glucose oxidation agreed with biopsy sucrase activity. The breath test also documented the return to normal of sucrose digestion and oxidation after supplementation of CSID patients with Sucraid.13C-sucrose BT is an accurate and specific noninvasive confirmatory test for CSID and for enzyme replacement management.

Starch is the major source of food glucose and its digestion requires small intestinal alpha-glucosidic activities provided by the 2 soluble amylases and 4 enzymes bound to the mucosal surface of enterocytes. Two of these mucosal activities are associated with sucrase-isomaltase complex, while another 2 are named maltase-glucoamylase (Mgam) in mice. Because the role of Mgam in alpha-glucogenic digestion of starch is not well understood, the Mgam gene was ablated in mice to determine its role in the digestion of diets with a high content of normal corn starch (CS) and resulting glucose homeostasis. Four days of unrestricted ingestion of CS increased intestinal alpha-glucosidic activities in wild-type (WT) mice but did not affect the activities of Mgam-null mice. The blood glucose responses to CS ingestion did not differ between null and WT mice; however, insulinemic responses elicited in WT mice by CS consumption were undetectable in null mice. Studies of the metabolic route followed by glucose derived from intestinal digestion of (13)C-labeled and amylase-predigested algal starch performed by gastric infusion showed that, in null mice, the capacity for starch digestion and its contribution to blood glucose was reduced by 40% compared with WT mice. The reduced alpha-glucogenesis of null mice was most probably compensated for by increased hepatic gluconeogenesis, maintaining prandial glucose concentration and total flux at levels comparable to those of WT mice. In conclusion, mucosal alpha-glucogenic activity of Mgam plays a crucial role in the regulation of prandial glucose homeostasis.

Human maltase-glucoamylase (MGAM) is one of the two enzymes responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM is anchored to the small-intestinal brush-border epithelial cells and contains two homologous glycosyl hydrolase family 31 catalytic subunits: an N-terminal subunit (NtMGAM) found near the membrane-bound end and a C-terminal luminal subunit (CtMGAM). In this study, we report the crystal structure of the human NtMGAM subunit in its apo form (to 2.0 A) and in complex with acarbose (to 1.9 A). Structural analysis of the NtMGAM-acarbose complex reveals that acarbose is bound to the NtMGAM active site primarily through side-chain interactions with its acarvosine unit, and almost no interactions are made with its glycone rings. These observations, along with results from kinetic studies, suggest that the NtMGAM active site contains two primary sugar subsites and that NtMGAM and CtMGAM differ in their substrate specificities despite their structural relationship. Additional sequence analysis of the CtMGAM subunit suggests several features that could explain the higher affinity of the CtMGAM subunit for longer maltose oligosaccharides. The results provide a structural basis for the complementary roles of these glycosyl hydrolase family 31 subunits in the bioprocessing of complex starch structures into glucose.