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Biochemical, Molecular, and Genetic Mechanisms

Contribution of Mucosal Maltase-Glucoamylase Activities to Mouse Small Intestinal Starch -Glucogenesis^1–3,

⁴ CIEP-Facultad de Ciencias Quimicas, Universidad Autonoma de San Luis Potosi, Zona Universitaria, San Luis Potosí, S.L.P., Mexico 78360; ⁵ USDA, Agricultural Research Service, Children's Nutrition Research Center and Department of Pediatrics, Baylor College of Medicine, Houston, TX, 77030-2600; ⁶ Whistler Center for Carbohydrate Research and Department of Food Science, Purdue University, West Lafayette, Indiana 47907-2009; ⁷ Division of Biological Sciences, Section of Physiology, Cornell University, Ithaca, New York 14853 ⁸ Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver V6T 1Z3, Canada; ⁹ Ingenium Pharmaceuticals AG, 82152 Martinsried, Germany; and ¹⁰ Institute of Biochemistry and Molecular Medicine, University of Berne, Berne CH-3012, Switzerland

^* To whom correspondence should be addressed. E-mail: bnichols{at}bcm.tmc.edu.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Digestion of starch requires activities provided by 6 interactive small intestinal enzymes. Two of these are luminal endo-glucosidases named -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 -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 -glucogenic activities of the jejunal mucosa with and without added recombinant pancreatic -amylase, using a range of food starch substrates. Compared with wild-type mucosa, null mucosa or -amylase alone had little -glucogenic activity. -Amylase amplified wild-type and null mucosal -glucogenesis. -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 -glucogenesis. Maltase-glucoamylase determined rates of digestion of starch in normal mice and -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 -glucogenesis to a wide range of food starches.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

The digestion of starch to free glucose is called -glucogenesis. This process varies in each species but generally uses a consortium of 6 -glucogenic enzymes. Two enzymes are secreted salivary and pancreatic luminal activities called -amylases (AMY),¹¹ which hydrolyze internal -1,4 glucose bonds of linear starch polymers and polymer branches (2). The genes for the -amylases, which are 98% homologous, are located within the same chromosomal locus. The gene has been found in all Coelomata sequenced. The mammalian AMY enzymes and their mechanisms of action are well understood (2–7). Additional enzyme activities are bound to the luminal surface of small intestinal enterocytes (8–13). These activities were first described as maltases, to distinguish the activities from AMY, which poorly digests maltose (14–17). Subsequent investigations distinguished 4 different maltase activities (11–13). Two activities were associated with the activities of a twined catalytic complex named sucrase-isomaltase (SI) (18–22). Two other activities were not associated with any identifying substrate specificities and were named maltase-glucoamylase (MGAM) (14–17). It has become recognized that these 4 maltase activities extend to other linear glucosides of infinite length and are thus better described as -glucosidases (18–22). All 4 hydrolyze nonreducing terminal -1,4 glucose bonds. The membrane bound catalytic unit of SI also has a -1,6 activity that can hydrolyze branched starches (20). There is no amino acid sequence homology between the -glucosidases and the AMY, but there is about a 60% homology within the tandem terminals and between the SI and MGAM complexes. These amino acid sequences also showed that the 2 enzymes have homology with Family 31 Gluco-hydrolases, found in nearly all eukaryotic organisms reported to date (14–16). Similarity of sequences makes it difficult to discriminate between SI and MGAM complexes at the genomic level, but the expression of the genes from 2 different chromosomes is characteristic. SI is only expressed in the intestine but 3' sequences of MGAM and its activities have been detected in many tissues (14).

The apparent redundancies of the -glucosidases have resulted in confusion about their individual roles. As an example, the in vitro hydrolysis of starch substrates by AMY produces some free glucose; this has led some to classify the enzyme as the major -glucosidase (2). Others have thought that the mucosal -glucosidases serve only as a backup for the luminal AMY enzymes (2). To resolve this lingering uncertainty, we ablated the MGAM gene (called Mgam in mice) to determine its function and whether the residual SI activity (called Si in mice) is in fact redundant. The absence of Mgam allowed the determination of the function of Si activities on starch digestion to glucose. The availability of human recombinant pancreatic amylase (rhpAMY) permitted clarification of its role in starch digestion (6,7). The -glucogenic activities of the Mgam null and wild-type (WT) mucosa were assayed in vitro with a variety of starches and starch hydrolysates. The mucosal assay results were confirmed in vivo. Because of the potential roles of starch digestion on glucose-driven disorders such as type II diabetes, we also tested the effects of the -glucosidase inhibitor acarbose in vitro (23).

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Mutation of Mgam by homologous recombination. Three genomic clones were identified in a mouse library screened with 5' PCR primer sets previously used in cloning and sequencing the human MGAM cDNA (15). Sequencing of the mouse genomic clones revealed a conserved Mgam exon 2. A targeting vector containing the Lac Z -MC1neo selection marker and the flanking thymidine kinase genes was designed to replace parts of exon 2 (Figure 1). The construct was electrophorated into strain Sv/129 stem cells. Three positive stem cell clones that were identified by Southern analysis using 3'or 5' external probes were implanted in prepared foster dams. The F1 generation was back-crossed with the same Sv/129 strain of mice and heterozygotes were identified by Southern analysis. After germ line transmission, the Mgam genotypes were identified by quantitative PCR (qPCR) of tail DNA. The control strains of mice were purchased from Jackson Laboratory. All mouse experiments were approved under the Baylor College of Medicine Institutional Animal Care and Use Committee protocol AN-1577.

View larger version (6K): [in this window] [in a new window]   FIGURE 1  Ablation of Mgam by homologous recombination. Genomic clones representing exon 2 were isolated and parts of exon 2 were replaced by insertion with an LacZ-MC1 neocassette. The selection cassette introduced a new HindIII restriction site that was used to screen ES cells by Southern analysis. After germ line transmission, the Mgam genotypes were identified by qPCR.

    Phenotyping of mice by Western blots and histochemistry. Mgam is internally duplicated with catalytic sites at both the N-terminal and C-terminal (14,15). A peptide was designed from translated Mgam nucleotide sequences to be specific for exon 25, located in the trefoil region at the beginning of the C-terminus. Peptide FFTREEERIDC (located at Mgam-893) was used for immunizing rabbits. The peptide was conjugated to KLH and used with Freund's adjuvant to immunize rabbit 14807. Antibody (Ab) titers were tested after 77 d and were >20,000 against the immunizing peptide. The resulting 14807 Ab were then immunopurified and used to resolve Western blot of Mgam null and WT jejunum homogenates that were developed by chemoluminiscence. Monoclonal Ab (mAb) DBB 2/68 against N-terminal subunit of rat Mgam confirmed (24,25) the results obtained with the polyclonal Ab. After centrifuging jejunal homogenates for 100,000 x g for 60 min, the 14807 Ab and mAb DBB 2/68 were used to test for presence of soluble Mgam in supernatants (25).

The homologous recombination cassette contained the E. coli ß-glucosidase knock-in gene. Localization of the expressed ß-glucosidase, driven by the intact Mgam promoter, was evaluated in OCT-embedded frozen jejunal sections of Mgam null mice using 5-bromo-4-chloro-3-indolyl -D-galactopyranoside (X-gal, Sigma-Aldrich) substrate.

    Starch digestion and oxidation to CO₂. Starch digesting phenotypes were investigated in 4 null and 4 WT mice by assaying the extent of conversion of fed ¹³C-starch substrate to ¹³CO₂ levels in breath. Universally labeled ¹³C-starch of algal source (Sigma-Aldrich) was exhaustively digested with porcine AMY (Type VI-B, Sigma-Aldrich) to make ¹³C-labeled limit dextrin (LD), which is insensitive to further luminal digestion. The soluble fraction of ¹³C-LD was freeze-dried and used for testing starch digestion. Based upon exhaustive AMG (Megazyme International) digestion followed by the glucose oxidase reaction, the ¹³C-LD solution contained 3.48 ± 0.12 g/L glucose equivalents, of which only 0.21 ± 0.01 g/L (6%) was free glucose. Genotyped mice were fed a low ¹³C-nonpurified diet (Harlan Teklad; TD.0689) for 2 wk before the experiment. The mice were then fasted overnight and placed in a 0.24 L glass breath collection chamber. Airflow to the chamber was 0.24 L/min. The airflow was interrupted for 7.5 min and then restarted for 2.5 min. The initial 0.24 L of effluent air was collected into a CO₂ impervious bag and the chamber was washed out. The collected effluent samples were analyzed with a POCone IR spectrophotometer (Otsuka Electronics) for total CO₂ and ratios of ¹³CO₂/¹²CO₂. After baseline samples were obtained, each mouse received 0.2 mL of the ¹³C-LD solution by gavage. Effluent breath samples were collected at 7 min after the gavage and at 10 min intervals thereafter. Total CO₂ never exceeded 2% in the samples. Results are reported as ¹³CO₂ delta over baseline (OB) breath enrichments.

    Dahlqvist -glucogenesis assays. The sucrase, maltase, and lactase assays were those described by Dahlqvist (10,26) at 16-mol/L concentrations and 60-min incubations at 37°C. The -glucosidase assay was a modification previously described (27) with 20 g/L maltodextrin (MD) used as substrate. Activity was reported as international enzyme units (U/g protein; 1 U = 1 µmol of glucose released per minute of reaction).

    Measures of catalytic properties. Because Dahlqvist assay measurements of human disaccharidase activities fail to differentiate the different maltase activities (16), we took measurements of the kinetic parameters of Michaelis constants (Km), and maximum rate of reaction (Vmax) of jejunal mucosa on maltose, sucrose, isomaltose, and palatinose substrates using the Tris glucose-oxidase (TGO) technique adapted to 96-well plates (28). Briefly, 10 µL of jejunal mucosa scraping homogenates were mixed with 10 µL of substrate solutions at concentrations ranging from 0.1 to 50 mmol/L for maltose and isomaltose, 1.1 to 100 mmol/L for sucrose, or 0.5 to 25 mmol/L for palatinose and incubated at 37°C for 30 min for maltase and isomaltase activities, or 60 min for sucrase and palatinase activities. To determine the inhibitory constants (Ki) of acarbose, before the incubation period, 10 µL of acarbose (Toronto Research Chemicals) solutions were added to the reaction mixtures to attain final concentrations of 1, 10, 100, and 1000 µmol/L . After incubations, 200 µL of TGO reagent were added to each well, the plates were incubated for an additional 60 min at 37°C, and then the OD at 450 nm of each well was measured in a SpectraMax190 microplate spectrophotometer (Molecular Devices). A standard curve of glucose concentration was run in parallel for each assay. Protein concentration in homogenates was measured by the Bradford method. Activities are reported as international units of activity per mg of protein.

    Kinetic -glucogenesis assay. Soluble substrates (maltose, isomaltose, MD, and LD) were dissolved at 5 g/L in PBS (10 mmol/L phosphates pH 6.8 with 150 mmol/L NaCl). Insoluble starches were wetted and then dissolved in DMSO at 10 g/L, by heating at 90°C for 30 min. Solubilized starches were precipitated with 4 volumes of 100% ethanol, sedimented by centrifugation, dried, and dissolved in warm (40°C) PBS at 5 g/L. Real-time glucose release was measured by a modification of the assay described above (28). Phosphate glucose-oxidase developing reagent (190 µL containing 150 U/L of glucose oxidase, 750 U/L of horseradish peroxidase, 0.2% Triton x100, and 50 mg/L of O-dianosidine-HCl, all in PBS), were placed in each well. Next, 10 µL of individual substrate were added to the wells (240 mg/L final concentration), the mixtures were preincubated for 10 min at 37°C, and then 10 µL of enzyme preparations were added to each well. OD at 450 nm was measured in the microplate spectrophotometer at 37°C at 2 min intervals with a 3 s shaking before each reading. rhpAMY (6,7) was added in a volume of 10 µL to obtain a final concentration of 1.8 mg/L. Solubilized mouse jejunal lysates were prepared by mixing 1 volume of a 10% sodium deoxycholate with 10% Nonidet P40 solution and 10 volumes of homogenate (100 g/L in PBS). After 1 h mixing by rotation at 4°C, lysates were centrifuged at 15,600 x g and supernatants recovered and diluted 1:10 with PBS. For glucogenic assays, 10 µL of diluted lysate supernatants were added to appropriate wells. All measurements were in triplicate. Blanks for substrate and enzyme preparations were included in each assay.

    Maltodextrin and -limit–dextrin characterizations. The food grade MD used (Polycose, Ross Laboratories) was manufactured by partial fungal AMY -1,4-digestion of cornstarch (1). The composition of MD, as determined by MALDI-TOF analysis before and after digestion with porcine pancreatic AMY, is described elsewhere (16). Data of mass fraction and MW for each oligomer, determined by MALDI-TOF analysis, were used to calculate the mean MW of MD. Reagent grades of maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentose (G5), sucrose, palatinose, whole starch, amylopectin, and amylose substrates were purchased from Sigma-Aldrich.

    Resistant starch production. The methods of production and properties of resistant starches from normal cornstarch are fully described elsewhere (29). Normal cornstarch was a gift from Tate and Lyle, Inc. Four products were prepared: ß-Amylase from barley (Optimalt)-treated normal cornstarch (BBA); BBA and transglucosidase L-500 from Aspergillus-treated cornstarch (BBA/TG); Maltogenic -AMY (Novamyl: 10000 BG: MA)-treated cornstarch (MA); and MA and transglucosidase L-500 from Aspergillus-treated cornstarch (MA/TG). The chemical and structural characteristics of these 4 resistant starches are fully described elsewhere (29).

    Statistics. Differences in the rate of breath release of ¹³CO₂ between strains were determined by ANOVA by the General Lineal Model, using strains as classifying factor and time as a covariate. Pairs of data (strain x time) with significant differences were determined by pairwise comparisons by the simultaneous test of Dunnett's (family error rate = 0.01) with WT mice as the control group. One-way ANOVA and Dunnett's test were used to determine differences in the levels of enzymatic activities measured by Dahlqvist's method between WT and heterozygous and null mice, with WT as a control group. Fisher's test (individual error rate = 0.01) was used to identify differences between heterozygous and null mice. Apparent Vmax, Km, and, when necessary, Ki values were calculated using nonlinear regression with the Marquardt-Levenberg algorithm, and models were adjusted to a single substrate Michaelis-Menten, a competitive inhibition, or to substrate inhibition kinetics. Differences between calculated kinetic parameters of the respective strains were determined pairwise by the Z test. Rates of reaction for real time glucogenesis assays were calculated under steady-state conditions by linear regression, during intervals from 10 to 30 min of reaction. Indices of digestion for different starches and derived substrates were calculated as the ratio of the rates of reaction at steady state, divided by the isomaltase activity observed after 60 min incubations for each enzyme mixture. Indices of digestion were analyzed by ANOVA and the General Lineal Model, with time as a covariate and enzyme mixture and/or substrate as classifying factors. Differences between indexes for each enzyme mixture and starch product were determined by 1-way ANOVA and Tukey's test (family error rate = 0.01). Statistical significance was considered at P < 0.05, unless otherwise noted.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Confirmation of Mgam KO by Southern blotting of DNA from embryonic stem cells. The selection cassette introduced a new HindIII site into the Mgam locus. A Southern blot of 93 embryonic stem cells revealed that 3 heterozygous lines, B2, B6, and F6, were produced. One of these heterozygous lines (B6) was successfully used to produce Mgam heterozygous offspring.

    Real time RT/PCR. The ablation of Mgam exon 2 in Mgam null mice was confirmed by qPCR from tail DNA. These mice with identified genotypes were used to breed experimental mice. After 10 generations of breeding, 44 random current-generation experimental mice were experimentally genotyped, and complete agreement was documented between the breeding and qPCR genotypes.

    Western blot and histochemistry for ß-glucosidase expression. Rabbit polyclonal Ab 14807 against Mgam exon 25 stained the characteristic 130 and 250 kDa bands (30,31) of Mgam in WT mice, but these bands were absent in null mouse jejunal homogenate (Fig. 2). This finding was confirmed by Western blot, using an anti-rat mAb recognizing the Mgam N-terminal subunit, and revealed deletion of the Mgam null mouse C-terminal as well as N-terminal subunits (25). No Mgam was detectable on blots of jejunal homogenate supernatants from Mgam null or WT mice with either 14807 antibodies or mAb DBB 2/68.

View larger version (95K): [in this window] [in a new window]   FIGURE 2  Phenotyping of mice by Western blot. Polyclonal Ab 14807, designed against a peptide fragment from mouse exon 25, stained with the characteristic 125 and 250 kDa bands of Mgam in jejunal mucosa homogenates (Ho) of WT. Characteristic Mgam protein bands were absent in null (Null) mouse jejunal mucosa homogenates. No Mgam peptides were detected in the supernatant (Sup) of null or WT homogenate after centrifugation at 100,000 x g. MW markers are shown in the right lane.

    Starch digesting phenotype. The response of mouse breath ¹³CO₂ concentrations to a ¹³C-starch load varied according to Mgam genotype (Fig. 3). WT mice reached a mean enrichment of 230 OB at 60 min, whereas the null mice reached a plateau at 100 OB after 30 min, following the ¹³C-LD bolus feeding.

View larger version (11K): [in this window] [in a new window]   FIGURE 3  Phenotyping of mice by starch substrate breath test. Digestion and oxidation to breath 13CO2 following a 13C-LD bolus feeding by mice with different Mgam phenotypes. Values are means ± SD, n = 4 Mgam null () and 4 WT (•) mice. *Different from Mgam null at that time, P < 0.01 (Dunnett's test).

    Dahlqvist assay mucosal enzyme activities. Mucosal homogenates from jejunum and ileum were assayed by the Dahlqvist method for total production of free glucose. The in vitro jejunal enzyme activity assays (Table 1) for 3 -glucosidase substrates (MD, maltose, and amylose) were reduced in Mgam null but not in heterozygous mice. All 3 of these hydrolytic activities in homogenates were significantly reduced by ANOVA analysis in the jejunum, and maltose activity was also reduced in the ileum of Mgam null mice (Table 1). Hydrolysis of sucrose and lactose was significantly increased by ANOVA analysis in the jejunum and ileum of the null mice. Assays with ileal mucosa (Table 1) demonstrated similar differences in -glucosidase activities in Mgam null, heterozygous, and WT mice to those observed in jejunum. In summary, -glucosidases showed an 50% reduction in the Mgam null jejunal homogenates, and sucrase and lactase activities were increased. The Mgam heterozygous null mouse had brush border enzyme activity levels identical to the WT mouse.

    Kinetics of activities of -glucogenic substrates. The enzyme kinetic parameters of the Mgam WT, null, and jejunal homogenates with congenital deficiencies of Mgam and Si (CBA/CaJ) mouse strains (Jackson Laboratory) are summarized in Table 2. The Km for maltose substrate was increased 5 to 10 times in the Mgam null and CBA/CaJ mouse jejunum, indicating that it was caused only by Si subunits and that this was reflected by a >60% reduction of the corresponding Vmax in the deficient strains. In contrast, the Km for sucrose, isomaltose, and palatinose hydrolysis were comparable across all 3 genotypes. However, the Vmax for isomaltase activities were increased by 50% in the Mgam null and CBA/CaJ mouse jejunums. Interestingly, although sucrase activity was also increased by 50% in Null mice, in CBA/CaJ mice it was decreased 50%, with respect to WT mice. These observations suggest that the synthesis of Si molecules is enhanced as a compensatory effect of the Mgam deficiency. The low sucrase activity in CBA/CaJ mice was expected, because we previously found a faster rate of inactivation of the sucrase subunit in this strain (28).

View this table: [in this window] [in a new window]   TABLE 2 Kinetic assays of mouse jejunal -glucosidase activities by Mgam genotype1

    Rates of -glucogenesis.

View larger version (12K): [in this window] [in a new window]   FIGURE 4  -Glucogenesis by jejunal homogenates from Mgam Null and WT mice. Real-time rates of -glucogenesis of Null and WT jejunal homogenates with maltose and isomaltase as substrates. Values are means of triplicate samples; SE are within the symbols.

View larger version (16K): [in this window] [in a new window]   FIGURE 5  -Glucogenic activity by jejunal homogenates from Mgam null and WT mice with MD as substrate, in the presence or absence of rhpAMY (Amy). (Left Y axis) Means of triplicate values for the ratios of glucose release from MD at different times, divided by glucose release from isomaltose at 60 min, caused by WT or Null jejunal homogenates in the absence or presence of rhpAMY. (Right Y axis) Glucogenic activity of rhpAMY alone from MD. Error bars represent the SD, n = 3. The slopes (dashed lines) were calculated by linear regression.

View this table: [in this window] [in a new window]   TABLE 3 Indexes of -glucogenesis from starch products from Mgam WT and Null mouse intestinal mucosa in the presence and absence of rhpAMY1

In contrast to null mice, WT intestinal mucosa homogenates containing Mgam and Si displayed substantial -glucogenic activity with all assayed substrates and in the absence of rhpAMY (Table 3). As expected, O2–5 was the most susceptible substrate for digestion by WT mucosa, whereas amylose was the most resistant substrate, probably due to the relatively low number of nonreducing ends and short-length glucose oligomers susceptible to the exo-glucosidic activities of Mgam and Si, respectively. Density of -1,6 diminished the digestibility of the substrates by WT mucosa. Corn amylopectin and all -1,6 transglycosilated substrates showed lower digestibility than normal cornstarch, indicating that this is a structural feature affecting digestibility. However, waxy starch with high amylopectin content was very digestible by WT mucosa, suggesting that it contained easily digestible molecules, probably formed by large number of relatively small starch molecules with short branched chains. rhpAMY showed an intense synergism with the -glucogenic activity of WT mucosa against most unpredigested substrates. Little or no change was observed in the -glucogenesis of WT mucosa with LD and O2–5 when assayed in conjunction with rhpAMY, suggesting that molecules susceptible to further rhpAMY digestion were exhausted during the predigestion. Amylopectin, MD, and waxy corn starch (WX) showed some resistance to the synergic effect of rhpAMY, probably due to a limited number of internal regions susceptible to the endo-glucosidic activity of rhpAMY and the steric hindrance caused by the high density of -1,6 linkages. All other substrates showed a 2- to 8-fold increment in -glucogenesis by WT mucosa when assayed with rhpAMY. Surprisingly, starch substrates subject to -1,6 linkage enrichment or -1,4 chain shortening were markedly more sensitive to the synergic activities of rhpAMY and WT small intestinal mucosa (Table 3).

    -Glucosidase inhibition by acarbose. As mentioned earlier, acarbose is sometimes used to inhibit -glucosidase activities in the management of type II diabetes (23). To understand this inhibition in more detail, we examined acarbose inhibition of -glucosidase activities in Mgam null and WT mice, as well as in mice from the CBA/CaJ strain (Table 2). The Ki for maltose substrate in the WT homogenate was much lower than that of the Mgam null or CBA/CaJ strain mice (Fig, 6A–C, Table 2). The Ki for sucrose substrate was equal among the strains of mice and in the range of the Ki for maltose in the WT mouse. The Ki of acarbose for isomaltose and palatinose substrates was much higher than that for sucrose and did not differ among the strains of mice. In summary, acarbose was most inhibitory of Mgam and less inhibitory of Si.

View larger version (15K): [in this window] [in a new window]   FIGURE 6  Relative inhibition of jejunal maltase activities from WT, Null, and CBA/CaJ mice by acarbose. Relative activities (with or without inhibitor) at variable substrate and inhibitor concentrations are means ± SD, n = 3. The vertical dashed lines represent the respective values of Ki ± SD for acarbose. Comparative Ki values by mouse strain are shown in Table 2.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

    Phenotype of Mgam deficiency. There were no visible signs of gastrointestinal insufficiency in the null mice. Ablation of intestinal Mgam caused depression of -glucosidic activity against all -1,4 substrates assayed. Thus, the ability to generate free absorbable glucose from these substrates was markedly impaired in null mice. Maltase activity in null mice was only about one-third of the activity present in WT. In addition, the value of Km for maltose was >500% higher for null than for WT mice. Similarly, using varieties of starches and derived products as substrate and in the absence of rhpAMY, the -glucosidic activity of null mice was always at least 95% lower than that of WT. Even in the presence of rhpAMY, the efficiency for -glucogenesis of null mice remained much lower than that of WT mice. Thus, as a result of the Mgam KO, the null mice are severely deprived of starch derived glucose, particularly on low-starch diets where Si is ineffective and the missing Mgam, with a low Km, would be the only enzyme capable of releasing glucose by digestion of food starch.

Is Mgam a necessary enzyme for starch digestion? The null mouse jejunal and ileal mucosa had persistent -glucosidase activities, but the Km of the residual activities in the null mice were 500% greater than WT. This was the consequence of slower Km of Si in null mice. To determine the in vivo significance of the changed activities in the small intestinal mucosa, a breath test of integrated starch digestion and oxidation was conducted in null and WT mice. A standard feeding of ¹³C-LD was given by gavage, and breath enrichment of ¹³CO₂ was measured at intervals. The WT mice with Mgam had a 2-fold larger increment in breath ¹³CO₂ than null mice following the feeding. The use of ¹³C-LD substrate, which was fully predigested by -amylase, limits starch digestion to the mucosal -glucosidase phase. This test confirmed the dominant role of Mgam in WT starch digestion and confirmed persistence of less active Si -glucosidase in the null mouse.

    Contribution of Si to starch digestion. The activities of Si were upregulated by 50% in null mice, probably by enhancement of the concentration of the Si protein molecules. To our knowledge, this is the first time that induction of Si activities has been documented as a direct result of Mgam deficiency. The upregulation of Si thus suggests existence of mechanisms for sensing total intestinal -glucosidic activities or concentrations of produced glucose in blood or body tissues.

    Contributions of human pancreatic AMY to starch digestion. Endo-glucosidic rhpAMY had a poor -glucogenic activity but had an important synergic effect on mucosal enzymes by increasing the numbers of short glucose oligomer substrates for Mgam and Si exo-glucosidic activities. The endo-glucosidic activity of rhpAMY is inhibited by increased -1,6 branching linkages or reduced length of -1,4 chains.

    Complexity of starch substrates. Our results revealed expected variations of glucose production from different food starches (Table 3) and several unifying conclusions were evident. The first is that ablation of Mgam reduces mucosal -glucogenesis for all starches by 20 fold. The second is that rhpAMY has little primary -glucogenic activity but amplifies the mucosal -glucogenic activities of Mgam null and WT mice from 1.5 to 4-fold. Prior full digestion with porcine pancreatic AMY to LD blocked all amplification of mucosal -glucogenic activities by the luminal enzyme. LD is therefore an exclusive substrate for mucosal -glucogenesis. The third conclusion is that the digestion of different food starches and starch products used varying combinations of the 6 -glucogenic activities. Amylopectins and their hydrolytic products mostly are digested by WT jejunal activities (Mgam), whereas amylose and "resistant" starches are very dependant on luminal AMY amplification for full -glucogenesis by WT mucosal activities.

    Inhibition of starch digestion by acarbose. Studies of the inhibitory mechanism of acarbose on rhpAMY have revealed that it behaves as a mixed, competitive, and uncompetitive inhibitor. In this work, using WT, Mgam null, and CBA/CaJ mice with mixed Mgam and Si deficiency, we analyzed the differential inhibitory effects of acarbose on the mucosal -glucogenic enzymes of these mouse strains. This strategy avoided the uncertainty caused by cross-contamination of Si and Mgam molecules that are present in samples purified by biochemical methods. We found differences of one order of magnitude among the Ki of acarbose on maltase activity measured in mucosal homogenates of WT (0.34 mmol/L), null (2.5 mmol/L), and CBA/Ca (13.3 mmol/L) mice. These differences suggest that Mgam is the most sensitive enzyme to the inhibitory effect of acarbose, whereas Si subunits require relatively high concentration of the inhibitor to inhibit their activity. The data obtained on catalytic properties of the enzymes and on acarbose inhibitory patterns suggest that, in vivo, this inhibitor impairs most of the -glucogenic process from glucose oligomers digested by Mgam.

    Significance of mouse Mgam null phenotype to human starch digestion. These studies of Mgam in the null and WT mouse are confirmatory of a parallel study of -glucosidase activities in the human duodenal biopsies (16). In the human study, the activities were immunoprecipitated from duodenal biopsy homogenates. By contrast, in this study the Mgam was ablated by KO technology. The immunoprecipitated human MGAM activity was inhibited by luminal concentrations of LD, MD, and G3, but SI activity was uninhibited. The MGAM inhibition was obscured in the Dahlqvist assay of human -glucosidases because of the 20-fold greater concentration of SI. The conclusion from our human studies was that MGAM primes intestinal glucogenesis and is important for low-starch diets and that SI sustains slower glucogenesis (16). The present mouse studies confirm the differences of Mgam and SI Km reported in humans. The null mice had about one-half of the ¹³C-LD fed breath ¹³CO₂ enrichment of the WT. The breath test confirmed that the 5- to 10-fold greater mucosal activity of Mgam dominates normal mucosal starch digestion.

In summary, maltase-glucoamylase, present in the WT mice, regulates the total rate of starch -glucogenesis. The absence of this enzyme in the mucosa of Mgam null mice reduced in vivo starch digestion by one-half. The in vitro mucosal sucrase-isomaltase activity of the null mouse sustained total -glucogenesis at half of the WT activity. The Km of jejunal -glucogenesis null mice was 500% greater and total starch digestion was slowed by half, proving that upregulated mucosal Si cannot fully replace the ablated Mgam activities. The ablation of Mgam was mimicked by the -glucosidase inhibitor acarbose, which was 700% more potent for Mgam activity of the WT mouse. -Amylase was poorly -glucogenic but was a synergistic amplifier of mucosal -glucogenic activities. The -amylase amplification was probably due to increased nonreducing ends following its internal hydrolysis of long-starch chains to short -glucoside substrates. The -amylase amplification was lost with -limit-dextrin substrates. There are many known variations of food starch chemical composition. We investigated the integrated roles of Mgam, Si, and AMY with model starches. The interacting activities of the lumenal and mucosal enzyme varied with starch substrate composition, but Mgam remained as the dominant -glucogenic activity. This enzyme interaction leads to the belief that the activities are not redundant but are part of an adaptive mechanism to allow small intestinal digestion of a broad range of food starch compositions.

	ACKNOWLEDGMENTS

 
R.Q.C. was on sabbatical from UASLP as a Visiting Professor in the Nichols Laboratory from 2004-2006. S.W. and M.C.N. were located at Lexicon Genetics, Inc. (The Woodlands, TX) in 1999 when they produced the Mgam ablation. The contents of this publication do not necessarily reflect the views or policies of the U. S. Department of Agriculture, the Swiss National Science Foundation, or the Canadian Institutes of Health, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S., Swiss, or Canadian governments. Ursula Luginbuehl, Stephen E. Avery, and David Petros provided expert technical support.

	FOOTNOTES

 
¹ Supported in part by federal funds from the USDA, Agricultural Research Service, under Cooperative Agreement Number 58-6250-1-003 (B.L.N.), Swiss National Science Foundation, Grant Number 3100A0-100772 (E.E.S.), and a grant from the Canadian Institutes of Health Research, number MOP-13338 (G.D.B.).

² Author disclosures: R. Quezada-Calvillo, C. C. Robayo-Torres, A. R. Opekun, P. Sen, Z. Ao, B. R. Hamaker, A. Quaroni, G. D. Brayer, S. Wattler, M. C. Nehls, E. E. Sterchi, and B. L. Nichols, no conflicts of interest.

³ Supplemental Table 1 is available with the online posting of this paper at jn.nutrition.org.

¹¹ Abbreviations used: AMY, -amylase; Ab, antibody; BBA, barley ß-Amylase; CBA/CaJ, jejunal homogenate with congenital deficiencies of Mgam and Si; OB, ¹³CO₂-delta over baseline; G2, maltose; G3, maltotriose; G4, maltotetraose; G5, maltopentose; Ki, inhibitory constant; Km, Michaelis constant; LD, corn -limit-dextrin; MA, maltogenic -amylase; mAb, monoclonal antibody; MD, corn maltodextrin; Mgam, mouse maltase-glucoamylase; MGAM, human maltase-glucoamylase; O2–5, glucosides from 2 to 5 glucose units long; qPCR, quantitative PCR; rhpAMY, recombinant human pancreatic amylase; Si, mouse sucrase-isomaltase; SI, human sucrase-isomaltase; TG, Aspergillus transglucosidase; TGO, Tris glucose-oxidase; Vmax, maximum rate of reaction; WT, wild-type; WX, waxy cornstarch.

Manuscript received 26 February 2007. Initial review completed 26 March 2007. Revision accepted 14 May 2007.

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