Insight into the combinatorial transcriptional regulation on α-amylase gene in animal groups with different dietary nutrient content.

Gene expression is generally regulated by multiple transcription factors (TFs). Despite previous findings of individual TFs regulating pancreatic α-amylase gene expression, the combinatorial transcriptional regulation is not fully understood. To gain insight into multiple TF regulation for pancreatic α-amylase gene, we employed a function conservation approach to predict interacting TFs regulating pancreatic α-amylase gene for 3 dietary animal groups. To this end, we have identified 77, 25, and 118 interacting TFs for herbivore, omnivore, and carnivore, respectively. Computational modeling of TF regulatory networks demonstrated that known pancreas-specific TFs (e.g. GR, NFAT, and PR) may play important roles in recruiting non pancreas-specific TFs to the TF-TF interaction networks, offering specificity and flexibility for controlling pancreatic α-amylase gene expression in different dietary animal groups. The findings from this study indicate that combinatorial transcriptional regulation could be a critical component controlling pancreatic α-amylase gene expression.

α-Amylase expressed in human small intestinal epithelial cells is essential for cell proliferation and differentiation.

α-Amylase, which plays an essential role in starch degradation, is expressed mainly in the pancreas and salivary glands. Human α-amylase is also detected in other tissues, but it is unclear whether the α-amylase is endogenously expressed in each tissue or mixed exogenously with one expressed by the pancreas or salivary glands. Furthermore, the biological significance of these α-amylases detected in tissues other than the pancreas and salivary glands has not been elucidated. We discovered that human α-amylase is expressed in intestinal epithelial cells and analyzed the effects of suppressing α-amylase expression. α-Amylase was found to be expressed at the second-highest messenger RNA level in the duodenum in human normal tissues after the pancreas. α-Amylase was detected in the cell extract of Caco-2 intestinal epithelial cells but not secreted into the culture medium. The amount of α-amylase expressed increased depending on the length of the culture of Caco-2 cells, suggesting that α-amylase is expressed in small intestine epithelial cells rather than the colon because the cells differentiate spontaneously upon reaching confluence in culture to exhibit the characteristics of small intestinal epithelial cells rather than colon cells. The α-amylase expressed in Caco-2 cells had enzymatic activity and was identified as AMY2B, one of the two isoforms of pancreatic α-amylase. The suppression of α-amylase expression by small interfering RNA inhibited cell differentiation and proliferation. These results demonstrate for the first time that α-amylase is expressed in human intestinal epithelial cells and affects cell proliferation and differentiation. This α-amylase may induce the proliferation and differentiation of small intestine epithelial cells, supporting a rapid turnover of cells to maintain a healthy intestinal lumen.

Central Nervous System Delivery and Biodistribution Analysis of an Antibody-Enzyme Fusion for the Treatment of Lafora Disease.

Lafora disease (LD) is a fatal juvenile epilepsy characterized by the accumulation of aberrant glucan aggregates called Lafora bodies (LBs). Delivery of protein-based therapeutics to the central nervous system (CNS) for the clearance of LBs remains a unique challenge in the field. Recently, a humanized antigen-binding fragment (hFab) derived from a murine systemic lupus erythematosus DNA autoantibody (3E10) has been shown to mediate cell penetration and proposed as a broadly applicable carrier to mediate cellular targeting and uptake. We report studies on the efficacy and CNS delivery of VAL-0417, an antibody-enzyme fusion composed of the 3E10 hFab and human pancreatic α-amylase, in a mouse model of LD. An enzyme-linked immunosorbent assay has been developed to detect VAL-0417 post-treatment as a measure of delivery efficacy. We demonstrate the robust and sensitive detection of the fusion protein in multiple tissue types. Using this method, we measured biodistribution in different methods of delivery. We found that intracerebroventricular administration provided robust CNS delivery when compared to intrathecal administration. These data define critical steps in the translational pipeline of VAL-0417 for the treatment of LD.

[Carbohydrate-related nutritional and genetic risks of obesity for indigenous northerners].

By the end of the 2010-s the prevalence of obesity among the indigenous people of the North approached to the all-Russia one and the speed of the spread of other metabolic disorders exceeded the average all-country levels. Aim of this review is to analyze data on the increase in consumption and variety of sugars coupled with a genetic specificity of regulation of saccharidase activity and their possible impact on the matters. Results. It have been shown that the traditional protein-lipid-based northern type diet has substantially changed and now contains a high proportion of carbohydrates. The carbohydrate per capita consumption among the indigenous people of the North has reached the all-Russia average level (40 kg per year) which exceeds the European average of 36.2 kg per year. The variety of food disaccharides has also considerably increased. The daily consumption of sucrose, at the beginning of the 20th century it was the only sugar contained in the store-bought foods, increased from 30 g in the 1930s to 63-65 g in the 1990s. In addition, the proportion of sucrose dropped to 60-70 per cent, while the contribution of other disaccharides (lactose, trehalose) reached 30-40 per cent. Daily starch consumption has also increased and got close to the national average (males 228.5 g, females 157.5 g per day). Such a diet in itself increases the risk of metabolic disorders and obesity. The high prevalence of the genotypes that determine reduced levels or inability to produce sucrase-isomaltase, lactase, trehalase, salivary and pancreatic amylases among northerners becomes a negative cofactor. The evolutionary driven and embodied in genotype reduced ability of the indigenous Arctic people to digest complex carbohydrates is in a conflict with the growing consumption of sugars and starchy foods in modern conditions. The northern people have a high proportion of carriers of the AG deletion in SI gene (3.5-14.3% against 0.05-0.2% among Europeans) which determines malabsorption of sucrose. The CC/LCT genotype (96.6% in northerners, 36-49% in Russians) presumes lactose intolerance and is associated with the risk of childhood obesity. The occurrence of A allele in the rs2276064 locus of TREH gene (trehalose intolerance; 31.3-58.9% in northerners, 1.9% in Europeans) increases the probability of the onset of type 2 diabetes mellitus. According to preliminary estimates, 28-52% of the northerners completely lost AMY gene that precludes or drastically reduces the ability to digest starch. A reduction in the number of copies of AMY gene (the average number of copies AMY2A - 4, in, in northerners it is 1.0-1.4) is associated with overweight and obesity. Conclusion. The analysis shows that, in the case of the modern indigenous northerners, nutritional and genetic risks of metabolic disorders accumulate.

Copy number variation of human AMY1 is a minor contributor to variation in salivary amylase expression and activity.

BACKGROUND: Salivary amylase in humans is encoded by the copy variable gene AMY1 in the amylase gene cluster on chromosome 1. Although the role of salivary amylase is well established, the consequences of the copy number variation (CNV) at AMY1 on salivary amylase protein production are less well understood. The amylase gene cluster is highly structured with a fundamental difference between odd and even AMY1 copy number haplotypes. In this study, we aimed to explore, in samples from 119 unrelated individuals, not only the effects of AMY1 CNV on salivary amylase protein expression and amylase enzyme activity but also whether there is any evidence for underlying difference between the common haplotypes containing odd numbers of AMY1 and even copy number haplotypes. RESULTS: AMY1 copy number was significantly correlated with the variation observed in salivary amylase production (11.7% of variance, P < 0.0005) and enzyme activity (13.6% of variance, P < 0.0005) but did not explain the majority of observed variation between individuals. AMY1-odd and AMY1-even haplotypes showed a different relationship between copy number and expression levels, but the difference was not statistically significant (P = 0.052). CONCLUSIONS: Production of salivary amylase is correlated with AMY1 CNV, but the majority of interindividual variation comes from other sources. Long-range haplotype structure may affect expression, but this was not significant in our data.

Effect of different salinities on gene expression and activity of digestive enzymes in the thick-lipped grey mullet (Chelon labrosus).

The effects of different environmental salinities (0, 12, 40, and 55 ppt) on pepsinogen 2 (pga2), trypsinogen 2 (try2), chymotrypsinogen (ctr), and pancreatic alpha-amylase (amy2a) gene expression, and on the total activities of their corresponding enzymes, were assessed in Chelon labrosus juveniles, after their corresponding full-complementary DNA sequences were cloned. Furthermore, the quantitative effect of different salinities on the hydrolysis of feed protein by fish digestive enzymes was evaluated using an in vitro system. Relative pga2 expression levels were significantly higher in animals maintained at 12 ppt, while a significantly higher gene expression level for ctr and try2 was observed at 40 ppt. amy2a gene expression showed its maximum level at 40 ppt and the lowest at 55 ppt. A significant reduction in the activity of amylase with the increase in salinity was observed, whereas the maximum activity for alkaline proteases was observed in individuals maintained at 40 ppt. A negative effect of high salinity on the action of proteases was confirmed by the in vitro assay, indicating a decreased efficiency in the digestive function in C. labrosus when maintained at high environmental salinities. Nevertheless, individuals can live under different environmental salinities, even though gene expression is different and the enzymatic activities are not maintained at the highest studied salinity. Therefore, compensatory mechanisms should be in place. Results are discussed on the light of the importance as a new species for aquaculture.

Obesity, starch digestion and amylase: association between copy number variants at human salivary (AMY1) and pancreatic (AMY2) amylase genes.

The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in association with body mass index. In this work, we use paralogue ratio tests, microsatellite analysis, read depth and fibre-FISH to demonstrate that human amylase CNV is not a smooth continuum, but is instead partitioned into distinct haplotype classes. There is a fundamental structural distinction between haplotypes containing odd or even numbers of AMY1 gene units, in turn coupled to CNV in pancreatic amylase genes AMY2A and AMY2B. Most haplotypes have one copy each of AMY2A and AMY2B and contain an odd number of copies of AMY1; consequently, most individuals have an even total number of AMY1. In contrast, haplotypes carrying an even number of AMY1 genes have rearrangements leading to CNVs of AMY2A/AMY2B. Read-depth and experimental data show that different populations harbour different proportions of these basic haplotype classes. In Europeans, the copy numbers of AMY1 and AMY2A are correlated, so that phenotypic associations caused by variation in pancreatic amylase copy number could be detected indirectly as weak association with AMY1 copy number. We show that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures we use and that qPCR data in other studies have been further compromised by systematic miscalibration. Our results uncover new patterns in human amylase variation and imply a potential role for AMY2 CNV in functional associations.

Complex Copy Number Variation of AMY1 does not Associate with Obesity in two East Asian Cohorts.

The human amylase gene locus at chromosome 1p21.1 is structurally complex. This region contains two pancreatic amylase genes, AMY2B, AMY2A, and a salivary gene AMY1. The AMY1 gene harbors extensive copy number variation (CNV), and recent studies have implicated this variation in adaptation to starch-rich diets and in association to obesity for European and Asian populations. In this study, we showed that by combining quantitative PCR and digital PCR, coupled with careful experimental design and calibration, we can improve the resolution of genotyping CNV with high copy numbers (CNs). In two East Asian populations of Chinese and Malay ethnicity studied, we observed a unique non-normal distribution of AMY1 diploid CN genotypes with even:odd CNs ratio of 4.5 (3.3-4.7), and an association between the common AMY2A CN = 2 genotype and odd CNs of AMY1, that could be explained by the underlying haplotypic structure. In two further case-control cohorts (n = 932 and 145, for Chinese and Malays, respectively), we did not observe the previously reported association between AMY1 and obesity or body mass index. Improved methods for accurately genotyping multiallelic CNV loci and understanding the haplotype complexity at the AMY1 locus are necessary for population genetics and association studies.

Diet adaptation in dog reflects spread of prehistoric agriculture.

Adaptations allowing dogs to thrive on a diet rich in starch, including a significant AMY2B copy number gain, constituted a crucial step in the evolution of the dog from the wolf. It is however not clear whether this change was associated with the initial domestication, or represents a secondary shift related to the subsequent development of agriculture. Previous efforts to study this process were based on geographically limited data sets and low-resolution methods, and it is therefore not known to what extent the diet adaptations are universal among dogs and whether there are regional differences associated with alternative human subsistence strategies. Here we use droplet PCR to investigate worldwide AMY2B copy number diversity among indigenous as well as breed dogs and wolves to elucidate how a change in dog diet was associated with the domestication process and subsequent shifts in human subsistence. We find that AMY2B copy numbers are bimodally distributed with high copy numbers (median 2nAMY2B=11) in a majority of dogs but no, or few, duplications (median 2nAMY2B=3) in a small group of dogs originating mostly in Australia and the Arctic. We show that this pattern correlates geographically to the spread of prehistoric agriculture and conclude that the diet change may not have been associated with initial domestication but rather the subsequent development and spread of agriculture to most, but not all regions of the globe.

Establishment of a novel, eco-friendly transgenic pig model using porcine pancreatic amylase promoter-driven fungal cellulase transgenes.

Competition between humans and livestock for cereal and legume grains makes it challenging to provide economical feeds to livestock animals. Recent increases in corn and soybean prices have had a significant impact on the cost of feed for pig producers. The utilization of byproducts and alternative ingredients in pig diets has the potential to reduce feed costs. Moreover, unlike ruminants, pigs have limited ability to utilize diets with high fiber content because they lack endogenous enzymes capable of breaking down nonstarch polysaccharides into simple sugars. Here, we investigated the feasibility of a transgenic strategy in which expression of the fungal cellulase transgene was driven by the porcine pancreatic amylase promoter in pigs. A 2,488 bp 5'-flanking region of the porcine pancreatic amylase gene was cloned by the genomic walking technique, and its structural features were characterized. Using GFP as a reporter, we found that this region contained promoter activity and had the potential to control heterologous gene expression. Transgenic pigs were generated by pronuclear microinjection. Founders and offspring were identified by PCR and Southern blot analyses. Cellulase mRNA and protein showed tissue-specific expression in the pancreas of F1 generation pigs. Cellulolytic enzyme activity was also identified in the pancreas of transgenic pigs. These results demonstrated the establishment of a tissue-specific promoter of the porcine pancreatic amylase gene. Transgenic pigs expressing exogenous cellulase may represent a way to increase the intake of low-cost, fiber-rich feeds.

Ligand-bound thyroid hormone receptor contributes to reprogramming of pancreatic acinar cells into insulin-producing cells.

One goal of diabetic regenerative medicine is to instructively convert mature pancreatic exocrine cells into insulin-producing cells. We recently reported that ligand-bound thyroid hormone receptor α (TRα) plays a critical role in expansion of the ß-cell mass during postnatal development. Here, we used an adenovirus vector that expresses TRα driven by the amylase 2 promoter (AdAmy2TRα) to induce the reprogramming of pancreatic acinar cells into insulin-producing cells. Treatment with l-3,5,3-triiodothyronine increases the association of TRα with the p85α subunit of phosphatidylinositol 3-kinase (PI3K), leading to the phosphorylation and activation of Akt and the expression of Pdx1, Ngn3, and MafA in purified acinar cells. Analyses performed with the lectin-associated cell lineage tracing system and the Cre/loxP-based direct cell lineage tracing system indicate that newly synthesized insulin-producing cells originate from elastase-expressing pancreatic acinar cells. Insulin-containing secretory granules were identified in these cells by electron microscopy. The inhibition of p85α expression by siRNA or the inhibition of PI3K by LY294002 prevents the expression of Pdx1, Ngn3, and MafA and the reprogramming to insulin-producing cells. In immunodeficient mice with streptozotocin-induced hyperglycemia, treatment with AdAmy2TRα leads to the reprogramming of pancreatic acinar cells to insulin-producing cells in vivo. Our findings suggest that ligand-bound TRα plays a critical role in ß-cell regeneration during postnatal development via activation of PI3K signaling.

Alpha-amylase inhibitor, CS-1036 binds to serum amylase in a concentration-dependent and saturable manner.

(2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-ß-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036), which is an α-amylase inhibitor, exhibited biphasic and sustained elimination with a long t1/2 (18.4-30.0 hours) in rats and monkeys, but exhibited a short t1/2 (3.7-7.9 hours) in humans. To clarify the species differences in the t1/2, the plasma protein binding of CS-1036 was evaluated by ultrafiltration. A concentration-dependent and saturable plasma protein binding of CS-1036 was observed in rats and monkeys with the dissociation rate constant (KD) of 8.95 and 27.2 nM, and maximal binding capacity (Bmax) of 52.8 and 22.1 nM, respectively. By the assessments of the recombinant amylase and immunoprecipitation, the major binding protein of CS-1036 in rats was identified as salivary amylase (KD 5.64 nM). CS-1036 also showed concentration-dependent and saturable binding to human salivary and pancreatic amylase, with similar binding affinity in rats. However, the protein binding of CS-1036 was constant in human plasma (≤10.2%) due to the lower serum amylase level compared with rats and monkeys. From the calculation of the unbound fraction (fu) in plasma based on in vitro KD and Bmax, the dose-dependent increase in fu after oral administration is speculated to lead to a dose-dependent increase in total body clearance and a high area under the curve/dose at lower doses, such as 0.3 mg/kg in rats.

Variation in salivary and pancreatic alpha-amylase genes in Italian horse breeds.

The dietary demand of the modern horse relies on high-cereal feeding and limited forage compared with natural grazing conditions, predisposing the horse to several important diseases. Salivary and pancreatic alpha-amylases (coded by AMY1 and AMY2 genes, respectively) play a crucial role in carbohydrate digestion in nonruminants, but little is known about these 2 genes in the horse. Aim of this work has been to distinguish genomic sequences of horse AMY1 and AMY2 genes and to analyze any polymorphisms in breeds historically characterized by marked differences in nutritional management. A single nucleotide polymorphism detection was performed and 7 novel single nucleotide polymorphisms were found. Three single nucleotide polymorphisms are in exons and were genotyped in 112 horses belonging to 6 breeds. One single nucleotide polymorphism in AMY1 gene distinguished Haflinger and the Italian native Murgese from the other breeds, whereas both the single nucleotide polymorphisms in AMY2 gene showed different allelic frequencies in Friesian compared with the other breeds. These differences are confirmed by quite high fixation index (Fst) values for these 2 nonsynonymous single nucleotide polymorphisms. These preliminary results highlight marked divergences in allele frequencies of AMY1 and AMY2 genes, involved in starch digestion, between horse breeds characterized by different histories of selection, thus providing first indications of possible relations between genetics and nutritional management.

Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication, diet and diabetes.

High amylase activity in dogs is associated with a drastic increase in copy numbers of the gene coding for pancreatic amylase, AMY2B, that likely allowed dogs to thrive on a relatively starch-rich diet during early dog domestication. Although most dogs thus probably digest starch more efficiently than do wolves, AMY2B copy numbers vary widely within the dog population, and it is not clear how this variation affects the individual ability to handle starch nor how it affects dog health. In humans, copy numbers of the gene coding for salivary amylase, AMY1, correlate with both salivary amylase levels and enzyme activity, and high amylase activity is related to improved glycemic homeostasis and lower frequencies of metabolic syndrome. Here, we investigate the relationship between AMY2B copy numbers and serum amylase activity in dogs and show that amylase activity correlates with AMY2B copy numbers. We then describe how AMY2B copy numbers vary in individuals from 20 dog breeds and find strong breed-dependent patterns, indicating that the ability to digest starch varies both at the breed and individual level. Finally, to test whether AMY2B copy number is strongly associated with the risk of developing diabetes mellitus, we compare copy numbers in cases and controls as well as in breeds with varying diabetes susceptibility. Although we see no such association here, future studies using larger cohorts are needed before excluding a possible link between AMY2B and diabetes mellitus.

Structures of human pancreatic α-amylase in complex with acarviostatins: Implications for drug design against type II diabetes.

Human pancreatic α-amylase (HPA) catalyzes the hydrolysis of α-d-(1,4) glycosidic linkages in starch and is one of the major therapeutic targets for type II diabetes. Several acarviostatins isolated from Streptomyces coelicoflavus var. nankaiensis previously showed more potent inhibition of HPA than acarbose, which has been successfully used in clinical therapy. However, the molecular mechanisms by which acarviostatins inhibit HPA remains elusive. Here we determined crystal structures of HPA in complexes with a series of acarviostatin inhibitors (I03, II03, III03, and IV03). Structural analyses showed that acarviostatin I03 undergoes a series of hydrolysis and condensation reactions in the HPA active site, similar to acarbose, while acarviostatins II03, III03, and IV03 likely undergo only hydrolysis reactions. On the basis of structural analysis combined with kinetic assays, we demonstrate that the final modified product with seven sugar rings is best suited for occupying the full active site and shows the most efficient inhibition of HPA. Our high resolution structures reported here identify first time an interaction between an inhibitor and subsite-4 of the HPA active site, which we show makes a significant contribution to the inhibitory effect. Our results provide important information for the design of new drugs for the treatment of type II diabetes or obesity.

[High-throughput screening of human pancreatic alpha-amylase inhibitors].

OBJECTIVE: Targeting the important enzyme in human glucose metabolic pathway, we established a high throughput screening model for human pancreatic alpha-amylase inhibitors. METHODS: Pichia pastoris expression system was used to clone and express the human pancreatic alpha-amylase; we established the alpha-amylase inhibitor screening model using the catalytic properties of enzyme; this model was applied in screening of actinomycete' metabolites; the taxonomic status of positive strains were analyzed by constructing 16S rRNA phylogenetic tree. RESULTS: We cloned and expressed the intact gene of human pancreatic alpha-amylase successfully; the high-throughput screening model of alpha-amylase inhibitors was established; nearly 2000 actinomycete' metabolites were screened, 14 alpha-amylase inhibitor producing strains were obtained finally, and showed taxonomically rich diversity. CONCLUSION: The alpha-amylase inhibitor high-throughput screening model had high practical value for developing new hypoglycemic drugs.

Comparative analyses of the Pan lineage reveal selection on gene pathways associated with diet and sociality in bonobos.

Chimpanzees (Pan troglodytes) and bonobos (Pan paniscus) diverged into distinct species approximately 1.7 million years ago when the ancestors of modern-day bonobo populations were separated by the Congo River. This geographic boundary separates the two species today and the associated ecological factors, including resource distribution and feeding competition, have likely shaped the divergent social behavior of both species. The most striking behavioral differences pertain to between group interactions in which chimpanzees behave aggressively towards unfamiliar conspecifics, while bonobos display remarkable tolerance. Several hypotheses attempt to explain how different patterns of social behavior have come to exist in the two species, some with specific genetic predictions, likening the evolution of bonobos to a process of domestication. Here, we utilize 73 ape genomes and apply linkage haplotype homozygosity and structure informed allele frequency differentiation methods to identify positively selected regions in bonobos since their split from a common pan ancestor to better understand the environment and processes that resulted in the behavioral differences observed today. We find novel evidence of selection in genetic regions that aid in starch digestion (AMY2) along with support for two genetic predictions related to self-domestication processes hypothesized to have occurred in the bonobo. We also find evidence for selection on neuroendocrine pathways associated with social behavior including the oxytocin, serotonin, and gonadotropin releasing hormone pathways.

Influence of AMY1A copy number variations on obesity and other cardiometabolic risk factors: A review of the evidence.

The rising incidence of obesity and type 2 diabetes is contributing to the escalating burden of disease globally. These metabolic disorders are closely linked with diet and in particular with carbohydrate consumption; hence, it is important to understand the underlying mechanisms that influence carbohydrate metabolism. Amylase, the enzyme responsible for the digestion of starch, is coded by the genes AMY1A, AMY1B, and AMY1C (salivary amylase) and AMY2A and AMY2B (pancreatic amylase). Previous studies demonstrate wide variations in AMY1A copy numbers, which can be attributed to several genetic, nutritional, and geographical diversities seen in populations globally. Current literature suggests that AMY1A copy number variations are important in obesity and other cardiometabolic disorders through their effects on glucose and lipid homeostasis, inflammatory markers, and the gut microbiome. This review synthesizes the available evidence to improve understanding of the role of AMY1A in obesity and related cardiometabolic risk factors and disorders including insulin resistance and type 2 diabetes, cardiovascular risk and inflammation, and the gut microbiome.

Association of AMY1A/AMY2A copy numbers and AMY1/AMY2 serum enzymatic activity with obesity in Mexican children.

BACKGROUND: Mexican children are characterized by a high-starch intake diet and high prevalence of obesity. OBJECTIVES: To investigate the association of AMY1A/AMY2A copy numbers (CNs) and AMY1/AMY2 serum enzymatic activity with childhood obesity in up to 427 and 337 Mexican cases and controls. METHODS: Anthropometric and dietary starch intake data were collected. CN of AMY1A/AMY2A and AMY1/AMY2 serum enzymatic activity were determined using droplet digital PCR (ddPCR) and enzymatic colorimetry, respectively. An individual participant level data meta-analysis of association between AMY1A CNVs and obesity was also performed. RESULTS: A positive association between AMY1A/AMY2A CNs and their corresponding AMY1/AMY2 serum enzyme activity was observed in children with normal weight and obesity. The serum enzyme activity of AMY1 and AMY2 was negatively associated with childhood obesity risk, and the association was restricted to kids eating medium/high amount of starch (Pinteraction = .004). While no association between AMY1A and AMY2A CNs and childhood obesity was observed in our sample, we confirmed a significant association between AMY1A CN and obesity in a meta-analysis of 3100 Mexican children. CONCLUSIONS: Our data suggest that genetically determined salivary and pancreatic amylase activity can increase/decrease the risk of obesity in Mexican children, this effect being blunted by a low-starch diet.