Digestive Enzyme Gene Expression Research Articles

Expression and characterization of digestive enzyme genes from hepatopancreatic transcripts from redclaw crayfish (Cherax quadricarinatus)

Redclaw crayfish, Cherax quadricarinatus, possess a number of biological and commercial attributes that make them ideal for commercial aquaculture. While some studies have investigated digestive enzyme activity and nutritional requirements of this species, little information exists about the expression of digestive enzyme genes and their role in regulating digestive capacity. The current study therefore sequenced and annotated a RNASeq library constructed from a redclaw hepatopancreas to identify genes involved in digestive enzyme production. We observed that most of the transcripts that were annotated as digestive enzyme genes are associated with carbohydrate metabolism, thus confirming that redclaw have an innate capacity to digest a range of carbohydrate substrates. While endoglucanases were the most abundant group of digestive enzymes found, a number of novel transcripts were also detected. Here, we provide the first report for the presence and expression of endo-b-mannanase in freshwater crayfish. This novel gene showed significant alignment with a GH5 family protein from marine Limnoriids, wood borers that do not possess symbiotic microbes in their gut system. Overall, the data generated here provide an important resource to better understand the suite of digestive enzymes in redclaw that are very useful to fully utilize the species’ digestive capacity and will assist development of specific artificial feeds.

The ontogeny of nutrient transporter and digestive enzyme gene expression in domestic pigeon (Columba livia) intestine and yolk sac membrane during pre- and posthatch development

To better understand the digestive capacity in domestic pigeons (Columba livia), this study was conducted to evaluate nutrient transporters and digestive enzymes gene expression in small intestine and yolk sac membrane (YSM) during pre- and posthatch development. We investigated the oligopeptide transporter Pept1, sodium glucose transporter SGLT1, glucose transporter GLUT2, aminopeptidase-N (APN), and sucrase-isomaltase (SI). Intestine was collected at embryo d 12, 14, and 16, day of hatch, and d 1, 3, 5, 8, and 14 posthatch. The YSM was collected at embryo d 12, 14, 16, and day of hatch. The cDNA fragments for Pept1, SGLT1, GLUT2, APN, and SI were isolated and cloned using reverse-transcription PCR. The sequences data showed that these genes were highly identical to the gene of chicken. The mRNA expression of each gene was assayed using real-time PCR. Expression of intestinal nutrient transporters increased linearly (P<0.001) with age whereas that of APN increased quadratically (P<0.001) and SI increased cubically (P=0.001). Levels of PepT1 mRNA were greatest in the duodenum (P=0.001), GLUT2 and SGLT1 mRNA were greatest in the jejunum (P=0.002) and APN were greatest in the ileum (P=0.004). The YSM expressed all the examined genes. The YSM-expressed genes decreased between embryo d 16 and day of hatch, whereas intestine-expressed genes increased. Our findings provide a comprehensive profile of gene expression patterns of nutrient transporter and digestive enzyme in the small intestine and YSM of pigeons and establish a foundation for future research on the nutrients requirements for young pigeons.

The chick embryo yolk sac membrane expresses nutrient transporter and digestive enzyme genes

The yolk sac membrane plays a major role in the transport of nutrients from the yolk contents to the chick embryo. We examined whether the yolk sac membrane expresses genes for nutrient digestion, enzymes, and nutrient transporters. We evaluated relative mRNA abundance of the digestive enzymes aminopeptidase N (APN) and sucrase-isomaltase (SI); the nutrient transporters oligopeptide transporter Pept1, cationic amino acid transporter CAT1, and sodium glucose transporter SGLT1; and the micronutrient transporters type IIb sodium phosphate cotransporter NPT2b, calcium transporter TRPV6, and zinc transporter ZnT-1 from embryonic d 11 (11E) to 21E (day of hatch) by real-time reverse-transcription PCR. The yolk sac membrane expressed all the examined genes, which exhibited several patterns of expression. Relative abundance of APN mRNA increased in the yolk sac membrane from 11E to 17E and decreased from 17E to 20E. Expression of PepT1 increased from 11E to 15E and decreased from 15E to 20E. In contrast, CAT1 expression decreased from 11E to 13E and increased from 15E to 17E. Expression of SGLT1 increased between 15E and 20E and decreased substantially between 20E and 21E. Expression of NPT2b increased during incubation and exhibited the highest relative expression of all the examined genes, particularly on 20E to 21E. Expression of TRPV6 decreased from 11E to 13E and increased substantially from 15E to 19E. No significant difference was found between the sampled days for ZnT-1 or SI expression, with the latter exhibiting the lowest relative expression of all the genes studied. These results present the first documentation of nutrient transporter and digestive enzyme gene-expression patterns in the yolk sac membrane, and provide a basis for future research on the capacity of the yolk sac membrane for nutrient digestion and transport.

Microarray analysis of high-glucose diet-induced changes in mRNA expression in jejunums of C57BL/6J mice reveals impairment in digestion, absorption

Long term intake of high-glucose diet (HGD) may induce many diseases such as dyslipidemia, fatty liver and diabetes disease. Most of the research for molecular mechanisms of the association between HGD and the above diseases focus on the metabolism of glucose and lipid. However, there are few studies on molecular mechanism of the effect of HGD on digestion and absorption. We used HGD (containing 20% glucose) to feed C57BL/6J mice for 4 weeks, detected the expressions of 13,098 genes in jejunums of C57BL/6J mice with DNA microarray. Microarray analysis showed the expression of genes related to digestive enzyme, gastrointestinal peptide and nutrient transporters were significantly changed, which indicated that HGD induced the suppression of digestive enzyme gene expression, attenuation of alimentary tract movement and nutrient transportation. In one word, the microarray analysis suggested that HGD impaired the function of digestion and absorption in jejunum of C57BL/6J mice. We validated our microarray findings by conducting real-time RT-PCR assays on selected genes and detecting the activities of disaccharidases such as lactase, maltase and sucrase in jejunum of C57BL/6J mice.

Effects of bombesin on pancreatic digestive enzyme gene expression.

We examined the effects of bombesin on rat pancreatic digestive enzyme gene expression using cloned complementary DNA probes for amylase, trypsinogen I, chymotrypsinogen B, and lysophospholipase. Rats were injected sc three times daily with 5 nmol/kg body wt bombesin. Pancreata were investigated after 6, 12, 24, 48, and 120 h of hormone treatment. Bombesin administration resulted in a time-dependent increase of pancreatic weight, as well as DNA and protein concentration. Cellular hypertrophy became evident after 48 h, and pancreatic hyperplasia occurred after 5 days of hormone treatment. Bombesin administration resulted in a time-dependent parallel decrease of amylase and lysophospholipase messenger RNA (mRNA) concentrations with maximal inhibition occurring after 120 h of bombesin treatment (13 +/- 1% and 14 +/- 3% of control, respectively, P less than 0.05, n = 6). In contrast, chymotrypsin and trypsin mRNA levels remained unaltered after bombesin treatment for up to 5 days. Amylase and chymotrypsin enzyme levels did not correlate with their respective mRNA concentrations. Both decreased to approximately 50% of control after 12 h and increased to 126 +/- 38% of control and 388 +/- 109% of control (P less than 0.05, n = 6), respectively, after 5 days of bombesin treatment. To test whether the bombesin regulation was mediated by the release of cholecystokinin (CCK), the specific CCK receptor antagonist L-364,718 (1 mg/kg body wt) was injected ip either alone, or 15 min before each bombesin injection for 5 days. Although the antagonist alone significantly reduced the mRNA concentrations for trypsin, chymotrypsin, and lysophospholipase to approximately 50%, it did not block the effects of bombesin on pancreatic digestive enzyme levels. These data therefore indicate that bombesin regulates pancreatic digestive enzyme mRNA and protein concentrations in a nonparallel manner; furthermore, CCK is not involved in mediating the bombesin effects on pancreatic gene expression.

Effects of bombesin on pancreatic digestive enzyme gene expression

Effects of bombesin on pancreatic digestive enzyme gene expression

Pancreatic digestive enzyme gene expression: effects of CCK and soybean trypsin inhibitor

Regulation of pancreatic gene expression by cholecystokinin (CCK) was examined in the rat using cloned cDNA probes to quantify changes in specific mRNAs (amylase, trypsinogen I, chymotrypsinogen B, and ribonuclease). Rats were administered intraduodenally an elemental liquid diet. Plasma CCK levels were raised to levels comparable to physiological postprandial levels either by intraduodenal perfusion with soybean trypsin inhibitor (SBTI) (6.9 +/- 1.0 pM, n = 8) or by continuous intravenous infusion with cholecystokinin octapeptide (CCK-8, 6.0 +/- 0.9 pM, n = 6). SBTI infusion resulted in fivefold increases in trypsinogen I and chymotrypsinogen B mRNA levels after 48 h. In contrast SBTI infusion had no effect on amylase mRNA levels and led to a decrease in ribonuclease mRNA levels to approximately 50% of control after 48 h. Intravenous infusion with CCK-8 for 24 h resulted in plasma levels of CCK comparable to those obtained with SBTI and had similar effects on digestive enzyme mRNA levels. These data suggested that SBTI acted via its ability to raise plasma CCK levels. To further test the specificity of these effects, animals were infused intraduodenally with the specific CCK receptor antagonist L364,718. Although the antagonist itself had no effect on digestive enzyme mRNA levels, antagonist treatment totally abolished the effects of both CCK infusion and SBTI treatment. These data therefore indicate that CCK regulates digestive enzyme gene expression at plasma concentrations comparable to physiological postprandial levels. Furthermore, the ability of SBTI infusion to increase plasma CCK accounts for its effects on pancreatic digestive enzyme mRNA levels.