Original Article

Obesity (2006) 14, 683–689; doi: 10.1038/oby.2006.77

Peptide YY and Proglucagon mRNA Expression Patterns and Regulation in the Gut*

Jun Zhou*, Maren Hegsted†, Kathleen L. McCutcheon*, Michael J. Keenan†, Xiaochun Xi*, Anne M. Raggio† and Roy J. Martin*,†

  1. *Pennington Biomedical Research Center, Baton Rouge, Louisiana
  2. †Human Ecology, Louisiana State University AgCenter, Baton Rouge, Louisiana.

Correspondence: Roy Martin Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808. E-mail: martinrj@PBRC.edu

*The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must, therefore, be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1 June 2005; Revised  0000; Accepted 24 January 2006.

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Abstract

Objective: Peptide YY (PYY) and glucagon-like peptide-1 are important in the control of energy homeostasis and are both secreted from the gut in response to ingested nutrients. However, more studies are needed on nutrient regulation of their gene expression patterns in specific areas of the gut. This study detailed PYY and proglucagon (the gene that encodes glucagon-like peptide-1) gene expression patterns and regulation in the gut. We further examined the regulation of PYY and proglucagon mRNA by a diet containing fermentation-resistant starch (in vivo) and butyrate (in vitro).

Research Methods and Procedures: Quantitative real time reverse transcriptase-polymerase chain reaction was used to measure PYY and proglucagon gene expression in epithelial cells collected from the duodenum, jejunum, cecum, and colon in normal Sprague-Dawley rats and in rats fed a resistant starch diet for 4 weeks. The same measurements were also performed in primary epithelial cells collected from the cecum and colon of normal rats after the cells were incubated with butyrate for 3 hours.

Results: The gene expression patterns for PYY and proglucagon are similar to their peptide distribution patterns in the gut. Also, PYY and proglucagon mRNA expression were up-regulated in the cecum and colon in resistant-starch–fed rats. Butyrate increased PYY and proglucagon gene expression in a dose-dependent manner in vitro.

Discussion: Our data provide evidence that the distal part of the gut has the ability to sense nutrients such as butyrate, resulting in the up-regulation of PYY and proglucagon gene expression.

Keywords:

gene, peptide YY, glucagon-like peptide-1, butyrate, resistant starch

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Introduction

Peptide YY (PYY)1 and glucagon-like peptide-1 (GLP-1) are proposed to have an important role in a number of physiological and disease processes. For example, PYY and GLP-1 have tropic effects on pancreatic and intestinal cells, a regulatory role in the ileal break to control stomach emptying and food intake. In addition, GLP-1 stimulates insulin secretion to lower blood glucose (1, 2). These beneficial effects are important in the prevention or treatment of obesity and diabetes.

Both PYY and GLP-1 are mainly synthesized in the intestinal epithelial L cells in response to ingested nutrients. Glucose and fatty acids are two nutrients that stimulate PYY and GLP-1 release (3, 4). Anatomically, the distal part of the gut is the site where PYY and GLP-1 are secreted, determined by immunocytochemistry (4, 5). However, the precise sites in the gut that express PYY and proglucagon (the gene that encodes GLP-1) genes are not clear. Furthermore, the in vivo regulation of these genes in the gut by nutrients needs additional study.

Here we examined the gene expression patterns for PYY and proglucagon in the duodenum, jejunum, cecum, and colon in rats and showed that their expression patterns are in agreement with their peptide distribution patterns in the gut. Because glucose and fatty acids are two major nutrients that influence PYY and GLP-1 secretion, we additionally tested the expression pattern for some genes that are related to glucose and fatty acid transport and metabolism in the same sites of the gut. These genes include glucose transport-2 (Glut-2), glucokinase (GK), monocarboxylate transporter (MCT1), fatty acid synthase (FAS), and carnitine palmitoyltransferase I (CPT-I). Glut-2 and GK are involved in glucose transport and metabolism. MCT1, FAS, and CPT-I are related to fatty acid transport and metabolism. Based on our results, we further studied the in vivo regulation of PYY and proglucagon mRNA expression by nutrients in rats fed a diet containing resistant starch. Resistant starch cannot be digested in the small intestine and, thus, largely escapes use in the small intestine. However, the undigested starch is fermented in the large intestine to produce short chain fatty acids (6). Our results show that resistant-starch–fed rats had higher PYY/proglucagon gene expression only in the large intestine, suggesting that the increased short chain fatty acids might be the nutrient signal. Because butyrate is one of the short chain fatty acids produced by fermentation (7), epithelial cells collected from the cecum and colon were incubated with butyrate in vitro to confirm that short chain fatty acids had a direct effect on up-regulation of PYY and proglucagon gene expression.

The results of this study provide new information on the current understanding of nutrient regulation of PYY/GLP-1. Moreover, for resistant-starch–fed rats, the nutrient sensing regulatory site seems to be the distal part of the gut.

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Research Methods and Procedures

Experimental Animal and Epithelial Cell Collection

Male Sprague-Dawley rats weighing 350 to 420 grams were individually housed in a humidity- and temperature-controlled room (22 plusminus 2 °C, 65% to 67% humidity) on a 12:12-hour light:dark cycle. Food and water were available ad libitum. The control diet was a standard AIN-93G powdered diet, and the resistant starch diet was the same as the control diet except that resistant starch (Hi-Maize cornstarch; National Starch & Chemical Co., Bridgewater, NJ) was used to replace the regular starch in the control diet. For determining the gene expression patterns of PYY and proglucagon in the gastrointestinal tract, six rats that were fed the control diet were used. For determining the effect of resistant starch on PYY and proglucagon mRNA expression, all rats were fed the control diet for a week before they were stratified by weight and randomly assigned to control or resistant starch diet groups for the 4-week study. To collect epithelial cells for the in vitro study, Sprague-Dawley rats weighing 200 to 250 grams were fed regular chow (Purina rat chow 5001; LabDiet, Richmond, IN). All rats were killed by decapitation. The gastrointestinal tract was removed, and its contents were washed out by sterilized saline. Epithelial cells from the duodenum, jejunum, cecum, and colon were collected by gently scraping the inner surface of the corresponding gastrointestinal tract components. The cells were frozen in liquid nitrogen immediately for RNA extraction or kept in a warm saline solution for later in vitro incubation with butyrate.

The Louisiana State University institutional animal care and use committee approved the animal protocols.

In Vitro Experiment

Epithelial cells were washed with 12 mL phosphate-buffered saline and allowed to sit for 2 minutes. The supernatants were collected and transferred into new tubes to remove the precipitated tissue scraps. The cells were further washed with 12 mL phosphate-buffered saline three times and with the incubation medium for the last two washes. The incubation medium was Dulbecco's modified Eagle's medium (1times liquid Dulbecco's modified Eagle's medium with high glucose; Invitrogen Corp., Carlsbad, CA) containing glutamine (2 mM), penicillin (200 U/mL), streptomycin (200 mug/mL), and gentamicin (50 mug/mL). The cells were resuspended in the medium and incubated with different concentrations of sodium butyrate (0, 0.02 muM, 0.5 muM, 50 muM, 1 mM, and 20 mM in phosphate-buffered saline solution) for 3 hours at 37 °C with 5% CO2. At the end of incubation, the cells were aspirated off, washed with phosphate-buffered saline once, and prepared for RNA extraction.

Measurement of mRNA Expression by Real-time Reverse Transcriptase-Polymerase Chain Reaction

PYY, proglucagon, MCT1, CPT-I, FAS, Glut2, and GK mRNA levels were determined by quantitative real-time reverse transcriptase-polymerase chain reaction. Cyclophilin mRNA levels from each sample were used as internal controls to normalize the mRNA levels. The sequences of TaqMan probes and primers for cyclophilin (GenBank no. M15933), PYY (M17523), CPT-I (NM_031559), FAS (NM-017,332), Glut2 (NM_012879), and GK (M25807) are available on request. The probe and primers for proglucagon (NM_012707, assay ID Rn00562293_ml) and MCT1 (NM_012716, assay ID Rn00562332_ml) were purchased from Applied Biosystems (Foster City, CA). The detailed real-time reverse transcriptase-polymerase chain reaction condition and data analyses are the same as we have published previously (8).

Data Analysis

Results are presented as mean plusminus standard error (SE) for each group. The data were analyzed by one-way ANOVA with post hoc Tukey's test for three independent experiments for the in vitro study.

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Results

The mRNA expression patterns for PYY and proglucagon in the gut are shown in Figure 1. PYY mRNA expression level was undetectable in the duodenum and was the highest in the colon. Proglucagon mRNA was expressed in the entire intestine with the highest expression in the jejunum and the second highest level in the colon. This is consistent with the peptide measurements in the same areas of the gut (9, 10, 11). For fatty acid transport– and metabolism-related genes (MCT1, CPT-I, and FAS), their mRNA was expressed in the entire intestine tract including duodenum, jejunum, cecum, and colon, with MCT1 and FAS having the highest expressions in the colon (Figure 2). In contrast, the two glucose transport– and metabolism-related genes, Glut-2 and GK, were mainly expressed in the duodenum and jejunum; Glut-2 mRNA was undetectable and GK mRNA had lower expression levels in the cecum and colon (Figure 3).

Figure 1.
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PYY and proglucagon gene expression patterns in epithelial cells from duodenum, jejunum, cecum, and colon in the gastrointestinal tract. Data are means plusminus SE of six normal Sprague-Dawley rats.

Full figure and legend (84K)

Figure 2.
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Fatty acid metabolism–related gene expression pattern in the gastrointestinal tract. Data are means plusminus SE of six normal Sprague-Dawley rats.

Full figure and legend (79K)

Figure 3.
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Glucose metabolism–related gene expression pattern in the gastrointestinal tract. Data are means plusminus SE of three normal Sprague-Dawley rats.

Full figure and legend (72K)

Based on gene expression patterns, it is likely that fatty acids, rather than glucose, can be sensed in the distal part of the gut and play a role in the regulation of PYY/proglucagon expression. Thus, we used a special diet (resistant starch) to increase short chain fatty acid concentrations in the distal part of the gut and tested whether the expressions of the PYY/proglucagon genes could be changed. PYY and proglucagon mRNA expressions were significantly higher in the resistant-starch–fed rats than the control rats (Figure 4). The increased mRNA expressions occurred in the cecum (p less than or equal to 0.01) and the colon (p less than or equal to 0.05) but not in the jejunum and duodenum. The expression of genes related to fatty acid transport and metabolism was also altered in resistant- starch–fed rats. However, those changes also occurred in the cecum and colon but not in the duodenum and jejunum (Figure 5). MCT1, a short chain fatty acid transporter, had significantly higher expression in the cecum (p < 0.01) and the colon (p < 0.05) for resistant-starch–fed rats than controls (Figure 5). The gene expression for FAS was also increased in both the cecum (p < 0.01) and colon (p < 0.01) for resistant-starch-fed rats. A long chain fatty acid transporter, CPT-I, did not show a difference in gene expression between the two diet groups in the duodenum, jejunum, cecum, or colon.

Figure 4.
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PYY (A) and proglucagon (B) gene expression in epithelial cells of jejunum, cecum, and colon from rats fed with control or resistant starch diet. Feeding resistant starch significantly increased PYY and proglucagon mRNA expression in cecum and colon compared with control rats. Data are means plusminus SE for a group of 7 to 9 rats.

Full figure and legend (88K)

Figure 5.
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Fatty acid metabolism–related gene expression in epithelial cells of the gastrointestinal tract from rats fed with control or resistant starch diet. Data are means plusminus SE for a group of 7 to 9 rats. Values that do not share a common letter are significantly different at p < 0.05.

Full figure and legend (55K)

Butyrate also increased PYY (Figure 6A and B) and proglucagon mRNA (Figure 6C and 6D) levels in epithelial cells directly collected from the cecum (Figure 6A and 6C) and colon of normal rats (Figure 6B and 6D). Both PYY and proglucagon gene expression were increased in a dose-dependent manner when incubated with butyrate.

Figure 6.
Figure 6 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

PYY (A and B) and proglucagon (C and D) gene expression in rat epithelial cells of cecum (A and C) and colon (B and D) after incubating with different concentrations of butyrate. Data are means plusminus SE. * p < 0.05 and ** p < 0.01 compared with control (no butyrate).

Full figure and legend (80K)

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Discussion

Besides digestion and absorption of nutrients, the gut also senses nutrients and sends feedback signals to the brain to control energy homeostasis (12). PYY and GLP-1 are likely among these regulatory signals. Both PYY and GLP-1 have been implicated in the "ileal brake" to regulate food intake (4, 13). PYY infusion resulted in decreased food intake in humans (14), monkeys (15), mice (16), and rats (17). GLP-1 injection also decreased food intake (18, 19) and improved insulin sensitivity. Thus, stimulation of endogenous PYY and GLP-1 secretion by nutrients in the gut is a promising approach for prevention and treatment of obesity-related diseases.

Our results show that resistant-starch–fed rats had higher gene expression of PYY and proglucagon. Resistant starch has been implicated in the amelioration of type 2 diabetes and increased satiety (20, 21). In several separate experiments, we also observed that resistant-starch–fed rats had significantly increased blood levels for PYY and GLP-1 (unpublished observations). Thus, the observed increase in PYY and proglucagon gene expression may indicate that these peptides could be involved in the satiety and antidiabetes effects induced by resistant starch. However, further study of this effect in mice lacking PYY and GLP-1 is needed to confirm this. Nevertheless, our data support the concept that resistant starch diets might be used to increase endogenous PYY/GLP-1 and reduce obesity and diabetes.

For the resistant-starch–fed rats, the increased PYY/proglucagon gene expressions occurred only in the cecum and colon, where the fermentation took place and the short chain fatty acid concentrations should be high (22, 23). In addition, the increased short chain fatty acids could up-regulate MCT1 expression (24). MCT1 transports monocarboxylates across the plasma membrane (25). Because the majority of monocarboxylates in the distal portion of the gut are short chain fatty acids, MCT1 could function as a short chain fatty acid transporter in cecum and colon cells (24). Our results show that the increased PYY/proglucagon gene expression was associated with increased MCT1 at the same sites of the gut. Thus, the increased short chain fatty acids might be the cause of up-regulation of PYY/proglucagon gene expression in the distal part of the gut in resistant-starch–fed rats.

Butyrate is one of the major short chain fatty acids produced by fermentation of resistant starch and is found to increase the secretion of gut peptides (13, 26, 27). We further showed that butyrate directly increased PYY/proglucagon gene expressions in vitro. Using the isolated vascularly perfused rat colon, luminal n-butyrate produced a dose-dependent release of PYY with a maximal response 300% of the basal value with 5 mM n-butyrate. Increasing the concentration of n-butyrate to 100 mM provoked a gradual decrease in PYY secretion (28). At high unphysiological levels of butyrate, gene expression was diminished, similar to what was found with similar high levels of butyrate and the secretion of PYY (28). Our results support these observations and provide new evidence that butyrate can directly increase the expression of PYY and proglucagon.

Traditionally, the upper parts of the gut are the focus of study on nutrient sensing in the gut, because that is the place where most nutrients are digested and absorbed. Here we give evidence that the lower part of the gut may also respond to short chain fatty acids and adjust the gene expression of the satiety peptides PYY and GLP-1. In resistant-starch–fed rats, the increased PYY/proglucagon gene expression was associated with increased MCT1 and FAS expression in both cecum and colon. If the increased gene expression is triggered by short chain fatty acids, the enteroendocrine L cells of the distal part of the gut might play a role in nutrient sensing. In the other nutrient-sensing organs, such as the hypothalamus, the ATP status and adenosine monophosphate-activated protein kinase signaling are directly related to appetite control peptide gene expression (29). Thus, further study is needed to determine whether the same mechanisms apply to the enteroendocrine L cells in the gut.

In summary, overall evidence supports the hypothesis that PYY and proglucagon gene expression patterns are the same as their peptide distribution in the gastrointestinal tract. These two genes can be modulated by feeding resistant starch in vivo and by butyrate in vitro. Mechanisms by which short-chain fatty acids influenced gene expression need additional study. The up-regulation of PYY/proglucagon by resistant starch and butyrate support the importance of nutrient sensing in the distal part of the gut and might represent a novel approach to increase endogenous PYY/GLP-1 secretion by nutrient–gene interaction.

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Notes

1 Nonstandard abbreviations: PYY, peptide YY; GLP-1, glucagon-like peptide-1; Glut-2, glucose transport-2; GK, glucokinase; MCT1, monocarboxylate transporter; SE, standard error; FAS, fatty acid synthase; CPT-I, carnitine palmitoyltransferase I.

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Acknowledgments

This study was supported by Pennington Biomedical Research Center and LSU Ag Center Biotechnology Education for Students and Teachers.

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