Original Article

International Journal of Obesity (2010) 34, 712–719; doi:10.1038/ijo.2009.277; published online 12 January 2010

Bifidobacterium as an oral delivery carrier of oxyntomodulin for obesity therapy: inhibitory effects on food intake and body weight in overweight mice

R T Long1, W S Zeng1, L Y Chen2, J Guo3, Y Z Lin4, Q S Huang5 and S Q Luo1

  1. 1Department of Cell Biology, Southern Medical University, Guangzhou, Guangdong Province, PR China
  2. 2School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong Province, PR China
  3. 3Department of Administration Office, Guangdong Pharmaceutical College, Guangzhou, Guangdong Province, PR China
  4. 4Department of Cell Biology, Guangdong Pharmaceutical College, Guangzhou, Guangdong Province, PR China
  5. 5Department of Biochemistry, Guangdong Pharmaceutical College, Guangzhou, Guangdong Province, PR China

Correspondence: Dr WS Zeng or Professor SQ Luo, Department of Cell Biology, Southern Medical University, Guangzhou, Guangdong Province 510515, PR China. E-mail: zengwsyh@yahoo.com.cn or luoshq888@163.com

Received 18 May 2009; Revised 30 September 2009; Accepted 1 November 2009; Published online 12 January 2010.

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Abstract

Introduction:

 

Oxyntomodulin (OXM) is a gut hormone released from intestinal L cell. Synthetic OXM and its analog reduce food intake and body weight in both rodents and human beings by being administered intravenously. However, people find intravenous administration difficult because of its side effects and inconvenience. The aim of this study is to develop a novel oral delivery system for OXM and its analog using genetically engineered Bifidobacterium as the carrier.

Methods:

 

An OXM gene expression vector pBBADs-OXM for the Bifidobacterium genus was constructed. Human OXM sequence was fused with extracellular exo-xylanase (XynF) signal peptide (Xs) from Bifidobacterium longum under the control of the pBAD promoter. B. longum NCC2705 was transformed with the recombinant plasmid pBBADs-OXM by electroporation, and the transformed B. longum was selected using MRS plates containing 60μgml−1 ampicillin. The OXM expression in vitro was identified by western blot and enzyme-linked immunosorbent assay (ELISA) assay after L-arabinose induction. Overweight BALB/c mice were treated with B. longum transformed with OXM after 0.2% L-arabinose induction every day for 4 weeks to investigate the effects of OXM-transformed B. longum on food intake and body weight by oral administration. The B. longum transformed with the green fluorescent protein (GFP) gene was used as negative control; orlistat, a gastrointestinal lipase inhibitor, was used as positive control; Normal saline (NS, 0.9% saline) was used as blank control. The food intakes of each group were measured every day, and body weights were measured once a week. Normal BALB/c (2 months old) mice were treated with OXM-transformed B. longum after induction by intragastric administration every day for 6 days to reveal the mechanism of transformed B. longum, with OXM exerting its biological function by oral administration. Plasma OXM, plasma ghrelin and the OXM of intestinal contents were detected by the ELISA method. Plasma glucose and triglyceride levels were analyzed using the Automatic Biochemistry Analyzer.

Results:

 

Transformed B. longum with OXM was selected and identified without biological and morphological alteration. An approximately 4–5kDa OXM peptide was detected in both the supernatant and the cell pellet of transformed B. longum after L-arabinose induction in vitro. The food intake, body weight and blood triglyceride level of overweight mice treated with OXM-transformed B. longum were all significantly reduced compared with that of the GFP negative control group and NS control group (P<0.01). Interestingly, the plasma triglyceride level of the GFP group was significantly decreased compared with that of the NS control group (P<0.01). The OXM level in the intestinal contents of the OXM group was significantly increased compared with that of the GFP negative control group and the NS group (P<0.05). The plasma ghrelin level of the OXM group was significantly decreased compared with that of the GFP and NS groups (P<0.01). Unexpectedly, the ghrelin level of the GFP group was significantly increased compared with that of the NS control group (P<0.01).

Conclusion:

 

A novel oral delivery system of Bifidobacterium for human OXM has been successfully established. The expression of recombinant OXM can be detected in the supernatant and cell pellet of transformed B. longum. OXM-transformed B. longum reduces food intake, body weight and plasma lipid level in overweight mice by oral administration.

Keywords:

oxyntomodulin (OXM); transformation; Bifidobacterium; oral administration; food intake; body weight

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Introduction

Oxyntomodulin (OXM) is a 37-amino acid peptide released from intestinal L cell and the central nervous system.1 OXM originates from posttranslational processing of proglucagon and other proglucagon-derived peptide members, such as glucagon-like peptide-1 (GLP-1), GLP-2 and glicentin.2, 3 It was named after its inhibitory action on the oxyntic glands of the stomach.4 OXM potently inhibits gastric acid secretion and pancreatic enzyme secretion.5 It is considered to be an effective regulator of appetite and body weight. The release of OXM is in response to nutrient intake and in proportion to the calories consumed.6 OXM levels are markedly elevated in tropic malabsorption and after jejuno-ileal bypass surgery for morbid obesity.7, 8, 9 Previous studies have shown that both intracerebroventricular and intraperitoneal administration of OXM reduce food intake and body weight gain in rodents.10, 11 Furthermore, intravenous infusion or subcutaneous injection of OXM has shown a double effect of suppressing appetite and increasing energy expenditure in overweight and obese humans without altering their enjoyment of food.12, 13 The weight loss rate of volunteers treated with OXM is much higher than that of any currently licensed antiobesity drugs.14 These studies have indicated that OXM is a potential therapeutic drug for obesity.

However, injection is still the main approach for peptide drug delivery till now. It is not easy for obese or overweight people to receive intravenous administration for a long time. A new delivery approach, such as oral administration, should be developed for OXM, GLP-1 and other peptide drugs. The genera Bifidobacterium are the dominant probiotic bacteria inhabiting the distal jejunum, ileum and large intestine of humans and other warm-blooded animals. Bifidobacterium spp has many beneficial effects on human health, including preventing infection, immunomodulation, reducing serum cholesterol, promoting lactose digestion and protecting against colon cancer.15, 16, 17, 18 Recently, genetically engineered Bifidobacterium has been used as an exogenous gene delivery carrier of cytosine deaminase and interleukin-10 for cancer gene therapy and bowel disease treatment.19, 20, 21 Moreover, Bifidobacterium longum was used as the host for recombinant pediocin PA-1 expression and secretion under the guide of α-amylase signal peptide.22 It suggests that Bifidobacterium spp may be the most suitable host (carriers) for human gut hormone gene expression and secretion in the intestinal tract for obesity and diabetes therapy.

In this study, we describe a novel oral delivery system of Bifidobacterium for OXM by constructing an Escherichia coliBifidobacterium shuttle vector. The expression characters of human OXM in Bifidobacteria in vitro were analyzed, and the therapeutic effects of OXM-transformed B. longum after oral administration on overweight mice were evaluated and confirmed.

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Materials and methods

Animals, bacterial strains, enzymes, reagents and plasmids

Female BALB/c mice (3 or 8 weeks old) were purchased from the Laboratory Animal Center (Southern Medical University, Guangzhou, PR China). Mice were maintained in a temperature-controlled room (21–23°C) in a 12-h light–dark cycle. B. longum strain NCC2705 was kindly provided by the Nestle Research Center (Lausanne, Switzerland). Restriction endonucleases, polymerase and T4 DNA ligase were purchased from MBI Fermentas Inc. (Vilnius, Lithuania). Human OXM DNA sequence was synthesized by Shanghai Sangon Biological Engineering Technology and Services Ltd (Shanghai, China). Rabbit anti-OXM IgG was purchased from Alphadiagnostic Inc. (San Antonio, TX, USA). OXM and ghrelin enzyme-linked immunosorbent assay (ELISA) kits were purchased from Adlitteram Diagnostic Laboratories Inc. (Fremont, CA, USA). BAD/gIII-A plasmid and L-arabinose were purchased from Invitrogen Inc. (Carlsbad, CA, USA). Synthesized OXM was purchased from Phoenix Biotech (Beijing, China). Orlistat (Xenical, a gastrointestinal lipase inhibitor) was purchased from Roche Inc. (South San Francisco, CA, USA). Ultra Low-Range protein Color Marker (molecular weight 1.06–26.6kDa) was purchased from Sigma-Aldrich Inc. (St Louis, MO, USA). Chemiluminescent substrate was purchased from ThemoFisher Inc. (Waltham, MA, USA). High-fat diet (40% fat) was purchased from Medical Laboratory Animal Center of Guangdong Province, (Guangzhou, PR China).

Vector construction

The E. coli–Bifidobacterium shuttle expression plasmid vector pBBAD/Xs was constructed previously by inserting the replicon and polymerase gene (BLP) sequence amplified from a B. longum strain23 into pBAD/gIII-A plasmid at the Bst1107 restriction site, and the gene III signal peptide sequence was replaced by an extracellular exo-xylanase (XynF) signal peptide (Xs) DNA sequence with an additional BpiI restriction site amplified from B. longum genomic DNA by the long primer overlap PCR method. The green fluorescent protein (GFP) report gene expression vector pBBADs-GFP was constructed by inserting GFP from pEGFP-N1 plasmid (Mountain View, CA, USA, Clontech) into NcoI and XbaI sites. The OXM expression vector pBBADs-OXM was constructed by inserting the human OXM gene into plasmid pBBADs-GFP to replace the GFP gene at the BpiI and XbaI sites (Figure 1). All constructed vectors were verified by restriction enzyme digestion and sequencing.

Figure 1.
Figure 1 - 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

Cloning strategy and plasmid map of oxyntomodulin (OXM) expression vector for Bifidobacterium. The details of vector construction are described in the ‘Materials and methods’ section. (a) The ligation adapter design of restriction enzyme BpiI for XynF signal OXM DNA sequence and XynF signal sequence. Small arrows indicate BpiI cleavage sites. (b) The amino-acid sequence of XynF signal and OXM fusion peptide; no redundant amino acid appeared after ligation through special BpiI adapter. The black triangle indicates the signal peptidase cleavage site. (c) Plasmid map of recombinant pBBADs-OXM and pBBADs-green fluorescent protein (GFP) expression vector. pBAD: araBAD promoter; Xs: XynF signal; Amp: ampicillin resistance gene; BLP: replicon and polymerase gene of Bifidobacterium plasmid; araC: araC gene.

Full figure and legend (98K)

Bifidobacteria transformation

B. longum NCC 2705 was used as the host to be transformed with recombinant plasmids pBBADs-OXM and pBBADs-GFP by electroporation. Competent B. longum cell preparation and electroporation were carried out using a Gene Pulser and Pulse Controller apparatus (Hercules, CA, USA, Bio-Rad) at 2.5kv, 25F and 200, as described previously.24 Bacteria were plated on MRS agar (1.5%, w/v) supplemented with 0.05% L-cysteine and 60μgml−1 ampicillin. Plates were incubated anaerobically at 37°C for 2 days, after which small- or full-size colonies were visible.

Transformed B. longum identification

Full-size colonies of transformed bacteria were selected and cultured in MRS broth with 60μgml−1 ampicillin for 2 days. The transformed bacteria were identified using PCR analysis to amplify target genes (OXM or GFP) and 16s RNA recombinant DNA from the extracted genomic DNA. The primers used for OXM gene and 16s recombinant DNA amplification were (1) OXM forward primer OXMF: 5′-CATTCACAGGGCACATTC-3′, reverse primer OXMR: 5′-TCATTAGGCAATGTTATTCCTG-3′; (2) 16s recombinant DNA forward primer 16SF 5′-TCCAGTTGATCGCATGGTC-3′, reverse primer: 16SR 5′-GGGAAGCCGTATCTC TACGA-3′. Gram staining was used to identify the morphology of transformed B. longum.

Gene expression induction in vitro

Transformed bacteria were cultured in MRS broth with 60μgml−1 ampicillin. L-arabinose was added to induce target gene expression after the bacterial suspension optical density (OD)695nm reached ~0.6. Culture supernatants and bacterium pellets were collected at 6, 12, 24 and 48h after 0.2% L-arabinose induction and stored at −70°C.

Preparation of overweight mice

A total of 60 female BALB/c mice were fed a high-fat diet (40% fat) without limit for food and water to prepare overweight mice. A group of mice (n=8) fed a standard rodent diet was used as chow-fed controls. Food intake and body weight were measured once a week. The overweight mouse was confirmed when its body weight was 20% heavier than that of the chow-fed mouse after 3–4 weeks of being fed a high-fat diet.

Animal experiment protocol

To investigate the effects of OXM-transformed B. longum on food intake and body weight, 32 overweight mice were chosen and divided randomly into four groups. The OXM group (n=8) was treated with 0.9ml (6 × 108 per ml) OXM-transformed B. longum after 12h of 0.2% L-arabinose induction by intragastric administration every day; the GFP group (n=8) was treated with 0.9ml (6 × 108 per ml) GFP-transformed B. longum and was used as the negative control; the orlistat group (n=8) was treated with 1.0gkg−1 (weight) orlistat, a gastrointestinal lipase inhibitor, dissolved in NS (0.9% saline) and was used as the positive control; the NS group (n=8) was treated with 0.9ml NS only and was considered as the blank control. The food intakes of each group were measured every day, and body weights were measured once a week. To investigate the effects of OXM-transformed B. longum on OXM and ghrelin levels in plasma, 36 normal BALB/c mice (8 weeks old) were randomly divided into three groups. The OXM group (n=12) was treated with 0.9ml (6 × 108 per ml) induced OXM-transformed B. longum by intragastric administration every day; the GFP group (n=12) was treated with 0.9ml (6 × 108 per ml) induced GFP-transformed B. longum and was considered as the negative control; the NS group (n=12) was treated with 0.9ml NS and was considered as the blank control. The animals were killed 6 days later. The intestinal contents of ileocecum and peripheral blood were collected. All animal experiments abided by the Administration Regulations of Experimental Animal of the South Medical University, PR China. Plasma levels of glucose and triglyceride were determined using a Hitachi7600-010 Automatic Biochemical Analyzer (Hitachi Ltd, Tokyo, Japan). The tissue slides of blood vessels, liver, kidney and colon were stained with hemotoxylin and eosin, and then examined using a microscope.

ELISA assay

The culture supernatant and serum samples were directly added to the microwell plate for ELISA detection. Whole-cell protein extract of pellets was prepared as follows: each pellet (1.5ml bacteria culture) was resuspended in 60μl TES lysis buffer (10mmoll−1 Tris-HCI, 1.0mmoll−1 EDTA, 150mmoll−1 NaCl, 0.1% Triton X-100, 20mgml−1 lysozyme, pH 8.0) containing protease inhibitors mix (Themo-Fisher, Waltham, MA, USA) for 20min at room temperature and then sonicated for 10min on ice using a Sonifier UP50H (Dr Hielscher Ltd, Teltow, Germany) at the following conditions: 0.5 cycle, 80% amplitude. The supernatants of intestinal content samples were prepared by adding 0.1mlg−1 (weight) 0.1moll−1 phosphate buffer and were centrifuged 10000 × g at 4°C. The test procedures were carried out according to the instruction manual provided in the kits. Serum sample (20μl), supernatant sample or 10μl pellet lysate was added to each well.

Western blot

Before electrophoresis, proteins in the supernatant sample were precipitated with trichloroacetic acid and washed twice with acetone. Whole-cell protein extract of pellets was prepared using the ELISA method with an additional 20μl 4 × SDS loading buffer. Sample proteins were separated in 15% (w/v) SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Pall Ltd, East Hills, NY, USA). The OXM expressions in supernatants and pellets were detected using the western blot method with rabbit anti-OXM IgG and monitored with enhanced chemiluminescence.

Statistical analysis

Combined data are presented as means±s.d. Multiple comparison of experimental groups was carried out by one-way ANOVA (analysis of variance), followed by the Student–Newman–Keuls test method using SPSS13.0 statistical software (SPSS Inc., IBM Company Headquarters, Chicago, IL, USA). When the equal variance test failed to produce results, the Dunnett T3 test was selected. In all cases, P<0.05 was considered to be statistically significant.

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Results

Transformed B. longum selection and morphological analysis

Transformed B. longum was selected in selective MRS plates supplemented with 60μgml−1 ampicillin after electroporation. Colonies of transformed B. longum appeared only on the transformation plates of pBBADs-OXM and pBBADs-GFP plasmids containing the BLP sequence. On the contrary, no colony was observed on the plate transformed with pBADs-GFP plasmids without the BLP sequence. It suggests that BLP is necessary for plasmid duplication and stable maintenance in B. longum. The results of Gram staining show that transformed B. longum could still maintain its typical morphology. No differences in morphology were observed in B. longum before and after transformation (pictures not presented).

Identification of transformed B. longum by PCR method

OXM-transformed B. longum was identified by PCR. A band of 120-bp fragment of the OXM gene was amplified from the genomic DNA of B. longum transformed with pBBADs-OXM (Figure 2a). No fragment was obtained from untransformed bacteria or bacteria transformed with pBBADs-GFP. The species of B. longum was identified by amplifying 16s RNA recombinant DNA by PCR. A band of 800-bp fragment was obtained from transformed bacteria, and negative result was obtained from E. coli (Figure 2b).

Figure 2.
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Identification of transformed B. longum by PCR method. (a) Electrophoresis of OXM gene PCR products in 2.0% agarose gel. Lane 1: 100-bp DNA ladder marker; lane 2: E. coli transformed with pBBADs-OXM plasmid; lanes 3–4: B. longum transformed with pBBADs-OXM plasmid. (b) Electrophoresis of 16s rDNA PCR products in 1.5% agarose gel. Lane 1: 100-bp DNA ladder marker; lane 2: product from E. coli transformed with pBBADs-OXM; lane 3–4 product from B. longum transformed with pBBADs-OXM.

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Detection of OXM expression in B. longum in vitro

The expression of recombinant OXM from OXM-transformed B. longum in vitro was detected by ELISA and western blot. OXM expression could be detected in supernatants and pellets of OXM-transformed bacteria. The concentrations of OXM in supernatants were extremely higher than that of cell pellets (three repeat expressions) (Figure 3b). The levels of recombinant OXM in both supernatants and pellets increased with induction time. The level of supernatant reached a maximum at 12h, whereas the level of pellet reached a maximum at 24h. The result of western blot showed that ~5-kDa protein bands were observed on the lanes of both supernatants and pellet lysates. It is concordant with the 4.7-kDa theoretical molecular weight of mature OXM. No reaction band presented on the lane of preinduced transformed bacteria (Figure 3a).

Figure 3.
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Recombinant OXM expression analysis. (a) Western blot analysis of OXM expression in vitro. A band of 5-kDa peptide was shown in culture supernatant and cell pellet of B. longum transformed with pBBADs-OXM plasmid after 0.2% L-arabinose induction. Lane 1: culture supernatant before induction; lanes 2–3: culture supernatant after 6 and 12h induction, respectively; lane 4: cell pellet after 6h induction. (b) Enzyme-linked immunosorbent assay (ELISA) assay of OXM expression in vitro after 0.2% L-arabinose induction at different time (three repeat expressions, mean±s.d.). *P<0.01.

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Effects on food intake and body weight

The daily intake of the OXM group (total intake of one group, n=8) began to decrease after 1-week oral administration of OXM-transformed B. longum. The daily intake in the fourth week decreased significantly compared with that of the negative control GFP group and that of the NS control group (7.79±0.44g vs GFP group 11.31±0.39g and NS group 11.23±0.70g, P<0.01), whereas it was almost similar (6.93±0.29g) in the chow-fed group. There was no difference between the daily intake of the OXM group and that of the orlistat group (P>0.05) (Figure 4). The body weight of the OXM group also began decreasing after 1-week oral administration of OXM-transformed B. longum. It decreased significantly compared with that of the GFP and NS groups (33.19±2.41g vs GFP group 38.98±1.28g and NS group 42.00±3.09g, P<0.01). No difference appeared between the body weight of the OXM group and that of the orlistat group (P>0.05); Interestingly, the body weight of the GFP group decreased slightly compared with that of the NS group (P<0.05) (Figure 5).

Figure 4.
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Effects of transformed B. longum on food intake of overweight mice. The mean of daily intake was the daily intake of a group of mice (n=8) in the fourth week (n=7, mean±s.d.). *P<0.01.

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Figure 5.
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Effects of transformed B. longum on body weight. Data presented are the mean body weigh of a group of mice (n=8) in the fourht week (mean±s.d.). +P<0.05 (GFP vs NS); *P<0.01 (OXM vs GFP or NS).

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Effects on plasma lipid

The effect of OXM-transformed B. longum on plasma lipid by oral administration was also investigated. Blood triglyceride levels of the OXM and orlistat groups decreased significantly compared with that of the NS group (1.66±0.34 and 1.41±0.64 vs 5.39±1.87mmoll−1, P<0.001). They were even lower than that (1.96±0.67mmoll−1) of the chow-fed group, although no statistical difference appeared. Interestingly, the blood triglyceride level of the GFP group also decreased significantly compared with that of the NS group (2.03±0.44 vs 5.39±1.87mmoll−1, P<0.001) (Figure 6). No differences in plasma glucose levels were found between the OXM and the GFP or NS groups. Pathological changes in blood vessel, liver and kidney tissues were not observed in any group by the hemotoxylin and eosin staining method (data not presented).

Figure 6.
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Effects of transformed B. longum on plasma triglyceride levels of overweight mice. The mean triglyceride level is presented as the mean of a group of mice (n=8, mean±s.d.). +P<0.001 (GFP vs NS); *P<0.001 (OXM or GFP or orlistat vs NS).

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OXM level in intestinal contents

After 6 days oral administration of OXM-transformed B. longum, the OXM level of the intestinal contents of the OXM group was significantly increased compared with that of the GFP negative control and NS control groups (50.24±15.29pgml−1 vs GFP group 36.31±12.29pgml−1 and NS group 33.21±8.01pgml−1, P<0.05) (Figure 7). No difference was observed between the GFP and NS groups. However, a high deviation of OXM levels appeared in all groups. It might be because of the high heterogeneity of liquid water content in intestinal contents.

Figure 7.
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OXM levels of intestinal contents in normal mice treated with transformed B. longum. OXM levels are presented as the mean of a group of mice (n=12, mean±s.d.). +P<0.05.

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Effects on plasma OXM and ghrelin

There were no differences in plasma OXM concentration between the OXM group and the GFP or NS groups (36.18±3.87pgml−1 vs GFP group 37.04±4.87pgml−1 and NS group 38.23±4.13pgml−1, P>0.05) (Figure 8). The plasma ghrelin level of the OXM group was significantly decreased compared with that of the GFP and NS groups (12.28±0.58pgml−1 vs GFP group 32.15±1.57pgml−1 and NS group 23.68±3.63pgml−1, P<0.01). Unexpectedly, the plasma ghrelin level of the GFP group was significantly higher than that of the NS group (P<0.001) (Figure 9).

Figure 8.
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Plasma OXM levels of normal mice treated with transformed B. longum. Data are presented as the mean of a group of mice (n=12, mean±s.d.).

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Figure 9.
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Plasma ghrelin levels of normal mice treated with transformed B. longum. Ghrelin level is presented as the mean of a group of mice (n=12, mean±s.d.). *P<0.01.

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Discussion

Previous studies including clinical trials have shown that OXM significantly reduces food intake and body weight in rats and human beings by systemic administration (intravenous, intraperitoneal or subcutaneous injection) or intracerebroventricular injection.10, 11, 12, 13 However, almost all peptide drugs including OXM are still administrated intravenously. Not only is intravenous administration inconvenient but it also causes many side effects, such as infection, allergy and angeitis.

In this study, we described a novel oral delivery system for OXM by using genetically engineered Bifidobacterium as the carrier. The results show that OXM-transformed B. longum reduces food intake, body weight and decreases blood lipid in overweight mice by oral administration. The benefits of OXM-transformed B. longum on reduction of food intake, body weight and blood lipid are similar to that of orlistat, a new gastrointestinal lipase inhibitor drug for obesity therapy. Furthermore, B. longum itself has many other beneficial functions for health without side effects (such as steatorrhea, nutrition imbalance, allergy, angeitis and so on), which are always caused by orlistat and intravenous administration.

The oral administration of GFP-transformed Bifidobacteria significantly decreases blood triglyceride levels in overweight mice than NS (P<0.001). It is concordant with other reports that wild Bifidobacteria reduce plasma cholesterol and lipid.25, 26, 27 The blood triglyceride level of the OXM group is slightly lower than that of the GFP group. It suggests that OXM may promote lipid reduction of wild Bifidobacterium, although no statistical difference emerged.

To reveal the mechanism of action of OXM-transformed B. longum by oral administration on food intake and body weight, we investigated the OXM and ghrelin levels in blood circulation in the experimental mice. Previous studies have shown that intact small protein and peptide can be absorbed by intestinal mucosa epithelium and be detected in the portal vein and peripheral blood.28, 29 We hypothesize that being absorbed into circulation is one of the probable pathways by which recombinant OXM of transformed B. longum exerts its biological functions. Although the OXM level of the intestinal contents of OXM-transformed B. longum-treated mice was higher than that of GFP-transformed B. longum-treated mice, no differences in plasma OXM levels were found between the OXM and GFP control groups. The reason may be the rapid degradation of OXM and insufficient numbers of experimental animals. We still believe that a small amount of OXM from B. longum may be absorbed by gastrointestinal mucosa epithelium and may enter into blood circulation to exert its effects on appetite and energy expenditure.

Acting directly on gastrointestinal epithelial cells may be the main probable pathway of recombinant OXM exerting its biological functions by oral administration. OXM can bind to both glucagon and GLP-1 receptor (GLP-1R).30, 31 GLP-1R is necessary for the anorectic effects of OXM, as well as for the anorectic effects of other GLP-1R agonists, namely, GLP-1 and exendin-4 (Ex-4). GLP-1R is widely expressed, being found in the gastrointestinal tract, pancreas, lung, heart and centrally in the hypothalamus.32, 33 Oral administration of OXM-transformed Bifidobacteria may directly act on GLP-1R or on an unidentified receptor in the gastrointestinal epithelium, and may then increase energy expenditure and decrease energy intake. It has been reported that GLP-1, CCK, PYY and other gut hormones can inhibit the release of ghrelin through vagus nerve stimulation.34, 35, 36 Recombinant OXM in the intestinal tract may also stimulate the vagus nerve, followed by inhibition of ghrelin release by acting on its receptors. Further experiments are required to elucidate the mechanisms by which OXM exerts its effects on food intake and energy expenditure by oral administration.

Ghrelin is a powerful signal molecule of appetite. It rises preprandially in the plasma and is considered to be a trigger for meal initiation.37, 38 The inhibitory effects of OXM on plasma ghrelin level by intravenous administration have already been reported.39, 40 In this study, oral administration of OXM-transformed B. longum decreases the ghrelin plasma level significantly. The result presented is concordant with previous data. Our results also confirm that oral administration of OXM-transformed B. longum does have definite biological effects on appetite. The mechanism by which GFP-transformed B. longum increases ghrelin plasma level is unclear. We hypothesize that the beneficial promotion of Bifidobacteria to gastrointestinal digestive function may be responsible for the increasing plasma ghrelin level.

The recombinant OXM levels in the supernatants of transformed B. longum culture at different times after induction in vitro are extremely higher than that inside cells (pellets). It demonstrates that OXM in transformed B. longum is expressed as a mature secretory peptide, and the XynF signal peptide coming from B. longum is suitable to guide exogenous protein secretion in engineering Bifidobacterium spp. More studies are required to elucidate the expression and absorption characters of recombinant OXM from engineering B. longum in intestinal tract, and to ensure the safety of transformed B. longum to gastrointestinal microecology in mammals.

In conclusion, an expression vector for Bifidobacterium spp has been established as a novel oral delivery carrier for OXM and other peptides. The OXM expression in transformed B. longum can be detected in supernatant and inside cells after L-arabinose induction. OXM-transformed B. longum reduces food intake, body weight and blood lipid levels in overweight mice by oral administration.

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Conflict of interest

The authors declare no conflict of interest.

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References

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Acknowledgements

We express our thanks to the Clinical Ecsomatic laboratory of the Southern Hospital for their invaluable contribution to the detection of some blood biochemical parameters. This research was funded by the National Natural Science Foundation of China, (NSFC:30472001), by the Science and Technology Planning Project of Guangdong Province (2006B3552005) by and the Technology Planning Project of Zhongshan city, Guangdong Province (2006A119).

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