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Effects of octreotide on nitric oxide synthase expression in the small intestine of high fat diet-induced obese rats
Obesity Research & Clinical Practice (2012) 6, e280—e287
Effects of octreotide on nitric oxide synthase expression in the small intestine of high fat diet-induced obese rats Yan Ou a,b, Rui Liu a,∗, Na Wei a,b, Xian Li a, Ou Qiang a, Wei Huang a,b, Chengwei Tang b a
Division of Peptides Related to Human Disease, National Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China b Department of Gastroenterology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China Received 28 July 2011 ; received in revised form 31 October 2011; accepted 22 November 2011
KEYWORDS Obesity; Neuronal nitric oxide synthase (nNOS); Endothelial nitric oxide synthase (eNOS); Inducible nitric oxide synthase (iNOS); Octreotide
∗
Summary Objective: To investigate whether obesity induced by high fat diet is associated with expression of neuronal, endothelial, and inducible nitric oxide synthase (nNOS, eNOS, and iNOS) in the intestine, and to test the effects of the somatostatin analog octreotide on this expression. Methods: The study included high fat diet-induced obese and normal control rats. The obese rats were further separated into an obese control group and an octreotide intervention group. Rats in the intervention group were injected with 40 g/kg octreotide every 12 h for 8 days. Expressions of nNOS, eNOS, and iNOS in the small intestine were analyzed by RT-PCR and immunohistochemistry. The NO level of small intestinal homogenate was measured with an ELISA kit. Results: The body weight; Lee’s index; small intestinal eNOS and iNOS mRNA and protein expression levels; nNOS protein expression levels; and small intestinal homogenate NO levels were all significantly higher in the obese control group than in the normal controls (p < 0.01); nNOS mRNA expression was also higher in the obese control group, but not significantly so. Octreotide intervention significantly reduced the body weight and small intestinal homogenate NO level of the obese rats relative to the obese control group (p < 0.05). The mRNA and protein expression levels of eNOS and iNOS; the protein expression level of nNOS in the small intestine were also significantly lower in the octreotide intervention group than in the obese control group (p < 0.01), while nNOS mRNA expression was lower but not significantly so.
Corresponding author. Tel.: +86 28 85164011. E-mail address: [email protected] (R. Liu).
1871-403X/$ — see front matter © 2011 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.orcp.2011.11.004
Effects of octreotide on nitric oxide synthase expression
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Conclusion: High fat diet-induced obesity is associated with elevated small intestinal nNOS, eNOS, and iNOS expression levels. Octreotide treatment can inhibit nNOS, eNOS, and iNOS expression and lead to weight loss. © 2011 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
Introduction In recent years, as living standards have risen, lifestyle and diet have undergone great changes. Physical inactivity and diets high in calories and fat are important factors leading to obesity, and the incidence of obesity is rising steeply [1]. Excessive accumulation and abnormal distribution of body fat are closely linked to type 2 diabetes, hypertension, dyslipidemia, and obstructive sleep apnea [2]. Obesity incidence is positively correlated with excessive calorie intake. The small intestine is a major absorption site and gateway of energy intake. Nitric oxide synthase (NOS) plays an important role in digestion and absorption in the small intestine [3,4]. NOS has three isoforms: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) [5]. The enzyme’s product, nitric oxide (NO), is a vascular endothelium relaxing factor [6,7] and acts as a non-adrenergic, non-cholinergic inhibitory neurotransmitter in the small intestine [8]. Somatostatin (SST) is a multifunctional gut peptide synthesized and released by intestinal endocrine cells (D cells). Its effects are mediated by the SST receptor, which is located in the cell membrane. SST regulates the physiological functions of intestinal epithelial cells and immune cells, as well as gastric motor activity [9]. Octreotide is an artificial synthetic analog of SST with a longer half-life and duration of activity. Many studies have shown that somatostatin can affect the intestinal absorption and nutritional functions [10,11]. However, there are no published studies investigating whether the effect of somatostatin on intestinal absorption is associated with NOS. We investigated whether obesity induced by a high fat diet is associated with expression of intestinal nNOS, eNOS, and iNOS. In addition, we tested the effects of octreotide on these expression levels.
Materials and methods Experimental animals and grouping All experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University (Chengdu, China).
We used 66 healthy male 21-day-old Sprague—Dawley (SD) rats that had been weaned for 3 days. All animals were obtained from the Animal Center of Sichuan University. After adaptive feeding for 3 days, the rats were placed into two groups, one group of 18 (the normal control group) fed with standard chow (290 kcal/100 g, in line with the People’s Republic of China National Standard GB 14924—2001), and another of 48 fed with high-fat chow (430 kcal/100 g). Food and water were supplied ad libitum, and the animals were housed in independently ventilated cages on a 12:12-h light:dark schedule and kept at 20—25 ◦ C. They were weighed and measured for body and tail length each week for 24 weeks. After 24 weeks, rats from the high-fat chow group with a mean body weight at least 1.4 times that of the controls were selected as obese rats. Obese rats were then placed into two groups, an obese control group of 16 rats, and an octreotidetreated group of 15 rats. The octreotide-treated group was injected with octreotide (40 g/kg body weight) every 12 h for 8 days. Rats were measured weekly for body weight to calculate Lee’s index [body weight (g)1/3 × 1000/body length (cm)].
Sample preparation At the end of the experiment, after fasting for 12 h, rats were anesthetized intraperitoneally with 2% sodium pentobarbital, and 2 cm of the small intestine was removed from the same position on each rat. The samples were fixed in 4% paraformaldehyde and then embedded in paraffin. Another 2 cm section of small intestine was kept at −140 ◦ C until total RNA extraction and NO level determination.
Reverse transcription PCR analysis Total RNA was extracted from the frozen small intestine using Trizol reagent (Takara BioEngineering Co., Ltd., Kyoto, Japan). First-strand cDNA was synthesized from 2 g of total RNA for each sample using reverse transcription kits (MBI, Fermentas Life Sciences Inc., Vilnius, Lithuania). Reverse transcription reactions were performed at 40 ◦ C for 60 min and 70 ◦ C for 5 min. Primer sequences are in Table 1. The thermal cycling parameters consisted of an initial denaturation
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Y. Ou et al. PCR primer sequences.
Gene
Primer sequences
Product size
Database
iNOS
Sense Anti-sense
5 -ATC CCG AAA CGC TAC ACT T-3 [30] 5 -TCT GGC GAA GAA CAA TCC-3
314 bp
NM 012611.3
nNOS
Sense Anti-sense
5 -GGC ACT GGC ATC GCA CCC TT-3 [31] 5 -CTT TGG CCT GTC CGG TTC CC-3
213 bp
NM 052799.1
eNOS
Sense Anti-sense
5 -TGC ACC CTT CCG GGG ATT CT-3 [31] 5 -GGA TCC CTG GAA AAG GCG GT-3
189 bp
NM 021838.2
GAPDH (1)
Sense Anti-sense
5 -CAT GAC CAC AGT CCA TGC CAT C-3 5 -CAC CCT GTT GCT GTA GCC ATA TTC-3
451 bp
NM 017008.3
GAPDH (2)
Sense Anti-sense
5 -TAT GAC AAC TCC CTC AAG AT-3 [32] 5 -AGA TCC ACA ACG GAT ACA TT-3
318 bp
NM 017008.3
step for 5 min at 94 ◦ C, then 40 s at 94 ◦ C, 40 s at 60 ◦ C for iNOS, 67 ◦ C for eNOS or 64 ◦ C for nNOS, 50 s at 72 ◦ C, totally 35, 37 and 39 cycles, respectively. The final extension step was for 7 min at 72 ◦ C. PCR products were resolved by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. Densitometry was carried out using a Bio-Rad GelDoc image acquisition system and Quantity One (v. 4.3) quantitation software (Bio-Rad, Hercules, CA, USA).
Immunohistochemistry of small intestine nNOS, iNOS, and eNOS For the immunohistochemical detection of nNOS, iNOS, and eNOS protein expression levels, 4 mthick small intestine tissue sections were cut from the formalin-fixed and paraffin-embedded tissues. The sections were deparaffinized, endogenous peroxidase was blocked with 3% H2 O2 , and antigen retrieval was performed at high temperature and pressure. For nonspecific blocking, 10% goat sera was added, each section was incubated for 30 min at 37 ◦ C, and then either nNOS, iNOS, or eNOS rabbit anti-rat polyclonal antibody was added (1:400, 1:400, 1:150; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China). After incubating overnight at 4 ◦ C and rewarming to 37 ◦ C for 40 min, each section was stained with a ready-to-use streptavidin—catalase immunohistochemical reagent system. The color reaction was developed with diaminobenzidine (DAB; Zhongshan Bioagent Company, Beijing, China). A semiquantitative immunohistochemical analysis of raw data was performed with Image-Pro Plus 4.0 software (Media Cybernetics, Silver Spring, MD, USA) to score integrated optical density (IOD) for positive reaction areas.
Immunohistochemistry positive standard Positive expression of iNOS was considered to have occurred if the cytoplasm of the small intestine macrophages in the mucosal and submucosal layers was stained yellow to brown yellow. We took a continuous measurement of IOD values of the three small intestinal villi, then divided by 3 to obtain the IOD average, which was used in statistical analysis. Positive expression of eNOS was indicated if the cytoplasms of vascular endothelial cells were stained yellow to brown in the full thickness of the small intestine. We counted the total number of positive vessels in each section and used this number in our statistical analysis. Positive expression of nNOS was indicated if the cytoplasms of nonadrenergic non-cholinergic nerve cells were stained yellow to brown in the submucosa and muscular layers of the small intestine. The total number of positive nerve cells in each section was used for our statistical analysis.
Determination of NO levels in the small intestine The small intestine samples homogenized in ice cold phosphate buffered saline (PBS, pH7.4). After centrifugation at 1050 × g for 15 min at 4 ◦ C, the supernatant was used to measure the NO levels by ELISA kit (R&D Systems, Minneapolis, MN, USA).
Statistical methods All data were analyzed with statistical software SPSS 16.0 (SPSS Inc., USA). Data are presented as means and standard deviations. Groups were compared with analysis of variance. All tests were two-tailed, and p values less than 0.05 were considered to indicate statistical significance. All data
Effects of octreotide on nitric oxide synthase expression met the assumptions of the tests used to analyze them.
Results Octreotide treatment and parameters associated with obesity From the third week, the body weight growth rate of the high fat-diet group was significantly higher than that of the normal control group. After 24 weeks, the rats with high fat diet-induced obesity showed typical features of obesity, such as higher body weight and Lee’s index, while those in the normal control group did not (p < 0.01) (Table 2). The octreotide-treated group had significantly lower body weight than did the obese control group (p < 0.05) (Table 2).
Octreotide treatment and NOS mRNA expression The mRNA expression levels of eNOS and iNOS in the obese control group were significantly higher than those in the normal control group (p < 0.01); there was an increasing tendency of nNOS mRNA expression in obese control group as compared with normal control group (Fig. 1 and Table 3). The eNOS and iNOS mRNA expression levels were significantly lower in the octreotide-treated group than in the obese control group (p < 0.01); there was a decreasing tendency of nNOS mRNA expression in octreotide intervention group as compared with obese control group (Fig. 1 and Table 3).
Octreotide treatment and NOS protein expression iNOS was expressed in the small intestinal lamina propria macrophages and other immune cells, expressed to a lesser extent in the submucosa, and almost completely absent in the muscular and serosa layers. iNOS-positive cells had yellow to brown staining in the cytoplasm, with only a small amount of nuclear staining. In the obese control group, small intestinal iNOS expression levels were significantly higher than those of the normal control group (p < 0.01) (Table 3 and Fig. 2). These expression levels were also significantly lower in the octreotide intervention group than in the obese control group (p < 0.01) (Table 3 and Fig. 2). eNOS mainly appeared in the small intestinal submucosal and muscular layer vascular endothelial cells, and was expressed in the lamina propria
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microvasculature. The cytoplasms of eNOS-positive cells were stained yellow to brown, with no expression in the nuclei. In the obese control group, the small intestine eNOS expression levels were significantly higher than those of the normal control group (p < 0.01) (Table 3 and Fig. 3). eNOS expression levels were significantly lower in the octreotide intervention group than in the obese control group (p < 0.01) (Table 3 and Fig. 3). nNOS was found in the small intestinal nonadrenergic, non-cholinergic nerve plexus and fibers. It was highly expressed in the myenteric plexus and weakly expressed in the submucosal plexus. The cytoplasms of nNOS-positive cells were stained yellow to brown. The small intestine nNOS expression levels of the obese control group were significantly higher than those of the normal control group (p < 0.01) (Table 3 and Fig. 4). Those levels were significantly lower in the octreotide intervention group than in the obese control group (p < 0.01) (Table 3 and Fig. 4).
NO levels in the small intestine The NO level in small intestine of obese control group was significantly higher than that in normal control group (p < 0.01). The NO level in octreotide intervention group was significantly lower than that in obese control group (p < 0.05) (Table 3).
Discussion The constitutive nitric oxide synthases nNOS and eNOS express under normal physiological conditions, while iNOS expresses in the pathological state [12]. nNOS is distributed in the small intestine nerve plexus and fibers [13], and is expressed strongly in the myenteric plexus and weakly in the submucosal plexus [14]. eNOS is expressed in vascular endothelial cells and lamina propria histiocytes [15,16]. iNOS is expressed in small intestinal lamina propria macrophages and other immune cells [17]. iNOS transcription and translation are activated by cytokines such as tumor necrosis factor-␣ (TNF-␣) [18]. We found that nNOS, eNOS, iNOS protein expression levels and eNOS, iNOS mRNA expression rose in the small intestines of high fat diet-induced obese rats. The resulting increased production of NO may have improved the absorption of nutrients and energy in the rats’ small intestines. nNOS can produce NO that acts as an enteric nervous system non-adrenergic, non-cholinergic inhibitory neurotransmitter [8]. This suggests that
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Table 2
Body weight and Lee’s index in each group.
Body weight (g) Lee’s Index *
Normal control (n = 18)
Obese control (n = 16)
Octreotide intervention (n = 15)
378.54 ± 111.75 318.73 ± 20.10
605.61 ± 141.00* 337.61 ± 10.82*
508.27 ± 94.39 334.67 ± 16.56
p < 0.01 vs. normal control group. p < 0.05 vs. obese control group.
Figure 1 Expression of iNOS, eNOS and nNOS mRNA in the small intestine. 1 and 2, normal control group; 3 and 4, obese control group; 5 and 6, octreotide intervention group. RT-PCR showed that iNOS and eNOS mRNA expression was higher in obese control group than normal control and octerotide-intervention group (p < 0.01). nNOS mRNA expression tended to be higher in the obese control group than in the normal controls and lower in the octreotide intervention group than in the obese control group. *p < 0.01 vs. normal control group; :, p < 0.01 vs. obese control group.
Figure 2 iNOS expression in small intestine mucosa. The expression of iNOS in small intestine mucosa was detected by immunohistochemistry. The images show the SD rat intestinal villi. iNOS was mainly expressed in the small intestine lamina propria which was stained yellow to brown. iNOS expression levels in obese control group were significantly higher than normal control and octreotide intervention group. Magnification: 400×.
Effects of octreotide on nitric oxide synthase expression
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Figure 3 eNOS expression in small intestine submucosal layer. The expression of eNOS in small intestine submucosal layer was detected by immunohistochemistry. eNOS mainly appeared in the small intestine submucosal layer vascular endothelial cells which were stained yellow. eNOS expression levels in obese control group were significantly higher than normal control and octreotide intervention group. Magnification: 400×.
Figure 4 nNOS expression in small intestine muscular layer. The expression of nNOS in small intestine muscular layer was detected by immunohistochemistry. nNOS was mainly found in the small intestine myenteric plexus which were stained yellow. nNOS expression levels in obese control group were significantly higher than normal control and octreotide intervention group. Magnification: 400×.
nNOS-produced NO inhibits intestine motility, increases the time that chyme stays in the small intestine, and enhances nutrient absorption. eNOS expresses in small intestine vascular endothelial cells and creates endothelium-derived relaxing factor NO that can relax blood vessels [6,7]. Thus, the
Table 3
increased small intestinal eNOS in the obese rat group would have increased small intestine blood flow, which is conducive to nutrition component transport. Our previous research found that TNF-␣ serum levels increased in obese rats than controls (p < 0.05) [19]. Elevated TNF-␣ can activate iNOS
Expression of NOS mRNA, protein and NO levels in small intestine.
iNOS RT-PCR (IOD ratio) nNOS RT-PCR (IOD ratio) eNOS RT-PCR (IOD ratio) iNOS IHC (IOD ratio) eNOS IHC (number/section) nNOS IHC (number/section) NO (mol/g protein)
Normal control (n = 18)
Obese control (n = 16)
Octreotide intervention (n = 15)
0.4978 ± 0.1475 0.5729 ± 0.2147 0.5390 ± 0.1580 14.30 ± 3.57 4.50 ± 1.58 11.33 ± 3.88 0.6382 ± 0.2435
0.7728 ± 0.1777 0.6714 ± 0.2564 0.7946 ± 0.1753* 42.26 ± 15.08* 14.13 ± 5.43* 20.56 ± 5.19* 0.9284 ± 0.2690*
0.5791 0.5863 0.5499 18.26 6.8 12.27 0.7584
* p < 0.01 vs. normal control group. : p < 0.01, : p < 0.05 vs. obese control group.
*
± ± ± ± ± ± ±
0.1453 0.2482 0.1277 4.70 3.43 3.99 0.2713
e286 transcription and translation [18], which in turn generates large amounts of NO that relax lamina propria vessels and improve small intestinal blood flow. NO generated by iNOS can have both protective and toxic effects, depending on its quantity [5,20]. We observed in the current experiment that the rats’ small intestine color was ruddy without congestion and edema, so we concluded that the increased NO generated by iNOS had a protective effect and did not cause pathological changes in small intestine vascular permeability. Immunohistochemistry and RT-PCR results showed that the octreotide intervention group’s nNOS, eNOS, iNOS protein expression levels and eNOS, iNOS mRNA expression were significantly lower than those of the obese control rats. In another study, we found that serum levels of TNF-␣ also decreased in octerotide intervention rats than obese control rats (p < 0.05) (a manuscript currently in revision). SST can inhibit TNF-␣ [21], which in turn decreases small intestine iNOS mRNA and protein expression. Many studies have shown that nutritional obesity causes overexpression of NF-B in vivo [22,23]. There are specific NF-B binding sites on the iNOS gene promoter and enhancer regions. At the transcriptional level NF-B regulates iNOS expression, causing rapid synthesis of iNOS mRNA [24]. Davis indicated that shear stress increased eNOS transcription by NF-B activation [25]. Somatostatin inhibits NF-B expression [26], thereby inhibiting activation of iNOS and eNOS transcription and reducing small intestine iNOS and eNOS mRNA and protein expression levels. Florio showed in in vitro experiments that somatostatin directly affects endothelial cell line proliferation by blocking growth factor-stimulated MAPK and eNOS activities [27]. We also found that octreotide inhibited eNOS protein expression in the small intestine. Somatostatin decreases intracellular calcium [28], and since nNOS and eNOS activation are both Ca2+ dependent, somatostatin should inhibit nNOS and eNOS activation and reduce their expression levels in the small intestine. High fat diet-induced obesity is associated with elevated small intestinal nNOS, eNOS, and iNOS protein expression levels. Octreotide can inhibit these effects. Octreotide-inhibited nitric oxide synthase protein expression and reduced NO production thereby reduce small intestine blood vessel dilation and endogenous neural inhibitory neurotransmitter to reduce the intestinal absorption of nutrients and energy intake. Next to tobacco, obesity is now the secondlargest preventable cause of death [29]. Weight loss is a proven, effective way to prevent future trouble. Octreotide has been in wide clinical use for
Y. Ou et al. many years, and has a good safety record. We found that the drug can effectively reduce the weight of high fat diet-induced obese rats, suggesting that it should be investigated further as a potential weight-loss drug.
Disclosure All authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Acknowledgments This work was supported by grants from the National Natural Sciences Foundation of China (No. 30870919) and Sichuan Provincial Department of Science and Technology (No. 2010SZ0176).
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Effects of octreotide on nitric oxide synthase expression in the small intestine of high fat diet-induced obese rats Yan Ou a,b, Rui Liu a,∗, Na Wei a,b, Xian Li a, Ou Qiang a, Wei Huang a,b, Chengwei Tang b a
Division of Peptides Related to Human Disease, National Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China b Department of Gastroenterology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China Received 28 July 2011 ; received in revised form 31 October 2011; accepted 22 November 2011
KEYWORDS Obesity; Neuronal nitric oxide synthase (nNOS); Endothelial nitric oxide synthase (eNOS); Inducible nitric oxide synthase (iNOS); Octreotide
∗
Summary Objective: To investigate whether obesity induced by high fat diet is associated with expression of neuronal, endothelial, and inducible nitric oxide synthase (nNOS, eNOS, and iNOS) in the intestine, and to test the effects of the somatostatin analog octreotide on this expression. Methods: The study included high fat diet-induced obese and normal control rats. The obese rats were further separated into an obese control group and an octreotide intervention group. Rats in the intervention group were injected with 40 g/kg octreotide every 12 h for 8 days. Expressions of nNOS, eNOS, and iNOS in the small intestine were analyzed by RT-PCR and immunohistochemistry. The NO level of small intestinal homogenate was measured with an ELISA kit. Results: The body weight; Lee’s index; small intestinal eNOS and iNOS mRNA and protein expression levels; nNOS protein expression levels; and small intestinal homogenate NO levels were all significantly higher in the obese control group than in the normal controls (p < 0.01); nNOS mRNA expression was also higher in the obese control group, but not significantly so. Octreotide intervention significantly reduced the body weight and small intestinal homogenate NO level of the obese rats relative to the obese control group (p < 0.05). The mRNA and protein expression levels of eNOS and iNOS; the protein expression level of nNOS in the small intestine were also significantly lower in the octreotide intervention group than in the obese control group (p < 0.01), while nNOS mRNA expression was lower but not significantly so.
Corresponding author. Tel.: +86 28 85164011. E-mail address: [email protected] (R. Liu).
1871-403X/$ — see front matter © 2011 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.orcp.2011.11.004
Effects of octreotide on nitric oxide synthase expression
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Conclusion: High fat diet-induced obesity is associated with elevated small intestinal nNOS, eNOS, and iNOS expression levels. Octreotide treatment can inhibit nNOS, eNOS, and iNOS expression and lead to weight loss. © 2011 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
Introduction In recent years, as living standards have risen, lifestyle and diet have undergone great changes. Physical inactivity and diets high in calories and fat are important factors leading to obesity, and the incidence of obesity is rising steeply [1]. Excessive accumulation and abnormal distribution of body fat are closely linked to type 2 diabetes, hypertension, dyslipidemia, and obstructive sleep apnea [2]. Obesity incidence is positively correlated with excessive calorie intake. The small intestine is a major absorption site and gateway of energy intake. Nitric oxide synthase (NOS) plays an important role in digestion and absorption in the small intestine [3,4]. NOS has three isoforms: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) [5]. The enzyme’s product, nitric oxide (NO), is a vascular endothelium relaxing factor [6,7] and acts as a non-adrenergic, non-cholinergic inhibitory neurotransmitter in the small intestine [8]. Somatostatin (SST) is a multifunctional gut peptide synthesized and released by intestinal endocrine cells (D cells). Its effects are mediated by the SST receptor, which is located in the cell membrane. SST regulates the physiological functions of intestinal epithelial cells and immune cells, as well as gastric motor activity [9]. Octreotide is an artificial synthetic analog of SST with a longer half-life and duration of activity. Many studies have shown that somatostatin can affect the intestinal absorption and nutritional functions [10,11]. However, there are no published studies investigating whether the effect of somatostatin on intestinal absorption is associated with NOS. We investigated whether obesity induced by a high fat diet is associated with expression of intestinal nNOS, eNOS, and iNOS. In addition, we tested the effects of octreotide on these expression levels.
Materials and methods Experimental animals and grouping All experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University (Chengdu, China).
We used 66 healthy male 21-day-old Sprague—Dawley (SD) rats that had been weaned for 3 days. All animals were obtained from the Animal Center of Sichuan University. After adaptive feeding for 3 days, the rats were placed into two groups, one group of 18 (the normal control group) fed with standard chow (290 kcal/100 g, in line with the People’s Republic of China National Standard GB 14924—2001), and another of 48 fed with high-fat chow (430 kcal/100 g). Food and water were supplied ad libitum, and the animals were housed in independently ventilated cages on a 12:12-h light:dark schedule and kept at 20—25 ◦ C. They were weighed and measured for body and tail length each week for 24 weeks. After 24 weeks, rats from the high-fat chow group with a mean body weight at least 1.4 times that of the controls were selected as obese rats. Obese rats were then placed into two groups, an obese control group of 16 rats, and an octreotidetreated group of 15 rats. The octreotide-treated group was injected with octreotide (40 g/kg body weight) every 12 h for 8 days. Rats were measured weekly for body weight to calculate Lee’s index [body weight (g)1/3 × 1000/body length (cm)].
Sample preparation At the end of the experiment, after fasting for 12 h, rats were anesthetized intraperitoneally with 2% sodium pentobarbital, and 2 cm of the small intestine was removed from the same position on each rat. The samples were fixed in 4% paraformaldehyde and then embedded in paraffin. Another 2 cm section of small intestine was kept at −140 ◦ C until total RNA extraction and NO level determination.
Reverse transcription PCR analysis Total RNA was extracted from the frozen small intestine using Trizol reagent (Takara BioEngineering Co., Ltd., Kyoto, Japan). First-strand cDNA was synthesized from 2 g of total RNA for each sample using reverse transcription kits (MBI, Fermentas Life Sciences Inc., Vilnius, Lithuania). Reverse transcription reactions were performed at 40 ◦ C for 60 min and 70 ◦ C for 5 min. Primer sequences are in Table 1. The thermal cycling parameters consisted of an initial denaturation
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Y. Ou et al. PCR primer sequences.
Gene
Primer sequences
Product size
Database
iNOS
Sense Anti-sense
5 -ATC CCG AAA CGC TAC ACT T-3 [30] 5 -TCT GGC GAA GAA CAA TCC-3
314 bp
NM 012611.3
nNOS
Sense Anti-sense
5 -GGC ACT GGC ATC GCA CCC TT-3 [31] 5 -CTT TGG CCT GTC CGG TTC CC-3
213 bp
NM 052799.1
eNOS
Sense Anti-sense
5 -TGC ACC CTT CCG GGG ATT CT-3 [31] 5 -GGA TCC CTG GAA AAG GCG GT-3
189 bp
NM 021838.2
GAPDH (1)
Sense Anti-sense
5 -CAT GAC CAC AGT CCA TGC CAT C-3 5 -CAC CCT GTT GCT GTA GCC ATA TTC-3
451 bp
NM 017008.3
GAPDH (2)
Sense Anti-sense
5 -TAT GAC AAC TCC CTC AAG AT-3 [32] 5 -AGA TCC ACA ACG GAT ACA TT-3
318 bp
NM 017008.3
step for 5 min at 94 ◦ C, then 40 s at 94 ◦ C, 40 s at 60 ◦ C for iNOS, 67 ◦ C for eNOS or 64 ◦ C for nNOS, 50 s at 72 ◦ C, totally 35, 37 and 39 cycles, respectively. The final extension step was for 7 min at 72 ◦ C. PCR products were resolved by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. Densitometry was carried out using a Bio-Rad GelDoc image acquisition system and Quantity One (v. 4.3) quantitation software (Bio-Rad, Hercules, CA, USA).
Immunohistochemistry of small intestine nNOS, iNOS, and eNOS For the immunohistochemical detection of nNOS, iNOS, and eNOS protein expression levels, 4 mthick small intestine tissue sections were cut from the formalin-fixed and paraffin-embedded tissues. The sections were deparaffinized, endogenous peroxidase was blocked with 3% H2 O2 , and antigen retrieval was performed at high temperature and pressure. For nonspecific blocking, 10% goat sera was added, each section was incubated for 30 min at 37 ◦ C, and then either nNOS, iNOS, or eNOS rabbit anti-rat polyclonal antibody was added (1:400, 1:400, 1:150; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China). After incubating overnight at 4 ◦ C and rewarming to 37 ◦ C for 40 min, each section was stained with a ready-to-use streptavidin—catalase immunohistochemical reagent system. The color reaction was developed with diaminobenzidine (DAB; Zhongshan Bioagent Company, Beijing, China). A semiquantitative immunohistochemical analysis of raw data was performed with Image-Pro Plus 4.0 software (Media Cybernetics, Silver Spring, MD, USA) to score integrated optical density (IOD) for positive reaction areas.
Immunohistochemistry positive standard Positive expression of iNOS was considered to have occurred if the cytoplasm of the small intestine macrophages in the mucosal and submucosal layers was stained yellow to brown yellow. We took a continuous measurement of IOD values of the three small intestinal villi, then divided by 3 to obtain the IOD average, which was used in statistical analysis. Positive expression of eNOS was indicated if the cytoplasms of vascular endothelial cells were stained yellow to brown in the full thickness of the small intestine. We counted the total number of positive vessels in each section and used this number in our statistical analysis. Positive expression of nNOS was indicated if the cytoplasms of nonadrenergic non-cholinergic nerve cells were stained yellow to brown in the submucosa and muscular layers of the small intestine. The total number of positive nerve cells in each section was used for our statistical analysis.
Determination of NO levels in the small intestine The small intestine samples homogenized in ice cold phosphate buffered saline (PBS, pH7.4). After centrifugation at 1050 × g for 15 min at 4 ◦ C, the supernatant was used to measure the NO levels by ELISA kit (R&D Systems, Minneapolis, MN, USA).
Statistical methods All data were analyzed with statistical software SPSS 16.0 (SPSS Inc., USA). Data are presented as means and standard deviations. Groups were compared with analysis of variance. All tests were two-tailed, and p values less than 0.05 were considered to indicate statistical significance. All data
Effects of octreotide on nitric oxide synthase expression met the assumptions of the tests used to analyze them.
Results Octreotide treatment and parameters associated with obesity From the third week, the body weight growth rate of the high fat-diet group was significantly higher than that of the normal control group. After 24 weeks, the rats with high fat diet-induced obesity showed typical features of obesity, such as higher body weight and Lee’s index, while those in the normal control group did not (p < 0.01) (Table 2). The octreotide-treated group had significantly lower body weight than did the obese control group (p < 0.05) (Table 2).
Octreotide treatment and NOS mRNA expression The mRNA expression levels of eNOS and iNOS in the obese control group were significantly higher than those in the normal control group (p < 0.01); there was an increasing tendency of nNOS mRNA expression in obese control group as compared with normal control group (Fig. 1 and Table 3). The eNOS and iNOS mRNA expression levels were significantly lower in the octreotide-treated group than in the obese control group (p < 0.01); there was a decreasing tendency of nNOS mRNA expression in octreotide intervention group as compared with obese control group (Fig. 1 and Table 3).
Octreotide treatment and NOS protein expression iNOS was expressed in the small intestinal lamina propria macrophages and other immune cells, expressed to a lesser extent in the submucosa, and almost completely absent in the muscular and serosa layers. iNOS-positive cells had yellow to brown staining in the cytoplasm, with only a small amount of nuclear staining. In the obese control group, small intestinal iNOS expression levels were significantly higher than those of the normal control group (p < 0.01) (Table 3 and Fig. 2). These expression levels were also significantly lower in the octreotide intervention group than in the obese control group (p < 0.01) (Table 3 and Fig. 2). eNOS mainly appeared in the small intestinal submucosal and muscular layer vascular endothelial cells, and was expressed in the lamina propria
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microvasculature. The cytoplasms of eNOS-positive cells were stained yellow to brown, with no expression in the nuclei. In the obese control group, the small intestine eNOS expression levels were significantly higher than those of the normal control group (p < 0.01) (Table 3 and Fig. 3). eNOS expression levels were significantly lower in the octreotide intervention group than in the obese control group (p < 0.01) (Table 3 and Fig. 3). nNOS was found in the small intestinal nonadrenergic, non-cholinergic nerve plexus and fibers. It was highly expressed in the myenteric plexus and weakly expressed in the submucosal plexus. The cytoplasms of nNOS-positive cells were stained yellow to brown. The small intestine nNOS expression levels of the obese control group were significantly higher than those of the normal control group (p < 0.01) (Table 3 and Fig. 4). Those levels were significantly lower in the octreotide intervention group than in the obese control group (p < 0.01) (Table 3 and Fig. 4).
NO levels in the small intestine The NO level in small intestine of obese control group was significantly higher than that in normal control group (p < 0.01). The NO level in octreotide intervention group was significantly lower than that in obese control group (p < 0.05) (Table 3).
Discussion The constitutive nitric oxide synthases nNOS and eNOS express under normal physiological conditions, while iNOS expresses in the pathological state [12]. nNOS is distributed in the small intestine nerve plexus and fibers [13], and is expressed strongly in the myenteric plexus and weakly in the submucosal plexus [14]. eNOS is expressed in vascular endothelial cells and lamina propria histiocytes [15,16]. iNOS is expressed in small intestinal lamina propria macrophages and other immune cells [17]. iNOS transcription and translation are activated by cytokines such as tumor necrosis factor-␣ (TNF-␣) [18]. We found that nNOS, eNOS, iNOS protein expression levels and eNOS, iNOS mRNA expression rose in the small intestines of high fat diet-induced obese rats. The resulting increased production of NO may have improved the absorption of nutrients and energy in the rats’ small intestines. nNOS can produce NO that acts as an enteric nervous system non-adrenergic, non-cholinergic inhibitory neurotransmitter [8]. This suggests that
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Table 2
Body weight and Lee’s index in each group.
Body weight (g) Lee’s Index *
Normal control (n = 18)
Obese control (n = 16)
Octreotide intervention (n = 15)
378.54 ± 111.75 318.73 ± 20.10
605.61 ± 141.00* 337.61 ± 10.82*
508.27 ± 94.39 334.67 ± 16.56
p < 0.01 vs. normal control group. p < 0.05 vs. obese control group.
Figure 1 Expression of iNOS, eNOS and nNOS mRNA in the small intestine. 1 and 2, normal control group; 3 and 4, obese control group; 5 and 6, octreotide intervention group. RT-PCR showed that iNOS and eNOS mRNA expression was higher in obese control group than normal control and octerotide-intervention group (p < 0.01). nNOS mRNA expression tended to be higher in the obese control group than in the normal controls and lower in the octreotide intervention group than in the obese control group. *p < 0.01 vs. normal control group; :, p < 0.01 vs. obese control group.
Figure 2 iNOS expression in small intestine mucosa. The expression of iNOS in small intestine mucosa was detected by immunohistochemistry. The images show the SD rat intestinal villi. iNOS was mainly expressed in the small intestine lamina propria which was stained yellow to brown. iNOS expression levels in obese control group were significantly higher than normal control and octreotide intervention group. Magnification: 400×.
Effects of octreotide on nitric oxide synthase expression
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Figure 3 eNOS expression in small intestine submucosal layer. The expression of eNOS in small intestine submucosal layer was detected by immunohistochemistry. eNOS mainly appeared in the small intestine submucosal layer vascular endothelial cells which were stained yellow. eNOS expression levels in obese control group were significantly higher than normal control and octreotide intervention group. Magnification: 400×.
Figure 4 nNOS expression in small intestine muscular layer. The expression of nNOS in small intestine muscular layer was detected by immunohistochemistry. nNOS was mainly found in the small intestine myenteric plexus which were stained yellow. nNOS expression levels in obese control group were significantly higher than normal control and octreotide intervention group. Magnification: 400×.
nNOS-produced NO inhibits intestine motility, increases the time that chyme stays in the small intestine, and enhances nutrient absorption. eNOS expresses in small intestine vascular endothelial cells and creates endothelium-derived relaxing factor NO that can relax blood vessels [6,7]. Thus, the
Table 3
increased small intestinal eNOS in the obese rat group would have increased small intestine blood flow, which is conducive to nutrition component transport. Our previous research found that TNF-␣ serum levels increased in obese rats than controls (p < 0.05) [19]. Elevated TNF-␣ can activate iNOS
Expression of NOS mRNA, protein and NO levels in small intestine.
iNOS RT-PCR (IOD ratio) nNOS RT-PCR (IOD ratio) eNOS RT-PCR (IOD ratio) iNOS IHC (IOD ratio) eNOS IHC (number/section) nNOS IHC (number/section) NO (mol/g protein)
Normal control (n = 18)
Obese control (n = 16)
Octreotide intervention (n = 15)
0.4978 ± 0.1475 0.5729 ± 0.2147 0.5390 ± 0.1580 14.30 ± 3.57 4.50 ± 1.58 11.33 ± 3.88 0.6382 ± 0.2435
0.7728 ± 0.1777 0.6714 ± 0.2564 0.7946 ± 0.1753* 42.26 ± 15.08* 14.13 ± 5.43* 20.56 ± 5.19* 0.9284 ± 0.2690*
0.5791 0.5863 0.5499 18.26 6.8 12.27 0.7584
* p < 0.01 vs. normal control group. : p < 0.01, : p < 0.05 vs. obese control group.
*
± ± ± ± ± ± ±
0.1453 0.2482 0.1277 4.70 3.43 3.99 0.2713
e286 transcription and translation [18], which in turn generates large amounts of NO that relax lamina propria vessels and improve small intestinal blood flow. NO generated by iNOS can have both protective and toxic effects, depending on its quantity [5,20]. We observed in the current experiment that the rats’ small intestine color was ruddy without congestion and edema, so we concluded that the increased NO generated by iNOS had a protective effect and did not cause pathological changes in small intestine vascular permeability. Immunohistochemistry and RT-PCR results showed that the octreotide intervention group’s nNOS, eNOS, iNOS protein expression levels and eNOS, iNOS mRNA expression were significantly lower than those of the obese control rats. In another study, we found that serum levels of TNF-␣ also decreased in octerotide intervention rats than obese control rats (p < 0.05) (a manuscript currently in revision). SST can inhibit TNF-␣ [21], which in turn decreases small intestine iNOS mRNA and protein expression. Many studies have shown that nutritional obesity causes overexpression of NF-B in vivo [22,23]. There are specific NF-B binding sites on the iNOS gene promoter and enhancer regions. At the transcriptional level NF-B regulates iNOS expression, causing rapid synthesis of iNOS mRNA [24]. Davis indicated that shear stress increased eNOS transcription by NF-B activation [25]. Somatostatin inhibits NF-B expression [26], thereby inhibiting activation of iNOS and eNOS transcription and reducing small intestine iNOS and eNOS mRNA and protein expression levels. Florio showed in in vitro experiments that somatostatin directly affects endothelial cell line proliferation by blocking growth factor-stimulated MAPK and eNOS activities [27]. We also found that octreotide inhibited eNOS protein expression in the small intestine. Somatostatin decreases intracellular calcium [28], and since nNOS and eNOS activation are both Ca2+ dependent, somatostatin should inhibit nNOS and eNOS activation and reduce their expression levels in the small intestine. High fat diet-induced obesity is associated with elevated small intestinal nNOS, eNOS, and iNOS protein expression levels. Octreotide can inhibit these effects. Octreotide-inhibited nitric oxide synthase protein expression and reduced NO production thereby reduce small intestine blood vessel dilation and endogenous neural inhibitory neurotransmitter to reduce the intestinal absorption of nutrients and energy intake. Next to tobacco, obesity is now the secondlargest preventable cause of death [29]. Weight loss is a proven, effective way to prevent future trouble. Octreotide has been in wide clinical use for
Y. Ou et al. many years, and has a good safety record. We found that the drug can effectively reduce the weight of high fat diet-induced obese rats, suggesting that it should be investigated further as a potential weight-loss drug.
Disclosure All authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Acknowledgments This work was supported by grants from the National Natural Sciences Foundation of China (No. 30870919) and Sichuan Provincial Department of Science and Technology (No. 2010SZ0176).
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