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Published-Ahead-of-Print February 21, 2007, DOI:10.2164/jandrol.106.001602
Journal of Andrology, Vol. 28, No. 4, July/August 2007
Copyright © American Society of Andrology
DOI: 10.2164/jandrol.106.001602

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Metformin Restores the Penile Expression of Nitric Oxide Synthase in High-Fat–Fed Obese Rats

YONG-WOON KIM*, SO-YOUNG PARK*, JONG-YEON KIM*, JUNG-YOON HUH*, WOO-SUNG JEON{dagger}, CHANG-JUN YOON{dagger}, SUNG-SU YUN{ddagger} AND KI-HAK MOON{dagger}

From the * Department of Physiology, {dagger} Department of Urology, and {ddagger} Department of Surgery, College of Medicine, Yeungnam University, Daegu, Republic of Korea.

Correspondence to: Dr Ki-Hak Moon, Department of Urology, College of Medicine, Yeungnam University, 317-1 Daemyung-Dong, Nam-Gu, Daegu, 705-035, Republic of Korea (e-mail: khmoon{at}med.yu.ac.kr).
Received for publication September 4, 2006; accepted for publication February 19, 2007.

   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Obesity is a well-known risk factor for erectile dysfunction, which is associated with reduced penile nitric oxide synthase (NOS) expression. Recently it was reported that metformin activates AMP-activated protein kinase (AMPK), which increases the expression of neuronal (n) NOS and endothelial (e) NOS. Thus, to evaluate whether metformin restores NOS expression in penile tissue, we measured penile expression of nNOS and eNOS after 4 weeks of metformin treatment (300 mg/kg/d) in 5-month-old high-fat–fed obese (HFO) rats. HFO rats have increased fat accumulation in visceral areas and marked suppression of nNOS and eNOS expression in penile tissue. However, metformin treatment decreased visceral fat deposition and restored nNOS and eNOS expression in penile tissue. The levels of AMPK and phosphorylated AMPK were also decreased in HFO rats but were subsequently elevated by metformin treatment. These results suggest that expression of NOS was suppressed by the high-fat diet but restored by metformin treatment. The effect of metformin on the expression of NOS may be associated with its activation of AMPK.

     Key words: AMP-activated protein kinase, penile tissue


Arecent 14-year prospective study concluded that obesity is a risk factor for erectile dysfunction (Bacon et al, 2006). Moreover, erectile function scores in obese men improved with weight loss and increased physical activity (Esposito et al, 2004). In another study, rats fed a high-energy diet for 12 weeks reportedly had decreased erectile function (Yu et al, 2006). One of the possible mechanisms for erectile dysfunction in obesity is endothelial dysfunction associated with suppression of nitric oxide synthase (NOS) expression (Giugliano et al, 2004; Fonseca and Jawa, 2005; Roberts et al, 2005).

NO, synthesized from its precursor L-arginine via NOS, was originally described as a vasodilator. In addition, it is widely accepted that NO is important in the relaxation of corpus cavernosum smooth muscle and vasculature. NO generated by neuronal NOS (nNOS) is considered the main factor responsible for the immediate relaxation of corpus cavernosum, while NO from endothelial NOS (eNOS) is essential for maintaining relaxation (Andersson, 2003). Conditions associated with reduction of nNOS and eNOS levels can cause circulatory and structural changes in penile tissues, resulting in erectile dysfunction. Moreover, NOS levels are decreased in various tissues including kidney, aorta, heart, and hypothalamus in obese animal models (Morley and Mattammal, 1996; Li et al, 2005; Roberts et al, 2005).

Metformin is an oral biguanide insulin-sensitizing agent that inhibits hepatic glucose production, enhances the effects of insulin on glucose uptake in skeletal muscles and adipocytes, and decreases intestinal absorption of glucose (Hundal et al, 1992; Nestler et al, 1998; Wiernsperger and Bailey, 1999). Another phenomenon commonly associated with metformin treatment is a subsequent decrease in body weight (Paolisso et al, 1998; Kay et al, 2001; Kim et al, 2006). Recent reports have indicated that metformin targets AMP-activated protein kinase (AMPK) (Zhou et al, 2001; Hawley et al, 2002), which activates nNOS and eNOS (Fryer et al, 2000). Moreover, metformin increases NOS in obese mice and cultured bovine aortic endothelial cells (Kumar et al, 2001; Davis et al, 2006). However, the effect of metformin on penile NOS expression and/or erectile function has not been evaluated to date.

To test the effects of metformin on the expression of nNOS and eNOS in penile tissue in an obese animal model, we treated obese rats fed a high-fat diet with metformin for 4 weeks and measured the subsequent penile expression of nNOS and eNOS, as well as levels of phosphorylated AMPK (pAMPK).


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animal Care

One-month-old male Sprague-Dawley rats were purchased from Daehan Experimental Animal Center (Seoul, Korea) after weaning. After 1 week of adaptation, the rats were randomly divided into 2 groups: the standard chow (SC) and high-fat diet (HFO) groups. For 16 weeks the SC rats were fed a standard chow diet, while the HFO rats were fed a high-fat diet (60% of calories from fat). The high-fat diet consisted of butter, corn oil, sucrose, and casein (38%, 21%, 23%, and 17%, respectively, of total calories), supplemented with vitamins (0.8%), minerals (1.9%), and methionine (0.15%). The animals were cared for in accordance with the principles outlined in the Guide to the Care and Use of Experimental Animals of the Yeungnam Medical Center. The rats were housed individually with a 12:12-hour light:dark cycle (0700 to 1900 hours).

Experimental Design

To evaluate the effects of metformin on penile NOS expression, SC and HFO rats were further divided into both metformin-treated and untreated control groups. Metformin (300 mg/kg/d) was dissolved in the drinking water and administered orally for 4 weeks. We determined metformin dosage according to Sartoretto et al (2005), which indicates that metformin restores altered microvascular reactivity in diabetic rats. Metformin concentrations in the water were readjusted twice a week after measuring the daily water intake. The control group received drinking water without metformin ad libitum.

Tissue Harvesting and Preparation

The rats were anesthetized with 85 mg/kg pentobarbital (intraperitoneally). Blood samples were collected through the heart puncture, and serum was harvested by a 10-minute centrifugation in serum separator tubes. The circulatory system was perfused with 50 mL of cold saline, and the visceral fat (inguinal, perirenal, and retroperitoneal white adipose tissues), soleus muscle, and penis were excised and weighed. The tissues and serum were quick-frozen with liquid nitrogen and stored at –70°C.

Reverse Transcriptase Polymerase Chain Reaction

Total RNA was extracted from the penile tissue using a modification of the method of Chomczynski and Sacchi (1987). The RNA concentration was determined using spectrophotometry (OD260). The reverse transcriptase (RT) reaction was performed using a QIAGEN one-step RT-polymerase chain reaction (PCR) kit (Hilden, Germany) as previously described (Kim et al, 2005). One microgram of total RNA was reverse transcribed into cDNA using Omniscript RT, Sensiscript RT, and primers. The sense primer sequence for eNOS was 5'-TGCACCCTTCCGGGGATTCT-3' and the antisense was 5'-GGATCCCTGGAAAAGGCGGT-3'. The sense primer sequence for nNOS was 5'-GGCAC TGGCATCGCACCCTT-3' and the antisense was 5'-CTTTGGCCTGTCCGGTTCCC-3'. Amplification was initiated at 50°C for 30 minutes, followed by 30 cycles consisting of denaturation at 94°C for 1 minute, annealing at the appropriate primer-pair annealing temperature for 1 minute, and extension at 72°C for 1 minute, and then a final extension step of 10 minutes at 72°C. ß-actin (sense: 5'-TCTACAAT GAGCTGCGTGTG-3' and antisense: 5'-GGTCAGGATC TTCATGAGGT-3') was used as an internal control and standard. The RT-PCR products were electrophoresed on a 1.5% agarose gel and visualized by staining with ethidium bromide.

Western Blot Analysis

The proteins (30 µg) from the lysates of the middle part of the penis and soleus muscle were separated on 10% polyacrylamide gels and then transferred onto nitrocellulose membranes. pAMPK and total AMPK were determined by Western blotting with the corresponding specific rabbit polyclonal antibodies (Cell Signaling Technology Inc, Boston, Mass) (1:1000 dilution). Glyceraldehyde-3-phosphate dehydrogenase served as an internal standard. In each case, an antirabbit IgG antibody linked to horseradish peroxidase was used as the secondary antibody. Blots were developed by enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, United Kingdom); quantification was performed with Scion Image (Scion Corp, Frederick, Md).

Measurement of Serum Biochemicals

Serum biochemicals (glucose, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides) were measured by an automatic analyzer (AU5400; Olympus, Tokyo, Japan). Serum insulin concentrations were measured with a rat insulin enzyme immunoassay kit (SPI-BIO, Montigny le Bretonneux, France).

Statistical Analysis

Individual groups were compared by Student's t test, and the data were analyzed. A value of P < .05 was considered significant.


   Results
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 Results
Discussion
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Body Weight and Visceral Fat Mass

After 4 months, HFO rats exhibited increased body weight and visceral fat mass compared with the control SC rats. Metformin treatment decreased body weight and visceral fat mass of both SC and HFO rats, although these effects were more prominent in HFO rats (Table 1).


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  		Table 1. Body weight and total visceral fat mass in the experimental groups*
 

Effect of Metformin on Penile Expression of NOS

Penile expression of nNOS was markedly suppressed in HFO rats compared with SC rats. However, metformin restored nNOS expression to 65% of that in SC rats. The penile expression of eNOS was also noticeably reduced in HFO rats and recovered to 87% of that in SC rats by metformin treatment (Figure 1).


Figure 1
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  		Figure 1. The mRNA expressions of neuronal and endothelial nitric oxide synthase in the penile tissue of metformin- or vehicle-treated standard chow (SC) and high-fat–fed obese (HFO) rats. Representative blots are shown in each panel. Data are presented as the mean ± SE. *P < .05 vs vehicle-treated SC rats, {dagger}P < .05 vs vehicle-treated HFO rats.

 
Effect of Metformin on AMPK and pAMPK Levels in Penile Tissue and Soleus Muscle

pAMPK levels were decreased in the penile tissue of HFO rats; however, metformin treatment increased pAMPK levels in both SC and HFO rats. AMPK levels were also elevated in both metformin-treated SC and HFO rats (Figure 2A). We measured pAMPK and AMPK levels in soleus muscle as a reference. There was also a decrease in pAMPK levels in the soleus muscle of HFO rats compared with that of SC rats; however, it was recovered by metformin treatment. The AMPK level in soleus muscle revealed a decreasing tendency in HFO rats, and it was elevated by metformin treatment (Figure 2B).


Figure 2
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  		Figure. 2. The levels of AMP-activated protein kinase (AMPK) and phosphorylated AMPK in the penile tissue (A) and soleus muscle (B) of metformin- or vehicle-treated standard chow (SC) and high-fat–fed obese (HFO) rats. Representative blots are shown in each panel. Data are presented as the mean ± SE. *P < .05 vs vehicle-treated SC rats, {dagger}P < .05 vs vehicle-treated HFO rats.

 
Effect of Metformin on Blood Biochemicals

Serum glucose, as measured at the fed state, was not elevated in rats fed the high-fat diet and did not change significantly with metformin treatment in either SC or HFO rats. On the other hand, total cholesterol and LDL cholesterol concentrations were significantly increased by the high-fat diet; these were not affected significantly by metformin treatment. Serum triglyceride and HDL cholesterol concentrations did not differ among the experimental groups. Serum insulin levels were increased in the HFO rats; however, metformin treatment decreased the serum insulin concentrations (Table 2).


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  		Table 2. Serum biochemicals in the experimental groups*
 


   Discussion
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 Results
 Discussion
References
 
In the present study, we were interested in restoring the penile expression of NOS in an obese animal model fed a high-fat diet, primarily because the suppressed NOS levels in these experimental rats might be associated with erectile dysfunction. Metformin was chosen as the inducer of NOS expression for the following reasons: 1) metformin activates the AMPK system and elevates NOS, 2) metformin is widely prescribed for type II diabetic patients in the clinical setting, and 3) metformin reduces body weight.

The present study demonstrated that penile expression of NOS mRNA is markedly suppressed in HFO rats compared with SC rats and that expression is subsequently elevated by metformin treatment.

Although the comprehensive molecular basis for metformin's mechanism of action has not been established yet, it has been recently reported that metformin targets AMPK (Zhou et al, 2001; Hawley et al, 2002). AMPK is an energy-sensing enzyme that is activated by an increase in the AMP:ATP ratio (Hardie and Carling, 1997). As a result, AMPK is activated by any stress that depletes cellular ATP, such as metabolic stress, oxidative stress, hypoxia, or nutrient deprivation (Hardie and Carling, 1997). Medical interest in the AMPK system is higher due to the fact that AMPK is activated by biguanides and thiazolidinediones, drugs currently used in the treatment of type II diabetes. In this way, changes in AMPK affect a variety of metabolic processes, including eNOS and nNOS expression (Fryer et al, 2000; Fryer et al, 2002). Moreover, it has also been reported that AMPK phosphorylation is reduced in the skeletal muscle of obese rats (Liu et al, 2006; Sriwijitkamol et al, 2006), and activation of AMPK by {alpha}-lipoic acid prevents endothelial dysfunction in obese rats (Lee et al, 2005). In the current study, we found that pAMPK levels were decreased in the penile tissue and soleus muscle of HFO rats and that AMPK levels in penile tissue of HFO rats were also decreased; these levels increased with metformin treatment. Similar results were obtained by Liu et al (2006), who found that the content of AMPK{alpha} and pAMPK{alpha} were decreased in the gastrocnemius muscle of rats fed a high-fat diet and that pAMPK{alpha} was restored by metformin treatment. Thus, we can easily speculate that there is a defect in the AMPK system in obesity; this defect may be associated with obesity-related metabolic complications, and activation of the AMPK system recovers these complications.

Although there is little information about the levels of pAMPK and its relationship to NOS expression in penile tissue available in the literature, our current results suggest that metformin may elevate NOS mRNA expression in the penile tissue through the activation of AMPK in HFO rats. Activation of the NOS system is a major process in the induction and maintenance of penile erection, and defects in NOS are associated with erectile dysfunction in aged rats (Musicki et al, 2005). Even though the relationship between obesity and defects in penile NOS expression has not been evaluated yet, the combination of our finding that the mRNA expressions of nNOS and eNOS in the penile tissue of HFO rats are decreased and the finding of Yu et al (2006) that erectile dysfunction is induced following 12 weeks of a high-energy diet in rats suggest that defects in the NOS system may be associated with obesity-related erectile dysfunction. In addition, Morrow et al (2003) demonstrated that AMPK activates eNOS, resulting in increased NO production in aortic endothelial cells. Moreover, Davis et al (2006) found that metformin improves endothelial functions by increasing AMPK-dependent eNOS activation. Thus, we speculate that restoration of NOS expression via metformin activation of AMPK could be a potential treatment strategy for obesity-associated erectile dysfunction. However, in the present study, we only measured the mRNA levels of NOS isoforms, and evaluation of phosphorylated NOS isoforms, activated forms of NOS, is needed to confirm the exact action of metformin on penile tissue.

In the present study, serum glucose levels were not elevated in the fed state of HFO rats as a result of a compensatory elevation in the concentration of serum insulin. Total cholesterol levels were elevated by the high-fat diet but did not decrease significantly following metformin treatment. Thus, the changes in NOS expression induced by metformin in HFO rats seemed to result from the effects of metformin on the AMPK and NOS systems but not from improvement in serum biochemical levels.

In summary, the mRNA expression of eNOS and nNOS in penile tissue was suppressed by a HF diet but was restored by metformin treatment. The penile levels of AMPK and pAMPK were also decreased in HFO rats and elevated by metformin treatment. These results suggest that metformin's effects on NOS expression may be associated with activation of AMPK, and metformin may be a new strategy for the treatment of obesity-associated erectile dysfunction.


   Footnotes
 
Supported by Research Grant 105098 from the Advanced Research Center, Yeungnam University and Korea Science and Engineering Foundation (R01-2005-000-10747-0).


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