archives of oral biology 55 (2010) 803808

available at www.sciencedirect.com

journal homepage: http://www.elsevier.com/locate/aob

Changes in the lingual muscles of obese rats induced by high-fat diet feeding
Takashi Saito a,*, Akira Yamane b, Syuhei Kaneko c, Takumi Ogawa d, Tomoko Ikawa d, Kaori Saito e, Masashi Sugisaki a
a

Department of Dentistry, Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo 105-8461, Japan Department of Biophysics, Tsurumi University School of Dental Medicine, Yokohama 230-8501, Japan c Department of Geriatric Dentistry, Tsurumi University School of Dental Medicine, Yokohama 230-8501, Japan d Department of Fixed Prosthetic Dentistry, Tsurumi University School of Dental Medicine, Yokohama 230-8501, Japan e Department of Orthodontics, Tsurumi University School of Dental Medicine, Yokohama 230-8501, Japan
b

article info
Article history: Accepted 14 July 2010 Keywords: Obesity Muscle Genioglossus Geniohyoid Triglyceride

abstract
Objective: To elucidate the inuences of obesity on the properties and volume of lingual (genioglossus and geniohyoid) muscles in obese rats. Methods: We analysed the accumulation of triacylglycerol and the diameter of myobres in the lingual muscles using histochemistry, and the MyHC composition using real-time PCR in rats fed a high-fat diet for 10 weeks. In the genioglossus and geniohyoid muscles, the percentage of oil droplet areas in the obesity group were 3.6 and 2.5 times greater than those in the control group, respectively ( p < 0.025). The diameters of the slow myobres in the genioglossus and geniohyoid muscles were approximately 20% greater in the obesity group than in the control group ( p < 0.0001), while that of the fast myobres in the geniohyoid muscle was approximately 10% greater in the obesity group than in the control group ( p < 0.0001). No signicant difference in the expressions of any of the MyHC isoforms studied was found in any of the muscles studied between the obesity and control groups. Conclusion: High-fat diet feeding induced the fat deposition in the myobre and inuenced the structure of the lingual (genioglossus and geniohyoid) muscles. # 2010 Elsevier Ltd. All rights reserved.

1.

Introduction

Obesity has been shown to have a signicant positive correlation with obstructive sleep apnoea syndrome (OSAS), a breathing disorder in sleep caused by repetitive upper airway obstruction.13 It is well known that the maintenance of upper airway patency depends on an equilibrium between the occluding and dilating forces of the lingual muscles such as the genioglossus and geniohyoid muscles.4 However, the role of the lingual muscles in OSAS induced by obesity is not clear. Myosin heavy chain (MyHC), a major contractile protein in skeletal muscles, is encoded by a family of genes

consisting of at least six isoforms, including the embryonic, neonatal, I, IIa, IIb, and IId isoforms.5,6 These isoforms are often used as indicators for properties of skeletal muscles. Obese humans possess fewer type I muscle bres and more type IIb muscle bres compared with lean humans in limb skeletal muscle.7,8 Genetically obese ob/ob mice show a marked reduction in muscle mass and a reduced ability to undergo hypertrophy.9,10 Abnormalities in the structure and function of the genioglossus muscle have been reported in OSAS patients with obesity, such that the muscle showed a higher proportion of type IIb bres compared with healthy controls.11

* Corresponding author. Tel.: +81 3 3433 1111x3641; fax: +81 3 3431 5449. E-mail address: takashi_s@jikei.ac.jp (T. Saito). 00039969/$ see front matter # 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2010.07.004

804

archives of oral biology 55 (2010) 803808

In this study, to investigate the possible link between fat deposition in muscle tissues and the properties and structures of lingual muscles, we analysed the accumulation of triglyceride, the diameter of the myobres, and the MyHC composition in the lingual muscles in the rats fed a high-fat diet.

BSW software, (OLYMPUS, Tokyo Japan) to measure the diameters of the slow and fast myobres chosen on a muscle section from each rat. Myobres, the cross-sectional shape of which was as oblong as possible, were selected and the minimal distance between the parallel sides of each oblong section was measured.

2.
2.1.

Materials and methods

Animals and muscle samples

2.4. Quantitative analysis of expression level of myosin heavy chain (MyHC) mRNAs
Total RNA extraction, treatment with deoxyribonuclease I, and reverse transcription were performed as previously described.12 SYBR Green real-time PCR was performed using an MX3000P instrument (Stratagene Japan, Tokyo, Japan) with SYBR1GreenERTM qPCR SuperMix (Invitrogen Japan, Tokyo, Japan). The thermal prole included the following cycle parameters: denaturation at 95 8C for 10 min, followed by 40 cycles of 95 8C for 15 s for denaturation, and 55 8C for 15 s for annealing and extension. The quantities of MyHC mRNAs were normalised by the quantity of S16 mRNA (i.e., target mRNA/S16 mRNA). The sequences of primers for MyHC I, IIa, IIb, and IId were described previously.13

Twelve male Wistar rats at 8 weeks of age were divided into two groups: obese and controls. The obesity group was fed a high-fat diet (protein:fat:carbohydrate = 20:57:23, 508 kcal/ 100 g) (High Fat Diet32, Nihon Clea, Tokyo, Japan), and the control group was fed a regular diet (protein:fat:carbohydrate = 29:13:58, 343 kcal/100 g) (CE-2, Nihon Clea, Tokyo, Japan). All animals were weighed at 10, 30, and 50 days after initiation of the high-fat diet. At 70 days after initiation of the high-fat diet, all animals were weighed and killed by exsanguination under anaesthesia with pentobarbital sodium (Nembutal, Abbott Laboratories, Chicago, IL, USA) at a dose of 50 mg/kg body weight. Both the left and right sides of the genioglossus, geniohyoid, and masseter muscles were removed. The right-side muscles were rapidly frozen in liquid nitrogen and stored at 85 8C until subsequent real-time PCR analysis; the left-side muscles were frozen in liquid nitrogen and stored at 80 8C until histological analysis.

2.5.

Statistical analysis

2.2.

Quantitative analysis of oil droplet area

The middle portion of each muscle was prepared at a 10 mm thickness with a cryostat (CM1900, Leica Microsystems, Nussloch, Germany) and conventional oil red O staining was performed. All sections were examined using a microscope (BX50, OLYMPUS, Tokyo, Japan) coupled to a colour CCD camera (DP72, OLYMPUS, Tokyo, Japan). Digitally captured images were processed and analysed using image J software (NIH image) to measure the area of oil droplet accumulations in three rectangular regions of 577,000 mm2 chosen randomly on a section from each rat. The area of the three rectangular regions was 41.4 35.3% of the cross-sectional area of the myobre. The percentage of the oil droplet area relative to the area of the rectangular region was calculated. The percentages from the three rectangular regions were averaged to obtain the mean value in each muscle for 45 rats.

The General Linear Model (GLM) Repeated Measures was used to analyse the interaction between body weights of the obesity and control groups as well as effects of time course. A Mann Whitney U test and Bonfferoni correction for post hoc analysis was used to compare the median values of the oil droplet area, myobre diameter, and levels of MHC mRNA expression in each muscle, between the obesity and control groups.

3.
3.1.

Results
Weight of animal

2.3.

Quantitative analysis of myobre diameter

The body weights in the obesity group gradually increased until 50 days after initiation of the high-fat diet feeding and, thereafter, did not change (Fig. 1). However, the body weights in the control group showed no signicant change during the experimental period. The body weights of rats in the obesity group were 1753% heavier than those of rats in the control group throughout the experimental period. GLM repeated measures investigated the existence of the interaction between body weight of obesity and control groups (F: 49.617, signicant level: 0.0001).

Staining for nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) was performed to visualise the slow (type IIa and I) and fast (IIb and IId) myobres. The cryosections were incubated at 37 8C for 30 min in staining solution containing 0.05 M TrisHCl (pH 7.4), 1 mg/ml nitroblue tetrazolium (NBT), and 0.8 mg/ml nicotinamide adenine dinucleotide (NADH). They were immersed in a graduated series of acetone (30, 60, and 90%) and observed under a light microscope. Sections were examined using the above-described microscope with the coupled camera. Digitally captured images were processed and analysed using DP2-

3.2.

Oil droplet area

Fig. 2 shows representative staining images of oil droplet with oil red O in the genioglossus (A, B), geniohyoid (D, E), and masseter (G, H) muscles of rats in the obesity and control groups. Oil droplets were observed in the three muscles studied, with the genioglossus muscles showing more droplets in both of the groups. In the genioglossus and geniohyoid muscles, the percentage of oil droplet areas in the obesity group were 3.6 and 2.5 times greater than those in the control group, respectively ( p < 0.025) (Fig. 2C, F), but in the

archives of oral biology 55 (2010) 803808

805

masseter muscle, no signicant difference in this measure was found between the two groups (Fig. 2I).

3.3.

Myobre diameter

Fig. 1 Changes in the body weight of rats in the obesity (solid box) and control (open box) groups during the feeding period of a high-fat diet. In these plots, the bar in the box represents the median value, the box represents the interquartile range and the whisker represents the full range of data. The numbers of rats in the obesity and control groups were six each.

To study the inuence of the accumulation of oil droplets on the size of myobres, we stained slow and fast myobres with NADH-TR (Fig. 3AD) and measured the diameter of the myobres (Fig. 3EL). We considered that the more-densely stained myobres were slow and that the less-densely stained myobres were fast. Since few slow and fast myobres were observed in the masseter and genioglossus muscles, respectively, we measured the diameter of only the fast myobres in the masseter muscle and only the slow myobres in the genioglossus muscles. The diameters of the slow myobres in the genioglossus and geniohyoid muscles were approximately 20% greater in the obesity group than the control group ( p < 0.0001), while those of the fast myobres in the geniohyoid and masseter muscles were approximately 10% greater in the obesity group than in the control group ( p < 0.0001).

3.4.

The expression of MyHC mRNAs

To study the inuence of the accumulation of oil droplets on muscle properties, we analysed the expression of MyHC I, IIa, IIb, and IId mRNAs, using real-time PCR (Fig. 4). No signicant difference in the expressions of any of the isoforms studied was found in any of the muscles studied between the obesity

Fig. 2 Oil red staining images of genioglossus (A, B), geniohyoid (D, E), and masseter (G, H) muscles in the obesity (A, D, G) and control (B, E, H) groups. The black dots indicate the stained lipids. The same magnification as A was used in the BH. Box and whisker plots of percentage of oil droplet area relative to the observed area of myofibres (C, F, I) of four to five rats. Significant difference between the obesity and control groups, *p < 0.025. GG, GH, and Mas indicate genioglossus, geniohyoid, and masseter muscles, respectively.

806

archives of oral biology 55 (2010) 803808

Fig. 3 Staining images (AD) of genioglossus, geniohyoid, and masseter muscles with nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) in the obesity (A, C) and control (B, D) groups. The same magnification as A was used in the BD. Frequency distribution of fibre diameters (EL) of genioglossus (E, F), geniohyoid (GJ), and masseter (K, L) muscles in the obesity (E, G, I, K) and control (F, H, J, L) groups. Median (the number of myofibres measured) and p value between the obesity and control groups were shown in the EL. GG, GH, and Mas indicate genioglossus, geniohyoid, and masseter muscles, respectively.

archives of oral biology 55 (2010) 803808

807

Increased oil droplet accumulation was observed in the genioglossus and geniohyoid muscles of the obese rat, but not in the masseter muscle, indicating that the effects of high-fat diet feeding on the oil droplet accumulation into the lingual muscles were greater than that in the masseter muscle. Brennick reported that fat deposition was marked in the tongue and soft palate but not in the masseter in the New Zealand obese mouse,2 which is consistent with the present results. Oil droplets have been found to selectively accumulate in the slow-type bres of limb skeletal muscles.1417 In the present study, the percentage of slow bres to total bres in the lingual muscles, such as the genioglossus and geniohyoid (about 50% slow bres), was much higher than that in the masseter muscle. The difference in the oil droplet accumulation between the lingual and masseter muscles is likely due to differences in the composition of the bre types between these muscles. In the present study, we observed no bre-type transition of the lingual or masseter muscles in the obese rat. Thus, neither oil droplet accumulation nor a concomitant alteration in fatty acid metabolism in the skeletal muscle appears to inuence the bre-type composition. It is reported that the transition of the bre type occurs in the lingual muscle of OSAS patient due to the hypoxic condition induced by upper airway obstruction.11,18 Thus it seems that the obese rats in the present study do not reach to sufcient hypoxic condition to induce the transition of the bre type. In conclusion, high-fat diet feeding induced the fat deposition in the myobre and inuenced the structure of the lingual (genioglossus and geniohyoid) muscles, but no alteration in the bre type composition.

Conicts of interest
None declared. Fig. 4 Box and whisker plots of expression levels of myosin heavy chain (MyHC) isoform IIb (A), IId (B), IIa (C), and I (D) in the obesity (solid box) and control (open box) groups of six rats, respectively. The vertical axis represents the ratio of each target gene to S16 mRNA quantity.

Ethical approval
The protocol of this study was approved by the Animal Care and Use Committee for Jikei University.

and control groups. These results suggest that the accumulation of oil droplets did not affect the properties of the lingual muscles.

Acknowledgements
This study was supported in part by grants-in-aid for funding scientic research (Nos. 19791550 to T.S. and 20592190 to A.Y.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

4.

Discussion

To our knowledge, this is the rst report to directly show a relationship between the accumulation of oil droplets and increased bre diameter. This was observed in the slow bres of the genioglossus and geniohyoid muscles, suggesting that an accumulation of oil droplets played a role in increasing the bre diameter of the lingual muscles in the obese rat. We assume that the oil droplets were incorporated into the myobres directly and/or indirectly through fatty acid metabolism, leading to increased myobre volume.

references

1. Douglas NJ, Polo O. Pathogenesis of obstructive sleep apnoea/hypopnoea syndrome. Lancet 1994;344:6535. 2. Brennick MJ, Pack AI, Ko K, Kim E, Pickup S, Maislin G, et al. Altered upper airway and soft tissue structures in the New Zealand Obese mouse. Am J Respir Crit Care Med 2009;179:15869.

808

archives of oral biology 55 (2010) 803808

3. Mortimore IL, Marshall I, Wraith PK, Sellar RJ, Douglas NJ. Neck and total body fat deposition in nonobese and obese patients with sleep apnea compared with that in control subjects. Am J Respir Crit Care Med 1998;157:2803. 4. Wiegand DA, Latz B, Zwillich CW, Wiegand L. Upper airway resistance and geniohyoid muscle activity in normal men during wakefulness and sleep. J Appl Physiol 1990;69:125261. 5. Schiafno S, Reggiani C. Molecular diversity of myobrillar proteins: gene regulation and functional signicance. Physiol Rev 1996;76:371423. 6. Pette D, Staron RS. Mammalian skeletal muscle ber type transitions. Int Rev Cytol 1997;170:143223. 7. Houmard JA, Tanner CJ, Yu C, Cunningham PG, Pories WJ, MacDonald KG, et al. Effect of weight loss on insulin sensitivity and intramuscular long-chain fatty acyl-CoAs in morbidly obese subjects. Diabetes 2002;51:295963. 8. Lillioja S, Young AA, Culter CL, Ivy JL, Abbott WG, Zawadzki JK, et al. Skeletal muscle capillary density and ber type are possible determinants of in vivo insulin resistance in man. J Clin Invest 1987;80:41524. 9. Almond RE, Enser M. A histochemical and morphological study of skeletal muscle from obese hyperglycaemic ob/ob mice. Diabetologia 1984;27:40713. 10. Warmington SA, Tolan R, McBennett S. Functional and histological characteristics of skeletal muscle and the effects of leptin in the genetically obese (ob/ob) mouse. Int J Obes Relat Metab Disord 2000;24:104050. 11. Carrera M, Barbe F, Sauleda J, Tomas M, Gomez C, Santos C, et al. Effects of obesity upon genioglossus structure and

12.

13.

14.

15.

16.

17.

18.

function in obstructive sleep apnoea. Eur Respir J 2004;23:4259. Yamane A, Mayo M, Shuler C, Crowe D, Ohnuki Y, Dalrymple K, et al. Expression of myogenic regulatory factors during the development of mouse tongue striated muscle. Arch Oral Biol 2000;45:718. Sartorius CA, Lu BD, Acakpo-Satchivi L, Jacobsen RP, Byrnes WC, Leinwand LA. Myosin heavy chains IIa and IId are functionally distinct in the mouse. J Cell Biol 1998;141:94353. Coen PM, Dube JJ, Amati F, Stefanovic-Racic M, Ferrell RE, Toledo FG, et al. Insulin resistance is associated with higher intramyocellular triglycerides in type I but not type II myocytes concomitant with higher ceramide content. Diabetes 2010;59:808. Guo ZK, Jensen MD. Accelerated intramyocellular triglyceride synthesis in skeletal muscle of high-fat-induced obese rats. Int J Obes Relat Metab Disord 2003;27:10149. Malenfant P, Joanisse DR, Theriault R, Goodpaster BH, Kelley DE, Simoneau JA. Fat content in individual muscle bers of lean and obese subjects. Int J Obes Relat Metab Disord 2001;25:131621. Koopman R, Schaart G, Hesselink MK. Optimisation of oil red O staining permits combination with immunouorescence and automated quantication of lipids. Histochem Cell Biol 2001;116:638. Pae EK, Wu J, Nguyen D, Monti R, Harper RM. Geniohyoid muscle properties and myosin heavy chain composition are altered after short-term intermittent hypoxic exposure. J Appl Physiol 2005;98:88994.