The Effect of STZ-Induced Diabetes on Lipid Structure, Function and Composition of Rat Skeletal Muscle: An FTIR Study
HACER ERAR1, SEVGI TURKER GORGULU2,SARA BANU AKKAS2, OKKES YILMAZ3
andFERIDE SEVERCAN2
1Department of Biophysics, Faculty of Medicine, OndokuzMayisUniversity, Samsun, TURKEY
2Department of Biology, Middle EastTechnicalUniversity, Ankara, TURKEY
3Department of Biology, FiratUniversity, Elazig, TURKEY
Abstract:Diabetes mellitus is a disorder in which blood levels of glucose are abnormally high due to an absolute deficiency of insulin secretion, or as a result of reduced effectiveness of insulin, or both. Patients with diabetes mellitus can experience a variety of disorders in many of the organs of the human body such as the heart, kidney, eyes and skeletal muscle. In the current study, the application of Fourier Transform Infrared spectroscopy on diabetic rat skeletal muscle was investigated and the promise of this technique in medical research was highlighted. Diabetes mellitus was induced in rats by streptozotocin injection, which is one of the most popular experimental models for the study of type I diabetes. Shifts in the peak position, changes in the bandwidth and intensity of the bands in the C-H stretching region were analyzed to attain valuable structural and functional information, which may have diagnostic value. In the diabetic tissues,a significant decrease in the concentration of unsaturated lipids was observed. The signal intensity of the CH2 symmetric stretching vibration was also dramatically decreased, which indicates a decrease in the saturated lipid content. In addition, an increase in lipid fluidity and protein to lipid ratio was observed in diabetic tissues.
Key-Words: Type I diabetes, skeletal muscle, FTIR spectroscopy, lipid fluidity
1 Introduction
Diabetes mellitus is a metabolic disorder associatedwith insulin, a hormone that allows blood glucose to enter the cells of the body and be used for energy. This disorder manifests itself in two ways:Type I or inherited diabetes, which usually occurs during childhood or adolescence, results from an autoimmune process in which the body’s immune system attacks and destroys the insulin-producing beta-islet cells of the pancreas. Therefore, the body cannot produce insulin.Skeletal muscle is the most insulin-sensitive tissue in the body and therefore, a primary target of insulin disorders [1].
Biophysical studies that give information about the effect of diabetes on the content, structure and function of macromolecular components of membranes and tissues are very limited [2-4]. Recently, diabetes induced lipidperoxidation was reported in rat microsomal membranes [3]. The effect of diabetes on lipid fluidity has been reported by a limited number of studies,which are not in agreement with each other [4-6].The effect of diabetes on the lipid and protein composition and lipid order of skeletal muscle has not been reported so far. These are important parameters that can facilitatethe differentiation of diabetes.
Fourier Transform Infrared (FTIR) spectroscopy possesses several potential advantages for the study of biological membrane structure and dynamic properties [2,3], since it requires neither the use of probe molecules nor the performance of chemical reactions prior to the determination of the reaction products. The method directly and simultaneously monitors specific functional groups in molecules.
In the current study, the effect of streptozotocin-induced diabetes (Type I) on lipid and protein content, lipid order and fluidity of rat skeletal muscle was investigated using FTIR spectroscopy, which to our best of knowledge was not reported previously.
2 Materials and Methods
2.1 Experimental Design for Diabetes Treatment to Rats
Sixteen male Wistar rats (250-300g) were divided into 2 groups as control (n=8) and diabetic (n=8) groups. Animals were housed at a density of two or three per cage, in a room that was maintained at a temperature of 221 °C and a 12 h light/dark cycle. All the animals were fed with standard diet and water. Diabetes induction was made by a single i.p. dose of STZ (50 mg/kg) dissolved in 0.05 M citrate buffer (pH 4.5). Control group received 50 mg/kg physiological saline solution. After two weeks, blood glucose levels were checked weekly and the rats that have blood glucose levels above 200 mg/dl were considered as diabetic. At the end of the eighth week, rats were weighed and decapitated; muscles were removed and stored at
-80°C until use.
2.2 Sample Preparation for FTIR study
The samples were ground with liquid nitrogen and dried in a MAXI dry lyo freeze drier overnight. Then the samples were ground with potassium bromide at 1/100 sample/KBr ratio. This powder was then compressed into a thin KBr disk under a pressure of 1000 kg/ cm2 for 5 minutes.
2.3 FTIR Spectroscopic Analysis
Infrared spectra were obtained using PERKIN ELMER(Spectrum one) spectrometer which was equipped with DTGS (deuterated triglycine sulfate) detector. Water vapour and carbon dioxide interference were automatically subtracted. The FTIR spectra of samples were recorded in the
4000-400 cm-1spectral region at room temperature. 400 scans were taken for each interferogram at
4 cm-1resolution. Each sample was scanned three times, which gave identical results, and were averaged using GRAMS/32 software program, for further analysis. For other data analysis Perkin Elmer software was used.
2.4 Statistics
Mann-Whitney-U-test performed on the groups to test the significance of the differences between the control group and infected group. P value of < 0.05 was considered to be statistically significant.
3 Results and Discussion
In the present work the C-H stretching region located at 3025-2800 cm-1 was investigated. This region contains several bands that arise form the vibration of functional groups belong to lipids andproteins. Although the normalized average spectra are presented in the figure to visually demonstrate the comparative changes, for the precise determination of variations in the frequency, bandwith, signal intensity and area values, all spectra belonging to the control and diabetic samples were considered, and mean values and statistical significances were calculated [2-4].
Figure 1 shows the average FTIR spectra of control and diabetic rat skeletal muscle tissue in the 4000-400 cm-1 region. The spectra were normalized with respect to the CH2 asymmetric mode, which is observed at 2925 cm-1. The assignment of the bands waspresented in Table 1[4-8]. As seen from the figure, there are dramatic differences between the FTIR spectra of control and diabetic samples. The variations in the signal intensity, bandwidth, and the ratio of the bands are clearly seen from the figure.
Figure 1. The average FTIR spectra of control and diabetic rat skeletal muscle tissue in the 4000-400 cm-1 region (The spectra were normalized with respect to the CH2 asymmetric mode, which is observed at 2925 cm-1).
Table 1. General band assignment of an FTIR spectrum of rat skeletal muscle tissue in the C-H stretching region.
# / Frequency(cm-1) / Definition1 / 3012 / Olefinic =CH strecthing: unsaturated lipids
2 / 2955 / CH3 asymmetric stretching: mainly lipids and little protein
3 / 2925 / CH2 asymmetric stretching: mainly lipids and little protein
4 / 2872 / CH3 symmetric stretching: mainly protein and little lipids
5 / 2854 / CH2 symmetric stretching: mainly lipids and little protein
The peak area of the olefinic=CH band (3012 cm-1) can be used as an index of relative concentration of double bonds in the lipid structure form unsaturated lipids [3]. There is a decrease in peak area of olefinic band in diabeticsamples, indicating a decrease in the concentration of unsaturated lipids in the diabetic tissues.This value decreased from 2.06 ± 0.20 in control to 1.36± 0.14(p<0.05) in diabetic group. The reason for this can be lipid peroxidation, which attacks unsaturated lipids more than saturated lipids and thus may lead to a decrease in olefinic band area. Supporting this finding Sills et al. also observed a decrease in the intensity of olefinic band[9]. This seems to contrastthe previous studies on diabetic patients’ platelets [5] and diabetic rat liver microsomal membranes [3] because they reported an increase in the intensity of the olefinic band. In this latter study, Severcan and co-workers [3] stated that this situation can be explained to bea result of the lipid peroxidation end products that also carry double bonds [3].Therefore, in the current skeletal muscle study, the observed decrease in the olefinic band area can be directly the indication of lipid peroxidation.
Important results can be obtained from the investigation of the CH2symmetricstretching vibrations. As seen from the figure, a decrease in the intensity of this band occured as a result of diabetes induction. This implies a decrease in the lipid content of diabetic samples. This result also supports the previous finding because previously a decreade in the intensity of the CH2 strectching band was proposed as an indication of lipidperoxidation.The absorbances for the CH2 stretching vibrations are expected to decrease when there is lipid peroxidation [10].
Theshift in the frequency of the CH2 stretching bands gives information about the order/disorder status of lipids [9]. Since the frequencies of these bands were not shifted, lipid order was found not to be changed for diabetic samples.
Membrane dynamic information is obtained from bandwidth of the CH2 asymmetric stretching band [2, 6]. An increase in the bandwidth of the CH2 asymmetric vibration mode was observed in diabetic samples indicating higher fluidity in diabetic membranes. The bandwidth increased from 20.00±0.44 for control to 26.14±0.70for diabetes (p<0.001) groups. The result of the current study was in agreement with previous studies on diabetic heart membranes [2,4]. In contrast tothese studies, Liu et al., [5]reported a decrease in membrane fluidity in diabetic platelets.This confliction might be due to different action of diabetes on different systems.
Since, the CH2 symmetric stretching arises mainly from lipids and the CH3 stretching arises mainly from proteins, the peak area ratio of these absorptions gives information about protein to lipid content ofthe system. There is an increase in the protein to lipid ratio (intensity ratio of the CH3 to the CH2 symmetric stretching vibrations). This ratio increased from 0.9484±0.0168in control to 1.0077±0.0049 in diabetic group (p=0.018*). As seen from the figure, this increase resulted from the increase of protein concentration (CH3symmetric stretching vibration) and decrease of lipid concentration (CH2 symmetric stretching vibration).
4 Conclusion
This FTIR study clearly shows that diabetes induces significant alterations in rat skeletal muscle by changing the saturated and unsaturated lipid concentration, lipid to protein ratio and lipid fluidity. This information is important because in future, these kind of changes may be used in patient monitoring for early diagnosis of diabetes and determining the role of drugs in the therapy of diabetes.
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