THE EFFECT OF STZ-INDUCED DIABETES ON LIPIDS …



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

and FERIDE SEVERCAN2

1Department of Biophysics, Faculty of Medicine, Ondokuz Mayis University, Samsun, TURKEY

2Department of Biology, Middle East Technical University, Ankara, TURKEY

3Department of Biology, Firat University, 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 associated with 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 lipid peroxidation 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 facilitate the 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 22 ( 1 °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-1 spectral region at room temperature. 400 scans were taken for each interferogram at

4 cm-1 resolution. 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 and proteins. 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 was presented 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.

[pic]

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) |Definition |

|1 |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 diabetic samples, 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 ................
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