Molecular and Cellular Biochemistry https://doi.org/10.1007/s11010-018-3361-5
The role of mitochondrial DNA damage at skeletal muscle oxidative stress on the development of type 2 diabetes Julia Matzenbacher dos Santos3,4,5 · Denise Silva de Oliveira1 · Marcos Lazaro Moreli3 · Sandra Aparecida Benite‑Ribeiro1,2,3 Received: 3 November 2017 / Accepted: 16 April 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract Reduced cellular response to insulin in skeletal muscle is one of the major components of the development of type 2 diabetes (T2D). Mitochondrial dysfunction involves in the accumulation of toxic reactive oxygen species (ROS) that leads to insulin resistance. The aim of this study was to verify the involvement of mitochondrial DNA damage at ROS generation in skeletal muscle during development of T2D. Wistar rats were fed a diet containing 60% fat over 8 weeks and at day 14 a single injection of STZ (25 mg/kg) was administered (T2D-induced). Control rats received standard food and an injection of citrate buffer. Blood and soleus muscle were collected. Abdominal fat was quantified as well as glucose, triglyceride, LDL, HDL, and total cholesterol in plasma and mtDNA copy number, cytochrome b (cytb) mRNA, 8-hydroxyguanosine, and 8-isoprostane (a marker of ROS) in soleus muscle. T2D-induced animal presented similar characteristics to humans that develop T2D such as changes in blood glucose, abdominal fat, LDL, HDL and cholesterol total. In soleus muscle 8-isoprostane, mtDNA copy number and 8-hydroxyguanosine were increased, while cytb mRNA was decreased in T2D. Our results suggest that in the development of T2D, when risks factors of T2D are present, intracellular oxidative stress increases in skeletal muscle and is associated with a decrease in cytb transcription. To overcome this process mtDNA increased but due to the proximity of ROS generation, mtDNA remains damaged by oxidation leading to an increase in ROS in a vicious cycle accounting to the development of insulin resistance and further T2D. Keywords Oxidative stress · MtDNA damage · Skeletal muscle · 8-Hydroxyguanosine
Introduction
* Julia Matzenbacher dos Santos
[email protected];
[email protected] * Sandra Aparecida Benite‑Ribeiro
[email protected] 1
Instituto de Biociências, Federal University of Jataí, Jataí, Goias, Brazil
2
Pós‑Graduação em Biociência Animal, Federal University of Jataí, Jataí, Goias, Brazil
3
Pós‑Graduação em Ciência da Saúde, Federal University of Jataí, Jataí, Goias, Brazil
4
Detroit R&D, Inc, 2727 2nd street, 4113, Detroit, Michigan 48201, USA
5
Science and Math Department, Henry Ford College, Dearborn, MI, USA
Reduced cellular response to insulin in skeletal muscle, also known as insulin resistance, is the main factor in the development of type 2 diabetes (T2D) [1, 2]. A combination of environmental and genetic factors underlie insulin resistance, and reactive oxygen stress (ROS) appears to be linked with this process [3]. Substantial evidences have shown that mitochondrial dysfunction plays a key role in the accumulation of toxic reactive oxygen species that leads to insulin resistance [4–6]. The circular mammalian mitochondrial genome, known as mitochondrial DNA (mtDNA) is present in multiple (often 2–10) copies in each mitochondrion. Physiological and pathological conditions modify the number of copy of mtDNA, which might affect mitochondrial homeostasis [7, 8]. MtDNA encodes 13 polypeptides, all of which are involved in oxidative phosphorylation through the electron transport chain (ETC) [7, 8]. Cytochrome b (cytb), one of
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mtDNA-encoded polypeptides, is a component of complex III. Superoxide is generated in its majority by dysfunction of complex III, and decreased expression of cytb seems to be involved in this process [9]. Due to its proximity to the ETC and lack of histone, mtDNA is very vulnerable to certain kinds of damage, in particular reactive oxygen species (ROS)-mediated lesions [8, 9]. Despite cutting edge research in this field, the mechanism by which damage to mtDNA and ROS generation affects development of T2D is not yet completely identified. Therefore, the aim of this study was to verify if skeletal muscle mtDNA damage and increased ROS are involved in the development of T2D.
Methods Animals Wistar rats (8–10 weeks old) were assigned to control and T2D-induced group. The protocol for T2D was previously standardized by several laboratories [13–15]. Rats (n = 11) were fed with a manipulated high-fat diet for 3 weeks from Pragsoluções Biociências® (Jaú, SP, Brazil) containing (5205 cal/Kg, 20% kcal protein, 20% kcal carbohydrate, and 60% fat). At day 14, after overnight fasting, T2D animals received a single intraperitoneal injection of streptozotocin (STZ- 25 mg/kg of body weight) diluted in citrate buffer (pH 4.5). The feeding of the high-fat diet continued for 6 weeks. Control animals (n = 11) received manipulated chow (3868 cal/Kg, 20% kcal protein, 70% kcal carbohydrate e 10% kcal fat) from the same vendor, and injection of citrate buffer instead of STZ. Body weight and fasting blood glucose were assessed at the beginning and at the end of the protocol. Rats were euthanized after 10 weeks of experiment, and blood, omental fat, and soleus muscle were collected. The protocol was approved by the ethical and research committee of Federal University of Goias (Protocol Number 34/2014).
Blood analyses Plasma was prepared using anticoagulant (EDTA 6 g/dL, potassium fluoride 12 g/dL) (Labtest®-Lagoa Santa, MG, Brazil) and blood glucose was determined by oxide glucose test (Labtest®) while levels of triglyceride, LDL, HDL, and total cholesterol were determinated by colorimetric assays (Labtest®).
Oxidative stress 8-Isoprostane, a marker for oxidative stress, was extracted from soleus muscle and plasma and assessed using an
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Molecular and Cellular Biochemistry
8-isoprostane ELISA kit from Detroit R&D, Inc (Detroit, MI) according to the manufacture’s protocol.
Mitochondrial isolation Mitochondria were isolated using isolation buffer (250 mM sucrose, 2 mM EDTA, and 25 mM Tris–HCl, pH 7.4) and protease inhibitor using a Dounce homogenizer. Samples were centrifuged at 700 g/5 min/4 °C and supernatant was further centrifuged at 12,000×g/15 min/4 °C. Pellets were washed and protein was determined by Lowry method.
DNA isolation Whole muscle and mitochondrial extract DNA were isolated using the DNA Blood Isolation Kit (Mo Bio, Carlsbad, Ca) according to the manufacture’s protocol. DNA was quantified using a Nanodrop 2000 Spectrometry (Thermo Scientific).
MtDNA copy number DNA from whole muscle was used to assess mtDNA copy number by rtPCR using the mtDNA Copy Number Kit (MCN2) from Detroit R&D, Inc (Detroit, MI).
Oxidized mtDNA MtDNA (4 ng) was spotted on a nitrocellulose membrane and 8-hydroxydeoxyguanosine (8-OHdG) was accessed by dot blot analysis using an 8-OHdG antibody from VWR Bioscience (Radnor, PA).
RNA analyses RNA, isolated by TRIzol reagent (Invitrogen), was employed to make cDNA using High Capacity DNA Reverse Transcription Kit (Applied Biosystem, Foster City, CA). Gene expression was performed by conventional PCR using ratspecific primers for identifying mitochondrial transcript gene Cytb (Forward 5′-TGACCTTCCCGCCCCATCCA-3′ Reverse 5′-AGCCGTAGTTTACGTCTCGGCA-3′) and the internal control β-actin (Forward 5′-AGCGAGCCGGAG CCAATCAG-3′ Reverse 5′-TGCGCCGCCGGGTTTTAT AGG-3′).
Statistical analysis Statistical analysis was carried out using commercially available software (Salstat2). Shapiro–Wilk tests were used to test for normal distribution of the data. t test was used for data that present normal distribution and Mann–Whitney U
Molecular and Cellular Biochemistry Table 1 Effect of T2D induction protocol on metabolic changes Control
T2D-induced Significance (p)
Blood glucose (mg/dL) 125 ± 86 204 ± 88 Fasting blood glucose 138 ± 11 147 ± 14 (mg/dL) Body weight (g) 360 ± 77 406 ± 85 Abdominal fat (g) 23 ± 14 37 ± 17 Triglyceride (mg/dL) 95 ± 46 76 ± 60 LDL (mg/dL) 30 ± 34 49 ± 17 HDL (mg/dL) 19 ± 9 15 ± 12 Total cholesterol (mg/dL) 73 ± 23 98 ± 13
