Combined Administration of Metformin and Propionate Reduces the Degree of Oxidative/Nitrosative Damage of Hypothalamic Neurons in Rat Model of Type 2 Diabetes Mellitus

Abstract

Many complications associated with type 2 diabetes mellitus (T2DM) are closely linked with the generation of reactive species or free radicals leading to oxidative/nitrosative stress. The aim of this study was to investigate the effect of combined administration of metformin with propionate on the degree of oxidative/nitrosative damage in the brain of rats with an experimental model of T2DM. Male Wistar rats were divided into control (healthy rats); rats with T2DM and no further therapy; rats with T2DM that received: metformin, propionate, propionate + metformin. Ventromedial hypothalamus samples were analyzed by transmission electron microscopy, gas-liquid chromatography, Western blotting, RT-PCR and electron paramagnetic resonance. Combined treatment resulted in normalization of the neuronal NOS levels and reduction of mRNA level of induced nitric oxide synthase (NOS) and superoxide radicals compared to untreated T2DM rats. A decrease was also observed in the level of 8-oxyguanine with normalization of fatty acids distribution. The combined treatment partially mitigated ultrastructural alterations resulting from oxidative/nitrosative damage in neurons' mitochondria in T2DM. Thus, we demonstrated a positive effect of the combined use of metformin and propionate on all indicators of oxidative/nitrosative stress in T2DM.

Keywords: Diabetes mellitus; Metformin; Oxidative/Nitrosative stress; Propionate.

Fig. 1

Effects of metformin and propionate (PA) administration on nNOS in rat brain. A Immunoblot analysis of nNOS in rat VMH: representative immunoblots quantified using tubulin as a loading control for hypothalamic lysates are shown. B nNOS data are presented as mean ± SD (n = 6 rats per group), C relative expression of nNOS mRNA in rat brain: data are normalized to β-actin, results of 3 independent measurement (n = 6 rats per group). All data are presented as mean ± SD; * P < 0.05 vs control, ** P < 0.05 vs T2DM, # P < 0.05 vs metformin administration and @ P < 0.05 vs PA administration

Fig. 2

Effect of metformin and propionate (PA) administration on the rate of SR formation (A) and 8-oxoguanine level (B) in rat brain. The results of 3 independent measurement (n = 6 rats per group) are presented. All data are shown as means ± SD; ∗ P < 0.05 vs. control, ∗  ∗ P < 0.05 vs. T2DM

Fig. 3

Percentage amount of each FA type: of saturated (C14:0), (C15:0), (C16:0), (C17:0), (C15:0), unsaturated (C18:1), (C18:2), (C18:3), (C20:4) and polyunsaturated (C18:2), (C18:3), (C20:4) fatty acids measured in the homogenates of brain tissue after metformin and/or propionate (PA) administration on the background of T2DM (A). A diagram showing the ratio PUFA/SFA, UFA/SFA (B). Data are shown as means ± SD (n = 6 rats per group); * P < 0.05 vs. control, ** P < 0.05 vs. T2DM

Fig. 4

Ultrastructural changes of VMH neurons and glial cell were assessed by electron microscopy observations. Gallery of micrographs obtained by scanning electron microscope (n = 12 neurons for each group): A, B, C – control rats; D, E, F – rats from the T2DM group rats. Representative images of neurons are presented on A, C, D, E, F panels, and glial cells – B panels. The yellow arrows indicate mitochondria, green arrows – lipofuscin granules. Scale bar: 200 nm

Fig. 5

Structural parameters of the mitochondria’s after metformin and propionate administration on the background of T2DM: ImageJ software was used to calculate the S_mitoch—areas of the mitochondria and the S_crists—area of crists in each cell based on the electronic microscopic microphotographs. We counted at least 7–10 mitochondria in each neuron, while in each group we studied 6 neurons. Values are given as mean ± SD. *p < 0.05 compared with control

Fig. 6

Ultrastructural changes of VMH neurons were assessed by electron microscopy observations. Gallery of micrographs obtained by scanning electron microscope (n = 12 neurons for each group): A, B – metformin group; C, D – propionate group; E, F – metformin and propionate group. The arrows indicate mitochondria. Scale bar: 200 nm

Fig. 7

Each experimental group consisted of 6 animals. These included healthy animals as a control group, a group with T2DM without correction (which received water), and 3 groups with treatment. T2DM was induced by a high-fat diet followed by a single injection of streptozotocin (STZ, 25 mg/kg of b.w.). The first group of animals T2DM-induced group treated with metformin (GLUKOFAGE, Merck Sante, France), at a dose 60 mg/kg of b.w., for 14 days, orally on the background of T2DM. The other group treated with propionate (PROPICUM®, Flexopharm Brain GmbH & Co, Germany) at a dose 60 mg/kg of b.w., for 14 days, orally on the background of T2DM. The third group receiving both metformin and propionate concurrently while having T2DM. Following the two-week treatment, the rats were euthanized by decapitation under deep anesthesia induced by Sodium thiopental (200 mg/kg)

Fig. 8

The animal body parameters: weight (A), body mass index (B), blood glucose (C), and HbA1C (D) after diabetes induction and metformin and propionate (PA) administration. Test results: oral glucose tolerance tests (E), intraperitoneal insulin tolerance test (F) on control rats (n = 6, solid line) and rats with T2DM (n = 6, dashed line). Blood samples were collected from the tail at the indicated time points and analyzed for glucose concentration (mmol/l). Values are means ± SD. *p < 0.05 compared with control

Fig. 9

Process of ventromedial hypothalamus area (VMH) identification. A—macroscopic brain landmarks; B—The Rat Brainin Stereotaxic Coordinates George Paxinos Charles Watson. International Standard Book Number: 0–12-547,623-X (Bregma -2,4 mm); C—IGH staining of the brain slice