Prolonged metformin treatment leads to reduced transcription of Nrf2 and neurotrophic factors without cognitive impairment in older C57BL/6J mice

Abstract

Long-term use of anti-diabetic agents has become commonplace as rates of obesity, metabolic syndrome and diabetes continue to escalate. Metformin, a commonly used anti-diabetic drug, has been shown to have many beneficial effects outside of its therapeutic regulation of glucose metabolism and insulin sensitivity. Studies on metformin's effects on the central nervous system are limited and predominantly consist of in vitro studies and a few in vivo studies with short-term treatment in relatively young animals; some provide support for metformin as a neuroprotective agent while others show evidence that metformin may be deleterious to neuronal survival. In this study, we examined the effect of long-term metformin treatment on brain neurotrophins and cognition in aged male C57Bl/6 mice. Mice were fed control (C), high-fat (HF) or a high-fat diet supplemented with metformin (HFM) for 6 months. Metformin decreased body fat composition and attenuated declines in motor function induced by a HF diet. Performance in the Morris water maze test of hippocampal based memory function, showed that metformin prevented impairment of spatial reference memory associated with the HF diet. Quantitative RT-PCR on brain homogenates revealed decreased transcription of BDNF, NGF and NTF3; however protein levels were not altered. Metformin treatment also decreased expression of the antioxidant pathway regulator, Nrf2. The decrease in transcription of neurotrophic factors and Nrf2 with chronic metformin intake, cautions of the possibility that extended metformin use may alter brain biochemistry in a manner that creates a vulnerable brain environment and warrants further investigation.

Keywords: BDNF; Metformin; NGF; Neurotrophin 3; Nrf2; Water maze.

Figure 1. Effect of high fat diet and metformin on body weight and body composition

(A) Bodyweight changes during the 6-month dietary treatment *= significantly different from C. # = significantly different from HF. p<0.05 (B) Body fat percentage taken at month 4 of dietary treatment. **=significantly different from HF and C, p=0.022(HFM vs C), p<0.001 (HFM vs. HF). * = significantly from HF, p< 0.001 Error bar = ± standard error of mean (n=9,8, and 7 for C HF and HFM groups respectively).

Figure 2. Effect of high fat diet and metformin on fasting glucose levels and glucose tolerance

(A)Fasting blood glucose levels were measured as part of the oral glucose tolerance test, after 5 months of dietary treatment. (B) Blood glucose levels at various time points after an oral dose of 2g/kg(body weight) of glucose by gavage. * Significantly different from corresponding time point of C and HF groups, p=0.025. Error bar = ± standard error of mean. (C) Analysis of area under the curve showed no significant differences between groups p=0.225 (n= 6, 7 and 7 for C, HF and HFM groups respectively).

Figure 3. Effect of high fat diet and metformin treatment on motor activity

(A) Average daily voluntary wheel running activity. (B) Motor coordination via rodarod performance. * HFM significantly different from HF, p=0.003. Error bar = ± standard error of mean225 (n= 15, 13 and 11 for C, HF and HFM groups respectively).

Figure 4. Effect of high fat diet and metformin treatment on Morris water maze performance

(A) Latencies to reach the hidden escape platform. (B) Duration of time spent in target and opposite quadrants of the maze. * = significantly different compared to time spent in the opposite quadrant. (p=0.002 for C, p=0.04 for HFM). C=control, HF= high-fat, HFM = High-fat plus metformin. Error bar = ± standard error of mean (n=7,4,5 in C, HF and HFM groups respectively).

Figure 5. Effect of high fat diet and metformin treatment on neurotrophic factor expression in brain

(A) BDNF mRNA levels. (B) Protein levels of preBDNF and mature BDNF. (C) NGF mRNA levels (D) NGF protein levels, (E) NTF3 mRNA levels. (F) NTF3 Protein levels. * = significantly different from C and HF, p < 0.001. All mRNA levels were normalized to GAPDH and protein levels were normalized to β-actin protein or ponsceau S. Error bar = ± standard error of mean.

Figure 6. Effect of metformin treatment on Nrf2 and AMPK expression

(A) Nrf2 mRNA levels. (n=9,10,7 for C, HF and HFM respectively). (B) Protein levels of pNrf2 relative to total Nrf2 protein (n= 8,12,6 for C, HF and HFM respectively) ** = significantly different from C (p=0.007) and HF (p=0.049). (C) AMPK mRNA levels (n=14,13,7 for C, HF and HFM respectively); * = significantly different from C and HF (p=0.01). (D) Protein levels of pAMPK relative to total AMPK protein (n=9,9,10 for C, HF and HFM respectively). P=0.068. Error bar = ± standard error of mean.