Aging induced by D-galactose aggravates cardiac dysfunction via exacerbating mitochondrial dysfunction in obese insulin-resistant rats

Abstract

The prevalence of obesity and an aging population are increasing worldwide. Both obesity and aging are independently known to be associated with cardiac dysfunction. However, in obese insulin-resistant subjects, the effects of aging on metabolic status and cardiac and mitochondrial functions are not completely understood. We hypothesized that in the obese insulin-resistant condition, aging induced by D-galactose increases cardiac senescence markers and aggravates the impairment of metabolic parameters, cardiac and mitochondrial function, and increases oxidative stress, inflammation, apoptosis, and autophagy. Sixty-four male Wistar rats were fed with either normal diet (ND) or high-fat diet (HFD) for 12 weeks. Then, rats were divided into vehicle groups (0.9% NSS, subcutaneous injection (SC)) or D-galactose groups (150 mg/kg/day, SC). After 0.9%NSS or D-galactose treatment for 4 weeks and 8 weeks, metabolic and cardiac functions were determined. The heart was then removed to determine mitochondrial functions and enable biochemical studies. After 4 weeks of D-galactose injection, ND rats treated with D-galactose (NDD4), HFD rats treated with vehicle (HFV4), and HFD rats treated with D-galactose (HFD4) had reduced cardiac function, impaired cardiac mitochondrial function and autophagy, and increased oxidative stress, inflammation, and apoptosis. Interestingly, after 8 weeks, HFD rats treated with D-galactose (HFD8) had the worst impairment of cardiac and mitochondrial function, autophagy, and apoptosis in comparison to the other groups. Aging induced by D-galactose aggravated cardiac dysfunction in obese insulin-resistant rats through the worsening of cardiac mitochondrial function, autophagy, and increased apoptosis in a time-dependent manner.

Keywords: Aging; D-galactose; Heart; Mitochondria; Obesity.

Fig. 1

a Study protocol. b Effect of D-galactose on senescence marker expression. *p < 0.05 vs NDV at same week, †p < 0.05 vs NDD at same week, ‡p < 0.05 vs HFV at same week, #p < 0.05 vs 4 weeks within group. N = 4 per group. NDV normal diet vehicle, NDD normal diet D-galactose, HFV high-fat vehicle, HFD high-fat diet D-galactose, SA-β-gal senescence-associated β-galactosidase

Fig. 2

Baseline cardiac functions and the effect of D-galactose-induced aging on echocardiographic parameters, heart rate variability, and P-V loop analysis. a Baseline fractional shortening. b Fractional shortening. c Baseline heart rate variability. d Heart rate variability. e P-V loop analysis. *p < 0.05 vs NDV at same week, †p < 0.05 vs NDD at same week, ‡p < 0.05 vs HFV at same week, #p < 0.05 vs 4 weeks within group. N = 8 per group. ND normal diet-fed rats, HF high-fat diet-fed rats, NDV normal diet vehicle, NDD normal diet D-galactose, HFV high-fat vehicle, HFD high-fat diet D-galactose, FS fractional shortening, LF/HF ratio low-frequency/high-frequency ratio

Fig. 3

Baseline blood pressure and the effect of D-galactose-induced aging on blood pressure. *p < 0.05 vs NDV at same week, †p < 0.05 vs NDD at same week, ‡p < 0.05 vs HFV at same week, #p < 0.05 vs 4 weeks within group. N = 8 per group. ND normal diet-fed rats, HF high-fat diet-fed rats, SBP systolic blood pressure, DBP diastolic blood pressure, MAP mean arterial pressure

Fig. 4

a The effect of D-galactose administration at different time points on cardiac tissue oxidative stress. b Inflammation. c Cleaved caspase-3/caspase-3 with representative images of Western blotting band at 4 weeks after D-galactose administration. d Cleaved caspase-3/caspase-3 with representative images of Western blotting band at 8 weeks after D-galactose administration in obese insulin-resistant rats. *p < 0.05 vs NDV at same week, †p < 0.05 vs NDD at same week, ‡p < 0.05 vs HFV at same week, #p < 0.05 vs 4 weeks within group. N = 5 per group. NDV normal diet vehicle, NDD normal diet D-galactose, HFV high-fat vehicle, HFD high-fat diet D-galactose, MDA malondialdehyde, TNF-α tumor necrosis factor-alpha

Fig. 5

The effect of D-galactose-induced aging on cardiac apoptosis using the TUNEL-positive cells in obese insulin-resistant rats. a Representative figure of TUNEL-positive cells at 4-week D-galactose. b Representative figure of TUNEL-positive cells at 8-week D-galactose. c Percentage of TUNEL-positive cells at both 4- and 8-week D-galactose. *p < 0.05 vs NDV at same week, †p < 0.05 vs NDD at same week, ‡p < 0.05 vs HFV at same week, #p < 0.05 vs 4 weeks within group. N = 3 per group. NDV normal diet vehicle, NDD normal diet D-galactose, HFV high-fat vehicle, HFD high-fat diet D-galactose

Fig. 6

The effect of D-galactose-induced aging on cardiac mitochondrial function in obese insulin-resistant rats. a Cardiac mitochondrial ROS production. b Cardiac mitochondrial membrane potential. c Cardiac mitochondrial swelling. d TEM representative images of cardiac mitochondria. *p < 0.05 vs NDV at same week, †p < 0.05 vs NDD at same week, ‡p < 0.05 vs HFV at same week, #p < 0.05 vs 4 weeks within group. N = 8 per group. NDV normal diet vehicle, NDD normal diet D-galactose, HFV high-fat vehicle, HFD high-fat diet D-galactose, ROS reactive oxygen species, TEM transmission electron microscopy

Fig. 7

The effect of D-galactose-induced aging on cardiac mitochondrial fusion and fission proteins in obese insulin-resistant rats. a Mitochondrial MFN1 level. b Mitochondrial MFN2 level. c Phosphorylated Drp1 at serine 616 in cytosol. d Mitochondrial Drp1 level. *p < 0.05 vs NDV at same week, †p < 0.05 vs NDD at same week, ‡p < 0.05 vs HFV at same week, #p < 0.05 vs 4 weeks within group. N = 5 per group. NDV normal diet vehicle, NDD normal diet D-galactose, HFV high-fat vehicle, HFD high-fat diet D-galactose, MFN1 mitofusin 1, MFN2 mitofusin 2, Drp1 dynamin-related protein 1, VDAC voltage-dependent anion channels

Fig. 8

The effect of D-galactose-induced aging on autophagy in obese insulin-resistant rats. a Beclin-1 expression at 4-week D-galactose. b Beclin-1 expression at 8-week D-galactose. c p62 and d LC3II with representative Western blotting bands. *p < 0.05 vs NDV at same week, †p < 0.05 vs NDD at same week, ‡p < 0.05 vs HFV at same week, #p < 0.05 vs 4 weeks within group. N = 5 per group. NDV normal diet vehicle, NDD normal diet D-galactose, HFV high-fat vehicle, HFD high-fat diet D-galactose