Glycemic control with empagliflozin, a novel selective SGLT2 inhibitor, ameliorates cardiovascular injury and cognitive dysfunction in obese and type 2 diabetic mice

Abstract

Background: There has been uncertainty regarding the benefit of glycemic control with antidiabetic agents in prevention of diabetic macrovascular disease. Further development of novel antidiabetic agents is essential for overcoming the burden of diabetic macrovascular disease. The renal sodium glucose co-transporter 2 (SGLT2) inhibitor is a novel antihyperglycemic agent for treatment of type 2 diabetes. This work was performed to determine whether empagliflozin, a novel SGLT2 inhibitor, can ameliorate cardiovascular injury and cognitive decline in db/db mouse, a model of obesity and type 2 diabetes.

Methods: (1) Short-term experiment: The first experiment was performed to examine the effect of 7 days of empagliflozin treatment on urinary glucose excretion and urinary electrolyte excretion in db/db mice. (2) Long-term experiment: The second experiment was undertaken to examine the effect of 10 weeks of empagliflozin treatment on cardiovascular injury, vascular dysfunction, cognitive decline, and renal injury in db/db mice.

Results: (1) Short-term experiment: Empagliflozin administration significantly increased urinary glucose excretion, urine volume, and urinary sodium excretion in db/db mice on day 1, but did not increase these parameters from day 2. However, blood glucose levels in db/db mice were continuously decreased by empagliflozin throughout 7 days of the treatment. (2) Long-term experiment: Empagliflozin treatment caused sustained decrease in blood glucose in db/db mice throughout 10 weeks of the treatment and significantly slowed the progression of type 2 diabetes. Empagliflozin significantly ameliorated cardiac interstitial fibrosis, pericoronary arterial fibrosis, coronary arterial thickening, cardiac macrophage infiltration, and the impairment of vascular dilating function in db/db mice, and these beneficial effects of empagliflozin were associated with attenuation of oxidative stress in cardiovascular tissue of db/db mice. Furthermore, empagliflozin significantly prevented the impairment of cognitive function in db/db mice, which was associated with the attenuation of cerebral oxidative stress and the increase in cerebral brain-derived neurotrophic factor. Empagliflozin ameliorated albuminuria, and glomerular injury in db/db mice.

Conclusions: Glycemic control with empagliflozin significantly ameliorated cardiovascular injury and remodeling, vascular dysfunction, and cognitive decline in obese and type 2 diabetic mice. Thus, empagliflozin seems to be potentially a promising therapeutic agent for diabetic macrovascular disease and cognitive decline.

Figures

Figure 1

The design of Experiment II. Abbreviations used: BG, measurement of blood glucose; BP, measurement of blood pressure; OGTT, oral glucose tolerance test; MWM, Moris water maze test.

Figure 2

Effects of short-term (7 days) empagliflozin administration on non-fasting blood glucose (A), 24 hr-urinary glucose excretion (B), 24 hr-urine volume (C) and 24 hr-urinary sodium excretion (D) in db/db mice. Twenty four-hour urine of each mouse was collected with metabolic cages every day before and throughout the experiment. Abbreviations used: UGE, urinary glucose excretion; control, control (untreated) db/db mice; Empa, empagliflozin-treated db/db mice; pre, the data obtained before start of drug treatment. *p < 0.05, †p < 0.01 vs control db/db mice. Values are mean ± SEM (n = 10-11).

Figure 3

Effects of short-term (7 days) empagliflozin administration on body weight gain (A), food intake (B) and water intake(C) of db/db mice. Abbreviations used are the same as in Figure 2. *p < 0.05, †p < 0.01 vs control db/db mice. Values are mean ± SEM (n = 10-11).

Figure 4

Effects of short-term (7 days) empagliflozin administration on non-fasting blood glucose (A), 24 hr-urinary glucose excretion (B), 24 hr-urine volume (C), 24 hr-urinary sodium excretion (D), body weight gain (E), food intake (F), water intake (G), and serum insulin level (H) of db/m mice. Abbreviations: db/m, non-diabetic db/m mice; Control, control (untreated) db/m mice; Empa, empagliflozin-treated db/m mice. *p < 0.05, †p < 0.01, vs control db/m mice. Values are mean ± SEM (n = 10).

Figure 5

Effects of long-term (10 weeks) empagliflozin administration on non-fasting blood glucose (A), oral glucose tolerance test (B), serum insulin levels (C), and body weight (D) of db/db mice. Abbreviations: db/m, non-diabetic db/m mice; Control, control (untreated) db/db mice; Empa, empagliflozin-treated db/db mice. *p < 0.05, †p < 0.01, vs control db/db mice. Values are mean ± SEM (n = 9-11).

Figure 6

Effects of long-term empagliflozin administration on urinary glucose excretion (UGE) (A), urine volume (B), urinary sodium excretion (C), food intake (D), water intake (E), and blood pressure (F) of db/db mice. Abbreviations used are the same as in Figure 5. *p < 0.05, †p < 0.01 vs control db/db mice. Values are mean ± SEM (n = 9-11).

Figure 7

Effect of long-term empagliflozin treatment on cardiac interstitial fibrosis (A), peri-coronary arterial fibrosis and coronary arterial thickening (B), cardiac macrophage infiltration (C) and cardiac superoxide (D) in db/db mice. Upper panels in (A) and (B) indicate representative photomicrographs of cardiac sections stained with Sirius red. Upper panels in (C) and (D) indicate representative photomicrographs of cardiac sections stained with CD68 antibody and dihydroethidium, respectively. Abbreviations used are the same as in Figure 5. *p < 0.05, †p < 0.01 vs control db/db mice. Values are mean ± SEM (n = 9-11). Bar = 100 μm in (A), (C), and (D). Bar = 50 μm in (B).

Figure 8

Effect of long-term empagliflozin treatment on vascular relaxation by acetylcholine (A) and by SNAP (B), and vascular superoxide (C) in thoracic aortas of db/db mice. Upper panels in (C) are representative photomicrographs of dihydroethidium-stained aortic section. Bar = 50 μm. Abbreviations used are the same as in Figure 5. *p < 0.05, †p < 0.01, vs control db/db mice. Values are mean ± SEM (n = 9-11).

Figure 9

Effect of long-term empagliflozin treatment on cognitive function of db/db mice estimated by Morris water maze test. Cognitive function was evaluated by Morris water maze test at 9 weeks after the treatment. (A) indicates escape latency of the hidden platform test on 4 consecutive days (1-20 sessions). (B) indicates number of times across the platform in the probe test. (C) indicates escape latency in the visible test. (D) indicates swimming speeds. Abbreviations used are the same as in Figure 5. Values are mean ± SEM (n = 9-11).

Figure 10

Effect of empagliflozin treatment on cerebral superoxide ((A)-(C)), cerebral 8-hydroxy-deoxyguanosine (8-OHdG) (D), gp91phox (E), p67phox (F), and brain-derived neurotrophic factor (BDNF)(G) of db/db mice. Upper panels in (A), (B), and (C) indicate representative photomicrograph of DHE-stained cerebral sections (hippocampus, cortex, and white matter, respectively). Upper panels in (E), (F), and (G) indicate representative Western blot band. Abbreviations used are the same as in Figure 1. Values are mean ± SEM (n = 11). *p < 0.05, †p < 0.01, vs control db/db mice.

Figure 11

Effects of empagliflozin on urinary albumin/creatinine ratio (A), glomerular sclerosis (B), glomerular macrophage infiltration (C) and glomerular superoxide (D) of db/db mice. Urinary albumin/creatinine ratio in (A) was measured at 4 and 8 weeks after start of drug treatment. Upper panels in (B), (C), and (D) indicate representative photomicrographs of renal sections stained with PAS, CD68 antibody, and dihydroethidium, respectively. Abbreviations used are the same as in Figure 5. *p < 0.05, †p < 0.01 vs control db/db mice. Values are mean ± SEM (n = 9-11). Bar = 50 μm in (B), (C), and (D).