Treatment of diabetic mice with the SGLT2 inhibitor TA-1887 antagonizes diabetic cachexia and decreases mortality

Abstract

A favorable effect of an inhibitor of the sodium-glucose cotransporter 2 (SGLT2i) on mortality of diabetic patients was recently reported, although mechanisms underlying that effect remained unclear. Here, we examine SGLT2i effects on survival of diabetic mice and assess factors underlying these outcomes. To examine SGLT2i treatment effects in a model of severe diabetes, we fed genetically diabetic db/db mice a high-fat diet and then assessed outcomes including diabetic complications between SGLT2i TA-1887-treated and control mice. We also compare effects of SGLT2i TA-1887 with those of lowering blood glucose levels via insulin treatment. Untreated db/db mice showed remarkable weight loss, or cachexia, while TA-1887-treated mice did not but rather continued to gain weight at later time points and decreased mortality. TA-1887 treatment prevented pancreatic beta cell death, enhanced preservation of beta cell mass and endogenous insulin secretion, and increased insulin sensitivity. Moreover, TA-1887 treatment attenuated inflammation, oxidative stress, and cellular senescence, especially in visceral white adipose tissue, and antagonized endothelial dysfunction. Insulin treatment of db/db mice also prevented weight loss and antagonized inflammation and oxidative stress. However, insulin treatment had less potent effects on survival and prevention of cellular senescence and endothelial dysfunction than did TA-1887 treatment. SGLT2i treatment prevents diabetic cachexia and death by preserving function of beta cells and insulin target organs and attenuating complications. SGLT2i treatment may be a promising therapeutic strategy for type 2 diabetes patients with morbid obesity and severe insulin resistance.

Conflict of interest statement

The authors declare that they have no competing financial interests.

Figures

Fig. 1

TA-1887-treated or insulin-treated severely diabetic mice show enhanced survival and increased body weight but does not alter food intake or energy expenditure. a left: Changes in body weight in db/db mice fed a high-fat diet (HF) or comparable mice treated with saline (HF + saline) or with TA-1887(HF + TA) or insulin (HF + Ins) (n = 20–30). right: AUC for body weight at indicated periods. b Food intake of each group at 13, 37, and 72 days (n = 6). c Proportion of animals surviving 4 months (n = 20–30). d Representative appearance of mice in indicated groups at day 80. e Energy expenditure (EE) after 9 weeks of each treatment, as determined by indirect calorimetry (n = 6). f Volume of lean body mass, total fat, visceral fat (v fat) and subcutaneous fat (s fat), as measured by computed tomography (CT) after 3 months of each treatment (n = 6). Values shown are means ± SEM. *p < 0.05, **p < 0.01 versus HF, and #p < 0.05, ##p < 0.01 versus HF + saline, and ¶¶p < 0.01 versus HF + TA and ††p < 0.01 versus HF + Ins

Fig. 2

Protective effect of TA-1887 on pancreas and insulin sensitivity in db/db mice fed a high-fat (HF) diet. a, b Blood glucose and plasma insulin (n = 8) levels at 0, 1, and 2 months of indicated treatments. c Image showing insulin immunostaining in pancreatic tissue of representative mice after 4 months of indicated treatment. Squares in upper panels are magnified in corresponding lower panels. Scale bars: 500 μm (upper), 200 μm (lower). d Analysis of INS1 and INS2 mRNA expression in mouse pancreatic tissue after 4 months of treatment (n = 5–9). e Representative double immunostaining of insulin and active caspase 3 in pancreatic tissue after 4 months of treatment. Scale bars: 100 μm. f Insulin tolerance test after 10 weeks of drug treatment and corresponding AUC (n = 5–9). g levels of mRNAs encoding glycolytic enzymes (n = 5–9) in eWAT, iWAT, MG, MS and liver after 4 months of treatment. DAPI, 4',6-diamidino-2-phenylindole; INS1, insulin I; INS2, insulin II; HK2, hexokinase 2; Eno1, enolase 1; PFKFB1, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1; Gpi1, glucose phosphate isomerase 1. Values shown are means ± SEM. *p < 0.05 and **p < 0.01 versus HF

Fig. 3

Expression of inflammatory and senescence markers in TA-1887-treated db/db mice fed a high-fat diet. a mRNA levels of inflammatory mediators in eWAT, iWAT, MG and MS after 4 months of indicated treatment (n = 5-9). b Plasma IL-6 concentration after 2 months of drug treatment, as determined by ELISA (n = 8). c mRNA levels of the senescence markers p21 and p16INK4a in eWAT, iWAT, MG and MS after 4 months of indicated treatment (n = 5–9). d Senescence-associated staining for SPiDER beta-Gal in frozen sections of eWAT, iWAT and MG tissues after 4 months of treatment. Scale bars: 200 μm. IL-6, interleukin-6; IL-1b, interleukin-1b; PAI-1, plasminogen activator inhibitor-1; MCP1, monocyte chemoattractant protein-1; mmp9, matrix metalloproteinase 9. Values shown are means ± SEM. *p < 0.05 and **p < 0.01 versus HF

Fig. 4

Effect of TA-1887 on urinary excretion and tissue expression of 8-OHdG, expression of antioxidative enzymes and vascular function in db/db mice fed a high-fat diet. a Urinary excretion of 8-OHdG in db/db mice fed a high-fat (HF) diet and treated for 4 months as indicated, as measured by ELISA (n = 5). b Immunohistochemistry with an 8-OHdG antibody of representative eWAT, iWAT and MG samples of mice treated 4 months as indicated. Scale bars: 200 μm (eWAT, iWAT), 100 μm (MG). c levels of mRNAs encoding the antioxidative enzymes MnSOD and catalase in eWAT, iWAT, MG and MS after 4 months of treatment (n = 5–9). d Endothelium-dependent vasorelaxation in response to acetylcholine in aorta after 4 months of treatment (n = 5–9). e Expression of transcripts associated with vascular inflammation in aorta of mice treated as indicated for 4 months (n = 5–9). f Expression of senescence marker transcripts in aorta after 4 months of indicated treatment (n = 5–9). g Expression of transcripts encoding antioxidative enzymes in aorta after 4 months of indicated treatment (n = 5–9). Values shown are means ± SEM. *p < 0.05 and **p < 0.01 versus HF

Fig. 5

Effects of insulin on tissue composition, pancreatic beta cell, glucose metabolism and inflammation in db/db mice fed a high-fat (HF) diet. a Volume of lean body mass, total fat, visceral fat (v fat) and subcutaneous fat (s fat), as measured by Computed Tomography after 3 months of indicated treatment (n = 5). b Analysis of INS1 and INS2 mRNAs in samples of pancreatic tissue after 4 months of indicated treatment (n = 5–7). c Immunostaining for insulin in representative pancreatic tissue samples after 4 months of indicated treatment. Squares in upper panels are magnified in corresponding lower panels. Scale bars: 200 μm. d Double immunostaining for insulin and active caspase 3 in representative pancreatic tissues after 4 months of treatment. Scale bars: 100 μm. e, f Blood glucose and plasma insulin levels at 0, 1, and 2 months of drug treatment (n = 8). g levels of mRNAs encoding glycolytic enzymes in eWAT, iWAT, MG and MS after 4 months of indicated treatment (n = 5–7). h Plasma IL-6 concentrations as measured by ELISA after 2 months of drug treatment (n = 8). i levels of inflammatory mRNAs in eWAT, iWAT, MG and MS after 4 months of indicated treatment (n = 5–7). DAPI, 4',6-diamidino-2-phenylindole; INS1, insulin I; INS2, insulin II; HK2, hexokinase 2; Eno1, enolase 1; PFKFB1, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1; Gpi1, glucose phosphate isomerase 1; IL-6, interleukin-6; IL-1b, interleukin-1b; PAI-1, plasminogen activator inhibitor-1; MCP1, monocyte chemoattractant protein-1; mmp9, matrix metalloproteinase 9. Values shown are means ± SEM. *p < 0.05 and **p < 0.01 versus HF + saline

Fig. 6

Effects of insulin treatment on senescence and oxidative stress markers and on vascular function in db/db mice fed a high-fat (HF) diet. a left: 8-OHdG concentration in urine as determined by ELISA after 4 months of treatment. right: Immunohistochemistry with an 8-OHdG antibody of representative eWAT, iWAT and MG samples after 4 months of indicated treatment. Scale bars: 200 μm (eWAT, iWAT), 100 μm (MG). b Levels of transcripts encoding the antioxidative enzymes MnSOD or catalase in mouse eWAT, iWAT, MG and MS following 4 months of indicated treatments (n = 5–7). c mRNA levels of the senescence markers p21 and p16INK4a in eWAT, iWAT, MG and MS after 4 months of indicated treatment (n = 5–7). d Senescence-associated staining for SPiDER beta-Gal in frozen sections of eWAT, iWAT and MG tissues after 4 months of indicated treatment. Scale bars: 200 μm. e Endothelium-dependent vasorelaxation in response to acetylcholine in aorta after 4 months of treatment (n = 5–7). f Expression of transcripts associated with vascular inflammation in aorta after 4 months of treatment (n = 5–7). g Expression of transcripts of senescence markers in aorta after 4 months of indicated treatment (n = 5–7). h Expression of transcripts encoding antioxidative enzymes in aorta after 4 months of indicated treatment (n = 5–7). Values shown are means ± SEM. *p < 0.05 and **p < 0.01 versus HF + saline