Sodium-Glucose Cotransporter 2 Inhibitor and a Low Carbohydrate Diet Affect Gluconeogenesis and Glycogen Content Differently in the Kidney and the Liver of Non-Diabetic Mice

Abstract

A low carbohydrate diet (LCHD) as well as sodium glucose cotransporter 2 inhibitors (SGLT2i) may reduce glucose utilization and improve metabolic disorders. However, it is not clear how different or similar the effects of LCHD and SGLT2i are on metabolic parameters such as insulin sensitivity, fat accumulation, and especially gluconeogenesis in the kidney and the liver. We conducted an 8-week study using non-diabetic mice, which were fed ad-libitum with LCHD or a normal carbohydrate diet (NCHD) and treated with/without the SGLT-2 inhibitor, ipragliflozin. We compared metabolic parameters, gene expression for transcripts related to glucose and fat metabolism, and glycogen content in the kidney and the liver among the groups. SGLT2i but not LCHD improved glucose excursion after an oral glucose load compared to NCHD, although all groups presented comparable non-fasted glycemia. Both the LCHD and SGLT2i treatments increased calorie-intake, whereas only the LCHD increased body weight compared to the NCHD, epididimal fat mass and developed insulin resistance. Gene expression of certain gluconeogenic enzymes was simultaneously upregulated in the kidney of SGLT2i treated group, as well as in the liver of the LCHD treated group. The SGLT2i treated groups showed markedly lower glycogen content in the liver, but induced glycogen accumulation in the kidney. We conclude that LCHD induces deleterious metabolic changes in the non-diabetic mice. Our results suggest that SGLT2i induced gluconeogenesis mainly in the kidney, whereas for LCHD it was predominantly in the liver.

Fig 1. The effect of LCHD and SGLT2i on body weight and glycemia.

Body weight (a), non-fasted glycemia (b), average mean daily food intake per mouse (c), average mean daily carbohydrate intake per mouse (d), average mean daily fat intake per mouse (e), average mean daily protein intake per mouse (f), urinary glucose excretion at day 40 (g). n = 6–10. Data are presented as means ± SEM. *p < 0.05, **p<0.01, ****p < 0.0005 vs NC. White squares, NC; black squares, LC; white circles, NC+Ipra; black circles, LC+Ipra; white bars, NC; hatched white bars, LC; hatched grey bars, NC+Ipra; black bars, LC+Ipra.

Fig 2. LCHD induced impaired glucose tolerance, insulin resistance and fat accumulation.

Blood glucose levels were measured after an oral glucose load (a). Blood glucose AUC in OGTT (b). Glucose stimulated insulin secretion (c) measured during OGTT (Δ-insulin = Insulin_15min−Insulin _0min). HOMA IR (d) were calculated according to the formula: HOMA IR = Glucose (mmol/L) X Insulin (mU/L)/22.5. Blood glucose levels decrease after i.p. injection of insulin presented in % (e). Epididymal fat masses were measured at day 56 (f). n = 6–10. Data are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0005 vs NC. White squares, NC; black squares, LC; white circles, NC+Ipra; black circles, LC+Ipra; white bars, NC; hatched white bars, LC; hatched grey bars, NC+Ipra; black bars LC+Ipra.

Fig 3. Relative mRNA expression in the liver and the kidney of control mice.

mRNA expression in the liver was taken as 100

Fig 4. SGLT2i activates gluconeogenesis gene expression in the kidney, while LCHD activates expression in the liver.

mRNA expression of mouse G6pc, Pck, Fbp in the kidney (a, b, c) and the liver (d, e, f,) were determined by quantitative RT-PCR. The means ± SEM of mRNA levels related to the NC group are presented. n = 6–10. *p < 0.05, **p < 0.01, ****p < 0.0005 vs NC. White bars, NC; hatched white bars, LC; hatched grey bars, Ipra; black bars LC+Ipra.

Fig 5. Ipragliflozin upregulated both Foxo1 and Creb1 genes in the kidney, while LCHD upregulated in the liver.

Foxo1 and Creb1 mRNA expression in the kidney (a, b) and liver (c, d) were determined by quantitative RT-PCR. The means ± SEM of mRNA related to NC group are presented. n = 6–10. *p < 0.05, **p < 0.01 vs NC. White bars, NC; hatched white bars, LC; hatched grey bars, NC+Ipra; black bars LC+Ipra.

Fig 6. Ipragliflozin treatment reduced liver, but increased kidney glycogen content.

Glycogen content in the liver and kidney (a) measured in the NC group. Glycogen content in the kidney (b) and liver (c) in non-fasted mice at day 56. (d) Best carmine staining indicating glycogen storage in the liver. n = 6–10. Data are presented as means ± SEM. Scale bars = 100 μm. *p < 0.05, ****p < 0.0005 vs NC. White bars, NC; hatched white bars, LC; hatched grey bars, Ipra; black bars LC+Ipra.

Fig 7. Ipragliflozin increased Pygl expression in the kidney, while LCHD increased in the liver.

Pygl and Gys1/Gys2 mRNA expression in the kidney (a, b) and in the liver (c, d) were determined by quantitative RT-PCR. The means ± SEM of mRNA related to NC group are presented. n = 6–10. *p < 0.05, **p < 0.01, ***p<0.005, ****p<0.0005 vs NC. White bars, NC; hatched white bars, LC; hatched grey bars, NC+Ipra; black bars LC+Ipra.

Fig 8. Ipragliflozin enhanced Acad11 and Fasn mRNA expression in the kidney.

Acad11 and Fasn mRNA expression in the kidney (a, b) and liver (c, d) were determined by quantitative RT-PCR. The means ± SEM of mRNA related to NC group are presented. TG content in the kidney and the liver (e) measured in the NC group. TG content in the kidney (f) and liver (g) in non-fasted mice. n = 6–10. *p < 0.05, **p < 0.01, ***p<0.005 vs NC. White bars, NC; hatched white bars, LC; hatched grey bars, NC+Ipra; black bars LC+Ipra.