Loss of function of renal Glut2 reverses hyperglycaemia and normalises body weight in mouse models of diabetes and obesity

Abstract

Aims/hypothesis: Renal GLUT2 is increased in diabetes, thereby enhancing glucose reabsorption and worsening hyperglycaemia. Here, we determined whether loss of Glut2 (also known as Slc2a2) specifically in the kidneys would reverse hyperglycaemia and normalise body weight in mouse models of diabetes and obesity.

Methods: We used the tamoxifen-inducible CreERT2-Lox system in mice to knockout Glut2 specifically in the kidneys (Ks-Glut2 KO) to establish the contribution of renal GLUT2 to systemic glucose homeostasis in health and in insulin-dependent as well as non-insulin-dependent diabetes. We measured circulating glucose and insulin levels in response to OGTT or IVGTT under different experimental conditions in the Ks-Glut2 KO and their control mice. Moreover, we quantified urine glucose levels to explain the phenotype of the mice independently of insulin actions. We also used a transcription factor array to identify mechanisms underlying the crosstalk between renal GLUT2 and sodium-glucose cotransporter 2 (SGLT2).

Results: The Ks-Glut2 KO mice exhibited improved glucose tolerance and massive glucosuria. Interestingly, this improvement in blood glucose control was eliminated when we knocked out Glut2 in the liver in addition to the kidneys, suggesting that the improvement is attributable to the lack of renal GLUT2. Remarkably, induction of renal Glut2 deficiency reversed hyperglycaemia and normalised body weight in mouse models of diabetes and obesity. Longitudinal monitoring of renal glucose transporters revealed that Sglt2 (also known as Slc5a2) expression was almost abolished 3 weeks after inducing renal Glut2 deficiency. To identify a molecular basis for this crosstalk, we screened for renal transcription factors that were downregulated in the Ks-Glut2 KO mice. Hnf1α (also known as Hnf1a) was among the genes most downregulated and its recovery restored Sglt2 expression in primary renal proximal tubular cells isolated from the Ks-Glut2 KO mice.

Conclusions/interpretation: Altogether, these results demonstrate a novel crosstalk between renal GLUT2 and SGLT2 in regulating systemic glucose homeostasis via glucose reabsorption. Our findings also indicate that inhibiting renal GLUT2 is a potential therapy for diabetes and obesity.

Keywords: Diabetes; GLUT2; Glucose homeostasis; Glucose transporters; Mouse models; Obesity; SGLT2.

Fig. 1

Validation of inducible kidney-specific loss of Glut2 in mice. (a) Representative genotypes of mice used in this study. Glut2^loxP mouse model: Glut2^loxP heterozygous (705 and 566 bp); Glut2^loxP homozygous (705 bp). KspCad^CreERT2 mouse model: KspCad^CreERT2 heterozygous (700 bp); WT (no band). (b–e) Expression of renal cortical and hepatic Glut2 (b), and immunofluorescence staining of renal GLUT2 and SGLT2 (c), together with their quantification (d, e). Scale bar, 100 μm. (f–h) Expression of renal Sglt2 (f), Glut1 (g) and Sglt1 (h). Data are shown as means ± SEM for 8- to 12-week-old male mice within 14 days of inducing renal Glut2 deficiency, n=5–7. ***p<0.001 vs Ctrl group (one-way ANOVA followed by Bonferroni’s post hoc test or two-tailed unpaired Student’s t test). Ctrl, control group; Het, heterozygous; Hom, homozygous; LTL, Lotus tetragonolobus lectin, a marker of renal proximal tubules; NC, negative control; WT, wild-type

Fig. 2

Lack of renal Glut2 improves glucose tolerance and elevates glucosuria in mice. (a) Body weight. (b) Blood glucose levels during OGTT, with corresponding AUC. (c) Blood glucose levels during IVGTT. (d) Plasma insulin levels during GSIS. (e) Blood glucose levels during ITT, with corresponding AUC. (f) 24 h urine glucose concentration. (g) 24 h urine volume. (h) 24 h amount of glucose in urine. Data are shown as means ± SEM for 8- to 12-week-old male mice within 14 days of inducing renal Glut2 deficiency, n=5–7. **p<0.01 and ***p<0.001 vs Ctrl group (two-tailed unpaired Student’s t test or repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test). Ctrl, control group

Fig. 3

Mice lacking Glut2 in the liver in addition to the kidneys show impaired glucose tolerance despite elevated glucosuria. (a) Representative genotypes of mice used in this study. Glut2^loxP mouse model: Glut2^loxP heterozygous (705 and 566 bp); and Glut2^loxP homozygous (705 bp). Ggt1^Cre mouse model: Ggt1^Cre homozygous (100 bp); and WT (no band). (b) Renal cortical and hepatic Glut2 expression. (c) Blood glucose levels during OGTT, with corresponding AUC. (d) 24 h urine glucose concentration. (e) 24 h urine volume. (f) 24 h amount of glucose in urine. (g) Plasma insulin levels during oral glucose challenge. (h) Serum glucagon levels. Data are shown as means ± SEM for 8- to 12-week-old male mice, n=4–6. *p<0.05 and ***p<0.001 vs Ctrl group (two-tailed unpaired Student’s t test, or one-way ANOVA or repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test). Ctrl, control group; Het, heterozygous; Hom, homozygous; K+L Glut2 KO, mice with Glut2 deficiency in the kidneys and liver; NC, negative control; PC, positive control; WT, wild-type

Fig. 4

Renal Glut2 deficiency reverses hyperglycaemia and normalises body weight in mouse models of diabetes and obesity. (a) Fasting blood glucose levels at baseline, after inducing type 1 diabetes by STZ and on the seventh day after knocking out renal Glut2 by tamoxifen. (b–e) Body weight (b), 24 h urine glucose concentration (c), 24 h urine volume (d) and 24 h amount of glucose in urine (e) in 8- to 12-week-old female mice with STZ-induced type 1 diabetes before, and on the 12th day after, knocking out renal Glut2 using tamoxifen. (f, g) Expression of renal Glut2 (f) and Sglt2 (g) in female mice with STZ-induced type 1 diabetes before, and on the 14th day after, knocking out renal Glut2. (h) Changes in body weight during 25 weeks of regular diet or HFSD feeding. (i) Changes in the weight of epididymal adipose tissue 4 weeks after inducing renal Glut2 deficiency. (j, k) Blood glucose levels during OGTT at 1 week pre-renal Glut2 deficiency (j) and on the fifth day post-renal Glut2 deficiency (k). (l) Plasma insulin levels three days before and on eighth day after renal Glut2 deficiency. (m–o) 24 h urine glucose concentration (m), 24 h urine volume (n) and 24 h amount of glucose in urine (o) on the tenth day after inducing renal Glut2 deficiency in male mice fed a regular diet or HFSD. Data are shown as means ± SEM, n=5. *p<0.05 and ***p<0.001 vs baseline (a) or Ctrl + Sal (f, g); †p<0.05 and ^†††p<0.001 vs STZ (a) or Ctrl + STZ (c–f); ^‡p<0.05, ^‡‡p<0.01 and ^‡‡‡p<0.001 vs Ctrl + RD; ^§p<0.05, ^§§p<0.01 and ^§§§p<0.001 vs Ctrl + HFSD; (two-tailed unpaired Student’s t test, or one-way ANOVA or repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test). Ctrl, control Glut2^loxP/loxP mice; RD, regular diet; TAM, tamoxifen

Fig. 5

Dapagliflozin improves glucose tolerance in the absence and presence of diabetes in mice lacking renal Glut2. (a) Blood glucose levels during OGTT, with corresponding AUC, in 8- to 12-week-old female mice on the seventh day after inducing renal Glut2 deficiency. (b–d) 24 h urine glucose concentration (b), 24 h urine volume (c) and 24 h amount of glucose in urine (d) on the 12th day after inducing renal Glut2 deficiency in 8- to 12-week-old female mice. (e) Blood glucose levels during OGTT, with corresponding AUC, on the eighth day after inducing renal Glut2 deficiency in 8-week-old female mice with STZ-induced type 1 diabetes. (f–i) Blood glucose levels during OGTT, with corresponding AUC (f), 24 h urine glucose concentration (g), 24 h urine volume (h) and 24 h amount of glucose in urine (i) on the 14th day after inducing renal Glut2 deficiency in HFSD-fed mice. Data are shown as means ± SEM, n=5 or 6. *p<0.05, **p<0.01 and ***p<0.001 vs Ctrl + Sal; ^†p<0.05, ^††p<0.01 and ^†††p<0.001 vs Ctrl + DAPA; ‡p<0.05, ^‡‡p<0.01 and ^‡‡‡p<0.001 vs Ks-Glut2 KO + Sal; ^§p<0.05, ^§§p<0.01 and ^§§§p<0.001 vs Ctrl + HFSD + Sal; ^¶p<0.05 and ^¶¶p<0.01 vs Ctrl + HFSD + DAPA; ^¥p<0.05 and ^¥¥ p<0.01 vs Ks-Glut2 KO + HFSD +Sal; (repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test). Ctrl, control group; DAPA, dapagliflozin; Sal, saline

Fig. 6

Long-term renal Glut2 deficiency almost abolishes the expression of Sglt2 by downregulating Hnf1α. (a, b) Expression of renal cortical Glut2 (a) and Sglt2 (b). (c, d) Immunofluorescence staining of renal SGLT2, with its quantification. Scale bar, 100 μm. (e–i) Expression of renal cortical Glut1 (e), Sglt1 (f) and Hnf1α (g), and immunofluorescence staining of renal HNF1α as well as its quantification (h, i), on the 21st day following renal Glut2 deficiency in 8- to 12-week-old male mice. Scale bar, 100 μm. (j) mRNA levels of Hnf1α, Sglt2 and Glut2 in primary renal proximal tubular epithelial cells isolated from 8- to 12-week-old male control (Glut2^loxP/loxP) mice, or mice with kidney-specific loss of Glut2 (Ks-Glut2 KO) on the 21st day following renal Glut2 deficiency. Data are shown as means ± SEM, n=5 or 6. ***p<0.001 vs Ctrl; ^‡‡‡p<0.001 vs Ks-Glut2 KO + Glut2; ^†††p<0.001 vs Ks-Glut2 KO + control plasmid group (two-tailed unpaired Student’s t test or two-way ANOVA followed by Bonferroni’s multiple comparison test). Ctrl, control Glut2^loxP/loxP group; HNF1α, hepatocyte nuclear factor-1α; Ks-Glut2 KO, Glut2 knocked out specifically in the kidneys; Ks-Glut2 KO + Hnf1α or Glut2, primary renal proximal tubular epithelial cells isolated from Ks-Glut2 KO mice and transfected with Hnf1α or Glut2 expressing plasmids; LTL, Lotus tetragonolobus lectin, a marker of renal proximal tubules