Journal: Cells

Article Title: Canagliflozin, an Inhibitor of the Na + -Coupled D-Glucose Cotransporter, SGLT2, Inhibits Astrocyte Swelling and Brain Swelling in Cerebral Ischemia.

doi: 10.3390/cells12182221

Figure Lengend Snippet: Figure 2. SGLT2 is upregulated in astrocyte cell bodies and astrocyte endfeet following cerebral ischemia. (a–e) Double immunolabeling for SGLT2 (green) and NeuN (a,b) or GFAP (c–e) (red) in contralateral (Contra) and ipsilateral (Ipsi) brain sections from mice post-MCAo/R (2/6 h); merged images are shown in a, b, d, e; individual labelings are shown in c, d—inset, e—inset; the images shown are representative of findings in 3 mice; scale bars, 25 µm except insert, 10 µm.

Article Snippet: We studied SGLT2 expression using a commercial rabbit polyclonal anti-SGLT2 antibody (#NBP1-92384; Novus Biologicals, Centennial, CO, USA).

Techniques: Immunolabeling

Journal: Cells

Article Title: Canagliflozin, an Inhibitor of the Na + -Coupled D-Glucose Cotransporter, SGLT2, Inhibits Astrocyte Swelling and Brain Swelling in Cerebral Ischemia.

doi: 10.3390/cells12182221

Figure Lengend Snippet: Figure 3. Slc5a2 mRNA and SGLT2 protein are upregulated in astrocytes following cerebral ischemia. (a–c) Images (a) and quantification (b,c) of RNAscope for Slc5a2 (red) and Aqp4 (green) mRNA in contralateral (Contra) and ipsilateral (Ipsi) brain sections from mice post-MCAo/R (2/6 h); scale bar, 25 µm; data from 3 mice per group; **, p < 0.01. (d) qPCR for Slc5a2 mRNA in astrocytes and neurons isolated from the contralateral (Contra) and ipsilateral (Ipsi) MCA territory of mice following MCAo/R (2/6 h) and uninjured controls (naïve), expressed as fold-change; 5 mice per group; *, p < 0.05. (e) Quantitative immunohistochemistry for SGLT2 expressed within regions of interest determined by NeuN; data from 7 mice per group; ns, not significant. (f,g) Immunoblot (f) and quantification of immunoblot (g) for SGLT2 in astrocytes isolated from the contralateral (Contra) and ipsilateral (Ipsi) MCA territory of mice following MCAo/R (2/6 h) and uninjured controls (naïve), expressed as fold-change; PC, positive control (kidney lysate); β-actin used as a loading control; 3 mice per group.

Article Snippet: We studied SGLT2 expression using a commercial rabbit polyclonal anti-SGLT2 antibody (#NBP1-92384; Novus Biologicals, Centennial, CO, USA).

Techniques: RNAscope, Isolation, Immunohistochemistry, Western Blot, Positive Control, Control

Journal: Cells

Article Title: Canagliflozin, an Inhibitor of the Na + -Coupled D-Glucose Cotransporter, SGLT2, Inhibits Astrocyte Swelling and Brain Swelling in Cerebral Ischemia.

doi: 10.3390/cells12182221

Figure Lengend Snippet: Figure 4. D-glucose-induced Na+ influx in post-ischemic astrocytes is inhibited by canagliflozin and by the astrocyte-specific deletion of Slc5a2/SGLT2. (a) Live cell image of a tdTomato-expressing astrocyte in an ex vivo brain slice from an MCAo/R (2/6 h) mouse, following incubation with ING-2; scale bar, 25 µm. (b–e) Time course of Na+ influx with step change in D-glucose from 2 to 10 mM in ipsilateral (Ipsi) vs. contralateral (Contra) astrocytes (b); in ipsilateral astrocytes without and with pre-incubation with canagliflozin (5 µM) (c); in ipsilateral astrocytes without and with the addition of canagliflozin (5 µM) at the time indicated by the bar (d); in ipsilateral astrocytes from Ast-Slc5a2WT

Article Snippet: We studied SGLT2 expression using a commercial rabbit polyclonal anti-SGLT2 antibody (#NBP1-92384; Novus Biologicals, Centennial, CO, USA).

Techniques: Expressing, Ex Vivo, Slice Preparation, Incubation

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Renal denervation ameliorates cardiac metabolic remodeling in diabetic cardiomyopathy rats by suppressing renal SGLT2 expression.

doi: 10.1038/s41374-021-00696-1

Figure Lengend Snippet: Fig. 1 RDN effectively promoted urinary glucose excretion in DCM rats. A RDN removed renal sympathetic nerves and attenuated sympathetic outflow. a Representative images of TH immunohistochemical staining in the renal vessels (magnification, 100×) and renal cortex (magnification, ×200). b Quantitative analysis of TH-positive areas in the renal cortex. c ELISA quantification of renal norepinephrine levels. B RDN facilitated UGE by regulating renal SGLT2 expression. a Representative images of SGLT2 immunofluorescence staining in the kidney (magnification, ×200). b WB analysis of protein levels of SGLT2 in the kidney. c Quantitative analysis of SGLT2 fluorescence intensity in the kidney. d The relative levels of SGLT2 calculated from the WB. e The 24-h urine volume of rats from three groups collected in the metabolic cage study. f Urinary glucose concentration of rats from three groups. (P < 0.05; n = 6, 9, and 10 in the control, DCM, and RDN groups, respectively; five sections were randomly selected per sample.) *P < 0.05 vs. the control group; #P < 0.05 vs. the DCM group. RDN renal denervation, DCM diabetic cardiomyopathy, TH tyrosine hydroxylase, UGE urinary glucose excretion, SGLT2 sodium-glucose cotransporter 2.

Article Snippet: Immunofluorescence Immunofluorescence (IF) staining with an anti-SGLT2 antibody (Proteintech, Rosemont, Illinois, USA) was performed to detect the distribution of SGLT2 in the kidney.

Techniques: Immunohistochemical staining, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Concentration Assay, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Renal denervation ameliorates cardiac metabolic remodeling in diabetic cardiomyopathy rats by suppressing renal SGLT2 expression.

doi: 10.1038/s41374-021-00696-1

Figure Lengend Snippet: Fig. 6 The protective effects of RDN were mediated by regulation of renal SGLT2 expression. A The combination of RDN and SGLT2i did not further ameliorate glycotoxicity. a Representative images of AGEs staining in the myocardium (magnification, ×400) of rats from the four groups. b WB analysis of protein levels of O-GlcNAcylation in the hearts. c Quantitative analysis of AGEs-positive regions in the heart. d The relative levels of O-GlcNAcylation calculated from WB. B The combination of RDN and SGLT2i did not further ameliorate lipotoxicity. a Representative images of oil red O staining in the hearts (magnification, ×400) of rats from the four groups. b Quantitative analysis of lipid accumulation in the heart. c ELISA quantification of myocardial ceramide levels. d ELISA quantification of myocardial triglyceride levels. C The combination of RDN and SGLT2i did not further ameliorate mitochondrial dysfunction. a Representative images of mitochondria (magnification, ×7000) of rats from the four groups. (b) ELISA quantification of myocardial ATP levels. c The analysis of mitochondrial membrane potential. (P < 0.05; n = 6, 10, 8, and 9 in the DCM, RDN, SGLT2i and RDN + SGLT2i groups, respectively; five sections were randomly selected per sample.) *P < 0.05 vs. the DCM group. AGEs advanced glycation end products.

Article Snippet: Immunofluorescence Immunofluorescence (IF) staining with an anti-SGLT2 antibody (Proteintech, Rosemont, Illinois, USA) was performed to detect the distribution of SGLT2 in the kidney.

Techniques: Expressing, Staining, Enzyme-linked Immunosorbent Assay, Membrane

Journal: Medicina

Article Title: Effects of Lactobacillus Plantarum and Lactobacillus Helveticus on Renal Insulin Signaling, Inflammatory Markers, and Glucose Transporters in High-Fructose-Fed Rats

doi: 10.3390/medicina55050207

Figure Lengend Snippet: The protein expression levels of insulin receptor substrate (IRS-1) ( a ), protein kinase B (AKT) ( b ), endothelial nitric oxide synthase (eNOS) ( c ), sodium-glucose cotransporter-2 (SGLT2) ( d ), and glucose transporter type-5 (GLUT5) ( e ) in kidney tissues of Control, Fruc, Fruc + LP, and Fruc + LH groups. The protein levels were quantified using densitometry and normalized with GAPDH. Representative Western blot images are included above the corresponding figures. Each bar represents at least six rats. * p < 0.05, significantly different from control; # p < 0.05, significantly different from fructose. Fruc: Fructose; LP: L. Plantarum ; LH: L. Helveticus; IRS-1: insulin receptor substrate 1; AKT: protein kinase B; eNOS: endothelial nitric oxide synthase; SGL2: sodium-glucose cotransporter-2.

Article Snippet: Primary antibodies were utilized for priming the respective IRS-1 (anti-IRS-1 rabbit IgG, Santa Cruz, CA, USA, 1:1000), eNOS (anti- eNOS rabbit IgG, Santa Cruz, CA, USA, 1:500), AKT (anti- AKT rabbit IgG, Santa Cruz, CA, USA, 1:1000), SGLT2 (anti-SGLT2 rabbit IgG, BosterBio, Wuhan, China, 1:1000), and GLUT5 (anti-GLUT5 rabbit IgG, BosterBio, Wuhan, China, 1:1000) proteins for 2 h at room temperature or overnight at +4 °C.

Techniques: Expressing, Control, Western Blot

Journal: Circulation

Article Title: Metabolic Communication by SGLT2 Inhibition

doi: 10.1161/CIRCULATIONAHA.123.065517

Figure Lengend Snippet: Overview of study design and phenotypes. A , Overall discovery strategy. Wild-type (WT) and hyperglycemic type 1 diabetic Akita mice were put on a high-fat, high-carbohydrate Western diet for 4 weeks, followed by SGLT2i (sodium-glucose cotransporter 2 inhibitor), dapagliflozin (10 mg/kg of Western diet), or vehicle (repelleted Western diet) for 1 week. B , Organs and body fluids were snap-frozen and subjected to multi-omics analysis using mass spectrometry. SGLT2 interactome was determined from kidney. C , Main findings. D , Overview of functional follow-up studies and key findings. GDF15 indicates growth/differentiation factor 15; RBC, red blood cell; and WAT, white adipose tissue.

Article Snippet: The antibodies used were rabbit anti-murine SGLT2 (20802; BiCell), specificity for SGLT2 previously confirmed using Sglt2 KO mice, rabbit anti-human SGLT2 (142195; Cell Signaling; amino terminus targeted), and, for isotype control, rabbit immunoglobulin G (Sigma).

Techniques: Western Blot, Biomarker Discovery, Mass Spectrometry, Functional Assay

Journal: Circulation

Article Title: Metabolic Communication by SGLT2 Inhibition

doi: 10.1161/CIRCULATIONAHA.123.065517

Figure Lengend Snippet: Integrated proteome/interactome analysis indicates broad remodeling of kidney metabolite transport by SGLT2i. A , Volcano plot quantification of proteomics analysis of kidney cortex. Overlapping significant proteins (false discovery rate <0.05) between wild-type (WT) and Akita mice upon SGLT2i (sodium-glucose cotransporter 2 inhibitor) treatment are labeled with their gene name. B , Enrichment of Gene Ontology Biological Process (GOBP) terms in WT kidney cortex (kidney cortex proteome was used as background). C , Nephron localization of altered transmembrane transporters of the solute carrier group (SLC). D , Mass spectrometry–based interactome analysis of mouse SGLT2i. SGLT2 interacts with membrane transporters that mediate the reabsorption or secretion of sodium, amino acids, urate, protons, anions, and multiple other compounds. E , Human tissue atlas of selected putative human SGLT2 interactors (source: The Human Proteome Atlas; https://www.proteinatlas.org ).

Article Snippet: The antibodies used were rabbit anti-murine SGLT2 (20802; BiCell), specificity for SGLT2 previously confirmed using Sglt2 KO mice, rabbit anti-human SGLT2 (142195; Cell Signaling; amino terminus targeted), and, for isotype control, rabbit immunoglobulin G (Sigma).

Techniques: Labeling, Mass Spectrometry, Membrane

Journal: Circulation

Article Title: Metabolic Communication by SGLT2 Inhibition

doi: 10.1161/CIRCULATIONAHA.123.065517

Figure Lengend Snippet: Untargeted metabolome analysis reveals SGLT2i-induced alterations in amino acid and organic anion metabolism. A , Overview of untargeted metabolomics results in wild-type (WT) and diabetic Akita mice. Significant metabolites from >30 000 quantified features with breakdown of altered metabolites by organ and genotype. B , The metabolite pool is sensitive to changes in production as well as kidney secretion and reabsorption. C , Illustration of interorgan communication by linking solute carrier group (SLCs) downregulated in the kidney to enhanced urinary metabolites and depleted metabolites in other organs. D , SGLT2 (sodium-glucose cotransporter 2)–dependent changes in plasma:urine ratios indicate increased urinary excretion of glucose and glucose metabolites, and less secretion of substrates of the kidney organic anion transporters (see text for details). Urine metabolites were normalized to creatinine. E , Immunoblot analysis of OAT1 (organic anion transporter 1) in SGLT2i-treated WT mice shows reduction of OAT1 (Slc22a6; normalized to b-actin; 2-sided t test), consistent with proteomics results. F , Targeted analysis of microbiota-derived organic anions (uremic toxins) in the plasma of WT and diabetic Akita mice. RBC indicates red blood cell.

Article Snippet: The antibodies used were rabbit anti-murine SGLT2 (20802; BiCell), specificity for SGLT2 previously confirmed using Sglt2 KO mice, rabbit anti-human SGLT2 (142195; Cell Signaling; amino terminus targeted), and, for isotype control, rabbit immunoglobulin G (Sigma).

Techniques: Clinical Proteomics, Western Blot, Derivative Assay

Journal: Circulation

Article Title: Metabolic Communication by SGLT2 Inhibition

doi: 10.1161/CIRCULATIONAHA.123.065517

Figure Lengend Snippet: Metaproteomics analysis demonstrates reshaping and attenuation of the microbiome fermentation of amino acids to uremic toxins by SGLT2i. A , Taxonomic analysis of metaproteome of gut microbiota with SGLT2i (sodium-glucose cotransporter 2 inhibitor) on family level. B , Cumulative histogram analysis of cresol- and phenol-producing bacteria species on metaproteome level, as well as tryptophan-metabolizing bacteria. C , Murine fecal metabolomics results of key metabolites. D , Targeted metabolomics of human fecal microbiota fermentation (48 hours) in the presence or absence of SGLT2i dapagliflozin (100 nM). Metabolites with >0.5-fold change (48 hours fermentation versus control) and P <0.05 between both conditions are labeled with an open circle (3 donors, with 3 fermentations each). IAA indicates indoleacrylic acid; and IL, indolelactate.

Article Snippet: The antibodies used were rabbit anti-murine SGLT2 (20802; BiCell), specificity for SGLT2 previously confirmed using Sglt2 KO mice, rabbit anti-human SGLT2 (142195; Cell Signaling; amino terminus targeted), and, for isotype control, rabbit immunoglobulin G (Sigma).

Techniques: Metaproteomics, Bacteria, Control, Labeling

Journal: Circulation

Article Title: Metabolic Communication by SGLT2 Inhibition

doi: 10.1161/CIRCULATIONAHA.123.065517

Figure Lengend Snippet: Using Sglt2 KO mice to probe for metabolic off-target effects of SGLT2i. A , Study design. B , Quantification of glucosuria. C , Quantification of plasma:urine ratios in dapagliflozin-treated mice and Sglt2 knockout (KO) mice as compared with untreated wild-type (WT) mice. D , Rank of changes in plasma:urine ratios with SGLT2i (sodium-glucose cotransporter 2 inhibitor; all P <0.05). E , Analysis of off-target effects on mouse plasma, defined as significant changes in the presence of SGLT2i in Sglt2 KO mice. F , Quantification of p-cresol sulfate abundance in response to SGLT2i in both WT and Sglt2 KO mice. B and F , 2-way ANOVA to probe for a significant effect of Sglt2 genotype ( Sglt2 ), dapagliflozin (dapa), or the interaction between the 2 factors ( P interaction ). If the interaction was statistically significant, then a pairwise multiple comparison procedure (Holm-Sidak method) identified the significant effects. * P <0.05 versus vehicle; # P <0.05 versus WT.

Article Snippet: The antibodies used were rabbit anti-murine SGLT2 (20802; BiCell), specificity for SGLT2 previously confirmed using Sglt2 KO mice, rabbit anti-human SGLT2 (142195; Cell Signaling; amino terminus targeted), and, for isotype control, rabbit immunoglobulin G (Sigma).

Techniques: Clinical Proteomics, Knock-Out, Comparison

Journal: Circulation

Article Title: Metabolic Communication by SGLT2 Inhibition

doi: 10.1161/CIRCULATIONAHA.123.065517

Figure Lengend Snippet: SGLT2i-induced reduction in uremic toxins translates to humans. A and D , Design of decompensated heart failure (HF) cohort and randomized controlled trial in patients with diabetes. B and C , Decompensated HF: heatmaps of SGLT2i (sodium-glucose cotransporter 2 inhibitor)–induced log2 fold changes for comparisons at discharge ( B ) and between admission and discharge ( C ). D , Randomized control trial of patients with diabetes: heatmaps of SGLT2i-induced log2 fold changes for SGLT2i versus placebo ( E ) and treatment versus baseline ( F ). SGLT2i treatment reduces uremic toxins originating from aromatic amino acid fermentation.

Article Snippet: The antibodies used were rabbit anti-murine SGLT2 (20802; BiCell), specificity for SGLT2 previously confirmed using Sglt2 KO mice, rabbit anti-human SGLT2 (142195; Cell Signaling; amino terminus targeted), and, for isotype control, rabbit immunoglobulin G (Sigma).

Techniques: Control

Journal: Circulation

Article Title: Metabolic Communication by SGLT2 Inhibition

doi: 10.1161/CIRCULATIONAHA.123.065517

Figure Lengend Snippet: Effects of SGLT2i-modulated uremic toxin, p-cresol, on human engineered heart tissue. A , Summary of metabolite changes in response to SGLT2i (sodium-glucose cotransporter 2 inhibitor) in mouse, rat, and human. B , Human engineered heart tissue (EHT) was exposed to either DMSO (black) or 300 µM p-cresol (PCL; green) for 5 days. Bar charts show force (mN), and relaxation time from peak to 80% relaxation (RT 80%; seconds) in spontaneously contracting EHTs. C , Time course of spontaneous force 1 day before exposure to 3 mM PCL (−1; green) or DMSO (black) until 5 days of daily treatment. After 3 days of treatment, PCL was replaced by DMSO (green arrows) for another 48 hours’ incubation (n=16–22 per group, derived from 4 [300 µM] and 5 [3 mM] independent rounds of cardiac differentiation unpaired, parametric T test [** = P<0.01 and **** = P<0.0001]). D and E , Proteomic effects of incubation of EHT with p-cresol (300 µM) or other SGLT2i-modulated metabolites for 5 days. F , GDF15 (growth/differentiation factor 15) in patients with heart failure (HF) with new SGLT2i treatment (patients from Figure 6A). FDR indicates false discovery rate; HTN, hypertension; and WT, wild type.

Article Snippet: The antibodies used were rabbit anti-murine SGLT2 (20802; BiCell), specificity for SGLT2 previously confirmed using Sglt2 KO mice, rabbit anti-human SGLT2 (142195; Cell Signaling; amino terminus targeted), and, for isotype control, rabbit immunoglobulin G (Sigma).

Techniques: Incubation, Derivative Assay

Journal: Circulation

Article Title: Metabolic Communication by SGLT2 Inhibition

doi: 10.1161/CIRCULATIONAHA.123.065517

Figure Lengend Snippet: Integrated views on the reconfiguration by SGLT2i of the early proximal tubule and systemic metabolic communication. A , Summary of findings and metabolic communication of SGLT2i (sodium-glucose cotransporter 2 inhibitor) in an early proximal tubule centric view in wild-type (WT) mice. Proteins with red lining were upregulated and proteins with blue lining were downregulated by SGLT2i. Proteins showing green filling have been shown to interact with SGLT2 in affinity purification analysis (see also Figure 2D). For further discussion, see text and Figure S8 . B , Summary of findings and metabolic communication of SGLT2i on a systemic level in WT mice. C , Chemical structures of SGLT2 inhibitors dapagliflozin and empagliflozin and p-cresol. ACAT3 indicates acetyl-coenzyme A acetyltransferase 3; AGE, advanced glycation end products; AMPK, 5′-adenosine monophosphate–activated protein kinase; CKD, chronic kidney disease; ER, endoplasmic reticulum; GLUT2, glucose transporter 2; HGD, homogentisate 1,2-dioxygenase; NHE3, Na + /H + exchanger 3; OAT1, organic anion transporter 1; PDZK1IP1, PDZK1 interacting protein 1; SLC, solute carrier; TMEM27, transmembrane protein 27; and WAT, white adipose tissue.

Article Snippet: The antibodies used were rabbit anti-murine SGLT2 (20802; BiCell), specificity for SGLT2 previously confirmed using Sglt2 KO mice, rabbit anti-human SGLT2 (142195; Cell Signaling; amino terminus targeted), and, for isotype control, rabbit immunoglobulin G (Sigma).

Techniques: Affinity Purification