Dapagliflozin mitigates cellular stress and inflammation through PI3K/AKT pathway modulation in cardiomyocytes, aortic endothelial cells, and stem cell-derived β cells

Abstract

Dapagliflozin (DAPA), a sodium-glucose cotransporter 2 (SGLT2) inhibitor, is well-recognized for its therapeutic benefits in type 2 diabetes (T2D) and cardiovascular diseases. In this comprehensive in vitro study, we investigated DAPA's effects on cardiomyocytes, aortic endothelial cells (AECs), and stem cell-derived beta cells (SC-β), focusing on its impact on hypertrophy, inflammation, and cellular stress. Our results demonstrate that DAPA effectively attenuates isoproterenol (ISO)-induced hypertrophy in cardiomyocytes, reducing cell size and improving cellular structure. Mechanistically, DAPA mitigates reactive oxygen species (ROS) production and inflammation by activating the AKT pathway, which influences downstream markers of fibrosis, hypertrophy, and inflammation. Additionally, DAPA's modulation of SGLT2, the Na+/H + exchanger 1 (NHE1), and glucose transporter (GLUT 1) type 1 highlights its critical role in maintaining cellular ion balance and glucose metabolism, providing insights into its cardioprotective mechanisms. In aortic endothelial cells (AECs), DAPA exhibited notable anti-inflammatory properties by restoring AKT and phosphoinositide 3-kinase (PI3K) expression, enhancing mitogen-activated protein kinase (MAPK) activation, and downregulating inflammatory cytokines at both the gene and protein levels. Furthermore, DAPA alleviated tumor necrosis factor (TNFα)-induced inflammation and stress responses while enhancing endothelial nitric oxide synthase (eNOS) expression, suggesting its potential to preserve vascular function and improve endothelial health. Investigating SC-β cells, we found that DAPA enhances insulin functionality without altering cell identity, indicating potential benefits for diabetes management. DAPA also upregulated MAFA, PI3K, and NRF2 expression, positively influencing β-cell function and stress response. Additionally, it attenuated NLRP3 activation in inflammation and reduced NHE1 and glucose-regulated protein GRP78 expression, offering novel insights into its anti-inflammatory and stress-modulating effects. Overall, our findings elucidate the multifaceted therapeutic potential of DAPA across various cellular models, emphasizing its role in mitigating hypertrophy, inflammation, and cellular stress through the activation of the AKT pathway and other signaling cascades. These mechanisms may not only contribute to enhanced cardiac and endothelial function but also underscore DAPA's potential to address metabolic dysregulation in T2D.

Keywords: AKT signaling; Beta cells; Cardiomyocyte; Dapagliflozin; Endothelial cells; Inflammation; Sodium-glucose cotransporter.

Fig. 1

DAPA reduces ISO-induced cardiomyocyte hypertrophy by decreasing ROS in both co-treatment and post-stimulation conditions. A Cardiomyocytes were stimulated for hypertrophy by treating them with ISO at concentrations ranging from 2.5 to 20 µM for 24 and 48 h, followed by cell viability assessment. B The impact of DAPA was investigated by treating cardiomyocytes with DAPA at concentrations ranging from 5 to 20 µM for 24 and 48 h, followed by cell viability assessment C and D Immunofluorescence (IF) data were collected to measure cell areas of ISO-treated cardiomyocytes at ISO concentrations ranging from 10 to 20 µM for 24 and 48 h (D), as well as DAPA-treated cardiomyocytes at concentrations ranging from 10 to 20 µM for the same duration (E and F). Cell area measurements were conducted for cardiomyocytes subjected to co-treatment with ISO + DAPA for 24 h (G and H). I-J mRNA gene expression study for hypertrophy markers ANP and BNP, (K, L, M, and N) protein expression study and immunoblot for ANP and BNP. (O) ROS levels were measured in cardiomyocytes exposed to ISO stimulation for 24 h, with and without prior treatment with DAPA for the same duration. The measurements were taken at 2- and 4-hours post-stimulation. P The pre-protective effect of DAPA was evaluated by administering it to cardiomyocytes for 24 h before ISO stimulation, followed by ROS measurements at 2 and 4 h after ISO stimulation. In this protocol, cardiomyocytes were first stimulated with ISO for 24 h to induce hypertrophy, followed by treatment with DAPA for an additional 24 h. Immunofluorescence staining images were captured at 10x magnification, with scale bars set at 100 μm. Error bars represent the standard error of the mean. Statistical significance is denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

DAPA reduces ISO-induced cardiomyocytes by Activating the AKT Pathway, enhancing GLUT1 expression, and downregulating pro-fibrotic markers. A-I qPCR and J-R Western blot analyses were performed to assess the gene and protein expression levels of PI3K/AKT, fibrosis markers (pSMAD2, ASMA), the apoptosis marker (P-p38), sodium-glucose cotransporters (SGLT-1 and SGLT-2), the sodium-hydrogen exchanger (NHE-1), and the glucose transporter protein (GLUT-1). S Representative Western blot images for the proteins. Statistical significance is represented as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 3

DAPA inhibits ISO-induced cardiomyocyte inflammation and modulates antioxidant responses. A-E qPCR and F-J Western blot analyses were performed to evaluate the gene and protein expression levels of inflammatory markers (NLRP3), antioxidant markers (NRF2, NQO1, and HO-1), and endothelial nitric oxide synthase (eNOS). S Representative Western blot images for these proteins are presented. K-N ELISA assays were conducted to measure IL-1β, IL-6, and TNFα levels in the cell supernatant. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 4

DAPA reduces TNFα-induced inflammation in AECs by modulating the AKT pathway. A-D MTT assays were performed with various treatments: A TNFα alone, B DAPA alone, C pre-treatment with TNFα for 24 h followed by DAPA, and D co-treatment with TNFα + DAPA for 24 h. E-U mRNA gene expression studies for AKT, PI3K, mitochondrial marker (GRP78), inflammation and antioxidant markers (MAPK, NF-κB, NRF2, NLRP3, IL-1β, TNFα, IL-6), adhesion molecules (ICAM, VCAM), SGLT1, SGLT2, NHE-1, GLUT-1 and eNOS. V-Y protein expression study for pAKT/AKT, SGLT1, SGLT2 and TNFα and Z Representative Western blot images for the proteins. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5

DAPA effects on extracellular matrix (ECM) remodeling, vascular function, and inflammation in AECs. A-B Cell counting and representative images of immunofluorescence staining for SGLT2 C-D ICAM and E-F collagen expression. G-N ELISA measurement of ICAM-1, VCAM-1, eNOS, iNOS, NO, IL-1β, IL-6 and TNFα. Immunofluorescence staining images were captured at 20x magnification, with scale bars set at 100 μm. Statistical significance is denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 6

DAPA improves the insulin functionality of SC-β cells. A Representative image of the directed differentiation protocol for SC-β cells in-vitro. B A representative image of a fully developed SC-β cluster at step 6 (4x magnification). C-D Flow cytometry and immunofluorescence (IF) images for all stages, from embryonic stem cells (ESC), definitive endoderm (DE), gut tube endoderm (GTE), pancreatic progenitor 1, pancreatic progenitor 2, endocrine (ED), to stem cell-derived beta (SC-β) cells. E Glucose-stimulated insulin secretion (GSIS) of SC-β clusters. F Counting of insulin and NKX6.1-positive cells using flow cytometry. G-H GSIS ELISA measurement of SC-β between control and DAPA-treated groups, along with stimulation index measurement. I GSIS of human islets in control and DAPA treatment measured by ELISA. Error bars represent the standard error of the mean. Statistical significance is denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 7

DAPA modulates SC-β cell function, inflammation, and stress response. A-B Representative images of cell counting and immunofluorescence and for KI67. C-F mRNA gene expression of β-cell-related markers (Insulin, MAFA, GLUT1, and GLUT2) (C), AKT pathway-related genes (PI3K, NRF2) (D), inflammation-related pathways (MAPK, NLRP3, IL-6) (E), and mitochondria-related genes (XBP1, IRE-1, and GRP78) (F) in control and DAPA-treated groups. (G-K) mRNA gene expression of β-cell-related markers (Insulin, MAFA, GLUT1, and GLUT2) (G), AKT pathway-related genes (PI3K, AKT, and NRF2) (H), inflammatory and antioxidant genes (MAPK, NF-κB, NLRP3, IL-6, and TNFα) (I), GRP78 (J), and NHE-1 (K) comparing control, DAPA-treated, LPS-stimulated, and LPS + DAPA co-treated groups. Immunofluorescence staining images were captured at 10x and 20x magnification, with scale bars set at 100 μm. Error bars represent the standard error of the mean. Statistical significance is denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001