SGLT1 Expression Research Articles

Inhibition of SGLT2 protects podocytes in diabetic kidney disease by rebalancing mitochondria-associated endoplasmic reticulum membranes

BackgroundSodium-glucose cotransporter 2 (SGLT2) inhibitors have changed the therapeutic landscape for diabetic kidney disease (DKD) patients, but their underlying mechanisms are complicated and not fully understood. Mitochondria-associated endoplasmic reticulum membranes (MAMs), the dynamic contact sites between mitochondria and the endoplasmic reticulum (ER), serve as intracellular platforms important for regulating cellular fate and function. This study explored the roles and mechanisms of SGLT2 inhibitors in regulating MAMs formation in diabetic podocytes.MethodsWe assessed MAMs formation in podocytes from DKD patients’ renal biopsy samples and induced an increase in MAMs formation in cultured human podocytes by transfecting OMM-ER linker plasmid to investigate the effects of MAMs imbalance on podocyte injury. Empagliflozin-treated diabetic mice and podocyte-specific SGLT2 knockout diabetic mice (diabetic states were induced by streptozotocin and a high-fat diet), empagliflozin-treated podocytes, SGLT2-downregulated podocytes, and SGLT2-overexpressing podocytes were used to investigate the effects and mechanisms of SGLT2 inhibitors on MAMs formation in diabetic podocytes.ResultsMAMs were increased in podocytes and were associated with renal dysfunction in DKD patients. Increased MAMs aggravated HG-induced podocyte injury. The expression of SGLT2 was increased in diabetic podocytes. In addition, empagliflozin-treatment and podocyte-specific SGLT2 knockout attenuated MAMs formation and podocyte injury in diabetic mice. Empagliflozin treatment and SGLT2 knockdown decreased podocyte MAMs formation by activating the AMP-activated protein kinase (AMPK) pathway, while SGLT2 overexpression had the opposite effect.ConclusionsInhibition of SGLT2 attenuates MAMs imbalance in diabetic podocytes by activating the AMPK pathway. This study expands our knowledge of the roles of SGLT2 inhibitors in improving DKD podocyte injury and provides new insights into DKD treatment.

Uninephrectomy and sodium-glucose cotransporter 2 inhibitor administration delay the onset of hyperglycemia.

The kidneys are essential for glucose homeostasis, as they perform gluconeogenesis, utilize glucose, and reabsorb glucose. Reabsorption is performed by SGLT2, which is responsible for about 90%. However, little is known about how renal glucose handling is altered in patients with chronic kidney disease (CKD). SGLT2 inhibitors have demonstrated efficacy in suppressing CKD progression in clinical trials, but their mechanisms are not fully understood. Therefore, this study aimed to evaluate how each uninephrectomy (UNx) and SGLT2 inhibitor affects blood glucose concentrations and SGLTs dynamics in rats with type 2 diabetes mellitus. Male rats were divided into four treatment groups: sham + placebo, sham + dapagliflozin, UNx + placebo, and UNx + dapagliflozin. There were few group differences in food intake or body weight, but blood glucose concentrations continued to rise in the sham + placebo, whereas this rise was delayed for several weeks in the UNx + placebo, and largely suppressed by dapagliflozin. SGLT2 mRNA expression was significantly lower in the UNx group, but SGLT1 mRNA expression did not significantly differ. Dapagliflozin did not alter SGLT1 or SGLT2 mRNA expression. In animal models of diabetes, renal glucose reabsorption appears likely to be a major contributor to the development of hyperglycemia.

Human epicardial adipose tissue-derived factors promote atrial endothelial dysfunction: role of pro-inflammatory cytokines and AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway

Abstract Introduction Epicardial adipose tissue (EAT) has been suggested to contribute to left atrium dysfunction via paracrine mechanisms to induce endothelial dysfunction, pro-arrhythmogenic and pro-remodeling responses thereby favoring atrial fibrillation (AF) onset. Recently, sodium-glucose co-transporter2 inhibitors (SGLT2i) have been shown to reduce AF incidence, EAT volume, differentiation and inflammation, however, the underlying mechanisms are unknown. Purpose This study investigates the impact of human EAT-derived mediators on atrial endothelial cell function and, if so, to characterize the role of SGLT2 using the inhibitor empagliflozin (EMPA). Methods Epicardial and subcutaneous adipose tissues (SAT) were collected from 70 cardiac patients at a university hospital. Conditioned medium (CM, 24 h) from fats were applied to porcine atrial endothelial cells (AECs, first passage) for 24 h. Histology of tissues was assessed by haematoxylin and eosin staining, protein expression by Western blot analysis or immunofluorescence staining, mRNA levels by RT-qPCR, formation of reactive oxygen species (ROS) and nitric oxide (NO) by fluorescent probes, and pro-inflammatory cytokine levels by ELISA. Results Compared to SAT, EATs were more vascularized with increased infiltration of M1-like macrophages, expression of proinflammatory cytokines (IL-1ß, IL-6, TNF-α), markers of ECs VCAM-1 and eNOS, fibrosis TGF-β, NADPH oxidases NOX-1 and SGLT1/2. These effects were associated with a pro-oxidant response that was inhibited by neutralizing Abs directed against IL-1ß or IL-6, and by inhibition of either NOS, NADPH oxidases, ACE, AT1R or SGLT2. Levels of SGLT2 and ROS in EATs were higher in AF compared to non-AF patients and progressively increasing with age. CM of EATs showed higher concentrations of IL-1ß, IL-6, TNF-α and MCP-1 than that of SAT. Exposure of AECs to CM of EATs increased the level of oxidative stress that was positively correlated to the level of pro-inflammatory cytokines, and reduced basal and bradykinin-induced NO formation that was prevented by EMPA. It also resulted in upregulation of p53 and SGLT2 expression, and nuclear translocation of NF-kB by CM of the top quartile inflamed EATs but not by the lowest quartile of EATs or SATs, prevented by EMPA. Conclusion The findings indicate that compared to SATs, the EATs showed higher in situ expression and release of pro-inflammatory cytokines that contributed to oxidative stress involving the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway. CMs of inflamed EATs induced AECs dysfunction, oxidative stress and activation of NF-kB involving pro-inflammatory cytokines and SGLT2. Thus, SGLT2i appear as an interesting strategy to decrease EAT inflammation and improve endothelial function contributing to prevent cardiac remodeling and fibrotic responses.

Impact of plasma from patients with severe aortic stenosis on valvular endothelial cells

Abstract Background The natural history of aortic stenosis (AS) encompasses a spectrum of pathophysiological stages, including endothelial dysfunction, inflammation, fibrosis and calcification. Accumulating data underscore the detrimental impact of the haemostatic system on AS, contributing to disease progression and the development of a prothrombotic phenotype within the valve. Understanding these mechanisms is pivotal for the exploration of novel therapeutic avenues, particularly in the context of transcatheter aortic valve replacement (TAVR). Following TAVR, the native diseased aortic valve persists, maintaining its pathological activity and thrombogenic effects on the bioprosthetic valve, potentially influencing its durability and patient outcomes. The precise effects of plasma and its various components on valvular endothelial cells (VECs) dysfunction remains inadequately explored. Purpose To evaluate the influence of plasma derived from individuals with severe AS on aortic VECs. Methods Plasma samples obtained from patients with severe AS undergoing TAVR (n=100) were collected immediately before the procedure, alongside plasma samples from healthy individuals. Factor (F)Xa activity within the plasma samples was quantified using fluorometer-based method. Porcine VECs were then exposed to either plasma (at 10% concentration) or isolated FXa (1 nM), with or without different pharmacological modulators. Protein expression levels were assessed by Western blot analyses. The level of reactive oxygen species (ROS) was evaluated by dihydroethidium staining. Results Exposure of VECs to plasma from severe AS patients promoted ROS formation compared to plasma from healthy individuals (1402 ± 438 AU vs 732 ± 137 AU, p=0.001) (Figure 1). The heightened oxidative stress elicited by plasma from severe AS patients was attenuated by empagliflozin, perindoprilat, losartan, neutralizing antibodies directed against IL-1, IL-6 and TNF-α. Plasma from severe AS patients showed elevated FXa activity compared to plasma from healthy individuals (179.4 ± 101.3 ng/mL vs 63.2 ± 87.2 ng/mL, p=0.02). Stimulation of VECs with isolated FXa resulted in a sustained formation of ROS, which was blunted by empagliflozin, perindoprilat, losartan, rivaroxaban, dabigatran and PAR-1, 2 and 4 antagonists. Quantification of protein expression in VECs stimulated by isolated FXa revealed an endothelial dysfunction phenotype characterized by increased expression of SGLT2, VCAM-1 and COX-1, and decreased expression of eNOS. Conclusions Plasma from patients with severe AS exerts a detrimental effect on aortic VECs, characterized by a pronounced oxidative stress response. This adverse impact is mediated by multiple mechanisms, including activation of the AT1R/NADPH oxidase/SGLT2 pathway, upregulation of pro-inflammatory cytokines, and activation of PAR. FXa plays a pivotal role by promoting oxidative stress in VECs and contributing to valvular endothelial dysfunction.Pro-oxidant state in VECs.

Regulation of Enterocyte Brush Border Membrane Primary Na-Absorptive Transporters in Human Intestinal Organoid-Derived Monolayers.

In the small intestine, sodium (Na) absorption occurs primarily via two apical transporters, Na-hydrogen exchanger 3 (NHE3) and Na-glucose cotransporter 1 (SGLT1). The two primary Na-absorptive pathways were previously shown to compensatorily regulate each other in rabbit and rat intestinal epithelial cells. However, whether NHE3 and SGLT1 regulate one another in normal human enterocytes is unknown, mainly due to a lack of appropriate experimental models. To investigate this, we generated 2D enterocyte monolayers from human jejunal 3D organoids and used small interfering RNAs (siRNAs) to knock down NHE3 or SGLT1. Molecular and uptake studies were performed to determine the effects on NHE3 and SGLT1 expression and activity. Knockdown of NHE3 by siRNA in enterocyte monolayers was verified by qPCR and Western blot analysis and resulted in reduced NHE3 activity. However, in NHE3 siRNA-transfected cells, SGLT1 activity was significantly increased. siRNA knockdown of SGLT1 was confirmed by qPCR and Western blot analysis and resulted in reduced SGLT1 activity. However, in SGLT1 siRNA-transfected cells, NHE3 activity was significantly increased. These results demonstrate for the first time the functionality of siRNA in patient-derived organoid monolayers. Furthermore, they show that the two primary Na absorptive pathways in human enterocytes reciprocally regulate one another.

Salmon Milt Extract Suppresses Glucose Uptake by Downregulating SGLT1 and GLUT2 Expression in Caco-2 Cells.

Salmon milt extract (SME) is rich in nucleotides, especially deoxyribonucleoside monophosphates (dNMPs), which has the potential to exert anti-obesity effects. Sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2) are responsible for absorbing sugar from the small intestine. The purpose of this study was to examine the effects of SME on the functions of SGLT1 and GLUT2 and elucidate the mechanisms underlying the inhibition of glucose absorption by SME. We investigated the effect of SME on the expression and function of intestinal glucose transporters, using differentiated Caco-2 cells. SME treatment decreased the expression SGLT1 and GLUT2 mRNA and protein in Caco-2 cells. [14C]-Labelled methyl-α-D-glucopyranoside and [3H]-labelled 2-deoxy-D-glucose (DG) uptake into Caco-2 cells was significantly reduced by SME treatment. Similarly, the dNMP mixture containing the four mononucleotides 2'-deoxyadenosine 5'-monophosphate (dAMP), 2'-deoxyguanosine 5'-monophosphate (dGMP), 2'-deoxycytidine 5'-monophosphate (dCMP), and 2'-deoxythymidine 5'-monophosphate (dTMP) decreased SGLT1 and GLUT2 expression. dNMP mixture-induced reduction in the mRNA expression of these transporters was suppressed when exposed to the mixture without dTMP. Furthermore, dNMP mixture-induced alterations in the expression of hepatocyte nuclear factor (HNF)-1α and HNF1β, which have been characterized as modulators of both transporters also showed a similar trend. dTMP treatment alone decreased GLUT2 expression, resulting in reduced [3H] DG uptake by Caco-2 cells. SME decreased the expression of HNF1α, HNF1β, and its targets SGLT1 and GLUT2, resulting in reduced glucose uptake by Caco-2 cells. In addition, our results revealed that dTMP plays an important role in suppressing the expression of intestinal glucose transporters.

Abstract P138: Diet Influences the Expression of Renal Sodium-Glucose Cotransporters in Juvenile Male Rats

Poor nutrition and metabolic dysfunction are becoming more prevalent among children, as evident by increasing childhood obesity in recent years. The kidney sodium-glucose cotransporter 2 (SGLT2) is a contributor to overall metabolic homeostasis, with inhibition of SGLT2 significantly reducing circulating glucose and lipids. The study objective was to determine if diet influences the expression of renal glucose transporters in young rats. Weanling male Wistar Kyoto rats received 1) standard laboratory chow, 2) AIN-93G diet, or 3) a high-fat diet (60% kcal, HFD). Each dietary group was then further subdivided to receive either filtered water or 5% (w/v) sucrose solution ad libitum . Four weeks later, animals were euthanized and kidneys were collected and processed. mRNA expression of Sglt1 , Sglt2 , Sqstm1 , and Nlrp3 was determined using gene specific primers and RT-PCR. Renal SGLT2 protein expression was visualized using immunohistochemistry and quantified using Imagej software. Diet significantly influenced the renal expression of both Sglt1 ( P =0.005) and Sglt2 (P=0.046), while beverage did not have a significant effect on either ( P =0.7 and P =0.4, respectively). Rats fed the HFD had more than 30% reduction in Sglt1 mRNA expression as compared to rats fed chow, and ~20% reduction in Sglt2 as compared to rats fed AIN. Preliminary data suggest that diet and beverage significantly altered renal protein expression of SGLT2, with rats fed chow having the lowest renal expression that was slightly increased with sugar consumption (1.5% versus 2.7%, respectively). Rats fed AIN had the greatest expression, but was drastically reduced in rats consuming sugar (4% versus 1.2%, respectively). To determine if autophagy and inflammasome activation is involved, the expression of Sqstm1 and Nlrp3 was measured by RT-PCR. Similarly, rats fed HFD had the lowest expression of both Sqstm1 ( P<0.001) and Nlrp3 ( P=0.03). Sqstm1 and Nlrp3 mRNA expression was decreased with sugar consumption in rats fed chow and AIN, whereas Sqstm1 ( P =0.01) and Nlrp3 ( P <0.001) mRNA expression was increased in rats fed HFD with sugar beverage. Our data suggest that diet significantly influences renal glucose transporter expression in juvenile rats in a way that may be mediated by autophagic impairment. The modulatory effect of sugar consumption on SGLT2 protein, Sqstm1, and Nlrp3 was diet-dependent, highlighting the importance of nutrition to renal physiology within weeks after weaning.

Fufang Zhenzhu Tiaozhi (FTZ) capsule ameliorates diabetic kidney disease in mice via inhibiting the SGLT2/glycolysis pathway

Ethnopharmacological relevanceFufang Zhenzhu Tiaozhi (FTZ) capsule is a hospital preparation of a patented traditional Chinese medicine compound. FTZ has been clinically used for nearly 13 years in the treatment of diabetes and glycolipid metabolic diseases. With the significant benefits of SGLT2 inhibitor in patients with diabetic kidney disease (DKD), it provides a research avenue to explore the mechanism of FTZ in treating this disease based on glycolysis pathway. Aim of the studyTo explore the pharmacological characteristics of FTZ in DKD mice and its impact on the glycolysis pathway. Materials and methodsWe induced a DKD model in C57BL/6 mice by injection of streptozotocin (STZ) combined with long-term high-fat diet. We administered three doses of FTZ for 12 weeks of treatment. Kidney function, blood lipid levels, glucose tolerance, and key glycolytic enzymes were evaluated. Renal pathological changes were observed using HE, MASSON, and PAS staining. The potential targets of the active ingredients of FTZ in the glycolysis pathway were predicted using network pharmacology and molecular docking. Validation was performed using immunohistochemistry and Western blotting. ResultsFTZ effectively reduces blood glucose, total cholesterol, triglyceride, low density lipoprotein cholesterol, 24 h proteinuria, serum creatinine, blood urea nitrogen, and increases urinary glucose levels. Glucose tolerance and renal pathological changes were significantly improved by FTZ treatment. Pinusolidic acid, a component of FTZ, shows good binding affinity with three active pockets of SGLT2. WB and immunohistochemistry revealed that FTZ significantly inhibits the expression of SGLT2 and its glycolytic related proteins (GLUT2/PKM2/HK2). Hexokinase, pyruvate kinase, and lactate dehydrogenase in the kidney were also significantly inhibited by FTZ in a dose-dependent manner. ConclusionFTZ may alleviate the progression of DKD by inhibiting the activation of the SGLT2/glycolytic pathway. Our study provides new insights into the clinical application of FTZ in DKD.

Quercetin slow-release system delays starch digestion via inhibiting transporters and enzymes

This study investigates the potential of a quercetin-based emulsion system to moderate starch digestion and manage blood glucose levels, addressing the lack of in vivo research. By enhancing quercetin bioaccessibility and targeting release in the small intestine, the emulsion system demonstrates significant inhibition of starch digestion and glucose spikes through both in vitro and in vivo experiments. The system inhibits α-amylase and α-glucosidase via competitive and mixed inhibition mechanisms, primarily involving hydrogen bonds and van der Waals forces, leading to static fluorescence quenching. Additionally, this system downregulates the protein expression and gene transcription of SGLT1 and GLUT2. These findings offer a novel approach to sustaining glucose equilibrium, providing a valuable foundation for further application of quercetin emulsion in food science.

SGLT2i Alleviates Atherosclerosis by Inhibiting NHE1 Activation to Protect against Macrophage Senescence Induced by Angiotensin II.

Sodium-dependent glucose transporter (SGLT2) inhibitors (SGLT2i) have been found to have anti-atherosclerotic effects in clinical treatment. The aim of this study was to explore whether angiotensin II (Ang II) induces changes in the expression of Na+/H+ exchanger of cytoplasmic membrane channel proteins (NHE1) and SGLT2 in macrophages and whether dapagliflozin (DAPA), an SGLT2i, protects against Ang II induced macrophage senescence by inhibiting NHE1 activation to alleviate Atherosclerosis (AS). After intervention with DAPA plus gavage or feeding them a high-fat diet, the mice's aortas were dissected, and oil red O staining was performed. Cell proliferation and toxicity detection, western blot, immunofluorescence, and β-galactosidase staining methods were adopted to detect cell activity, expressions of senescence-related genes, and number of senescent cells after different concentrations of Ang II or DAPA or plasmid NHE1 were treated with RAW264.7 cells. (1) The formation of AS plaques in ApoE -/- mice showed a downward trend under DAPA. (2) After the intervention of Ang II, the cell activity of RAW264.7 decreased, and the expression of senescent cells and related genes increased. (3) Under the Ang II condition, the expression of SGLT2 and NHE1 increased, and SGLT2, NHE1, and senescence-related genes decreased with the addition of DAPA. (4) The expression of NHE1, senescent cells and related genes decreased in RAW264.7 cells after DAPA treatment with plasmid NHE1 intervention. SGLT2i alleviates atherosclerosis by inhibiting NHE1 activation to protect against macrophage senescence induced by Ang II.

#2940 Expression and modulation of SGLT2 in human podocytes. Not all in tubule

Abstract Background and Aims Inhibitors of the sodium-glucose cotransporter type 2 (iSGLT2) modify important non-glycemic pathways that lead to the protection of target organs. Its renoprotective pleiotropic effects in CKD, associated or not with diabetes, justifies further studies to elucidate the mechanisms of action (MoA) of these drugs. Although the co-transporter SGLT2 is mostly expressed in the proximal tubule, its location and function in other cells, that can be potential therapeutic targets for inhibitor drugs, has not been well studied. We explored the presence of SGLT2 in human podocyte cells and whether it is modified in different situations of cellular stress. Method Differentiated immortalized human podocytes were cultured under different experimental conditions for 24 or 48 h: Normoxia (Control); Normoxia + High concentration of glucose (HG); Hypoxia(1%)+2 h Reoxygenation(H) and the combination H + HG. Cells were also incubated with 0.1 uM Dihydrotestosterone (DHT). SGLT2 gene expression was determined by quantitative PCR and SGLT2 protein was detected by Western Blot (WB) and immunofluorescence (IF). Results Sglt2 gene expression was detected in human podocytes in culture, with an increase of expression after 24 h of exposure to HG. Protein expression by WB showed a trend to increase after 48h of exposure to HG and hypoxia, which was significant with co-incubation with DHT. HG plus DHT also increased the protein expression of SGLT2 under normoxic conditions. The IF measured by intensity of area did not show an increase in the protein expression of the transporter under HG incubation. However, protein expression increased under all hypoxic conditions: Control 50.05 ± 3, Hypoxia 140 ± 16.3 (p < 0.05 vs control) and HG + Hypoxia 253 ± 21.29 (p < 0.001 vs control). Conclusion Our data suggest that podocyte may be one of the therapeutic targets on which the iSGLT2 is acting. The experimental conditions described did not show differences in the expression of the transporter before the single stimulus of glucose, but they did under hypoxia and the combination Hypoxia-high glucose. In addition, co-incubation with testosterone seems to enhance the expression. Studying the MoA of iSGLT2 will open better comprehension of the kidney pathophysiology, particularly in the podocytopathies, regardless of diabetes.

#1870 Uninephrectomized diabetic rats delay onset of hyperglycemia via down-regulation of SGLT2 mRNA

Abstract Background and Aims The kidney is essential for glucose metabolism. Reabsorption of almost all the glucose filtered by the glomerulus is performed by the sodium-glucose cotransporters (the SGLTs) in the proximal tubules, with SGLT2 responsible for about 90%. In addition, the kidney, along with the liver, plays an important role in gluconeogenesis, especially in the long fasting state. SGLT2 inhibitors originally developed for the treatment of type 2 diabetes mellitus (DM) are clinically very effective drugs halting chronic kidney disease progression. The underlying mechanisms are, however, not fully understood. Furthermore, SGLT2 expression in diabetic patients has been reported to increase and decrease but remains unclear. Therefore, this study aimed to evaluate how uninephrectomy (UNx) and SGLT2 inhibitor treatment affects the dynamics of blood glucose and SGLTs in SDT fatty rats with obese type 2 DM (leptin receptor mutation), which is hyperglycemic from an early stage. Method 24 male SDT fatty rats were divided into four groups: sham + placebo group (n=6); sham + dapagliflozin (dapa, 1.5 mg/kg/day) group (n=6); UNx + placebo group (n=6); UNx + dapa group (n=6). UNx or sham operations were undergone at week 6. Measurements included body weight, food intake, water consumption, urine output, blood pressure, serum creatinine, and blood glucose in the experiment. At week 12, the rats were sacrificed, and the kidneys were sampled to measure SGLT1 and SGLT2 mRNA in the renal cortex by real-time PCR quantification. Results Blood glucose levels continued to rise from week 7 in the sham + placebo group, but the rate of increase was still slower in the UNx + placebo group, and the increase in blood glucose levels was mostly suppressed with dapa administration (Fig. 1). Only the Sham + placebo group showed increased urinary volume and weight loss after week 10 due to hyperglycemia. There were no significant differences in serum creatinine or blood pressure during the study. Real-time PCR results showed that SGLT2 mRNA in the renal cortex was significantly decreased by 26% in the UNx group (P=0.0386), but SGLT1 mRNA was not significantly different. Conclusion Uninephrectomized diabetic rats delayed onset of hyperglycemia and reduced SGLT2 mRNA; SGLT2 inhibitor treatment further delayed the onset of hyperglycemia but did not alter SGLT2 mRNA expression. In animal models of diabetes, renal glucose reabsorption appeared to be a major contributor to the development of hyperglycemia.

#1324 The antiproteinuric effect of Gliflozins is associated with the mitochondrial density of endothelial cells in diabetic patients

Abstract Background and Aims The sodium-glucose co-transporter type 2 inhibitors (SGLT2i) such as dapagliflozin and empagliflozin, are a new class of nephro- and cardio-protective drugs in diabetic and non-diabetic patients. SGLT2 is greatly expressed in the proximal tubule cells, and therefore the protective effects of gliflozins are thought to be mediated by a hemodynamic mechanism on glomerular hyperfiltration. However, they could be acting also in other cell types, particularly in diabetic patients, because hyperglycemia induces the expression of SGLT2 in endothelial cells, mesangial cells, cardiomyocytes, etc. Because SGLT2 changes the intracellular concentration of sodium and glucose, gliflozins may indirectly change mitochondrial swelling and activity. In this study, we have tested the hypothesis that gliflozins act on mitochondria in patients suffering from diabetes. Method We have analyzed kidney biopsies from diabetic patients in treatment with a gliflozin (n = 10) or without gliflozin treatment (n = 10) stained with fuchsin red, which gives a color intensity proportional to mitochondrial content. Clinical variables (albumin-creatinine ratio (ACR), blood pressure, eGFR, glycated albumin) have been measured before and after 4 months of treatment. Images have been acquired using a Zeiss Axioscope with 100x objective and analyzed using ImageJ software to quantify staining intensity (expressed as optical density) after background subtraction. Two different cell types have been considered: proximal tubule cells and endothelial cells in glomeruli. Only non-sclerotic glomeruli have been considered. Results The systolic blood pressure was linearly correlated to the intensity of staining of mitochondria in the proximal tubule cells in all patients irrespective of gliflozin treatment. In non-treated diabetic patients, the mitochondrial content of endothelial cells was not related to the extent of ACR reduction over time. In diabetic patients treated with gliflozins, the extent of ACR reduction after treatment was correlated with the intensity of mitochondrial staining in glomerular endothelial cells. This correlation was not present in proximal tubule cells. Gliflozin treatment may induce a change in the mitochondrial content of endothelial cells, particularly in patients responding with a reduction in proteinuria. Conclusion Proteinuria in diabetic patients depends on damage in both the endothelial and podocyte compartments. Gliflozins may improve proteinuria by a hemodynamic mechanism; however, our data suggest an effect of gliflozins specific on mitochondria of the endothelial cells in the glomeruli, which is proportional to their anti-proteinuric effect. Though we do not analyze the mechanism of gliflozins on mitochondria, it is possible to speculate that the changes of intracellular sodium-induced by gliflozins may induce an altered mitochondrial activity.

The off-target NHE1 inhibitory effect of SGLT2 inhibitors in cardiac remodeling

Sodium-glucose cotransporter-2 (SGLT-2) inhibitors, approved for the treatment of diabetes mellitus, have gained attention for their cardioprotective effect. The exact mechanism by which SGLT-2 inhibitors exert their cardioprotective effect remains unclear. Recent studies have suggested that empagliflozin (EMPA), an SGLT inhibitor, exerts its car¬dioprotective effect by inhibiting the Na+/H+ exchanger (NHE); a group of membrane proteins that regulate intracel¬lular pH and cell volume. Increased activity and expression of NHE isoform 1 (NHE1), the predominant isoform ex¬pressed in the heart, leads to cardiac hypertrophy. Our research group investigates the indirect mechanisms by which SGLT inhibitors exert their cardioprotective effect and have demonstrated that angiotensin II (ANG)-induced hyper¬trophy of H9c2 cardiomyoblasts is accompanied with increased SGLT-1 and NHE1 protein expression; an effect which is reversed in the presence of EMPA. In addition, we demonstrated that dapagliflozin improved survival of transgenic mice expressing cardiac-specific NHE1.

Clock and Bmal1 inhibit expression of Sglt1 in mouse Kidneys and glucose reabsorption

Aberrantly elevated sodium/glucose cotransporter 1 (Sglt1) expression and increased glucose reabsorption contribute to obesity or lean-associated metabolic complications, however, the precise mechanisms underlying the high expression of Sglt1 and increased glucose absorption are unclear. Clock (circadian locomotor output cycles kaput protein) is known as the master clock gene; Bmal1 (basic helix-loop-helix arnt like 1) is a known heterodimer of Clock that is a critical regulator of circadian metabolism and has the potential for regulating obesity-related diseases. We examined whether circadian clock genes involving Clock/Bmal1 and the orphan nuclear receptor Shp (the small heterodimer partner) regulate the expression of Sglt1 to maintain glucose reabsorption and glucose homeostasis. Renal Sglt1 levels were increased in Clock-mutant or Bmal1-knockout (KO) mice and were decreased by activation of Bmal1 in an SHP-dependent manner. Mechanistically, the transactivation of Sglt1 by HNF1α (hepatic nuclear factor 1 alpha) was inhibited by SHP. In wild-type mice fed a western diet, activation of Shp by Nobiletin reduced Sglt1 levels and improved glucose reabsorption and glucose handling, but these beneficial effects were not seen in Clock−mutant and Bmal1 KO mice. These results demonstrate that normal inhibition of Sglt1 by Clock/Bmal1-SHP is defective in obesity, resulting in increased Sglt1 levels, glucose reabsorption, and renal steatosis. This Clock/Bmal1-SHP-Sglt1 pathway may present novel targets for treating obesity. This work was supported in part by National Institutes of Health (NIH) grant R56 HL137912 and American Heart Association Scientist Development Grant 2300158 and Grant-in-Aid 16GRNT30960027 to XP; by U.S. NIH grants DK121490, HL137202, HL158054 and HD094778, and VA Merit Award BX004113 to MMH; and NIH grants R01DK112042, R01HL142814, RF1AG061296, the UAB/UCSD O’Brien Center of Acute Kidney Injury NIH-P30DK079337, and the Department of Veterans Affairs to V Vallon. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Long-term intermittent fasting induces sex-dependent changes in small intestine from SAMP8 aged mice

Background: Intermittent fasting (IF) is a popular and effective method to decrease body weight. Yet, the effects of IF on aged intestine is unknown. SAMP8 (Senescence Accelerated Mouse-Prone 8) mice are used as a model for studying aging. This study aimed to examine the potential for IF to ameliorate age-associated disturbances in murine jejunum. Methods: Two-month-old male and female SAMP8 mice were randomly assigned to either an IF protocol or an ad-libitum diet (AL) for the 8-month study (n=7-10 group). Fasted mice followed a 24-hour alternate day fasting protocol. At the end of the study mice were euthanized and jejunum stored at −80°C until use for western blot or other assay, and a segment was fixed for histology. Results: IF resulted in significantly less weight gain in both males (22% less, n=7-10, p<0.05) and females (33% less, n=7-8, p<0.05) and response to glucose load (glucose tolerance testing) was comparable in both sexes: area under curve for AL > IF. Interestingly, IF significantly reduced fasting serum glucose levels in males (30% less, n=6, p<0.05), but IF had no effect on fasting serum glucose in females. In jejunum, we assessed: protein expression, health, and morphology. In females, IF led to a 57% decrease in GLUT2 expression (n=5-6, p<0.05) versus AL. GLUT2 moves glucose/galactose and fructose out of the enterocyte into the blood, suggesting restructuring of monosaccharide transport due to IF. IF had no effect on jejunum GLUT5 or SGLT1 expression. Cleaved Caspase-3 expression was decreased by IF in both sexes versus AL. This reduction was greater in females, suggesting a sex-dependent protective role of IF against apoptosis. Moreover, IF induced a significant increase 1.4-fold (n=5=6, p<0.05) in jejunum superoxide dismutase (SOD) activity in both sexes. Jejunum morphology was significantly changed in females only: IF induced both a decrease in villi length (by 21%, n=4/group, p<0.05) and a decrease in wall thickness (by 7%, n=4/group, p<0.05). Conclusions: In both sexes, IF decreased body weight, yet in females resting glucose levels were unchanged by IF. In females, IF reduced monosaccharide absorption out of intestines, yet improved overall intestinal health, increasing antioxidant levels with SOD. These findings collectively highlight the complex effects of IF on cellular processes and demonstrate significant sex-dependent aged intestinal responses. Unraveling the underlying molecular mechanisms driving these sex-dependent IF-induced effects is currently under evaluation. Midwestern-Arizona Alzheimer’s Consortium Grant, Midwestern University Intramural Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cardioprotective Effects of Sodium-Glucose Cotransporter Subtype Inhibition on Ischemic and Pharmacological Preconditioning.

Sodium-glucose cotransporter (SGLT) 2 inhibitors partially inhibit SGLT1 expression; however, whether a clinical dose of SGLT2 inhibitor abrogates ischemic preconditioning (IPC) is unknown, and the pharmacological cardioprotective effect under SGLT1 inhibition has not been examined. In this study, we investigated whether a clinical dose of tofogliflozin abrogates IPC and whether pharmacological preconditioning with olprinone has cardioprotective effects under SGLT1 inhibition. Male Wistar rats were divided into seven groups (seven rats per group) and subjected to the following treatments before inducing ischemia/reperfusion (I/R; 30 minutes of coronary artery occlusion followed by 120 minutes of reperfusion): saline infusion control treatment (Con); ischemic preconditioning(IPC); IPC after phlorizin infusion (IPC+Phl); IPC after low-dose tofogliflozin infusion (IPC+L-Tof); IPC after high-dose tofogliflozin infusion (IPC+H-Tof); olprinone infusion (Olp); and Olp infusion after phlorizin infusion (Olp+Phl). The infarct size was significantly decreased in the IPC group, but not in the IPC+Phl group. In contrast, the infarct size decreased in the IPC+L-Tof and IPC+H-Tof groups. Additionally, Olp reduced the infarct size, and the effect was preserved in Olp+Phl groups. Phosphorylated AMP-activated protein kinase (AMPK) expression was lower in the IPC+Phl group compared to that in the IPC group. The cardioprotective effect of IPC was attenuated by strong SGLT1 inhibition, but the effect was preserved under a clinical dose of highly selective SGLT2 inhibitor. Olprinone exerts a cardioprotective effect even under strong SGLT1 inhibition.

Mechanical regulation of lipid and sugar absorption by Piezo1 in enterocytes

Obesity is primarily caused by excessive intake as well as absorption of sugar and lipid. Postprandial surge in distention pressure and intestinal motility accelerates the absorption of nutrients. The response of intestinal epithelial cells to mechanical stimulation is not fully understood. Piezo1, a mechanosensitive ion channel, is widely expressed throughout the digestive tract. However, its function in intestinal nutrient absorption is not yet clear. In our study, excessive lipid deposition was observed in the duodenum of obese patients, while duodenal Piezo1–CaMKK2–AMPKα was decreased when compared to normal-weight individuals. Under high-fat diet condition, the Piezo1iKO mice exhibited abnormally elevated sugar and lipid absorption as well as severe lipid deposition in the duodenum and liver. These phenotypes were mainly caused by the inhibition of duodenal CaMKK2–AMPKα and the upregulation of SGLT1 and DGAT2. In contrast, Yoda1, a Piezo1 agonist, was found to reduce intestinal lipid absorption in diet induced obese mice. Overexpression of Piezo1, stretch and Yoda1 inhibited lipid accumulation and the expression of DGAT2 and SGLT1, whereas knockdown of Piezo1 stimulated lipid accumulation and DGAT2 in Caco-2 cells. Our study reveals a previously unexplored mechanical regulation of nutrient absorption in intestinal epithelial cells, which may shed new light on the therapy of obesity.

Canagliflozin inhibits PASMCs proliferation via regulating SGLT1/AMPK signaling and attenuates artery remodeling in MCT-induced pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) was a devastating disease characterized by artery remodeling, ultimately resulting in right heart failure. The aim of this study was to investigate the effects of canagliflozin (CANA), a sodium-glucose cotransporter 2 inhibitor (SGLT2i) with mild SGLT1 inhibitory effects, on rats with PAH, as well as its direct impact on pulmonary arterial smooth muscle cells (PASMCs). PAH rats were induced by injection of monocrotaline (MCT) (40 mg/kg), followed by four weeks of treatment with CANA (30 mg/kg/day) or saline alone. Pulmonary artery and right ventricular (RV) remodeling and dysfunction in PAH were alleviated with CANA, as assessed by echocardiography. Hemodynamic parameters and structural of pulmonary arteriole, including vascular wall thickness and wall area, were reduced by CANA. RV hypertrophy index, cardiomyocyte hypertrophy, and fibrosis were decreased with CANA treatment. PASMCs proliferation was inhibited by CANA under stimulation by platelet-derived growth factor (PDGF)-BB or hypoxia. Activation of AMP kinase (AMPK) was induced by CANA treatment in cultured PASMCs in a time- and concentration-dependent manner. These effects of CANA were attenuated when treatment with compound C, an AMPK inhibitor. Abundant expression of SGLT1 was observed in PASMCs and pulmonary arteries, while SGLT2 expression was undetectable. SGLT1 increased in response to PDGF-BB or hypoxia stimulation, while PASMCs proliferation was inhibited and beneficial effects of CANA were counteracted by knockdown of SGLT1. Our research demonstrated for the first time that CANA inhibited the proliferation of PASMCs by regulating SGLT1/AMPK signaling and thus exerted an anti-proliferative effect on MCT-induced PAH.

Duck compound probiotics fermented diet alters the growth performance by shaping the gut morphology, microbiota and metabolism

Dietary absorption and digestion are influenced by the microbiota, morphology, and digestive enzymes of intestines, and fermentation is a popular and effective technique to enhance animal rearing growth performance. This study aims to explore the pivotal role of Muscovy duck probiotics fermented feedstuff (FF) in altering the growth performance by reshaping gut morphology, microorganisms and metabolism. The findings showed that FF considerably raised the levels of fatty acids (FA) and small peptides (7-19AA) in the diet. Further feeding trial data reveals that FF greatly increased the Muscovy duck average daily gain (ADG) but had no effect on their daily feed intake (DFI), and the FCR significantly dropped (P < 0.05). Additionally, it was evident that FF improved the integrity of the intestinal mucosa in Muscovy duck by increasing villus height, villus height-to-crypt depth ratio, and lowering crypt depth. Then, in comparison to the control group (NC), there was a significant increase in the gene expression of the mucosal tight junction proteins Occludin, Claudin-1, and Zo-1 in the intestine of Muscovy duck. Additionally, there was higher expression of the mucosal transport channels SGLT-1, PepT1, AQP2, AQP3, and AQP10 in the similarly colon site, jejunum, and duodenum. Furthermore, in AB-PAS/PAS-stained duodenum, jejunum, ileum, and similarly colon site, FF markedly increased relative mucus output and goblet cells while decreasing epithelial cell apoptosis. Following 16S sequencing data indicated that the intestinal microbiota was altered and the diversity and richness of gut microbes was greatly enhanced by FF. Particularly, the boost of core probiotics, such as Rothia of duodenum, Limosilactobacillus and Lentilactobacillus of jejunum, Lactococcus and Rothia of ileum, Ligilactobacillus and Entocuccus of similarly colon site, Gallibacterium of caecum. And reduced potentially pathogenic bacteria (Campylobacter, Prevotellaceae, Clostridia-vadinBB60, and Oscillospira). Nontargeted metabolomics assay for intestinal content confirmed an increased organic acids (oxidanesulfonic acid, cholic acid, gallic acid, coumaric acid, pipecollc acid, 13s-hydroxyoctadecadienoic acid) and glycosides metabolites (5-hydroxydantrolene, 3-hydroxyguanfacine glucuronide, acetylleucine, astragalin, xanthosine, taxiphylin, sinapine, denudatine, penylalanyl-tyrosine and phenylalanyl-valine). These findings demonstrated that FF, a viable option to improve Muscovy duck growth performance through reconstructed intestinal morphology, microorganisms, and metabolism, subsequently promoted the gut health and increased diet digestion and absorption. The study that is being presented offers scientific proof that FF might be a useful strategy for improving Muscovy duck growth performance.