Effects of Empagliflozin-Induced Glycosuria on Weight Gain, Food Intake and Metabolic Indicators in Mice Fed a High-Fat Diet.

BACKGROUND: Sodium glucose-linked transporter 2 (SGLT2) inhibitors promote glucose, and therefore calorie, excretion in the urine. Patients taking SGLT2 inhibitors typically experience mild weight loss, but the amount of weight loss falls short of what is expected based on caloric loss. Understanding the mechanisms responsible for this weight loss discrepancy is imperative, as strategies to improve weight loss could markedly improve type 2 diabetes management and overall metabolic health. METHODS: Two mouse models of diet-induced obesity were administered the SGLT2 inhibitor empagliflozin in the food for 3 months. Urine glucose excretion, body weight, food intake and activity levels were monitored. In addition, serum hormone measurements were taken, and gene expression analyses were conducted. RESULTS: In both mouse models, mice receiving empagliflozin gained the same amount of body weight as their diet-matched controls despite marked glucose loss in the urine. No changes in food intake, serum ghrelin concentrations or activity levels were observed, but serum levels of fibroblast growth factor 21 (FGF21) decreased after treatment. A decrease in the levels of deiodinase 2 (Dio2) was also observed in the white adipose tissue, a primary target tissue of FGF21. CONCLUSION: These findings suggest that compensatory metabolic adaptations, other than increased food intake or decreased physical activity, occur in response to SGLT2 inhibitor-induced glycosuria that combats weight loss, and that reductions in FGF21, along with subsequent reductions in peripheral Dio2, may play a role.

Glucose/Fructose Delivery to the Distal Nephron Activates the Sodium-Chloride Cotransporter via the Calcium-Sensing Receptor.

BACKGROUND: The calcium-sensing receptor (CaSR) in the distal convoluted tubule (DCT) activates the NaCl cotransporter (NCC). Glucose acts as a positive allosteric modulator of the CaSR. Under physiologic conditions, no glucose is delivered to the DCT, and fructose delivery depends on consumption. We hypothesized that glucose/fructose delivery to the DCT modulates the CaSR in a positive allosteric way, activating the WNK4-SPAK-NCC pathway and thus increasing salt retention. METHODS: We evaluated the effect of glucose/fructose arrival to the distal nephron on the CaSR-WNK4-SPAK-NCC pathway using HEK-293 cells, C57BL/6 and WNK4-knockout mice, ex vivo perfused kidneys, and healthy humans. RESULTS: HEK-293 cells exposed to glucose/fructose increased SPAK phosphorylation in a WNK4- and CaSR-dependent manner. C57BL/6 mice exposed to fructose or a single dose of dapagliflozin to induce transient glycosuria showed increased activity of the WNK4-SPAK-NCC pathway. The calcilytic NPS2143 ameliorated this effect, which was not observed in WNK4-KO mice. C57BL/6 mice treated with fructose or dapagliflozin showed markedly increased natriuresis after thiazide challenge. Ex vivo rat kidney perfused with glucose above the physiologic threshold levels for proximal reabsorption showed increased NCC and SPAK phosphorylation. NPS2143 prevented this effect. In healthy volunteers, cinacalcet administration, fructose intake, or a single dose of dapagliflozin increased SPAK and NCC phosphorylation in urinary extracellular vesicles. CONCLUSIONS: Glycosuria or fructosuria was associated with increased NCC, SPAK, and WNK4 phosphorylation in a CaSR-dependent manner.

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

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.

Lipid Accumulation Product Combined With Urine Glucose Excretion Improves the Efficiency of Diabetes Screening in Chinese Adults.

Background: To compare the efficacy of lipid accumulation product (LAP) and urine glucose excretion (UGE) in predicting diabetes and evaluate whether the combination of LAP and UGE would help to improve the efficacy of using LAP alone or UGE alone in identifying diabetes. Methods: Data from 7485 individuals without prior history of diabetes who participated in a cross-sectional survey in Jiangsu, China, were analyzed. Each participant underwent an oral glucose-tolerance test. Operating characteristic curves (ROC) and logistic regression analyses were used to evaluate the performance of LAP and UGE in identification of newly diagnosed diabetes (NDM) and prediabetes (PDM). Results: For subjects with NDM, the area under the ROC curve was 0.72 for LAP and 0.85 for UGE, whereas for PDM, these values were 0.62 and 0.61, respectively. Furthermore, LAP exhibited a comparable sensitivity with UGE in detecting NDM (76.4% vs 76.2%, p = 0.31). In predicting PDM, LAP showed a higher sensitivity than UGE (66.4% vs 42.8%, p < 0.05). The combination of LAP and UGE demonstrated a significantly higher sensitivity than that of LAP alone and UGE alone for identification of NDM (93.6%) and PDM (80.1%). Moreover, individuals with both high LAP and high UGE had significantly increased risk of NDM and PDM than those with both low LAP and low UGE. Conclusions: The combination of LAP and UGE substantially improved the efficacy of using LAP and using UGE alone in detecting diabetes, and may be a novel approach for mass screening in the general population.

High urinary excretion rate of glucose attenuates serum uric acid level in type 2 diabetes with normal renal function.

AIMS/INTRODUCTION: The relationship between urinary excretion rate of glucose (UEGL) and uric acid (UA) metabolism in adults with type 2 diabetes (T2D) remains unclear to date. This study aimed to investigate the relationships of UEGL with serum UA (SUA), urinary excretion rate of uric acid (UEUA), and renal clearance of uric acid (CLUA) in adults with T2D. We hypothesised that high UEGL increases UA excretion, which in turn leads to lower SUA. MATERIALS AND METHODS: This was a cross-sectional study of 635 inpatients with T2D recruited between 2018 and 2019. The relationships of UEGL with UEUA, CLUA, and hyperuricaemia were assessed using analysis of covariance and multivariate regression analysis. RESULTS: Patients in the higher quartile of UEGL tended to have lower SUA levels than those in the lower quartile. In contrast, patients in the higher quartile of UEGL tended to have higher CLUA (p for trend < 0.0001), and a similar trend was observed for UEUA. In adjusted multivariable linear regression model, UEGL was negatively correlated with SUA (ß = - 0.023, 95% CI - 0.034 to - 0.013, p < 0.0001). However, positive correlations of UEGL with UEUA (ß = 0.046, 95% CI 0.018-0.074, p = 0.001) and CLUA (ß = 0.063, 95% CI 0.042-0.085, p < 0.0001) were found. Furthermore, consistent significant inverse associations were observed between quartiles of UEGL and hyperuricaemia in the adjusted multivariate logistic regression model. CONCLUSIONS: A high UEGL level was positively correlated with UEUA and CLUA. Moreover, it was inversely associated with SUA level, and a consistently increased UEGL level reduced the risk of hyperuricaemia in patients with T2D.

A role for tubular Na+/H+ exchanger NHE3 in the natriuretic effect of the SGLT2 inhibitor empagliflozin.

Inhibitors of proximal tubular Na+-glucose cotransporter 2 (SGLT2) are natriuretic, and they lower blood pressure. There are reports that the activities of SGLT2 and Na+-H+ exchanger 3 (NHE3) are coordinated. If so, then part of the natriuretic response to an SGLT2 inhibitor is mediated by suppressing NHE3. To examine this further, we compared the effects of an SGLT2 inhibitor, empagliflozin, on urine composition and systolic blood pressure (SBP) in nondiabetic mice with tubule-specific NHE3 knockdown (NHE3-ko) and wild-type (WT) littermates. A single dose of empagliflozin, titrated to cause minimal glucosuria, increased urinary excretion of Na+ and bicarbonate and raised urine pH in WT mice but not in NHE3-ko mice. Chronic empagliflozin treatment tended to lower SBP despite higher renal renin mRNA expression and lowered the ratio of SBP to renin mRNA, indicating volume loss. This effect of empagliflozin depended on tubular NHE3. In diabetic Akita mice, chronic empagliflozin enhanced phosphorylation of NHE3 (S552/S605), changes previously linked to lesser NHE3-mediated reabsorption. Chronic empagliflozin also increased expression of genes involved with renal gluconeogenesis, bicarbonate regeneration, and ammonium formation. While this could reflect compensatory responses to acidification of proximal tubular cells resulting from reduced NHE3 activity, these effects were at least in part independent of tubular NHE3 and potentially indicated metabolic adaptations to urinary glucose loss. Moreover, empagliflozin increased luminal α-ketoglutarate, which may serve to stimulate compensatory distal NaCl reabsorption, while cogenerated and excreted ammonium balances urine losses of this "potential bicarbonate." The data implicate NHE3 as a determinant of the natriuretic effect of empagliflozin.

Renal proximal tubulopathy in an HIV-infected patient treated with tenofovir alafenamide and gentamicin: a case report.

BACKGROUND: The nucleotide reverse transcriptase inhibitor Tenofovir Alafenamide (TAF) is a novel pro-drug of tenofovir (TFV) and possesses a superior renal safety profile compared with tenofovir disoproxil fumerate (TDF). Due to unique pharmacokinetic characteristics, treatment with TAF is not associated with significant renal proximal tubular accumulation of TFV. TAF is associated with a lower risk of acute kidney injury, chronic kidney disease, proteinuria and renal proximal tubular dysfunction than treatment with TDF. No cases of Fanconi syndrome have been reported in clinical trials of TAF. It is unknown whether treatment with TAF can lead to accumulation of TFV in proximal tubular cells and cause nephrotoxicity under certain clinical circumstances. CASE PRESENTATION: Here we report the case of a patient on stable TAF-based antiretroviral therapy with for HIV-1 infection who developed proximal tubulopathy when treated with gentamicin for febrile neutropenia in the context of relapsed Hodgkin lymphoma. Eighteen days after commencing chemotherapy for relapsed Hodgkin lymphoma the patient presented to hospital with fevers, hypotension and neutropenia. The patient was commenced on piperacillin, tazobactam and gentamicin. Within 24 h the patient developed marked hypokalaemia and hypophosphataemia requiring intravenous replacement therapy. There was proteinuria, glycosuria and evidence of marked urinary electrolyte wasting, consistent with acute proximal tubular dysfunction. Eleven days after the gentamicin was stopped the serum biochemistry normalised. The urinary electrolyte wasting and proteinuria had improved, and the glycosuria had resolved. CONCLUSION: This is the first case report to describe acute renal proximal tubulopathy in an HIV-infected patient treated with TAF and gentamicin. As the number of patients prescribed TAF outside the clinical trial setting increases, so too does the potential for previously unreported drug interactions and adverse events. Clinicians need to be aware of potential unreported adverse drug reactions as the use of TAF becomes increasingly common in clinical practice.

Relationship between renal capacity to reabsorb glucose and renal status in patients with diabetes.

AIMS: Interindividual variability in capacity to reabsorb glucose at the proximal renal tubule could contribute to risk of diabetic kidney disease. Our present study investigated, in patients with diabetes, the association between fractional reabsorption of glucose (FRGLU) and degree of renal disease as assessed by urinary albumin excretion (UAE) and estimated glomerular filtration rate (eGFR). METHODS: FRGLU [1-(glucose clearance/creatinine clearance)] was assessed in 637 diabetes patients attending our tertiary referral centre, looking for correlations between FRGLU and UAE (normo-, micro-, macro-albuminuria) and Kidney Disease: Improving Global Outcomes (KDIGO) eGFR categories: >90 (G1); 90-60 (G2); 59-30 (G3); and<30-16 (G4) mL/min/1.73 m2. Patients were stratified by admission fasting plasma glucose (FPG) into three groups: low (<6mmol/L); intermediate (6-11mmol/L); and high (>11mmol/L). RESULTS: Median (interquartile range, IQR) FRGLU levels were blood glucose-dependent: 99.90% (0.05) for low (n=106); 99.90% (0.41) for intermediate (n=288); and 96.36% (12.57) for high (n=243) blood glucose categories (P<0.0001). Also, FRGLU increased with renal disease severity in patients in the high FPG group: normoalbuminuria, 93.50% (17.74) (n=135); microalbuminuria, 96.56% (5.94) (n=77); macroalbuminuria, 99.12% (5.44) (n=31; P<0.001); eGFR G1, 94.13% (16.24) (n=111); G2, 96.35% (11.94) (n=72); G3 98.88% (7.59) (n=46); and G4, 99.11% (2.20) (n=14; P<0.01). On multiple regression analyses, FRGLU remained significantly and independently associated with UAE and eGFR in patients in the high blood glucose group. CONCLUSION: High glucose reabsorption capacity in renal proximal tubules is associated with high UAE and low eGFR in patients with diabetes and blood glucose levels>11mmol/L.

Increase in Endogenous Glucose Production With SGLT2 Inhibition Is Unchanged by Renal Denervation and Correlates Strongly With the Increase in Urinary Glucose Excretion.

OBJECTIVE: Sodium-glucose cotransporter 2 (SGLT2) inhibition causes an increase in endogenous glucose production (EGP). However, the mechanisms are unclear. We studied the effect of SGLT2 inhibitors on EGP in subjects with type 2 diabetes (T2D) and without diabetes (non-DM) in kidney transplant recipients with renal denervation. RESEARCH DESIGN AND METHODS: Fourteen subjects who received a renal transplant (six with T2D [A1C 7.2 ± 0.1%] and eight non-DM [A1C 5.6 ± 0.1%) underwent measurement of EGP with [3-3H]glucose infusion following dapagliflozin (DAPA) 10 mg or placebo. Plasma glucose, insulin, C-peptide, glucagon, and titrated glucose-specific activity were measured. RESULTS: Following placebo in T2D, fasting plasma glucose (FPG) (143 ± 14 to 124 ± 10 mg/dL; P = 0.02) and fasting plasma insulin (12 ± 2 to 10 ± 1.1 µU/mL; P < 0.05) decreased; plasma glucagon was unchanged, and EGP declined. After DAPA in T2D, FPG (143 ± 15 to 112 ± 9 mg/dL; P = 0.01) and fasting plasma insulin (14 ± 3 to 11 ± 2 µU/mL; P = 0.02) decreased, and plasma glucagon increased (all P < 0.05 vs. placebo). EGP was unchanged from baseline (2.21 ± 0.19 vs. 1.96 ± 0.14 mg/kg/min) in T2D (P < 0.001 vs. placebo). In non-DM following DAPA, FPG and fasting plasma insulin decreased, and plasma glucagon was unchanged. EGP was unchanged from baseline (1.85 ± 0.10 to 1.78 ± 0.10 mg/kg/min) after DAPA, whereas EGP declined significantly with placebo. When the increase in EGP production following DAPA versus placebo was plotted against the difference in urinary glucose excretion (UGE) for all patients, a strong correlation (r = 0.824; P < 0.001) was observed. CONCLUSIONS: Renal denervation in patients who received a kidney transplant failed to block the DAPA-mediated stimulation of EGP in both individuals with T2D and non-DM subjects. The DAPA-stimulated rise in EGP is strongly related to the increase in UGE, blunting the decline in FPG.

Pharmacokinetics and pharmacodynamics of rongliflozin, a novel selective inhibitor of sodium-glucose co-transporter-2, in people with type 2 diabetes mellitus.

AIMS: To evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of rongliflozin in a cohort of healthy Chinese people and people with type 2 diabetes mellitus (T2DM). MATERIALS AND METHODS: We examined the effects of a single ascending dose (SAD) of rongliflozin (10-200 mg) in combination with food (20 mg) in 50 healthy people, and a multiple ascending dose (MAD) of rongliflozin (10-50 mg once daily for 12 days) in 36 people with T2DM. RESULTS: No serious adverse events (AEs) or discontinuations as a result of AEs (related to rongliflozin) occurred in either study. In healthy participants and those with T2DM, rongliflozin was rapidly absorbed, with a time to maximum plasma concentration of 0.63 to 1.75 hours. Systemic exposure (maximum observed serum concentration and area under the curve) to rongliflozin and its inactive major metabolites (T1444, T1454 and T1830) increased in proportion to dose. In the SAD and MAD studies, there was a dose-related increase in urinary glucose excretion (UGE) ranging from 10 to 50 mg rongliflozin. This increase in UGE was associated with dose-related decreases in serum glucose values in people with T2DM in the MAD group. In the SAD group, UGE plateaued at 50 to 200 mg. CONCLUSIONS: Rongliflozin was well tolerated in all participants. The PK and PD measurements obtained for rongliflozin demonstrate a dose-response relationship when the drug is administered at doses ranging from 10 to 50 mg in healthy people and in people with T2DM.

Tubular Deficiency of Heterogeneous Nuclear Ribonucleoprotein F Elevates Systolic Blood Pressure and Induces Glycosuria in Mice.

We reported previously that overexpression of heterogeneous nuclear ribonucleoprotein F (Hnrnpf) in renal proximal tubular cells (RPTCs) suppresses angiotensinogen (Agt) expression, and attenuates systemic hypertension and renal injury in diabetic Hnrnpf-transgenic (Tg) mice. We thus hypothesized that deletion of Hnrnpf in the renal proximal tubules (RPT) of mice would worsen systemic hypertension and kidney injury, perhaps revealing novel mechanism(s). Tubule-specific Hnrnpf knockout (KO) mice were generated by crossbreeding Pax8-Cre mice with floxed Hnrnpf mice on a C57BL/6 background. Both male and female KO mice exhibited elevated systolic blood pressure, increased urinary albumin/creatinine ratio, tubulo-interstitial fibrosis and glycosuria without changes in blood glucose or glomerular filtration rate compared with control littermates. However, glycosuria disappeared in male KO mice at the age of 12 weeks, while female KO mice had persistent glycosuria. Agt expression was elevated, whereas sodium-glucose co-transporter 2 (Sglt2) expression was down-regulated in RPTs of both male and female KO mice as compared to control littermates. In vitro, KO of HNRNPF in human RPTCs (HK-2) by CRISPR gRNA up-regulated AGT and down-regulated SGLT2 expression. The Sglt2 inhibitor canagliflozin treatment had no effect on Agt and Sglt2 expression in HK-2 and in RPTCs of wild-type mice but induced glycosuria. Our results demonstrate that Hnrnpf plays a role in the development of hypertension and glycosuria through modulation of renal Agt and Sglt2 expression in mice, respectively.

Comparison of the urinary glucose excretion contributions of SGLT2 and SGLT1: A quantitative systems pharmacology analysis in healthy individuals and patients with type 2 diabetes treated with SGLT2 inhibitors.

AIM: To develop a quantitative drug-disease systems model to investigate the paradox that sodium-glucose co-transporter (SGLT)2 is responsible for >80% of proximal tubule glucose reabsorption, yet SGLT2 inhibitor treatment results in only 30% to 50% less reabsorption in patients with type 2 diabetes mellitus (T2DM). MATERIALS AND METHODS: A physiologically based four-compartment model of renal glucose filtration, reabsorption and excretion via SGLT1 and SGLT2 was developed as a system of ordinary differential equations using R/IQRtools. SGLT2 inhibitor pharmacokinetics and pharmacodynamics were estimated from published concentration-time profiles in plasma and urine and from urinary glucose excretion (UGE) in healthy people and people with T2DM. RESULTS: The final model showed that higher renal glucose reabsorption in people with T2DM versus healthy people was associated with 54% and 28% greater transporter capacity for SGLT1 and SGLT2, respectively. Additionally, the analysis showed that UGE is highly dependent on mean plasma glucose and estimated glomerular filtration rate (eGFR) and that their consideration is critical for interpreting clinical UGE findings. CONCLUSIONS: Quantitative drug-disease system modelling revealed mechanistic differences in renal glucose reabsorption and UGE between healthy people and those with T2DM, and clearly showed that SGLT2 inhibition significantly increased glucose available to SGLT1 downstream in the tubule. Importantly, we found that the findings of lower than expected UGE with SGLT2 inhibition are explained by the shift to SGLT1, which recovered additional glucose (~30% of total).

Diuretic Effects of Sodium Glucose Cotransporter 2 Inhibitors and Their Influence on the Renin-Angiotensin System.

The renin-angiotensin system (RAS) plays an important role in regulating body fluids and blood pressure. However, inappropriate activation of the RAS contributes to the pathogenesis of cardiovascular and renal diseases. Recently, sodium glucose cotransporter 2 (SGLT2) inhibitors have been used as anti-diabetic agents. SGLT2 inhibitors induce glycosuria and improve hyperglycemia by inhibiting urinary reabsorption of glucose. However, in the early stages of treatment, these inhibitors frequently cause polyuria and natriuresis, which potentially activate the RAS. Nevertheless, the effects of SGLT2 inhibitors on RAS activity are not straightforward. Available data indicate that treatment with SGLT2 inhibitors transiently activates the systemic RAS in type 2 diabetic patients, but not the intrarenal RAS. In this review article, we summarize current evidence of the diuretic effects of SGLT2 inhibitors and their influence on RAS activity.

Comparison of HbA1c and OGTT for the diagnosis of type 2 diabetes in children at risk of diabetes.

BACKGROUND: The aim of this study was to evaluate the correlation between plasma glucose and HbA1c and the diagnostic accuracy of HbA1c as a screening tool to identify asymptomatic diabetes mellitus in children and adolescents with obesity or asymptomatic glucosuria. METHODS: A total of 190 subjects who underwent an oral glucose tolerance test (OGTT) to confirm diabetes were categorized into normal glucose tolerance (NGT; n = 117), impaired glucose tolerance (IGT; n = 33), and diabetes (DM; n = 40) according to the OGTT. Forty-seven patients with DM were diagnosed by either OGTT or HbA1c levels. The diagnostic accuracy for the detection of diabetes is based on 47 patients. Laboratory tests were performed after 12 h of fasting. RESULTS: According to the HbA1c criterion, 107 (55.3%) subjects were in the NGT group, 41 (21.6%) were in the IGT group, and 42 (22.1%) were in the DM group. Diagnostic sensitivities of HbA1c and 2-hour plasma glucose level following OGTT (2-h OGTT) for DM were significantly higher than that of fasting plasma glucose, FPG (89.4, 85.1 vs. 63.8%). In addition, the area under the curves of diagnostic criteria was 0.970 for HbA1c, 0.939 for FPG and 0.977 for 2-h OGTT. Mean FPG and 2-h OGTT for HbA1c level >6.5% were 115.2 mg/dL and 181.8 mg/dL, respectively. The optimal HbA1c level cut-off point for predicting DM is 6.15%, with a sensitivity of 95.7% in Korean children and adolescents. CONCLUSION: The HbA1c criterion ≥6.5% was adequate to detect DM among Korean children and adolescents with obesity or asymptomatic glucosuria. We also recommend HbA1c level of 6.15% as the optimal cut-off point for detecting DM in Korean children and adolescents.

SGLT1 in pancreatic α cells regulates glucagon secretion in mice, possibly explaining the distinct effects of SGLT2 inhibitors on plasma glucagon levels.

OBJECTIVES: It is controversial whether sodium glucose transporter (SGLT) 2 inhibitors increase glucagon secretion via direct inhibition of SGLT2 in pancreatic α cells. The role of SGLT1 in α cells is also unclear. We aimed to elucidate these points that are important not only for basic research but also for clinical insight. METHODS: Plasma glucagon levels were assessed in the high-fat, high-sucrose diet (HFHSD) fed C57BL/6J mice treated with dapagliflozin or canagliflozin. RT-PCR, RNA sequence, and immunohistochemistry were conducted to test the expression of SGLT1 and SGLT2 in α cells. We also used αTC1 cells and mouse islets to investigate the molecular mechanism by which SGLT1 modulates glucagon secretion. RESULTS: Dapagliflozin, but not canagliflozin, increased plasma glucagon levels in HFHSD fed mice. SGLT1 and glucose transporter 1 (GLUT1), but not SGLT2, were expressed in αTC1 cells, mouse islets and human islets. A glucose clamp study revealed that the plasma glucagon increase associated with dapagliflozin could be explained as a response to acute declines in blood glucose. Canagliflozin suppressed glucagon secretion by inhibiting SGLT1 in α cells; consequently, plasma glucagon did not increase with canagliflozin, even though blood glucose declined. SGLT1 effect on glucagon secretion depended on glucose transport, but not glucose metabolism. Islets from HFHSD and db/db mice displayed higher SGLT1 mRNA levels and lower GLUT1 mRNA levels than the islets from control mice. These expression levels were associated with higher glucagon secretion. Furthermore, SGLT1 inhibitor and siRNA against SGLT1 suppressed glucagon secretion in isolated islets. CONCLUSIONS: These data suggested that a novel mechanism regulated glucagon secretion through SGLT1 in α cells. This finding possibly explained the distinct effects of dapagliflozin and canagliflozin on plasma glucagon levels in mice.

Renal tubule insulin receptor modestly promotes elevated blood pressure and markedly stimulates glucose reabsorption.

Although the cause of hypertension among individuals with obesity and insulin resistance is unknown, increased plasma insulin, acting in the kidney to increase sodium reabsorption, has been proposed as a potential mechanism. Insulin may also stimulate glucose uptake, but the contributions of tubular insulin signaling to sodium or glucose transport in the setting of insulin resistance is unknown. To directly study the role of insulin signaling in the kidney, we generated inducible renal tubule-specific insulin receptor-KO mice and used high-fat feeding and mineralocorticoids to model obesity and insulin resistance. Insulin receptor deletion did not alter blood pressure or sodium excretion in mice on a high-fat diet alone, but it mildly attenuated the increase in blood pressure with mineralocorticoid supplementation. Under these conditions, KO mice developed profound glucosuria. Insulin receptor deletion significantly reduced SGLT2 expression and increased urinary glucose excretion and urine flow. These data demonstrate a direct role for insulin receptor-stimulated sodium and glucose transport and a functional interaction of insulin signaling with mineralocorticoids in vivo. These studies uncover a potential mechanistic link between preserved insulin sensitivity and renal glucose handling in obesity and insulin resistance.

Physiology of renal glucose handling via SGLT1, SGLT2 and GLUT2.

The concentration of glucose in plasma is held within narrow limits (4-10 mmol/l), primarily to ensure fuel supply to the brain. Kidneys play a role in glucose homeostasis in the body by ensuring that glucose is not lost in the urine. Three membrane proteins are responsible for glucose reabsorption from the glomerular filtrate in the proximal tubule: sodium-glucose cotransporters SGLT1 and SGLT2, in the apical membrane, and GLUT2, a uniporter in the basolateral membrane. 'Knockout' of these transporters in mice and men results in the excretion of filtered glucose in the urine. In humans, intravenous injection of the plant glucoside phlorizin also results in excretion of the full filtered glucose load. This outcome and the finding that, in an animal model, phlorizin reversed the symptoms of diabetes, has stimulated the development and successful introduction of SGLT2 inhibitors, gliflozins, in the treatment of type 2 diabetes mellitus. Here we summarise the current state of our knowledge about the physiology of renal glucose handling and provide background to the development of SGLT2 inhibitors for type 2 diabetes treatment.

Ascorbic acid-A black hole of urine chemistry screening.

BACKGROUND: Study was performed in order: (i) to assess the comparability of glucose, bilirubin, hemoglobin, leukocyte esterase, and protein; (ii) to assess accuracy of glucose, bilirubin, hemoglobin, leukocyte esterase, and protein; and (iii) to evaluate interference of ascorbic acid on the glucose, bilirubin, hemoglobin, and nitrite determination using 2 different dipsticks: iChem Velocity, Iris Diagnostics and Combur-10M, Roche Diagnostics. METHODS: Random urine specimens were included in the study. Comparability, accuracy, and ascorbic acid interference testing were performed. RESULTS: Obtained results have shown almost perfect agreement for all parameters between 2 dipsticks in samples with negative ascorbic acid. Agreement in samples with positive ascorbic acid was not acceptable for bilirubin, protein, nitrite, and hemoglobin. Accuracy was not acceptable for hemoglobin and leukocyte esterase on both dipsticks. Ascorbic acid interference examination has shown that intensity of interference differs between dipsticks. Ascorbic acid interferes with glucose, hemoglobin, nitrite, and bilirubin at different concentrations causing false-negative results. CONCLUSION: Obtained results indicate that it is necessary to determine diagnostic accuracy of used dipstick in order to define purpose of urinalysis. It is very important to choose dipstick with ascorbic acid indicator and to examine ascorbic acid impact on dipstick analytes independently of manufacturer claims.

Organic anion transporter OAT3 enhances the glucosuric effect of the SGLT2 inhibitor empagliflozin.

The sodium-glucose cotransporter SGLT2 inhibitor empagliflozin (plasma protein binding ~88%) may reach its target in the brush border of the early proximal tubule by glomerular filtration and tubular secretion. Here we determined whether empagliflozin is secreted by renal tubules in mice and whether genetic knockout of the basolateral organic anion transporter 3 ( Oat3-/-) affects its tubular secretion or glucosuric effect. Renal clearance studies in wild-type (WT) mice showed that tubular secretion accounted for 50-70% of empagliflozin urinary excretion. Immunostaining indicated that SGLT2 and OAT3 localization partially overlapped in proximal tubule S1 and S2 segments. Glucosuria in metabolic cage studies was reduced in Oat3-/- vs. WT mice for acute empagliflozin doses of 1, 3, and 10 mg/kg, whereas 30 mg/kg induced similar maximal glucosuria in both genotypes. Chronic application of empagliflozin (~25 mg·kg-1 ·day-1) in Oat3-/- mice was associated with lower urinary glucose-to-creatinine ratios despite maintaining slightly higher blood glucose levels than WT. On a whole kidney level, renal secretion of empagliflozin was largely unchanged in Oat3-/- mice. However, the absence of OAT3 attenuated the influence of empagliflozin on fractional glucose excretion; higher levels of plasma or filtered empagliflozin were needed to induce similar increases in fractional renal glucose excretion. We conclude that empagliflozin is excreted into the urine to similar extent by glomerular filtration and tubular secretion. The latter can occur largely independent of OAT3. However, OAT3 increases the glucosuric effect of empagliflozin, which may relate to the partial overlap of its localization with SGLT2 and thus OAT3-mediated tubular secretion of empagliflozin in the early proximal tubule.

Empagliflozin Treatment Is Associated With Improved ß-Cell Function in Type 2 Diabetes Mellitus.

Objective: To examine whether lowering plasma glucose concentration with the sodium-glucose transporter-2 inhibitor empagliflozin improves ß-cell function in patients with type 2 diabetes mellitus (T2DM). Methods: Patients with T2DM (N = 15) received empagliflozin (25 mg/d) for 2 weeks. ß-Cell function was measured with a nine-step hyperglycemic clamp (each step, 40 mg/dL) before and at 48 hours and at 14 days after initiating empagliflozin. Results: Glucosuria was recorded on days 1 and 14 [mean ± standard error of the mean (SEM), 101 ± 10 g and 117 ± 11 g, respectively] after initiating empagliflozin, as were reductions in fasting plasma glucose levels (25 ± 6 mg/dL and 38 ± 8 mg/dL, respectively; both P < 0.05). After initiating empagliflozin and during the stepped hyperglycemic clamp, the incremental area under the plasma C-peptide concentration curve increased by 48% ± 12% at 48 hours and 61% ± 10% at 14 days (both P < 0.01); glucose infusion rate increased by 15% on day 3 and 16% on day 14, compared with baseline (both P < 0.05); and ß-cell function, measured with the insulin secretion/insulin resistance index, increased by 73% ± 21% at 48 hours and 112% ± 20% at 14 days (both P < 0.01). ß-cell glucose sensitivity during the hyperglycemic clamp was enhanced by 42% at 14 hours and 54% at 14 days after initiating empagliflozin (both P < 0.01). Conclusion: Lowering the plasma glucose concentration with empagliflozin in patients with T2DM augmented ß-cell glucose sensitivity and improved ß-cell function.