SGLT2 inhibition reprograms systemic metabolism via FGF21-dependent and -independent mechanisms.
ABSTRACT: Pharmacologic inhibition of the renal sodium/glucose cotransporter-2 induces glycosuria and reduces glycemia. Given that SGLT2 inhibitors (SGLT2i) reduce mortality and cardiovascular risk in type 2 diabetes, improved understanding of molecular mechanisms mediating these metabolic effects is required. Treatment of obese but nondiabetic mice with the SGLT2i canagliflozin (CANA) reduces adiposity, improves glucose tolerance despite reduced plasma insulin, increases plasma ketones, and improves plasma lipid profiles. Utilizing an integrated transcriptomic-metabolomics approach, we demonstrate that CANA modulates key nutrient-sensing pathways, with activation of 5' AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin (mTOR), independent of insulin or glucagon sensitivity or signaling. Moreover, CANA induces transcriptional reprogramming to activate catabolic pathways, increase fatty acid oxidation, reduce hepatic steatosis and diacylglycerol content, and increase hepatic and plasma levels of FGF21. Given that these phenotypes mirror the effects of FGF21 to promote lipid oxidation, ketogenesis, and reduction in adiposity, we hypothesized that FGF21 is required for CANA action. Using FGF21-null mice, we demonstrate that FGF21 is not required for SGLT2i-mediated induction of lipid oxidation and ketogenesis but is required for reduction in fat mass and activation of lipolysis. Taken together, these data demonstrate that SGLT2 inhibition triggers a fasting-like transcriptional and metabolic paradigm but requires FGF21 for reduction in adiposity.
Project description:We assessed the change in hepatic transciptional pattern after treatment with SGLT-2 inhibitors canagliflozin in a mice model of diet-induced obesity. Pharmacologic inhibition of the renal sodium/glucose cotransporter-2 induces glycosuria and reduces glycemia. Given that SGLT2 inhibitors (SGLT2i) reduce mortality and CV risk in T2D, improved understanding of molecular mechanisms mediating these metabolic effects is required. Treatment of obese but nondiabetic mice with the SGLT2i canagliflozin (CANA) reduces adiposity, improves glucose tolerance despite reduced plasma insulin, increases plasma ketones, and improves plasma lipid profiles. We utilized an integrated transcriptomic-metabolomics approach to demonstrate that CANA modulates key nutrient-sensing pathways, with activation of AMPK and inhibition of mTOR, independent of insulin or glucagon sensitivity or signaling. Moreover, CANA induces transcriptional reprogramming to activate catabolic pathways, increase fatty acid oxidation, reduce hepatic steatosis and diacylglycerol content, and increase hepatic and plasma levels of FGF21. Taken together, these data demonstrate that SGLT-2 inhibition triggers a fasting-like transcriptional and metabolic paradigm.
Project description:BACKGROUND:Enthusiasm for the benefits of sodium-glucose cotransporter 2 inhibitors (SGLT2i) as an adjunctive treatment in type 1 diabetes (T1D) has been offset by the possible increased risk of diabetic ketoacidosis (DKA). Since pump-treated T1D patients are susceptible to DKA due to infusion site problems, this study was undertaken to assess how treatment with SGLT2i affects patterns of early metabolic decompensation following suspension of basal insulin. METHODS:Ten T1D participants (age 19-35 years, duration 10?±?8 years, A1c 7.4%?±?0.8%) underwent overnight pump suspension studies before and after treatment with canagliflozin (CANA). On both nights, basal insulin was suspended at 3 AM and plasma glucose (PG), ?-hydroxybutyrate (BHB), free fatty acids (FFA), plasma insulin (PI), and glucagon were measured. Studies were terminated 6?h after suspension or if PG rose to >350?mg/dL or BHB >2.5?mmol/L. RESULTS:PI levels at the start of suspension were reduced by 30% after CANA treatment (44?±?11?uU/mL vs. 31?±?10?uU/mL, P?<?0.01), but baseline PG, BHB, FFA, and glucagon levels were not significantly different. During the suspension, PG rose from 104?±?10 to 301?±?21?mg/dL before treatment, but only from 109?±?8 to 195?±?14?mg/dL after treatment (P?=?0.002 vs. pretreatment values). On the other hand, CANA treatment did not significantly affect the magnitude of increases in FFA, BHB, and glucagon levels during the suspension study. CONCLUSION:These data indicate that SGLT2i do not accelerate the rate of ketogenesis following the interruption of basal insulin infusion in T1D. Rather, the failure of patients to promptly recognize early metabolic decompensation relates to the much more gradual rise in PG levels.
Project description:<h4>Aims/hypothesis</h4>Sodium-glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i) constitute a novel class of glucose-lowering (type 2) kidney-targeted agents. We recently reported that the SGLT2i empagliflozin (EMPA) reduced cardiac cytosolic Na<sup>+</sup> ([Na<sup>+</sup>]<sub>c</sub>) and cytosolic Ca<sup>2+</sup> ([Ca<sup>2+</sup>]<sub>c</sub>) concentrations through inhibition of Na<sup>+</sup>/H<sup>+</sup> exchanger (NHE). Here, we examine (1) whether the SGLT2i dapagliflozin (DAPA) and canagliflozin (CANA) also inhibit NHE and reduce [Na<sup>+</sup>]<sub>c</sub>; (2) a structural model for the interaction of SGLT2i to NHE; (3) to what extent SGLT2i affect the haemodynamic and metabolic performance of isolated hearts of healthy mice.<h4>Methods</h4>Cardiac NHE activity and [Na<sup>+</sup>]<sub>c</sub> in mouse cardiomyocytes were measured in the presence of clinically relevant concentrations of EMPA (1 μmol/l), DAPA (1 μmol/l), CANA (3 μmol/l) or vehicle. NHE docking simulation studies were applied to explore potential binding sites for SGTL2i. Constant-flow Langendorff-perfused mouse hearts were subjected to SGLT2i for 30 min, and cardiovascular function, O<sub>2</sub> consumption and energetics (phosphocreatine (PCr)/ATP) were determined.<h4>Results</h4>EMPA, DAPA and CANA inhibited NHE activity (measured through low pH recovery after NH<sub>4</sub><sup>+</sup> pulse: EMPA 6.69 ± 0.09, DAPA 6.77 ± 0.12 and CANA 6.80 ± 0.18 vs vehicle 7.09 ± 0.09; p < 0.001 for all three comparisons) and reduced [Na<sup>+</sup>]<sub>c</sub> (in mmol/l: EMPA 10.0 ± 0.5, DAPA 10.7 ± 0.7 and CANA 11.0 ± 0.9 vs vehicle 12.7 ± 0.7; p < 0.001). Docking studies provided high binding affinity of all three SGLT2i with the extracellular Na<sup>+</sup>-binding site of NHE. EMPA and CANA, but not DAPA, induced coronary vasodilation of the intact heart. PCr/ATP remained unaffected.<h4>Conclusions/interpretation</h4>EMPA, DAPA and CANA directly inhibit cardiac NHE flux and reduce [Na<sup>+</sup>]<sub>c</sub>, possibly by binding with the Na<sup>+</sup>-binding site of NHE-1. Furthermore, EMPA and CANA affect the healthy heart by inducing vasodilation. The [Na<sup>+</sup>]<sub>c</sub>-lowering class effect of SGLT2i is a potential approach to combat elevated [Na<sup>+</sup>]<sub>c</sub> that is known to occur in heart failure and diabetes.
Project description:Sodium-glucose transport protein 2 (SGLT2) inhibitors are a class of anti-diabetic agents; however, concerns have been raised about their potential to induce euglycemic ketoacidosis and to increase both glucose production and glucagon secretion. The mechanisms behind these alterations are unknown. Here we show that the SGLT2 inhibitor (SGLT2i) dapagliflozin promotes ketoacidosis in both healthy and type 2 diabetic rats in the setting of insulinopenia through increased plasma catecholamine and corticosterone concentrations secondary to volume depletion. These derangements increase white adipose tissue (WAT) lipolysis and hepatic acetyl-CoA content, rates of hepatic glucose production, and hepatic ketogenesis. Treatment with a loop diuretic, furosemide, under insulinopenic conditions replicates the effect of dapagliflozin and causes ketoacidosis. Furthermore, the effects of SGLT2 inhibition to promote ketoacidosis are independent from hyperglucagonemia. Taken together these data in rats identify the combination of insulinopenia and dehydration as a potential target to prevent euglycemic ketoacidosis associated with SGLT2i.
Project description:The acute phase response (APR) produces marked alterations in lipid and carbohydrate metabolism including decreasing plasma ketone levels. Fibroblast growth factor 21 (FGF21) is a recently discovered hormone that regulates lipid and glucose metabolism and stimulates ketogenesis. Here we demonstrate that lipopolysaccharide (LPS), zymosan, and turpentine, which induce the APR, increase serum FGF21 levels 2-fold. Although LPS, zymosan, and turpentine decrease the hepatic expression of FGF21, they increase FGF21 expression in adipose tissue and muscle, suggesting that extrahepatic tissues account for the increase in serum FGF21. After LPS administration, the characteristic decrease in plasma ketone levels is accentuated in FGF21-/- mice, but this is not due to differences in expression of carnitine palmitoyltransferase 1? or hydroxymethyglutaryl-CoA synthase 2 in liver, because LPS induces similar decreases in the expression of these genes in FGF21-/- and control mice. However, in FGF21-/- mice, the ability of LPS to increase plasma free fatty acid levels is blunted. This failure to increase plasma free fatty acid could contribute to the accentuated decrease in plasma ketone levels because the transport of fatty acids from adipose tissue to liver provides the substrate for ketogenesis. Treatment with exogenous FGF21 reduced the number of animals that die and the rapidity of death after LPS administration in leptin-deficient ob/ob mice and to a lesser extent in control mice. FGF21 also protected from the toxic effects of cecal ligation and puncture-induced sepsis. Thus, FGF21 is a positive APR protein that protects animals from the toxic effects of LPS and sepsis.
Project description:Glucagon is classically described as a counterregulatory hormone that plays an essential role in the protection against hypoglycemia. In addition to its role in the regulation of glucose metabolism, glucagon has been described to promote ketosis in the fasted state. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are a new class of glucose-lowering drugs that act primarily in the kidney, but some reports have described direct effects of SGLT2i on α-cells to stimulate glucagon secretion. Interestingly, SGLT2 inhibition also results in increased endogenous glucose production and ketone production, features common to glucagon action. Here, we directly test the ketogenic role of glucagon in mice, demonstrating that neither fasting- nor SGLT2i-induced ketosis is altered by interruption of glucagon signaling. Moreover, any effect of glucagon to stimulate ketogenesis is severely limited by its insulinotropic actions. Collectively, our data suggest that fasting-associated ketosis and the ketogenic effects of SGLT2 inhibitors occur almost entirely independent of glucagon.
Project description:The hepatocyte-derived hormone fibroblast growth factor 21 (FGF21) is a hormone-like regulator of metabolism. The nicotinamide adenine dinucleotide-dependent deacetylase SIRT1 regulates fatty acid metabolism through multiple nutrient sensors. Hepatic overexpression of SIRT1 reduces steatosis and glucose intolerance in obese mice. We investigated mechanisms by which SIRT1 controls hepatic steatosis in mice.Liver-specific SIRT1 knockout (SIRT1 LKO) mice and their wild-type littermates (controls) were divided into groups that were placed on a normal chow diet, fasted for 24 hours, or fasted for 24 hours and then fed for 6 hours. Liver tissues were collected and analyzed by histologic examination, gene expression profiling, and real-time polymerase chain reaction assays. Human HepG2 cells were incubated with pharmacologic activators of SIRT1 (resveratrol or SRT1720) and mitochondrion oxidation consumption rate and immunoblot analyses were performed. FGF21 was overexpressed in SIRT1 LKO mice using an adenoviral vector. Energy expenditure was assessed by indirect calorimetry.Prolonged fasting induced lipid deposition in livers of control mice, but severe hepatic steatosis in SIRT1 LKO mice. Gene expression analysis showed that fasting up-regulated FGF21 in livers of control mice but not in SIRT1 LKO mice. Decreased hepatic and circulating levels of FGF21 in fasted SIRT1 LKO mice were associated with reduced hepatic expression of genes involved in fatty acid oxidation and ketogenesis, and increased expression of genes that control lipogenesis, compared with fasted control mice. Resveratrol or SRT1720 each increased the transcriptional activity of the FGF21 promoter (-2070/+117) and levels of FGF21 messenger RNA and protein in HepG2 cells. Surprisingly, SIRT1 LKO mice developed late-onset obesity with impaired whole-body energy expenditure. Hepatic overexpression of FGF21 in SIRT1 LKO mice increased the expression of genes that regulate fatty acid oxidation, decreased fasting-induced steatosis, reduced obesity, increased energy expenditure, and promoted browning of white adipose tissue.SIRT1-mediated activation of FGF21 prevents liver steatosis caused by fasting. This hepatocyte-derived endocrine signaling appears to regulate expression of genes that control a brown fat-like program in white adipose tissue, energy expenditure, and adiposity. Strategies to activate SIRT1 or FGF21 could be used to treat fatty liver disease and obesity.
Project description:OBJECTIVE:Sodium-glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i), or gliflozins, are anti-diabetic drugs that lower glycemia by promoting glucosuria, but they also stimulate endogenous glucose and ketone body production. The likely causes of these metabolic responses are increased blood glucagon levels, and decreased blood insulin levels, but the mechanisms involved are hotly debated. This study verified whether or not SGLT2i affect glucagon and insulin secretion by a direct action on islet cells in three species, using multiple approaches. METHODS:We tested the in vivo effects of two selective SGLT2i (dapagliflozin, empagliflozin) and a SGLT1/2i (sotagliflozin) on various biological parameters (glucosuria, glycemia, glucagonemia, insulinemia) in mice. mRNA expression of SGLT2 and other glucose transporters was assessed in rat, mouse, and human FACS-purified ?- and ?-cells, and by analysis of two human islet cell transcriptomic datasets. Immunodetection of SGLT2 in pancreatic tissues was performed with a validated antibody. The effects of dapagliflozin, empagliflozin, and sotagliflozin on glucagon and insulin secretion were assessed using isolated rat, mouse and human islets and the in situ perfused mouse pancreas. Finally, we tested the long-term effect of SGLT2i on glucagon gene expression. RESULTS:SGLT2 inhibition in mice increased the plasma glucagon/insulin ratio in the fasted state, an effect correlated with a decline in glycemia. Gene expression analyses and immunodetections showed no SGLT2 mRNA or protein expression in rodent and human islet cells, but moderate SGLT1 mRNA expression in human ?-cells. However, functional experiments on rat, mouse, and human (29 donors) islets and the in situ perfused mouse pancreas did not identify any direct effect of dapagliflozin, empagliflozin or sotagliflozin on glucagon and insulin secretion. SGLT2i did not affect glucagon gene expression in rat and human islets. CONCLUSIONS:The data indicate that the SGLT2i-induced increase of the plasma glucagon/insulin ratio in vivo does not result from a direct action of the gliflozins on islet cells.
Project description:Fibroblast growth factor 21 (FGF21) is a key metabolic regulator. Expressed primarily in liver and adipose tissue, FGF21 is induced via peroxisome proliferator-activated receptor (PPAR) pathways during states requiring increased fatty acid oxidation including fasting and consumption of a ketogenic diet. To test the hypothesis that FGF21 is a physiological regulator that plays a role in lipid oxidation, we generated mice with targeted disruption of the Fgf21 locus (FGF21 knockout). Mice lacking FGF21 had mild weight gain and slightly impaired glucose homeostasis, indicating a role in long-term energy homeostasis. Furthermore, FGF21KO mice tolerated a 24-h fast, indicating that FGF21 is not essential in the early stages of starvation. In contrast to wild-type animals in which feeding KD leads to dramatic weight loss, FGF21KO mice fed KD gained weight, developed hepatosteatosis, and showed marked impairments in ketogenesis and glucose control. This confirms the physiological importance of FGF21 in the adaptation to KD feeding. At a molecular level, these effects were accompanied by lower levels of expression of PGC1alpha and PGC1beta in FGF21KO mice, strongly implicating these key transcriptional regulators in the action of FGF21. Furthermore, within the liver, the maturation of the lipogenic transcription factor sterol regulatory element-binding protein-1c was increased in FGF21KO mice, implicating posttranscriptional events in the maladaptation of FGF21KO mice to KD. These data reinforce the role of FGF21 is a critical regulator of long-term energy balance and metabolism. Mice lacking FGF21 cannot respond appropriately to a ketogenic diet, resulting in an impaired ability to mobilize and utilize lipids.
Project description:The diabetes medication canagliflozin (Cana) is a sodium glucose cotransporter 2 (SGLT2) inhibitor acting by increasing urinary glucose excretion and thus reducing hyperglycaemia. Cana treatment also reduces body weight. However, it remains unclear whether Cana could directly work on adipose tissue. In the present study, the pharmacological effects of Cana and the associated mechanism were investigated in adipocytes and mice. Stromal-vascular fractions (SVFs) were isolated from subcutaneous adipose tissue and differentiated into mature adipocytes. Our results show that Cana treatment directly increased cellular energy expenditure of adipocytes by inducing mitochondrial biogenesis independently of SGLT2 inhibition. Along with mitochondrial biogenesis, Cana also increased mitochondrial oxidative phosphorylation, fatty acid oxidation and thermogenesis. Mechanistically, Cana promoted mitochondrial biogenesis and function via an Adenosine monophosphate-activated protein kinase (AMPK) - silent information regulator 1 (Sirt1) - peroxisome proliferator-activated receptor γ coactivator-1α (Pgc-1α) signalling pathway. Consistently, <i>in vivo</i> study demonstrated that Cana increased AMPK phosphorylation and the expression of Sirt1 and Pgc-1α. The present study reveals a new therapeutic function for Cana in regulating energy homoeostasis.<h4>Abbreviations</h4>Ucp-1, uncoupling protein 1; cAMP, cyclic adenosine monophosphate; PKA, cAMP-dependent protein kinase A; SGLT, sodium glucose cotransporter; Cana, canagliflozin; T2DM: type 2 diabetes; Veh, vehicle; Pgc-1α, peroxisome proliferator-activated receptor γ coactivator-1α; SVFs, stromal-vascular fractions; FBS, bovine serum; Ad, adenovirus; mtDNA, mitochondrial DNA; COX2, cytochrome oxidase subunit 2; RT-PCR, real-time PCR; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; Prdm16, PR domain zinc finger protein 16; Cidea, cell death inducing DFFA-like effector A; Pgc-1β, peroxisome proliferator-activated receptor γ coactivator-1β; NRF1, nuclear respiratory factor 1; Tfam, mitochondrial transcription factor A; OXPHOS, oxidative phosphorylation; FAO, fatty acid oxidation; AMPK, Adenosine monophosphate-activated protein kinase; p-AMPK, phosphorylated AMPK; Sirt1, silent information regulator 1; mTOR, mammalian target of rapamycin; WAT, white adipose tissue; Fabp4, fatty acid binding protein 4; Lpl, lipoprotein lipase; Slc5a2, solute carrier family 5 member 2; ERRα, oestrogen related receptor α; Uqcrc2, ubiquinol-cytochrome c reductase core protein 2; Uqcrfs1, ubiquinol-cytochrome c reductase, Rieske iron-sulphur polypeptide 1; Cox4, cytochrome c oxidase subunit 4; Pparα, peroxisome proliferator activated receptor α; NAD<sup>+</sup>, nicotinamide adenine dinucleotide; Dio2, iodothyronine deiodinase 2; Tmem26, transmembrane protein 26; Hoxa9, homeobox A9; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Rot/AA, rotenone/antimycin A; OCR, oxygen consumption rate; Pparγ, peroxisome proliferator activated receptor γ; C/ebp, CCAAT/enhancer binding protein; LKB1, liver kinase B1; AUC, area under the cure; Vd, apparent volume of distribution.