Effects of dapagliflozin and dapagliflozin-saxagliptin on erythropoiesis, iron and inflammation markers in patients with type 2 diabetes and chronic kidney disease: data from the DELIGHT trial.

BACKGROUND: This post-hoc analysis of the DELIGHT trial assessed effects of the SGLT2 inhibitor dapagliflozin on iron metabolism and markers of inflammation. METHODS: Patients with type 2 diabetes and albuminuria were randomized to dapagliflozin, dapagliflozin and saxagliptin, or placebo. We measured hemoglobin, iron markers (serum iron, transferrin saturation, and ferritin), plasma erythropoietin, and inflammatory markers (urinary MCP-1 and urinary/serum IL-6) at baseline and week 24. RESULTS: 360/461 (78.1%) participants had available biosamples. Dapagliflozin and dapagliflozin-saxagliptin, compared to placebo, increased hemoglobin by 5.7 g/L (95%CI 4.0, 7.3; p < 0.001) and 4.4 g/L (2.7, 6.0; p < 0.001) and reduced ferritin by 18.6% (8.7, 27.5; p < 0.001) and 18.4% (8.7, 27.1; p < 0.001), respectively. Dapagliflozin reduced urinary MCP-1/Cr by 29.0% (14.6, 41.0; p < 0.001) and urinary IL-6/Cr by 26.6% (9.1, 40.7; p = 0.005) with no changes in other markers. CONCLUSIONS: Dapagliflozin increased hemoglobin and reduced ferritin and urinary markers of inflammation, suggesting potentially important effects on iron metabolism and inflammation. TRIAL REGISTRATION: ClinicalTrials.gov NCT02547935.

Enhanced expression of ß cell CaV3.1 channels impairs insulin release and glucose homeostasis.

Voltage-gated calcium 3.1 (CaV3.1) channels are absent in healthy mouse ß cells and mediate minor T-type Ca2+ currents in healthy rat and human ß cells but become evident under diabetic conditions. Whether more active CaV3.1 channels affect insulin secretion and glucose homeostasis remains enigmatic. We addressed this question by enhancing de novo expression of ß cell CaV3.1 channels and exploring the consequent impacts on dynamic insulin secretion and glucose homeostasis as well as underlying molecular mechanisms with a series of in vitro and in vivo approaches. We now demonstrate that a recombinant adenovirus encoding enhanced green fluorescent protein-CaV3.1 subunit (Ad-EGFP-CaV3.1) efficiently transduced rat and human islets as well as dispersed islet cells. The resulting CaV3.1 channels conducted typical T-type Ca2+ currents, leading to an enhanced basal cytosolic-free Ca2+ concentration ([Ca2+]i). Ad-EGFP-CaV3.1-transduced islets released significantly less insulin under both the basal and first phases following glucose stimulation and could no longer normalize hyperglycemia in recipient rats rendered diabetic by streptozotocin treatment. Furthermore, Ad-EGFP-CaV3.1 transduction reduced phosphorylated FoxO1 in the cytoplasm of INS-1E cells, elevated FoxO1 nuclear retention, and decreased syntaxin 1A, SNAP-25, and synaptotagmin III. These effects were prevented by inhibiting CaV3.1 channels or the Ca2+-dependent phosphatase calcineurin. Enhanced expression of ß cell CaV3.1 channels therefore impairs insulin release and glucose homeostasis by means of initial excessive Ca2+ influx, subsequent activation of calcineurin, consequent dephosphorylation and nuclear retention of FoxO1, and eventual FoxO1-mediated down-regulation of ß cell exocytotic proteins. The present work thus suggests an elevated expression of CaV3.1 channels plays a significant role in diabetes pathogenesis.

Effects of a selective glucocorticoid receptor modulator (AZD9567) versus prednisolone in healthy volunteers: two phase 1, single-blind, randomised controlled trials.

BACKGROUND: Glucocorticoids are highly effective and widely used anti-inflammatory drugs, but their use is limited by serious side-effects, including glucocorticoid-induced hyperglycaemia and diabetes. AZD9567 is a non-steroidal, selective glucocorticoid receptor modulator that aims to reduce side-effects. We aimed to assess the safety, tolerability, and pharmacokinetics of AZD9567 in healthy volunteers. METHODS: Two phase 1 clinical studies were done. First, a randomised, placebo-controlled, single-blind, single-ascending dose study was done in healthy men who received single oral doses of AZD9567 2 mg, 10 mg, 20 mg, 40 mg, 80 mg, 100 mg, 125 mg, or 155 mg, or prednisolone 60 mg (n=8 per dose group, randomly assigned [6:2] to receive active drug or placebo). Second, a randomised, active-controlled, single-blind, multiple-ascending dose study was done, in which men and women received oral AZD9567 or prednisolone once daily for 5 days. One cohort of volunteers with prediabetes received AZD9567 10 mg (n=7) or prednisolone 20 mg (n=2). All other cohorts comprised healthy volunteers, receiving AZD9567 20 mg, 40 mg, 80 mg, or 125 mg (n=7 per dose group), or prednisolone 5 mg (n=13), 20 mg (n=16), or 40 mg (n=13). Participants and study centre staff were masked to treatment assignment for each cohort, although data were unmasked for safety review between cohorts. The primary outcome of the single-ascending dose study was the safety, tolerability, and pharmacokinetics of single ascending doses of AZD9567; for the multiple-ascending dose study it was the safety and tolerability of AZD9567 following multiple ascending doses. As a secondary outcome, effects on glycaemic control were ascertained with oral glucose tolerance tests (OGTTs) done at baseline and on day 1 of the single-ascending dose study, and at baseline and on day 4 of the multiple-ascending dose study. These trials are registered at ClinicalTrials.gov, NCT02512575 and NCT02760316. FINDINGS: In the single-ascending dose study, between Nov 18, 2015, and Sept 26, 2016, 72 healthy white men were enrolled, and all completed the study. In the multiple-ascending dose study, between May 2, 2016, and Sept 13, 2017, 77 predominantly white male volunteers (including nine individuals with prediabetes and eight women) were enrolled and 75 completed the study. All doses of AZD9567 and prednisolone were well tolerated, with no serious adverse events or events suggesting adrenal insufficiency. In the single-ascending dose study, nine adverse events of mild intensity were reported (five with AZD9567 and four with placebo); no adverse event was reported by more than one person. In the multiple-ascending dose study, 44 adverse events of mild or moderate intensity were reported (18 with AZD9567 and 26 with prednisolone). The most common were headache and micturition. Apparent clearance, volume of distribution, and half-life of AZD9567 were consistent across doses and for single versus repeated dosing. In the multiple-ascending dose study, OGTTs showed no significant difference with AZD9567 doses up to 80 mg compared with prednisolone 5 mg in glucose area under the curve from 0 h to 4 h post-OGTT (AUC0-4h) from baseline to day 4; the increase in glucose AUC0-4h from baseline to day 4 was significantly lower with all AZD9567 doses versus prednisolone 20 mg (AZD9567 20 mg p<0·0001, 40 mg p=0·0001, 80 mg p=0·0001, and 125 mg p=0·0237). INTERPRETATION: AZD9567 appears to be safe and well tolerated in healthy, predominantly white male volunteers and shows promising initial evidence for improved post-prandial glucose control. Studies of longer duration, with a greater proportion of women and other ethnic groups, and in patients requiring anti-inflammatory treatment are needed to characterise the clinical efficacy and safety profile of AZD9567. FUNDING: AstraZeneca.

Exploring the insulin secretory properties of the PGD2-GPR44/DP2 axis in vitro and in a randomized phase-1 trial of type 2 diabetes patients.

AIMS/HYPOTHESIS: GPR44 (DP2, PTGDR2, CRTh2) is the receptor for the pro-inflammatory mediator prostaglandin D2 (PGD2) and it is enriched in human islets. In rodent islets, PGD2 is produced in response to glucose, suggesting that the PGD2-GPR44/DP2 axis may play a role in human islet function during hyperglycemia. Consequently, the aim of this work was to elucidate the insulinotropic role of GPR44 antagonism in vitro in human beta-cells and in type 2 diabetes (T2DM) patients. METHODS: We determined the drive on PGD2 secretion by glucose and IL-1beta, as well as, the impact on insulin secretion by pharmacological GPR44/DP2 antagonism (AZD1981) in human islets and beta-cells in vitro. To test if metabolic control would be improved by antagonizing a hyperglycemia-driven increased PGD2 tone, we performed a proof-of-mechanism study in 20 T2DM patients (average 54 years, HbA1c 9.4%, BMI 31.6 kg/m2). The randomized, double-blind, placebo-controlled cross-over study consisted of two three-day treatment periods (AZD1981 or placebo) separated by a three-day wash-out period. Mixed meal tolerance test (MMTT) and intravenous graded glucose infusion (GGI) was performed at start and end of each treatment period. Assessment of AZD1981 pharmacokinetics, glucose, insulin, C-peptide, glucagon, GLP-1, and PGD2 pathway biomarkers were performed. RESULTS: We found (1) that PGD2 is produced in human islet in response to high glucose or IL-1beta, but likely by stellate cells rather than endocrine cells; (2) that PGD2 suppresses both glucose and GLP-1 induced insulin secretion in vitro; and (3) that the GPR44/DP2 antagonist (AZD1981) in human beta-cells normalizes insulin secretion. However, AZD1981 had no impact on neither glucose nor incretin dependent insulin secretion in humans (GGI AUC C-peptide 1-2h and MMTT AUC Glucose 0-4h LS mean ratios vs placebo of 0.94 (80% CI of 0.90-0.98, p = 0.12) and 0.99 (90% CI of 0.94-1.05, p = 0.45), despite reaching the expected antagonist exposure. CONCLUSION/INTERPRETATION: Pharmacological inhibition of the PGD2-GPR44/DP2 axis has no major impact on the modulation of acute insulin secretion in T2DM patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT02367066.

Human pancreatic islet-derived extracellular vesicles modulate insulin expression in 3D-differentiating iPSC clusters.

It has been suggested that extracellular vesicles (EVs) can mediate crosstalk between hormones and metabolites within pancreatic tissue. However, the possible effect of pancreatic EVs on stem cell differentiation into pancreatic lineages remains unknown. Herein, human islet-derived EVs (h-Islet-EVs) were isolated, characterized and subsequently added to human induced pluripotent stem cell (iPSC) clusters during pancreatic differentiation. The h-islet-EVs had a mean size of 117±7 nm and showed positive expression of CD63 and CD81 EV markers as measured by ELISA. The presence of key pancreatic transcription factor mRNA, such as NGN3, MAFA and PDX1, and pancreatic hormone proteins such as C-peptide and glucagon, were confirmed in h-Islet-EVs. iPSC clusters were differentiated in suspension and at the end stages of the differentiation protocol, the mRNA expression of the main pancreatic transcription factors and pancreatic hormones was increased. H-Islet-EVs were supplemented to the iPSC clusters in the later stages of differentiation. It was observed that h-Islet-EVs were able to up-regulate the intracellular levels of C-peptide in iPSC clusters in a concentration-dependent manner. The effect of h-Islet-EVs on the differentiation of iPSC clusters cultured in 3D-collagen hydrogels was also assessed. Although increased mRNA expression for pancreatic markers was observed when culturing the iPSC clusters in 3D-collagen hydrogels, delivery of EVs did not affect the insulin or C-peptide intracellular content. Our results provide new information on the role of h-Islet-EVs in the regulation of insulin expression in differentiating iPSC clusters, and are highly relevant for pancreatic tissue engineering applications.

Single-Cell Transcriptome Profiling of Human Pancreatic Islets in Health and Type 2 Diabetes.

Hormone-secreting cells within pancreatic islets of Langerhans play important roles in metabolic homeostasis and disease. However, their transcriptional characterization is still incomplete. Here, we sequenced the transcriptomes of thousands of human islet cells from healthy and type 2 diabetic donors. We could define specific genetic programs for each individual endocrine and exocrine cell type, even for rare δ, γ, ε, and stellate cells, and revealed subpopulations of α, ß, and acinar cells. Intriguingly, δ cells expressed several important receptors, indicating an unrecognized importance of these cells in integrating paracrine and systemic metabolic signals. Genes previously associated with obesity or diabetes were found to correlate with BMI. Finally, comparing healthy and T2D transcriptomes in a cell-type resolved manner uncovered candidates for future functional studies. Altogether, our analyses demonstrate the utility of the generated single-cell gene expression resource.

Inhibition of intestinal bile acid transporter Slc10a2 improves triglyceride metabolism and normalizes elevated plasma glucose levels in mice.

Interruption of the enterohepatic circulation of bile acids increases cholesterol catabolism, thereby stimulating hepatic cholesterol synthesis from acetate. We hypothesized that such treatment should lower the hepatic acetate pool which may alter triglyceride and glucose metabolism. We explored this using mice deficient of the ileal sodium-dependent BA transporter (Slc10a2) and ob/ob mice treated with a specific inhibitor of Slc10a2. Plasma TG levels were reduced in Slc10a2-deficient mice, and when challenged with a sucrose-rich diet, they displayed a reduced response in hepatic TG production as observed from the mRNA levels of several key enzymes in fatty acid synthesis. This effect was paralleled by a diminished induction of mature sterol regulatory element-binding protein 1c (Srebp1c). Unexpectedly, the SR-diet induced intestinal fibroblast growth factor (FGF) 15 mRNA and normalized bile acid synthesis in Slc10a2-/- mice. Pharmacologic inhibition of Slc10a2 in diabetic ob/ob mice reduced serum glucose, insulin and TGs, as well as hepatic mRNA levels of Srebp1c and its target genes. These responses are contrary to those reported following treatment of mice with a bile acid binding resin. Moreover, when key metabolic signal transduction pathways in the liver were investigated, those of Mek1/2-Erk1/2 and Akt were blunted after treatment of ob/ob mice with the Slc10a2 inhibitor. It is concluded that abrogation of Slc10a2 reduces hepatic Srebp1c activity and serum TGs, and in the diabetic ob/ob model it also reduces glucose and insulin levels. Hence, targeting of Slc10a2 may be a promising strategy to treat hypertriglyceridemia and diabetes.