CDKN2C expression in adipose tissue is reduced in type II diabetes and central obesity: impact on adipocyte differentiation and lipid storage?

CDKN2C/p18 (Cyclin-Dependent Kinase Inhibitor 2C) is a cell growth regulator that controls cell cycle progression and has previously been associated with increased risk for type II diabetes (T2D) and reduced peripheral adipose tissue (AT) storage capacity. This study explored the role of CDKN2C in AT lipid and glucose metabolism in T2D. Expression of CDKN2C and other genes was analyzed by transcriptomics, or real-time PCR in subcutaneous AT (SAT) samples obtained from T2D and control subjects matched for sex, age and BMI and also in paired SAT and omental AT (OAT) samples. Functional studies included adipocyte glucose uptake and lipolysis rates. CRISPR/Cas9 CDKN2C gene knockdown was performed in human preadipocytes to assess adipogenesis. CDKN2C mRNA expression in SAT and OAT was reduced in T2D and obese subjects compared to controls. CDKN2C expression in SAT was inversely correlated with measures of hyperglycemia, insulin resistance and visceral adiposity and positively correlated with expression of genes in several metabolic pathways, including insulin signaling and fatty acid and carbohydrate metabolism. CDKN2C protein was mainly expressed in adipocytes compared to stromal vascular cells, and its gene and protein expression was up-regulated during adipocyte differentiation. Knockdown of CDKN2C did not affect the percentage of differentiating cells compared to wild type cultures. However, CDKN2C knockdown cultures had significantly lower expression of differentiation markers CEBPA, ADIPOQ and FASN and transiently reduced lipid accumulation per adipocyte during differentiation. Our findings suggest that adipose CDKN2C expression might be reduced as a consequence of insulin resistance and obesity, and this can further contribute to impairment of SAT lipid storage.

Impaired HMG-CoA Reductase Activity Caused by Genetic Variants or Statin Exposure: Impact on Human Adipose Tissue, ß-Cells and Metabolome.

Inhibition of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase is associated with an increased risk of new-onset type 2 diabetes. We studied the association of genetic or pharmacological HMG-CoA reductase inhibition with plasma and adipose tissue (AT) metabolome and AT metabolic pathways. We also investigated the effects of statin-mediated pharmacological inhibition of HMG-CoA reductase on systemic insulin sensitivity by measuring the HOMA-IR index in subjects with or without statin therapy. The direct effects of simvastatin (20-250 nM) or its active metabolite simvastatin hydroxy acid (SA) (8-30 nM) were investigated on human adipocyte glucose uptake, lipolysis, and differentiation and pancreatic insulin secretion. We observed that the LDL-lowering HMGCR rs12916-T allele was negatively associated with plasma phosphatidylcholines and sphingomyelins, and HMGCR expression in AT was correlated with various metabolic and mitochondrial pathways. Clinical data showed that statin treatment was associated with HOMA-IR index after adjustment for age, sex, BMI, HbA1c, LDL-c levels, and diabetes status in the subjects. Supra-therapeutic concentrations of simvastatin reduced glucose uptake in adipocytes and normalized fatty acid-induced insulin hypersecretion from ß-cells. Our data suggest that inhibition of HMG-CoA reductase is associated with insulin resistance. However, statins have a very mild direct effect on AT and pancreas, hence, other tissues as the liver or muscle appear to be of greater importance.

Tissue-specific glucose partitioning and fat content in prediabetes and type 2 diabetes: whole-body PET/MRI during hyperinsulinemia.

OBJECTIVE: To obtain direct quantifications of glucose turnover, volumes and fat content of several tissues in the development of type 2 diabetes (T2D) using a novel integrated approach for whole-body imaging. DESIGN AND METHODS: Hyperinsulinemic-euglycemic clamps and simultaneous whole-body integrated [18F]FDG-PET/MRI with automated analyses were performed in control (n = 12), prediabetes (n = 16) and T2D (n = 13) subjects matched for age, sex and BMI. RESULTS: Whole-body glucose uptake (Rd) was reduced by approximately 25% in T2D vs control subjects, and partitioning to brain was increased from 3.8% of total Rd in controls to 7.1% in T2D. In liver, subcutaneous AT, thigh muscle, total tissue glucose metabolic rates (MRglu) and their % of total Rd were reduced in T2D compared to control subjects. The prediabetes group had intermediate findings. Total MRglu in heart, visceral AT, gluteus and calf muscle was similar across groups. Whole-body insulin sensitivity assessed as glucose infusion rate correlated with liver MRglu but inversely with brain MRglu. Liver fat content correlated with MRglu in brain but inversely with MRglu in other tissues. Calf muscle fat was inversely associated with MRglu only in the same muscle group. CONCLUSIONS: This integrated imaging approach provides detailed quantification of tissue-specific glucose metabolism. During T2D development, insulin-stimulated glucose disposal is impaired and increasingly shifted away from muscle, liver and fat toward the brain. Altered glucose handling in the brain and liver fat accumulation may aggravate insulin resistance in several organs.

Identification of human glucocorticoid response markers using integrated multi-omic analysis from a randomized crossover trial.

Background: Glucocorticoids are among the most commonly prescribed drugs, but there is no biomarker that can quantify their action. The aim of the study was to identify and validate circulating biomarkers of glucocorticoid action. Methods: In a randomized, crossover, single-blind, discovery study, 10 subjects with primary adrenal insufficiency (and no other endocrinopathies) were admitted at the in-patient clinic and studied during physiological glucocorticoid exposure and withdrawal. A randomization plan before the first intervention was used. Besides mild physical and/or mental fatigue and salt craving, no serious adverse events were observed. The transcriptome in peripheral blood mononuclear cells and adipose tissue, plasma miRNAomic, and serum metabolomics were compared between the interventions using integrated multi-omic analysis. Results: We identified a transcriptomic profile derived from two tissues and a multi-omic cluster, both predictive of glucocorticoid exposure. A microRNA (miR-122-5p) that was correlated with genes and metabolites regulated by glucocorticoid exposure was identified (p=0.009) and replicated in independent studies with varying glucocorticoid exposure (0.01 ≤ p≤0.05). Conclusions: We have generated results that construct the basis for successful discovery of biomarker(s) to measure effects of glucocorticoids, allowing strategies to individualize and optimize glucocorticoid therapy, and shedding light on disease etiology related to unphysiological glucocorticoid exposure, such as in cardiovascular disease and obesity. Funding: The Swedish Research Council (Grant 2015-02561 and 2019-01112); The Swedish federal government under the LUA/ALF agreement (Grant ALFGBG-719531); The Swedish Endocrinology Association; The Gothenburg Medical Society; Wellcome Trust; The Medical Research Council, UK; The Chief Scientist Office, UK; The Eva Madura's Foundation; The Research Foundation of Copenhagen University Hospital; and The Danish Rheumatism Association. Clinical trial number: NCT02152553.

Several diseases, including asthma, arthritis, some skin conditions, and cancer, are treated with medications called glucocorticoids, which are synthetic versions of human hormones. These drugs are also used to treat people with a condition call adrenal insufficiency who do not produce enough of an important hormone called cortisol. Use of glucocorticoids is very common, the proportion of people in a given country taking them can range from 0.5% to 21% of the population depending on the duration of the treatment. But, like any medication, glucocorticoids have both benefits and risks: people who take glucocorticoids for a long time have an increased risk of diabetes, obesity, cardiovascular disease, and death. Because of the risks associated with taking glucocorticoids, it is very important for physicians to tailor the dose to each patient's needs. Doing this can be tricky, because the levels of glucocorticoids in a patient's blood are not a good indicator of the medication's activity in the body. A test that can accurately measure the glucocorticoid activity could help physicians personalize treatment and reduce harmful side effects. As a first step towards developing such a test, Chantzichristos et al. identified a potential way to measure glucocorticoid activity in patient's blood. In the experiments, blood samples were collected from ten patients with adrenal insufficiency both when they were on no medication, and when they were taking a glucocorticoid to replace their missing hormones. Next, the blood samples were analyzed to determine which genes were turned on and off in each patient with and without the medication. They also compared small molecules in the blood called metabolites and tiny pieces of genetic material called microRNAs that turn genes on and off. The experiments revealed networks of genes, metabolites, and microRNAs that are associated with glucocorticoid activity, and one microRNA called miR-122-5p stood out as a potential way to measure glucocorticoid activity. To verify this microRNA's usefulness, Chantzichristos et al. looked at levels of miR-122-5p in people participating in three other studies and confirmed that it was a good indicator of the glucocorticoid activity. More research is needed to confirm Chantzichristos et al.'s findings and to develop a test that can be used by physicians to measure glucocorticoid activity. The microRNA identified, miR-122-5p, has been previously linked to diabetes, so studying it further may also help scientists understand how taking glucocorticoids may increase the risk of developing diabetes and related diseases.

Improved Urinary Cortisol Metabolome in Addison Disease: A Prospective Trial of Dual-Release Hydrocortisone.

CONTEXT: Oral once-daily dual-release hydrocortisone (DR-HC) replacement therapy has demonstrated an improved metabolic profile compared to conventional 3-times-daily (TID-HC) therapy among patients with primary adrenal insufficiency. This effect might be related to a more physiological cortisol profile, but also to a modified pattern of cortisol metabolism. OBJECTIVE: This work aimed to study cortisol metabolism during DR-HC and TID-HC. DESIGN: A randomized, 12-week, crossover study was conducted. INTERVENTION AND PARTICIPANTS: DC-HC and same daily dose of TID-HC were administered to patients with primary adrenal insufficiency (n = 50) vs healthy individuals (n = 124) as controls. MAIN OUTCOME MEASURES: Urinary corticosteroid metabolites were measured by gas chromatography/mass spectrometry at 24-hour urinary collections. RESULTS: Total cortisol metabolites decreased during DR-HC compared to TID-HC (P < .001) and reached control values (P = .089). During DR-HC, 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) activity measured by tetrahydrocortisol + 5α-tetrahydrocortisol/tetrahydrocortisone ratio was reduced compared to TID-HC (P < .05), but remained increased vs controls (P < .001). 11ß-HSD2 activity measured by urinary free cortisone/free cortisol ratio was decreased with TID-HC vs controls (P < .01) but normalized with DR-HC (P = .358). 5α- and 5ß-reduced metabolites were decreased with DR-HC compared to TID-HC. Tetrahydrocortisol/5α-tetrahydrocortisol ratio was increased during both treatments, suggesting increased 5ß-reductase activity. CONCLUSIONS: The urinary cortisol metabolome shows striking abnormalities in patients receiving conventional TID-HC replacement therapy, with increased 11ß-HSD1 activity that may account for the unfavorable metabolic phenotype in primary adrenal insufficiency. Its change toward normalization with DR-HC may mediate beneficial metabolic effects. The urinary cortisol metabolome may serve as a tool to assess optimal cortisol replacement therapy.

In Vitro and In Vivo Evaluation of 3D Printed Capsules with Pressure Triggered Release Mechanism for Oral Peptide Delivery.

In this study a 3D printed capsule designed to break from the physiological pressures in the antropyloric region was evaluated for its ability to deliver the synthetic octapeptide octreotide in beagle dogs when co-formulated with the permeation enhancer sodium caprate. The pressure sensitive capsules were compared to traditional enteric coated hard gelatin capsules and enteric coated tablets. Paracetamol, which is completely absorbed in dogs, was included in the formulations and used as an absorption marker to give information about the in vivo performance of the dosage forms. The pressure sensitive capsules released drug in 50% of the dogs. In the cases where drug was released, there was no difference in octreotide bioavailability or Cmax compared to the enteric coated dosage forms. When comparing all dosage forms, a correlation was seen between paracetamol Cmax and octreotide bioavailability, suggesting that a high drug release rate may be beneficial for peptide absorption when delivered together with sodium caprate.

Impact of CKD Progression on Cardiovascular Disease Risk in a Contemporary UK Cohort of Individuals With Diabetes.

INTRODUCTION: It remains unclear whether an increased progression rate of chronic kidney disease (CKD) adds predictive information regarding cardiovascular disease (CVD) risk. The aim of this study was to evaluate the association between CKD progression, based on estimated glomerular filtration rate (eGFR) slope estimates and the risk for CVD. METHODS: We compared the updated eGFR slope calculated over multiple overlapping 2-year periods and the updated mean eGFR. Incident CKD subjects were selected from a prevalent population with diabetes (T2DM). Subjects from the UK Clinical Practice Research Data Link GOLD (CPRD) were followed from CKD diagnosis (n = 30,222) until heart failure (HF), myocardial infarction (MI), ischemic stroke (IS), or a composite end point including all 3 event types (MACE plus), mortality, database dropout, or end of study follow-up. RESULTS: Both the updated eGFR slope and updated mean eGFR were associated with MACE plus and HF. Updated eGFR slope decline of > -3 ml/min/1.73 m2 increased the risk for MACE plus (adjusted hazard ratio [HR] = 1.45; 95% confidence interval [CI], 1.26-1.67), HF (HR = 1.50; 95% CI, 1.27-1.76), and MI (HR = 1.39; 95% CI, 1.01-1.91). CONCLUSIONS: This study strongly supports current evidence that CKD is an independent risk factor for CVD. From a clinical perspective, both rate of progression and cumulative status of CKD describe distinct aspects of the cardiorenal risk among persons with diabetes. This evidence is essential to enable more timely and improved use of treatments in this population.

Inhibition of the prostaglandin D2-GPR44/DP2 axis improves human islet survival and function.

AIMS/HYPOTHESIS: Inflammatory signals and increased prostaglandin synthesis play a role during the development of diabetes. The prostaglandin D2 (PGD2) receptor, GPR44/DP2, is highly expressed in human islets and activation of the pathway results in impaired insulin secretion. The role of GPR44 activation on islet function and survival rate during chronic hyperglycaemic conditions is not known. In this study, we investigate GPR44 inhibition by using a selective GPR44 antagonist (AZ8154) in human islets both in vitro and in vivo in diabetic mice transplanted with human islets. METHODS: Human islets were exposed to PGD2 or proinflammatory cytokines in vitro to investigate the effect of GPR44 inhibition on islet survival rate. In addition, the molecular mechanisms of GPR44 inhibition were investigated in human islets exposed to high concentrations of glucose (HG) and to IL-1ß. For the in vivo part of the study, human islets were transplanted under the kidney capsule of immunodeficient diabetic mice and treated with 6, 60 or 100 mg/kg per day of a GPR44 antagonist starting from the transplantation day until day 4 (short-term study) or day 17 (long-term study) post transplantation. IVGTT was performed on mice at day 10 and day 15 post transplantation. After termination of the study, metabolic variables, circulating human proinflammatory cytokines, and hepatocyte growth factor (HGF) were analysed in the grafted human islets. RESULTS: PGD2 or proinflammatory cytokines induced apoptosis in human islets whereas GPR44 inhibition reversed this effect. GPR44 inhibition antagonised the reduction in glucose-stimulated insulin secretion induced by HG and IL-1ß in human islets. This was accompanied by activation of the Akt-glycogen synthase kinase 3ß signalling pathway together with phosphorylation and inactivation of forkhead box O-1and upregulation of pancreatic and duodenal homeobox-1 and HGF. Administration of the GPR44 antagonist for up to 17 days to diabetic mice transplanted with a marginal number of human islets resulted in reduced fasting blood glucose and lower glucose excursions during IVGTT. Improved glucose regulation was supported by increased human C-peptide levels compared with the vehicle group at day 4 and throughout the treatment period. GPR44 inhibition reduced plasma levels of TNF-α and growth-regulated oncogene-α/chemokine (C-X-C motif) ligand 1 and increased the levels of HGF in human islets. CONCLUSIONS/INTERPRETATION: Inhibition of GPR44 in human islets has the potential to improve islet function and survival rate under inflammatory and hyperglycaemic stress. This may have implications for better survival rate of islets following transplantation.

Integration of whole-body [18F]FDG PET/MRI with non-targeted metabolomics can provide new insights on tissue-specific insulin resistance in type 2 diabetes.

Alteration of various metabolites has been linked to type 2 diabetes (T2D) and insulin resistance. However, identifying significant associations between metabolites and tissue-specific phenotypes requires a multi-omics approach. In a cohort of 42 subjects with different levels of glucose tolerance (normal, prediabetes and T2D) matched for age and body mass index, we calculated associations between parameters of whole-body positron emission tomography (PET)/magnetic resonance imaging (MRI) during hyperinsulinemic euglycemic clamp and non-targeted metabolomics profiling for subcutaneous adipose tissue (SAT) and plasma. Plasma metabolomics profiling revealed that hepatic fat content was positively associated with tyrosine, and negatively associated with lysoPC(P-16:0). Visceral adipose tissue (VAT) and SAT insulin sensitivity (Ki), were positively associated with several lysophospholipids, while the opposite applied to branched-chain amino acids. The adipose tissue metabolomics revealed a positive association between non-esterified fatty acids and, VAT and liver Ki. Bile acids and carnitines in adipose tissue were inversely associated with VAT Ki. Furthermore, we detected several metabolites that were significantly higher in T2D than normal/prediabetes. In this study we present novel associations between several metabolites from SAT and plasma with the fat fraction, volume and insulin sensitivity of various tissues throughout the body, demonstrating the benefit of an integrative multi-omics approach.

rs953413 Regulates Polyunsaturated Fatty Acid Metabolism by Modulating ELOVL2 Expression.

Long-chain polyunsaturated fatty acids (LC-PUFAs) influence human health in several areas, including cardiovascular disease, diabetes, fatty liver disease, and cancer. ELOVL2 encodes one of the key enzymes in the in vivo synthesis of LC-PUFAs from their precursors. Variants near ELOVL2 have repeatedly been associated with levels of LC-PUFA-derived metabolites in genome-wide association studies (GWAS), but the mechanisms behind these observations remain poorly defined. In this study, we found that rs953413, located in the first intron of ELOVL2, lies within a functional FOXA and HNF4α cooperative binding site. The G allele of rs953413 increases binding of FOXA1/FOXA2 and HNF4α to an evolutionarily conserved enhancer element, conferring allele-specific upregulation of the rs953413-associated gene ELOVL2. The expression of ELOVL2 was significantly downregulated by both FOXA1 and HNF4α knockdown and CRISPR/Cas9-mediated direct mutation to the enhancer element. Our results suggest that rs953413 regulates LC-PUFAs metabolism by altering ELOVL2 expression through FOXA1/FOXA2 and HNF4α cooperation.

Phase 1 Pharmacokinetic Study of AZD5718 in Healthy Volunteers: Effects of Coadministration With Rosuvastatin, Formulation and Food on Oral Bioavailability.

AZD5718 is a first-in-class small-molecule anti-inflammatory drug with the potential to reduce the residual risk of cardiovascular events after myocardial infarction in patients receiving lipid-lowering statin therapy. Leukotrienes are potent proinflammatory and vasoactive mediators synthesized in leukocytes via 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP). AZD5718 is a FLAP inhibitor that dose-dependently reduced leukotriene biosynthesis in a first-in-human study. We enrolled 12 healthy men in a randomized, open-label, crossover, single-dose phase 1 pharmacokinetic study of AZD5718 to investigate a potential drug-drug interaction with rosuvastatin, and the effects of formulation and food intake (ClinicalTrials.gov identifier: NCT02963116). Rosuvastatin (10 mg) were absorbed more rapidly when coadministered with AZD5718 (200 mg), probably owing to weak inhibition of hepatic statin uptake, but relative bioavailability was unaffected (geometric least-squares mean ratio [GMR], 100%; 90% confidence interval [CI], 86%-116%). AZD5718 pharmacokinetics were unaffected by coadministration of rosuvastatin. AZD5718 (200 mg) was absorbed less rapidly when formulated as tablets than oral suspension, with reduced relative bioavailability (GMR, 72%; 90%CI, 64%-80%). AZD5718 absorption was slower when 200-mg tablets were taken after a high-fat breakfast than after fasting, but relative bioavailability was unaffected (GMR, 96%; 90%CI, 87%-106%). In post hoc pharmacodynamic simulations, plasma leukotriene B4 levels were inhibited by >90% throughout the day following once-daily AZD5718, regardless of formulation or administration with food. AZD5718 was well tolerated, with no severe or serious adverse events. These data supported the design of a phase 2a efficacy study of AZD5718 in patients with coronary artery disease.

Author Correction: Intra- and inter-individual metabolic profiling highlights carnitine and lysophosphatidylcholine pathways as key molecular defects in type 2 diabetes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Intra- and inter-individual metabolic profiling highlights carnitine and lysophosphatidylcholine pathways as key molecular defects in type 2 diabetes.

Type 2 diabetes (T2D) mellitus is a complex metabolic disease commonly caused by insulin resistance in several tissues. We performed a matched two-dimensional metabolic screening in tissue samples from 43 multi-organ donors. The intra-individual analysis was assessed across five key metabolic tissues (serum, visceral adipose tissue, liver, pancreatic islets and skeletal muscle), and the inter-individual across three different groups reflecting T2D progression. We identified 92 metabolites differing significantly between non-diabetes and T2D subjects. In diabetes cases, carnitines were significantly higher in liver, while lysophosphatidylcholines were significantly lower in muscle and serum. We tracked the primary tissue of origin for multiple metabolites whose alterations were reflected in serum. An investigation of three major stages spanning from controls, to pre-diabetes and to overt T2D indicated that a subset of lysophosphatidylcholines was significantly lower in the muscle of pre-diabetes subjects. Moreover, glycodeoxycholic acid was significantly higher in liver of pre-diabetes subjects while additional increase in T2D was insignificant. We confirmed many previously reported findings and substantially expanded on them with altered markers for early and overt T2D. Overall, the analysis of this unique dataset can increase the understanding of the metabolic interplay between organs in the development of T2D.

Comparative quantitative systems pharmacology modeling of anti-PCSK9 therapeutic modalities in hypercholesterolemia.

Since the discovery of proprotein convertase subtilisin/kexin type 9 (PCSK9) as an attractive target in the treatment of hypercholesterolemia, multiple anti-PCSK9 therapeutic modalities have been pursued in drug development. The objective of this research is to set the stage for the quantitative benchmarking of two anti-PCSK9 pharmacological modality classes, monoclonal antibodies (mAbs) and small interfering RNA (siRNA). To this end, we developed an integrative mathematical model of lipoprotein homeostasis describing the dynamic interplay between PCSK9, LDL-cholesterol (LDL-C), VLDL-cholesterol, HDL-cholesterol (HDL-C), apoB, lipoprotein a [Lp(a)], and triglycerides (TGs). We demonstrate that LDL-C decreased proportionally to PCSK9 reduction for both mAb and siRNA modalities. At marketed doses, however, treatment with mAbs resulted in an additional ∼20% LDL-C reduction compared with siRNA. We further used the model as an evaluation tool and determined that no quantitative differences were observed in HDL-C, Lp(a), TG, or apoB responses, suggesting that the disruption of PCSK9 synthesis would provide no additional effects on lipoprotein-related biomarkers in the patient segment investigated. Predictive model simulations further indicate that siRNA therapies may reach reductions in LDL-C levels comparable to those achieved with mAbs if the current threshold of 80% PCSK9 inhibition via siRNA could be overcome.

Studies of liver tissue identify functional gene regulatory elements associated to gene expression, type 2 diabetes, and other metabolic diseases.

BACKGROUND: Genome-wide association studies (GWAS) of diseases and traits have found associations to gene regions but not the functional SNP or the gene mediating the effect. Difference in gene regulatory signals can be detected using chromatin immunoprecipitation and next-gen sequencing (ChIP-seq) of transcription factors or histone modifications by aligning reads to known polymorphisms in individual genomes. The aim was to identify such regulatory elements in the human liver to understand the genetics behind type 2 diabetes and metabolic diseases. METHODS: The genome of liver tissue was sequenced using 10X Genomics technology to call polymorphic positions. Using ChIP-seq for two histone modifications, H3K4me3 and H3K27ac, and the transcription factor CTCF, and our established bioinformatics pipeline, we detected sites with significant difference in signal between the alleles. RESULTS: We detected 2329 allele-specific SNPs (AS-SNPs) including 25 associated to GWAS SNPs linked to liver biology, e.g., 4 AS-SNPs at two type 2 diabetes loci. Two hundred ninety-two AS-SNPs were associated to liver gene expression in GTEx, and 134 AS-SNPs were located on 166 candidate functional motifs and most of them in EGR1-binding sites. CONCLUSIONS: This study provides a valuable collection of candidate liver regulatory elements for further experimental validation.

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.

The development of a GPR44 targeting radioligand [11C]AZ12204657 for in vivo assessment of beta cell mass.

BACKGROUND: The G-protein-coupled receptor 44 (GPR44) is a beta cell-restricted target that may serve as a marker for beta cell mass (BCM) given the development of a suitable PET ligand. METHODS: The binding characteristics of the selected candidate, AZ12204657, at human GPR44 were determined using in vitro ligand binding assays. AZ12204657 was radiolabeled using 11C- or 3H-labeled methyl iodide ([11C/3H]CH3I) in one step, and the conversion of [11C/3H]CH3I to the radiolabeled product [11C/3H]AZ12204657 was quantitative. The specificity of radioligand binding to GPR44 and the selectivity for beta cells were evaluated by in vitro binding studies on pancreatic sections from human and non-human primates as well as on homogenates from endocrine and exocrine pancreatic compartments. RESULTS: The radiochemical purity of the resulting radioligand [11C]AZ12204657 was > 98%, with high molar activity (MA), 1351 ± 575 GBq/µmol (n = 18). The radiochemical purity of [3H]AZ12204657 was > 99% with MA of 2 GBq/µmol. Pancreatic binding of [11C/3H]AZ12204657 was co-localized with insulin-positive islets of Langerhans in non-diabetic individuals and individuals with type 2 diabetes (T2D). The binding of [11C]AZ12204657 to GPR44 was > 10 times higher in islet homogenates compared to exocrine homogenates. In human islets of Langerhans GPR44 was co-expressed with insulin, but not glucagon as assessed by co-staining and confocal microscopy. CONCLUSION: We radiolabeled [11C]AZ12204657, a potential PET radioligand for the beta cell-restricted protein GPR44. In vitro evaluation demonstrated that [3H]AZ12204657 and [11C]AZ12204657 selectively target pancreatic beta cells. [11C]AZ12204657 has promising properties as a marker for human BCM.

FKBP5 expression in human adipose tissue: potential role in glucose and lipid metabolism, adipogenesis and type 2 diabetes.

PURPOSE: Here, we explore the involvement of FKBP51 in glucocorticoid-induced insulin resistance (IR) in human subcutaneous adipose tissue (SAT), including its potential role in type 2 diabetes (T2D). Moreover, we assess the metabolic effects of reducing the activity of FKBP51 using the specific inhibitor SAFit1. METHODS: Human SAT was obtained by needle biopsies of the lower abdominal region. FKBP5 gene expression was assessed in fresh SAT explants from a cohort of 20 T2D subjects group-wise matched by gender, age and BMI to 20 non-diabetic subjects. In addition, human SAT was obtained from non-diabetic volunteers (20F/9M). SAT was incubated for 24 h with or without the synthetic glucocorticoid dexamethasone and SAFit1. Incubated SAT was used to measure the glucose uptake rate in isolated adipocytes. RESULTS: FKBP5 gene expression levels in SAT positively correlated with several indices of IR as well as glucose area under the curve during oral glucose tolerance test (r = 0.33, p < 0.05). FKBP5 gene expression levels tended to be higher in T2D subjects compared to non-diabetic subjects (p = 0.088). Moreover, FKBP5 gene expression levels were found to inversely correlate with lipolytic, lipogenic and adipogenic genes. SAFit1 partly prevented the inhibitory effects of dexamethasone on glucose uptake. CONCLUSIONS: FKBP5 gene expression in human SAT tends to be increased in T2D subjects and is related to elevated glucose levels. Moreover, FKBP5 gene expression is inversely associated with the expression of lipolytic, lipogenic and adipogenic genes. SAFit1 can partly prevent glucose uptake impairment by glucocorticoids, suggesting that FKBP51 might be a key factor in glucocorticoid-induced IR.

Altered Glucose Uptake in Muscle, Visceral Adipose Tissue, and Brain Predict Whole-Body Insulin Resistance and may Contribute to the Development of Type 2 Diabetes: A Combined PET/MR Study.

We assessed glucose uptake in different tissues in type 2 diabetes (T2D), prediabetes, and control subjects to elucidate its impact in the development of whole-body insulin resistance and T2D. Thirteen T2D, 12 prediabetes, and 10 control subjects, matched for age and BMI, underwent OGTT and abdominal subcutaneous adipose tissue (SAT) biopsies. Integrated whole-body 18F-FDG PET and MRI were performed during a hyperinsulinemic euglycemic clamp to asses glucose uptake rate (MRglu) in several tissues. MRglu in skeletal muscle, SAT, visceral adipose tissue (VAT), and liver was significantly reduced in T2D subjects and correlated positively with M-values (r=0.884, r=0.574, r=0.707 and r=0.403, respectively). Brain MRglu was significantly higher in T2D and prediabetes subjects and had a significant inverse correlation with M-values (r=-0.616). Myocardial MRglu did not differ between groups and did not correlate with the M-values. A multivariate model including skeletal muscle, brain and VAT MRglu best predicted the M-values (adjusted r2=0.85). In addition, SAT MRglu correlated with SAT glucose uptake ex vivo (r=0.491). In different stages of the development of T2D, glucose uptake during hyperinsulinemia is elevated in the brain in parallel with an impairment in peripheral organs. Impaired glucose uptake in skeletal muscle and VAT together with elevated glucose uptake in brain were independently associated with whole-body insulin resistance, and these tissue-specific alterations may contribute to T2D development.

Secretagogin is increased in plasma from type 2 diabetes patients and potentially reflects stress and islet dysfunction.

Beta cell dysfunction accompanies and drives the progression of type 2 diabetes mellitus (T2D), but there are few clinical biomarkers available to assess islet cell stress in humans. Secretagogin, a protein enriched in pancreatic islets, demonstrates protective effects on beta cell function in animals. However, its potential as a circulating biomarker released from human beta cells and islets has not been studied. In this study primary human islets, beta cells and plasma samples were used to explore secretion and expression of secretagogin in relation to the T2D pathology. Secretagogin was abundantly and specifically expressed and secreted from human islets. Furthermore, T2D patients had an elevated plasma level of secretagogin compared with matched healthy controls, which was confirmed in plasma of diabetic mice transplanted with human islets. Additionally, the plasma secretagogin level of the human cohort had an inverse correlation to clinical assessments of beta cell function. To explore the mechanism of secretagogin release in vitro, human beta cells (EndoC-ßH1) were exposed to elevated glucose or cellular stress-inducing agents. Secretagogin was not released in parallel with glucose stimulated insulin release, but was markedly elevated in response to endoplasmic reticulum stressors and cytokines. These findings indicate that secretagogin is a potential novel biomarker, reflecting stress and islet cell dysfunction in T2D patients.