Abstract Current research on metabolic disorders and diabetes relies on animal models because multi-organ diseases cannot be well studied with standard in vitro assays. Here, we have connected cell models of key metabolic organs, the pancreas and liver, on a microfluidic chip to enable diabetes research in a human-based in vitro system. Aided by mechanistic mathematical modeling, we demonstrate that hyperglycemia and high cortisone concentration induce glucose dysregulation in the pancreas-liver microphysiological system (MPS), mimicking a diabetic phenotype seen in patients with glucocorticoid-induced diabetes. In this diseased condition, the pancreas-liver MPS displays beta-cell dysfunction, steatosis, elevated ketone-body secretion, increased glycogen storage, and upregulated gluconeogenic gene expression. Conversely, a physiological culture condition maintains glucose tolerance and beta-cell function. This method was reproducible in two laboratories and was effective in multiple pancreatic islet donors. The model also provides a platform to identify new therapeutic proteins, as demonstrated with a combined transcriptome and proteome analysis.

Homeostasis

Steatosis

10.1038/s42003-024-06514-w

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

Diabetologia (2020)

Shadab Abadpour Björn Tyrberg Simen W. Schive Charlotte Wennberg Huldt Peter Gennemark

Abstract Aims/hypothesis Inflammatory signals and increased prostaglandin synthesis play a role during the development of diabetes. The prostaglandin D 2 (PGD 2 ) 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 PGD 2 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-1β. 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 PGD 2 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-1β in human islets. This was accompanied by activation of the Akt–glycogen synthase kinase 3β 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.

Proinflammatory cytokine

Pancreatic Islets

10.1007/s00125-020-05138-z

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Functional coupling of human pancreatic islets and liver spheroids on-a-chip: Towards a novel human ex vivo type 2 diabetes model

Scientific Reports (2017)

Sophie Bauer Charlotte Wennberg Huldt Kajsa P. Kanebratt Isabell Durieux Daniela Gunne

Human in vitro physiological models studying disease and drug treatment effects are urgently needed as more relevant tools to identify new drug targets and therapies. We have developed a human microfluidic two-organ-chip model to study pancreatic islet-liver cross-talk based on insulin and glucose regulation. We have established a robust co-culture of human pancreatic islet microtissues and liver spheroids maintaining functional responses up to 15 days in an insulin-free medium. Functional coupling, demonstrated by insulin released from the islet microtissues in response to a glucose load applied in glucose tolerance tests on different days, promoted glucose uptake by the liver spheroids. Co-cultures maintained postprandial glucose concentrations in the circulation whereas glucose levels remained elevated in both single cultures. Thus, insulin secreted into the circulation stimulated glucose uptake by the liver spheroids, while the latter, in the absence of insulin, did not consume glucose as efficiently. As the glucose concentration fell, insulin secretion subsided, demonstrating a functional feedback loop between the liver and the insulin-secreting islet microtissues. Finally, inter-laboratory validation verified robustness and reproducibility. Further development of this model using tools inducing impaired glucose regulation should provide a unique in vitro system emulating human type 2 diabetes mellitus.

Ex vivo

Pancreatic Islets

10.1038/s41598-017-14815-w

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Diseased human pancreas and liver microphysiological system for preclinical diabetes research

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Sophie Rigal Belén Casas Kajsa P. Kanebratt Charlotte Wennberg Huldt Lisa U. Magnusson

ABSTRACT Current research on metabolic disorders such as type 2 diabetes relies on animal models because multi-organ diseases cannot be well studied with the standard in vitro assays. Here, we connect models of key metabolism organs, pancreas and liver, on a microfluidic chip to enable diabetes research in a human-based preclinical system. Aided by mechanistic mathematical modelling, we developed a two-organ microphysiological system (MPS) that replicates clinically-relevant phenotypes of diabetic dysregulation both in the liver and pancreas compartments. Exposure to hyperglycemia and high cortisone created a diseased pancreas-liver MPS which displayed beta-cell dysfunction, steatosis, elevated ketone-body secretion, increased glycogen storage, and upregulated gluconeogenic machinery. In turn, normoglycemia and physiological cortisone concentration maintained glucose tolerance and stable liver and beta-cell functions. This method was evaluated for repeatability in two laboratories and was effective in multiple pancreatic islet donors. The model also provides a platform to identify new therapeutic targets as demonstrated with a liver-secreted IL-1R2 protein that induced islet proliferation.

Steatosis

10.1101/2023.07.03.547412

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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

PLoS ONE (2018)

Stanko Skrtic Björn Tyrberg Malin A. Broberg Hans Ericsson Volker Schnecke

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.

10.1371/journal.pone.0208998

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Integrated experimental-computational analysis of a liver-islet microphysiological system for human-centric diabetes research

bioRxiv (Cold Spring Harbor Laboratory) (2021)

Belén Casas Liisa Vilén Sophie Bauer Kajsa P. Kanebratt Charlotte Wennberg Huldt

Abstract Microphysiological systems (MPS) are powerful tools for emulating human physiology and replicating disease progression in vitro . MPS could be better predictors of human outcome than current animal models, but mechanistic interpretation and in vivo extrapolation of the experimental results remain significant challenges. Here, we address these challenges using an integrated experimental-computational approach. This approach allows for in silico representation and predictions of glucose metabolism in a previously reported MPS with two organ compartments (liver and pancreas) connected in a closed loop with circulating medium. We developed a computational model describing glucose metabolism over 15 days of culture in the MPS. The model was calibrated on an experiment-specific basis using data from seven experiments, where single-liver or liver-islet cultures were exposed to both normal and hyperglycemic conditions resembling high blood glucose levels in diabetes. The calibrated models reproduced the fast (i.e. hourly) variations in glucose and insulin observed in the MPS experiments, as well as the long-term (i.e. over weeks) decline in both glucose tolerance and insulin secretion. We also investigated the behavior of the system under hypoglycemia by simulating this condition in silico , and the model could correctly predict the glucose and insulin responses measured in new MPS experiments. Last, we used the computational model to translate the experimental results to humans, showing good agreement with published data of the glucose response to a meal in healthy subjects. The integrated experimental-computational framework opens new avenues for future investigations toward disease mechanisms and the development of new therapies for metabolic disorders.

Computational model

Artificial Pancreas

10.1101/2021.08.18.456693

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A novel role of the checkpoint kinase ATR in leptin signaling

Molecular and Cellular Endocrinology (2015)

Elke Ericson Charlotte Wennberg Huldt Maria Strömstedt Peter Brodin

In a world with increasing incidences of obesity, it becomes critical to understand the detailed regulation of appetite. To identify novel regulators of the signaling mediated by one of the key hormones of energy homeostasis, leptin, we screened a set of compounds for their effect on the downstream Signal Transducer and Activator of Transcription 3 (STAT3) signaling. Interestingly, cells exposed to inhibitors of the Ataxia Telangiectasia and RAD3-related protein ATR increased their leptin dependent STAT3 activity. This was due to failure of the cells to induce the negative feedback mediator Suppressor of Cytokine Signaling 3 (SOCS3), suggesting that ATR has a previously unknown role in the negative feedback regulation of leptin signaling. This is an important finding not only because it sheds light on additional genes involved in leptin signaling, but also because it brings forward a new potential therapeutic intervention point for increasing leptin signaling in obese individuals.

Mediator

Cell Signaling

10.1016/j.mce.2015.04.034

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Citations (3)

Publisher Correction: Functional coupling of human pancreatic islets and liver spheroids on-a-chip: Towards a novel human ex vivo type 2 diabetes model

Scientific Reports (2018)

Sophie Bauer Charlotte Wennberg Huldt Kajsa P. Kanebratt Isabell Durieux Daniela Gunne

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Ex vivo

Spheroid

Pancreatic Islets

Human liver

10.1038/s41598-018-20340-1

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A Versatile and Convenient Synthesis of ³⁴S‐Labeled Phosphorothioate Oligonucleotides

ChemBioChem (2018)

Rouven Stulz Johan Meuller Dženita Baždarević Charlotte Wennberg Huldt Roger Strömberg

Abstract A synthetic protocol for 34 S‐labeled phosphorothioate oligonucleotides (PS ONs) was developed to facilitate MS‐based assay analysis. This was enabled by a highly efficient, two‐step, one‐pot synthesis of 34 S‐labeled phenylacetyl disulfide ( 34 S‐PADS), starting from 34 S‐enriched elemental sulfur ( 34 S 8 ). 34 S‐PADS was subsequently used for stable isotope labeling (SIL) of oligonucleotides containing a phosphorothioate backbone. The 34 S‐SIL PS ONs are shown to retain the same melting temperature, antisense activity, and secondary structure as those of the corresponding unlabeled 32 S PS ONs.

Oligonucleotide synthesis

10.1002/cbic.201800417

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Citations (7)