Publications by authors named "Per-Olof Berggren"

225 Publications

Tissue-specific expression of insulin receptor isoforms in obesity/type 2 diabetes mouse models.

J Cell Mol Med 2021 Mar 19. Epub 2021 Mar 19.

Department of Molecular Medicine and Surgery, The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.

The two insulin receptor (IR) isoforms IR-A and IR-B are responsible for the pleiotropic actions of insulin and insulin-like growth factors. Consequently, changes in IR isoform expression and in the bioavailability of their ligands will impact on IR-mediated functions. Although alteration of IR isoform expression has been linked to insulin resistance, knowledge of IR isoform expression and mechanisms underlying tissue/cell-type-specific changes in metabolic disease are lacking. Using mouse models of obesity/diabetes and measuring the mRNA of the IR isoforms and mRNA/protein levels of total IR, we provide a data set of IR isoform expression pattern that documents changes in a tissue-dependent manner. Combining tissue fractionation and a new in situ mRNA hybridization technology to visualize the IR isoforms at cellular resolution, we explored the mechanism underlying the change in IR isoform expression in perigonadal adipose tissue, which is mainly caused by tissue remodelling, rather than by a shift in IR alternative splicing in a particular cell type, e.g. adipocytes.
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http://dx.doi.org/10.1111/jcmm.16452DOI Listing
March 2021

Lowering apolipoprotein CIII protects against high-fat diet-induced metabolic derangements.

Sci Adv 2021 Mar 12;7(11). Epub 2021 Mar 12.

The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital L1, SE-171 76 Stockholm, Sweden.

Increased levels of apolipoprotein CIII (apoCIII), a key regulator of lipid metabolism, result in obesity-related metabolic derangements. We investigated mechanistically whether lowering or preventing high-fat diet (HFD)-induced increase in apoCIII protects against the detrimental metabolic consequences. Mice, first fed HFD for 10 weeks and thereafter also given an antisense (ASO) to lower apoCIII, already showed reduced levels of apoCIII and metabolic improvements after 4 weeks, despite maintained obesity. Prolonged ASO treatment reversed the metabolic phenotype due to increased lipase activity and receptor-mediated hepatic uptake of lipids. Fatty acids were transferred to the ketogenic pathway, and ketones were used in brown adipose tissue (BAT). This resulted in no fat accumulation and preserved morphology and function of liver and BAT. If ASO treatment started simultaneously with the HFD, mice remained lean and metabolically healthy. Thus, lowering apoCIII protects against and reverses the HFD-induced metabolic phenotype by promoting physiological insulin sensitivity.
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http://dx.doi.org/10.1126/sciadv.abc2931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954448PMC
March 2021

Human Islet Microtissues as an In Vitro and an In Vivo Model System for Diabetes.

Int J Mol Sci 2021 Feb 11;22(4). Epub 2021 Feb 11.

The Rolf Luft Research Center for Diabetes and Endocrinology, Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska Sjukhuset L1:03, 17176 Stockholm, Sweden.

Loss of pancreatic β-cell function is a critical event in the pathophysiology of type 2 diabetes. However, studies of its underlying mechanisms as well as the discovery of novel targets and therapies have been hindered due to limitations in available experimental models. In this study we exploited the stable viability and function of standardized human islet microtissues to develop a disease-relevant, scalable, and reproducible model of β-cell dysfunction by exposing them to long-term glucotoxicity and glucolipotoxicity. Moreover, by establishing a method for highly-efficient and homogeneous viral transduction, we were able to monitor the loss of functional β-cell mass in vivo by transplanting reporter human islet microtissues into the anterior chamber of the eye of immune-deficient mice exposed to a diabetogenic diet for 12 weeks. This newly developed in vitro model as well as the described in vivo methodology represent a new set of tools that will facilitate the study of β-cell failure in type 2 diabetes and would accelerate the discovery of novel therapeutic agents.
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http://dx.doi.org/10.3390/ijms22041813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918101PMC
February 2021

Apolipoprotein CIII Is an Important Piece in the Type-1 Diabetes Jigsaw Puzzle.

Int J Mol Sci 2021 Jan 19;22(2). Epub 2021 Jan 19.

The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital, Anna Steckséns gata 53, SE-171 76 Stockholm, Sweden.

It is well known that type-2 diabetes mellitus (T2D) is increasing worldwide, but also the autoimmune form, type-1 diabetes (T1D), is affecting more people. The latest estimation from the International Diabetes Federation (IDF) is that 1.1 million children and adolescents below 20 years of age have T1D. At present, we have no primary, secondary or tertiary prevention or treatment available, although many efforts testing different strategies have been made. This review is based on the findings that apolipoprotein CIII (apoCIII) is increased in T1D and that in vitro studies revealed that healthy β-cells exposed to apoCIII became apoptotic, together with the observation that humans with higher levels of the apolipoprotein, due to mutations in the gene, are more susceptible to developing T1D. We have summarized what is known about apoCIII in relation to inflammation and autoimmunity in in vitro and in vivo studies of T1D. The aim is to highlight the need for exploring this field as we still are only seeing the top of the iceberg.
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http://dx.doi.org/10.3390/ijms22020932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7832341PMC
January 2021

Integration of Primary Endocrine Cells and Supportive Cells Using Functionalized Silk Promotes the Formation of Prevascularized Islet-like Clusters.

ACS Biomater Sci Eng 2020 02 8;6(2):1186-1195. Epub 2020 Jan 8.

Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

Pancreatic islet transplantation has not yet succeeded as an overall treatment for type 1 diabetes because of limited access to donor islets, as well as low efficacy and poor reproducibility of the current procedure. Herein, a method to create islets-like composite clusters (coclusters) from dispersed endocrine cells and supportive cells is described, attempting to improve compatibility with the recipient and more efficiently make use of the donor-derived material. To mimic the extracellular matrix environment, recombinant spider silk functionalized with cell binding motifs are used as 3D support for the coclusters. A cell binding motif derived from fibronectin (FN) was found superior in promoting cell adherence, while a plain RGD-motif incorporated in the repetitive part of the silk protein (2R) increased the mobility and cluster formation of endocrine cells. Self-assembly of a mixture of FN/2R silk is utilized to integrate endocrine cells together with endothelial and mesenchymal cells into islet-like coclusters. Both xenogenic and allogenic versions of these coclusters were found to be viable and were able to respond to dynamic glucose stimulation with insulin release. Moreover, the endothelial cells were found to be colocalized with the endocrine cells, showing that the silk combined with supportive cells may promote vascularization. This method to engineer combined islet-like coclusters allows donor-derived endocrine cells to be surrounded by supportive cells from the recipient, which have the potential to further promote engraftment in the host and considerably reduce risk of rejection.
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http://dx.doi.org/10.1021/acsbiomaterials.9b01573DOI Listing
February 2020

Glucokinase intrinsically regulates glucose sensing and glucagon secretion in pancreatic alpha cells.

Sci Rep 2020 11 19;10(1):20145. Epub 2020 Nov 19.

The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska Sjukhuset L1:03, 17176, Stockholm, Sweden.

The secretion of glucagon by pancreatic alpha cells is regulated by a number of external and intrinsic factors. While the electrophysiological processes linking a lowering of glucose concentrations to an increased glucagon release are well characterized, the evidence for the identity and function of the glucose sensor is still incomplete. In the present study we aimed to address two unsolved problems: (1) do individual alpha cells have the intrinsic capability to regulate glucagon secretion by glucose, and (2) is glucokinase the alpha cell glucose sensor in this scenario. Single cell RT-PCR was used to confirm that glucokinase is the main glucose-phosphorylating enzyme expressed in rat pancreatic alpha cells. Modulation of glucokinase activity by pharmacological activators and inhibitors led to a lowering or an increase of the glucose threshold of glucagon release from single alpha cells, measured by TIRF microscopy, respectively. Knockdown of glucokinase expression resulted in a loss of glucose control of glucagon secretion. Taken together this study provides evidence for a crucial role of glucokinase in intrinsic glucose regulation of glucagon release in rat alpha cells.
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http://dx.doi.org/10.1038/s41598-020-76863-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7678872PMC
November 2020

Islet vascularization is regulated by primary endothelial cilia via VEGF-A-dependent signaling.

Elife 2020 11 17;9. Epub 2020 Nov 17.

The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska University Hospital L1, Stockholm, Sweden.

Islet vascularization is essential for intact islet function and glucose homeostasis. We have previously shown that primary cilia directly regulate insulin secretion. However, it remains unclear whether they are also implicated in islet vascularization. At eight weeks, murine islets show significantly lower intra-islet capillary density with enlarged diameters. Transplanted islets exhibit delayed re-vascularization and reduced vascular fenestration after engraftment, partially impairing vascular permeability and glucose delivery to β-cells. We identified primary cilia on endothelial cells as the underlying cause of this regulation, via the vascular endothelial growth factor-A (VEGF-A)/VEGF receptor 2 (VEGFR2) pathway. In vitro silencing of ciliary genes in endothelial cells disrupts VEGF-A/VEGFR2 internalization and downstream signaling. Consequently, key features of angiogenesis including proliferation and migration are attenuated in human silenced endothelial cells. We conclude that endothelial cell primary cilia regulate islet vascularization and vascular barrier function via the VEGF-A/VEGFR2 signaling pathway.
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http://dx.doi.org/10.7554/eLife.56914DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695455PMC
November 2020

Topologically selective islet vulnerability and self-sustained downregulation of markers for β-cell maturity in streptozotocin-induced diabetes.

Commun Biol 2020 Sep 30;3(1):541. Epub 2020 Sep 30.

Umeå Centre for Molecular Medicine, Umeå University, Umeå, Sweden.

Mouse models of Streptozotocin (STZ) induced diabetes represent the most widely used preclinical diabetes research systems. We applied state of the art optical imaging schemes, spanning from single islet resolution to the whole organ, providing a first longitudinal, 3D-spatial and quantitative account of β-cell mass (BCM) dynamics and islet longevity in STZ-treated mice. We demonstrate that STZ-induced β-cell destruction predominantly affects large islets in the pancreatic core. Further, we show that hyperglycemic STZ-treated mice still harbor a large pool of remaining β-cells but display pancreas-wide downregulation of glucose transporter type 2 (GLUT2). Islet gene expression studies confirmed this downregulation and revealed impaired β-cell maturity. Reversing hyperglycemia by islet transplantation partially restored the expression of markers for islet function, but not BCM. Jointly our results indicate that STZ-induced hyperglycemia results from β-cell dysfunction rather than β-cell ablation and that hyperglycemia in itself sustains a negative feedback loop restraining islet function recovery.
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http://dx.doi.org/10.1038/s42003-020-01243-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527346PMC
September 2020

Alpha cell regulation of beta cell function.

Diabetologia 2020 10 7;63(10):2064-2075. Epub 2020 Sep 7.

The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska Sjukhuset L1:03, 17176, Stockholm, Sweden.

The islet of Langerhans is a complex endocrine micro-organ consisting of a multitude of endocrine and non-endocrine cell types. The two most abundant and prominent endocrine cell types, the beta and the alpha cells, are essential for the maintenance of blood glucose homeostasis. While the beta cell produces insulin, the only blood glucose-lowering hormone of the body, the alpha cell releases glucagon, which elevates blood glucose. Under physiological conditions, these two cell types affect each other in a paracrine manner. While the release products of the beta cell inhibit alpha cell function, the alpha cell releases factors that are stimulatory for beta cell function and increase glucose-stimulated insulin secretion. The aim of this review is to provide a comprehensive overview of recent research into the regulation of beta cell function by alpha cells, focusing on the effect of alpha cell-secreted factors, such as glucagon and acetylcholine. The consequences of differences in islet architecture between species on the interplay between alpha and beta cells is also discussed. Finally, we give a perspective on the possibility of using an in vivo imaging approach to study the interactions between human alpha and beta cells under in vivo conditions. Graphical abstract.
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http://dx.doi.org/10.1007/s00125-020-05196-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476996PMC
October 2020

XPR1 Mediates the Pancreatic β-Cell Phosphate Flush.

Diabetes 2021 Jan 21;70(1):111-118. Epub 2020 Aug 21.

The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden

Glucose-stimulated insulin secretion is the hallmark of the pancreatic β-cell, a critical player in the regulation of blood glucose concentration. In 1974, the remarkable observation was made that an efflux of intracellular inorganic phosphate (P) accompanied the events of stimulated insulin secretion. The mechanism behind this "phosphate flush," its association with insulin secretion, and its regulation have since then remained a mystery. We recapitulated the phosphate flush in the MIN6m9 β-cell line and pseudoislets. We demonstrated that knockdown of XPR1, a phosphate transporter present in MIN6m9 cells and pancreatic islets, prevented this flush. Concomitantly, XPR1 silencing led to intracellular P accumulation and a potential impact on Ca signaling. XPR1 knockdown slightly blunted first-phase glucose-stimulated insulin secretion in MIN6m9 cells, but had no significant impact on pseudoislet secretion. In keeping with other cell types, basal P efflux was stimulated by inositol pyrophosphates, and basal intracellular P accumulated following knockdown of inositol hexakisphosphate kinases. However, the glucose-driven phosphate flush occurred despite inositol pyrophosphate depletion. Finally, while it is unlikely that XPR1 directly affects exocytosis, it may protect Ca signaling. Thus, we have revealed XPR1 as the missing mediator of the phosphate flush, shedding light on a 45-year-old mystery.
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http://dx.doi.org/10.2337/db19-0633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881847PMC
January 2021

Effectiveness of Antivirals in a Type 1 Diabetes Model and the Move Toward Human Trials.

Viral Immunol 2020 11 4;33(9):594-599. Epub 2020 Aug 4.

Division of Infectious Diseases, Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden.

A Picornavirus (Ljungan virus [LV]) originally found in bank voles has been associated with type 1 diabetes (T1D) in its wild rodent reservoir, but also associated with T1D in a laboratory rat model for the disease, the diabetes prone (DP) Bio Breeding (BB) rat. Successful treatment of diabetes in this rat model, using experimental antiviral compounds directed against picornavirus, has been reported. In the present study we show significant clinical response in DP-BB rats using antiviral compounds available for human use (Pleconaril, Efavirenz, and Ribavirin). Presence of LV picornavirus antigen has been detected in islets of Langerhans from both human and the T1D rat model with clear morphological similarity. Based on these data it would be of interest to test antiviral treatment in patients with newly diagnosed T1D. Successful outcome will offer both proof of concept regarding the role of virus involvement in the disease and possibly a first generation treatment interrupting a persistent infection and stopping -cell destruction.
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http://dx.doi.org/10.1089/vim.2020.0039DOI Listing
November 2020

Studying the biology of cytotoxic T lymphocytes in vivo with a fluorescent granzyme B-mTFP knock-in mouse.

Elife 2020 07 22;9. Epub 2020 Jul 22.

Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), Saarland University, Homburg, Germany.

Understanding T cell function in vivo is of key importance for basic and translational immunology alike. To study T cells in vivo, we developed a new knock-in mouse line, which expresses a fusion protein of granzyme B, a key component of cytotoxic granules involved in T cell-mediated target cell-killing, and monomeric teal fluorescent protein from the endogenous locus. Homozygous knock-ins, which are viable and fertile, have cytotoxic T lymphocytes with endogeneously fluorescent cytotoxic granules but wild-type-like killing capacity. Expression of the fluorescent fusion protein allows quantitative analyses of cytotoxic granule maturation, transport and fusion in vitro with super-resolution imaging techniques, and two-photon microscopy in living knock-ins enables the visualization of tissue rejection through individual target cell-killing events in vivo. Thus, the new mouse line is an ideal tool to study cytotoxic T lymphocyte biology and to optimize personalized immunotherapy in cancer treatment.
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http://dx.doi.org/10.7554/eLife.58065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375811PMC
July 2020

Platelet factor 4 enhances CD4 T effector memory cell responses via Akt-PGC1α-TFAM signaling-mediated mitochondrial biogenesis.

J Thromb Haemost 2020 10 28;18(10):2685-2700. Epub 2020 Aug 28.

Department of Medicine-Solna, Clinical Epidemiology Unit, Clinical Pharmacology Group, Karolinska Institutet, Stockholm, Sweden.

Background: Cell metabolism drives T cell functions, while platelets regulate overall CD4 T cell immune responses.

Objective: To investigate if platelets influence cell metabolism and thus regulate CD4 T effector memory cell (Tem) responses.

Methods: Human CD4 Tem cells were activated with αCD3/αCD28 and cultured without or with platelets or platelet-derived mediators.

Results: Polyclonal stimulation induced rapid and marked Th1 and Treg cell activation of CD4 Tem cells. Platelet co-culture enhanced Th1 response transiently, while it persistently enhanced Treg cell activation of Tem cells, with an enhancement that plateaued by day 3. Platelet factor 4 (PF4) was the key platelet-derived mediator regulating CD4 Tem cell responses, which involved cellular metabolisms as indicated by mass spectrometric analyses. PF4 exerted its effects via its receptor CXCR3, attenuated Akt activity, and reduced PGC1α phosphorylation, and resulted in elevations of PGC1α function and mitochondrial transcription factor A (TFAM) synthesis. The latter increased mitochondrial biogenesis, and subsequently enhanced Th1 and Treg responses. Consistent with these observations, inhibition of mitochondrial function by rotenone counteracted the enhancements by recombinant PF4, and TFAM overexpression by TFAM-adenovirus infection mimicked PF4 effects. Furthermore, increased mitochondrial mass elevated oxygen consumption, and enhanced adenosine triphosphate and reactive oxygen species production, which, in turn, stimulated Th1 (T-bet) and Treg (FoxP3) transcription factor expression and corresponding CD4 T effector cell responses.

Conclusions: Platelets enhance CD4 T cell responses of Tem cells through PF4-dependent and Akt-PGC1α-TFAM signaling-mediated mitochondrial biogenesis. Hence, PF4 may be a promising intervention target of platelet-regulated immune responses.
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http://dx.doi.org/10.1111/jth.15005DOI Listing
October 2020

Mechanism and effects of pulsatile GABA secretion from cytosolic pools in the human beta cell.

Nat Metab 2019 11 15;1(11):1110-1126. Epub 2019 Nov 15.

J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, USA.

Pancreatic beta cells synthesize and secrete the neurotransmitter γ-aminobutyric acid (GABA) as a paracrine and autocrine signal to help regulate hormone secretion and islet homeostasis. Islet GABA release has classically been described as a secretory vesicle-mediated event. Yet, a limitation of the hypothesized vesicular GABA release from islets is the lack of expression of a vesicular GABA transporter in beta cells. Consequentially, GABA accumulates in the cytosol. Here we provide evidence that the human beta cell effluxes GABA from a cytosolic pool in a pulsatile manner, imposing a synchronizing rhythm on pulsatile insulin secretion. The volume regulatory anion channel (VRAC), functionally encoded by LRRC8A or Swell1, is critical for pulsatile GABA secretion. GABA content in beta cells is depleted and secretion is disrupted in islets from type 1 and type 2 diabetic patients, suggesting that loss of GABA as a synchronizing signal for hormone output may correlate with diabetes pathogenesis.
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http://dx.doi.org/10.1038/s42255-019-0135-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236889PMC
November 2019

"PoZdro!" as an example of a successful multicenter programme for obesity management and healthy lifestyle promotion in children and adolescents - programme protocol and preliminary results from the first intervention site.

Pediatr Endocrinol Diabetes Metab 2020 ;26(1):22-26

"PoZdro!" Programme Scientific Board, Medicover Foundation, Poland.

Introduction: Overweight and obesity rates in children and adolescents increase worldwide for over 30 years, what leads to an increased risk of de-veloping metabolic diseases.

Aim Of The Study: Protocol description and preliminary results of the largest to-date obesity management programme for children in Poland - "PoZdro!"

Material And Methods: The Programme is based on three main steps: screening, parental engagement and long-term behavioral intervention (medical, dietetic, psychological, physical activity intervention panels) and education activities in secondary schools. Over 30.000 children were screened in four big Polish cities and over 2.000 engaged in the programme.

Results: Preliminary results from the first city show that, since 2014, 3998 of the 6346 (63%) eligible students from 24 schools (first grade of the secondary school) were screened. 16% of the children were overweight (body mass index - BMI ≥ 85th < 95th centile) and 4.7% were obese (BMI ≥95th centile). 603 children fulfilling the qualification (QUA) criteria (BMI > 90th centile) were offered the Individual Integrated Care (IIC). 470 (77.94% of QUA) began and 253 (41.96% of QUA, 53.82% of IIC) finished the full interventional pro-gramme with the mean BMI centile decrease of 3.04 in two following years.

Conclusions: "PoZdro!" is a unique obesity prevention and management programme in Poland operating simultaneously in several Polish big cities. The programme incorporates parental engagement and long-term behavioral intervention. Preliminary results show that it may result in meaningful decrease in BMI in two following years. Further data analyses are necessary to show the influence of the programme on metabolic risk in studied group.
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http://dx.doi.org/10.5114/pedm.2020.94393DOI Listing
March 2021

Islet Transplantation to the Anterior Chamber of the Eye-A Future Treatment Option for Insulin-Deficient Type-2 Diabetics? A Case Report from a Nonhuman Type-2 Diabetic Primate.

Cell Transplant 2020 Jan-Dec;29:963689720913256

Translational Pre-Clinical Model Platform, Singapore Eye Research Institute (SERI), Singapore.

Replacement of the insulin-secreting beta cells through transplantation of pancreatic islets to the liver is a promising treatment for type-1 diabetes. However, low oxygen tension, shear stress, and the induction of inflammation lead to significant islet dysfunction and loss. The anterior chamber of the eye (ACE) has gained considerable interest and represents an alternative therapeutic islet transplantation site because of its accessibility, high oxygen tension, and immune-privileged milieu. We have previously demonstrated the feasibility of intraocular islet transplant in mouse and nonhuman primate models of type-1 diabetes and are now assessing its efficacy on glucose homeostasis in a nonhuman primate model of type-2 diabetes. We transplanted allogeneic donor islets (1,500 islet equivalents/kg) into the anterior chamber of one eye in a cynomolgus monkey with high-fat-diet-induced type-2 diabetes. Repeated examinations of the anterior and posterior segments of both eyes were done to monitor the engrafted islets and assess the overall ocular health. Fasting blood glucose level, blood biochemistry, and other metabolic parameters were routinely evaluated to determine the function of the islet graft and diabetes status. The transplanted islets were rapidly engrafted onto the iris and became vascularized 1 month after transplantation. We did not detect changes in intraocular pressure, cataract formation, ophthalmitis, or retinal vessel deformation. A significant lower fasting blood glucose level was observed while the graft was in place, and the transplantation reverts the progression of diabetes. The metabolic markers, hemoglobin A and fructosamine, demonstrated improvement following islet transplantation. As a conclusion, intraocular islet transplantation in one eye of a cynomolgus monkey with type-2 diabetes improved its overall plasma glucose homeostasis, as evidenced by short-term measures and long-term metabolic markers. These results further support the future application of the ACE as an alternative site for clinical islet transplants in the context of type-2 diabetes.
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http://dx.doi.org/10.1177/0963689720913256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7444223PMC
April 2020

Modelling of dysregulated glucagon secretion in type 2 diabetes by considering mitochondrial alterations in pancreatic α-cells.

R Soc Open Sci 2020 Jan 22;7(1):191171. Epub 2020 Jan 22.

Faculty of Natural Sciences and Mathematics, University of Maribor, 2000 Maribor, Slovenia.

Type 2 diabetes mellitus (T2DM) has been associated with insulin resistance and the failure of β-cells to produce and secrete enough insulin as the disease progresses. However, clinical treatments based solely on insulin secretion and action have had limited success. The focus is therefore shifting towards α-cells, in particular to the dysregulated secretion of glucagon. Our qualitative electron-microscopy-based observations gave an indication that mitochondria in α-cells are altered in Western-diet-induced T2DM. In particular, α-cells extracted from mouse pancreatic tissue showed a lower density of mitochondria, a less expressed matrix and a lower number of cristae. These deformities in mitochondrial ultrastructure imply a decreased efficiency in mitochondrial ATP production, which prompted us to theoretically explore and clarify one of the most challenging problems associated with T2DM, namely the lack of glucagon secretion in hypoglycaemia and its oversecretion at high blood glucose concentrations. To this purpose, we constructed a novel computational model that links α-cell metabolism with their electrical activity and glucagon secretion. Our results show that defective mitochondrial metabolism in α-cells can account for dysregulated glucagon secretion in T2DM, thus improving our understanding of T2DM pathophysiology and indicating possibilities for new clinical treatments.
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http://dx.doi.org/10.1098/rsos.191171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029933PMC
January 2020

In vivo Ca dynamics in single pancreatic β cells.

FASEB J 2020 01 28;34(1):945-959. Epub 2019 Nov 28.

The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden.

The dynamics of cytoplasmic free Ca concentration ([Ca]) in pancreatic β cells is central to our understanding of β-cell physiology and pathology. In this context, there are numerous in vitro studies available but existing in vivo data are scarce. We now critically evaluate the anterior chamber of the eye as an in vivo, non-invasive, imaging site for measuring [Ca] dynamics longitudinally in three dimensions and at single-cell resolution. By applying a fluorescently labeled glucose analogue 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose in vivo, we followed how glucose almost simultaneously distributes to all cells within the islet volume, resulting in [Ca] changes. We found that almost all β cells in healthy mice responded to a glucose challenge, while in hyperinsulinemic, hyperglycemic mice about 80% of the β cells could not be further stimulated from fasting basal conditions. This finding indicates that our imaging modality can resolve functional heterogeneity within the β-cell population in terms of glucose responsiveness. Importantly, we demonstrate that glucose homeostasis is markedly affected using isoflurane compared to hypnorm/midazolam anesthetics, which has major implications for [Ca] measurements. In summary, this setup offers a powerful tool to further investigate in vivo pancreatic β-cell [Ca] response patterns at single-cell resolution in health and disease.
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http://dx.doi.org/10.1096/fj.201901302RRDOI Listing
January 2020

Enhanced expression of β cell Ca3.1 channels impairs insulin release and glucose homeostasis.

Proc Natl Acad Sci U S A 2020 01 23;117(1):448-453. Epub 2019 Dec 23.

The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, SE-171 76 Stockholm, Sweden;

Voltage-gated calcium 3.1 (Ca3.1) channels are absent in healthy mouse β cells and mediate minor T-type Ca currents in healthy rat and human β cells but become evident under diabetic conditions. Whether more active Ca3.1 channels affect insulin secretion and glucose homeostasis remains enigmatic. We addressed this question by enhancing de novo expression of β cell Ca3.1 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-Ca3.1 subunit (Ad-EGFP-Ca3.1) efficiently transduced rat and human islets as well as dispersed islet cells. The resulting Ca3.1 channels conducted typical T-type Ca currents, leading to an enhanced basal cytosolic-free Ca concentration ([Ca]). Ad-EGFP-Ca3.1-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-Ca3.1 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 Ca3.1 channels or the Ca-dependent phosphatase calcineurin. Enhanced expression of β cell Ca3.1 channels therefore impairs insulin release and glucose homeostasis by means of initial excessive Ca influx, subsequent activation of calcineurin, consequent dephosphorylation and nuclear retention of FoxO1, and eventual FoxO1-mediated down-regulation of β cell exocytotic proteins. The present work thus suggests an elevated expression of Ca3.1 channels plays a significant role in diabetes pathogenesis.
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http://dx.doi.org/10.1073/pnas.1908691117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955371PMC
January 2020

IgGs from patients with amyotrophic lateral sclerosis and diabetes target Caαδ1 subunits impairing islet cell function and survival.

Proc Natl Acad Sci U S A 2019 Dec 11. Epub 2019 Dec 11.

The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital L1, SE-171 76 Stockholm, Sweden;

Patients with amyotrophic lateral sclerosis (ALS) often show hallmarks of type 2 diabetes mellitus (T2DM). However, the causal link between ALS and T2DM has remained a mystery. We now demonstrate that 60% of ALS patients with T2DM (ALS-T2DM) have sera that exaggerated K-induced increases in cytosolic free Ca concentration ([Ca]) in mouse islet cells. The effect was attributed to the presence of pathogenic immunoglobulin Gs (IgGs) in ALS-T2DM sera. The pathogenic IgGs immunocaptured the voltage-dependent Ca (Ca) channel subunit Caαδ1 in the plasma membrane enhancing Ca1 channel-mediated Ca influx and [Ca], resulting in impaired mitochondrial function. Consequently, impairments in [Ca] dynamics, insulin secretion, and cell viability occurred. These data reveal that patients with ALS-T2DM carry cytotoxic ALS-T2DM-IgG autoantibodies that serve as a causal link between ALS and T2DM by immunoattacking Caαδ1 subunits. Our findings may lay the foundation for a pharmacological treatment strategy for patients suffering from a combination of these diseases.
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http://dx.doi.org/10.1073/pnas.1911956116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936400PMC
December 2019

Intraocular Pressure Monitoring Following Islet Transplantation to the Anterior Chamber of the Eye.

Nano Lett 2020 03 21;20(3):1517-1525. Epub 2019 Nov 21.

Nano Science Technology Institute, Department of Materials Science and Engineering, Yonsei University, Seoul 03722, Republic of Korea.

Intraocular islet transplantation was investigated as a new procedure to treat diabetes. The development of this procedure requires close monitoring of the function of both eye and islet graft. We developed a soft, smart contact lens to monitor the intraocular pressure and applied this for noninvasive monitoring in association with the intraocular islet transplantation in diabetes. A strain sensor inside the lens can detect detailed changes in intraocular pressure by focusing the strain only in the desired, selective area of the contact lens. In addition, this smart contact lens can transmit the real-time value of the intraocular pressure wirelessly using an antenna. The wireless measurement of intraocular pressure that was obtained using this contact lens had a high correlation with the intraocular pressure measured by a rebound tonometer, thereby proving the good accuracy of the contact lens sensor. In the initial period, a slight elevation of intraocular pressure was observed, but the pressure returned to normal in the initial period after the transplantation. This type of monitoring will provide important information on potential changes in the intraocular pressure associated with the transplantation procedure, and it enables appropriate clinical safety steps to be taken, if needed.
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http://dx.doi.org/10.1021/acs.nanolett.9b03605DOI Listing
March 2020

Noninvasive intravital high-resolution imaging of pancreatic neuroendocrine tumours.

Sci Rep 2019 10 10;9(1):14636. Epub 2019 Oct 10.

Division of Vascular Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, 17165, Sweden.

Preclinical trials of cancer drugs in animal models are important for drug development. The Rip1Tag2 (RT2) transgenic mouse, a model of pancreatic neuroendocrine tumours (PNET), has provided immense knowledge about PNET biology, although tumour progression occurs in a location inaccessible for real-time monitoring. To overcome this hurdle we have developed a novel platform for intravital 3D imaging of RT2 tumours to facilitate real-time studies of cancer progression. Pre-oncogenic islets retrieved from RT2 mice were implanted into the anterior chamber of the eye (ACE) of host mice, where they engrafted on the iris, recruited blood vessels and showed continuous growth. Noninvasive confocal and two-photon laser-scanning microscopy through the transparent cornea facilitated high-resolution imaging of tumour growth and angiogenesis. RT2 tumours in the ACE expanded up to 8-fold in size and shared hallmarks with tumours developing in situ in the pancreas. Genetically encoded fluorescent reporters enabled high-resolution imaging of stromal cells and tumour cell migration. Sunitinib treatment impaired RT2 tumour angiogenesis and growth, while overexpression of the vascular endothelial growth factor (VEGF)-B increased tumour angiogenesis though tumour growth was impaired. In conclusion, we present a novel platform for intravital high-resolution and 3D imaging of PNET biology and cancer drug assessment.
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http://dx.doi.org/10.1038/s41598-019-51093-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6787246PMC
October 2019

Islet macrophages are associated with islet vascular remodeling and compensatory hyperinsulinemia during diabetes.

Am J Physiol Endocrinol Metab 2019 12 1;317(6):E1108-E1120. Epub 2019 Oct 1.

Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore.

β-Cells respond to peripheral insulin resistance by first increasing circulating insulin during diabetes. Islet remodeling supports this compensation, but its drivers remain poorly understood. Infiltrating macrophages have been implicated in late-stage type 2 diabetes, but relatively little is known on islet resident macrophages, especially during compensatory hyperinsulinemia. We hypothesized that islet resident macrophages would contribute to islet vascular remodeling and hyperinsulinemia during diabetes, the failure of which results in a rapid progression to frank diabetes. We used chemical (clodronate), genetics (CD169-diphtheria toxin receptor mice), or antibody-mediated (colony-stimulating factor 1 receptor α) macrophage ablation methods in diabetic (db/db) and diet-induced models of compensatory hyperinsulinemia to investigate the role of macrophages in islet remodeling. We transplanted islets devoid of macrophages into naïve diabetic mice and assessed the impact on islet vascularization. With the use of the above methods, we showed that macrophage depletion significantly and consistently compromised islet remodeling in terms of size, vascular density, and insulin secretion capacity. Depletion of islet macrophages reduced VEGF-A secretion in both human and mouse islets ex vivo, and this functionally translated to delayed revascularization upon transplantation in vivo. We revealed that islet resident macrophages were associated with islet remodeling and increased insulin secretion during diabetes. This suggests utility in harnessing islet macrophages during this phase to promote islet vascularization, remodeling, and insulin secretion.
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http://dx.doi.org/10.1152/ajpendo.00248.2019DOI Listing
December 2019

Structural basis for delta cell paracrine regulation in pancreatic islets.

Nat Commun 2019 08 16;10(1):3700. Epub 2019 Aug 16.

Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, 636921, Singapore.

Little is known about the role of islet delta cells in regulating blood glucose homeostasis in vivo. Delta cells are important paracrine regulators of beta cell and alpha cell secretory activity, however the structural basis underlying this regulation has yet to be determined. Most delta cells are elongated and have a well-defined cell soma and a filopodia-like structure. Using in vivo optogenetics and high-speed Ca imaging, we show that these filopodia are dynamic structures that contain a secretory machinery, enabling the delta cell to reach a large number of beta cells within the islet. This provides for efficient regulation of beta cell activity and is modulated by endogenous IGF-1/VEGF-A signaling. In pre-diabetes, delta cells undergo morphological changes that may be a compensation to maintain paracrine regulation of the beta cell. Our data provides an integrated picture of how delta cells can modulate beta cell activity under physiological conditions.
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http://dx.doi.org/10.1038/s41467-019-11517-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697679PMC
August 2019

Phospholipase C-β1 potentiates glucose-stimulated insulin secretion.

FASEB J 2019 10 3;33(10):10668-10679. Epub 2019 Jul 3.

School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, South Korea.

PLC-β exerts biologic influences through GPCR. GPCRs are involved in regulating glucose-stimulated insulin secretion (GSIS). Previous studies have suggested that PLC-βs might play an important role in pancreatic β cells. However, because of a lack of the specific inhibitors of PLC-β isozymes and appropriate genetic models, the function of specific PLC-β isozymes in pancreatic β cells and their physiologic relevance in the regulation of insulin secretion have not been studied so far. The present study showed that PLC-β1 was crucial for β-cell function by generation of each PLC-β conditional knockout mouse. Mice lacking PLC-β1 in β cells exhibited a marked defect in GSIS, leading to glucose intolerance. In studies, the secreted insulin level and Ca response in ; pancreas/duodenum homeobox protein 1 ()-Cre recombinase-estrogen receptor T2 () islets was lower than those in the islets under the high-glucose condition. PLC-β1 led to potentiate insulin secretion stimulation of particular G-protein-coupled receptors. ; mice fed a high-fat diet developed more severe glucose intolerance because of a defect in insulin secretion. The present study identified PLC-β1 as an important molecule that regulates β cell insulin secretion and can be considered a candidate for therapeutic intervention in diabetes mellitus.-Hwang, H.-J., Yang, Y. R., Kim, H. Y., Choi, Y., Park, K.-S., Lee, H., Ma, J. S., Yamamoto, M., Kim, J., Chae, Y. C., Choi, J. H., Cocco, L., Berggren, P.-O., Jang, H.-J., Suh, P.-G. Phospholipase Cβ1 potentiates glucose-stimulated insulin secretion.
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http://dx.doi.org/10.1096/fj.201802732RRDOI Listing
October 2019

Translational assessment of a genetic engineering methodology to improve islet function for transplantation.

EBioMedicine 2019 Jul 29;45:529-541. Epub 2019 Jun 29.

The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden; Diabetes Research Institute, Miller School of Medicine, University of Miami, Miami, USA; Lee Kong Chian School of Medicine, Nanyang Technological University, Imperial College London, Novena Campus, Singapore, Singapore.

Background: The functional quality of insulin-secreting islet beta cells is a major factor determining the outcome of clinical transplantations for diabetes. It is therefore of importance to develop methodological strategies aiming at optimizing islet cell function prior to transplantation. In this study we propose a synthetic biology approach to genetically engineer cellular signalling pathways in islet cells.

Methods: We established a novel procedure to modify islet beta cell function by combining adenovirus-mediated transduction with reaggregation of islet cells into pseudoislets. As a proof-of-concept for the genetic engineering of islets prior to transplantation, this methodology was applied to increase the expression of the V1b receptor specifically in insulin-secreting beta cells. The functional outcomes were assessed in vitro and in vivo following transplantation into the anterior chamber of the eye.

Findings: Pseudoislets produced from mouse dissociated islet cells displayed basic functions similar to intact native islets in terms of glucose induced intracellular signalling and insulin release, and after transplantation were properly vascularized and contributed to blood glucose homeostasis. The synthetic amplification of the V1b receptor signalling in beta cells successfully modulated pseudoislet function in vitro. Finally, in vivo responses of these pseudoislet grafts to vasopressin allowed evaluation of the potential benefits of this approach in regenerative medicine.

Interpretation: These results are promising first steps towards the generation of high-quality islets and suggest synthetic biology as an important tool in future clinical islet transplantations. Moreover, the presented methodology might serve as a useful research strategy to dissect cellular signalling mechanisms of relevance for optimal islet function.
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http://dx.doi.org/10.1016/j.ebiom.2019.06.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6642289PMC
July 2019

Correction to: In vivo imaging of type 1 diabetes immunopathology using eye-transplanted islets in NOD mice.

Diabetologia 2019 Aug;62(8):1517

Diabetes Research Institute and Cell Transplant Center, University of Miami Miller School of Medicine, 1450 NW 10th Ave, Miami, FL, 33136, USA.

Unfortunately there is a mistake in the presentation of the affiliations in this paper.
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http://dx.doi.org/10.1007/s00125-019-4922-1DOI Listing
August 2019

In vivo imaging of type 1 diabetes immunopathology using eye-transplanted islets in NOD mice.

Diabetologia 2019 07 14;62(7):1237-1250. Epub 2019 May 14.

Diabetes Research Institute and Cell Transplant Center, University of Miami Miller School of Medicine, 1450 NW 10th Ave, Miami, FL, 33136, USA.

Aims/hypothesis: Autoimmune attack against the insulin-producing beta cells in the pancreatic islets results in type 1 diabetes. However, despite considerable research, details of the type 1 diabetes immunopathology in situ are not fully understood mainly because of difficult access to the pancreatic islets in vivo.

Methods: Here, we used direct non-invasive confocal imaging of islets transplanted in the anterior chamber of the eye (ACE) to investigate the anti-islet autoimmunity in NOD mice before, during and after diabetes onset. ACE-transplanted islets allowed longitudinal studies of the autoimmune attack against islets and revealed the infiltration kinetics and in situ motility dynamics of fluorescence-labelled autoreactive T cells during diabetes development. Ex vivo immunostaining was also used to compare immune cell infiltrations into islet grafts in the eye and kidney as well as in pancreatic islets of the same diabetic NOD mice.

Results: We found similar immune infiltration in native pancreatic and ACE-transplanted islets, which established the ACE-transplanted islets as reliable reporters of the autoimmune response. Longitudinal studies in ACE-transplanted islets identified in vivo hallmarks of islet inflammation that concurred with early immune infiltration of the islets and preceded their collapse and hyperglycaemia onset. A model incorporating data on ACE-transplanted islet degranulation and swelling allowed early prediction of the autoimmune attack in the pancreas and prompted treatments to intercept type 1 diabetes.

Conclusions/interpretation: The current findings highlight the value of ACE-transplanted islets in studying early type 1 diabetes pathogenesis in vivo and underscore the need for timely intervention to halt disease progression.
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http://dx.doi.org/10.1007/s00125-019-4879-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561836PMC
July 2019