Islet cells in human type 1 diabetes: from recent advances to novel therapies - a symposium-based roadmap for future research.

There is a growing understanding that the early phases of type 1 diabetes (T1D) are characterised by a deleterious dialogue between the pancreatic beta cells and the immune system. This, combined with the urgent need to better translate this growing knowledge into novel therapies, provided the background for the JDRF-DiabetesUK-INNODIA-nPOD symposium entitled 'Islet cells in human T1D: from recent advances to novel therapies', which took place in Stockholm, Sweden, in September 2022. We provide in this article an overview of the main themes addressed in the symposium, pointing to both promising conclusions and key unmet needs that remain to be addressed in order to achieve better approaches to prevent or reverse T1D.

G protein-coupled receptor (GPR)40-dependent potentiation of insulin secretion in mouse islets is mediated by protein kinase D1.

AIMS/HYPOTHESIS: Activation of the G protein-coupled receptor (GPR)40 by long-chain fatty acids potentiates glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells, and GPR40 agonists are in clinical development for type 2 diabetes therapy. GPR40 couples to the G protein subunit Gα(q/11) but the signalling cascade activated downstream is unknown. This study aimed to determine the mechanisms of GPR40-dependent potentiation of GSIS by fatty acids. METHODS: Insulin secretion in response to glucose, oleate or diacylglycerol (DAG) was assessed in dynamic perifusions and static incubations in islets from wild-type (WT) and Gpr40 (-/-) mice. Depolymerisation of filamentous actin (F-actin) was visualised by phalloidin staining and epifluorescence. Pharmacological and molecular approaches were used to ascertain the roles of protein kinase D (PKD) and protein kinase C delta in GPR40-mediated potentiation of GSIS. RESULTS: Oleate potentiates the second phase of GSIS, and this effect is largely dependent upon GPR40. Accordingly, oleate induces rapid F-actin remodelling in WT but not in Gpr40 (-/-) islets. Exogenous DAG potentiates GSIS in both WT and Gpr40 (-/-) islets. Oleate induces PKD phosphorylation at residues Ser-744/748 and Ser-916 in WT but not Gpr40 (-/-) islets. Importantly, oleate-induced F-actin depolymerisation and potentiation of GSIS are lost upon pharmacological inhibition of PKD1 or deletion of Prkd1. CONCLUSIONS/INTERPRETATION: We conclude that the signalling cascade downstream of GPR40 activation by fatty acids involves activation of PKD1, F-actin depolymerisation and potentiation of second-phase insulin secretion. These results provide important information on the mechanisms of action of GPR40, a novel drug target for type 2 diabetes.

SU-E-T-477: Influence of Eye Size on Radiation Absorbed Dose Delivered to Non- Targeted Tissues during Stereotactic Radiosurgery for Age-Related Macular Degeneration.

PURPOSE: This work determines how variations in eye size will influence the radiation absorbed dose delivered to non-targeted tissues within the eye during stereotactic radiosurgery of age-related macular degeneration (AMD) using the IRay™ treatment. METHODS: Stylized models of the eye were created with axial lengths of 20, 22, 24, 26, and 28mm. Each model was based upon the reference eye model from NCRP Report 130 and then scaled appropriately for each axial length. Models were incorporated with MCNPX radiation transport code in order to simulate the three beam IRay™ delivery system. Simulation results were assessed for both the mean absorbed dose and dose-volume histograms (DVH) for both target (macula) and non- targeted eye tissues, including the lens, retina, central retinal artery, and optic nerve. RESULTS: For each of the three beams, an average dose of 8Gy was delivered to the macula resulting in a total average dose of 24Gy for each eye model. The lens of the eye received a total average dose ranging from 146 to 189mGy, with the larger doses occurring in the smaller eye models since the beams traverse through the sciera closer to the limbus. The distal tip (1.5mm) of the central retinal artery received a total average dose ranging from 499 to 567mGy, with the larger doses occurring in the larger eye models due to increased scatter resulting from longer tissue path length to the nominal target. The optic nerve received a total average dose ranging from 207 to 225mGy, with the larger doses occurring in the smaller eye models. CONCLUSIONS: The small variation in dose to the lens, central retinal artery, and optic nerve suggests that eye size does not significantly affect radiation dose to non-targeted eye tissues. This work was sponsored by Oraya Therapeutics.

Time-dependent effects of Prkce deletion on glucose homeostasis and hepatic lipid metabolism on dietary lipid oversupply in mice.

AIMS/HYPOTHESIS: We examined the time-dependent effects of deletion of the gene encoding protein kinase C epsilon (Prkce) on glucose homeostasis, insulin secretion and hepatic lipid metabolism in fat-fed mice. METHODS: Prkce(-/-) and wild-type (WT) mice were fed a high-fat diet for 1 to 16 weeks and subjected to i.p. glucose tolerance tests (ipGTT) and indirect calorimetry. We also investigated gene expression and protein levels by RT-PCR, quantitative protein profiling (isobaric tag for relative and absolute quantification; iTRAQ) and immunoblotting. Lipid levels, mitochondrial oxidative capacity and lipid metabolism were assessed in liver and primary hepatocytes. RESULTS: While fat-fed WT mice became glucose intolerant after 1 week, Prkce(-/-) mice exhibited normal glucose and insulin levels. iTRAQ suggested differences in lipid metabolism and oxidative phosphorylation between fat-fed WT and Prkce(-/-) animals. Liver triacylglycerols were increased in fat-fed Prkce(-/-) mice, resulting from altered lipid partitioning which promoted esterification of fatty acids in hepatocytes. In WT mice, fat feeding elevated oxygen consumption in vivo and in isolated liver mitochondria, but these increases were not seen in Prkce(-/-) mice. Prkce(-/-) hepatocytes also exhibited reduced production of reactive oxygen species (ROS) in the presence of palmitate. After 16 weeks of fat feeding, however, the improved glucose tolerance in fat-fed Prkce(-/-) mice was instead associated with increased insulin secretion during ipGTT, as we have previously reported. CONCLUSIONS/INTERPRETATION: Prkce deletion ameliorates diet-induced glucose intolerance via two temporally distinct phenotypes. Protection against insulin resistance is associated with changes in hepatic lipid partitioning, which may reduce the acute inhibitory effects of fatty acid catabolism, such as ROS generation. In the longer term, enhancement of glucose-stimulated insulin secretion prevails.

Deletion of protein kinase Cδ in mice modulates stability of inflammatory genes and protects against cytokine-stimulated beta cell death in vitro and in vivo.

AIMS/HYPOTHESIS: Proinflammatory cytokines contribute to beta cell destruction in type 1 diabetes, but the mechanisms are incompletely understood. The aim of the current study was to address the role of the protein kinase C (PKC) isoform PKCδ, a diverse regulator of cell death, in cytokine-stimulated apoptosis in primary beta cells. METHODS: Islets isolated from wild-type or Prkcd(-/-) mice were treated with IL-1ß, TNF-α and IFNγ and assayed for apoptosis, nitric oxide (NO) generation and insulin secretion. Activation of signalling pathways, apoptosis and endoplasmic reticulum (ER) stress were determined by immunoblotting. Stabilisation of mRNA transcripts was measured by RT-PCR following transcriptional arrest. Mice were injected with multiple low doses of streptozotocin (MLD-STZ) and fasting blood glucose monitored. RESULTS: Deletion of Prkcd inhibited apoptosis and NO generation in islets stimulated ex vivo with cytokines. It also delayed the onset of hyperglycaemia in MLD-STZ-treated mice. Activation of ERK, p38, JNK, AKT1, the ER stress markers DDIT3 and phospho-EIF2α and the intrinsic apoptotic markers BCL2 and MCL1 was not different between genotypes. However, deletion of Prkcd destabilised mRNA transcripts for Nos2, and for multiple components of the toll-like receptor 2 (TLR2) signalling complex, which resulted in disrupted TLR2 signalling. CONCLUSIONS/INTERPRETATION: Loss of PKCδ partially protects against hyperglycaemia in the MLD-STZ model in vivo, and against cytokine-mediated apoptosis in vitro. This is accompanied by reduced NO generation and destabilisation of Nos2 and components of the TLR2 signalling pathway. The results highlight a mechanism for regulating proinflammatory gene expression in beta cells independently of transcription.

The hypoxia response pathway and ß-cell function.

ß-cells sense glucose and secrete appropriate amounts of insulin by coupling glucose uptake and glycolysis with quantitative ATP production via mitochondrial oxidative pathways. Therefore, oxidative phosphorylation is essential for normal ß-cell function. Multiple cell types adapt to hypoxia by inducing a transcriptional programme coordinated by the transcription factor hypoxia-inducible factor (HIF). HIF activity is regulated by the von Hippel-Lindau (Vhl) protein, which targets the HIFα subunit for proteasomal degradation in the presence of oxygen. Several recent studies have shown that Vhl deletion in ß-cells results in Hif1α activation, impaired glucose-stimulated insulin secretion (GSIS) and glucose intolerance. This was found to be because of alterations in ß-cell gene expression inducing a switch from aerobic glucose metabolism to anaerobic glycolysis, thus disrupting the GSIS triggering pathway. Situations in which islets may become hypoxic are discussed, in particular islet transplantation which has been reported to cause islet hypoxia because of an inadequate blood supply post-transplant. Aside from this principal role for HIF in negatively regulating ß-cell glucose sensing, other aspects of hypoxia signalling are discussed including ß-cell differentiation, development and vascularization. In conclusion, recent studies clearly show that hypoxia response mechanisms can negatively impact on glucose sensing mechanisms in the ß-cell and this has the potential to impair ß-cell function in a number of physiological and clinical situations.

Pancreatic deletion of insulin receptor substrate 2 reduces beta and alpha cell mass and impairs glucose homeostasis in mice.

AIMS/HYPOTHESIS: Insulin signalling pathways regulate pancreatic beta cell function. Conditional gene targeting using the Cre/loxP system has demonstrated that mice lacking insulin receptor substrate 2 (IRS2) in the beta cell have reduced beta cell mass. However, these studies have been complicated by hypothalamic deletion when the RIPCre (B6.Cg-tg(Ins2-cre)25Mgn/J) transgenic mouse (expressing Cre recombinase under the control of the rat insulin II promoter) is used to delete floxed alleles in insulin-expressing cells. These features have led to marked insulin resistance making the beta cell-autonomous role of IRS2 difficult to determine. To establish the effect of deleting Irs2 only in the pancreas, we generated PIrs2KO mice in which Cre recombinase expression was driven by the promoter of the pancreatic and duodenal homeobox factor 1 (Pdx1, also known as Ipf1) gene. MATERIALS AND METHODS: In vivo glucose homeostasis was examined in PIrs2KO mice using glucose tolerance and glucose-stimulated insulin secretion tests. Endocrine cell mass was determined by morphometric analysis. Islet function was examined in static cultures and by performing calcium imaging in Fluo3am-loaded beta cells. Islet gene expression was determined by RT-PCR. RESULTS: The PIrs2KO mice displayed glucose intolerance and impaired glucose-stimulated insulin secretion in vivo. Pancreatic insulin and glucagon content and beta and alpha cell mass were reduced. Glucose-stimulated insulin secretion and calcium mobilisation were attenuated in PIrs2KO islets. Expression of the Glut2 gene (also known as Slc2a2) was also reduced in PIrs2KO mice. CONCLUSIONS/INTERPRETATION: These studies suggest that IRS2-dependent signalling in pancreatic islets is required not only for the maintenance of normal beta and alpha cell mass but is also involved in the regulation of insulin secretion.

Liver-specific deletion of insulin receptor substrate 2 does not impair hepatic glucose and lipid metabolism in mice.

AIMS/HYPOTHESIS: Hepatic insulin resistance is thought to be a critical component in the pathogenesis of type 2 diabetes but the role of intrinsic insulin signalling pathways in the regulation of hepatic metabolism remains controversial. Global gene targeting in mice and in vitro studies have suggested that IRS2 mediates the physiological effects of insulin in the liver. Reduced hepatic production of IRS2 is found in many cases of insulin resistance. To investigate the role of IRS2 in regulating liver function in vivo, we generated mice that specifically lack Irs2 in the liver (LivIrs2KO). MATERIALS AND METHODS: Hepatic insulin signalling events were examined in LivIrs2KO mice by western blotting. Glucose homeostasis and insulin sensitivity were assessed by glucose tolerance tests and hyperinsulinaemic-euglycaemic clamp studies. The effects of high-fat feeding upon glucose homeostasis were also determined. Liver function tests were performed and expression of key metabolic genes in the liver was determined by RT-PCR. RESULTS: Proximal insulin signalling events and forkhead box O1 and A2 function were normal in the liver of LivIrs2KO mice, which displayed minimal abnormalities in glucose and lipid homeostasis, hepatic gene expression and liver function. In addition, hepatic lipid homeostasis and the metabolic response to a high-fat diet did not differ between LivIrs2KO and control mice. CONCLUSIONS/INTERPRETATION: Our findings suggest that liver IRS2 signalling, surprisingly, is not required for the long-term maintenance of glucose and lipid homeostasis, and that extra-hepatic IRS2-dependent mechanisms are involved in the regulation of these processes.