Pancreatic β-cells release insulin in response to fluctuations in plasma glucose, amino acids, and free fatty acids (FFA). Clonal cell lines and isolated islets serve as essential early models for studying the impact of nutrients and evaluating potential therapies to address β-cell dysfunction. Acute and chronic changes in FFA levels have been shown to have positive and negative effects on β-cell function both in vivo and in vitro. A key problem in comparing islet lipid studies from different laboratories is that a wide variety of methods are used to isolate, culture, and assess islet function. The current study compares bovine serum albumin (BSA) types and lipid preparation methods in clonal 832/13 cells and human islets. Changing the percentage and culture conditions when using FFA-free BSA can negatively affect β-cell function compared to regular BSA. Preparing palmitate with FFA-free BSA can rescue insulin secretion compared to treating cells alone with FFA-free BSA. Different methods of preparing palmitate can have unique effects on insulin secretion. Overall, interpreting the effects of lipids on β-cell function is complicated by a number of variables that need to be controlled for in islet experiments.

Pancreatic β-cells secrete insulin in response to nutrient stimulation. Glucose, amino acids and free fatty acids (FFA) have all been shown to stimulate insulin release in vivo. In vitro, isolated islet studies have also demonstrated similar results to those seen in vivo. It has also been shown that high-fat diet-fed mice or chronic treatment of isolated islets to high glucose and FFA can lead to glucolipotoxicity and impaired β-cell function. Isolated islet studies are a standard assay for preliminary testing of novel ideas and drugs related to islet function. Interpreting and comparing in vitro islet results from acute and chronic treatment of nutrients can be difficult since a wide variety of methods are used to isolate and culture islets and assess islet function. In this review, we compare in vivo and in vitro FFA absorption, transport and metabolism and discuss in vitro methods and concepts related to islet responses to nutrients, focusing on the effects of fatty acids on insulin secretion and β-cell function. This review also discusses FFA levels and transport seen in type 2 diabetes and compares them to how isolated islets are treated with FFA in vitro.

Ketone supplementation has been gaining interest in improving health and treating some diseases, such as diabetes. However, the mechanism of action of how these ketone supplements work is not fully understood. In a recent paper, Banerjee et al. (2024) showed that physiological concentrations of βHB can affect hormone secretion and signalling within pancreatic islets. They showed that acute treatment with βHB increases insulin secretion and decreases glucagon secretion at physiological glucose concentrations. Their studies also suggest chronic βHB treatment may protect human islet cells from cytokine-induced cell death. Although more work is needed, it is possible that physiological concentrations of βHB may influence hormone secretion and signalling within islets.

Objective: Lipotoxic injury from renal lipid accumulation in obesity and type 2 diabetes (T2D) is implicated in associated kidney damage. However, models examining effects of renal ectopic lipid accumulation independent of obesity or T2D are lacking. We generated renal tubule specific adipose triglyceride lipase knockout (RT-SAKO) mice to determine if this targeted triacylglycerol (TAG) over-storage affects glycemic control and kidney health. Methods: Male and female RT-SAKO mice and their control littermates were tested for changes in glycemic control at 10-12 and 16-18 weeks of age. Markers of kidney health and blood lipid and hormone concentrations were analyzed. Kidney and blood lysophosphatidic acid (LPA) levels were measured, and a role for LPA in mediating impaired glycemic control was evaluated using the LPA receptor 1/3 inhibitor Ki-16425. Results: All groups remained insulin sensitive, but 16- to 18 -week-old male RT-SAKO mice became glucose intolerant, without developing kidney inflammation or fibrosis. Rather, these mice displayed lower circulating insulin and glucagon-like peptide 1 (GLP-1) levels. Impaired firstphase glucose -stimulated insulin secretion was detected and restored by Exendin-4. Kidney and blood LPA levels were elevated in older male but not female RT-SAKO mice, associated with increased kidney diacylglycerol kinase epsilon. Inhibition of LPA-mediated signaling restored serum GLP-1 levels, first -phase insulin secretion, and glucose tolerance. Conclusions: TAG over -storage alone is insufficient to cause renal tubule lipotoxicity. This work is the first to show that endogenously derived LPA modulates GLP-1 levels in vivo, demonstrating a new mechanism of kidney-gut -pancreas crosstalk to regulate insulin secretion and glucose homeostasis. (c) 2024 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

The phosphorylation of the transmembrane protein nephrin has been shown to play an important role in signaling in kidney podocytes, and it has now been shown to also play a key role in regulating pancreatic β-cell function. Williamson et al have recently shown that the loss of nephrin tyrosine phosphorylation on its 3 cytoplasmic YDxV motifs can enhance insulin release in aged female mice. These studies suggest that blocking nephrin phosphorylation may be an effective treatment option for improving β-cell function.

Abstract Insulin, a key hormone in the regulation of glucose homoeostasis, is secreted by pancreatic β‐cells in response to elevated glucose levels. Insulin is released in a biphasic manner in response to glucose metabolism in β‐cells. The first phase of insulin secretion is triggered by an increase in the ATP:ADP ratio; the second phase occurs in response to both a rise in ATP:ADP and other key metabolic signals, including a rise in the NADPH:NADP+ ratio. Experimental evidence indicates that pyruvate‐cycling pathways play an important role in the elevation of the NADPH:NADP+ ratio in response to glucose. The authors developed a kinetic model for the tricarboxylic acid cycle and pyruvate cycling pathways. The authors successfully validated the model against experimental observations and performed a sensitivity analysis to identify key regulatory interactions in the system. The model predicts that the dicarboxylate carrier and the pyruvate transporter are the most important regulators of pyruvate cycling and NADPH production. In contrast, the analysis showed that variation in the pyruvate carboxylase flux was compensated by a response in the activity of mitochondrial isocitrate dehydrogenase (ICDm) resulting in minimal effect on overall pyruvate cycling flux. The model predictions suggest starting points for further experimental investigation, as well as potential drug targets for the treatment of type 2 diabetes.

Objectives: Multiple limb amputations are an uncommon complication from inotrope-induced peripheral gangrene. Case Presentation: A 20-year-old lady with valvular heart disease had septic shock secondary to infective endocarditis and required prolonged cardiopulmonary resuscitation. Despite aggressive fluid resuscitation, the patient had triple strength intravenous noradrenaline to maintain hemodynamic stability. On day 5 of post-shock, dry gangrene occurred in distal parts of all limbs, and inotrope was stopped. Although the gangrenous changes were non-progressive, she required a significant degree of assistance with mobility and daily function performance. The patient was counseled for multiple limb amputations to promote walking and hand function through prosthetic restoration. Five months after the event, she had a right transtibial amputation, left Chopart amputation, left wrist disarticulation, and right second, third, fourth, and fifth fingers amputation. Three specific goals for inpatient rehabilitation were independent short-distance ambulation with prostheses, performing basic activities of daily living with adaptive devices, and independent wheelchair propulsion for long-distance mobility using a right-sided transtibial prosthesis, left-sided Syme's prosthesis with Kingsley's foot, right-sided silicone-based cosmetic glove and left-sided body-powered transradial prosthesis. Discussion: Although an uncommon complication, inotrope may lead to multiple limb amputations secondary to peripheral gangrene. Following amputation, the ultimate rehabilitative goal is to restore the mobility and capacity to perform daily functions through prosthetic restoration, whether walking for lower amputees or functioning hand for upper limb amputees. Prescribing prosthesis in a single limb loss is relatively straightforward, but restoring multiple limb amputations bears many challenges toward successful recovery of walking and functions.

Legend for Supplementary Figures S1-S9.

Supplementary Figures S1-S9. Avocatin B is the most active avocado lipid analogue (S1); Kinetics of avocatin B-induced death (S2); Cell cycle analysis of avocatin B treated TEX cells (S3); Avocatin B increases DCFH-DA and DHE (S4); Avocatin B's activity is neutralized by co-incubation with PEG-SOD (S5); Jurkat T cells cultured in ethidium bromide medium have reduced mitochondria with decreased function (S6); Avocatin B is absent in mitochondria and cytosolic fractions of vehicle control treated TEX cells (S7); Avocatin B calibration curves and estimated concentraiton in cytosolic and mitochondrial fractions (S8); Avocatin B decreases levels of NADH and NADPH and increases ROS in OCI-AML2 cells (S9).

Supplementary Tables S1-S3. Summary of EC50 values for avocatin B in a panel of AML cell lines (S1); AML patient sample details used for annexin/PI (S2); AML patient sample details used for colony formation assays (S3).

Supplementary Methods and References. Description of additional methods and procedures used in the study. Also includes Supplementary References.

Pancreatic β-cells can secrete insulin via 2 pathways characterized as KATP channel -dependent and -independent. The KATP channel-independent pathway is characterized by a rise in several potential metabolic signaling molecules, including the NADPH/NADP+ ratio and α-ketoglutarate (αKG). Prolyl hydroxylases (PHDs), which belong to the αKG-dependent dioxygenase superfamily, are known to regulate the stability of hypoxia-inducible factor α. In the current study, we assess the role of PHDs in vivo using the pharmacological inhibitor dimethyloxalylglycine (DMOG) and generated β-cell-specific knockout (KO) mice for all 3 isoforms of PHD (β-PHD1 KO, β-PHD2 KO, and β-PHD3 KO mice). DMOG inhibited in vivo insulin secretion in response to glucose challenge and inhibited the first phase of insulin secretion but enhanced the second phase of insulin secretion in isolated islets. None of the β-PHD KO mice showed any significant in vivo defects associated with glucose tolerance and insulin resistance except for β-PHD2 KO mice which had significantly increased plasma insulin during a glucose challenge. Islets from both β-PHD1 KO and β-PHD3 KO had elevated β-cell apoptosis and reduced β-cell mass. Isolated islets from β-PHD1 KO and β-PHD3 KO had impaired glucose-stimulated insulin secretion and glucose-stimulated increases in the ATP/ADP and NADPH/NADP+ ratio. All 3 PHD isoforms are expressed in β-cells, with PHD3 showing the most distinct expression pattern. The lack of each PHD protein did not significantly impair in vivo glucose homeostasis. However, β-PHD1 KO and β-PHD3 KO mice had defective β-cell mass and islet insulin secretion, suggesting that these mice may be predisposed to developing diabetes.

Oxidative stress caused by the exposure of pancreatic ß-cells to high levels of fatty acids impairs insulin secretion. This lipotoxicity is thought to play an important role in ß-cell failure in type 2 diabetes and can be prevented by antioxidants. Gamma-hydroxybutyrate (GHB), an endogenous antioxidant and energy source, has previously been shown to protect mice from streptozotocin and alloxan-induced diabetes; both compounds are generators of oxidative stress and yield models of type-1 diabetes. We sought to determine whether GHB could protect mouse islets from lipotoxicity caused by palmitate, a model relevant to type 2 diabetes. We found that GHB prevented the generation of palmitate-induced reactive oxygen species and the associated lipotoxic inhibition of glucose-stimulated insulin secretion while increasing the NADPH/NADP+ ratio. GHB may owe its antioxidant and insulin secretory effects to the formation of NADPH.

The NAD-dependent deacetylase SIRT1 improves β cell function. Accordingly, nicotinamide mononucleotide (NMN), the product of the rate-limiting step in NAD synthesis, prevents β cell dysfunction and glucose intolerance in mice fed a high-fat diet. The current study was performed to assess the effects of NMN on β cell dysfunction and glucose intolerance that are caused specifically by increased circulating free fatty acids (FFAs). NMN was intravenously infused, with or without oleate, in C57BL/6J mice over a 48-h-period to elevate intracellular NAD levels and consequently increase SIRT1 activity. Administration of NMN in the context of elevated plasma FFA levels considerably improved glucose tolerance. This was due not only to partial protection from FFA-induced β cell dysfunction but also, unexpectedly, to a significant decrease in insulin clearance. However, in conditions of normal FFA levels, NMN impaired glucose tolerance due to decreased β cell function. The presence of this dual action of NMN suggests caution in its proposed therapeutic use in humans.

The α-ketoglutarate-dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is an HIF target that uses molecular oxygen to hydroxylate peptidyl prolyl residues. Although PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about the effects of this highly conserved enzyme in insulin-secreting β cells in vivo. Here, we show that the deletion of PHD3 specifically in β cells (βPHD3KO) was associated with impaired glucose homeostasis in mice fed a high-fat diet. In the early stages of dietary fat excess, βPHD3KO islets energetically rewired, leading to defects in the management of pyruvate fate and a shift from glycolysis to increased fatty acid oxidation (FAO). However, under more prolonged metabolic stress, this switch to preferential FAO in βPHD3KO islets was associated with impaired glucose-stimulated ATP/ADP rises, Ca2+ fluxes, and insulin secretion. Thus, PHD3 might be a pivotal component of the β cell glucose metabolism machinery in mice by suppressing the use of fatty acids as a primary fuel source during the early phases of metabolic stress.

8 1 Institute of Metabolism and Systems Research (IMSR), University of Birmingham, 9 Birmingham, UK. 10 2 Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, 11 Birmingham, UK. 12 3 Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham, 13 Birmingham, UK. 14 4 Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, 15 UK. 16 5 Oxford NIHR Biomedical Research Centre, Churchill Hospital, Oxford, UK. 17 6 Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, Oxford, 18 UK. 19 7 Mitochondrial Profiling Centre, School of Sport, Exercise and Rehabilitation Sciences, 20 University of Birmingham, Birmingham, UK. 21 8 School of Pharmacy, University of Waterloo, Kitchener, Ontario, Canada. 22 9 Division of Systems Medicine, School of Medicine, University of Dundee, Dundee, UK. 23 24 These authors contributed equally 25

Anaplerosis and the associated mitochondrial metabolite transporters generate unique cytosolic metabolic signaling molecules that can regulate insulin release from pancreatic β-cells. It has been shown that mitochondrial metabolites, transported by the citrate carrier (CIC), dicarboxylate carrier (DIC), oxoglutarate carrier (OGC), and mitochondrial pyruvate carrier (MPC) play a vital role in the regulation of glucose-stimulated insulin secretion (GSIS). Metabolomic studies on static and biphasic insulin secretion, suggests that several anaplerotic derived metabolites, including α-ketoglutarate (αKG), are strongly associated with nutrient regulated insulin secretion. Support for a role of αKG in the regulation of insulin secretion comes from studies looking at αKG dependent enzymes, including hypoxia-inducible factor-prolyl hydroxylases (PHDs) in clonal β-cells, and rodent and human islets. This review will focus on the possible link between defective anaplerotic-derived αKG, PHDs, and the development of type 2 diabetes (T2D).

Efforts to prevent or lessen the functional impact of psychosis can be informed by a better understanding of the neurobiological underpinnings at the earliest stages of the disorder. Understanding these processes early in the psychosis spectrum will in turn allow more targeted efforts to prevent or minimize functional limitations among patients with psychosis. Advances in technology have enabled the study of a host of biomarkers implicated in the neurobiology of psychosis offering unique avenues to investigate mechanisms of disease while at the same time shedding some light on more patient-tailored treatments and setting the foundation for personalized medicine in psychosis. Insights into the neurobiology of psychosis are reviewed, including findings from neuroimaging, neurocognitive, and electrophysiologic studies and findings related to the role of hypothalamic-pituitary axis activity and neuroinflammation in the emergence of psychosis. Biomarker-informed treatments are discussed, and potential promising biomarkers and related treatments are proposed. This review contains 5 figures, 13 tables, and 85 references. Key words: attenuated risk syndrome, biomarkers, prodrome, psychosis, schizophrenia, treatment

Pancreatic β-cells secrete insulin in a biphasic manner via KATP-channel dependent and independent pathways. The KATP-channel independent pathway produces a rise in the NADPH/NADP+ ratio and cytosolic α-ketoglutarate (α-KG). Prolyl hydroxylase domain proteins (PHDs) belong to the α-KG-dependent dioxygenase superfamily and regulates stability of hypoxia-inducible factor α (HIF-α). Prolyl hydroxylation of HIF-α for ubiquitin-mediated proteasomal degradation requires sufficient levels of oxygen, iron and α-KG. We have previously shown pharmacological inhibition of PHDs reduces glucose-stimulated insulin secretion (GSIS), glucose utilization, and mitochondrial function in clonal β-cells. Glucose-induced changes in cytosolic α-KG may regulate PHD and thus β-cell function. In mouse pancreatic β-cells, PHD isoforms express unique subcellular localization: PHD1 is expressed exclusively in the cytosol, PHD2 is expressed in the cytosol and nucleus, and PHD3 is mainly expressed in the nucleus. To further explore the isoform-specific roles of PHD, we employed a β-cell specific knockout (β-PHD KO) mouse model for each of the PHD isoforms. β-PHD1 KO, β-PHD2 KO and β-PHD3 KO. β-PHD KO mice show no significant in vivo defects associated with glucose tolerance and insulin resistance; however, β-PHD1 KO and β-PHD2 KO mice have significantly increased plasma insulin compared to wild type (WT) controls (p<0.05). Consistent with our cell culture model of PHD knockdown, PHD1 and PHD3 regulate insulin secretion as both β-PHD1 KO and β-PHD3 KO mice show impaired GSIS. Only β-PHD1 KO mice have reduced beta-cell mass (p<0.01). In conclusion, there are significant and unique effects observed with isoform-specific suppression of PHD. Disclosure M. Hoang: None. S.M. Janssen: None. J.W. Joseph: None. Funding Natural Sciences and Engineering Research Council of Canada; Canadian Institutes of Health Research

Diabetes is high in the agenda of public health issues with significant prevalence of diabetic-related amputations. Prosthetic restoration post-amputation is imperative to reduce disability, but its success is influenced by several factors. This study analysed the practice, and the determining factors affecting prosthetic restoration among major lower limb amputees with diabetes in Sabah. Methods: Retrospective cross-sectional study among 65 major lower limb amputees with diabetes referred for rehabilitation medicine services at Queen Elizabeth Hospital from0 1st January 2015 to 31st December 2017. Demographics data, pre-morbid diseases, levels of amputation and practice on prosthetic restoration (suitability for restoration, duration from prosthetic application to restoration, funding issue) are primary evaluated factors. Results: Forty-nine, fifteen and one amputees had below knee amputation, above knee amputation and hip disarticulation respectively. Forty-three amputees (66.2%) were deemed suitable for prosthetic restoration but only 27 were prosthetically restored, with mean duration from prosthetic application to restoration of 5.92±2.189 months. Having additional pre-morbid diseases did not reduced the likelihood of suitability for prosthetic restoration compared to those only with diabetes (p=0.082). Funding issue is the key factor affecting prosthetic restoration with higher likelihood for restoration among those eligible through governmental agencies funding (p=0.027). Conclusion: In Sabah, low rate of suitability for prosthetic restoration is observed among major lower limb amputees with diabetes. A larger study is warranted to investigate causes of such low rate of suitability for prosthetic restoration among this specific population in the effort to reduce public health burden from major lower limb amputation-related disability.