The N-methyl-D-aspartate (NMDA) receptor antagonist dextrorphan (DXO) protects pancreatic islets from cell death induced by streptozotocin (STZ), inflammatory cytokines, reactive oxygen species (ROS), as well as in multiple mouse models of diabetes. Here we show that cytoprotective concentrations of DXO upregulate the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), driving the expression of its downstream target genes. Paradoxically, DXO also increases the expression of the pro-apoptotic factor CHAC1 (chaC glutathione-specific γ-glutamylcyclotransferase 1). To investigate its role, we silenced Chac1 in islets, which significantly enhanced DXO-mediated cell protection. In contrast, both pharmacological inhibition and genetic knockdown of Nfe2l2 diminished the protective effects of DXO, confirming the cytoprotective role of NRF2. Conversely, pharmacological activation of NRF2 did not further enhance DXO-induced protection. Together, our data indicate that while DXO-mediated islet protection requires induction of NRF2, its full therapeutic efficacy is limited by the simultaneous upregulation of CHAC1.

Obesity-driven pathological expansion of white adipose tissue (WAT) is a key driver of endothelial dysfunction. However, early vascular alterations associated with over-nutrition also serve to exacerbate WAT dysfunction. Here, we conduct a single-cell transcriptomic analysis of WAT endothelium to delineate endothelial heterogeneity and elucidate vascular alterations and its consequence in a male murine model of obesity. We demarcate depot-specific differences in subcutaneous (sWAT) and visceral WAT (vWAT) endothelium through in sillico analysis and further corroboration of our findings. Moreover, we identify a sWAT-specific fenestrated endothelial cell (EC) subtype, which declines in obese conditions. Utilizing systemic anti-VEGFA blockade and genetic Vegfa manipulation, we demonstrate that VEGFA is necessary for maintaining fenestration in sWAT. Additionally, we detect this fenestrated EC subtype in male human WAT, which undergoes reduction in individuals with obesity. Collectively, this atlas serves as a valuable tool for future studies to decipher the functional significance of different WAT EC subtypes.

Recently, we have shown that after partial hepatectomy (PHx), an increased hepatic blood flow initiates liver growth in mice by vasodilation and mechanically-triggered release of angiocrine signals. Here, we use mass spectrometry to identify a mechanically-induced angiocrine signal in human hepatic endothelial cells, that is, myeloid-derived growth factor (MYDGF). We show that it induces proliferation and promotes survival of primary human hepatocytes derived from different donors in two-dimensional cell culture, via activation of mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3). MYDGF also enhances proliferation of human hepatocytes in three-dimensional organoids. In vivo, genetic deletion of MYDGF decreases hepatocyte proliferation in the regenerating mouse liver after PHx; conversely, adeno-associated viral delivery of MYDGF increases hepatocyte proliferation and MAPK signaling after PHx. We conclude that MYDGF represents a mechanically-induced angiocrine signal and that it triggers growth of, and provides protection to, primary mouse and human hepatocytes.

For treatment of type 1 diabetes mellitus, a combination of immune-based interventions and medication to promote beta-cell survival and proliferation has been proposed. Dextromethorphan (DXM) is an N-methyl-D-aspartate receptor antagonist with a good safety profile, and to date, preclinical and clinical evidence for blood glucose-lowering and islet-cell-protective effects of DXM have only been provided for animals and individuals with type 2 diabetes mellitus. Here, we assessed the potential anti-diabetic effects of DXM in the non-obese diabetic mouse model of type 1 diabetes. More specifically, we showed that DXM treatment led to five-fold higher numbers of pancreatic islets and more than two-fold larger alpha- and beta-cell areas compared to untreated mice. Further, DXM treatment improved glucose homeostasis and reduced diabetes incidence by 50%. Our data highlight DXM as a novel candidate for adjunct treatment of preclinical or recent-onset type 1 diabetes.

The BiP co-chaperone DNAJC3 protects cells during ER stress. In mice, the deficiency of DNAJC3 leads to beta-cell apoptosis and the gradual onset of hyperglycemia. In humans, biallelic DNAJC3 variants cause a multisystem disease, including early-onset diabetes mellitus. Recently, hyperinsulinemic hypoglycemia (HH) has been recognized as part of this syndrome. This report presents a case study of an individual with HH caused by DNAJC3 variants and provides an overview of the metabolic phenotype of individuals with HH and DNAJC3 variants. The study demonstrates that HH may be a primary symptom of DNAJC3 deficiency and can persist until adolescence. Additionally, glycemia and insulin release were analyzed in young DNACJ3 knockout (K.O.) mice, which are equivalent to human infants. In the youngest experimentally accessible age group of 4-week-old mice, the in vivo glycemic phenotype was already dominated by a reduced total insulin secretion capacity. However, on a cellular level, the degree of insulin release of DNAJC3 K.O. islets was higher during periods of increased synthetic activity (high-glucose stimulation). We propose that calcium leakage from the ER into the cytosol, due to disrupted DNAJC3-controlled gating of the Sec61 channel, is the most likely mechanism for HH. This is the first genetic mechanism explaining HH solely by the disruption of intracellular calcium homeostasis. Clinicians should screen for HH in DNAJC3 deficiency and consider DNAJC3 variants in the differential diagnosis of congenital hyperinsulinism.

Prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease, increases worldwide and associates with type 2 diabetes and other cardiometabolic diseases. Here we demonstrate that Sema3a is elevated in liver sinusoidal endothelial cells of animal models for obesity, type 2 diabetes and MASLD. In primary human liver sinusoidal endothelial cells, saturated fatty acids induce expression of SEMA3A, and loss of a single allele is sufficient to reduce hepatic fat content in diet-induced obese mice. We show that semaphorin-3A regulates the number of fenestrae through a signaling cascade that involves neuropilin-1 and phosphorylation of cofilin-1 by LIM domain kinase 1. Finally, inducible vascular deletion of Sema3a in adult diet-induced obese mice reduces hepatic fat content and elevates very low-density lipoprotein secretion. Thus, we identified a molecular pathway linking hyperlipidemia to microvascular defenestration and early development of MASLD. Eberhard et al. show that SEMA3A regulates liver sinusoidal endothelial cell fenestrations by signaling through NRP1 and LIMK1, revealing a pathway that connects hyperlipidemia to the development of steatotic liver disease.

Type 2 diabetes (T2D) is the most common metabolic disease worldwide and is characterized by insulin hypersecretion, followed by reduced glucose-stimulated insulin secretion (GSIS). Even though much has been discovered about insulin producing beta cell physiology, the molecular mechanisms behind beta cell decay in T2D are still unknown and no anti-diabetic drug is available to fully halt or reverse this process. Here, we show that short-term in vitro stimulation of mouse pancreatic islets with insulin secretagogue dextrorphan (DXO) enhances GSIS, but does not protect islets from cell death. In contrast, long-term insulin hypersecretion induced by DXO reduces GSIS, but protects islets from cell death. Bulk RNA sequencing of islets reveals increased expression of genes encoding enzymes of the serine-linked mitochondrial one-carbon metabolism (OCM) after long-term, but not short-term stimulation. In long-term stimulated islets, more glucose was metabolized to serine than citrate, and mitochondrial concentrations of ATP decreased, despite unaltered oxygen consumption, while NAPDH increased. Activating transcription factor-4 (Atf4) is shown to be required and sufficient to activate serine-linked mitochondrial OCM genes in islets, and gain- and loss-of-function experiments revealed that Atf4 reduces GSIS and is necessary, but not solely sufficient for full DXO-mediated islet cell protection. Furthermore, we show that de novo serine synthesis enzyme phosphoglycerate dehydrogenase (Phgdh) and enzymes of the mitochondrial OCM, such as serine hydroxymethyltransferase 2 (Shmt2) and methylenetetrahydrofolate dehydrogenase 2 (Mthfd2), reduce GSIS and regulate islet cell death. In summation, we identified a reversible metabolic pathway associated with islet cell protection at expense of secretory function.

Type 2 diabetes is characterized by insulin hypersecretion followed by reduced glucose-stimulated insulin secretion (GSIS). Here we show that acute stimulation of pancreatic islets with the insulin secretagogue dextrorphan (DXO) or glibenclamide enhances GSIS, whereas chronic treatment with high concentrations of these drugs reduce GSIS but protect islets from cell death. Bulk RNA sequencing of islets shows increased expression of genes for serine-linked mitochondrial one-carbon metabolism (OCM) after chronic, but not acute, stimulation. In chronically stimulated islets, more glucose is metabolized to serine than to citrate, and the mitochondrial ATP/ADP ratio decreases, whereas the NADPH/NADP+ ratio increases. Activating transcription factor-4 (Atf4) is required and sufficient to activate serine-linked mitochondrial OCM genes in islets, with gain- and loss-of-function experiments showing that Atf4 reduces GSIS and is required, but not sufficient, for full DXO-mediated islet protection. In sum, we identify a reversible metabolic pathway that provides islet protection at the expense of secretory function.

Dextromethorphan (DXM) acts as cough suppressant via its central action. Cell-protective effects of this drug have been reported in peripheral tissues, making DXM potentially useful for treatment of several common human diseases, such as type 2 diabetes mellitus (T2DM). Pancreatic islets are among the peripheral tissues that positively respond to DXM, and anti-diabetic effects of DXM were observed in two placebo-controlled, randomized clinical trials in humans with T2DM. Since these effects were associated with central side effects, we here developed chemical derivatives of DXM that pass the blood-brain barrier to a significantly lower extent than the original drug. We show that basic nitrogen-containing residues block central adverse events of DXM without reducing its anti-diabetic effects, including the protection of human pancreatic islets from cell death. These results show how to chemically modify DXM, and possibly other morphinans, as to exclude central side effects, while targeting peripheral tissues, such as pancreatic islets.

As part of early drug development, preformulation studies are used to comprehensively explore the properties of new drugs. In particular, this includes the biopharmaceutical characterization and evaluation of impacting factors (e.g. excipients, microenvironmental conditions etc.) by permeation studies. To overcome the limitations of current studies, a novel standardized ex vivo procedure using esophageal mucosa as surrogate has been established successfully and applied to preformulation studies for oromucosal delivery of cyclobenzaprine hydrochloride, a tricyclic muscle relaxant with potential for psychopharmacotherapeutic use. By using the standardized ex vivo permeation process, a twofold enhancement of permeability (0.98 ± 0.16 to 1.96 ± 0.10 * 10-5 cm/s) was observed by adjustment and controlling of microenvironmental pH, empowering a targeted and effective development of sublingual formulations. Predictivity and suitability were superior compared to in vitro experiments using artificial biomimetic membranes, revealing a determination coefficient (R2) of 0.995 vs. 0.322 concerning pH-dependent permeability of cyclobenzaprine. In addition, diffusion properties were extensively examined (e.g. influence of mucosal thicknesses, tissue freezing etc.). The alignment of the study design regarding physiologically/clinically relevant conditions resulted in ex vivo data that allowed for the estimation of plasma AUC levels in the extend of reported in vivo ranges.

We have recently shown that the N-Methyl-D-Aspartate receptor (NMDAR) antagonist and over-the-counter drug dextromethorphan (DXM) and its active metabolite dextrorphan (DXO) have antidiabetic and islet cell protective properties. However, both molecules are associated with central nervous system related side effects. To reduce these effects, we are currently developing and testing derivates of DXO that have a reduced blood-brain barrier (BBB) permeability while having a preserved antidiabetic potential.

An acute increase in blood flow triggers flow-mediated dilation (FMD), which is mainly mediated by endothelial nitric oxide synthase (eNOS). A long-term increase in blood flow chronically enlarges the arterial lumen, a process called arteriogenesis. In several common human diseases, these processes are disrupted for as yet unknown reasons. Here, we asked whether β1 integrin, a mechanosensory protein in endothelial cells, is required for FMD and arteriogenesis in the ischemic hindlimb. Permanent ligation of the femoral artery in C57BL/6 J mice enlarged pre-existing collateral arteries and increased numbers of arterioles in the thigh. In the lower leg, the numbers of capillaries increased. Notably, injection of β1 integrin-blocking antibody or tamoxifen-induced endothelial cell-specific deletion of the gene for β1 integrin (Itgb1) inhibited both arteriogenesis and angiogenesis. Using high frequency ultrasound, we demonstrated that β1 integrin-blocking antibody or endothelial cell-specific depletion of β1 integrin attenuated FMD of the femoral artery, and blocking of β1 integrin function did not further decrease FMD in eNOS-deficient mice. Our data suggest that endothelial β1 integrin is required for both acute and chronic widening of the arterial lumen in response to hindlimb ischemia, potentially via functional interaction with eNOS.

[This corrects the article DOI: 10.1371/journal.pone.0013650.].

Angiocrine signals derived from endothelial cells are an important component of intercellular communication and have a key role in organ growth, regeneration and disease 1 – 4 . These signals have been identified and studied in multiple organs, including the liver, pancreas, lung, heart, bone, bone marrow, central nervous system, retina and some cancers 1 – 4 . Here we use the developing liver as a model organ to study angiocrine signals 5 , 6 , and show that the growth rate of the liver correlates both spatially and temporally with blood perfusion to this organ. By manipulating blood flow through the liver vasculature, we demonstrate that vessel perfusion activates β1 integrin and vascular endothelial growth factor receptor 3 (VEGFR3). Notably, both β1 integrin and VEGFR3 are strictly required for normal production of hepatocyte growth factor, survival of hepatocytes and liver growth. Ex vivo perfusion of adult mouse liver and in vitro mechanical stretching of human hepatic endothelial cells illustrate that mechanotransduction alone is sufficient to turn on angiocrine signals. When the endothelial cells are mechanically stretched, angiocrine signals trigger in vitro proliferation and survival of primary human hepatocytes. Our findings uncover a signalling pathway in vascular endothelial cells that translates blood perfusion and mechanotransduction into organ growth and maintenance.

Angiopoietin-like 4 (ANGPTL4) is an important regulator of triacylglycerol metabolism, carrying out this role by inhibiting the enzymes lipoprotein lipase and pancreatic lipase. ANGPTL4 is a potential target for ameliorating cardiometabolic diseases. Although ANGPTL4 has been implicated in obesity, the study of the direct role of ANGPTL4 in diet-induced obesity and related metabolic dysfunction is hampered by the massive acute-phase response and development of lethal chylous ascites and peritonitis in Angptl4−/− mice fed a standard high-fat diet. The aim of this study was to better characterise the role of ANGPTL4 in glucose homeostasis and metabolic dysfunction during obesity.

Diabetes mellitus is characterized by chronically elevated blood glucose levels accelerated by a progressive decline of insulin‐producing β‐cells in the pancreatic islets. Although medications are available to transiently adjust blood glucose to normal levels, the effects of current drugs are limited when it comes to preservation of a critical mass of functional β‐cells to sustainably maintain normoglycemia. In this review, we recapitulate recent evidence on the role of pancreatic N‐methyl‐D‐aspartate receptors (NMDARs) in β‐cell physiology, and summarize effects of morphinan‐based NMDAR antagonists that are beneficial for insulin secretion, glucose tolerance and islet cell survival. We further discuss NMDAR‐mediated molecular pathways relevant for neuronal cell survival, which may also be important for the preservation of β‐cell function and mass. Finally, we summarize the literature for evidence on the role of NMDARs in the development of diabetic long‐term complications, and highlight beneficial pharmacologic aspects of NMDAR antagonists in diabetic nephropathy, retinopathy as well as neuropathy.

Type 2 diabetes mellitus (T2DM) is a worldwide escalating health disorder resulting from insulin resistance and functional loss of insulin-producing beta cells that finally cause chronically elevated blood glucose concentrations. Here we review the role of ubiquitously expressed antioxidant protein DJ-1 in the pathogenesis of T2DM. In beta cells, DJ-1 protects against oxidative stress, endoplasmic reticulum stress, and streptozotocin- and cytokine-induced stress and preserves beta cell viability and insulin secretion. In skeletal muscle, DJ-1 controls energy metabolism and efficient fuel utilization, whereas in adipose tissue a role in adipogenesis and obesity-induced inflammation has been reported. This suggests that DJ-1 plays multiple roles in many cell types under metabolically challenging conditions as seen in obesity, insulin resistance, and T2DM.