The Role of G Protein-Coupled Receptors and Receptor Kinases in Pancreatic ß-Cell Function and Diabetes.

Type 2 diabetes (T2D) mellitus has emerged as a major global health concern that has accelerated in recent years due to poor diet and lifestyle. Afflicted individuals have high blood glucose levels that stem from the inability of the pancreas to make enough insulin to meet demand. Although medication can help to maintain normal blood glucose levels in individuals with chronic disease, many of these medicines are outdated, have severe side effects, and often become less efficacious over time, necessitating the need for insulin therapy. G protein-coupled receptors (GPCRs) regulate many physiologic processes, including blood glucose levels. In pancreatic ß cells, GPCRs regulate ß-cell growth, apoptosis, and insulin secretion, which are all critical in maintaining sufficient ß-cell mass and insulin output to ensure euglycemia. In recent years, new insights into the signaling of incretin receptors and other GPCRs have underscored the potential of these receptors as desirable targets in the treatment of diabetes. The signaling of these receptors is modulated by GPCR kinases (GRKs) that phosphorylate agonist-activated GPCRs, marking the receptor for arrestin binding and internalization. Interestingly, genome-wide association studies using diabetic patient cohorts link the GRKs and arrestins with T2D. Moreover, recent reports show that GRKs and arrestins expressed in the ß cell serve a critical role in the regulation of ß-cell function, including ß-cell growth and insulin secretion in both GPCR-dependent and -independent pathways. In this review, we describe recent insights into GPCR signaling and the importance of GRK function in modulating ß-cell physiology. SIGNIFICANCE STATEMENT: Pancreatic ß cells contain a diverse array of G protein-coupled receptors (GPCRs) that have been shown to improve ß-cell function and survival, yet only a handful have been successfully targeted in the treatment of diabetes. This review discusses recent advances in our understanding of ß-cell GPCR pharmacology and regulation by GPCR kinases while also highlighting the necessity of investigating islet-enriched GPCRs that have largely been unexplored to unveil novel treatment strategies.

Diet-induced glial insulin resistance impairs the clearance of neuronal debris in Drosophila brain.

Obesity significantly increases the risk of developing neurodegenerative disorders, yet the precise mechanisms underlying this connection remain unclear. Defects in glial phagocytic function are a key feature of neurodegenerative disorders, as delayed clearance of neuronal debris can result in inflammation, neuronal death, and poor nervous system recovery. Mounting evidence indicates that glial function can affect feeding behavior, weight, and systemic metabolism, suggesting that diet may play a role in regulating glial function. While it is appreciated that glial cells are insulin sensitive, whether obesogenic diets can induce glial insulin resistance and thereby impair glial phagocytic function remains unknown. Here, using a Drosophila model, we show that a chronic obesogenic diet induces glial insulin resistance and impairs the clearance of neuronal debris. Specifically, obesogenic diet exposure down-regulates the basal and injury-induced expression of the glia-associated phagocytic receptor, Draper. Constitutive activation of systemic insulin release from Drosophila insulin-producing cells (IPCs) mimics the effect of diet-induced obesity on glial Draper expression. In contrast, genetically attenuating systemic insulin release from the IPCs rescues diet-induced glial insulin resistance and Draper expression. Significantly, we show that genetically stimulating phosphoinositide 3-kinase (Pi3k), a downstream effector of insulin receptor (IR) signaling, rescues high-sugar diet (HSD)-induced glial defects. Hence, we establish that obesogenic diets impair glial phagocytic function and delays the clearance of neuronal debris.

Mitochondrial fusion and altered beta-oxidation drive muscle wasting in a Drosophila cachexia model.

Cancer cachexia is a tumour-induced wasting syndrome, characterised by extreme loss of skeletal muscle. Defective mitochondria can contribute to muscle wasting; however, the underlying mechanisms remain unclear. Using a Drosophila larval model of cancer cachexia, we observed enlarged and dysfunctional muscle mitochondria. Morphological changes were accompanied by upregulation of beta-oxidation proteins and depletion of muscle glycogen and lipid stores. Muscle lipid stores were also decreased in Colon-26 adenocarcinoma mouse muscle samples, and expression of the beta-oxidation gene CPT1A was negatively associated with muscle quality in cachectic patients. Mechanistically, mitochondrial defects result from reduced muscle insulin signalling, downstream of tumour-secreted insulin growth factor binding protein (IGFBP) homologue ImpL2. Strikingly, muscle-specific inhibition of Forkhead box O (FOXO), mitochondrial fusion, or beta-oxidation in tumour-bearing animals preserved muscle integrity. Finally, dietary supplementation with nicotinamide or lipids, improved muscle health in tumour-bearing animals. Overall, our work demonstrates that muscle FOXO, mitochondria dynamics/beta-oxidation and lipid utilisation are key regulators of muscle wasting in cancer cachexia.

A STIM dependent dopamine-neuropeptide axis maintains the larval drive to feed and grow in Drosophila.

Appropriate nutritional intake is essential for organismal survival. In holometabolous insects such as Drosophila melanogaster, the quality and quantity of food ingested as larvae determines adult size and fecundity. Here we have identified a subset of dopaminergic neurons (THD') that maintain the larval motivation to feed. Dopamine release from these neurons requires the ER Ca2+ sensor STIM. Larvae with loss of STIM stop feeding and growing, whereas expression of STIM in THD' neurons rescues feeding, growth and viability of STIM null mutants to a significant extent. Moreover STIM is essential for maintaining excitability and release of dopamine from THD' neurons. Optogenetic stimulation of THD' neurons activated neuropeptidergic cells, including median neuro secretory cells that secrete insulin-like peptides. Loss of STIM in THD' cells alters the developmental profile of specific insulin-like peptides including ilp3. Loss of ilp3 partially rescues STIM null mutants and inappropriate expression of ilp3 in larvae affects development and growth. In summary we have identified a novel STIM-dependent function of dopamine neurons that modulates developmental changes in larval feeding behaviour and growth.

AMPK activates the Nrf2-Keap1 pathway to govern dendrite pruning via the insulin pathway in Drosophila.

During Drosophila metamorphosis, the ddaC dendritic arborisation sensory neurons selectively prune their larval dendrites in response to steroid hormone ecdysone signalling. The Nrf2-Keap1 pathway acts downstream of ecdysone signalling to promote proteasomal degradation and thereby dendrite pruning. However, how the Nrf2-Keap1 pathway is activated remains largely unclear. Here, we demonstrate that the metabolic regulator AMP-activated protein kinase (AMPK) plays a cell-autonomous role in dendrite pruning. Importantly, AMPK is required for Mical and Headcase expression and for activation of the Nrf2-Keap1 pathway. We reveal that AMPK promotes the Nrf2-Keap1 pathway and dendrite pruning partly via inhibition of the insulin pathway. Moreover, the AMPK-insulin pathway is required for ecdysone signalling to activate the Nrf2-Keap1 pathway during dendrite pruning. Overall, this study reveals an important mechanism whereby ecdysone signalling activates the Nrf2-Keap1 pathway via the AMPK-insulin pathway to promote dendrite pruning, and further suggests that during the nonfeeding prepupal stage metabolic alterations lead to activation of the Nrf2-Keap1 pathway and dendrite pruning.

Activation of mucosal insulin receptor exacerbates intestinal inflammation by promoting tissue resident memory T cells differentiation through EZH2.

BACKGROUND: Inflammatory Bowel Diseases (IBD), an autoimmune disease characterised by abnormal intestinal immunity, are related to vital morbidity around the world. However, therapeutic agents for IBD have not achieved desired benefit. Exploring new therapeutic targets for IBD, especially based on its abnormally intestinal immunity, could alleviate the flare-up and worsening of IBD. Tissue resident memory T cells (TRM) are core of multiple autoimmune diseases, including IBD. However, the mechanism of TRM differentiation remains to be investigated. METHODS: The alterations in mRNA and lncRNA profile of intestinal intraepithelial lymphocytes (IELs), the largest component of intestinal TRM, were analyzed in DSS-induced chronic colitis. Based on it, we examined the function of rectal insulin instillation in a dextran sodium sulfate (DSS) induced chronic colitis. Furthermore, we investigated the downstream-target of the insulin pathway-EZH2 and the crucial role of EZH2 in intestinal tissue resident memory T cell differentiation by utilizing EZH2fl/flCD4cre mice. RESULTS: Insulin receptor (INSR) expression was found to be significantly reduced. Activation of mucosal insulin pathway by rectal insulin instillation exacerbated colitis by disrupting IELs subgroups and up-regulating TNF-ɑ and IL-17 expression. Rectal insulin instillation promoted EZH2 expression and EZH2 inhibition alleviated chronic colitis. EZH2fl/flCD4cre mice restored the normal IEL subgroups and suppressed TNF-ɑ and IL-17 expression, exhibiting alleviated colitis. IELs from EZH2fl/flCD4cre mice exhibit significant changes in TRM related phenotype. CD4+TRM was significantly increased in chronic colitis and decreased in EZH2fl/flCD4cre mice. CONCLUSION: Insulin receptor of intestinal mucosal T-cells could promote intestinal TRM differentiation via EZH2. Our discoveries suggest that therapies targeting colonic INSR and EZH2 could be potential treatment for IBD based on its regulatory effects on TRM. Insulin receptor inhibitors rather than insulin should be applied during colitis-active phase. In addition, EZH2 shows to be a downstream signal of the insulin pathway and EZH2 inhibitor could alleviating intestinal inflammation. However, the critical role of EZH2 in TRM differentiation restricts the anti-tumor effects of EZH2 inhibitor in vivo.

Elucidating the Morphogenic and Signaling Roles of Defined Growth Factors Controlling Human Endothelial Cell Lumen Formation Versus Sprouting Behavior.

Five growth factors [ie, insulin, fibroblast growth factor-2 (FGF-2), stem cell factor, IL-3, and stromal-derived factor 1α] in combination are necessary for human endothelial cells (ECs) to undergo tube morphogenesis, a process requiring both lumen formation and sprouting behavior. This study investigated why these factors are required by subdividing the factors into 4 separate groups: insulin-only, insulin and FGF-2, no FGF-2 (all factors but without FGF-2), and all factors. The study found that the insulin-only condition failed to support EC morphogenesis or survival, the insulin and FGF-2 condition supported primarily EC lumen formation, and the no FGF-2 condition supported EC sprouting behavior. By comparison, the all-factors condition more strongly stimulated both EC lumen formation and sprouting behavior, and signaling analysis revealed prolonged stimulation of multiple promorphogenic signals coupled with inhibition of proregressive signals. Pharmacologic inhibition of Jak kinases more selectively blocked EC sprouting behavior, whereas inhibition of Raf, phosphatidylinositol 3-kinase, and Akt kinases showed selective blockade of lumen formation. Inhibition of Src family kinases and Notch led to increased sprouting coupled to decreased lumen formation, whereas inhibition of Pak, Mek, and mammalian target of rapamycin kinases blocked both sprouting and lumen formation. These findings reveal novel downstream biological and signaling activities of defined factors that are required for the assembly of human EC-lined capillary tube networks.

Trophectoderm biopsy of blastocysts following IVF and embryo culture increases epigenetic dysregulation in a mouse model.

STUDY QUESTION: Does trophectoderm biopsy (TEBx) of blastocysts for preimplantation genetic testing in the clinic affect normal placental and embryo development and offspring metabolic outcomes in a mouse model? SUMMARY ANSWER: TEBx impacts placental and embryonic health during early development, with some alterations resolving and others worsening later in development and triggering metabolic changes in adult offspring. WHAT IS KNOWN ALREADY: Previous studies have not assessed the epigenetic and morphological impacts of TEBx either in human populations or in animal models. STUDY DESIGN, SIZE, DURATION: We employed a mouse model to identify the effects of TEBx during IVF. Three groups were assessed: naturally conceived (Naturals), IVF, and IVF + TEBx, at two developmental timepoints: embryonic day (E)12.5 (n = 40/Naturals, n = 36/IVF, and n = 36/IVF + TEBx) and E18.5 (n = 42/Naturals, n = 30/IVF, and n = 35/IVF + TEBx). Additionally, to mimic clinical practice, we assessed a fourth group: IVF + TEBx + Vitrification (Vit) at E12.5 (n = 29) that combines TEBx and vitrification. To assess the effect of TEBx in offspring health, we characterized a 12-week-old cohort (n = 24/Naturals, n = 25/IVF and n = 25/IVF + TEBx). PARTICIPANTS/MATERIALS, SETTING, METHODS: Our mouse model used CF-1 females as egg donors and SJL/B6 males as sperm donors. IVF, TEBx, and vitrification were performed using standardized methods. Placenta morphology was evaluated by hematoxylin-eosin staining, in situ hybridization using Tpbpa as a junctional zone marker and immunohistochemistry using CD34 fetal endothelial cell markers. For molecular analysis of placentas and embryos, DNA methylation was analyzed using pyrosequencing, luminometric methylation assay, and chip array technology. Expression patterns were ascertained by RNA sequencing. Triglycerides, total cholesterol, high-, low-, and very low-density lipoprotein, insulin, and glucose were determined in the 12-week-old cohort using commercially available kits. MAIN RESULTS AND THE ROLE OF CHANCE: We observed that at E12.5, IVF + TEBx had a worse outcome in terms of changes in DNA methylation and differential gene expression in placentas and whole embryos compared with IVF alone and compared with Naturals. These changes were reflected in alterations in placental morphology and blood vessel density. At E18.5, early molecular changes in fetuses were maintained or exacerbated. With respect to placentas, the molecular and morphological changes, although different compared to Naturals, were equivalent to the IVF group, except for changes in blood vessel density, which persisted. Of note is that most differences were sex specific. We conclude that TEBx has more detrimental effects in mid-gestation placental and embryonic tissues, with alterations in embryonic tissues persisting or worsening in later developmental stages compared to IVF alone, and the addition of vitrification after TEBx results in more pronounced and potentially detrimental epigenetic effects: these changes are significantly different compared to Naturals. Finally, we observed that 12-week IVF + TEBx offspring, regardless of sex, showed higher glucose, insulin, triglycerides, lower total cholesterol, and lower high-density lipoprotein compared to IVF and Naturals, with only males having higher body weight compared to IVF and Naturals. Our findings in a mouse model additionally support the need for more studies to assess the impact of new procedures in ART to ensure healthy pregnancies and offspring outcomes. LARGE SCALE DATA: Data reported in this work have been deposited in the NCBI Gene Expression Omnibus under accession number GSE225318. LIMITATIONS, REASONS FOR CAUTION: This study was performed using a mouse model that mimics many clinical IVF procedures and outcomes observed in humans, where studies on early embryos are not possible. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights the importance of assaying new procedures used in ART to assess their impact on placenta and embryo development, and offspring metabolic outcomes. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by a National Centers for Translational Research in Reproduction and Infertility grant P50 HD068157-06A1 (M.S.B., C.C., M.M.), Ruth L. Kirschstein National Service Award Individual Postdoctoral Fellowship F32 HD107914 (E.A.R.-C.) and F32 HD089623 (L.A.V.), and National Institutes of Health Training program in Cell and Molecular Biology T32 GM007229 (C.N.H.). No conflict of interest.

Blockade of endogenous insulin receptor signaling in the nucleus tractus solitarius potentiates exercise pressor reflex function in healthy male rats.

Insulin not only regulates glucose and/or lipid metabolism but also modulates brain neural activity. The nucleus tractus solitarius (NTS) is a key central integration site for sensory input from working skeletal muscle and arterial baroreceptors during exercise. Stimulation of the skeletal muscle exercise pressor reflex (EPR), the responses of which are buffered by the arterial baroreflex, leads to compensatory increases in arterial pressure to supply blood to working muscle. Evidence suggests that insulin signaling decreases neuronal excitability in the brain, thus antagonizing insulin receptors (IRs) may increase neuronal excitability. However, the impact of brain insulin signaling on the EPR remains fully undetermined. We hypothesized that antagonism of NTS IRs increases EPR function in normal healthy rodents. In decerebrate rats, stimulation of the EPR via electrically induced muscle contractions increased peak mean arterial pressure (MAP) responses 30 min following NTS microinjections of an IR antagonist (GSK1838705, 100 µM; Pre: Δ16 ± 10 mmHg vs. 30 min: Δ23 ± 13 mmHg, n = 11, p = .004), a finding absent in sino-aortic baroreceptor denervated rats. Intrathecal injections of GSK1838705 did not influence peak MAP responses to mechano- or chemoreflex stimulation of the hindlimb muscle. Immunofluorescence triple overlap analysis following repetitive EPR stimulation increased c-Fos overlap with EPR-sensitive nuclei and IR-positive cells relative to sham operation (p < .001). The results suggest that IR blockade in the NTS potentiates the MAP response to EPR stimulation. In addition, insulin signaling in the NTS may buffer EPR stimulated increases in blood pressure via baroreflex-mediated mechanisms during exercise.

Targeting phospholipid remodeling pathway improves insulin resistance in diabetic mouse models.

Previous studies have revealed that membrane phospholipid composition controlled by lysophosphatidylcholine acyltransferase 3 (LPCAT3) is involved in the development of insulin resistance in type 2 diabetes. In this study, we aimed to investigate the therapeutic potential of targeting Lpcat3 in the treatment of insulin resistance in diabetic mouse models. Lpcat3 expression was suppressed in the whole body by antisense oligonucleotides (ASO) injection or in the liver by adeno-associated virus (AAV)-encoded Cre in high-fat diet (HFD)-induced and genetic ob/ob type 2 diabetic mouse models. Glucose tolerance test (GTT), insulin tolerance test (ITT), fasting blood glucose, and insulin levels were used to assess insulin sensitivity. Lipid levels in the liver and serum were measured. The expression of genes involved in de novo lipogenesis was analyzed by real-time RT-PCR. Metabolic rates were measured by indirect calorimetry using the Comprehensive Lab Animal Monitoring System (CLAMS). Our data demonstrate that acute knockout of hepatic Lpcat3 by AAV-Cre improves both hyperglycemia and hypertriglyceridemia in HFD-fed mice. Similarly, whole-body ablation of Lpcat3 by ASO administration improves obesity and insulin resistance in both HFD-fed and ob/ob mice. These findings demonstrate that targeting LPCAT3 could be a novel therapy for insulin resistance.

M1-derived extracellular vesicles polarize recipient macrophages into M2-like macrophages and alter skeletal muscle homeostasis in a hyper-glucose environment.

BACKGROUND: Macrophages release not only cytokines but also extracellular vesicles (EVs). which are small membrane-derived nanovesicles with virus-like properties transferring cellular material between cells. Until now, the consequences of macrophage plasticity on the release and the composition of EVs have been poorly explored. In this study, we determined the impact of high-glucose (HG) concentrations on macrophage metabolism, and characterized their derived-EV subpopulations. Finally, we determined whether HG-treated macrophage-derived EVs participate in immune responses and in metabolic alterations of skeletal muscle cells. METHODS: THP1-macrophages were treated with 15mM (MG15) or 30mM (MG30) glucose. Then, M1/M2 canonical markers, pro- and anti-inflammatory cytokines, activities of proteins involved in glycolysis or oxidative phosphorylation were evaluated. Macrophage-derived EVs were characterized by TEM, NTA, MRSP, and 1H-Nuclear magnetic resonance spectroscopy for lipid composition. Macrophages or C2C12 muscle cells were used as recipients of MG15 and MG30-derived EVs. The lipid profiles of recipient cells were determined, as well as proteins and mRNA levels of relevant genes for macrophage polarization or muscle metabolism. RESULTS: Untreated macrophages released small and large EVs (sEVs, lEVs) with different lipid distributions. Proportionally to the glucose concentration, glycolysis was induced in macrophages, associated to mitochondrial dysfunction, triacylglycerol and cholesterol accumulation. In addition, MG15 and MG30 macrophages had increased level of CD86 and increase release of pro-inflammatory cytokines. HG also affected macrophage sphingolipid and phospholipid compositions. The differences in the lipid profiles between sEVs and lEVs were abolished and reflected the lipid alterations in MG15 and MG30 macrophages. Interestingly, MG15 and MG30 macrophages EVs induced the expression of CD163, Il-10 and increased the contents of triacylglycerol and cholesterol in recipient macrophages. MG15 lEVs and sEVs induced insulin-induced AKT hyper-phosphorylation and accumulation of triacylglycerol in myotubes, a state observed in pre-diabetes. Conversely, MG30 lEVs and sEVs induced insulin-resistance in myotubes. CONCLUSIONS: As inflammation involves first M1 macrophages, then the activation of M2 macrophages to resolve inflammation, this study demonstrates that the dialog between macrophages through the EV route is an intrinsic part of the inflammatory response. In a hyperglycemic context, EV macrophages could participate in the development of muscle insulin-resistance and chronic inflammation.

Low Density Lipoprotein Receptor-related Protein 2 Expression and Function in Cultured Astrocytes and Microglia.

Activation of glial cells, astrocytes and microglia, has been observed in neurodegenerative diseases including Alzheimer's disease (AD). Amyloid ß (Aß), which is aggregated and the aggregation is detected as characteristic pathology in AD brain, is known to be produced by neurons and to activate glial cells. Clearance of Aß from the brain via active transport system is important to prevent the accumulation and aggregation. Low density lipoprotein receptor-related protein 2 (LRP2/megalin) is an Aß transporter. However, expression and contribution of LRP2 in astrocytes and microglia remain to be clarified. In the present study, we examined the expression of LRP2 and its roles in cultured astrocytes prepared from rat embryonic brain cortex and mouse microglial cell line BV-2. Both cultured rat astrocytes and BV-2 cells expressed LRP2 mRNA detected by RT-PCR. When lipopolysaccharide (LPS) or all-trans retinoic acid (ATRA) were added to BV-2 cells, LRP2 mRNA expression and uptake of microbeads, Aß and insulin were increased. On the other hand, LPS decreased LRP2 expression and uptake of Aß and insulin in cultured astrocytes. Knockdown of LRP2 using siRNA attenuated the LPS- or ATRA-increased uptake of microbeads, Aß and insulin in BV-2 cells. These results suggest that LRP2 was expressed in both astrocytes and microglia and might be involved in endocytosis activities. Adequate control of LRP2 expression and function in astrocytes and microglia might regulate Aß and insulin levels in brain and would be a potential target in AD pathology.

Effects of mirabegron on brown adipose tissue and metabolism in humans: A systematic review and meta-analysis.

BACKGROUND: Brown adipose tissue (BAT) has emerged as a potential therapeutic target for metabolic disorders due to its thermogenic and anti-obesity properties. ß3-adrenergic receptor (ß3-AR) agonists have also gained attention as potential agents for BAT activation and metabolic regulation. Mirabegron, a selective ß3-AR-agonist used clinically for overactive bladder syndrome, has been explored for its utility in metabolic disorders. However, the controversy surrounding the ability of mirabegron to activate BAT to accelerate metabolism requires further investigation. The aim of this systematic review is to characterize comprehensively the impact of mirabegron on human BAT and its metabolism. METHODS: We searched PubMed Central, Web of Science, Embase, and Cochrane Library databases for relevant papers published from the date of database inception to March 2023 for systematic reviews and meta-analyses. We extracted data on primary outcome indicators such as BAT volume, BAT activity, body temperature, and resting energy expenditure (REE), as well as secondary outcome indicators such as heart rate (HR), diastolic blood pressure (DBP), systolic blood pressure (SBP), non-esterified fatty acids (NEFA), blood glucose, and blood insulin from relevant studies. For studies that did not provide suitable data for meta-analysis, we used narrative data synthesis. For studies that provided suitable data for meta-analysis, we conducted meta-analysis using RevMan 5.4 software. RESULTS: We reviewed 10 papers and included 6 in our meta-analysis. Our findings revealed no significant changes in BAT volume (p = 0.72) or blood glucose (p = 0.52) with mirabegron when compared to the placebo or pre-dose population. However, patients showed significant increases in BAT activity (p < 0.01), blood NEFA (p < 0.01), body temperature (p < 0.01), REE (p < 0.01), HR (p < 0.01), DBP (p < 0.01), SBP (p = 0.25), and blood insulin (p < 0.01). CONCLUSION: Through our meta-analysis of 6 papers, we found that mirabegron has the potential to increase human BAT activity, REE, NEFA content, body temperature, HR, blood pressure, and blood insulin content. These effects may lead to reductions in blood glucose levels in obese/overweight and diabetic patients. Additionally, the activation of BAT by mirabegron could represent a novel approach for treating obesity, diabetes, and cardiovascular disease. TRIAL REGISTRATION NUMBER AND DATE: CRD42023413446, 04/11/2023.

Transgenic human C-reactive protein affects oxidative stress but not inflammation biomarkers in the aorta of spontaneously hypertensive rats.

BACKGROUND: C-reactive protein (CRP) is an acute inflammatory protein detected in obese patients with metabolic syndrome. Moreover, increased CRP levels have been linked with atherosclerotic disease, congestive heart failure, and ischemic heart disease, suggesting that it is not only a biomarker but also plays an active role in the pathophysiology of cardiovascular diseases. Since endothelial dysfunction plays an essential role in various cardiovascular pathologies and is characterized by increased expression of cell adhesion molecules and inflammatory markers, we aimed to detect specific markers of endothelial dysfunction, inflammation, and oxidative stress in spontaneously hypertensive rats (SHR) expressing human CRP. This model is genetically predisposed to the development of the metabolic syndrome. METHODS: Transgenic SHR male rats (SHR-CRP) and non-transgenic SHR (SHR) at the age of 8 months were used. Metabolic profile (including serum and tissue triglyceride (TAG), serum insulin concentrations, insulin-stimulated incorporation of glucose, and serum non-esterified fatty acids (NEFA) levels) was measured. In addition, human serum CRP, MCP-1 (monocyte chemoattractant protein-1), and adiponectin were evaluated by means of ELISA, histological analysis was used to study morphological changes in the aorta, and western blot analysis of aortic tissue was performed to detect expression of endothelial, inflammatory, and oxidative stress markers. RESULTS: The presence of human CRP was associated with significantly decreased insulin-stimulated glycogenesis in skeletal muscle, increased muscle and hepatic accumulation of TAG and decreased plasmatic cGMP concentrations, reduced adiponectin levels, and increased monocyte chemoattractant protein-1 (MCP-1) levels in the blood, suggesting pro-inflammatory and presence of multiple features of metabolic syndrome in SHR-CRP animals. Histological analysis of aortic sections did not reveal any visible morphological changes in animals from both SHR and SHR-CRP rats. Western blot analysis of the expression of proteins related to the proper function of endothelium demonstrated significant differences in the expression of p-eNOS/eNOS in the aorta, although endoglin (ENG) protein expression remained unaffected. In addition, the presence of human CRP in SHR in this study did not affect the expression of inflammatory markers, namely p-NFkB, P-selectin, and COX2 in the aorta. On the other hand, biomarkers related to oxidative stress, such as HO-1 and SOD3, were significantly changed, indicating the induction of oxidative stress. CONCLUSIONS: Our findings demonstrate that CRP alone cannot fully induce the expression of endothelial dysfunction biomarkers, suggesting other risk factors of cardiovascular disorders are necessary to be involved to induce endothelial dysfunction with CRP.

Elevated insulin growth factor-1 in dentate gyrus induces cognitive deficits in pre-term newborns.

Prematurely born infants are deprived of maternal hormones and cared for in the stressful environment of Neonatal Intensive Care Units (NICUs). They suffer from long-lasting deficits in learning and memory. Here, we show that prematurity and associated neonatal stress disrupt dentate gyrus (DG) development and induce long-term cognitive deficits and that these effects are mediated by insulin growth factor-1 (IGF1). Nonmaternal care of premature rabbits increased the number of granule cells and interneurons and reduced neurogenesis, suggesting accelerated premature maturation of DG. However, the density of glutamatergic synapses, mature dendritic spines, and synaptic transmission were reduced in preterm kits compared with full-term controls, indicating that premature synaptic maturation was abnormal. These findings were consistent with cognitive deficits observed in premature rabbits and appeared to be driven by transcriptomic changes in the granule cells. Preterm kits displayed reduced weight, elevated serum cortisol and growth hormone, and higher IGF1 expression in the liver and DG relative to full-term controls. Importantly, blocking IGF-1 receptor in premature kits restored cognitive deficits, increased the density of glutamatergic puncta, and rescued NR2B and PSD95 levels in the DG. Hence, IGF1 inhibition alleviates prematurity-induced cognitive dysfunction and synaptic changes in the DG through modulation of NR2B and PSD95. The study identifies a novel strategy to potentially rescue DG maldevelopment and cognitive dysfunction in premature infants under stress in NICUs.

Evaluation of a compounding phospholipid base for the percutaneous absorption of high molecular weight drugs using the Franz finite dose model.

BACKGROUND: Permeation-enhancing compounding bases are aimed to facilitate the penetration of the active pharmaceutical ingredients (APIs) across the skin barrier. OBJECTIVES: The purpose of this study was to evaluate the percutaneous absorption of radiolabeled human insulin (14 C-isototpe) when incorporated in a proprietary phospholipid base designed to deliver APIs with high molecular weights (HMW). The aim was not to claim the transdermal delivery of insulin with potential therapeutic applications in diabetes but, instead, to evaluate the ability of the compounding phospholipid base to deliver HMW drugs. METHODS: The percutaneous absorption of 14 C-insulin was determined using human torso skin and the Franz skin finite dose model. Two topical test formulations were prepared for in vitro evaluation: insulin 1% in phospholipid base (standard) and insulin 1% in phospholipid base HMW. The rate of percutaneous absorption (mean flux) and the distribution of 14 C-insulin through the skin were evaluated for both topical test formulations. A two-way ANOVA was used to determine statistical differences. RESULTS: The 14 C-insulin was distributed into the stratum corneum, epidermis and dermis. Mean flux values showed a rapid penetration upon application and the maximum flux was achieved at 30 min, followed by a slow decline. Subsequently, a slower decline was observed for the topical test formulation including the phospholipid base HMW. CONCLUSION: The phospholipid base HMW facilitates the percutaneous absorption of HMW drugs across human cadaver skin and, therefore, it may potentially be a useful option for compounding pharmacists and practitioners when considering the skin for the percutaneous delivery of large drugs.

Genetic and Physiological Effects of Insulin-Like Growth Factor-1 (IGF-1) on Human Urate Homeostasis.

SIGNIFICANCE STATEMENT: Hyperinsulinemia induces hyperuricemia by activating net renal urate reabsorption in the renal proximal tubule. The basolateral reabsorptive urate transporter GLUT9a appears to be the dominant target for insulin. By contrast, IGF-1 infusion reduces serum urate (SU), through mechanisms unknown. Genetic variants of IGF1R associated with reduced SU have increased IGF-1R expression and interact with genes encoding the GLUT9 and ABCG2 urate transporters, in a sex-specific fashion, which controls the SU level. Activation of IGF-1/IGF-1R signaling in Xenopus oocytes modestly activates GLUT9a and inhibits insulin's stimulatory effect on the transporter, which also activates multiple secretory urate transporters-ABCG2, ABCC4, OAT1, and OAT3. The results collectively suggest that IGF-1 reduces SU by activating secretory urate transporters and inhibiting insulin's action on GLUT9a. BACKGROUND: Metabolic syndrome and hyperinsulinemia are associated with hyperuricemia. Insulin infusion in healthy volunteers elevates serum urate (SU) by activating net urate reabsorption in the renal proximal tubule, whereas IGF-1 infusion reduces SU by mechanisms unknown. Variation within the IGF1R gene also affects SU levels. METHODS: Colocalization analyses of a SU genome-wide association studies signal at IGF1R and expression quantitative trait loci signals in cis using COLOC2, RT-PCR, Western blotting, and urate transport assays in transfected HEK 293T cells and in Xenopus laevis oocytes. RESULTS: Genetic association at IGF1R with SU is stronger in women and is mediated by control of IGF1R expression. Inheritance of the urate-lowering homozygous genotype at the SLC2A9 locus is associated with a differential effect of IGF1R genotype between men and women. IGF-1, through IGF-1R, stimulated urate uptake in human renal proximal tubule epithelial cells and transfected HEK 293T cells, through activation of IRS1, PI3/Akt, MEK/ERK, and p38 MAPK; urate uptake was inhibited in the presence of uricosuric drugs, specific inhibitors of protein tyrosine kinase, PI3 kinase (PI3K), ERK, and p38 MAPK. In X. laevis oocytes expressing ten individual urate transporters, IGF-1 through endogenous IGF-1R stimulated urate transport mediated by GLUT9, OAT1, OAT3, ABCG2, and ABCC4 and inhibited insulin's stimulatory action on GLUT9a and OAT3. IGF-1 significantly activated Akt and ERK. Specific inhibitors of PI3K, ERK, and PKC significantly affected IGF-1 stimulation of urate transport in oocytes. CONCLUSIONS: The combined results of infusion, genetics, and transport experiments suggest that IGF-1 reduces SU by activating urate secretory transporters and inhibiting insulin's action.

Expression of Insl3 Protein in Adult Danio rerio.

Insulin-like peptide 3 (INSL3) is a biomarker for Leydig cells in the testes of vertebrates, and it is principally involved in spermatogenesis through specific binding with the RXFP2 receptor. This study reports the insl3 gene transcript and the Insl3 prepropeptide expression in both non-reproductive and reproductive tissues of Danio rerio. An immunohistochemistry analysis shows that the hormone is present at a low level in the Leydig cells and germ cells at all stages of Danio rerio testis differentiation. Considering that the insl3 gene is transcribed in Leydig cells, our results highlight an autocrine and paracrine function of this hormone in the Danio rerio testis, adding new information on the Insl3 mode of action in reproduction. We also show that Insl3 and Rxfp2 belonging to Danio rerio and other vertebrate species share most of the amino acid residues involved in the ligand-receptor interaction and activation, suggesting a conserved mechanism of action during vertebrate evolution.

Regulated translocation of neutral sphingomyelinase-2 to the plasma membrane drives insulin resistance in steatotic hepatocytes.

Obesity-associated diabetes is linked to the accumulation of ceramide in various organs, including the liver. The exact mechanisms by which ceramide contributes to diabetic pathology are unclear, but one proposed scenario is that ceramide accumulation may inhibit insulin signaling pathways. It is unknown however whether the excess ceramide is generated proximal to the insulin receptor, that is, at the plasma membrane (PM), where it could affect the insulin signaling pathway directly, or the onset of insulin resistance is due to ceramide-induced mitochondrial dysfunction and/or lipotoxicity. Using hepatic cell lines and primary cultures, gain- and loss- of function approach, and state-of-the art lipid imaging, this study shows that PM-associated neutral sphingomyelinase 2 (nSMase2) regulates ceramide homeostasis in fat-loaded hepatocytes and drives the onset of insulin resistance. Our results provide evidence of a regulated translocation of nSMase2 to the PM which leads to local generation of ceramide and insulin resistance in cells treated with palmitic acid (PAL), a type of fat commonly found in diabetogenic diets. Oleic acid, which also causes accumulation of lipid droplets, does not induce nSMase2 translocation and insulin resistance. Experiments using the acyl-biotin exchange method to quantify protein palmitoylation show that cellular PAL abundance regulates the rate of nSMase2 palmitoylation. Furthermore, while inhibition of nSMase2 with GW4869 prevents PAL-induced insulin resistance, the overexpression of wild type nSMase2 but not palmitoylation-defective mutant protein potentiates the suppressive effect of PAL on insulin signaling. Overall, this study identifies nSMase2 as a novel component of the mechanism of insulin resistance onset in fat-loaded hepatocytes, that is, cell-autonomous and driven by PAL.

Delta-6 desaturase (Fads2) deficiency alters triacylglycerol/fatty acid cycling in murine white adipose tissue.

The Δ-6 desaturase (D6D) enzyme is not only critical for the synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from α-linolenic acid (ALA), but recent evidence suggests that it also plays a role in adipocyte lipid metabolism and body weight; however, the mechanisms remain largely unexplored. The goal of this study was to investigate if a D6D deficiency would inhibit triacylglycerol storage and alter lipolytic and lipogenic pathways in mouse white adipose tissue (WAT) depots due to a disruption in EPA and DHA production. Male C57BL/6J D6D knockout (KO) and wild-type (WT) mice were fed either a 7% w/w lard or flax (ALA rich) diet for 21 weeks. Energy expenditure, physical activity, and substrate utilization were measured with metabolic caging. Inguinal and epididymal WAT depots were analyzed for changes in tissue weight, fatty acid composition, adipocyte size, and markers of lipogenesis, lipolysis, and insulin signaling. KO mice had lower body weight, higher serum nonesterified fatty acids, smaller WAT depots, and reduced adipocyte size compared to WT mice without altered food intake, energy expenditure, or physical activity, regardless of the diet. Markers of lipogenesis and lipolysis were more highly expressed in KO mice compared to WT mice in both depots, regardless of the diet. These changes were concomitant with lower basal insulin signaling in WAT. Collectively, a D6D deficiency alters triacylglycerol/fatty acid cycling in WAT by promoting lipolysis and reducing fatty acid re-esterification, which may be partially attributed to a reduction in WAT insulin signaling.