High Doses of Exogenous Glucagon Stimulate Insulin Secretion and Reduce Insulin Clearance in Healthy Humans.

Glucagon is generally defined as a counterregulatory hormone with a primary role to raise blood glucose concentrations by increasing endogenous glucose production (EGP) in response to hypoglycemia. However, glucagon has long been known to stimulate insulin release, and recent preclinical findings have supported a paracrine action of glucagon directly on islet ß-cells that augments their secretion. In mice, the insulinotropic effect of glucagon is glucose dependent and not present during basal euglycemia. To test the hypothesis that the relative effects of glucagon on hepatic and islet function also vary with blood glucose, a group of healthy subjects received glucagon (100 ng/kg) during fasting glycemia or experimental hyperglycemia (∼150 mg/dL) on 2 separate days. During fasting euglycemia, administration of glucagon caused blood glucose to rise due to increased EGP, with a delayed increase of insulin secretion. When given during experimental hyperglycemia, glucagon caused a rapid, threefold increase in insulin secretion, as well as a more gradual increase in EGP. Under both conditions, insulin clearance was decreased in response to glucagon infusion. The insulinotropic action of glucagon, which is proportional to the degree of blood glucose elevation, suggests distinct physiologic roles in the fasting and prandial states.

Plantar osteochondral fragments in young Standardbreds are associated with minimal joint inflammation at the time of surgical removal.

BACKGROUND: Plantar osteochondral fragments (POF) are common but their effect on joint health of young Standardbreds in race training is largely unknown. OBJECTIVES: Evaluate the inflammatory effects of POF in metatarsophalangeal joints of young Standardbreds as a step towards developing evidence-based recommendations for surgical removal. STUDY DESIGN: Cohort study. METHODS: Forty-nine Standardbred horses (age 11-33 months) presented for surgical removal of POF from 56 metatarsophalangeal joints. Synovial tissue collected at arthroscopy was subjected to histopathology. IL-1ß, TNF-α, and PGE-2 were measured in synovial fluid using ELISA. Digital arthroscopy images were scored for inflammation. Racing performance data were retrieved from a public database. RESULTS: Median time in race training prior to surgery was 8 weeks (IQR 4-12; range 0-40). There was minimal evidence of synovial inflammation as assessed by histopathology (median total score 2/20, IQR 0-2, range 0-5) or arthroscopy (median average total score 2.67/15, IQR 1.79-4, range 0-8.83). IL-1ß was not detected in any sample. TNF-α (median 0 pg/mL, IQR 0-0) and PGE-2 (median 56.6 pg/mL, IRQ 40.5-99.8) were measured at low levels. Weeks in training prior to surgery was associated with the number of starts in the season after surgery (incidence rate ratio 1.02, 95% CI 1.00, 1.04, P = .03). MAIN LIMITATIONS: Small sample size from a single breed with a relatively short training time prior to surgery. CONCLUSIONS: There was minimal evidence of synovial inflammation in the metatarsophalangeal joints in this population of young Standardbred horses with POF. It is possible that POF may result in a different inflammatory response than other fragments because they are generally well-embedded in situ. These findings suggest that, in Standardbreds, race training can commence several weeks prior to surgical removal of POF with minimal detrimental effects on joint health, although further investigation of long-term effects of POF on joint health is warranted.

INTRODUCTION/CONTEXTE: Les fragments plantaires ostéochondraux (POF) sont communs mais leur effet au niveau sur la santé articulaire chez les jeunes Standardbreds en entraînement de course demeure inconnu. OBJECTIFS: Évaluer les effets inflammatoires des POF des articulations métatarsophalangiennes chez les jeunes Standardbreds dans le but d'ajouter à l'évidence disponible concernant les recommandations pour leur retrait chirurgical. TYPE D'ÉTUDE: Étude de cohorte descriptive clinique. MÉTHODES: Quarante-neuf chevaux Standardbreds (âgés 11-33 mois) ont été présentés pour retrait chirurgical de POF en provenance de 56 articulations métatarsophalangiennes. Un échantillon de membrane synoviale recueilli au moment de l'arthroscopie a été soumis en histopathologie. IL-1ß, TNF-α, and PGE-2 ont été mesurés dans le liquide synovial par ELISA. Les images digitales d'arthroscopie ont été évaluées pour la présence d'inflammation. Les données de performance en course ont été retrouvées via une base de données publique. RÉSULTATS: Le temps médian de retour à l'entraînement suivant la procédure chirurgicale était de 8 semaines (IQR 4-12; étendu 0-40). Peu d'inflammation synoviale a été détectée en histopathologie (score médian total 2/20, IQR 0-2, étendu 0-5) ou arthroscopie (score médian total 2.67/15, IQR 1.79-4, étendu 0-8.83). IL-1ß a été détectée dans aucun échantillon. TNF-α (médiane 0 pg/mL, IQR 0-0) et PGE-2 (médiane 56.6 pg/mL, IQR 40.5-99.8) ont été détectés en faible quantité. Le nombre de semaines à l'entraînement avant la procédure chirurgicale était associé au nombre de départs pour la saison suivant la chirurgie (IRR 1.02, P = 0.03). LIMITES PRINCIPALES: Petite taille d'échantillon provenant d'une seule race de chevaux ayant une période d'entraînement relativement courte avant la procédure chirurgicale. CONCLUSIONS: Il y a peu d'évidence d'inflammation synoviale dans les articulations métatarsophalangiennes chez cette population de jeunes chevaux Standardbreds ayant des POF. Il est possible que les POF entraînent une réponse inflammatoire différente des autres fragments puisqu'ils sont généralement bien attachés dans l'articulation. Ces résultats suggèrent que chez les Standardbreds, l'entraînement de course puisse commencer plusieurs semaines avant le retrait chirurgical des POF en ayant des effets délétères minimaux pour la santé articulaire. Ceci dit, davantage de recherche est nécessaire pour établir les effets à long-terme de ces POF sur la santé articulaire.

GLP-1 Receptor Blockade Reduces Stimulated Insulin Secretion in Fasted Subjects With Low Circulating GLP-1.

CONTEXT: Glucagon-like peptide 1 (GLP-1), an insulinotropic peptide released into the circulation from intestinal enteroendocrine cells, is considered a hormonal mediator of insulin secretion. However, the physiological actions of circulating GLP-1 have been questioned because of the short half-life of the active peptide. Moreover, there is mounting evidence for localized, intra-islet mediation of GLP-1 receptor (GLP-1r) signaling including a role for islet dipeptidyl-peptidase 4 (DPP4). OBJECTIVE: To determine whether GLP-1r signaling contributes to insulin secretion in the absence of enteral stimulation and increased plasma levels, and whether this is affected by DPP4. METHODS: Single-site study conducted at an academic medical center of 20 nondiabetic subjects and 13 subjects with type 2 diabetes. This was a crossover study in which subjects received either a DPP4 inhibitor (DPP4i; sitagliptin) or placebo on 2 separate days. On each day they received a bolus of intravenous (IV) arginine during sequential 60-minute infusions of the GLP-1r blocker exendin[9-39] (Ex-9) and saline. The main outcome measures were arginine-stimulated secretion of C-Peptide (C-PArg) and insulin (InsArg). RESULTS: Plasma GLP-1 remained at fasting levels throughout the experiments and IV arginine stimulated both α- and ß-cell secretion in all subjects. Ex-9 infusion reduced C-PArg in both the diabetic and nondiabetic groups by ~14% (P < .03 for both groups). Sitagliptin lowered baseline glycemia but did not affect the primary measures of insulin secretion. However, a significant interaction between sitagliptin and Ex-9 suggested more GLP-1r activation with DPP4i treatment in subjects with diabetes. CONCLUSION: GLP-1r activation contributes to ß-cell secretion in diabetic and nondiabetic people during α-cell activation, but in the absence of increased circulating GLP-1. These results are compatible with regulation of ß-cells by paracrine signals from α-cells. This process may be affected by DPP4 inhibition.

The effect of repeated freezing and thawing on the suture pull-out strength in equine arytenoid and cricoid cartilages.

OBJECTIVE: To assess the effect of repeated freezing and thawing on the suture pull-out strength in arytenoid and cricoid cartilages subjected to the laryngoplasty (LP) procedure. STUDY DESIGN: Ex vivo experimental study. SAMPLE POPULATION: Ten grossly normal equine cadaveric larynges. METHODS: Bilateral LP constructs were created using a standard LP technique. One hemilarynx was randomly allocated to the single freeze and thaw group and the other allocated to the repeated freeze and thaw (3 complete cycles) group. The suture ends of each LP construct were attached to a load frame and subjected to monotonic loading until construct failure. Mean load (N) and displacement (mm) at LP construct failure were compared between groups. RESULTS: All LP constructs failed by suture pull through the arytenoid cartilage. The mean load at failure was similar between groups (118.9 ± 25.5 N in the single freeze and thaw group and 113.4 ± 20.5 N in the repeated freeze and thaw group, P = .62). The mean displacement at failure was similar between groups (54.4 ± 15.1 mm in the single freeze and thaw group and 54.4 ± 15.4 mm in the repeated freeze and thaw group, P = .99). CONCLUSION: Repeated freezing and thawing did not affect the suture pullout strength of the arytenoid and cricoid cartilages. CLINICAL SIGNIFICANCE: Laryngeal specimens that have been subjected to repeated freezing and thawing can be utilized in the experimental evaluation of LP procedures because there is no alteration of the suture pull-out strength of the relevant cartilages.

Exploring Microbiome Functional Dynamics through Space and Time with Trait-Based Theory.

Microbiomes play essential roles in the health and function of animal and plant hosts and drive nutrient cycling across ecosystems. Integrating novel trait-based approaches with ecological theory can facilitate the prediction of microbial functional traits important for ecosystem functioning and health. In particular, the yield-acquisition-stress (Y-A-S) framework considers dominant microbial life history strategies across gradients of resource availability and stress. However, microbiomes are dynamic, and spatial and temporal shifts in taxonomic and trait composition can affect ecosystem functions. We posit that extending the Y-A-S framework to microbiomes during succession and across biogeographic gradients can lead to generalizable rules for how microbiomes and their functions respond to resources and stress across space, time, and diverse ecosystems. We demonstrate the potential of this framework by applying it to the microbiomes hosted by the carnivorous pitcher plant Sarracenia purpurea, which have clear successional trajectories and are distributed across a broad climatic gradient.

Evaluation of the airway mechanics of modified toggle laryngoplasty constructs using a vacuum chamber airflow model.

OBJECTIVE: To evaluate the airway mechanics of modified toggle LP constructs in an airflow chamber model and compare these to the airway mechanics of standard LP constructs. STUDY DESIGN: Ex-vivo experimental study. SAMPLE POPULATION: Fifty-one equine cadaveric larynges. METHODS: Bilateral LP constructs were performed using a modified toggle (n = 23) or a standard (n = 21) LP technique. Constructs were tested in an airflow model before and after cyclic loading which was designed to mimic postoperative swallowing. The cross-sectional area (CSA), peak translaryngeal airflow (L/s), and impedance (cmH2 0/L/s) were determined and compared between LP constructs before and after cycling. RESULTS: The mean CSA of the rima glottidis of the modified toggle LP constructs was 15.2 ± 2.6 cm2 before and 14.7 ± 2.6 cm2 after cyclic loading, and the mean CSA of the rima glottidis of the standard LP constructs was 16.4 ± 2.9 cm2 before and 15.7 ± 2.8 cm2 after cyclic loading. The modified toggle LP constructs had similar peak translaryngeal impedance before and after cyclic loading (p = .13); however, the standard LP constructs had higher peak translaryngeal impedance after cyclic loading (p = .02). CONCLUSION: The modified toggle and standard LP constructs had comparable airway mechanics in an ex-vivo model. CLINICAL SIGNIFICANCE: Further investigation is warranted to determine the extent to which the modified toggle LP technique restores normal airway function in horses with RLN.

A novel Streptococcus species causing clinical mastitis in a pregnant donkey.

An 8-y-old jenny was presented because of anorexia and mild depression. The jenny had weaned her colt 10 d before the admission. Upon arrival at the University of Illinois Veterinary Teaching Hospital, the heart rate was elevated, and the right udder was painful and swollen on palpation. Milk stripping of the affected side revealed purulent content; the contralateral udder had normal-appearing milk. Cytology of mammary gland secretions from the affected side revealed a large number of hypersegmented reactive neutrophils with phagocytized bacteria. Complete blood count, serum chemistry, and fibrinogen were within normal limits. A diagnosis of clinical mastitis was made, and the jenny was started on a 5-d course of broad-spectrum antibiotics, a non-steroidal anti-inflammatory, hydrotherapy, and milk stripping. Clinical signs reduced over time, and the cure was attained by 96 h post-admission. Aerobic culture and subsequent MALDI-TOF MS analysis identified a bacterium of the Streptococcus genus but not the species. Whole-genome analysis was performed, and 16S rDNA sequencing and analysis determined that our isolate 20-37394 clustered with 2 other Streptococcus strains (27284-01 and 28462). Single-nucleotide variations and phylogenetic tree analysis revealed that Streptococcus 20-37394 had 96.8% and 94.9% identities to Streptococcus strains 27284-01 and 28462, respectively; therefore, the bacteria isolated in our case was deemed as a new Streptococcus species.

Distance to native climatic niche margins explains establishment success of alien mammals.

One key hypothesis explaining the fate of exotic species introductions posits that the establishment of a self-sustaining population in the invaded range can only succeed within conditions matching the native climatic niche. Yet, this hypothesis remains untested for individual release events. Using a dataset of 979 introductions of 173 mammal species worldwide, we show that climate-matching to the realized native climatic niche, measured by a new Niche Margin Index (NMI), is a stronger predictor of establishment success than most previously tested life-history attributes and historical factors. Contrary to traditional climatic suitability metrics derived from species distribution models, NMI is based on niche margins and provides a measure of how distant a site is inside or, importantly, outside the niche. Besides many applications in research in ecology and evolution, NMI as a measure of native climatic niche-matching in risk assessments could improve efforts to prevent invasions and avoid costly eradications.

Reductive TCA cycle metabolism fuels glutamine- and glucose-stimulated insulin secretion.

Metabolic fuels regulate insulin secretion by generating second messengers that drive insulin granule exocytosis, but the biochemical pathways involved are incompletely understood. Here we demonstrate that stimulation of rat insulinoma cells or primary rat islets with glucose or glutamine + 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (Gln + BCH) induces reductive, "counter-clockwise" tricarboxylic acid (TCA) cycle flux of glutamine to citrate. Molecular or pharmacologic suppression of isocitrate dehydrogenase-2 (IDH2), which catalyzes reductive carboxylation of 2-ketoglutarate to isocitrate, results in impairment of glucose- and Gln + BCH-stimulated reductive TCA cycle flux, lowering of NADPH levels, and inhibition of insulin secretion. Pharmacologic suppression of IDH2 also inhibits insulin secretion in living mice. Reductive TCA cycle flux has been proposed as a mechanism for generation of biomass in cancer cells. Here we demonstrate that reductive TCA cycle flux also produces stimulus-secretion coupling factors that regulate insulin secretion, including in non-dividing cells.

Discordance between GLP-1R gene and protein expression in mouse pancreatic islet cells.

The insulinotropic actions of glucagon-like peptide 1 receptor (GLP-1R) in ß-cells have made it a useful target to manage type 2 diabetes. Metabolic stress reduces ß-cell sensitivity to GLP-1, yet the underlying mechanisms are unknown. We hypothesized that Glp1r expression is heterogeneous among ß-cells and that metabolic stress decreases the number of GLP-1R-positive ß-cells. Here, analyses of publicly available single-cell RNA-Seq sequencing (scRNASeq) data from mouse and human ß-cells indicated that significant populations of ß-cells do not express the Glp1r gene, supporting heterogeneous GLP-1R expression. To check these results, we used complementary approaches employing FACS coupled with quantitative RT-PCR, a validated GLP-1R antibody, and flow cytometry to quantify GLP-1R promoter activity, gene expression, and protein expression in mouse α-, ß-, and δ-cells. Experiments with Glp1r reporter mice and a validated GLP-1R antibody indicated that >90% of the ß-cells are GLP-1R positive, contradicting the findings with the scRNASeq data. α-cells did not express Glp1r mRNA and δ-cells expressed Glp1r mRNA but not protein. We also examined the expression patterns of GLP-1R in mouse models of metabolic stress. Multiparous female mice had significantly decreased ß-cell Glp1r expression, but no reduction in GLP-1R protein levels or GLP-1R-mediated insulin secretion. These findings suggest caution in interpreting the results of scRNASeq for low-abundance transcripts such as the incretin receptors and indicate that GLP-1R is widely expressed in ß-cells, absent in α-cells, and expressed at the mRNA, but not protein, level in δ-cells.

The effects of temperature and dispersal on species diversity in natural microbial metacommunities.

Dispersal is key for maintaining biodiversity at local- and regional scales in metacommunities. However, little is known about the combined effects of dispersal and climate change on biodiversity. Theory predicts that alpha-diversity is maximized at intermediate dispersal rates, resulting in a hump-shaped diversity-dispersal relationship. This relationship is predicted to flatten when competition increases. We anticipate that this same flattening will occur with increased temperature because, in the rising part of the temperature performance curve, interspecific competition is predicted to increase. We explored this question using aquatic communities of Sarracenia purpurea from early- and late-successional stages, in which we simulated four levels of dispersal and four temperature scenarios. With increased dispersal, the hump shape was observed consistently in late successional communities, but only in higher temperature treatments in early succession. Increased temperature did not flatten the hump-shape relationship, but decreased the level of alpha- and gamma-diversity. Interestingly, higher temperatures negatively impacted small-bodied species. These metacommunity-level extinctions likely relaxed interspecific competition, which could explain the absence of flattening of the diversity-dispersal relationship. Our findings suggest that climate change will cause extinctions both at local- and global- scales and emphasize the importance of intermediate levels of dispersal as an insurance for local diversity.

Intraislet Ghrelin Signaling Does Not Regulate Insulin Secretion From Adult Mice.

Exogenous ghrelin reduces glucose-stimulated insulin secretion and endogenous ghrelin protects against hypoglycemia during starvation. Islet ε-cells produce ghrelin and δ-cells express growth hormone secretagogue receptor (GHSR), suggesting the possibility of a paracrine mechanism for islet ghrelin to reach high local concentrations and affect insulin secretion. GHSR has high constitutive activity and may act independently of ghrelin. The objective in this study was to determine whether an intraislet ghrelin-GHSR axis modulates insulin secretion and glucose metabolism using mouse models lacking ghrelin (Ghrl-/- ) or GHSR (Ghsr-/- ). Ghsr-/- and Ghsr+/+ mice had comparable islet ghrelin concentrations. Exogenous ghrelin decreased insulin secretion in perifused isolated islets in a GHSR-dependent manner. Islets isolated from Ghrl-/- or Ghsr-/- mice did not differ from controls in glucose-, alanine-, or GLP-1-stimulated insulin secretion during perifusion. Consistent with this finding, Ghrl-/- and Ghsr-/- male mice studied after either 6 or 16 h of fasting had blood glucose concentrations comparable with those of controls following intraperitoneal glucose, or insulin tolerance tests, or after mixed nutrient meals. Collectively, our data provide strong evidence against a paracrine ghrelin-GHSR axis mediating insulin secretion or glucose tolerance in lean, chow-fed adult mice.

Ghrelin regulation of glucose metabolism.

Ghrelin and its receptor, the growth hormone secretagogue receptor 1a (GHSR1a), are implicated in the regulation of glucose metabolism via direct actions in the pancreatic islet, as well as peripheral insulin-sensitive tissues and the brain. Although many studies have explored the role of ghrelin in glucose tolerance and insulin secretion, a complete mechanistic understanding remains to be clarified. This review highlights the local expression and function of ghrelin and GHSR1a in pancreatic islets and how this axis may modulate insulin secretion from pancreatic ß-cells. Additionally, we discuss the effect of ghrelin on in vivo glucose metabolism in rodents and humans, as well as the metabolic circumstances under which the action of ghrelin may predominate.

Partial arytenoidectomy in 14 standing horses (2013-2017).

OBJECTIVE: To report our experience with partial arytenoidectomy in sedated standing horses. STUDY DESIGN: Retrospective study. ANIMALS: Fourteen client-owned adult horses. METHODS: The medical records (2013-2017) of horses treated with unilateral partial arytenoidectomy while standing and sedated were reviewed. Demographics, endoscopic findings, previous treatments, and outcome after surgery were investigated and recorded. RESULTS: Thirteen horses had unilateral left-sided recurrent laryngeal neuropathy (RLN) and 1 horse had bilateral RLN. Five horses had a previous failed prosthetic laryngoplasty. Left-sided partial arytenoidectomy without mucosal closure was successfully completed in all horses under sedation and local anesthesia. Report of long-term outcome was obtained via telephone conversations for 12 horses, of which 9 also had an endoscopic reevaluation performed; 3 horses had granulomas at the surgical site, of which 2 eventually required a permanent tracheostomy. Nine horses returned to athletic use without respiratory noise, 2 horses returned to athletic use with noise during exercise that was reduced compared with preoperative levels, and 1 horse continued to be used as a broodmare. CONCLUSION: Partial arytenoidectomy in standing horses was achieved with adequate sedation and local anesthesia. CLINICAL SIGNIFICANCE: Partial arytenoidectomy on standing sedated horses could be considered as an alternative to eliminate the risks associated with general anesthesia.

Increased Temperature Disrupts the Biodiversity-Ecosystem Functioning Relationship.

Gaining knowledge of how ecosystems provide essential services to humans is of primary importance, especially with the current threat of climate change. Yet little is known about how increased temperature will impact the biodiversity-ecosystem functioning (BEF) relationship. We tackled this subject theoretically and experimentally. We developed a BEF theory based on mechanistic population dynamic models, which allows the inclusion of the effect of temperature. Using experimentally established relationships between attack rate and temperature, the model predicts that temperature increase will intensify competition, and consequently the BEF relationship will flatten or even become negative. We conducted a laboratory experiment with natural microbial microcosms, and the results were in agreement with the model predictions. The experimental results also revealed that an increase in both temperature average and variation had a more intense effect than an increase in temperature average alone. Our results indicate that under climate change, high diversity may not guarantee high ecosystem functioning.

Gene Signature of the Human Pancreatic ε Cell.

The ghrelin-producing ε cell represents the fifth endocrine cell type in human pancreatic islets. The abundance of ε cells in adult pancreas is extremely low, which has hampered the investigation on the molecular pathways regulating the development and the function of this cell type. In this study, we explored the molecular features defining the function of pancreatic ε cells isolated from adult nondiabetic donors using single-cell RNA sequencing technology. We focus on transcription factors, cell surface receptors, and genes involved in metabolic pathways that contribute to regulation of cellular function. Furthermore, the genes that separate ε cells from the other islet endocrine cell types are presented. This study expands prior knowledge about the genes important for ε cell functioning during development and provides a resource to interrogate the transcriptome of this rare human islet cell type.

Interaction of GLP-1 and Ghrelin on Glucose Tolerance in Healthy Humans.

Emerging evidence supports the importance of ghrelin to defend against starvation-induced hypoglycemia. This effect may be mediated by inhibition of glucose-stimulated insulin secretion as well as reduced insulin sensitivity. However, administration of ghrelin during meal consumption also stimulates the release of glucagon-like peptide 1 (GLP-1), an incretin important in nutrient disposition. The objective of this study was to evaluate the interaction between ghrelin and GLP-1 on parameters of glucose tolerance following a mixed-nutrient meal. Fifteen healthy men and women completed the study. Each consumed a standard meal on four separate occasions with a superimposed infusion of 1) saline, 2) ghrelin, 3) the GLP-1 receptor antagonist exendin(9-39) (Ex9), or 4) combined ghrelin and Ex9. Similar to previous studies, infusion of ghrelin caused glucose intolerance, whereas Ex9 had a minimal effect. However, combined ghrelin and Ex9 resulted in greater postprandial glycemia than either alone, and this effect was associated with impaired ß-cell function and decreased glucose clearance. These findings suggest that in the fed state, stimulation of GLP-1 mitigates some of the effect of ghrelin on glucose tolerance. This novel interaction between gastrointestinal hormones suggests a system that balances insulin secretion and glucose disposal in the fed and fasting states.

Insulin transport into the brain.

While there is a growing consensus that insulin has diverse and important regulatory actions on the brain, seemingly important aspects of brain insulin physiology are poorly understood. Examples include: what is the insulin concentration within brain interstitial fluid under normal physiologic conditions; whether insulin is made in the brain and acts locally; does insulin from the circulation cross the blood-brain barrier or the blood-CSF barrier in a fashion that facilitates its signaling in brain; is insulin degraded within the brain; do privileged areas with a "leaky" blood-brain barrier serve as signaling nodes for transmitting peripheral insulin signaling; does insulin action in the brain include regulation of amyloid peptides; whether insulin resistance is a cause or consequence of processes involved in cognitive decline. Heretofore, nearly all of the studies examining brain insulin physiology have employed techniques and methodologies that do not appreciate the complex fluid compartmentation and flow throughout the brain. This review attempts to provide a status report on historical and recent work that begins to address some of these issues. It is undertaken in an effort to suggest a framework for studies going forward. Such studies are inevitably influenced by recent physiologic and genetic studies of insulin accessing and acting in brain, discoveries relating to brain fluid dynamics and the interplay of cerebrospinal fluid, brain interstitial fluid, and brain lymphatics, and advances in clinical neuroimaging that underscore the dynamic role of neurovascular coupling.

Unravelling the regulation of insulin transport across the brain endothelial cell.

AIMS/HYPOTHESIS: For circulating insulin to act on the brain it must cross the blood-brain barrier (BBB). Remarkably little is known about how circulating insulin crosses the BBB's highly restrictive brain endothelial cells (BECs). Therefore, we examined potential mechanisms regulating BEC insulin uptake, signalling and degradation during BEC transcytosis, and how transport is affected by a high-fat diet (HFD) and by astrocyte activity. METHODS: 125I-TyrA14-insulin uptake and transcytosis, and the effects of insulin receptor (IR) blockade, inhibition of insulin signalling, astrocyte stimulation and an HFD were tested using purified isolated BECs (iBECs) in monoculture and co-cultured with astrocytes. RESULTS: At physiological insulin concentrations, the IR, not the IGF-1 receptor, facilitated BEC insulin uptake, which required lipid raft-mediated endocytosis, but did not require insulin action on phosphoinositide-3-kinase (PI3K) or mitogen-activated protein kinase kinase (MEK). Feeding rats an HFD for 4 weeks decreased iBEC insulin uptake and increased NF-κB binding activity without affecting insulin PI3K signalling, IR expression or content, or insulin degrading enzyme expression. Using an in vitro BBB (co-culture of iBECs and astrocytes), we found insulin was not degraded during transcytosis, and that stimulating astrocytes with L-glutamate increased transcytosis, while inhibiting nitric oxide synthase decreased insulin transcytosis. CONCLUSIONS/INTERPRETATION: Insulin crosses the BBB intact via an IR-specific, vesicle-mediated transport process in the BECs. HFD feeding, nitric oxide inhibition and astrocyte stimulation can regulate BEC insulin uptake and transcytosis.