Empagliflozin improves vascular insulin sensitivity and muscle perfusion in persons with type 2 diabetes.

Sodium glucose cotransporter 2 inhibitors (SGLT2is) improved major adverse cardiovascular events (MACE), heart failure, and renal outcomes in large trials; however, a thorough understanding of the vascular physiological changes contributing to these responses is lacking. We hypothesized that SGLT2i therapy would diminish vascular insulin resistance and improve hemodynamic function, which could improve clinical outcomes. To test this, we treated 11 persons with type 2 diabetes for 12 wk with 10 mg/day empagliflozin and measured vascular stiffness, endothelial function, peripheral and central arterial pressures, skeletal and cardiac muscle perfusion, and vascular biomarkers before and at 120 min of a euglycemic hyperinsulinemic clamp at weeks 0 and 12. We found that before empagliflozin treatment, insulin infusion lowered peripheral and central aortic systolic pressure (P < 0.05) and muscle microvascular blood flow (P < 0.01), but showed no effect on other vascular measures. Following empagliflozin, insulin infusion improved endothelial function (P = 0.02), lowered peripheral and aortic systolic (each P < 0.01), diastolic (each P < 0.05), mean arterial (each P < 0.01), and pulse pressures (each P < 0.02), altered endothelial biomarker expression, and decreased radial artery forward and backward pressure amplitude (each P = 0.02). Empagliflozin also improved insulin-mediated skeletal and cardiac muscle microvascular perfusion (each P < 0.05). We conclude that empagliflozin enhances insulin's vascular actions, which could contribute to the improved cardiorenal outcomes seen with SGLT2i therapy.NEW & NOTEWORTHY The physiological underpinnings of the cardiovascular benefits of SGLT2 inhibitors remain uncertain. We tested whether empagliflozin mitigates vascular insulin resistance in patients with type 2 diabetes. Aortic and peripheral systolic, diastolic, mean and pulse pressures, endothelial function, vascular stiffness, and heart and muscle microvascular perfusion were measured before and during an insulin infusion at baseline and after 12 wk of empagliflozin. After empagliflozin, vascular responses to insulin improved dramatically.

Impact of Free Fatty Acids on Vascular Insulin Responses Across the Arterial Tree: A Randomized Crossover Study.

CONTEXT: Vascular insulin resistance is commonly observed in obesity and diabetes; yet, insulin action across the vascular tree and the relationship between insulin responses at different vascular locations remains incompletely defined. OBJECTIVE: To elucidate the impact of elevated free fatty acids (FFAs) on insulin action across the arterial tree and define the relationship among insulin actions in the different arterial segments. METHODS: This randomized crossover study assigned healthy lean adults to 2 separate admissions with euglycemic insulin clamp superimposed for the final 120 minutes of 5-hour lipid or matched-volume saline infusion. Vascular measures including peripheral and central arterial blood pressure, brachial artery flow-mediated dilation (FMD), carotid femoral pulse wave velocity (cfPWV), augmentation index (AIx), pulse wave separation analysis, subendocardial viability ratio (SEVR), and skeletal and cardiac muscle microvascular perfusion were determined before and after insulin clamp. Insulin-mediated whole body glucose disposal was calculated. RESULTS: Insulin enhanced FMD, AIx, reflection magnitude, and cardiac and skeletal muscle microvascular perfusion. Elevation of plasma FFA concentrations to the levels seen in the postabsorptive state in people with insulin resistance suppressed SEVR, blunted insulin-induced increases in FMD and cardiac and skeletal muscle microvascular blood volume, and lowered insulin's ability to reduce AIx and reflection magnitude. In multivariate regression, insulin-mediated muscle microvascular perfusion was independently associated with insulin-mediated FMD and cfPWV. CONCLUSION: Clinically relevant elevation of plasma FFA concentrations induces pan-arterial insulin resistance, the vascular insulin resistance outcomes are interconnected, and insulin-mediated muscle microvascular perfusion associates with cardiovascular disease predictors. Our data provide biologic plausibility whereby a causative relationship between FFAs and cardiovascular disease could exist, and suggest that further attention to interventions that block FFA-mediated vascular insulin resistance may be warranted.

Liraglutide and Exercise Synergistically Attenuate Vascular Inflammation and Enhance Metabolic Insulin Action in Early Diet-Induced Obesity.

Inflammation-induced vascular insulin resistance is an early event in diet-induced obesity and contributes to metabolic insulin resistance. To examine whether exercise and glucagon-like peptide 1 (GLP-1) receptor agonism, alone or in combination, modulate vascular and metabolic insulin actions during obesity development, we performed a euglycemic insulin clamp in adult male rats after 2 weeks of high-fat diet feeding with either access to a running wheel (exercise), liraglutide, or both. Rats exhibited increased visceral adiposity and blunted microvascular and metabolic insulin responses. Exercise and liraglutide alone each improved muscle insulin sensitivity, but their combination fully restored insulin-mediated glucose disposal rates. The combined exercise and liraglutide intervention enhanced insulin-mediated muscle microvascular perfusion, reduced perivascular macrophage accumulation and superoxide production in the muscle, attenuated blood vessel inflammation, and improved endothelial function, along with increasing endothelial nucleus translocation of NRF2 and increasing endothelial AMPK phosphorylation. We conclude that exercise and liraglutide synergistically enhance the metabolic actions of insulin and reduce vascular oxidative stress and inflammation in the early stage of obesity development. Our data suggest that early combination use of exercise and GLP-1 receptor agonism might be an effective strategy in preventing vascular and metabolic insulin resistance and associated complications during the development of obesity. ARTICLE HIGHLIGHTS: Inflammation-induced vascular insulin resistance occurs early in diet-induced obesity and contributes to metabolic insulin resistance. We examined whether exercise and GLP-1 receptor agonism, alone or in combination, modulate vascular and metabolic insulin actions during obesity development. We found that exercise and liraglutide synergistically enhanced the metabolic actions of insulin and reduced perimicrovascular macrophage accumulation, vascular oxidative stress, and inflammation in the early stage of obesity development. Our data suggest that early combination use of exercise and a GLP-1 receptor agonist might be an effective strategy in preventing vascular and metabolic insulin resistance and associated complications during the development of obesity.

Metformin prevents endothelial oxidative stress and microvascular insulin resistance during obesity development in male rats.

Insulin increases muscle microvascular perfusion, which contributes to its metabolic action in muscle, but this action is impaired in obesity. Metformin improves endothelial function beyond its glucose lowering effects. We aim to examine whether metformin could prevent microvascular insulin resistance and endothelial dysfunction during the development of obesity. Adult male rats were fed a high-fat diet (HFD) with or without simultaneous metformin administration for either 2 or 4 wk. Insulin's metabolic and microvascular actions were determined using a combined euglycemic-hyperinsulinemic clamp and contrast-enhanced ultrasound approach. Compared with chow-fed controls, HFD feeding increased body adiposity without excess body weight gain, and this was associated with a marked decrease in insulin-mediated whole body glucose disposal and abolishment of insulin-induced muscle microvascular recruitment. Simultaneous administration of metformin fully rescued insulin-induced muscle microvascular recruitment as early as 2 wk and normalized insulin-mediated whole body glucose disposal at week 4. The divergent responses between insulin's microvascular and metabolic actions seen at week 2 were accompanied with reduced endothelial oxidative stress and vascular inflammation, and improved endothelial function and vascular insulin signaling in metformin-treated rats. In conclusions, metformin could prevent the development of microvascular insulin resistance and endothelial dysfunction by alleviating endothelial oxidative stress and vascular inflammation during obesity development.NEW & NOTEWORTHY Muscle microvascular insulin action contributes to insulin-mediated glucose use. Microvascular insulin resistance is an early event in diet-induced obesity and is associated with vascular inflammation. Metformin effectively reduces endothelial oxidative stress, improves endothelial function, and prevents microvascular insulin resistance during obesity development. These may contribute to metformin's salutary diabetes prevention and cardiovascular protective actions.

Acute hyperglycaemia enhances both vascular endothelial function and cardiac and skeletal muscle microvascular function in healthy humans.

KEY POINTS: Multiple clinical studies report that acute hyperglycaemia (induced by mixed meal or oral glucose) decreases arterial vascular function in healthy humans. Feeding, however, impacts autonomic output, blood pressure, and insulin and incretin secretion, which may themselves alter vascular function. No prior studies have examined the effect of acute hyperglycaemia on both macro- and microvascular function while controlling plasma insulin concentrations. Macrovascular and microvascular functional responses to euglycaemia and hyperglycaemia were compared. Octreotide was infused throughout both protocols to prevent endogenous insulin release. Acute hyperglycaemia (induced by intravenous glucose) enhanced brachial artery flow-mediated dilatation, increased skeletal muscle microvascular blood volume and flow, and expanded cardiac muscle microvascular blood volume. Compared to other published findings, the results suggest that vascular responses to acute hyperglycaemia differ based on the study population (i.e. normal weight vs. overweight/obese) and/or glucose delivery method (i.e. intravenous vs. oral glucose). ABSTRACT: High glucose concentrations acutely provoke endothelial cell oxidative stress and are suggested to trigger diabetes-related macro- and microvascular injury in humans. Multiple clinical studies report that acute hyperglycaemia (induced by mixed meal or oral glucose) decreases arterial vascular function in healthy humans. Feeding, however, impacts autonomic output, blood pressure, and insulin and incretin secretion, which may each independently alter vascular function and obscure the effect of acute hyperglycaemia per se. Surprisingly, no studies have examined the acute effects of intravenous glucose-induced hyperglycaemia on both macro- and microvascular function while controlling plasma insulin concentrations. In this randomized study of healthy young adults, we compared macrovascular (i.e. brachial artery flow-mediated dilatation, carotid-femoral pulse wave velocity and post-ischaemic brachial artery flow velocity) and microvascular (heart and skeletal muscle perfusion by contrast-enhanced ultrasound) functional responses to euglycaemia and hyperglycaemia. Octreotide was infused throughout both protocols to prevent endogenous insulin release. Acute intravenous glucose-induced hyperglycaemia enhanced brachial artery flow-mediated dilatation (P = 0.004), increased skeletal muscle microvascular blood volume and flow (P = 0.001), and expanded cardiac muscle microvascular blood volume (P = 0.014). No measure of vascular function changed during octreotide-maintained euglycaemia. Our findings suggest that unlike meal-provoked acute hyperglycaemia, 4 h of intravenous glucose-induced hyperglycaemia enhances brachial artery flow-mediated dilatation, provokes cardiac and skeletal muscle microvascular function, and does not impair aortic stiffness. Previous findings of acute large artery vascular dysfunction during oral glucose or mixed meal ingestion may be due to differences in study populations and meal-induced humoral or neural factors beyond hyperglycaemia per se. (ClinicalTrials.gov number NCT03520569.).

Insulin increases central aortic stiffness in response to hyperglycemia in healthy humans: A randomized four-arm study.

INTRODUCTION: Increasing arterial stiffness is a feature of vascular aging that is accelerated by conditions that enhance cardiovascular risk, including diabetes mellitus. Multiple studies demonstrate divergence of carotid-femoral pulse wave velocity and augmentation index in persons with diabetes mellitus, though mechanisms responsible for this are unclear. MATERIALS AND METHODS: We tested the effect of acutely and independently increasing plasma glucose, plasma insulin, or both on hemodynamic function and markers of arterial stiffness (including carotid-femoral pulse wave velocity, augmentation index, forward and backward wave reflection amplitude, and wave reflection magnitude) in a four-arm, randomized study of healthy young adults. RESULTS: Carotid-femoral pulse wave velocity increased only during hyperglycemic-hyperinsulinemia (+0.36 m/s; p = 0.032), while other markers of arterial stiffness did not change (all p > 0.05). Heart rate (+3.62 bpm; p = 0.009), mean arterial pressure (+4.14 mmHg; p = 0.033), central diastolic blood pressure (+4.16 mmHg; p = 0.038), and peripheral diastolic blood pressure (+4.09 mmHg; p = 0.044) also significantly increased during hyperglycemic-hyperinsulinemia. CONCLUSIONS: Hyperglycemic-hyperinsulinemia acutely increased cfPWV, heart rate, mean arterial pressure, and diastolic blood pressure in healthy humans, perhaps reflecting enhanced sympathetic tone. Whether repeated bouts of hyperglycemia with hyperinsulinemia contribute to chronically-enhanced arterial stiffness remains unknown.

Adaptive thermogenesis in brown adipose tissue involves activation of pannexin-1 channels.

OBJECTIVE: Brown adipose tissue (BAT) is specialized in thermogenesis. The conversion of energy into heat in brown adipocytes proceeds via stimulation of ß-adrenergic receptor (ßAR)-dependent signaling and activation of mitochondrial uncoupling protein 1 (UCP1). We have previously demonstrated a functional role for pannexin-1 (Panx1) channels in white adipose tissue; however, it is not known whether Panx1 channels play a role in the regulation of brown adipocyte function. Here, we tested the hypothesis that Panx1 channels are involved in brown adipocyte activation and thermogenesis. METHODS: In an immortalized brown pre-adipocytes cell line, Panx1 currents were measured using patch-clamp electrophysiology. Flow cytometry was used for assessment of dye uptake and luminescence assays for adenosine triphosphate (ATP) release, and cellular temperature measurement was performed using a ratiometric fluorescence thermometer. We used RNA interference and expression plasmids to manipulate expression of wild-type and mutant Panx1. We used previously described adipocyte-specific Panx1 knockout mice (Panx1Adip-/-) and generated brown adipocyte-specific Panx1 knockout mice (Panx1BAT-/-) to study pharmacological or cold-induced thermogenesis. Glucose uptake into brown adipose tissue was quantified by positron emission tomography (PET) analysis of 18F-fluorodeoxyglucose (18F-FDG) content. BAT temperature was measured using an implantable telemetric temperature probe. RESULTS: In brown adipocytes, Panx1 channel activity was induced either by apoptosis-dependent caspase activation or by ß3AR stimulation via a novel mechanism that involves Gßγ subunit binding to Panx1. Inactivation of Panx1 channels in cultured brown adipocytes resulted in inhibition of ß3AR-induced lipolysis, UCP-1 expression, and cellular thermogenesis. In mice, adiponectin-Cre-dependent genetic deletion of Panx1 in all adipose tissue depots resulted in defective ß3AR agonist- or cold-induced thermogenesis in BAT and suppressed beigeing of white adipose tissue. UCP1-Cre-dependent Panx1 deletion specifically in brown adipocytes reduced the capacity for adaptive thermogenesis without affecting beigeing of white adipose tissue and aggravated diet-induced obesity and insulin resistance. CONCLUSIONS: These data demonstrate that Gßγ-dependent Panx1 channel activation is involved in ß3AR-induced thermogenic regulation in brown adipocytes. Identification of Panx1 channels in BAT as novel thermo-regulatory elements downstream of ß3AR activation may have therapeutic implications.

Hyperglycemia does not Inhibit Insulin's Effects on Microvascular Perfusion in Healthy Humans: A Randomized Crossover Study.

Diabetes mellitus accelerates vascular disease through multiple biochemical pathways driven by hyperglycemia, with insulin resistance and/or hyperinsulinemia also contributing. Persons with diabetes mellitus experience premature large vessel and microvascular disease when compared to normoglycemic controls. Currently there is a paucity of clinical data identifying how acutely the vasculature responds to hyperglycemia and whether other physiologic factors (e.g., vasoactive hormones) contribute. To our knowledge, no prior studies have examined the dynamic effects of acute hyperglycemia on insulin-mediated actions on both micro- and macrovascular function in the same subjects. In this randomized crossover trial, healthy young adults underwent two infusion protocols designed to compare the effects of insulin infusion during euglycemia and hyperglycemia on micro- and macrovascular function. Both euglycemic- and hyperglycemic-hyperinsulinemia increased skeletal (but not cardiac) muscle microvascular blood volume (each p<0.02) and blood flow significantly (each p<0.04), and these increases did not differ between protocols. Hyperglycemic-hyperinsulinemia trended towards increased carotid-femoral pulse wave velocity (indicating increased aortic stiffness; p= 0.065 after Bonferroni adjustment), while euglycemic-hyperinsulinemia did not. There were no changes in post-ischemic flow velocity or brachial artery flow-mediated dilation during either protocol. Plasma endothelin-1 levels significantly decreased during both protocols (each p<0.02). In this study, acute hyperglycemia for 4 hours did not inhibit insulin's ability to increase skeletal muscle microvascular perfusion but did provoke a slight increase in aortic stiffness. Hyperglycemia also did not adversely affect myocardial microvascular perfusion or endothelial function or prevent the decline of endothelin-1 during insulin infusion.

Brain Endothelial Cells Regulate Glucagon-Like Peptide 1 Entry Into the Brain via a Receptor-Mediated Process.

Glucagon-like peptide 1 (GLP-1) in addition to regulating glucose-dependent insulin and glucagon secretion exerts anorexic and neuroprotective effects. While brain-derived GLP-1 may participate in these central actions, evidence suggests that peripherally derived GLP-1 plays an important role and GLP-1 analogs are known to cross the blood brain barrier. To define the role of brain microvascular endothelial cells in GLP-1 entry into the brain, we infused labeled GLP-1 or exendin-4 into rats intravenously and examined their appearance and protein kinase A activities in various brain regions. We also studied the role of endothelial cell GLP-1 receptor and its signaling in endothelial cell uptake and transport of GLP-1. Systemically infused labeled GLP-1 or exendin-4 appeared rapidly in various brain regions and this was associated with increased protein kinase A activity in these brain regions. Pretreatment with GLP-1 receptor antagonist reduced labeled GLP-1 or exendin-4 enrichment in the brain. Sub-diaphragmatic vagus nerve resection did not alter GLP-1-mediated increases in protein kinase A activity in the brain. Rat brain microvascular endothelial cells rapidly took up labeled GLP-1 and this was blunted by either GLP-1 receptor antagonism or protein kinase A inhibition but enhanced through adenylyl cyclase activation. Using an artificially assembled blood brain barrier consisting of endothelial and astrocyte layers, we found that labeled GLP-1 time-dependently crossed the barrier and the presence of GLP-1 receptor antagonist blunted this transit. We conclude that GLP-1 crosses the blood brain barrier through active trans-endothelial transport which requires GLP-1 receptor binding and activation.

Vasodilatory Actions of Glucagon-Like Peptide 1 Are Preserved in Skeletal and Cardiac Muscle Microvasculature but Not in Conduit Artery in Obese Humans With Vascular Insulin Resistance.

OBJECTIVE: Obesity is associated with microvascular insulin resistance, which is characterized by impaired insulin-mediated microvascular recruitment. Glucagon-like peptide 1 (GLP-1) recruits skeletal and cardiac muscle microvasculature, and this action is preserved in insulin-resistant rodents. We aimed to examine whether GLP-1 recruits microvasculature and improves the action of insulin in obese humans. RESEARCH DESIGN AND METHODS: Fifteen obese adults received intravenous infusion of either saline or GLP-1 (1.2 pmol/kg/min) for 150 min with or without a euglycemic insulin clamp (1 mU/kg/min) superimposed over the last 120 min. Skeletal and cardiac muscle microvascular blood volume (MBV), flow velocity and blood flow, brachial artery diameter and blood flow, and pulse wave velocity (PWV) were determined. RESULTS: Insulin failed to change MBV or flow in either skeletal or cardiac muscle, confirming the presence of microvascular insulin resistance. GLP-1 infusion alone increased MBV by ∼30% and ∼40% in skeletal and cardiac muscle, respectively, with no change in flow velocity, leading to a significant increase in microvascular blood flow in both skeletal and cardiac muscle. Superimposition of insulin to GLP-1 infusion did not further increase MBV or flow in either skeletal or cardiac muscle but raised the steady-state glucose infusion rate by ∼20%. Insulin, GLP-1, and GLP-1 + insulin infusion did not alter brachial artery diameter and blood flow or PWV. The vasodilatory actions of GLP-1 are preserved in both skeletal and cardiac muscle microvasculature, which may contribute to improving metabolic insulin responses and cardiovascular outcomes. CONCLUSIONS: In obese humans with microvascular insulin resistance, GLP-1's vasodilatory actions are preserved in both skeletal and cardiac muscle microvasculature, which may contribute to improving metabolic insulin responses and cardiovascular outcomes.

Inhibiting myeloperoxidase prevents onset and reverses established high-fat diet-induced microvascular insulin resistance.

A high-fat diet (HFD) can rapidly recruit neutrophils to insulin target tissues and within days induce microvascular insulin resistance (IR). Myeloperoxidase (MPO) is highly enriched in neutrophils, can inhibit nitric oxide-mediated vasorelaxation in vitro and is associated with increased cardiovascular disease risk. AZD5904 irreversibly inhibits MPO and in human clinical trials. MPO knockout, or chemical inhibition, blunts HFD-induced metabolic IR in mice. Whether MPO affects microvascular IR or muscle metabolic insulin sensitivity in vivo is unknown. We used contrast-enhanced ultrasound and the euglycemic insulin clamp to test whether inhibiting MPO could prevent the development or reverse established HFD-induced metabolic and/or microvascular IR in Sprague-Dawley rats. Two weeks of HFD feeding blocked insulin-mediated skeletal muscle capillary recruitment, inhibited glucose utilization, and insulin signaling to muscle. Continuous subcutaneous AZD5904 infusion during the 2 wk selectively blocked HFD's microvascular effect. Furthermore, AZD5904 infusion during the last 2 of 4 wk of HFD feeding restored microvascular insulin sensitivity but not metabolic IR. We conclude that inhibiting MPO selectively improves vascular IR. This selective microvascular effect may connote a therapeutic potential for MPO inhibition in the prevention of vascular disease/dysfunction seen in IR humans.

GLP-1 and Insulin Recruit Muscle Microvasculature and Dilate Conduit Artery Individually But Not Additively in Healthy Humans.

CONTEXT: Glucagon-like peptide-1 (GLP-1) and insulin increase muscle microvascular perfusion, thereby increasing tissue endothelial surface area and nutrient delivery. OBJECTIVE: To examine whether GLP-1 and insulin act additively on skeletal and cardiac microvasculature and conduit artery. DESIGN: Healthy adults underwent three study protocols in random order. SETTING: Clinical Research Unit at the University of Virginia. METHODS: Overnight-fasted participants received an intravenous infusion of GLP-1 (1.2 pmol/kg/min) or normal saline for 150 minutes with or without a 2-hour euglycemic insulin clamp (1 mU/kg/min) superimposed from 30 minutes onward. Skeletal and cardiac muscle microvascular blood volume (MBV), flow velocity, and flow; brachial artery diameter, flow velocity, and blood flow; and pulse wave velocity (PWV) were measured. RESULTS: GLP-1 significantly increased skeletal and cardiac muscle MBV and microvascular blood flow (MBF) after 30 minutes; these remained elevated at 150 minutes. Insulin also increased skeletal and cardiac muscle MBV and MBF. Addition of insulin to GLP-1 did not further increase skeletal and cardiac muscle MBV and MBF. GLP-1 and insulin increased brachial artery diameter and blood flow, but this effect was not additive. Neither GLP-1, insulin, nor GLP-1 and insulin altered PWV. Combined GLP-1 and insulin infusion did not result in higher whole-body glucose disposal. CONCLUSION: GLP-1 and insulin at physiological concentrations acutely increase skeletal and cardiac muscle microvascular perfusion and dilate conduit artery in healthy adults; these effects are not additive. Thus, GLP-1 and insulin may regulate skeletal and cardiac muscle endothelial surface area and nutrient delivery under physiological conditions.

Direct Activation of Angiotensin II Type 2 Receptors Enhances Muscle Microvascular Perfusion, Oxygenation, and Insulin Delivery in Male Rats.

Angiotensin II receptors regulate muscle microvascular recruitment and the delivery of nutrients, oxygen, and insulin to muscle. Although angiotensin type 1 receptor antagonism increases muscle microvascular perfusion and insulin action, angiotensin type 2 receptor blockade markedly restricts muscle microvascular blood volume and decreases muscle delivery of insulin. To examine the effects of direct type 2 receptor stimulation using Compound 21 (C21) on microvascular perfusion, insulin delivery and action, and tissue oxygenation in muscle, overnight-fasted adult male rats were infused with C21 systemically. C21 potently increased microvascular blood volume without altering microvascular flow velocity or blood pressure, resulting in a net increase in microvascular blood flow in muscle. This was associated with a substantial increase in muscle interstitial oxygen saturation and insulin delivery into the skeletal and cardiac muscle. These effects were neutralized by coinfusion of the type 2 receptor antagonist or nitric oxide synthase inhibitor. Superimposing C21 infusion on insulin infusion increased insulin-mediated whole body glucose disposal by 50%. C21 significantly relaxed the preconstricted distal saphenous artery ex vivo. We have concluded that direct type 2 receptor stimulation markedly increases muscle microvascular perfusion through nitric oxide biosynthesis and enhances insulin delivery and action in muscle. These findings provide a physiologic mechanistic insight into type 2 receptor modulation of insulin action and suggest that type 2 receptor agonists might have therapeutic potential in the management of diabetes and its associated complications.

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.

Long-term high-fat diet induces hippocampal microvascular insulin resistance and cognitive dysfunction.

Insulin action on hippocampus improves cognitive function, and obesity and type 2 diabetes are associated with decreased cognitive function. Cerebral microvasculature plays a critical role in maintaining cerebral vitality and function by supplying nutrients, oxygen, and hormones such as insulin to cerebral parenchyma, including hippocampus. In skeletal muscle, insulin actively regulates microvascular opening and closure, and this action is impaired in the insulin-resistant states. To examine insulin's action on hippocampal microvasculature and parenchyma and the impact of diet-induced obesity, we determined cognitive function and microvascular insulin responses, parenchyma insulin responses, and capillary density in the hippocampus in 2- and 8-mo-old rats on chow diet and 8-mo-old rats on a long-term high-fat diet (6 mo). Insulin infusion increased hippocampal microvascular perfusion in rats on chow diet by ~80-90%. High-fat diet feeding completely abolished insulin-mediated microvascular responses and protein kinase B phosphorylation but did not alter the capillary density in the hippocampus. This was associated with a significantly decreased cognitive function assessed using both the two-trial spontaneous alternation behavior test and the novel object recognition test. As the microvasculature provides the needed endothelial surface area for delivery of nutrients, oxygen, and insulin to hippocampal parenchyma, we conclude that hippocampal microvascular insulin resistance may play a critical role in the development of cognitive impairment seen in obesity and diabetes. Our results suggest that improvement in hippocampal microvascular insulin sensitivity might help improve or reverse cognitive function in the insulin-resistant states.

Pathways for insulin access to the brain: the role of the microvascular endothelial cell.

Insulin affects multiple important central nervous system (CNS) functions including memory and appetite, yet the pathway(s) by which insulin reaches brain interstitial fluid (bISF) has not been clarified. Recent studies demonstrate that to reach bISF, subarachnoid cerebrospinal fluid (CSF) courses through the Virchow-Robin space (VRS) which sheaths penetrating pial vessels down to the capillary level. Whether insulin predominantly enters the VRS and bISF by local transport through the blood-brain barrier, or by being secreted into the CSF by the choroid plexus, is unknown. We injected 125I-TyrA14-insulin or regular insulin intravenously and compared the rates of insulin reaching subarachnoid CSF with its plasma clearance by brain tissue samples (an index of microvascular endothelial cell binding/uptake/transport). The latter process was more than 40-fold more rapid. We then showed that selective insulin receptor blockade or 4 wk of high-fat feeding each inhibited microvascular brain 125I-TyrA14-insulin clearance. We further confirmed that 125I-TyrA14-insulin was internalized by brain microvascular endothelial cells, indicating that the in vivo tissue association reflected cellular transport, not simply microvascular tracer binding.

Liraglutide prevents microvascular insulin resistance and preserves muscle capillary density in high-fat diet-fed rats.

Muscle microvasculature critically regulates endothelial exchange surface area to facilitate transendothelial delivery of insulin, nutrients, and oxygen to myocytes. Insulin resistance blunts insulin-mediated microvascular recruitment and decreases muscle capillary density; both contribute to lower microvascular blood volume. Glucagon-like peptide 1 (GLP-1) and its analogs are able to dilate blood vessels and stimulate endothelial cell proliferation. In this study, we aim to determine the effects of sustained stimulation of the GLP-1 receptors on insulin-mediated capillary recruitment and metabolic insulin responses, small arterial endothelial function, and muscle capillary density. Rats were fed a high-fat diet (HFD) for 4 wk with or without simultaneous administration of liraglutide and subjected to a euglycemic hyperinsulinemic clamp for 120 min after an overnight fast. Insulin-mediated muscle microvascular recruitment and muscle oxygenation were determined before and during insulin infusion. Muscle capillary density was determined and distal saphenous artery used for determination of endothelial function and insulin-mediated vasodilation. HFD induced muscle microvascular insulin resistance and small arterial vessel endothelial dysfunction and decreased muscle capillary density. Simultaneous treatment of HFD-fed rats with liraglutide prevented all of these changes and improved insulin-stimulated glucose disposal. These were associated with a significantly increased AMPK phosphorylation and the expressions of VEGF and its receptors. We conclude that GLP-1 receptor agonists may exert their salutary glycemic effect via improving microvascular insulin sensitivity and muscle capillary density during the development of insulin resistance, and early use of GLP-1 receptor agonists may attenuate metabolic insulin resistance as well as prevent cardiovascular complications of diabetes.

Insulin Enhances Endothelial Function Throughout the Arterial Tree in Healthy But Not Metabolic Syndrome Subjects.

CONTEXT: Insulin reportedly impairs endothelial function in conduit arteries but improves it in resistance and microvascular arterioles in healthy humans. No studies have assessed endothelial function at three arterial levels in healthy or metabolic syndrome (METSYN) subjects. OBJECTIVE: The objective of the study was to compare endothelial responsiveness of conduit arteries, resistance, and microvascular arterioles to insulin in healthy and METSYN subjects. DESIGN: We assessed conduit, resistance, and microvascular arterial function in the postabsorptive and postprandial states and during euglycemic hyperinsulinemia (insulin clamp). SETTING: The study was conducted at a clinical research unit. PARTICIPANTS: Age-matched healthy and METSYN subjects participated in the study. INTERVENTIONS: We used brachial flow-mediated dilation, forearm postischemic flow velocity, and contrast-enhanced ultrasound to assess the conduit artery, resistance arteriole, and microvascular arteriolar endothelial function, respectively. We also assessed the conduit artery stiffness (pulse wave velocity and augmentation index) and measured the plasma concentrations of 92 cardiovascular disease biomarkers at baseline and after the clamp. RESULTS: Postabsorptive and postprandial endothelial function was similar in controls and METSYN in all tested vessels. METSYN subjects were metabolically insulin resistant (P < .005). In controls, but not METSYN subjects, during euglycemic hyperinsulinemia, endothelial function improved at each level of arterial vasculature (P < .05 or less for each). Conduit vessel stiffness (pulse wave velocity) was increased in the METSYN group. Twelve of 92 biomarkers differed at baseline (P < .001) and remained different at the end of the insulin clamp. CONCLUSIONS: We conclude that insulin enhances arterial endothelial function in health but not in METSYN, and this vascular insulin resistance may underlie its increased cardiovascular disease risk.

Inflammation-induced microvascular insulin resistance is an early event in diet-induced obesity.

Endothelial dysfunction and vascular insulin resistance usually coexist and chronic inflammation engenders both. In the present study, we investigate the temporal relationship between vascular insulin resistance and metabolic insulin resistance. We assessed insulin responses in all arterial segments, including aorta, distal saphenous artery and the microvasculature, as well as the metabolic insulin responses in muscle in rats fed on a high-fat diet (HFD) for various durations ranging from 3 days to 4 weeks with or without sodium salicylate treatment. Compared with controls, HFD feeding significantly blunted insulin-mediated Akt (protein kinase B) and eNOS [endothelial nitric oxide (NO) synthase] phosphorylation in aorta in 1 week, blunted vasodilatory response in small resistance vessel in 4 weeks and microvascular recruitment in as early as 3 days. Insulin-stimulated whole body glucose disposal did not begin to progressively decrease until after 1 week. Salicylate treatment fully inhibited vascular inflammation, prevented microvascular insulin resistance and significantly improved muscle metabolic responses to insulin. We conclude that microvascular insulin resistance is an early event in diet-induced obesity and insulin resistance and inflammation plays an essential role in this process. Our data suggest microvascular insulin resistance contributes to the development of metabolic insulin resistance in muscle and muscle microvasculature is a potential therapeutic target in the prevention and treatment of diabetes and its related complications.