ABSTRACT
Endothelial insulin resistance represents a causal factor in the pathogenesis of type 2 diabetes (T2D) and vascular disease, thus the need to identify molecular mechanisms underlying defects in endothelial insulin signaling. We previously have shown that a disintegrin and metalloproteinase-17 (ADAM17) is increased while insulin receptor α-subunit (IRα) is decreased in the vasculature of patients with T2D, leading to impaired insulin-induced vasodilation. We have also demonstrated that ADAM17 sheddase activity targets IRα; however, the mechanisms driving endothelial ADAM17 activity in T2D are largely unknown. Herein, we report that externalization of phosphatidylserine (PS) to the outer leaflet of the plasma membrane causes ADAM17-mediated shedding of IRα and blunting of insulin signaling in endothelial cells. Furthermore, we demonstrate that endothelial PS externalization is mediated by the phospholipid scramblase anoctamin-6 (ANO6) and that this process can be stimulated by neuraminidase, a soluble enzyme that cleaves sialic acid residues. Of note, we demonstrate that men and women with T2D display increased levels of neuraminidase activity in plasma, relative to age-matched healthy individuals, and this occurs in conjunction with increased ADAM17 activity and impaired leg blood flow responses to endogenous insulin. Collectively, this work reveals the neuraminidase-ANO6-ADAM17 axis as a novel potential target for restoring endothelial insulin sensitivity in T2D.NEW & NOTEWORTHY This work provides the first evidence that neuraminidase, an enzyme increased in the circulation of men and women with type 2 diabetes (T2D), promotes anoctamin-6 (ANO6)-dependent externalization of phosphatidylserine in endothelial cells, which in turn leads to activation of a disintegrin and metalloproteinase-17 (ADAM17) and consequent shedding of the insulin receptor-α from the cell surface. Hence, this work supports that consideration should be given to the neuraminidase-ANO6-ADAM17 axis as a novel potential target for restoring endothelial insulin sensitivity in T2D.
Subject(s)
Diabetes Mellitus, Type 2 , Insulin Resistance , Male , Humans , Female , Endothelial Cells/metabolism , Receptor, Insulin/metabolism , Phosphatidylserines/metabolism , Neuraminidase/metabolism , Insulin/metabolism , Disintegrins , ADAM17 Protein/metabolism , Anoctamins/metabolismABSTRACT
Neuraminidases cleave sialic acids from glycocalyx structures and plasma neuraminidase activity is elevated in type 2 diabetes (T2D). Therefore, we hypothesize circulating neuraminidase degrades the endothelial glycocalyx and diminishes flow-mediated dilation (FMD), whereas its inhibition restores shear mechanosensation and endothelial function in T2D settings. We found that compared with controls, subjects with T2D have higher plasma neuraminidase activity, reduced plasma nitrite concentrations, and diminished FMD. Ex vivo and in vivo neuraminidase exposure diminished FMD and reduced endothelial glycocalyx presence in mouse arteries. In cultured endothelial cells, neuraminidase reduced glycocalyx coverage. Inhalation of the neuraminidase inhibitor, zanamivir, reduced plasma neuraminidase activity, enhanced endothelial glycocalyx length, and improved FMD in diabetic mice. In humans, a single-arm trial (NCT04867707) of zanamivir inhalation did not reduce plasma neuraminidase activity, improved glycocalyx length, or enhanced FMD. Although zanamivir plasma concentrations in mice reached 225.8 ± 22.0 ng/mL, in humans were only 40.0 ± 7.2 ng/mL. These results highlight the potential of neuraminidase inhibition for ameliorating endothelial dysfunction in T2D and suggest the current Food and Drug Administration-approved inhaled dosage of zanamivir is insufficient to achieve desired outcomes in humans.NEW & NOTEWORTHY This work identifies neuraminidase as a key mediator of endothelial dysfunction in type 2 diabetes that may serve as a biomarker for impaired endothelial function and predictive of development and progression of cardiovascular pathologies associated with type 2 diabetes (T2D). Data show that intervention with the neuraminidase inhibitor zanamivir at effective plasma concentrations may represent a novel pharmacological strategy for restoring the glycocalyx and ameliorating endothelial dysfunction.
Subject(s)
Diabetes Mellitus, Experimental , Diabetes Mellitus, Type 2 , Vascular Diseases , Mice , Humans , Animals , Zanamivir/pharmacology , Neuraminidase/chemistry , Neuraminidase/pharmacology , Endothelial Cells , Diabetes Mellitus, Type 2/drug therapy , Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacologyABSTRACT
Adults presenting with transverse maxillomandibular discrepancies in Class III skeletal patterns, asymmetries and anterior open bite can be challenging to treatment plan and manage. Therefore, they often necessitate a multidisciplinary approach, with surgical input from the oral and maxillofacial team. It can be difficult to assess how much the maxilla needs to be expanded after surgery in these patients, as the upper and lower teeth cannot be brought into occlusion. We aim to discuss the use of a device we have come to call the 'Powerbite', which is used intra-orally to assess the expansion of the maxilla after surgically assisted rapid palatal expansion (SARPE) and establish when expansion is sufficient to accommodate the mandibular arch. This device is not novel, last described in 1986 in the Journal of Orthodontics.
Subject(s)
Maxilla , Palatal Expansion Technique , Adult , Humans , Maxilla/surgeryABSTRACT
Aging of the vasculature is characterized by endothelial dysfunction and arterial stiffening, two key events in the pathogenesis of cardiovascular disease (CVD). Treatment with sodium glucose transporter 2 (SGLT2) inhibitors is now known to decrease cardiovascular morbidity and mortality in type 2 diabetes. However, whether SGLT2 inhibition attenuates vascular aging is unknown. We first confirmed in a cohort of adult subjects that aging is associated with impaired endothelial function and increased arterial stiffness and that these two variables are inversely correlated. Next, we investigated whether SGLT2 inhibition with empagliflozin (Empa) ameliorates endothelial dysfunction and reduces arterial stiffness in aged mice with confirmed vascular dysfunction. Specifically, we assessed mesenteric artery endothelial function and stiffness (via flow-mediated dilation and pressure myography mechanical responses, respectively) and aortic stiffness (in vivo via pulse wave velocity and ex vivo via atomic force microscopy) in Empa-treated (14 mg/kg/day for 6 weeks) and control 80-week-old C57BL/6 J male mice. We report that Empa-treated mice exhibited improved mesenteric endothelial function compared with control, in parallel with reduced mesenteric artery and aortic stiffness. Additionally, Empa-treated mice had greater vascular endothelial nitric oxide synthase activation, lower phosphorylated cofilin, and filamentous actin content, with downregulation of pathways involved in production of reactive oxygen species. Our findings demonstrate that Empa improves endothelial function and reduces arterial stiffness in a preclinical model of aging, making SGLT2 inhibition a potential therapeutic alternative to reduce the progression of CVD in older individuals.
Subject(s)
Diabetes Mellitus, Type 2 , Sodium-Glucose Transporter 2 Inhibitors , Vascular Diseases , Actins/metabolism , Aged , Animals , Diabetes Mellitus, Type 2/drug therapy , Humans , Male , Mice , Mice, Inbred C57BL , Oxidative Stress , Pulse Wave Analysis , Sodium-Glucose Transporter 2/metabolism , Sodium-Glucose Transporter 2/therapeutic use , Sodium-Glucose Transporter 2 Inhibitors/pharmacology , Sodium-Glucose Transporter 2 Inhibitors/therapeutic useABSTRACT
Impaired endothelial insulin signaling and consequent blunting of insulin-induced vasodilation is a feature of type 2 diabetes (T2D) that contributes to vascular disease and glycemic dysregulation. However, the molecular mechanisms underlying endothelial insulin resistance remain poorly known. Herein, we tested the hypothesis that endothelial insulin resistance in T2D is attributed to reduced expression of heat shock protein 72 (HSP72). HSP72 is a cytoprotective chaperone protein that can be upregulated with heating and is reported to promote insulin sensitivity in metabolically active tissues, in part via inhibition of JNK activity. Accordingly, we further hypothesized that, in individuals with T2D, 7 days of passive heat treatment via hot water immersion to waist level would improve leg blood flow responses to an oral glucose load (i.e., endogenous insulin stimulation) via induction of endothelial HSP72. In contrast, we found that: 1) endothelial insulin resistance in T2D mice and humans was not associated with reduced HSP72 in aortas and venous endothelial cells, respectively; 2) after passive heat treatment, improved leg blood flow responses to an oral glucose load did not parallel with increased endothelial HSP72; and 3) downregulation of HSP72 (via small-interfering RNA) or upregulation of HSP72 (via heating) in cultured endothelial cells did not impair or enhance insulin signaling, respectively, nor was JNK activity altered. Collectively, these findings do not support the hypothesis that reduced HSP72 is a key driver of endothelial insulin resistance in T2D but provide novel evidence that lower-body heating may be an effective strategy for improving leg blood flow responses to glucose ingestion-induced hyperinsulinemia.
Subject(s)
Diabetes Mellitus, Type 2 , HSP72 Heat-Shock Proteins , Insulin Resistance , Animals , Diabetes Mellitus, Type 2/metabolism , Endothelial Cells/metabolism , Glucose/metabolism , HSP72 Heat-Shock Proteins/genetics , HSP72 Heat-Shock Proteins/metabolism , Insulin/metabolism , MiceABSTRACT
Herein we report in a sample of healthy young men (n = 14) and women (n = 12) that hyperinsulinemia induces time-dependent decreases in total peripheral resistance and its contribution to the maintenance of blood pressure. In the same participants, we observe profound vasodilatory effects of insulin in the lower limb despite concomitant activation of the sympathetic nervous system. We hypothesized that this prominent peripheral vasodilation is possibly due to the ability of the leg vasculature to escape sympathetic vasoconstriction during systemic insulin stimulation. Consistent with this notion, we demonstrate in a subset of healthy men (n = 9) and women (n = 7) that systemic infusion of insulin blunts sympathetically mediated leg vasoconstriction evoked by a cold pressor test, a well-established sympathoexcitatory stimulus. Further substantiating this observation, we show in mouse aortic rings that insulin exposure suppresses epinephrine and norepinephrine-induced vasoconstriction. Notably, we found that such insulin-suppressing effects on catecholamine-induced constriction are diminished following ß-adrenergic receptor blockade. In accordance, we also reveal that insulin augments ß-adrenergic-mediated vasorelaxation in isolated arteries. Collectively, these findings support the idea that sympathetic vasoconstriction can be attenuated during systemic hyperinsulinemia in the leg vasculature of both men and women and that this phenomenon may be in part mediated by potentiation of ß-adrenergic vasodilation neutralizing α-adrenergic vasoconstriction.
Subject(s)
Adrenergic Agents/pharmacology , Hyperinsulinism/drug therapy , Sympathetic Nervous System/drug effects , Vasoconstriction/drug effects , Adult , Blood Pressure/drug effects , Female , Humans , Male , Norepinephrine/pharmacology , Regional Blood Flow/drug effects , Regional Blood Flow/physiology , Sympathetic Nervous System/physiology , Vascular Resistance/drug effectsABSTRACT
A peptide with the ability to release insulin from the rat BRIN-BD11 clonal beta cell line was isolated from norepinephrine-stimulated skin secretions of the Lemur leaf frog Hylomantis lemur Boulenger,1882. Determination of the primary structure (FLSLIPHVISALSSL.NH(2)) demonstrated that the peptide belongs to the phylloseptin family whose members have previously been identified in other Phyllomedusinae species. A synthetic replicate of the peptide, termed phylloseptin-L2, produced a significant stimulation of insulin release (134% of basal rate, P<0.01) from BRIN-BD11 cells at a concentration of 30 nM, with a maximum response (301% of basal rate, P<0.001) at a concentration of 3 microM. Phylloseptin-L2 did not stimulate release of the cytosolic enzyme, lactate dehydrogenase at concentrations up to 3 microM, indicating that the integrity of the plasma membrane had been preserved. The stimulatory action was maintained in the absence of extracellular Ca(2+) and in the presence of verapamil (50 microM) and diazoxide (300 microM) suggesting that mechanism of action of the peptide did not primarily involve influx of Ca(2+) or closure of ATP-sensitive K(+) channels. Administration of phylloseptin-L2 (50 nmol/kgbody weight) into mice significantly (P<0.05) increased total release of insulin and improved glucose tolerance during the 60 min period following an intraperitoneal injection of glucose (18 mmol/kgbody weight). It is concluded that the peptide shows potential for development into a therapeutically valuable agent for the treatment of Type 2 diabetes.
Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Anura/metabolism , Insulin/metabolism , Skin/chemistry , Amino Acid Sequence , Animals , Calcium/metabolism , Calcium/pharmacology , Cell Line , Diazoxide/pharmacology , Exocytosis/drug effects , Extracellular Fluid/metabolism , Glucose/metabolism , Hydro-Lyases/metabolism , Insulin/biosynthesis , Insulin Secretion , Mice , Molecular Sequence Data , Rats , Verapamil/pharmacologyABSTRACT
Peptides in extract of the skin of the Asian frog Hylarana guntheri Boulenger,1882 were purified by reversed-phase HPLC and individual components analysed for their ability to release insulin from the rat BRIN-BD11 clonal beta cell line. The most potent peptide identified in the extract belonged to the brevinin-2 family (brevinin-2GUb; GVIIDTLKGAAKTVAAELLRKAHCKLTNSC). Other peptides with weaker insulin-releasing activity belonged to the brevinin-1 (2 peptides), brevinin-2 (2 peptides) and temporin (3 peptides) families. Only the brevinin-1 peptides showed cytolytic activity against the BRIN-BD11 cells, as demonstrated by an increased rate of release of the cytosolic enzyme, lactate dehydrogenase. A synthetic replicate of brevinin-2GUb produced a significant stimulation of insulin release (139% of basal rate; P<0.05) at a concentration of 100 nM with a maximum response of 373% of basal rate at a concentration of 3 microM) by a mechanism that did not involve mobilization of intracellular calcium. Brevinin-2GUb also inhibited the growth of microorganisms (MIC against Escherichia coli=32 microM, Staphylococcus aureus=64 microM, and Candida albicans=64 microM) but had only weak hemolytic activity against human erythrocytes (LC(50)=700 microM). Administration of brevinin-2GUb (75 nmol/kg body weight) into mice significantly (P<0.05) improved glucose tolerance following a intraperitoneal injection of glucose, thereby demonstrating that the peptide shows potential for development into a therapeutically valuable agent for the treatment of Type 2 diabetes.
Subject(s)
Amphibian Proteins/isolation & purification , Ranidae/metabolism , Amino Acid Sequence , Amphibian Proteins/genetics , Amphibian Proteins/pharmacology , Animals , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/isolation & purification , Antimicrobial Cationic Peptides/pharmacology , Blood Glucose/metabolism , Cell Line , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Male , Mice , Molecular Sequence Data , Ranidae/genetics , Rats , Skin/chemistryABSTRACT
Abstract Pseudin-2 is a cationic alpha-helical peptide that was first isolated from the skin of the paradoxical frog Pseudis paradoxa on the basis of its antimicrobial activity. We have investigated the insulin-releasing properties and cytotoxicity of the peptide, together with selected analogues with increased cationicity and hydrophobicity. At concentrations in the range 10(-9)-10(-6) m, pseudin-2, and its [Lys18], [Phe8], and [d-Lys3,d-Lys10,d-Lys14] derivatives, stimulated insulin release from the BRIN-BD11 clonal beta-cell line without increasing release of lactate dehydrogenase. The [Lys18] analogue was the most potent (46% increase in insulin release at 10(-9) m) and the most effective (215% increase in insulin release at 10(-6) m). The more cationic [Lys3,Lys10,Lys14] and [Lys3,Lys10,Lys14,Lys21] analogues lacked insulinotropic action and the more hydrophobic [Phe16] analogue was cytotoxic at concentrations > or =10(-7) m. Pseudin-2 and [Lys18]-pseudin-2 had no effect on intracellular calcium concentrations and stimulated insulin release in the absence of external calcium. [Lys18]-pseudin-2 (10(-8) m) stimulated insulin release in the presence of diazoxide and verapamil. Our results demonstrate that pseudin-2 stimulates insulin secretion from BRIN-BD11 cells by a mechanism involving Ca2+-independent pathways and identify [Lys18]-pseudin-2 as a peptide that may have potential for development as a therapeutically valuable insulinotropic agent for the treatment of type 2 diabetes.
Subject(s)
Amphibian Proteins/chemistry , Amphibian Proteins/pharmacology , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/pharmacology , Hypoglycemic Agents/chemistry , Insulin/agonists , Animals , Calcium , Cations , Cell Line , Hydrophobic and Hydrophilic Interactions , Hypoglycemic Agents/pharmacology , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Ranidae , Skin , Structure-Activity RelationshipABSTRACT
OBJECTIVES: N-AcGIP is a potent and dipeptidylpeptidase IV-resistant analogue of glucose-dependent insulinotropic polypeptide with significantly improved antidiabetic actions in type 2 diabetes. The present study investigated the effects of subchronic treatment with N-AcGIP on glucose homeostasis in a type 1 model, namely, streptozotocin (STZ)-induced diabetic mice. METHODS: Swiss TO mice given a single intraperitoneal injection of STZ (150 mg/kg body weight) received once-daily injection of N-AcGIP (25 nmol/kg body weight) or saline for 20 days and effects on metabolic parameters and islet architecture assessed. RESULTS: Daily injection of N-AcGIP for 20 days did not significantly alter the characteristic STZ-induced changes of pancreatic insulin content, body weight, food intake, glucose, and glycated hemoglobin levels. Glucose tolerance and insulin sensitivity were also unchanged by N-AcGIP treatment. Circulating insulin was undetectable, and the number of intact islets and insulin expression was greatly reduced in both groups. Some proliferative activity was identified by 5-bromo-2-deoxyuridine staining in the pancreas, but this and expression of glucagon and somatostatin were similar in the 2 groups. CONCLUSIONS: These data indicate that subchronic treatment with the long-acting glucose-dependent insulinotropic polypeptide receptor agonist, N-AcGIP, does not have beneficial effects in insulin-deficient STZ-diabetic mice. This supports the primary antidiabetic action of this analogue in type 2 diabetes as stimulation of beta-cell function and insulin secretion.