Differential Antimicrobial Efficacy of Multipurpose Solutions against Acanthamoeba Trophozoites.

SIGNIFICANCE: This investigation examines the effectiveness of several common contact lens solutions in the disinfection of Acanthamoeba, which causes a serious eye infection most often resulting from dysfunctional or improper use of contact lens products. PURPOSE: Acanthamoeba keratitis is an eye infection caused by a free-living amoeba, which can lead to extensive corneal damage and frequently blindness. Acanthamoeba keratitis is linked with contact lens use combined with noncompliance with contact lens care cleaning regimens. The patient's choice and use of multipurpose solutions (MPSs) continue to be a risk factor for Acanthamoeba keratitis. Thus, it is critical that the Acanthamoeba disinfection efficacy of the popular MPSs be determined. Here we compare the efficacy of seven major MPSs on the global market. METHODS: Using standard methods of Acanthamoeba disinfection and quantification, Acanthamoeba ATCC 30461, 30868, 50370, and 50676 trophozoites were inoculated into each MPS and held for the manufacturer's recommended disinfection time. Acanthamoeba recovery plates were incubated for 14 days, after which positive wells were identified and cell concentrations determined using the 50% endpoint method. RESULTS: Members of the OPTI-FREE products (Express, Replenish, and Puremoist [Alcon, Fort Worth, TX]) demonstrated significantly higher percentages of antimicrobial activity compared with the renu Advanced Formula (Bausch + Lomb, Rochester, NY), Biotrue (Bausch + Lomb), Acuvue RevitaLens (Johnson & Johnson, Santa Ana, CA), and Lite products (Cooper Vision, Scottsville, NY) for four of the trophozoite strains tested. CONCLUSIONS: Many of the popular MPS biocides maintain little or no antimicrobial activity against Acanthamoeba trophozoites, and the number of biocides in an MPS does not necessarily indicate its antimicrobial activity.

Dysregulation of insulin-sensitive glucose transporters during insulin resistance-induced atrial fibrillation.

Diabetes has been identified as major risk factor for atrial fibrillation (AF). Although glucose and insulin disturbances during diabetes may affect atrial function, little is known about the potential pathogenic role of glucose metabolism during AF. Glucose transport into the cell via glucose transporters (GLUTs) is the rate-limiting step of glucose utilization. Although GLUT4 is the major isoform, GLUT8 has emerged as a novel insulin-sensitive cardiac isoform. We hypothesized that atrial glucose homeostasis will be impaired during insulin resistance-induced AF. AF was induced by transesophageal atrial pacing in healthy mice and following a long-term high-fat-diet-induced insulin resistance. Active cell surface GLUT content was measured using the biotinylated photolabeling assay in the intact perfused heart. Atrial fibrosis, advanced glycation end products (AGEs) and glycogen were measured in the atria using histological analyses. Animals fed a high-fat-diet were obese and mildly hyperglycemic, and developed insulin resistance compared to controls. Insulin-resistant (IR) animals demonstrated an increased vulnerability to induced AF, as well as spontaneous AF. Insulin-stimulated translocation of GLUT4 and GLUT8 was down-regulated in the atria of IR animals, as well as their total protein expression. We also reported the absence of fibrosis, glycogen and AGE accumulation in the atria of IR animals. In the absence of structural remodeling and atrial fibrosis, these data suggest that insulin signaling dysregulation, resulting in impaired glucose transport in the atria, could provide a metabolic arrhythmogenic substrate and be a novel early pathogenic factor of AF.

Diabetes Causes Significant Alterations in Pulmonary Glucose Transporter Expression.

Diabetes has been identified as a significant and independent risk factor for the development or increased severity of respiratory infections. However, the role of glucose transport in the healthy and diseased lung has received little attention. Specifically, the protein expression of the predominant glucose transporter (GLUT) isoforms in the adult lung remains largely to be characterized in both healthy and diabetic states. Type 1 diabetes was induced via streptozotocin and rescued via subcutaneous semi-osmotic insulin pump for 8 weeks. The gene and/or protein expression of the most predominant GLUT isoforms from Classes I and III, including the major insulin-sensitive isoform (i.e., GLUT4) and novel isoforms (i.e., GLUT-8 and GLUT-12), was quantified in the lung of healthy and diabetic mice via qRT-PCR and/or Western blotting. Pulmonary cell surface GLUT protein was measured using a biotinylated photolabeling assay, as a means to evaluate GLUT trafficking. Diabetic mice demonstrated significant alterations of total pulmonary GLUT protein expression, which were isoform- and location-dependent. Long-term insulin treatment rescued the majority of GLUT protein expression alterations in the lung during diabetes, as well as GLUT-4 and -8 trafficking to the pulmonary cell surface. These alterations in glucose homeostasis during diabetes may contribute to an increased severity of pulmonary infection during diabetes and may point to novel metabolic therapeutic strategies for diabetic patients with concurrent respiratory infections.

Complete Recovery of Acanthamoeba Motility among Surviving Organisms after Contact Lens Care Disinfection.

Acanthamoeba keratitis is a sight-threatening infection of the cornea which is extremely challenging to treat. Understanding this organism's responses during contact lens contact and disinfection could enhance our understanding of how Acanthamoebae colonize contact lens cases, better inform us on contact lens care solution (CLC) efficacy, and help us better understand the efficacy required of CLC products. To explore this gap in knowledge, we used Acanthamoeba ATCC 30461 and ATCC 50370 trophozoites to examine Acanthamoeba behavior during and after CLC disinfection. Amoebae were added to sterile aluminum flow cells and flow cell solutions were changed to Ringer's solution (control), or one of four CLCs based on biocides (PHMB, PAPB/Polyquad, Polyquad/Aldox, or Polyquad/Alexidine) for 6 h. Each flow cell solution was then changed to axenic culture media (AC6) for 12 h to determine the behavior of amoebae following disinfection. Distance, speed, and displacement were calculated for each organism. As compared to the control of one-quarter Ringer's solution, each CLC significantly impacted Acanthamoeba motility in both the CLC and AC6 conditions. However, the amoebae challenged with the PHMB CLC traveled a significantly greater total distance than with the other three CLCs, indicating differences in effectiveness between biocides. Furthermore, amoebae regaining motility post-disinfection by CLCs were observed to travel considerable distances and thus could be considered dangerous to ocular health. We determined that while all CLCs produced a substantial or complete cessation of movement vs. the control condition during disinfection, those which relied on the Polyquad biocides were the most effective, and that any amoebae which survived disinfection were able to recover motility. Future examinations of these findings should include direct correlations between motility and viability, and how infectivity and motility may be related.

Microbial Adherence to Contact Lenses and Pseudomonas aeruginosa as a Model Organism for Microbial Keratitis.

Microbial keratitis (MK), the infection of the cornea, is a devastating disease and the fifth leading cause of blindness and visual impairment around the world. The overwhelming majority of MK cases are linked to contact lens wear combined with factors which promote infection such as corneal abrasion, an immunocompromised state, improper contact lens use, or failing to routinely disinfect lenses after wear. Contact lens-related MK involves the adherence of microorganisms to the contact lens. Therefore, this review discusses the information currently available regarding the disease pathophysiology, the common types of microorganisms causing MK, physical and organic mechanisms of adhesion, material properties which are involved in adhesion, and current antimicrobial strategies. This review also concludes that Pseudomonas aeruginosa is a model organism for the investigation of contact lens microbial adherence due to its prevalence in MK cases, its extremely robust adhesion, antimicrobial-resistant properties, and the severity of the disease it causes.

Investigating the Relationship Between Cardiac Function and Insulin Sensitivity in Horses: A Pilot Study.

Metabolic syndrome in humans is commonly associated with cardiovascular dysfunction, including atrial fibrillation and left ventricular diastolic dysfunction. Although many differences exist between human and equine metabolic syndrome, both of these conditions share some degree of insulin resistance. The aims of this pilot study were to investigate the relationship between insulin sensitivity and cardiac function. Seven horses (five mares, two geldings, aged 17.2 ± 4.2 years, weight 524 ± 73 kg) underwent insulin-modified frequently sampled intravenous glucose tolerance testing to determine insulin sensitivity (mean 2.21 ± 0.03 × 10-4 L/min/mU). Standard echocardiograms were performed on each horse, including two-dimensional, M-mode, and pulse-wave tissue Doppler imaging. Pearson and Spearman correlation analyses were used to determine the association of insulin sensitivity with echocardiographic measures of cardiac function in 5 horses. Insulin sensitivity was found to be significantly correlated with peak myocardial velocity during late diastole (r = 0.89, P = 0.0419), ratio between peak myocardial velocity in early and late diastole (r = -0.92, P = 0.0263), isovolumetric relaxation time (r = -0.97, P = 0.0072), and isovolumetric contraction time (ρ = -0.90, P = 0.0374). These preliminary data suggest that decreased insulin sensitivity is correlated with alterations in both systolic and diastolic function, as measured with tissue Doppler imaging (TDI). Due to the small sample size of this study, the relationship between insulin sensitivity and myocardial function in horses requires further investigation.

Differential Antimicrobial Efficacy of Preservative-Free Contact Lens Disinfection Systems against Common Ocular Pathogens.

Microbial keratitis is a devastating disease that can cause eye damage and blindness and can be the result of infections by several common ocular pathogens. Importantly, some of these pathogens, such as Acanthamoeba, are particularly unsusceptible to biocides in common contact lens care solutions. Therefore, the disinfection efficacy of preservative-free (PF) disinfection systems against bacteria, fungi, and Acanthamoeba trophozoites and cysts should be assessed as products with the most potential to be efficacious against resistant organisms. PF disinfection systems were analyzed for antimicrobial efficacy. These were the one-step (hydrogen peroxide-based) Clear Care and Clear Care Plus systems and the two-step (povidone-iodine-based) Cleadew system. Stand-alone challenges using bacteria, fungi, and Acanthamoeba were prepared according to the International Standards Organization method 14729. These same challenges were also conducted in the presence of the following contact lenses: Boston RGP, Acuvue Oasys, Biofinity, Ultra, and 2-week PremiO. All challenges were performed at the manufacturer's recommended disinfection time. All preservative-free disinfection systems demonstrated similarly high rates of antimicrobial efficacy when challenged with bacteria or fungi, with or without lenses. However, both Clear Care and Clear Care Plus demonstrated significantly greater disinfection efficacy against Acanthamoeba trophozoites and cysts, with and without lenses (P < 0.05). Cleadew efficacy was impacted by the addition of contact lenses, whereas Clear Care/Clear Care Plus maintained similar efficacies in the absence or presence of lenses. While both hydrogen peroxide and povidone-iodine are highly effective against bacteria and fungi, hydrogen peroxide maintains significantly greater disinfection capabilities than povidone-iodine against all forms of Acanthamoeba. IMPORTANCE Understanding the most efficacious products will allow clinicians to best communicate to patients and consumers the safest products on the market to reduce adverse events, including microbial keratitis, during contact lens use.

Reduction of disinfection efficacy of contact lens care products on the global market in the presence of contact lenses and cases.

OBJECTIVE: Sight-threatening infections can be caused by pathogenic micro-organisms colonising the cornea, leading to microbial keratitis (MK). These micro-organisms can be introduced to the eye via improper contact lens use and care. MK can also result from ineffective contact lens care solutions (CLCs), even if the patient is following best practice guidelines. Therefore, it is critical to understand the differences between the effectiveness of popular CLCs on the global market. METHODS AND ANALYSIS: Following the International Standards Organisation standards 14 729 and 18259, bacteria (Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus), fungi (Candida albicans, Fusarium strains) and Acanthamoeba strains were inoculated into each CLC with and without contact lenses, and held for the manufacturer's stated disinfection time. Plate counts were conducted to determine the number of surviving micro-organisms. RESULTS: All CLCs examined met the primary log reduction criteria during stand-alone testing for Pseudomonas, Staphylococcus, Candida and Fusarium. renu Multiplus, All Clean Soft, and Kombilösung Super did not meet the primary criteria when challenged with Serratia. Only OPTI-FREE Express exceeded 4 log reduction for both strains of Acanthamoeba tested. We noted a substantial reduction in disinfection efficacy when CLCs were challenged with Fusarium in the presence of lenses and cases versus stand-alone testing. OPTI-FREE Express demonstrated significantly less net log reduction loss than the other four CLCs tested. CONCLUSION: Of the popular CLCs on the global market, the product which relies on dual biocides polyquaternium-1 and myristamidopropyl dimethylamine demonstrated the highest disinfection efficacy in microbial disinfection challenges in the absence and presence of contact lenses.

Acanthamoeba spp. aggregate and encyst on contact lens material increasing resistance to disinfection.

Introduction: Acanthamoeba keratitis is often caused when Acanthamoeba contaminate contact lenses and infect the cornea. Acanthamoeba is pervasive in the environment as a motile, foraging trophozoite or biocide-resistant and persistent cyst. As contact lens contamination is a potential first step in infection, we studied Acanthamoeba's behavior and interactions on different contact lens materials. We hypothesized that contact lenses may induce aggregation, which is a precursor to encystment, and that aggregated encystment would be more difficult to disinfect than motile trophozoites. Methods: Six clinically and/or scientifically relevant strains of Acanthamoeba (ATCC 30010, ATCC 30461, ATCC 50370, ATCC 50702, ATCC 50703, and ATCC PRA-115) were investigated on seven different common silicone hydrogel contact lenses, and a no-lens control, for aggregation and encystment for 72 h. Cell count and size were used to determine aggregation, and fluorescent staining was used to understand encystment. RNA seq was performed to describe the genome of Acanthamoeba which was individually motile or aggregated on different lens materials. Disinfection efficacy using three common multi-purpose solutions was calculated to describe the potential disinfection resistance of trophozoites, individual cysts, or spheroids. Results: Acanthamoeba trophozoites of all strains examined demonstrated significantly more aggregation on specific contact lens materials than others, or the no-lens control. Fluorescent staining demonstrated encystment in as little as 4 hours on contact lens materials, which is substantially faster than previously reported in natural or laboratory settings. Gene expression profiles corroborated encystment, with significantly differentially expressed pathways involving actin arrangement and membrane complexes. High disinfection resistance of cysts and spheroids with multi-purpose solutions was observed. Discussion: Aggregation/encystment is a protective mechanism which may enable Acanthamoeba to be more disinfection resistant than individual trophozoites. This study demonstrates that some contact lens materials promote Acanthamoeba aggregation and encystment, and Acanthamoeba spheroids obstruct multi-purpose solutions from disinfecting Acanthamoeba.

Evaluating Alternate Methods of Determining the Antimicrobial Efficacy of Contact Lens Care Products against Acanthamoeba Trophozoites.

Acanthamoeba keratitis (AK) is a serious ocular infection caused by a ubiquitous free-living amoeba, Acanthamoeba. This infection often results in extensive corneal damage and blindness, and is notoriously difficult to cure. While Acanthamoeba is an abundant organism, AK is most associated with contact lens hygiene noncompliance and inadequate contact lens care (CLC) disinfection regimens. Thus, accurate and timely antimicrobial efficacy testing of CLC solutions is paramount. Published methods for antimicrobial efficacy testing of Acanthamoeba trophozoites requires 14 days for results. Presently, alternate and/or rapid methods for evaluating CLC products rarely demonstrate equivalent results compared to commonly-reported methods. Propidium iodide is a cellular stain that can only bind to cells with damaged outer membranes. We evaluated propidium iodide staining as an alternative method for determining the relative antimicrobial efficacy of 11 different CLC products against Acanthamoeba trophozoites. Following exposure to a CLC product, the fluorescence intensity of propidium iodide in an Acanthamoeba population demonstrated a strong correlation to the log reduction determined by established, growth-based Acanthamoeba testing used to evaluate the antimicrobial efficacy of CLC products. Thus, propidium iodide was found to be an effective rapid tool for determining cell death in Acanthamoeba trophozoites following exposure to CLC solutions.

Continuous Real-Time Motility Analysis of Acanthamoeba Reveals Sustained Movement in Absence of Nutrients.

Acanthamoeba keratitis is a serious ocular infection which is challenging to treat and can lead to blindness. While this pathogen is ubiquitous and can contaminate contact lenses after contact with water, its habits remain elusive. Understanding this organism's natural behavior will better inform us on how Acanthamoeba colonize contact lens care systems. Acanthamoeba trophozoites were allowed to adhere to either a glass coverslip or non-nutrient agar (NNA) within a flow cell with nutrients (Escherichia coli or an axenic culture medium (AC6)) or without nutrients (Ringer's solution). Images were taken once every 24 s over 12 h and compiled, and videos were analyzed using ImageJ Trackmate software. Acanthamoeba maintained continuous movement for the entire 12 h period. ATCC 50370 had limited differences between conditions and surfaces throughout the experiment. Nutrient differences had a noticeable impact for ATCC 30461, where E. coli resulted in the highest total distance and speed during the early periods of the experiment but had the lowest total distance and speed by 12 h. The Ringer's and AC6 conditions were the most similar between strains, while Acanthamoeba in the E. coli and NNA conditions demonstrated significant differences between strains (p < 0.05). These results indicate that quantifiable visual tracking of Acanthamoeba may be a novel and robust method for identifying the movement of Acanthamoeba in relation to contact lens care products. The present study indicates that Acanthamoeba can undertake sustained movement for at least 12 h with and without nutrients, on both rough and smooth surfaces, and that different strains have divergent behavior.

Antimicrobial Efficacy of Contact Lens Solutions Assessed by ISO Standards.

Microbial keratitis (MK) is an eye infection caused by opportunistic bacteria or fungi, which may lead to sight-threatening corneal ulcers. These microorganisms can be introduced to the eye via improper contact lens usage or hygiene, or ineffective multipurpose solutions (MPSs) to disinfect daily wear contact lenses. Thus, the patient's choice and use of these MPSs is a known risk factor for the development of MK. It is then critical to determine the efficacy of popular MPSs against ubiquitous ocular microorganisms. Therefore, we compare the efficacy of nine major MPSs on the global market against four different microorganism species, and with four different common contact lenses. In accordance with International Standards Organization protocol 14729 and 18259, the microorganisms were inoculated into each MPS with and without contact lenses, and held for the manufacturer's disinfection time, 24 h, and 7 days after challenge with Serratia marcescens or Fusarium spp. Plates were incubated for 2-7 days and plate counts were conducted to determine the number of surviving microorganisms. The majority of MPSs demonstrated significantly higher disinfection efficacies without contact lenses. Broadly, among the microorganisms tested, the OPTI-FREE products (Puremoist, Express, and Replenish) maintained the highest disinfection efficacies at the manufacturer's stated disinfection time when paired with any contact lens, compared with other MPSs. These were followed closely by RevitaLens and renu Advanced. MPSs containing dual biocides polyquaternium-1 and myristamidopropyl dimethylamine possessed the highest disinfection efficacy against multiple ocular pathogens.

Insulin Treatment Reduces Susceptibility to Atrial Fibrillation in Type 1 Diabetic Mice.

Diabetes has been identified as an independent risk factor for atrial fibrillation (AF), the most common chronic cardiac arrhythmia. Whether or not glucose and insulin disturbances observed during diabetes enhance arrhythmogenicity of the atria, potentially leading to AF, is not well-known. We hypothesized that insulin deficiency and impaired glucose transport provide a metabolic substrate for the development and maintenance of AF during diabetes. Transesophageal atrial pacing was used to induce AF in healthy, streptozotocin-induced insulin-deficient type 1 diabetic, and insulin-treated diabetic mice. Translocation of insulin-sensitive glucose transporters (GLUTs) to the atrial cell surface was measured using a biotinylated photolabeling assay in the perfused heart. Fibrosis and glycogen accumulation in the atrium were measured using histological analysis. Diabetic mice displayed mild hyperglycemia, increased duration and frequency of AF episodes vs. age-matched controls (e.g., AF duration: 19.7 ± 6.8 s vs. 1.8 ± 1.1 s, respectively, p = 0.032), whereas insulin-treated diabetic animals did not. The translocation of insulin-sensitive GLUT-4 and -8 to the atrial cell surface was significantly downregulated in the diabetic mice (by 67 and 79%, respectively; p ≤ 0.001), and rescued by insulin treatment. We did not observe fibrosis or glycogen accumulation in the atria of diabetic mice. Therefore, these data suggest that insulin and glucose disturbances were sufficient to induce AF susceptibility during mild diabetes.

Differential Proteomic Expression of Equine Cardiac and Lamellar Tissue During Insulin-Induced Laminitis.

Endocrinopathic laminitis is pathologically similar to the multi-organ dysfunction and peripheral neuropathy found in human patients with metabolic syndrome. Similarly, endocrinopathic laminitis has been shown to partially result from vascular dysfunction. However, despite extensive research, the pathogenesis of this disease is not well elucidated and laminitis remains without an effective treatment. Here, we sought to identify novel proteins and pathways underlying the development of equine endocrinopathic laminitis. Healthy Standardbred horses (n = 4/group) were either given an electrolyte infusion, or a 48-h euglycemic-hyperinsulinemic clamp. Cardiac and lamellar tissues were analyzed by mass spectrometry (FDR = 0.05). All hyperinsulinemic horses developed laminitis despite being previously healthy. We identified 514 and 709 unique proteins in the cardiac and lamellar proteomes, respectively. In the lamellar tissue, we identified 14 proteins for which their abundance was significantly increased and 13 proteins which were significantly decreased in the hyperinsulinemic group as compared to controls. These results were confirmed via real-time reverse-transcriptase PCR. A STRING analysis of protein-protein interactions revealed that these increased proteins were primarily involved in coagulation and complement cascades, platelet activity, and ribosomal function, while decreased proteins were involved in focal adhesions, spliceosomes, and cell-cell matrices. Novel significant differentially expressed proteins associated with hyperinsulinemia-induced laminitis include talin-1, vinculin, cadherin-13, fibrinogen, alpha-2-macroglobulin, and heat shock protein 90. In contrast, no proteins were found to be significantly differentially expressed in the heart of hyperinsulinemic horses compared to controls. Together, these data indicate that while hyperinsulinemia induced, in part, microvascular damage, complement activation, and ribosomal dysfunction in the lamellae, a similar effect was not seen in the heart. In brief, this proteomic investigation of a unique equine model of hyperinsulinemia identified novel proteins and signaling pathways, which may lead to the discovery of molecular biomarkers and/or therapeutic targets for endocrinopathic laminitis.

Adverse Metabolic Effects of Diltiazem Treatment During Diabetic Cardiomyopathy.

Diabetes is a global epidemic disease, which leads to multiorgan dysfunction, including heart disease. Diabetes results from the limited absorption of glucose into insulin-sensitive tissues. The heart is one of the main organs to utilize glucose as an energy substrate. Glucose uptake into striated muscle is regulated by a family of membrane proteins called glucose transporters (GLUTs). Although calcium channel blockers, including diltiazem, are widely prescribed drugs for cardiovascular diseases, including in patients with diabetes, their pharmacological effects on glucose metabolism are somewhat controversial. We hypothesized that diltiazem treatment will exhibit detrimental effects on whole body glucose homeostasis and glucose transport in the striated muscle of patients with diabetes. Healthy and streptozotocin-treated rats were randomly assigned to receive diltiazem treatment or a placebo for 8 weeks. Blood glucose was significantly increased in the untreated diabetic groups, which worsened after diltiazem treatment. Diabetes decreased protein content of both GLUT4 (the predominate insulin-sensitive glucose transporter) and AS160 (Akt Substrate at 160 kDa, the downstream protein in the signaling cascade that regulates GLUT4 trafficking) in striated muscle of diabetic rats, with a more pronounced alteration after diltiazem treatment. We additionally reported that diabetic rodents displayed marked systolic dysfunction, which was not rescued by diltiazem treatment. In conclusion, diltiazem treatment worsened the effects of diabetes-induced hyperglycemia and diabetes-induced alterations in the regulation of glucose transport in striated muscle.

Glial Growth Factor 2 Regulates Glucose Transport in Healthy Cardiac Myocytes and During Myocardial Infarction via an Akt-Dependent Pathway.

Neuregulin (NRG), a paracrine factor in myocytes, promotes cardiac development via the ErbB receptors. NRG-1ß also improves cardiac function and cell survival after myocardial infarction (MI), although the mechanisms underlying these cardioprotective effects are not well elucidated. Increased glucose uptake has been shown to be cardio-protective during MI. We hypothesized that treatment with a recombinant version of NRG-1ß, glial growth factor 2 (GGF2), will enhance glucose transport in the healthy myocardium and during MI. Cardiac myocytes were isolated from MI and healthy adult rats, and subsequently incubated with or without insulin or GGF2. Glucose uptake was measured using a fluorescent D-glucose analog. The translocation of glucose transporter (GLUT) 4 to the cell surface, the rate-limiting step in glucose uptake, was measured using a photolabeled biotinylation assay in isolated myocytes. Similar to insulin, acute in vitro GGF2 treatment increased glucose uptake in healthy cardiac myocytes (by 40 and 49%, respectively, P = 0.002). GGF2 treatment also increased GLUT4 translocation in healthy myocytes by 184% (P < 0.01), while ErbB 2/4 receptor blockade (by afatinib) abolished these effects. In addition, GGF2 treatment enhanced Akt phosphorylation (at both threonine and serine sites, by 75 and 139%, respectively, P = 0.029 and P = 0.01), which was blunted by ErbB 2/4 receptor blockade. GGF2 treatment increased the phosphorylation of AS160 (an Akt effector) by 72% (P < 0.05), as well as the phosphorylation of PDK-1 and PKC (by 118 and 92%, respectively, P < 0.05). During MI, cardiac GLUT4 translocation was downregulated by 44% (P = 0.004) and was partially rescued by both in vitro insulin and GGF2 treatment. Our data demonstrate that acute GGF2 treatment increased glucose transport in cardiac myocytes by activating the ErbB 2/4 receptors and subsequent key downstream effectors (i.e., PDK-1, Akt, AS160, and PKC). These findings highlight novel mechanisms of action of GGF2, which warrant further investigation in patients with heart failure.

Diabetes Alters the Expression and Translocation of the Insulin-Sensitive Glucose Transporters 4 and 8 in the Atria.

Although diabetes has been identified as a major risk factor for atrial fibrillation, little is known about glucose metabolism in the healthy and diabetic atria. Glucose transport into the cell, the rate-limiting step of glucose utilization, is regulated by the Glucose Transporters (GLUTs). Although GLUT4 is the major isoform in the heart, GLUT8 has recently emerged as a novel cardiac isoform. We hypothesized that GLUT-4 and -8 translocation to the atrial cell surface will be regulated by insulin and impaired during insulin-dependent diabetes. GLUT protein content was measured by Western blotting in healthy cardiac myocytes and type 1 (streptozotocin-induced, T1Dx) diabetic rodents. Active cell surface GLUT content was measured using a biotinylated photolabeled assay in the perfused heart. In the healthy atria, insulin stimulation increased both GLUT-4 and -8 translocation to the cell surface (by 100% and 240%, respectively, P<0.05). Upon insulin stimulation, we reported an increase in Akt (Th308 and s473 sites) and AS160 phosphorylation, which was positively (P<0.05) correlated with GLUT4 protein content in the healthy atria. During diabetes, active cell surface GLUT-4 and -8 content was downregulated in the atria (by 70% and 90%, respectively, P<0.05). Akt and AS160 phosphorylation was not impaired in the diabetic atria, suggesting the presence of an intact insulin signaling pathway. This was confirmed by the rescued translocation of GLUT-4 and -8 to the atrial cell surface upon insulin stimulation in the atria of type 1 diabetic subjects. In conclusion, our data suggest that: 1) both GLUT-4 and -8 are insulin-sensitive in the healthy atria through an Akt/AS160 dependent pathway; 2) GLUT-4 and -8 trafficking is impaired in the diabetic atria and rescued by insulin treatment. Alterations in atrial glucose transport may induce perturbations in energy production, which may provide a metabolic substrate for atrial fibrillation during diabetes.