Alterations of whole body glucose metabolism in a feline SARS-CoV-2 infection model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been especially devastating to patients with comorbidities, including metabolic and cardiovascular diseases. Elevated blood glucose during SARS-CoV-2 infection increased mortality of patients with COVID-19, although the mechanisms are not well understood. It has been previously demonstrated that glucose transport and utilization is a crucial pathway for other highly infectious RNA viruses. Thus, we hypothesized that SARS-CoV-2 infection could lead to alterations in cellular and whole body glucose metabolism. Specific pathogen-free domestic cats were intratracheally inoculated with USA-WA1/2020 (wild-type) SARS-CoV-2 or vehicle-inoculated, then euthanized at 4- and 8-days postinoculation (dpi). Blood glucose and cortisol concentrations were elevated at 4 and 8 dpi. Blood ketones, insulin, and angiotensin II concentrations remained unchanged throughout the experimental timeline. SARS-CoV-2 RNA was detected in the lung and heart, without changes in angiotensin-converting enzyme 2 (ACE2) RNA expression. In the lung, SARS-CoV-2 infection increased glucose transporter 1 (GLUT1) protein levels at 4 and 8 dpi, whereas GLUT4 level was only upregulated at 8 dpi. In the heart, GLUT-1 and -4 protein levels remained unchanged. Furthermore, GLUT1 level was upregulated in the skeletal muscle at 8 dpi, and AMPK was activated in the hearts of infected cats. SARS-CoV-2 infection increased blood glucose concentration and pulmonary GLUT protein levels. These findings suggest that SARS-CoV-2 infection induces metabolic reprogramming primarily in the lung to support viral replication. Furthermore, this translational feline model mimicked human COVID-19 and could be used to explore novel therapeutic targets to treat metabolic disease during SARS-CoV-2 infection.NEW & NOTEWORTHY Our study on a feline model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, mirroring human COVID-19, revealed alterations in whole body and cellular glucose metabolism. Infected cats developed mild hyperglycemia, increased protein levels of glucose transporters in the lung, and AMPK activation in the heart. These findings suggest that SARS-CoV-2 infection induces metabolic reprogramming in the cardiorespiratory system to support viral replication. Understanding these mechanisms could lead to novel antiviral therapeutic strategies.

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.

Sarcoplasmic reticulum Ca2+ ATPase pump is a major regulator of glucose transport in the healthy and diabetic heart.

Despite intensive research, the pathways that mediate calcium (Ca(2+))-stimulated glucose transport in striated muscle remain elusive. Since the sarcoplasmic reticulum calcium ATPase (SERCA) pump tightly regulates cytosolic [Ca(2+)], we investigated whether the SERCA pump is a major regulator of cardiac glucose transport. We used healthy and insulin-deficient diabetic transgenic (TG) mice expressing SERCA1a in the heart. Active cell surface glucose transporter (GLUT)-4 was measured by a biotinylated photolabeled assay in the intact perfused myocardium and isolated myocytes. In healthy TG mice, cardiac-specific SERCA1a expression increased active cell-surface GLUT4 and glucose uptake in the myocardium, as well as whole body glucose tolerance. Diabetes reduced active cell-surface GLUT4 content and glucose uptake in the heart of wild type mice, all of which were preserved in diabetic TG mice. Decreased basal AS160 and increased proportion of calmodulin-bound AS160 paralleled the increase in cell surface GLUT4 content in the heart of TG mice, suggesting that AS160 regulates GLUT trafficking by a Ca(2+)/calmodulin dependent pathway. In addition, cardiac-specific SERCA1a expression partially rescues hyperglycemia during diabetes. Collectively, these data suggested that the SERCA pump is a major regulator of cardiac glucose transport by an AS160 dependent mechanism during healthy and insulin-deficient state. Our data further indicated that cardiac-specific SERCA overexpression rescues diabetes induced-alterations in cardiac glucose transport and improves whole body glucose homeostasis. Therefore, findings from this study provide novel mechanistic insights linking upregulation of the SERCA pump in the heart as a potential therapeutic target to improve glucose metabolism during diabetes.

GLUT12 functions as a basal and insulin-independent glucose transporter in the heart.

Glucose uptake from the bloodstream is the rate-limiting step in whole body glucose utilization, and is regulated by a family of membrane proteins called glucose transporters (GLUTs). Although GLUT4 is the predominant isoform in insulin-sensitive tissues, there is recent evidence that GLUT12 could be a novel second insulin-sensitive GLUT. However, its physiological role in the heart is not elucidated and the regulation of insulin-stimulated myocardial GLUT12 translocation is unknown. In addition, the role of GLUT12 has not been investigated in the diabetic myocardium. Thus, we hypothesized that, as for GLUT4, insulin regulates GLUT12 translocation to the myocardial cell surface, which is impaired during diabetes. Active cell surface GLUT (-4 and -12) content was quantified (before and after insulin stimulation) by a biotinylated photolabeled assay in both intact perfused myocardium and isolated cardiac myocytes of healthy and type 1 diabetic rodents. GLUT localization was confirmed by immunofluorescent confocal microscopy, and total GLUT protein expression was measured by Western blotting. Insulin stimulation increased translocation of GLUT-4, but not -12, in the healthy myocardium. Total GLUT4 content of the heart was decreased during diabetes, while there was no difference in total GLUT12. Active cell surface GLUT12 content was increased in the diabetic myocardium, potentially as a compensatory mechanism for the observed downregulation of GLUT4. Collectively, our data suggest that, in contrast to GLUT4, insulin does not mediate GLUT12 translocation, which may function as a basal GLUT located primarily at the cell surface in the myocardium.

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.

Impact of Delta SARS-CoV-2 Infection on Glucose Metabolism: Insights on Host Metabolism and Virus Crosstalk in a Feline Model.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes enhanced mortality in people with metabolic and cardiovascular diseases. Other highly infectious RNA viruses have demonstrated dependence on glucose transport and utilization, so we hypothesized that SARS-CoV-2 infection could lead to alterations in cellular and whole-body glucose metabolism. Twenty-four healthy domestic cats were intratracheally inoculated with B.1.617.2 (delta) SARS-CoV-2 and samples were collected at 4- and 12-days post-inoculation (dpi). Blood glucose and circulating cortisol concentrations were elevated at 4 and 12 dpi. Serum insulin concentration was statistically significantly decreased, while angiotensin 2 concentration was elevated at 12 dpi. SARS-CoV-2 RNA was detected in the pancreas and skeletal muscle at low levels; however, no change in the number of insulin-producing cells or proinflammatory cytokines was observed in the pancreas of infected cats through 12 dpi. SARS-CoV-2 infection statistically significantly increased GLUT protein expression in both the heart and lungs, correlating with increased AMPK expression. In brief, SARS-CoV-2 increased blood glucose concentration and cardio-pulmonary GLUT expression through an AMPK-dependent mechanism, without affecting the pancreas, suggesting that SARS-CoV-2 induces the reprogramming of host glucose metabolism. A better understanding of host cell metabolism and virus crosstalk could lead to the discovery of novel metabolic therapeutic targets for patients affected by COVID-19.

Functional consequences of stably expressing a mutant calsequestrin (CASQ2D307H) in the CASQ2 null background.

The role of calsequestrin (CASQ2) in cardiac sarcoplasmic reticulum (SR) calcium (Ca(2+)) transport has gained significant attention since point mutations in CASQ2 were reported to cause ventricular arrhythmia. In the present study, we have critically evaluated the functional consequences of expressing the CASQ2(D307H) mutant protein in the CASQ2 null mouse. We recently reported that the mutant CASQ2(D307H) protein can be stably expressed in CASQ2 null hearts, and it targets appropriately to the junctional SR (Kalyanasundaram A, Bal NC, Franzini-Armstrong C, Knollmann BC, Periasamy M. J Biol Chem 285: 3076-3083, 2010). In this study, we found that introduction of CASQ2(D307H) protein in the CASQ2 null background partially restored triadin 1 levels, which were decreased in the CASQ2 null mice. Despite twofold expression (relative to wild-type CASQ2), the mutant protein failed to increase SR Ca(2+) load. We also found that the Ca(2+) transient decays slower in the CASQ2 null and CASQ2(D307H) cells. CASQ2(D307H) myocytes, when rhythmically paced and challenged with isoproterenol, exhibit spontaneous Ca(2+) waves similar to CASQ2 null myocytes; however, the stability of Ca(2+) cycling was increased in the CASQ2(D307H) myocytes. In the presence of isoproterenol, Ca(2+)-transient amplitude in CASQ2(D307H) myocytes was significantly decreased, possibly indicating an inherent defect in Ca(2+) buffering capacity and release from the mutant CASQ2 at high Ca(2+) concentrations. We also observed polymorphic ventricular tachycardia in the CASQ2(D307H) mice, although lesser than in the CASQ2 null mice. These data suggest that CASQ2(D307H) point mutation may affect Ca(2+) buffering capacity and Ca(2+) release. We propose that poor interaction between CASQ2(D307H) and triadin 1 could affect ryanodine receptor 2 stability, thereby increasing susceptibility to delayed afterdepolarizations and triggered arrhythmic activity.

Association between race, depression, and antiretroviral therapy adherence in a low-income population with HIV infection.

BACKGROUND: Racial disparities exist in many aspects of HIV/AIDS. Comorbid depression adds to the complexity of disease management. However, prior research does not clearly show an association between race and antiretroviral therapy (ART) adherence, or depression and adherence. It is also not known whether the co-existence of depression modifies any racial differences that may exist. OBJECTIVE: To examine racial differences in ART adherence and whether the presence of comorbid depression moderates these differences among Medicaid-enrolled HIV-infected patients. DESIGN: Retrospective cohort study. SETTING: Multi-state Medicaid database (Thomson Reuters MarketScan®). PARTICIPANTS: Data for 7,034 HIV-infected patients with at least two months of antiretroviral drug claims between 2003 and 2007 were assessed. MAIN MEASURES: Antiretroviral therapy adherence (90 % days covered) were measured for a 12-month period. The main independent variables of interest were race and depression. Other covariates included patient variables, clinical variables (comorbidity and disease severity), and therapy-related variables. KEY RESULTS: In this study sample, over 66 % of patients were of black race, and almost 50 % experienced depression during the study period. A significantly higher portion of non-black patients were able to achieve optimal adherence (≥90 %) compared to black patients (38.6 % vs. 28.7 %, p < 0.001). In fact, black patients had nearly 30 % decreased odds of being optimally adherent to antiretroviral drugs compared to non-black patients (OR = 0.70, 95 % CI: 0.63-0.78), and was unchanged regard less of whether the patient had depression. Antidepressant treatment nearly doubled the odds of optimal ART adherence among patients with depression (OR = 1.92, 95 % CI: 1.12-3.29). CONCLUSIONS: Black race was significantly associated with worse ART adherence, which was not modified by the presence of depression. Under-diagnosis and under-treatment of depression may hinder ART adherence among HIV-infected patients of all races.

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.

Equine insulin dysregulation causes tissue specific alterations of proinflammatory cytokines and acute phase proteins in a NF-kB independent manner.

Similar to human diabetes, equine metabolic syndrome (EMS) causes insulin dysregulation leading to debilitating sequela including laminitis. The pathophysiological mechanisms underlying EMS and laminitis are not well known. Therefore, using an insulin-resistant equine model, we hypothesized that insulin dysregulation induces an increased expression of inflammatory proteins in a tissue specific manner. Two groups of horses (n = -5/group) were categorized as insulin-resistant (IR) or insulin-sensitive (IS), using a frequently sampled intra-venous glucose tolerance test. Biopsies from skeletal muscle, and visceral and subcutaneous adipose tissues were collected in both groups. Protein expression was quantified via Western blotting in order to investigate HSP90, α 2 macroglobulin (A2M), Fibrinogen α, ß, γ isoforms as well as cytokines, including interleukin-1ß (IL-1ß) and interleukin-6 (IL-6), in muscle and adipose tissues. Protein expression of HSP90, A2M and IL1-ß was significantly greater in visceral adipose tissue of IR horses compared to IS horses. Fibrinogen (α and γ) expression was only significantly increased in subcutaneous adipose tissue of IR group compared to IS group. In contrast, no statistically significant difference in protein expression of proinflammatory cytokines and acute phase proteins was reported in skeletal muscle of IR vs. IS horses. Relative protein expression of total and phospho-NFκB protein expression was not statistically significantly changed in adipose tissues of IR horses compared to IS horses. In conclusion, proinflammatory cytokines and acute phase proteins were upregulated in adipose tissue, but not in skeletal muscle, through an NF-kB independent pathway. Insights from this study could reveal novel biomarkers and potential therapeutic targets for EMS.

Chronic heart failure selectively induces regional heterogeneity of insulin-responsive glucose transporters.

Glucose uptake across the sarcolemma is regulated by a family of membrane proteins called glucose transporters (GLUTs), which includes GLUT4 (the major cardiac isoform) and GLUT12 (a novel, second insulin-sensitive isoform). Potential regional patterns in glucose transport across the cardiac chambers have not been examined; thus, we hypothesized that insulin-responsive GLUT4 and -12 protein and gene expression would be chamber specific in healthy subjects and during chronic heart failure (HF). Using a canine model of tachypacing-induced, progressive, chronic HF, total GLUT protein and messenger RNA in both ventricles and atria (free wall and appendage) were investigated by immunoblotting and real-time PCR. In controls, GLUT4, but not GLUT12, protein content was significantly higher in the atria compared with the ventricles, with the highest content in the right atrium (RA; P < 0.001). GLUT4 and GLUT12 mRNA levels were similar across the cardiac chambers. During chronic HF, GLUT4 and GLUT12 protein content was highest in the left ventricle (LV; by 2.5- and 4.2-fold, respectively, P < 0.01), with a concomitant increase in GLUT4 and GLUT12 mRNA (P < 0.001). GLUT4, but not GLUT12, protein content was decreased in RA during chronic HF (P = 0.001). In conclusion, GLUT4 protein was differentially expressed across the chambers in the healthy heart, and this regional pattern was reversed during HF. Our data suggest that LV was the primary site dependent on both GLUT4 and GLUT12 during chronic HF. In addition, the paradoxical decrease in GLUT4 content in RA may induce perturbations in atrial energy production during chronic HF.

Medical utilization and costs associated with statin adherence in Medicaid enrollees with type 2 diabetes.

BACKGROUND: Statin adherence is a serious problem in patients with hyperlipidemia. However, it is not clear whether statin adherence is associated with medical utilization or health-care costs. OBJECTIVE: To study statin adherence and assess associated medical utilization and health-care costs in patients with type 2 diabetes, based on a national Medicaid database. METHODS: A retrospective claims-based study was conducted using the records of patients with type 2 diabetes with comorbid hyperlipidemia who were continuously enrolled in Medicaid from January 2004 to December 2006. All data were drawn from MarketScan Medicaid Database, including inpatient, outpatient, and drug claims. The eligible patients starting statins in 2005 were followed for 1 year to measure medication use, hospitalization, outpatient visits, emergency department (ED) visits, and health-care costs based on Medicaid medical and drug claims. Adherence was measured by medication possession ratio (MPR). Multiple regression analyses were implemented to assess statin adherence-associated outcomes, including medical utilization (risks for hospitalization and ED visits), all-cause costs, and hyperlipidemia-related medical costs. RESULTS: A total of 1705 eligible patients with type 2 diabetes and hyperlipidemia were identified. The average adherence rate to statins (MPR) at 1 year was 0.61, and 37% of the patients (n=624) were adherent to statins (MPR≥0.8). Regression analyses indicated that diabetic patients who were adherent to statins showed lower risks for hospitalization (OR 0.80; 95% CI 0.636 to 0.966) and ED visits (OR 0.71; 95% CI 0.519 to 0.812) and decreased all-cause medical costs by 15% (p<0.05) and hyperlipidemia-related medical costs by 12% (p<0.05). CONCLUSIONS: Our study found high prevalence of nonadherence to statins in Medicaid patients with type 2 diabetes. Adherence to statins (MPR≥0.8) was associated with reduced medical utilization and lower medical costs.

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.

Factors predictive of abnormal results for computed tomography of the head in horses affected by neurologic disorders: 57 cases (2001-2007).

OBJECTIVE: To determine neurologic indications associated with abnormal results for computed tomography (CT) imaging of the head of horses affected by neurologic disorders. DESIGN: Retrospective case series. ANIMALS: 57 horses. PROCEDURES: Signalment, history, clinical abnormalities, and clinicopathologic findings were obtained from medical records of horses examined because of neurologic disorders, and precontrast and postcontrast CT images of the head were reviewed. Data were analyzed by use of univariate and multivariate logistic regression. RESULTS: For a horse with abnormal mentation, odds of having abnormal results for CT imaging of the head was 30 times (95% confidence interval [CI], 2.36 to 374.63) the odds for a similar horse without abnormal mentation. For a horse with cranial nerve deficits, odds of having abnormal results for CT imaging of the head was 11 times (95% CI, 1.00 to 127.96) the odds for a similar horse without cranial nerve deficits. For a horse with seizure-like activity, odds of having abnormal results for CT imaging of the head was 0.05 times (95% CI, 0 to 0.90) the odds for a similar horse without seizures. CONCLUSIONS AND CLINICAL RELEVANCE: These results suggested that alterations in consciousness and cranial nerve deficits were strong predictors of abnormal CT findings for the head of affected horses. Thus, CT can be a useful complementary diagnostic test in horses with these neurologic deficits. In contrast, alternative diagnostic tests (eg, electroencephalography and magnetic resonance imaging) should be considered in horses with seizure-like activity that do not have head trauma or cranial nerve deficits.

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.

A mutation in calsequestrin, CASQ2D307H, impairs Sarcoplasmic Reticulum Ca2+ handling and causes complex ventricular arrhythmias in mice.

OBJECTIVE: A naturally-occurring mutation in cardiac calsequestrin (CASQ2) at amino acid 307 was discovered in a highly inbred family and hypothesized to cause Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). The goal of this study was to establish a causal link between CASQ2(D307H) and the CPVT phenotype using an in vivo model. METHODS AND RESULTS: Cardiac-specific expression of the CASQ2(D307H) transgene was achieved using the alpha-MHC promoter. Multiple transgenic (TG) mouse lines expressing CASQ2(D307H) from 2- to 6-fold possess structurally normal hearts without any sign of hypertrophy. The hearts displayed normal ventricular function. Myocytes isolated from TG mice had diminished I(Ca)-induced Ca2+ transient amplitude and duration, as well as increased Ca2+ spark frequency. These myocytes, when exposed to isoproterenol and caffeine, displayed disturbances in their rhythmic Ca2+ oscillations and membrane potential, and delayed afterdepolarizations. ECG monitoring revealed that TG mice challenged with isoproterenol and caffeine developed complex ventricular arrhythmias, including non-sustained polymorphic ventricular tachycardia. CONCLUSIONS: The findings of the present study demonstrate that expression of mutant CASQ2(D307H) in the mouse heart results in abnormal myocyte Ca2+ handling and predisposes to complex ventricular arrhythmias similar to the CPVT phenotype observed in human patients.

Quantification of Cell-Surface Glucose Transporters in the Heart Using a Biotinylated Photolabeling Assay.

The biotinylated photolabeling assay enables quantification of cell-surface glucose transporters (GLUTs). This technique has been successfully applied to quantify the cell-surface GLUT protein content in striated muscles and adipose tissue, as a means to evaluate GLUT trafficking. Here, we describe the detailed method of quantifying the cell-surface content of several GLUT isoforms (1, 4, 8, and 12) in isolated cardiac myocytes, as well as in the intact perfused atria and ventricle.

Ingestion of starch-rich meals after exercise increases glucose kinetics but fails to enhance muscle glycogen replenishment in horses.

Fatiguing exercise substantially decreases muscle glycogen concentration in horses, impairing athletic performance in subsequent exercise bouts. Our objective was to determine the effect of ingestion of starch-rich meals after exercise on whole body glucose kinetics and muscle glycogen replenishment. In a randomized, cross-over study seven horses with exercise-induced muscle glycogen depletion were either not fed for 8 h, fed half of the daily energy requirements ( approximately 15 Mcal DE) as hay, or fed an isocaloric amount of corn 15 min and 4 h after exercise. Starch-rich meals fed after exercise, when compared to feed withholding, resulted in mild to moderate hyperglycemia (5.7+/-0.3 vs. 4.7+/-0.3 mM, P<0.01) and hyperinsulinemia (79.9+/-9.3 vs. 39.0+/-1.9 pM, P<0.001), 3-fold greater whole body glucose kinetics (15.5+/-1.4 vs. 5.3+/-0.4 micromol kg(-1)min(-1), P<0.05), but these only minimally enhanced muscle glycogen replenishment (171+/-19 vs. 170+/-56 and 260+/-45 vs. 294+/-29 mmol/kg dry weight immediately and 24 h after exercise, P>0.05). It is concluded that after substantial exercise-induced muscle glycogen depletion, feeding status only minimally affects net muscle glycogen concentrations after exercise, despite marked differences in soluble carbohydrate ingestion and availability of glucose to skeletal muscle.