Advanced glycation end product cross-link breaker attenuates diabetes-induced cardiac dysfunction by improving sarcoplasmic reticulum calcium handling

Diabetic heart disease is a distinct clinical entity that can progress to heart failure and sudden death. However, the mechanisms responsible for the alterations in excitation-contraction coupling leading to cardiac dysfunction during diabetes are not well known. Hyperglycemia, the landmark of diabetes, leads to the formation of advanced glycation end products (AGEs) on long-lived proteins, including sarcoplasmic reticulum (SR) Ca2+ regulatory proteins. However, their pathogenic role on SR Ca2+ handling in cardiac myocytes is unknown. Therefore, we investigated whether an AGE cross-link breaker could prevent the alterations in SR Ca2+ cycling that lead to in vivo cardiac dysfunction during diabetes. Streptozotocin-induced diabetic rats were treated with alagebrium chloride (ALT-711) for 8 weeks and compared to age-matched placebo-treated diabetic rats and healthy rats. Cardiac function was assessed by echocardiographic examination. Ventricular myocytes were isolated to assess SR Ca2+ cycling by confocal imaging and quantitative Western blots. Diabetes resulted in in vivo cardiac dysfunction and ALT-711 therapy partially alleviated diastolic dysfunction by decreasing isovolumetric relaxation time and myocardial performance index (MPI) (by 27 and 41% vs. untreated diabetic rats, respectively, P < 0.05). In cardiac myocytes, diabetes-induced prolongation of cytosolic Ca2+ transient clearance by 43% and decreased SR Ca2+ load by 25% (P < 0.05); these parameters were partially improved after ALT-711 therapy. SERCA2a and RyR2 protein expression was significantly decreased in the myocardium of untreated diabetic rats (by 64 and 36% vs. controls, respectively, P < 0.05), but preserved in the treated diabetic group compared to controls. Collectively, our results suggest that, in a model of type 1 diabetes, AGE accumulation primarily impairs SR Ca2+ reuptake in cardiac myocytes and that long-term treatment with an AGE cross-link breaker partially normalized SR Ca2+ handling and improved diabetic cardiomyopathy.Peer reviewedPhysiological Science

Loss of Toll-Like Receptor 4 Function Partially Protects against Peripheral and Cardiac Glucose Metabolic Derangements During a Long-Term High-Fat Diet

We would like to acknowledge Matt Priest for excellent technical assistance.Diabetes is a chronic inflammatory disease that carries a high risk of cardiovascular disease. However, the pathophysiological link between these disorders is not well known. We hypothesize that TLR4 signaling mediates high fat diet (HFD)-induced peripheral and cardiac glucose metabolic derangements. Mice with a loss-of-function mutation in TLR4 (C3H/HeJ) and age-matched control (C57BL/6) mice were fed either a high-fat diet or normal diet for 16 weeks. Glucose tolerance and plasma insulin were measured. Protein expression of glucose transporters (GLUT), AKT (phosphorylated and total), and proinflammatory cytokines (IL-6, TNF-α and SOCS-3) were quantified in the heart using Western Blotting. Both groups fed a long-term HFD had increased body weight, blood glucose and insulin levels, as well as impaired glucose tolerance compared to mice fed a normal diet. TLR4-mutant mice were partially protected against long-term HFD-induced insulin resistance. In control mice, feeding a HFD decreased cardiac crude membrane GLUT4 protein content, which was partially rescued in TLR4-mutant mice. TLR4-mutant mice fed a HFD also had increased expression of GLUT8, a novel isoform, compared to mice fed a normal diet. GLUT8 content was positively correlated with SOCS-3 and IL-6 expression in the heart. No significant differences in cytokine expression were observed between groups, suggesting a lack of inflammation in the heart following a HFD. Loss of TLR4 function partially restored a healthy metabolic phenotype, suggesting that TLR4 signaling is a key mechanism in HFD-induced peripheral and cardiac insulin resistance. Our data further suggest that TLR4 exerts its detrimental metabolic effects in the myocardium through a cytokine-independent pathway.Yeshttp://www.plosone.org/static/editorial#pee

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

We would like to thank Dr. Emilie Martinez and Jill Murray for their excellent technical assistance and animal care.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.Yeshttp://www.plosone.org/static/editorial#pee

Validation of the insulin-deficient (type 1) diabetic animal model.

<p><b>A) Mean ± SE venous blood glucose concentration</b> obtained at baseline and up to 8 weeks in type 1 diabetic (T1Dx) and age-matched control (Con) mice (n = 9-11/group). <b>B) Mean ± SE body weight</b> obtained at baseline and up to 8 weeks after induction of type 1 diabetes (n = 9-11/group). <b>C) Mean ± SE serum insulin concentration</b> obtained at 8 weeks after induction of type 1 diabetes (n = 6-8/group). T1Dx: type 1 diabetic; Con: control; *P<0.05 vs. control; # P<0.05 vs. baseline.</p

Intact insulin signaling pathway in the atria of insulin-deficient diabetic animals.

<p><b>Type 1 diabetes (T1 Dx) did not alter A) Akt or B) AS160 phosphorylation in the atria</b>. Top panels: representative Western blot from total lysate of mouse atria; calsequestrin (CLSQ) was used as a loading control. Bottom panels: Mean ± SE of protein expression (values expressed relative to control; n = 4-5/group). <b>Insulin stimulates C) GLUT4 and D) GLUT8 trafficking to the atrial cell surface in type 1 diabetic (T1 Dx) subjects</b>. Top panels: representative Western blot. Bottom panels: Mean ± SE of cell surface GLUT protein content (values expressed relative to control basal labeled; n = 4-6/group). Methods: Intact mouse hearts were perfused with and without insulin, and photolabeled with bio-LC-ATB-BGPA to determine the amount of cell surface (L: labeled fraction) and intracellular (UL: unlabeled fraction) content. T1Dx: type 1 diabetic; *P<0.05 vs. control; # P<0.05 vs. basal.</p

Analysis of the downstream insulin signaling pathways in the healthy atria.

<p><b>A) Insulin stimulates phosphorylation of Akt at s473 and Th308 site</b> in atrial myocytes. Top panel: representative Western blot from total lysate of isolated rat atrial myocytes incubated with (0.01μM) and without (basal) insulin; calsequestrin (CLSQ) was used as a loading control. Bottom panel: Mean ± SE of protein expression (values expressed relative to basal; n = 5/group); # P<0.05 vs. basal. <b>B) Significant linear positive linear correlation between Akt phosphorylation (at s473 and Th308 site</b>) <b>and GLUT4 expression</b> in the healthy atria. <b>C) Insulin stimulates phosphorylation of AS160</b> in atrial myocytes. Top panel: representative Western blot from total lysate of isolated rat atrial myocytes; calsequestrin (CLSQ) was used as a loading control. Bottom panel: Mean ± SE of protein expression (values expressed relative to basal; n = 6-8/group); # P<0.05 vs. basal. <b>D) Significant linear correlation between AS160 phosphorylation and GLUT-4 and -8 expression</b> in the healthy atria.</p

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

TLR4-mutant mice were partially protected against obesity (A), hyperglycemia (B), peripheral glucose intolerance (C), and hyperinsulinemia (D) induced by a HFD.

<p>AUC = area under the curve for [glucose] measured during IPGTT. HFD = high-fat diet; ND = normal diet; * p<0.05 vs. control fed a ND, † p<0.05 vs. control fed a HFD, ‡ p<0.05 vs. TLR4-mutant fed a ND; n = 8–21 per group.</p

Correlations between pro-inflammatory cytokines and glucose transport in the heart.

<p>(A) Linear correlation between cardiac IL-6 protein expression and GLUT4 protein expression (R² = 0.029, p = 0.355). (B) Linear correlation between cardiac IL-6 protein expression and GLUT8 protein expression (R² = 0.138, p = 0.044). (C) Linear correlation between cardiac SOCS-3 protein expression and GLUT4 protein expression (R² = 0.044, p = 0.249). (D) Linear correlation between cardiac SOCS-3 protein content and GLUT8 protein content (R² = 0.132, p = 0.049). RU = relative units; HFD = high-fat diet; ND = normal diet. Trendlines show the line-of-best fit for all four groups taken together. n = 6–8 per group.</p