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15. Differential proteomic expression of equine cardiac and lamellar tissue during insulin-induced laminitis

Author

Campolo, Allison, Frantz, Matthew, de Laat, Melody A, Hartson, Steven, Furr, Martin, Lacombe, Veronique, Campolo, Allison, Frantz, Matthew, de Laat, Melody A, Hartson, Steven, Furr, Martin, and Lacombe, Veronique

Abstract

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 simi

Published
2020

27. Adverse Metabolic Effects of Diltiazem Treatment During Diabetic Cardiomyopathy.

Author

Parriman, Matt, Campolo, Allison, Waller, Amanda P., and Lacombe, Véronique A.

Subjects
DILTIAZEM, CARDIOMYOPATHIES, GLYCEMIC control
Abstract

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. [ABSTRACT FROM AUTHOR]

Published
2019
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29. Mechanisms underlying vascular and respiratory complications associated with diabetes

Author

Campolo, Allison

Abstract

Diabetes and metabolic syndrome has risen to epidemic proportions in both humans and veterinary species. Diabetes is characterized by a lack of insulin production (type 1) or action (type 2), with subsequent alterations in glucose transport and utilization in insulin-sensitive tissue. Importantly, diabetes is well known to cause multi-organ failure (including cardiovascular dysfunction). Additionally, diabetes has recently been shown to increase the risk of respiratory infection in diabetic patients. Thus, we here sought to describe the mechanisms underlying glucose dysregulation-derived organ dysfunction in several tissues (e.g., insulin-sensitive tissues, lamellae, and lung). While equine metabolic syndrome is well-known to be associated with endocrinopathic laminitis, the underlying cause of laminitic pathogenesis remains unknown. We determined that horses with laminitis induced by a 48-hour euglycemic hyperinsulinemic clamp had a significant upregulation of the insulin signaling pathway in the heart. Thus, horses with endocrinopathic laminitis were not insulin resistant. After proteomic analysis, we subsequently found an upregulation of heat shock protein 90, alpha-2-macroglobulin, and fibrinogen beta, and a downregulation of cadherin-13, vinculin, and talin-1 in lamellae of hyperinsulinemic horses. Separately, the regulation of glucose homeostasis in the lung, and how it may be affected during diabetes or during influenza infection, is under-investigated. We determined the protein and mRNA quantities of seven glucose transporter (GLUT) isoforms in the lung of healthy and diabetic mice. After infecting diabetic mice with H1N1 Influenza A (A/PR/8/34), infected and diabetic mice displayed an increased amount of glucose in BALF over control and non-infected counterparts. Insulin or metformin rescued these metabolic alterations in both non-infected and infected diabetic mice. While diabetes significantly altered protein expression of some GLUTs in the total lysate from the whole lung, influenza alone increased the cell-surface expression of insulin-sensitive GLUT4. Thus, overall, it can be concluded that alterations in glucose and insulin metabolism result in substantial alterations in the regulation of glucose transport in the lamellae of hyperinsulinemic horses, as well as in the lung of (type 1 and type 2) diabetic mice infected with influenza. Together, these findings underscore that glucose and insulin dysregulation contribute to multiple organ dysfunction during diabetes and metabolic syndrome.

Published
2019

30. Quantification of Acanthamoeba spp. Motility.

Author

Campolo A, Lara E, and Crary M

Subjects
Humans, Acanthamoeba physiology
Abstract

Acanthamoeba keratitis is a severe ocular infection that poses treatment challenges and can lead to blindness. Despite its ubiquity and potential contamination of contact lenses after water exposure, the natural behavior of this pathogen remains elusive. Understanding Acanthamoeba's movement patterns can inform us about how it colonizes contact lenses and contaminates the patient's cornea. It is fortunate that Acanthamoeba spp. are visible via brightfield microscopy starting at 4x magnification. Previous techniques have been developed to quantify Acanthamoeba motility in regard to cytopathic effects or under-exposure to the electric field. Here, we describe a method to track and quantify Acanthamoeba spp. motility long-term (hours to days), which is a protocol applicable to multiple amoeba strains, surfaces, and nutritional status of amoeba. This procedure is germane to determining many core motility quantifications, such as distance, speed, confinement, and directionality, which are necessary to monitor different stages of infection, proliferation, or behavioral change.

Published
2024
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