The impact of prolonged hyperinsulinaemia on glucose transport in equine skeletal muscle and digital lamellae

REASONS FOR PERFORMING STUDY An increased incidence of metabolic disease in horses has led to heightened recognition of the pathological consequences of insulin resistance (IR). Laminitis, failure of the weight-bearing digital lamellae, is an important consequence. Altered trafficking of specialised glucose transporters (GLUTs) responsible for glucose uptake, are central to the dysregulation of glucose metabolism and may play a role in laminitis pathophysiology. OBJECTIVES We hypothesised that prolonged hyperinsulinaemia alters the regulation of glucose transport in insulin-sensitive tissue and digital lamellae. Our objectives were to compare the relative protein expression of major GLUT isoforms in striated muscle and digital lamellae in healthy horses and during hyperinsulinaemia. STUDY DESIGN Randomised, controlled study. METHODS Prolonged hyperinsulinaemia and lamellar damage were induced by a prolonged-euglycaemic hyperinsulinaemic clamp (p-EHC) or a prolonged-glucose infusion (p-GI) and results were compared to electrolyte-treated controls. GLUT protein expression was examined with immunoblotting. RESULTS Lamellar tissue contained more GLUT1 protein than skeletal muscle (p = 0.002) and less GLUT4 than the heart (p = 0.037). During marked hyperinsulinaemia and acute laminitis (induced by the p-EHC), GLUT1 protein expression was decreased in skeletal muscle (p = 0.029) but unchanged in the lamellae, while novel GLUTs (8; 12) were increased in the lamellae (p = 0.03), but not skeletal muscle. However, moderate hyperinsulinaemia and subclinical laminitis (induced by the p-GI) did not cause differential GLUT protein expression in the lamellae vs. control horses. CONCLUSIONS The results suggest that lamellar tissue functions independently of insulin and that IR may not be an essential component of laminitis aetiology. Marked differences in GLUT expression exist between insulin-sensitive and insulin-independent tissues during metabolic dysfunction in horses. The different expression profiles of novel GLUTs during acute and subclinical laminitis may be important to disease pathophysiology and require further investigation.

Corrosion studies on the plasma-sprayed nanostructured titanium dioxide coating

Purpose: The purpose of this paper is to report the resistance of plasma-sprayed titanium dioxide (TiO2) nanostructured coatings in a corrosive environment.----- Design/methodology/approach: Weight loss studies are performed according to ASTM G31 specifications in 3.5?wt% NaCl. Electrochemical polarization resistance measurements are made according to ASTM G59-91 specifications. Corrosion resistance in a humid and corrosive environment is determined by exposing the samples in a salt spray chamber for 100?h. Microstructural studies are carried out using an atomic force microscope and scanning electron microscope.----- Findings: The nanostructured TiO2 coatings offer good resistance to corrosion, as shown by the results of immersion, electrochemical and salt spray studies. The corrosion resistance of the coating is dictated primarily by the geometry of splat lamellae, density of unmelted nanoparticles, magnitude of porosity and surface homogeneity.----- Practical implications: The TiO2 nanostructured coatings show promising potential for use as abrasion, wear-resistant and thermal barrier coatings for service in harsh environments.----- Originality/value: The paper relates the corrosion resistance of nanostructured TiO2 coatings to their structure and surface morphology.

Advanced glycation endproducts in horses with insulin-induced laminitis

Advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of cancer, inflammatory conditions and diabetic complications. An interaction of AGEs with their receptor (RAGE) results in increased release of pro-inflammatory cytokines and reactive oxygen species (ROS), causing damage to susceptible tissues. Laminitis, a debilitating foot condition of horses, occurs in association with endocrine dysfunction and the potential involvement of AGE and RAGE in the pathogenesis of the disease has not been previously investigated. Glucose transport in lamellar tissue is thought to be largely insulin-independent (GLUT-1), which may make the lamellae susceptible to protein glycosylation and oxidative stress during periods of increased glucose metabolism. Archived lamellar tissue from horses with insulin-induced laminitis (n=4), normal control horses (n=4) and horses in the developmental stages (6 h, 12 h and 24 h) of the disease (n=12) was assessed for AGE accumulation and the presence of oxidative protein damage and cellular lipid peroxidation. The equine-specific RAGE gene was identified in lamellar tissue, sequenced and is now available on GenBank. Lamellar glucose transporter (GLUT-1 and GLUT-4) gene expression was assessed quantitatively with qRT-PCR in laminitic and control horses and horses in the mid-developmental time-point (24 h) of the disease. Significant AGE accumulation had occurred by the onset of insulin-induced laminitis (48 h) but not at earlier time-points, or in control horses. Evidence of oxidative stress was not found in any group. The equine-specific RAGE gene was not expressed differently in treated and control animals, nor was the insulin-dependent glucose transporter GLUT-4. However, the glucose transporter GLUT-1 was increased in lamellar tissue in the developmental stages of insulin-induced laminitis compared to control horses and the insulin-independent nature of the lamellae may facilitate AGE formation. However, due to the lack of AGE accumulation during disease development and a failure to detect an increase in ROS or upregulation of RAGE, it appears unlikely that oxidative stress and protein glycosylation play a central role in the pathogenesis of acute, insulin-induced laminitis.

The developmental and acute phases of insulin-induced laminitis involve minimal metalloproteinase activity

Metalloproteinases have been implicated in the pathogenesis of equine laminitis and other inflammatory conditions, through their role in the degradation and remodelling of the extracellular matrix environment. Matrix metalloproteinases (MMPs) and their inhibitors are present in normal equine lamellae, with increased secretion and activation of some metalloproteinases reported in horses with laminitis associated with systemic inflammation. It is unknown whether these enzymes are involved in insulin-induced laminitis, which occurs without overt systemic inflammation. In this study, gene expression of MMP-2, MMP-9, MT1-MMP, ADAMTS-4 and TIMP-3 was determined in the lamellar tissue of normal control horses (n = 4) and horses that developed laminitis after 48 h of induced hyperinsulinaemia (n = 4), using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Protein concentrations of MMP-2 and MMP-9 were also examined using gelatin zymography in horses subject to prolonged hyperinsulinaemia for 6 h (n = 4), 12 h (n = 4), 24 h (n = 4) and 48 h (n = 4), and in normal control horses (n = 4). The only change in gene expression observed was an upregulation of MMP-9 (p < 0.05) in horses that developed insulin-induced laminitis (48 h). Zymographical analysis showed an increase (p < 0.05) in pro MMP-9 during the acute phase of laminitis (48 h), whereas pro MMP-2 was present in similar concentration in the tissue of all horses. Thus, MMP-2, MT1-MMP, TIMP-3 and ADAMTS-4 do not appear to play a significant role in the pathogenesis of insulin-induced laminitis. The increased expression of MMP-9 may be associated with the infiltration of inflammatory leukocytes, or may be a direct result of hyperinsulinaemia. The exact role of MMP-9 in basement membrane degradation in laminitis is uncertain as it appears to be present largely in the inactive form.

Histological and morphometric lesions in the pre-clinical, developmental phase of insulin-induced laminitis in Standardbred horses

Lamellar pathology in experimentally-induced equine laminitis associated with euglycaemic hyperinsulinaemia is substantial by the acute, clinical phase (∼48 h post-induction). However, lamellar pathology of the developmental, pre-clinical phase requires evaluation. The aim of this study was to analyse lamellar lesions both qualitatively and quantitatively, 6, 12 and 24 h after the commencement of hyperinsulinaemia. Histological and histomorphometrical analyses of lamellar pathology at each time-point included assessment of lamellar length and width, epidermal cell proliferation and death, basement membrane (BM) pathology and leucocyte infiltration. Archived lamellar tissue from control horses and those with acute, insulin-induced laminitis (48 h) was also assessed for cellular proliferative activity by counting the number of cells showing positive nuclear immuno labelling for TPX2. Decreased secondary epidermal lamellar (SEL) width and increased histomorphological evidence of SEL epidermal basal (and supra-basal) cell death occurred early in disease progression (6 h). Increased cellular proliferation in SELs, infiltration of the dermis with small numbers of leucocytes and BM damage occurred later (24 and 48 h). Some lesions, such as narrowing of the SELs, were progressive over this time period (6–48 h). Cellular pathology preceded leucocyte infiltration and BM pathology, indicating that the latter changes may be secondary or downstream events in hyperinsulinaemic laminitis.

A potential role for lamellar insulin-like growth factor-1 receptor in the pathogenesis of hyperinsulinaemic laminitis

The reason why a sustained high concentration of insulin induces laminitis in horses remains unclear. Cell proliferation occurs in the lamellae during insulin-induced laminitis and in other species high concentrations of insulin can activate receptors for the powerful cell mitogen, insulin-like growth factor (IGF)-1. The first aim of this study was to determine if IGF-1 receptors (IGF-1R) are activated in the hoof during insulin-induced laminitis. Gene expression for IGF-1R and the insulin receptor (InsR) was measured using qRT-PCR, in lamellar tissue from control horses and from horses undergoing a prolonged euglycaemic, hyperinsulinaemic clamp (p-EHC), during the mid-developmental (24 h) and acute (46 h) phases of insulin-induced laminitis. Gene expression for both receptors was decreased 13–32-fold (P < 0.05) at both time-points in the insulin-treated horses. A second aim was to determine if the down-regulation of the receptor genes could be accounted for by an increase in circulating IGF-1. Serum IGF-1 was measured at 0, 10, 25 and 46 h post-treatment in horses given a p-EHC for approximately 46 h, and in matched controls administered a balanced, electrolyte solution. There was no increase in serum IGF-1 concentrations during the p-EHC, consistent with down-regulation of both receptors by insulin. Stimulation of the IGF-1R by insulin may lead to inappropriate lamellar epidermal cell proliferation and lamellar weakening, a potential mechanism for hyperinsulinaemic laminitis. Targeting this receptor may provide insights into the pathogenesis or identify a novel therapy for hyperinsulinaemic laminitis.

Histopathological examination of chronic laminitis in Kaimanawa feral horses of New Zealand

AIMS: To investigate the prevalence, histopathological and histomorphometric presentation of chronic laminitis in a population of Kaimanawa feral horses. METHODS: Following the capture and euthanasia of feral horses from the Kaimanawa Ranges of New Zealand, the left forefoot of 28 stallions and 28 mares aged between 6 and 12 years were removed and processed for histology. Sections of lamellar samples from each horse were examined using light microscopy. The presence of laminitis was assessed and the histopathological lesions were described. Horses were grouped by histological diagnosis into laminitic and non-laminitic groups and histomorphometric analysis was conducted and compared between groups. The parameters examined were total length of primary epidermal lamellae (PEL), keratinised length of PEL, and the length of secondary epidermal lamellae (SEL) at the abaxial end and axial end of each PEL. RESULTS: Of the horses examined, 25 (45%) were diagnosed with chronic laminitis. The most prevalent histopathological features were the presence of excessive cap horn, and multi-branched and attenuated SEL. Histomorphometric assessment of the lamellar architecture revealed no difference in morphometric measurements between the normal and laminitic groups for any parameter measured (p>0.05). CONCLUSIONS: The current study found a high prevalence of laminitis in feral Kaimanawa horses. The reason for this in the Kaimanawa population is not known. Histomorphometric analysis may not be a good indicator of chronic laminitis in feral horses. CLINICAL RELEVANCE: It is an important finding that the feral horse lifestyle in the environment of the Kaimanawa Ranges in New Zealand offers no protection against foot disease. The finding suggests that horses are vulnerable to laminitis whether in domestic care or in a feral habitat.

Insulin and the insulin-like growth factor system : a novel theory for hyperinsulinemic laminitis pathogenesis

Research into hyperinsulinemic laminitis has progressed significantly in recent years with the use of the prolonged-euglycemic, hyperinsulinemic clamp (p-EHC). Previous investigations of laminitis pathophysiology have focused on digital vascular dysfunction, inflammation, altered glucose metabolism within the lamellae, and lamellar basement membrane breakdown by metalloproteinases. The etiopathogenesis of laminitis occurring in association with hyperinsulinemia is yet to be fully characterized, but it may not involve these mechanisms. Insulin stimulates cellular proliferation and can also affect other body systems, such as the insulin-like growth factor (IGF) system. Insulin-like growth factor-1 (IGF-1) is structurally homologous to insulin and, like insulin, binds with strong affinity to a specific tyrosine kinase receptor on the cell surface to produce its effects, which include promoting cell proliferation. Receptors for IGF-1 (IGF-1R) are present in the lamellar epidermis. An alternative theory for the pathogenesis of hyperinsulinemic laminitis is that uncontrolled cell proliferation, mediated through both the insulin receptor (InsR) and IGF-1R, leads to lengthening, weakening, and failure of the lamellae. An analysis of the proliferative activity of lamellar epidermal cells during the developmental and acute phases of hyperinsulinemic laminitis, and lamellar gene expression of the InsR and IGF-1R was undertaken.

Evaluation of primary epidermal lamellar density in the forefeet of near-term fetal Australian feral and domesticated horses

Objective: To investigate the density of the primary epidermal lamellae (PEL) around the solar circumference of the forefeet of near-term fetal feral and nonferal (ie, domesticated) horses. Sample: Left forefeet from near-term Australian feral (n = 14) and domesticated (4) horse fetuses. Procedures: Near-term feral horse fetuses were obtained from culled mares within 10 minutes of death; fetuses that had died in utero 2 weeks prior to anticipated birth date and were delivered from live Thoroughbred mares were also obtained. Following disarticulation at the carpus, the left forefoot of each fetus was frozen during dissection and data collection. In a standard section of each hoof, the stratum internum PEL density was calculated at the midline center (12 o'clock) and the medial and lateral break-over points (11 and 1 o'clock), toe quarters (10 and 2 o'clock), and quarters (4 and 6 o'clock). Values for matching lateral and medial zones were averaged and expressed as 1 density. Density differences at the 4 locations between the feral and domesticated horse feet were assessed by use of imaging software analysis. Results: In fetal domesticated horse feet, PEL density did not differ among the 4 locations. In fetal feral horse feet, PEL density differed significantly among locations, with a pattern of gradual reduction from the dorsal to the palmar aspect of the foot. The PEL density distribution differed significantly between fetal domesticated and feral horse feet. Conclusions and Clinical Relevance: Results indicated that PEL density distribution differs between fetal feral and domesticated horse feet, suggestive of an adaptation of feral horses to environment challenges.

Novel keratins identified by quantitative proteomic analysis as the major cytoskeletal proteins of equine (Equus caballus) hoof lamellar tissue

The dermo-epidermal interface that connects the equine distal phalanx to the cornified hoof wall withstands great biomechanical demands, but is also a region where structural failure often ensues as a result of laminitis. The cytoskeleton in this region maintains cell structure and facilitates intercellular adhesion, making it likely to be involved in laminitis pathogenesis, although it is poorly characterized in the equine hoof lamellae. The objective of the present study was to identify and quantify the cytoskeletal proteins present in the epidermal and dermal lamellae of the equine hoof by proteomic techniques. Protein was extracted from the mid-dorsal epidermal and dermal lamellae from the front feet of 5 Standardbred geldings and 1 Thoroughbred stallion. Mass spectrometry-based spectral counting techniques, PAGE, and immunoblotting were used to identify and quantify cytoskeletal proteins, and indirect immunofluorescence was used for cellular localization of K14 and K124 (where K refers to keratin). Proteins identified by spectral counting analysis included 3 actin microfilament proteins; 30 keratin proteins along with vimentin, desmin, peripherin, internexin, and 2 lamin intermediate filament proteins; and 6 tubulin microtubule proteins. Two novel keratins, K42 and K124, were identified as the most abundant cytoskeletal proteins (22.0 ± 3.2% and 23.3 ± 4.2% of cytoskeletal proteins, respectively) in equine hoof lamellae. Immunoreactivity to K14 was localized to the basal cell layer, and that to K124 was localized to basal and suprabasal cells in the secondary epidermal lamellae. Abundant proteins K124, K42, K14, K5, and α1-actin were identified on 1- and 2-dimensional polyacrylamide gels and aligned with the results of previous studies. Results of the present study provide the first comprehensive analysis of cytoskeletal proteins present in the equine lamellae by using mass spectrometry-based techniques for protein quantification and identification.

Molecular characterization of "Candidatus Parilichlamydia carangidicola," a novel Chlamydia-like epitheliocystis agent in yellowtail kingfish, Seriola lalandi (Valenciennes), and the proposal of a new family, "Candidatus Parilichlamydiaceae" fam. nov. (order Chlamydiales).

Three cohorts of farmed yellowtail kingfish (Seriola lalandi) from South Australia were examined for Chlamydia-like organisms associated with epitheliocystis. To characterize the bacteria, 38 gill samples were processed for histopathology, electron microscopy, and 16S rRNA amplification, sequencing, and phylogenetic analysis. Microscopically, the presence of membrane-enclosed cysts was observed within the gill lamellae. Also observed was hyperplasia of the epithelial cells with cytoplasmic vacuolization and fusion of the gill lamellae. Transmission electron microscopy revealed morphological features of the reticulate and intermediate bodies typical of members of the order Chlamydiales. A novel 1,393-bp 16S chlamydial rRNA sequence was amplified from gill DNA extracted from fish in all cohorts over a 3-year period that corresponded to the 16S rRNA sequence amplified directly from laser-dissected cysts. This sequence was only 87% similar to the reported "Candidatus Piscichlamydia salmonis" (AY462244) from Atlantic salmon and Arctic charr. Phylogenetic analysis of this sequence against 35 Chlamydia and Chlamydia-like bacteria revealed that this novel bacterium belongs to an undescribed family lineage in the order Chlamydiales. Based on these observations, we propose this bacterium of yellowtail kingfish be known as "Candidatus Parilichlamydia carangidicola" and that the new family be known as "Candidatus Parilichlamydiaceae."

Toll-like receptor and pro-inflammatory cytokine expression during prolonged hyperinsulinaemia in horses : implications for laminitis

Equine laminitis, a disease of the lamellar structure of the horse’s hoof, can be incited by numerous factors that include inflammatory and metabolic aetiologies. However, the role of inflammation in hyperinsulinaemic laminitis has not been adequately defined. Tolllike receptor (TLR) activation results in up-regulation of inflammatory pathways and the release of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-�), and may be a pathogenic factor in laminitis. The aim of this study was to determine whether TLR4 expression and subsequent pro-inflammatory cytokine production is increased in lamellae and skeletal muscle during equine hyperinsulinaemia. Standardbred horses were treated with either a prolonged, euglycaemic hyperinsulinaemic clamp (p-EHC) or a prolonged, glucose infusion (p-GI), which induced marked and moderate hyperinsulinaemia, respectively. Age-matched control horses were treated simultaneously with a balanced electrolyte solution. Treated horses developed clinical (p-EHC) or subclinical (p-GI) laminitis, whereas controls did not. Skeletal muscle and lamellar protein extracts were analysed by Western blotting for TLR4, IL-6, TNF-� and suppressor of cytokine signalling 3 (SOCS3) expression. Lamellar protein expression of TLR4 and TNF-�, but not IL-6, was increased by the p-EHC, compared to control horses. A significant positive correlation was found between lamellar TLR4 and SOCS3. Skeletal muscle protein expression of TLR4 signalling parameters did not differ between control and p-EHC-treated horses. Similarly, the p-GI did not result in up-regulation of lamellar protein expression of any parameter. The results suggest that insulin-sensitive tissues may not accurately reflect lamellar pathology during hyperinsulinaemia. While TLR4 is present in the lamellae, its activation appears unlikely to contribute significantly to the developmental pathogenesis of hyperinsulinaemic laminitis. However, inflammation may have a role to play in the later stages (e.g., repair or remodelling) of the disease.

Metabolic dysfunction and laminitis

Objective: This review focuses on laminitis that develops as a result of metabolic dysfunction and aims to provide a concise assessment of the current state of knowledge on this form of the disease. Outline: The most prevalent form of laminitis is associated with metabolic or endocrinopathic diseases, such as Equine Metabolic Syndrome and pituitary pars intermedia dysfunction, and the feeding of high-energy diets, particularly those rich in non-structural carbohydrates. Insulin dysregulation is the key hormonal imbalance implicated in causing this form of laminitis and hyperinsulinaemia is an important risk factor for the disease. Hyperinsulinaemia can occur in association with insulin resistance, obesity, regionalised adiposity, dysregulated cortisol metabolism and may also be related to other factors, such as breed and genetic predisposition. Recognition of hyperinsulinaemia is best achieved by using a dynamic oral glucose test that can be performed relatively easily under field conditions. Insulin produces a unique pathological lesion in the lamellae and the features of this lesion have informed investigations on the pathogenesis of the disease. Research into the mechanism of disease is continuing so that more targeted therapies than are currently available can be developed. However, dietary restriction and exercise remain effective management strategies for metabolic disease. Conclusions: Although the pathogenic mechanism/s of metabolic and endocrinopathic forms of laminitis remain the subject of intense research, ample data on risk factors for the disease are available. Efforts focussed on preventing the disease should aim to identify metabolic disease and reduce obesity and insulin resistance in at-risk individuals.