Martin N. Sillence

Equine laminitis: induced by 48 h hyperinsulinaemia in Standardbred horses.

REASONS FOR PERFORMING STUDY Hyperinsulinaemia is known to induce laminitis experimentally in healthy ponies with no history of the condition. Horses are more insulin sensitive than ponies and whether prolonged hyperinsulinaemia and euglycaemia would have a similar laminitogenic effect requires study. OBJECTIVES To determine if laminitis results when the prolonged euglycaemic hyperinsulinaemic clamp technique (p-EHC) is applied to clinically normal Standardbred horses, and to monitor hoof wall temperature seeking an association between vascular activity and laminitis development. METHODS Eight young, clinically normal Standardbred horses were assigned into 4 pairs and within each pair, one was assigned randomly to either treatment (n = 4) or control (n = 4) groups. Treated horses received continuous infusions of insulin and glucose until clinical signs of laminitis developed, at which point the horses were subjected to euthanasia. Control horses received an equivalent volume of a balanced electrolyte infusion for the same period. Hoof wall surface temperature (HWST) was monitored continuously throughout the experimental period. RESULTS All horses in the treatment group were calculated to have normal insulin sensitivity. All treated horses, and none in the control group, developed laminitis (P = 0.01). Pronounced digital pulses were a feature of the treatment group, while insignificant digital pulses occurred in control horses. HWST was higher and less variable in treated horses once hyperinsulinaemia was established. CONCLUSIONS Healthy Standardbred horses subjected to prolonged hyperinsulinaemia develop laminitis within 48 h, demonstrating that laminitis in horses can be triggered by insulin. POTENTIAL RELEVANCE Insulin resistance and the associated hyperinsulinaemia place horses and ponies at risk of developing laminitis. This study demonstrates a need for prompt management of the persistent hyperinsulinaemia seen in some endocrinopathies.

β2‐Agonist administration reverses muscle wasting and improves muscle function in aged rats

The β2‐adrenoceptor agonist (β2‐agonist) fenoterol has potent anabolic effects on rat skeletal muscle. We conducted an extensive dose–response study to determine the most efficacious dose of fenoterol for increasing skeletal muscle mass in adult rats and used this dose in testing the hypothesis that fenoterol may have therapeutic potential for ameliorating age‐related muscle wasting and weakness. We used adult (16‐month‐old) rats that had completed their growth and development, and old (28‐month‐old) rats that exhibited characteristic muscle wasting and weakness, and treated them daily with either fenoterol (1.4 mg kg−1, i.p), or saline vehicle, for 4 weeks. Following treatment, functional characteristics of fast‐twitch extensor digitorum longus (EDL) and predominantly slow‐twitch soleus muscles of the hindlimb were assessed in vitro. Untreated old rats exhibited a loss of skeletal muscle mass and a decrease in force‐producing capacity, in both fast and slow muscles, compared with adult rats (P < 0.05). However, there was no age‐associated decrease in skeletal muscle β‐adrenoceptor density, nor was the muscle response to chronic β‐agonist stimulation reduced with age. Thus, muscle mass and force‐producing capacity of EDL and soleus muscles from old rats treated with fenoterol was equivalent to, or greater than, untreated adult rats. The increase in mass and strength was attributed to a non‐selective increase in the cross‐sectional area of all muscle fibre types, in both the EDL and soleus. Fenoterol treatment caused a small increase in fatiguability due to a decrease in oxidative metabolism in both EDL and soleus muscles, with some cardiac hypertrophy. Further studies are needed to fully separate the desirable effects on skeletal muscle and the undesirable effects on the heart. Nevertheless, our results demonstrate that fenoterol is a powerful anabolic agent that can restore muscle mass and strength in old rats, and provide preliminary evidence of therapeutic potential for age‐related muscle wasting and weakness.

Systemic administration of β2‐adrenoceptor agonists, formoterol and salmeterol, elicit skeletal muscle hypertrophy in rats at micromolar doses

β2‐Adrenoceptor agonists provide a potential therapy for muscle wasting and weakness, but their use may be limited by adverse effects on the heart, mediated in part, by β1‐adrenoceptor activation. Two β2‐agonists, formoterol and salmeterol, are approved for treating asthma and have an extended duration of action and increased safety, associated with greater β2‐adrenoceptor selectivity. The pharmacological profiles of formoterol and salmeterol and their effects on skeletal and cardiac muscle mass were investigated in 12‐week‐old, male F344 rats. Formoterol and salmeterol were each administered via daily i.p. injection at one of seven doses (ranging from 1 to 2000 μg kg−1 day−1), for 4 weeks. Rats were anaesthetised and the EDL and soleus muscles and the heart were excised and weighed. Dose–response curves were constructed based on skeletal and cardiac muscle hypertrophy. Formoterol was more potent than salmeterol, with a significantly lower ED50 in EDL muscles (1 and 130 μg kg−1 day−1, P <0.05), whereas salmeterol had greater intrinsic activity than formoterol in both EDL and soleus muscles (12% greater hypertrophy than formoterol). The drugs had similar potency and intrinsic activity in the heart, with a smaller leftward shift for formoterol than seen in skeletal muscle. A dose of 25 μg kg−1 day−1 of formoterol elicited greater EDL and soleus hypertrophy than salmeterol, but resulted in similar β‐adrenoceptor downregulation. These results show that doses as low as 1 μg kg−1 day−1 of formoterol can elicit significant muscle hypertrophy with minimal cardiac hypertrophy and provide important information regarding the potential therapeutic use of formoterol and salmeterol for muscle wasting.

Evaluation of commercially available assays for the measurement of equine insulin

Determining circulating equine insulin concentrations is becoming increasingly important in equine clinical practice and research. Most available assays are optimized for human medicine, but there is strong equine cross-reactivity because of the highly conserved nature of insulin. To identify an accurate and reliable assay for equine insulin, 6 commercial immunoassays were evaluated for precision, accuracy, and specificity. Only 1 assay initially reached the requisite standard: Mercodia Equine Insulin Enzyme-linked Immunosorbent assay (ELISA). Plasma matrix interferences were identified when the provided assay buffer was used with the Siemens Count-a-Coat Insulin radioimmunoassay (RIA) but not when charcoal-stripped equine plasma was used as the diluent. This modified RIA and the Mercodia Equine Insulin ELISA were evaluated further by directly examining accuracy by comparing their results for 18 equine plasma samples with values obtained using liquid chromatography and high-resolution/high-accuracy mass spectrometry (LC-MS). Compared with LC-MS measurements, the modified Siemens Insulin RIA rendered a moderate Lins concordance coefficient (ρ(c)) of 0.41, whereas the Mercodia Equine Insulin ELISA rendered a very poor ρ(c) of 0.06. This suggests that the Siemens Insulin RIA is appropriate to use for routine evaluations when LC-MS is not available.

Equine laminitis: comparative histopathology 48 hours after experimental induction with insulin or alimentary oligofructose in standardbred horses.

Laminitis has many triggers and comparing the histopathology of lesions induced by different causes may help to establish whether a common mechanism or multiple pathologies are involved. The aim of this study was to describe the microscopical lesions and to quantify morphometric changes in the lamellae of horses with insulin-induced (n=4) and oligofructose (OF)-induced laminitis (n=4) compared with normal controls (n=4). Archived lamellar samples collected during two previous studies were used. Laminitis was induced within 48 h in standardbred horses with either a euglycaemic, hyperinsulinaemic clamp (EHC) technique or, in a separate experiment, with an overdose of alimentary OF. Normal tissue was obtained from control horses in the EHC experiment that received a balanced electrolyte solution intravenously for 48 h. Six measurements of lamellar length and width were recorded for each hoof. Leucocyte infiltration was assessed by immunolocalization of calprotectin. All control horses exhibited normal lamellar architecture, whereas treated horses developed clinical and histopathological changes consistent with laminitis. Laminitic samples displayed lengthening and narrowing of secondary epidermal lamellae (SELs), rounded epidermal basal cell (EBC) nuclei, mitosis and apoptosis. In the fore feet of laminitic horses, the length from the end of the keratinized axis to the axial tip of the primary epidermal lamellae (PELs) was increased (P<0.05). SELs were significantly longer (P<0.05) and narrower (P<0.05) in the treated horses compared with controls. The two treated groups did not differ from each other in SEL length or width. Calprotectin expression was absent in control horses, moderate in hyperinsulinaemic horses and marked in OF-treated horses. Laminitis induced experimentally with insulin or OF results in comparable lengthening and narrowing of the SELs and elongation of the axial end of the PELs at 48 h. Immunolocalization of calprotectin indicated that hyperinsulinaemia induces less leucocyte emigration than carbohydrate overload at 48 h. The microscopical lesion of laminitis is similar, but not identical in different forms of the disease.

Glucose transport in the equine hoof

REASONS FOR PERFORMING STUDY Several conditions associated with laminitis in horses are also associated with insulin resistance, which represents the failure of glucose uptake via the insulin-responsive glucose transport proteins in certain tissues. Glucose starvation is a possible mechanism of laminitis, but glucose uptake mechanisms in the hoof are not well understood. OBJECTIVES To determine whether glucose uptake in equine lamellae is dependent on insulin, to characterise the glucose transport mechanism in lamellae from healthy horses and ponies, and to compare this with ponies with laminitis. METHODS Study 1 investigated the effects of insulin (300 µU/ml; acute and 24 h) and various concentrations of glucose up to 24 mmol/l, on 2-deoxy-D-[2,6-(3)H] glucose uptake in hoof lamellar explants in vitro. Study 2 measured the mRNA expression of GLUT1 and GLUT4 transport proteins by PCR analysis in coronary band and lamellar tissue from healthy horses and ponies, ponies with insulin-induced laminitis, and ponies suffering from chronic laminitis as a result of equine Cushings syndrome. RESULTS Glucose uptake was not affected by insulin. Furthermore, the relationship between glucose concentration and glucose uptake was consistent with an insulin-independent glucose transport system. GLUT1 mRNA expression was strong in brain, coronary band and lamellar tissue, but was weak in skeletal muscle. Expression of GLUT4 mRNA was strong in skeletal muscle, but was either absent or barely detectable in coronary band and lamellar tissue. CONCLUSIONS The results do not support a glucose deprivation model for laminitis, in which glucose uptake in the hoof is impaired by reduced insulin sensitivity. Hoof lamellae rely on a GLUT1-mediated glucose transport system, and it is unlikely that GLUT4 proteins play a substantial role in this tissue. POTENTIAL RELEVANCE Laminitis associated with insulin resistance is unlikely to be due to impaired glucose uptake and subsequent glucose deprivation in lamellae.

Histopathology of insulin-induced laminitis in ponies.

REASONS FOR PERFORMING STUDY Ponies with laminitis associated with insulin resistance and hyperinsulinaemia lack systemic and/or intestinal inflammatory signs, suggesting a different pathogenesis potentially reflected in differing histopathology. OBJECTIVES To describe the histological appearance and quantify morphological changes in primary and secondary epidermal lamellae (PEL and SEL) of laminitis lesions from ponies with insulin-induced laminitis. METHODS Equine hoof lamellar tissue was obtained from 4 control ponies and 5 ponies with laminitis induced following infusion of insulin (1036 ± 55 µU/ml) while maintaining euglycaemia for 55.4 ± 5.5 h. Sections from all 4 hooves were stained and examined by a veterinary pathologist. Measurements of lamellar length (PEL and SEL) were made in mid-dorsal sections of the right forefeet by 2 blinded observers. Immunolabelling for calprotectin was performed using a monoclonal antibody. RESULTS No lesions were detected in normal ponies. Lesions detected in ponies with laminitis were variable in severity between ponies. Within ponies, SEL lesions were more severe along the axial region of PEL. Lesions included swelling, disorganisation and abnormal keratinisation of epidermal cells, increased mitotic activity and apoptosis. Separation of basement membranes was minimal. Immunostaining revealed inflammatory cells within the lamellar dermis. SEL were significantly elongated in laminitic hooves relative to controls, with the greatest elongation in those attached to abaxial and middle regions of PEL. CONCLUSIONS Laminitis induced by prolonged infusion of insulin lacked widespread basement membrane disintegration, and increases in epidermal cellular proliferation at axial aspects were marked for this acute stage of disease. POTENTIAL RELEVANCE Defining equine laminitis entirely in terms of separation of the basement membrane may not be appropriate for laminitis associated with hyperinsulinaemia.

Equine laminitis: Ultrastructural lesions detected in ponies following hyperinsulinaemia

REASONS FOR PERFORMING STUDY Anatomical changes in the hoof lamellar tissue induced by prolonged hyperinsulinaemia have not been described previously. Analysis of the induced lesions may promote understanding of hyperinsulinaemic laminitis pathogenesis and produce clinical benefit. OBJECTIVES To use light and transmission electron microscopy (TEM) to document hoof lamellar lesions in ponies clinically lame after prolonged hyperinsulinaemia. METHODS Nine clinically normal, mature ponies were allocated randomly to either a treatment group (n = 5) or control group (n = 4). The treatment group received insulin via a modified, prolonged euglycaemic hyperinsulinaemic clamp technique (EHCT) and were subjected to euthanasia when clinical signs of Obel grade II laminitis occurred. The control group was sham treated with an equivalent volume of 0.9% saline and killed at 72 h. Lamellar tissues of the right front feet were harvested and processed for TEM. RESULTS Lamellae from insulin treated ponies were attenuated and elongated with many epidermal basal cells (EBC) in mitosis. Unlike carbohydrate induced laminitis in horses there was no global separation at the lamellar dermal/epidermal interface among ponies. Sporadic EBC basement membrane (BM) separation was associated with the proximity of infiltrating leucocytes. In 2 ponies, the lamellar BM was thickened. The number of hemidesmosomes/microm of BM was decreased in all insulin treated ponies. CONCLUSIONS Prolonged hyperinsulinaemia causes unique lamellar lesions normally characteristic of acute and chronic laminitis. Lamellar proliferation may be an insulin effect through its mitogenic pathway. Aberrant lamellar mitosis may lengthen and weaken the lamellar, distal phalanx attachment apparatus and contribute to the clinical signs that developed. POTENTIAL RELEVANCE The study shows that insulin alone, in higher than normal circulating concentrations, induces profound, changes in lamellar anatomy. Medical control of insulin resistance and hyperinsulinaemia may ameliorate lesions and produce clinical benefit.

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 (∼48h 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 24h 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 (48h) 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 (6h). Increased cellular proliferation in SELs, infiltration of the dermis with small numbers of leucocytes and BM damage occurred later (24 and 48h). Some lesions, such as narrowing of the SELs, were progressive over this time period (6-48h). Cellular pathology preceded leucocyte infiltration and BM pathology, indicating that the latter changes may be secondary or downstream events in hyperinsulinaemic laminitis.

Equine hyperinsulinemia: investigation of the enteroinsular axis during insulin dysregulation

Compared with some other species, insulin dysregulation in equids is poorly understood. However, hyperinsulinemia causes laminitis, a significant and often lethal disease affecting the pedal bone/hoof wall attachment site. Until recently, hyperinsulinemia has been considered a counterregulatory response to insulin resistance (IR), but there is growing evidence to support a gastrointestinal etiology. Incretin hormones released from the proximal intestine, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide, augment insulin secretion in several species but require investigation in horses. This study investigated peripheral and gut-derived factors impacting insulin secretion by comparing the response to intravenous (iv) and oral d-glucose. Oral and iv tests were performed in 22 ponies previously shown to be insulin dysregulated, of which only 15 were classified as IR (iv test). In a more detailed study, nine different ponies received four treatments: d-glucose orally, d-glucose iv, oats, and commercial grain mix. Insulin, glucose, and incretin concentrations were measured before and after each treatment. All nine ponies showed similar iv responses, but five were markedly hyperresponsive to oral d-glucose and four were not. Insulin responsiveness to oral d-glucose was strongly associated with blood glucose concentrations and oral glucose bioavailability, presumably driven by glucose absorption/distribution, as there was no difference in glucose clearance rates. Insulin was also positively associated with the active amide of GLP-1 following d-glucose and grain. This study has confirmed a functional enteroinsular axis in ponies that likely contributes to insulin dysregulation that may predispose them to laminitis. Moreover, iv tests for IR are not reliable predictors of the oral response to dietary nonstructural carbohydrate.