Effects of systemic inflammation on insulin sensitivity in horses and inflammatory cytokine expression in adipose tissue.

OBJECTIVE: To determine whether an inflammatory challenge induces insulin resistance in horses and examine possible contributions of adipose tissue to inflammatory cytokine production. ANIMALS: 15 adult mares. PROCEDURES: Lipopolysaccharide (0.045 mug/kg, IV) or saline solution was administered, and insulin sensitivity was determined by means of the hyperinsulinemic, euglycemic clamp procedure or an adipose tissue biopsy was performed. Adipose tissue samples were collected, and mature adipocytes were obtained. Mature adipocytes were stimulated with lipopolysaccharide or dedifferentiated into preadipocytes and then stimulated with lipopolysaccharide. Interleukin-1, interleukin-6, and tumor necrosis factor A expression in blood, adipose tissue, and adipocytes was quantified with a real-time, reverse transcriptase- PCR assay. RESULTS: Lipopolysaccharide induced a transient increase in insulin sensitivity followed by a reduction in insulin sensitivity at 24 hours. Increased cytokine expression was observed in blood and adipose tissue following administration of lipopolysaccharide, and adipocytes and preadipocytes stimulated with lipopolysaccharide stained positive for tumor necrosis factor A. Expression of interleukin-1, interleukin-6, and tumor necrosis factor A was detected in preadipocytes stimulated with lipopolysaccharide, and interleukin-6 and tumor necrosis factor A were detected in mature adipocytes stimulated with lipopolysaccharide. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that insulin resistance develops following systemic inflammation in horses and suggested that adipose tissue may contribute to this inflammatory response. Methods to regulate insulin sensitivity may improve clinical outcome in critically ill patients.

Rapid phase adjustment of melatonin and core body temperature rhythms following a 6-h advance of the light/dark cycle in the horse.

BACKGROUND: Rapid displacement across multiple time zones results in a conflict between the new cycle of light and dark and the previously entrained program of the internal circadian clock, a phenomenon known as jet lag. In humans, jet lag is often characterized by malaise, appetite loss, fatigue, disturbed sleep and performance deficit, the consequences of which are of particular concern to athletes hoping to perform optimally at an international destination. As a species renowned for its capacity for athletic performance, the consequences of jet lag are also relevant for the horse. However, the duration and severity of jet lag related circadian disruption is presently unknown in this species. We investigated the rates of re-entrainment of serum melatonin and core body temperature (BT) rhythms following an abrupt 6-h phase advance of the LD cycle in the horse. METHODS: Six healthy, 2 yr old mares entrained to a 12 h light/12 h dark (LD 12:12) natural photoperiod were housed in a light-proofed barn under a lighting schedule that mimicked the external LD cycle. Following baseline sampling on Day 0, an advance shift of the LD cycle was accomplished by ending the subsequent dark period 6 h early. Blood sampling for serum melatonin analysis and BT readings were taken at 3-h intervals for 24 h on alternate days for 11 days. Disturbances to the subsequent melatonin and BT 24-h rhythms were assessed using repeated measures ANOVA and analysis of Cosine curve fitting parameters. RESULTS: We demonstrate that the equine melatonin rhythm re-entrains rapidly to a 6-h phase advance of an LD12:12 photocycle. The phase shift in melatonin was fully complete on the first day of the new schedule and rhythm phase and waveform were stable thereafter. In comparison, the advance in the BT rhythm was achieved by the third day, however BT rhythm waveform, especially its mesor, was altered for many days following the LD shift. CONCLUSION: Aside from the temperature rhythm disruption, rapid resynchronization of the melatonin rhythm suggests that the central circadian pacemaker of the horse may possess a particularly robust entrainment response. The consequences for athletic performance remain unknown.

Acute systemic inflammation transiently synchronizes clock gene expression in equine peripheral blood.

Peripheral clocks receive timing signals from the master mammalian pacemaker in the suprachiasmatic nucleus (SCN) and function to adaptively anticipate daily changes that influence local physiology. Evidence suggests that peripheral immune activation may act as a resetting signal for circadian clocks in peripheral tissues. We wished to investigate whether acute systemic inflammation could synchronize clock gene expression in equine peripheral blood, a tissue that does not normally oscillate in this species. We report that in vivo administration of lipopolysaccharide (LPS) results in significant upregulation of the core clock genes Per2 and Bmal1 in equine blood, in association with an acute rise in tumor necrosis factor (TNF) alpha and core body temperature compared to vehicle-treated control animals. Furthermore, co-administration of LPS and phenylbutazone, a non-steroidal anti-inflammatory drug (NSAID) known to inhibit prostaglandin (PG) E(2) synthesis in the horse, prevents both the febrile response and the synchronized increase in clock gene expression. However, the rise in Per2 and Bmal1 expression cannot be replicated in equine peripheral blood mononuclear cells (PBMCs) ex vivo by treatment with PGE(2), LPS or a heat shock mimicking the in vivo febrile response. These results may suggest an indirect communication pathway between immune modulators and the molecular machinery of cell clocks in peripheral blood. This potential immune feedback regulation of an equine peripheral clock implies a role for the circadian system in contributing to innate immune reactions and maintaining homeostasis in a tissue that acts as the first line of defense during an infectious challenge.

Evidence of an oscillating peripheral clock in an equine fibroblast cell line and adipose tissue but not in peripheral blood.

The master mammalian pacemaker in the brain controls numerous diverse physiological and behavioral processes throughout the organism. Timing information is continually transmitted from the master clock to peripheral organs to synchronize rhythmic daily oscillations of clock gene transcripts and control local physiology. To investigate the presence of peripheral clocks in the horse, quantitative real-time RT-PCR assays were designed to detect levels of equine clock genes. Expression profiles for Per2, Bmal1 and Cry1 were first determined in a synchronized equine cell line. Subsequently, expression in equine whole blood and adipose tissue was assessed. Robust circadian oscillations of Per2, Bmal1 and Cry1 were observed in vitro. A synchronized molecular clock was also demonstrated in equine adipose tissue although oscillation of Bmal1 was less robust than that of Per2 and Cry1. In contrast to previous studies in humans and rats however, there was no evidence of synchronized clock gene expression in equine peripheral blood. These studies suggest that synchronous control of clock gene oscillation in equine peripheral blood is not as tightly regulated as in other species and may reflect the influence of different evolutionary challenges modifying the function of a peripheral clock.