Mandi M. Vick

Obesity is associated with altered metabolic and reproductive activity in the mare: effects of metformin on insulin sensitivity and reproductive cyclicity

In mares, obesity is associated with continuous reproductive activity during the non-breeding season. To investigate the effect of obesity and associated alterations in metabolic parameters on the oestrous cycle, two related studies were conducted. In Experiment 1, obese (body condition score > 7) mares were fed ad libitum or were moderately feed restricted during the late summer and autumn months. Feed restriction did not alter the proportion of mares entering seasonal anoestrus. However, obese mares exhibited a significantly longer duration of the oestrous cycle, significant increases in circulating concentrations of leptin and insulin, and decreased insulin sensitivity and concentrations of thyroxine compared with feed-restricted mares throughout the experiment. Experiment 2 was designed to investigate the effects of administration of the insulin-sensitising drug metformin hydrochloride on insulin sensitivity and the characteristics of the oestrous cycle in obese mares. In a dose-response trial, metformin increased insulin sensitivity after 30 days following administration of 3 g day(-1), but not 6 or 9 g day(-1), compared with controls receiving vehicle only. However, there were no differences in insulin sensitivity or oestrous cycle characteristics between control and metformin-treated groups when the 3 g day(-1) dose was tested for a longer period of 2 months. These results demonstrate that obesity is associated with aberrations in the oestrous cycle and perturbations in several markers of metabolic status. The results also indicate that metformin is not an effective long-term monotherapy for increasing insulin sensitivity in horses at the doses tested. Additional studies are needed to examine possible effects of increasing insulin sensitivity on reproductive activity in obese mares.

Effect of body condition, body weight and adiposity on inflammatory cytokine responses in old horses

Advanced age is associated with a low-grade, systemic inflammatory response characterized by increased inflammatory cytokine production both in vitro and in vivo, termed inflamm-aging. It is also known that increased white adipose tissue, associated with obesity, leads to increased production of inflammatory cytokines. To date, it is unknown whether increased adiposity contributes to the age-related increased inflammatory status. Here we show that peripheral blood mononuclear cells (PBMC) from old horses compared to young horses have increased inflammatory cytokine production; moreover, fat old horses compared to thin old horses have even greater frequencies of lymphocytes and monocytes producing inflammatory cytokines. Therefore, we proposed that decreasing adiposity in old horses would reduce age-associated increases of inflammatory cytokines both in vitro and in vivo, and increasing adiposity in old horses would increase these measurements. To test this hypothesis further, eight old obese horses (20-28 year) were assigned to two consecutive treatments, dietary restriction (DR) during weeks 1-12 and increased dietary intake (DI) during weeks 13-30. Body weight, body condition score (BCS) and percent body fat were measured weekly. PBMC were stimulated in vitro and interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) production was measured by intracellular staining. Levels of nascent IFNgamma and TNFalpha mRNA expression were examined by RT-PCR. Serum concentrations of TNFalpha protein were also measured weekly. Reducing body weight and fat in old horses significantly reduced the percent of IFNgamma and TNFalpha positive lymphocytes and monocytes, and serum levels of TNFalpha protein. Further, when weight and fat increased in these old horses there was a significant increase in inflammatory cytokine production. Regression analysis also revealed significant relationships. These findings demonstrate that age-related obesity potentially plays a role in the dysregulation of inflammatory cytokine production by the immune system with age or inflamm-aging in the horse.

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.

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.

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.

Characterization of matrix metalloproteinase-2 and matrix metalloproteinase-9 and their inhibitors in equine granulosa cells in vivo and in vitro.

Matrix metalloproteinases (MMP) and tissue inhibitors of MMP (TIMP) regulate tissue remodeling events necessary for ovulation. Thus, changes in MMP and TIMP expression and protein enzyme activity were examined in vivo and in vitro during follicular development and atresia in the horse. Equine granulosa cells and follicular fluid from medium (15 to 29 mm) healthy and atretic follicles and from large (>30 mm) healthy and preovulatory follicles were collected by transvaginal aspiration. The cells were either snap-frozen (in vivo study) or cultured for 48 h (in vitro study) to determine gene expression and protein enzyme activity of MMP-2 and MMP-9 and TIMP-1 and TIMP-2. Concentrations of progesterone and estradiol were determined by RIA in follicular fluid and conditioned media and were used along with follicle dynamics to classify follicles. In vivo, expression of MMP-2 and TIMP-2 was increased (P < 0.05) in large-preovulatory follicles, whereas TIMP-1 was decreased. The ratio of MMP-2:TIMP-2 expression was decreased (P < 0.05) in medium-healthy and large-preovulatory follicles, whereas the MMP-9:TIMP-1 ratio was increased only in large-preovulatory follicles compared with large-healthy follicles. Estradiol was greatest (P < 0.05) in the fluid of large-healthy and large-preovulatory follicles. However, medium-atretic follicles were associated with the least estradiol concentrations, both in vivo and in vitro. Progesterone concentrations were greatest (P < 0.05) in large-preovulatory follicles both in vivo and in vitro. In healthy follicles in vivo, the diameter was correlated with estradiol concentration, the estradiol:progesterone ratio, MMP-9 and TIMP-1 expression, and MMP-2 and MMP-9 protein activity. In contrast to in vivo studies, the ratio of MMP-9:TIMP-1 expression was increased (P < 0.05) in medium-healthy follicles; TIMP-2 expression decreased in large-preovulatory follicles in vitro. In addition, MMP-9 protein activity was decreased (P < 0.05) in the media samples of cells from large-healthy follicles compared with those from medium-healthy follicles. These results indicate that changes in MMP-2 and MMP-9 activities may be essential to the tissue reorganization necessary for ovulation in the equine ovary.

Equine Neutrophils Respond to PGE2 by Activating Expression of Core Circadian Clock Genes

An emerging body of evidence supports a relationship between the circadian and immune systems during an innate immune response. Previously, we have demonstrated synchronized upregulation of core circadian clock genes, Per2 and Bmal1, in equine whole blood following lipopolysaccharide (LPS) administration in vivo. Subsequent experi- ments suggested a role for the febrile mediator, prostaglandin E2 (PGE2), in mediating this response. However, PGE2 failed to directly stimulate clock gene expression in equine PBMCs. This study demonstrates that ex vivo cultured equine neutrophils actively respond to PGE2 by upregulating Per2 and Bmal1 expression. In addition, we show that LPS induces marked neutrophilia and concomitant monocytopenia in equine peripheral blood at the time corresponding to the previ- ously observed clock gene rise. We further report that the peak in the PGE2 mediated endotoxic fever also occurs simulta- neously. Combined, our data suggest that neutrophils are the source of the rise in Per2 and Bmal1 expression previously observed in equine peripheral blood following LPS administration, and that this response is likely mediated by PGE2. These results provide the first evidence for a potential role of core circadian clock genes in neutrophil function following innate immune activation.