Leptin and ghrelin play an important role in controlling food intake and energy balance (1–3). Recent studies focusing on the pathophysiological mechanisms of sepsis have shown that leptin (4–12) and ghrelin (13–19) hormones may have a role as proinflammatory mediators during endotoxemia. Available data on plasma leptin response to endotoxin in various animal species and humans show considerable variation. Some authors reported that endotoxin did not affect circulating leptin levels in humans (20, 21) and farm animals (22, 23), whereas others showed that serum leptin levels were increased in humans (9, 16), nonhuman primates (16, 17), hamsters (5), mice (6–8), and rats (9, 10) by endotoxin. Contradictory data on ghrelin responses to endotoxin have also been reported, as in the case of leptin. Hataya et al. (13) and Wang et al. (16) have shown that a single endotoxin injection suppressed plasma ghrelin levels in rats. Chang et al. (15) reported that a single endotoxin injection elevated ghrelin levels in rats. No data are available yet, however, on whether endotoxin alters circulating ghrelin in humans or in a human model of endotoxemia other than those reported by the described studies (13–16) in rats. In addition, no study has evaluated the effects of endotoxemia on circulating leptin and ghrelin in a parallel manner, although we know that their circulating levels are regulated in opposite directions in most situations.
Thus, this study was designed to determine changes in circulating leptin and ghrelin levels in a parallel manner by using a canine model of endotoxemia, which is useful in replicating the signs and laboratory findings observed in human sepsis/endotoxemia (24–27). In addition to leptin and ghrelin, we also investigated the changes in circulating levels of nitric oxide (NO) and procalcitonin (PCT) in response to endotoxin. These proinflammatory mediators are thought to be involved in the body’s response to endotoxin (28–30). Further, we evaluated adrenocorticotropic hormone (ACTH), cortisol, and epinephrine responses to endotoxin because endotoxin administrations trigger the hypophyseal-pituitary-adrenal axis (25); glucocorticoids alter plasma leptin and ghrelin in dogs (31–33). Because our endotoxemia model in dogs was associated with damages in liver and kidney (25), the two organs involved in clearance of leptin (20) and ghrelin (34) from circulation, we finally examined the changes in levels of serum biochemical markers of hepatorenal dysfunctions in response to endotoxin.
MATERIALS AND METHODS
Animals and General Procedures.
A total of 16 adult healthy dogs (eight male and eight female, 15–19 kg) were used in this study. The dogs were housed in individual cages in a controlled room (18–24°C and 12:12-hr light/dark cycle) for 3 days before the experiment at Veterinary Teaching Hospital, Uludag University, Bursa, Turkey. All dogs were kept in similar conditions, provided ad libitum water, and were fed twice daily with extruded diet (IAMS-Propet, Istanbul, Turkey). The experimental protocol was approved by the Animal Care and Use Committee of the University of Uludag.
The dogs were assigned to the control (n = 8; four female and four male) or test group (n = 8, four female and four male). Dogs in the control group received an intravenous vehicle (0.9% NaCl, 0.2 mL/kg), whereas endotoxin was injected intravenously once at 1 mg/kg to the dogs in the test group. The dose (1 mg/kg) of endotoxin used in the present study was selected from our previous studies in dogs (25–27).
The experiment was initiated at 9:00 am, after 14 hrs of fasting in both groups of dogs. All dogs were monitored clinically for 48 hrs after the treatment. During this period, dogs were fed four times, each feeding lasting for 30 mins (at 12, 24, 36, and 48 hrs after the treatments). Three out of eight endotoxin-treated dogs did not eat, whereas the remaining 5 dogs ate much less (by about 50%) than saline-treated dogs at 12 hrs after endotoxin. At 24, 36, and 48 hrs, all endotoxin-treated dogs ate their food within 30 mins after it was offered. At all time points of feeding, saline-treated dogs finished eating their food within 10 mins after it was offered.
Assessment of Tissue Injury, Hypophyseal-Pituitary-Adrenal Axis Activation, and Inflammation.
Hepatorenal dysfunctions were assessed by measuring the rises in serum levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatinine, urea, and uric acid (25). Endotoxin-induced hypophyseal-pituitary-adrenal axis activation was assessed by measuring the rises in serum levels of ACTH, cortisol, and epinephrine. Inflammatory reactions were assessed by measuring the rises in serum levels of NO (as NO3 −/NO2 −) and PCT.
Sample Collection and Measurements.
Venous blood samples were collected into vacutainer tubes (BDV system, NJ) before (baseline) and at intervals of 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hrs posttreatment.
Plasma leptin and ghrelin were measured by radioimmunoassay using a commercially available kit (multispecies leptin radioimmunoassay kit and total ghrelin radioimmunoassay kit, LINCO Research, St. Charles, MO). Within- and between-run coefficients of variation were 3.1% or 7.4% and 7.3% or 11.6% for leptin or ghrelin assay kit, respectively. Validity and reliability of these radioimmunoassay kits for measuring plasma leptin levels in dog were determined in our previous study (32).
Serum NO (as NO3 −/NO2 −) was determined by colorimetric kit (RD Systems, Minneapolis, MN). Serum PCT was measured using an immunoluminometric assay (BRAHMS Diagnostica, Berlin, Germany). Serum cortisol and plasma ACTH were measured by a solid-phase chemiluminescent enzyme immunoassay system (Immulite 2000, BioDPC, Los Angeles, CA). Plasma epinephrine was measured by radioimmunoassay using a commercially available kit (Bio Source Europe, Nivelles, Belgium). Serum urea, uric acid, creatinine, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase were measured by an automated clinical chemistry analyzer (Architect 8200, Abbott GmbH, Wiesbaden, Germany).
Endotoxin (lipopolysaccharide, Escherichia coli serotype 055:B5, purity >97%) was purchased from Sigma Chemical (St. Louis, MO); it was dissolved in sterile saline (0.9% NaCl) solution immediately before the experiment. The volume of solution injected intravenously was 0.2 mL/kg.
Results are expressed as mean ± sem. Data were evaluated by repeated-measures analysis of variance, followed by Tukey test for pairwise comparisons (SPSS 10.0, SPSS Germany). Pearson’s correlation analysis was used to determine a relationship between the mean levels of plasma leptin and ghrelin. A p value of <.05 was considered significant.
Effects of Endotoxin on Plasma Leptin and Ghrelin in Dogs.
After overnight fasting, baseline levels in dogs of plasma leptin and ghrelin before intravenous injection of endotoxin or saline were 2.4 ± 0.1 ng/mL (n = 16) and 867 ± 58 pg/mL (n = 16), respectively. Basal plasma leptin or ghrelin levels in female and male dogs were similar (data not shown).
As seen in Figure 1, plasma leptin increased gradually in response to endotoxin, reached its maximum, 3.4 ± 0.1 ng/mL (p < .001), at 4 hrs, and remained elevated for 48 hrs after administration of endotoxin. Plasma ghrelin increased from a baseline value to 1530 ± 74 pg/mL (p < .001) within 0.5 hr after endotoxin and remained elevated at this level for 48 hrs after endotoxin. Statistical analysis revealed significant changes over time for plasma leptin (F9,70 = 6.05; p < .001) and ghrelin (F9,70 = 4.47; p < .001) after endotoxin administration. Saline treatment did not affect plasma leptin (F9,70 = 0.65; p = .749) or ghrelin levels (F9,70 = 1.17; p = .327) within 48 hrs of follow-up (Fig. 1). Repeated-measures two-way analysis of variance revealed no difference in plasma leptin (F1,54 = 0.759; p = .417) or ghrelin (F1,54 = 0.321; p = .964) response to endotoxin between male and female dogs.
Mean values of plasma leptin and ghrelin concentrations were positively correlated (r = .844; p < .001) in endotoxin-treated dogs.
Effects of Endotoxin on Inflammatory Markers and Stress Hormones.
Circulating NO and PCT increased from their baseline values within 0.5 hr after endotoxin, reached their maximum at 2 hrs, and remained elevated at 4–48 hrs after endotoxin (Fig. 2, a and b). Repeated-measures analysis of variance revealed significant changes over time for serum NO (F9,70 = 3.76; p < .001) and PCT (F9,70 = 3.26; p < .001) after endotoxin administration. In saline-treated animals, no significant changes were observed in serum NO (F9,70 = 1.05; p = .659) or PCT (F9,70 = 0.15; p = .998) levels within 48 hrs (Fig. 2, a and b).
After endotoxin administration, baseline plasma ACTH increased significantly (p < .001) within 0.5 hr and reached its maximum at 1–2 hrs, followed by a reduction to near baseline values at 8–48 hrs (Fig. 2 c). Serum cortisol level increased significantly (p < .001) within 0.5 hr after endotoxin and remained elevated at 1–48 hrs after endotoxin (Fig. 2 d). Plasma epinephrine level peaked at 0.5–2 hrs after endotoxin administration and then showed a gradual decline toward baseline values from 4 to 48 hrs after endotoxin (Fig. 2 e).
Effects of Endotoxin on Serum Hepatorenal Injury Markers.
Endotoxin increased serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, urea, uric acid, and creatinine in a time-dependent manner (Fig. 3). Concentrations of serum hepatic injury markers peaked at 4–12 hrs after endotoxin injection and they decreased gradually toward their initial values at 24–48 hrs after endotoxin treatment (Fig. 3, a, c, and e). Serum renal injury markers urea, creatinine, and uric acid increased at 0.5 hr (p < .001; vs. baselines) after endotoxin and remained elevated throughout the study (Fig. 3, b, d, and f).
Relations of Plasma Leptin and Ghrelin Levels with Circulating Levels of Proinflammatory Mediators, Stress Hormones, and Serum Hepatorenal Injury Markers.
Mean plasma leptin and ghrelin levels in endotoxin-treated dogs were positively correlated with circulating proinflammatory mediators, cortisol, and hepatorenal dysfunction markers (Table 1).
The present study shows that plasma leptin and ghrelin levels are increased in response to endotoxin administration in dogs. The increases in plasma leptin and ghrelin levels were associated and positively correlated with elevations in plasma levels of PCT, NO, stress hormones, and hepatorenal injury markers.
Previous studies have shown that endotoxin administration increases circulating levels of leptin in mice (6–8), rats (9, 10) hamsters (5), nonhuman primates (11, 12), and humans (11), but not in cows (22) and sheep (23). The present study extends these previous observations by demonstrating long-lasting elevations in serum leptin levels in dogs after endotoxin treatment. In experimental animals, it has been shown that leptin is involved in endotoxin-induced anorexia (6, 9), fever (9, 10), and sickness behavior (10), and leptin deficiency increases susceptibility to endotoxic shock (7). Hyperleptinemia increases resistance to endotoxin (8), and plasma leptin levels are found to be increased in survivors of acute sepsis (4). Taken together, it is likely that the observed increases in plasma leptin levels may confer benefit in endotoxemia but may also be involved in some of the observed clinical signs, such as decreased appetite and fever in endotoxin-treated dogs.
In our previous study (32), we found a negative correlation between plasma leptin and ghrelin levels in healthy fasted dogs, as has been reported for humans (31) and experimental animals (31). In the present study, intravenous endotoxin injection increased plasma ghrelin in parallel with the elevations in plasma leptin; moreover, plasma ghrelin levels were positively and highly correlated (r = .844; p < .001) with plasma leptin levels. These data indicate that endotoxemia apparently alters the inverse relation between circulating leptin and ghrelin levels in dogs. The elevations in plasma ghrelin levels during endotoxemia could reflect an increased entry of ghrelin into the blood stream due to stimulation of its secretion from ghrelin-producing cells directly by endotoxin (15, 35) or indirectly as part of adaptive processes to negative energy balance and decreased appetite during endotoxemia (15), or they could be caused by reduced clearance due to hepatorenal injury (34). Given the facts that ghrelin improves endothelial (36) and cardiac functions (37, 38), down-regulates proinflammatory cytokines (18), and improves tissue perfusion in sepsis (15), the observed elevations in circulating levels of ghrelin could be considered as an adaptive protective response to endotoxin, as suggested (15). This view is further supported by recent observations that the cardiovascular ghrelin receptors are upregulated in the hyperdynamic phase of sepsis (17), and exogenous ghrelin improves endotoxin-induced wasting syndrome (13) and normalizes endotoxin-induced altered digestive (16) and cardiovascular functions (15) and metabolic disturbance (15).
As expected and in accordance with previous reports (25, 28–30), endotoxemia was accompanied by a rapid rise in circulating levels of proinflammatory mediators PCT and NO (Fig. 2, a and b), stress-reactant hormones ACTH, cortisol, and epinephrine (Fig. 2, c, d, and e), and hepatorenal dysfunction markers (Fig. 3). The finding that plasma leptin or ghrelin levels were positively correlated with serum levels of PCT, NO, cortisol, and hepatorenal injury markers suggests that plasma leptin and ghrelin responses are parts of the body’s complex response to endotoxin. Although our present data do not indicate, by any means, that any of these changes may be causally interrelated, it is reasonable to assume that they may have influences, to some extent, on each other. For example, ghrelin could be involved, in part, in an increase in circulating NO (36) stress hormone (i.e., ACTH, cortisol, and epinephrine) levels (37). Also, elevations in circulating PCT, NO, and cortisol could stimulate leptin release (4, 32, 33), whereas epinephrine decreases its circulating levels (39). Because liver and kidney are both important for clearance of leptin (20) and ghrelin (34) from circulation, the observed hepatorenal dysfunctions could slow ghrelin and leptin clearance and hence be involved in the observed elevations in their plasma levels.
In conclusion, the data from the present study are the first to show that plasma leptin and ghrelin levels are both increased in response to endotoxin. The roles of these elevations in plasma leptin and ghrelin in pathophysiological mechanisms during endotoxemia require further investigation.
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Keywords:© 2008 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins
leptin; ghrelin; nitric oxide; procalcitonin; endotoxin; lipopolysaccharide; dog