Effect of cholecystokinin on cytokines during endotoxic shock in rats

AIM To study the effect of cholecystokinin-octapeptide(CCK-8)on systemic hypotension and cytokine productionin lipopolysaccharide(LPS)-induced endotoxic shock(ES)rats.METHODS The changes of blood pressure were observedusing physiological record instrument in four groups ofrats:LPS(8mg·kg~(-1),iv)induced ES;CCK-8(40μg·kg~1,iv)pretreatment 10min before LPS(8mg.kg~(-1));CCK-8(40μg·kg~(-1),iv)or normal saline(control)groups.Differences in tissue and circulating specificity of theproinflammatory cytokines(TNF-α,IL-1β and IL-6)wereassayed with ELISA kits.RESULTS CCK-8 reversed LPS-induced decrease of meanartery blood pressure(MABP)in rats.Compared withcontrol,LPS elevated the serum level of IL-6 significantly(3567±687ng·L~(-1) vs 128±22ng·L~1,P<0.01),whilecontents of TNF-α and IL-1β elevated significantly(277±86ng.L~1 vs not detectable and 43±9ng·L~1 vs notdetectable,P<0.01)but less extent than IL-6.CCK-8significantly inhibited the LPS-induced increase in serumTNF-α,IL-1β and IL-6.LPS elevated spleen and lungcontent of IL-1β significantly(5184±85ng·L~1 vs 1047±21ng·L~1 and 4050±614ng·L~1 vs not detectable.P<0.01),while levels of TNF-α and IL-6 also rosesignificantly but in less extent than IL-1β.CCK-8 inhibitedthe LPS-induced increase of the cytokines in spleen andlung.In the heart,CCK-8 significantly inhibited LPS-induced increase of TNF-α(864±123ng·L~1 in CCK-8 LPS group vs 1599±227ng·L~1 in LPS group,P<0.01),and IL-1β(282±93ng·L~1 in CCK-8 LPS group vs 621±145ng·L~1 in LPS group,P<0.01).CONCLUSION CCK-8 reverses ES,which may be relatedto its inhibitory effect on the overproduction of cytokines.     

Hypothalamic somatostatin mediates the suppression of growth hormone secretion by centrally administered thyrotropin-releasing hormone in conscious rats

The effect and mechanism of action of central TRH on the regulation of GH secretion was studied in conscious male rats with indwelling intraatrial and intracerebroventricular (icv) cannulae. Plasma GH was measured every 10-20 min from 1000 h-1400 h by repeated blood sampling. In animals that received saline iv or icv, GH secretion was pulsatile, with peak hormone levels occurring at 1120-1200 h. TRH (10 micrograms), injected icv at 1100 h, inhibited spontaneous GH secretion, and mean plasma GH levels remained suppressed (less than 20 ng/ml) for at least 3 h after injection. In contrast, an iv injection of the same dose of TRH at 1100 h did not significantly affect spontaneous GH secretion. Intravenous injection of human GH-releasing factor [1-40] (hGRF, 1 micrograms) at 1100 h in animals injected 5 min earlier with saline (10 microliters, icv) stimulated GH release, with peak values (748 +/- 63 ng/ml, mean +/- SE) observed 10 min after injection. However, animals injected icv with TRH (10 micrograms) 5 min before the iv injection of hGRF exhibited an attenuated GH response to hGRF (peak values, 115 +/- 28 ng/ml; P less than 0.001 vs. saline icv + hGRF). The inhibition of GH secretion by central TRH was abolished by pretreatment of animals with antisomatostatin serum (0.5 ml, iv) but not with normal serum (P less than 0.001). These results suggest an inhibitory role of central TRH in the regulation of spontaneous GH secretion in the rat that is mediated by stimulation of hypothalamic somatostatin.     

Mechanisms by which blood levels of interleukin-6 (IL-6) are elevated after intracerebroventricular injection of IL-1beta in the rat: neural versus humoral control

Intracerebroventricular (icv) injection of interleukin-1beta (IL-1beta) in rats induces elevated IL-6 levels in peripheral blood, exceeding those induced by iv or ip injection. Two hypotheses postulated to explain this phenomenon were tested. Mediation by peripheral sympathetic activation was excluded by showing that agents that blocked preganglionic cholinergic synapses (chlorisondamine), beta-adrenergic receptors (propanalol, butoxamine), and alpha-adrenergic receptors (phentolamine) did not prevent the IL-6 response. That the peripheral response was due to passage of the injected IL-1beta into blood from the brain was supported by several observations. Immunoreactive IL-1beta appeared in peripheral blood by 10 min after icv injection and remained constant between 10-100 min after injection; values after icv injection were virtually identical to those after iv injection at 60 and 80 min. Radioiodine-labeled IL-1beta appeared in blood as early as 5 min, and by phamacokinetic analysis was found to be transferred from the brain at a rate greater than 2% of brain content per min(-1). IL-1beta infused iv in a pattern mimicking brain to blood transfer induced IL-6 levels that were more than double the values induced by a single bolus injection and were not significantly different from the values observed after icv injection. Sustained levels of IL-1beta in blood over time contribute to the high peripheral IL-6 response. This was shown by administering the same total dose iv as a single bolus of 100 ng or in two doses of 50 ng 1 h apart. Rats given a divided dose had 6-10 times higher blood IL-6 levels at 2 h than those given a single injection. The high levels of IL-6 in blood after icv injection of IL-1beta are best explained by the reservoir function of the brain IL-1beta pool and the self-priming effect of IL-1beta in peripheral tissues.     

Mechanisms of calcitonin-induced growth hormone (GH) suppression: roles of somatostatin and GH-releasing factor

Calcitonin (CT) binds to specific receptors in the hypothalamus and has been localized in the pituitary, suggesting a potential neuroendocrine role for this peptide. We and others have previously shown that CT given centrally markedly suppresses pulsatile GH secretion. However, the mechanism mediating this response remains to be elucidated. In the present study, we assessed the involvement of the two hypothalamic GH-regulatory peptides, somatostatin (SRIF) and GH-releasing factor (GRF), using a combination of in vivo and in vitro techniques. Six-hour GH secretory profiles were obtained from eight groups of freely moving rats bearing chronic intracerebroventricular (icv) and intraatrial cannulae. In four groups, salmon (s) CT (250 ng/10 microliters) was administered icv, whereas the remaining four groups received either normal saline (NS) icv or sCT iv. Central injection of sCT caused a severe suppression in amplitude of spontaneous GH pulses compared to NS icv-treated control rats, whereas the same dose of sCT iv had no significant effect. Passive immunization of sCT icv-injected rats with a specific antiserum to SRIF failed to restore the amplitude of GH pulses to normal values. In addition, in vitro basal and 50 mM K+-stimulated SRIF release from incubated hypothalamic fragments was not altered by sCT in doses ranging from 10(-10) to 10(-6) M. The iv administration of a bolus of rat GRF (1-29)NH2 (1 microgram) 1 h after sCT icv injection also failed to augment plasma GH levels compared to sCT iv-treated rats (16.6 +/- 10.0 vs. 326.6 +/- 63.6 ng/ml; P less than 0.001) and NS icv controls (407.2 +/- 145.4 ng/ml; P less than 0.01). Blood calcium levels decreased similarly 1 h after iv and icv sCT administration. These results demonstrate that: sCT inhibits pulsatile GH secretion via a central nervous system site of action, GH suppression induced by sCT is apparently not due solely to increased hypothalamic SRIF release, and centrally administered sCT produces an acute loss of responsiveness of somatotrophs to GRF, which can be dissociated from peripheral blood calcium levels.     

Adrenocorticotropin release induced by intracerebroventricular injection of recombinant human interleukin-1 in rats: possible involvement of prostaglandin

ACTH release induced by iv and intracerebroventricular (icv) injection of recombinant human interleukin-1 beta (IL-1 beta) or alpha (IL-1 alpha) was studied in conscious, unrestrained rats. A dose as small as 3 ng of IL-1 beta injected icv induced a significant rise in plasma ACTH levels, whereas 100 ng/100 g body wt (approximately 300 ng/rat) was needed for a significant ACTH response when injected iv. Intracerebroventricular administration of 30 ng IL-1 alpha tended to increase plasma ACTH levels, but not significantly. Intravenous injection of 1000 ng/100 g IL-1 beta induced a maximal response with a pronounced elevation of plasma ACTH levels at 10 and 30 min after injection, but plasma ACTH levels fell at 60 min post injection. On the other hand, icv injection of 30 ng IL-1 beta raised plasma ACTH levels at 10 min, reaching peak values between 30 and 60 min post injection, and plasma ACTH levels remained elevated for 2-3 h after injection. Pretreatment with indomethacin completely prevented the ACTH response induced by either iv or icv injection of IL-1 beta. Administration of indomethacin did not alter the elevation of plasma ACTH levels induced by immobilization stress, however. On the other hand, vagotomy did not alter the ACTH response to iv administered IL-1 beta. Neither iv nor icv injection of IL-1 beta in a dose which induced a maximal ACTH response altered plasma PRL levels. These findings strongly suggest that the brain is the primary site of action of IL-1 beta, and that IL-1 beta transmits the message of the immune system to the brain and, possibly, CRF neurons. It is also suggested that prostaglandins may be involved in this central action of IL-1 beta.     

Agouti-related protein stimulates the hypothalamic-pituitary-adrenal (HPA) axis and enhances the HPA response to interleukin-1 in the primate

alpha-MSH antagonizes many of the immune and neuroendocrine effects induced by inflammatory cytokines. Studies have shown that alpha-MSH attenuates the stimulatory effect of IL-1 on the hypothalamic-pituitary-adrenal (HPA) axis and plays a physiological role in limiting the HPA response to IL-1. Recently an alpha-MSH antagonist, agouti-related protein (AGRP), has been identified in the hypothalamus, which stimulates food intake by antagonizing the effects of alpha-MSH at specific melanocortin receptors. It is unknown whether AGRP can also modulate neuroendocrine responses to inflammatory cytokines. We have therefore examined the effects of AGRP on the HPA axis and on prolactin (PRL) at baseline and in response to stimulation by IL-1 beta in nine ovariectomized rhesus monkeys. In the first study, the effects of intracerebroventricular (i.c.v) infusion of 20 microg (n = 6) and 50 micro g (n = 4) of human AGRP (83-132)-NH(2) were compared with icv saline infusion. There was a significant stimulatory effect of 20 microg AGRP on cortisol release over time (P < 0.001). The area under the hormone response curve (AUC) for cortisol increased by 29% after 20 microg AGRP vs. saline; the AUC for ACTH increased by 166% (P = 0.028); the AUC for PRL increased by 108% (P = 0.046). There was a significant stimulatory effect of 50 microg AGRP on ACTH (P < 0.001), cortisol (P < 0.001), and PRL (P < 0.001) release over time. The AUC for ACTH after 50 microg AGRP increased by 98%; the AUC for cortisol increased by 37%; the AUC for PRL increased by 161%. The effects of AGRP on ACTH, cortisol, and PRL release were prevented by alpha-MSH infusion. In the second study, animals received icv either 50 ng of human IL-1 beta or 20 microg of AGRP followed by 50 ng IL-1 beta. AGRP significantly enhanced the ACTH (P < 0.05) response to IL-1 beta. The peak ACTH response to IL-1 beta alone was 124 +/- 55 pg/ml vs. 430 +/- 198 pg/ml after IL-1 beta plus AGRP; the peak cortisol response was 70 +/- 8.2 microg/dl vs. 77 +/- 6.2 microg/dl, but this was not significantly different. In conclusion, AGRP stimulated ACTH, cortisol, and PRL release in the monkey and enhanced the ACTH response to IL-1 beta. These studies suggest that, in addition to its known orexigenic effects, AGRP may play a role in neuroendocrine regulation and specifically that AGRP may interact with alpha-MSH to modulate neuroendocrine responses to inflammation.     

Leptin(116-130) stimulates prolactin and luteinizing hormone secretion in fasted adult male rats.

Leptin is a hormone secreted by adipocytes with an important role in the control of feeding behavior and neuroendocrine function. Leptin stimulates in vivo LH secretion in fasted female rats and in vitro PRL secretion. Recent data indicate that leptin(116-130), an active fragment of the native molecule, exerts effects similar to those of the native peptide on body weight and food intake. The present study was carried out to determine whether this fragment is also able to stimulate LH and PRL secretion. Adult male rats fasted for 5 days were injected with saline or leptin(116-130) (15 microgram i.c.v.) and LH and PRL concentrations were measured thereafter at 15-min intervals during a 150-min period. Administration of leptin(116-130) increased the frequency of LH pulses (2.0 +/- 0.26 vs. 1.20 +/- 0. 37/150 min; p 相似文献   

Leptin, free leptin index, insulin resistance and liver fibrosis in children with non-alcoholic fatty liver disease

OBJECTIVE: Prevalence of non-alcoholic fatty liver disease (NAFLD) among children is increasing dramatically. It is unclear why some patients develop steatohepatitis (NASH), fibrosis and cirrhosis from steatosis, and others do not. A role for leptin has been claimed. This study aims to evaluate the relationship between leptin, insulin resistance (IR) and NAFLD in children. DESIGN AND METHODS: In 72 biopsy-proven NAFLD children (aged 9-18 years; 51M/21F), fasting leptin and its soluble receptor (sOB-R) were measured; free leptin index (FLI) was calculated as leptin/sOB-R; IR was estimated by homeostasis model assessment (HOMA-IR) and insulin sensitivity index (ISI-comp); glucose tolerance by oral glucose tolerance test (OGTT). Percentage of total body fat (TBF) by dual-energy X-ray absorptiometry (DXA) was available in 65 patients. RESULTS: Prevalence of diabetes, impaired fasting and/or after load glucose tolerance was 11%. HOMA-IR and ISI-comp values were 2.55 +/- 1.39 and 4.4 +/- 2. NASH was diagnosed in 38 and simple steatosis in 25 children; diagnosis was indeterminate in 29 children. Increased fibrosis, mostly of mild severity, was observed in 41 patients. Median NAFLD activity (NAS) score was 3.42 +/- 1.60. According to histology, levels of leptin and FLI increased as steatosis (leptin from 11.9 +/- 6.3 in score 1 to 17.4 +/- 6.9 in score 2 (P = 0.01) and 22.2 +/- 6.8 ng/ml in score 3 (P < 0.001); FLI 2.56 +/- 1.40, 3.57 +/- 0.34, 4.45 +/- 0.64 respectively (P = 0.05)); ballooning (from 13.7 +/- 6.7 in score 1 to 17 +/- 7.5 in score 2 (P = 0.001) and 22.1 +/- 7.1 ng/ml in score 3 (P = 0.01); FLI 2.81 +/- 1.50, 3.40 +/- 1.65, 4.57 +/- 1.67 (P = 0.01 between 0 and 2)); fibrosis (from 14.3 +/- 7 to18.3 +/- 6.9; P = 0.03; FLI 3.03 +/- 1.57 vs 3.92 +/- 077; P < 0.05) and NAS score (score 1-2: 12.9 +/- 6.9; score 3-4: 17 +/- 6.9 (P = 0.01); score 5-7: 22.9 +/- 7.5 ng/ml (P = 0.03); FLI 2.70 +/- 1.53, 3.12 +/- 1.53, 4.58 +/- 1.57 P = 0.01 and P = 0.05 between 1-2 vs 3-4 and 3-4 vs 5-7 respectively) worsened. Higher leptin correlated with more severe steatosis, ballooning and NAS score (r(0) = 0.6, 0.4 and 0.6 respectively; for all P < 0.001); FLI with ballooning (r(0) = 0.4, P < 0.0001), steatosis (r(0) = 0.5, P < 0.0001) and NAS score (r(0) = 0.5, P < 0.0001). CONCLUSIONS: Leptin and liver injury correlated independently of age, BMI and gender in the present study. Nevertheless, any causative role of leptin in NAFLD progression could be established. Thus, studies are needed to define whether the hormone plays a major role in the disease.     

Effect of a lipid-enriched diet on body composition and some regulatory hormones of food intake in growing rats

OBJECTIVE: The aim of the present study was to investigate the effects of a lipid-enriched diet on body composition and on main regulatory hormones of food intake (insulin, adiponectin, leptin, ghrelin). METHOD: Two groups of 16 rats, 35 days old, weighing 80+/-6 g, were constituted. One group (S) was given a standard diet during 10 weeks and served as control. The second group (L) was given a lipidic-enriched diet (containing: G: 41.5, L: 38.5, P: 20% calorie). Food and water were given "ad libitum". RESULTS: Total food intake, body weight, skeletal area and lean body mass of rats eating lipid-enriched diet were lowered (6694+/-178 vs. 8160+/-184 kcal, P=0.01; 431+/-38 vs. 468+/-25 g, P=0.003; 72.19+/-0.96 vs. 76.07+/-1.31 cm2, P=0.03; 369+/-18 vs. 409+/-23 g, P=0.0006), fat mass difference was not statistically significant (82.5+/-17 vs. 80+/-17 g, P=0.7). Blood ghrelin, adiponectin levels were lowered (1517+/-224 vs. 1915+/-579 pg/ml, P=0.03; 10+/-3 vs. 19+/-3 microg/ml, P=0.003) whereas insulin and leptin were unchanged (1.8+/-1.5 vs. 2.6+/-1.4 ng/ml, P=0.1; 16+/-11 vs. 13+/-10 ng/ml, P=0.4). CONCLUSION: A period of high fat diet in growing rats leads to a hypophagia, resulting in a lower lean body mass development. Some regulatory hormones of food intake did not change, while others significantly decreased, notably ghrelin being possible causal factor of the observed hypophagia linked to high fat diet.     

白细胞介素12重组腺病毒气道内应用对哮喘豚鼠治疗作用的研究

目的　探讨激发前气道内应用白细胞介素 12 (IL 12 )重组腺病毒对过敏性气道高反应的调节作用。方法　以C5 7BL/ 6小鼠经鸡卵蛋白 (OVA)免疫建立哮喘模型 ,实验分 6组 ,每组 6只。激发前气管内单次使用IL 12重组腺病毒 (10 8pfu/mouse) ,观察抗原激发后反应的变化。结果　 (1)小鼠气道内应用IL 12重组腺病毒在肺内可有效表达 ,48h血浆及肺泡灌洗液IL 12分别为 (5 40± 6 0 )U/ml和 (470 0± 80 0 )U/ml,对照病毒和PBS组未检出 ,两组比较差异有显著性 (P <0 0 1)。 (2 )在抗原激发阶段使用IL 12重组腺病毒 ,可明显抑制肺内IL 4[(3 5± 2 0 )ng/ml∶85 0± 2 5 0 )ng/ml]和IL 5[(6 5± 4 5 )ng/ml∶(5 4 0± 14 0 )ng/ml];γ干扰素 (IFN γ)的产生增加 [(6 90 0± 32 0 )ng/ml∶(12 5±3 2 )ng/ml];并明显抑制气道高反应性 [(36 0± 30 )cmH2 O∶(810± 5 0 )cmH2 O];抑制外周血 [(0 7±0 1) %∶(9 2± 0 5 ) % ]及肺泡灌洗液 [(3 5± 0 7)∶(2 1 6± 4 7)× 10 4 /ml]中的嗜酸细胞的水平 ;与对照组比较 (t分别 =7 97、7 92、5 1 6、18 9、9 33、47 1,P均 <0 0 1) ;但与总IgE[(6 5± 9) μg/ml∶(6 7± 10 )μg/ml]及抗原特异性IgE[(32± 8)∶(33± 8)U/ml]比较无明显影响 (P均 >0 0 5 )。     

Interleukin-1beta (IL-1beta) and IL-6 modulate insulin-like growth factor-binding protein (IGFBP) secretion in colon cancer epithelial (Caco-2) cells

Chronic inflammation is characterised by modifications in cytokine concentrations, whereas growth is mainly dependent on the GH-IGF axis. IGF-I bioavailability is modulated by a family of IGF-binding proteins (IGFBPs). The aim of the present study was to evaluate the interactions among interleukin-1beta (IL-1beta), IL-6 and IGFBP secretion by intestinal cells to assess whether cytokines modulate IGFBP secretion, and in turn IGF-I and IGF-II bioavailability. The human colon carcinoma derived cell line Caco-2 was used as an in vitro model for its capacity to differentiate spontaneously. Experiments were carried out on day 4 (undifferentiated state) and day 14 (differentiated state) after plating. Carcinoembryonic antigen (CEA) was used as a marker of differentiation and increased in the conditioned media (CM) from days 4 to 14 (0.2+/-0.01 ng/ml per 10(5) cells vs 3.3+/-0.2 ng/ml per 10(5) cells, P<0.05). IGFBP-2 and IGFBP-4 secretion decreased concomitantly. Cells were stimulated with IL-1beta and IL-6 at 1, 10 and 50 ng/ml, and with IL-1beta and IL-6 in combination at the same dose of 1 and 10 ng/ml. IGF-I at 50 ng/ml was used as a control. Caco-2 cells expressed and secreted mainly IGFBP-2 and IGFBP-4 into the CM. On day 4, IL-1beta (1 ng/ml) and IL-6 (10 and 50 ng/ml) reduced IGFBP-2 by 29+/-8%, and by 32+/-9 and 38+/-8% respectively (P<0.05). IGFBP-4 was also reduced by IL-1beta at 1 and 50 ng/ml (-14+/-4% and -46+/-11% vs serum free medium (SFM) respectively, P<0.05), and IL-6 at 50 ng/ml (-46+/-15%, P<0.05). Both IGFBP-2 and IGFBP-4 were reduced by IL-1beta and IL-6 in combination at 1 and 10 ng/ml (P<0.05). On day 14, IGFBP-2 band intensity was reduced at 10 ng/ml of IL-1beta (-22+/-15% vs SFM, P<0.05) and at 50 ng/ml of both cytokines (-33%+/-8% and -13%+/-13% vs baseline respectively, P<0.05). IGFBP-4 band intensity decreased with 10 and 50 ng/ml of IL-1beta (-35+/-11% and -46+/-15% vs SFM respectively) and IL-6 (-36%+/-10% and -46+/-15% vs SFM respectively). IL-1beta and IL-6 in combination at 1 and 10 ng/ml reduced both IGFBP-2 and IGFBP-4.In conclusion, IGFBP-2 and IGFBP-4 secretion in CM decreased with Caco-2 cell differentiation. IGFBP-2 and IGFBP-4 were significantly decreased by IL-1beta and IL-6 treatment in both the undifferentiated and differentiated state. Furthermore, these cytokines increased cell proliferation whereas total protein content was significantly reduced only at the higher concentrations of IL-6 and IL-1beta. These findings suggest that interleukins modulate the IGF-IGFBP system in Caco-2 cells in vitro.     

Regulation of growth hormone and somatomedin-C secretion in postmenopausal women: effect of physiological estrogen replacement

To determine the effects of estrogen deficiency and replacement on GH secretion, we measured the 22-h GH secretory pattern and response to 1 h of light exercise in 16 normal postmenopausal women before and after treatment replacement with ethinyl estradiol (20 micrograms/day for 15 days). To determine whether the changes found were due to pituitary sensitization by estrogen, the response to synthetic GH-releasing hormone (GHRH; 1.0 microgram/kg, iv) was measured. To assess the biological effectiveness of GH in estrogen-treated women, somatomedin-C (Sm-C) responses to GHRH were measured. Pre- and postestrogen GH secretion rates, expressed as mean areas circumscribed by plasma GH values, were as follows: 22-h study, 1.4 +/- 0.1 (+/- SEM) vs. 2.0 +/- 0.3 ng/ml X h (P = 0.04; n = 5); during 1 h of exercise, 2.3 +/- 0.4 vs. 3.2 +/- 0.4 ng/ml X h (P = 0.03; n = 16); after GHRH-(1-40), 6.7 +/- 1.7 vs. 8.5 +/- 1.5 ng/ml X h (P = 0.12; n = 16). There also was a modest but significant increase in resting plasma GH (1.5 +/- 0.2 vs. 2.3 +/- 0.5 ng/ml (P = 0.039). Pre- and postestrogen plasma Sm-C concentrations were 0.56 +/- 0.08 and 0.32 +/- 0.03 U/ml, respectively (P = 0.006; n = 16). Thus, estrogen therapy increased spontaneous and exercise-induced GH secretion in postmenopausal women and reduced Sm-C levels. The mechanisms of GH elevation by estrogen may include both central effects and a negative feedback linkage to reduced plasma Sm activity.     

Leptin regulates pulsatile luteinizing hormone and growth hormone secretion in the sheep

Administration of leptin during reduced nutrition improves reproductive activity in several monogastric species and reverses GH suppression in rodents. Whether leptin is a nutritional signal regulating neuroendocrine control of pituitary function in ruminant species is unclear. The present study examined the control of pulsatile LH and GH secretion in sheep. We determined whether exogenous leptin could prevent either the suppression of pulsatile LH secretion or the enhancement of GH secretion that occur during fasting. Recombinant human met-leptin (rhmet-leptin; 50 microg/kg BW; n = 8) or vehicle (n = 7) was administered s.c. every 8 h during a 78-h fast to estrogen-treated, castrated yearling males. LH and GH were measured in blood samples collected every 15 min for 6 h before fasting and during the last 6 h of fasting. Leptin was measured both by a universal leptin assay and by an assay specific for ovine leptin. During the fast, endogenous plasma leptin fell from 1.49 +/- 0.16 to 1.03 +/- 0.13 ng/ml. The average concentration of rhmet-leptin 8 h after leptin administration was 18.0 ng/ml. During fasting, plasma insulin, glucose, and insulin-like growth factor I levels declined, and nonesterified fatty acid concentrations increased similarly in vehicle-treated and leptin-treated animals. In vehicle-treated animals, LH pulse frequency declined markedly during fasting (5.6 +/- 0.5 vs. 1.1 +/- 0.5 pulses/6 h; fed vs. fasting; P < 0.0001). Leptin treatment prevented the fall in LH pulse frequency (5.0 +/- 0.4 vs. 4.9 +/- 0.4 pulses/6 h; P = 0.6). Neither fasting nor leptin administration altered GH pulse frequency. Fasting produced a modest increase in mean concentrations of circulating GH in control animals (2.4 +/- 0.5 vs. 3.4 +/- 0.6 ng/ml; P = 0.04), whereas there was a much greater increase in GH during leptin treatment (2.7 +/- 0.6 vs. 8.6 +/- 1.6 ng/ml; P = 0.0001). GH pulse amplitudes were also increased by fasting in control (P = 0.04) and leptin-treated sheep (P = 0.007). The finding that exogenous rhmet-leptin regulates LH and GH secretion in sheep indicates that this fat-derived hormone conveys information about nutrition to mechanisms controlling neuroendocrine function in ruminants.     

Effects of physiological leptin administration on markers of inflammation, platelet activation, and platelet aggregation during caloric deprivation

CONTEXT: Leptin is a nutritionally regulated adipocyte-derived cytokine. Previous studies in obese patients have demonstrated increased inflammatory markers and increased platelet aggregation in association with leptin. However, the effects of leptin administration on markers of inflammation and platelet aggregation in a human model of undernutrition have not previously been studied. OBJECTIVE: The objective of the study was to investigate markers of inflammation, platelet activation, and platelet aggregation in a model of caloric deprivation and increased leptin sensitivity. DESIGN: This study was a randomized, placebo-controlled study conducted between November 2002 and November 2003. SETTING: The study was conducted at an inpatient care setting at the General Clinical Research Center. PARTICIPANTS: Twenty healthy, young (18-35 yr old), normal-weight (body mass index, 20-26 kg/m2) women were recruited from local advertisements. No subjects withdrew due to adverse effects. INTERVENTION: The effects of physiological recombinant methionyl human leptin or identical placebo administration were investigated over a 4-d fast. MAIN OUTCOME MEASURES: The primary outcome measures for this study were C-reactive protein (CRP) and indices of platelet activity. RESULTS: Leptin administration prevented the fasting-induced decline in leptin (P < 0.05 vs. placebo at each time point). Leptin administration increased CRP (6.3 +/- 2.4 vs. 0.7 +/- 0.3 mg/liter; P = 0.04), circulating P-selectin (11.6 +/- 10.2 vs. -28.9 +/- 15.6 ng/ml; P = 0.04), and induction of platelet aggregation (5.8 +/- 2.6 vs. -2.7 +/- 2.9%, P = 0.04, percent maximum platelet aggregation) relative to placebo administration (change in leptin vs. change in placebo, respectively, for each variable). Leptin tended to increase serum amyloid A [0.1 +/- 0.2 vs. -0.3 +/- 0.1 log10 (ng/ml); P = 0.07], and the changes in serum amyloid A and CRP were highly correlated (r = 0.83; P < 0.0001). No changes in TNFalpha, IL-6, IL-10, plasminogen activator inhibitor-1, haptoglobin, intercellular adhesion molecule, or vascular cell adhesion molecule were seen between the groups. CONCLUSIONS: Our data provide evidence that physiological leptin administration stimulates inflammatory and platelet responses in humans during caloric deprivation.     

Disruption of the synchronous secretion of leptin, LH, and ovarian androgens in nonobese adolescents with the polycystic ovarian syndrome

The present study probes putative disruption of hypothalamic control of multihormone outflow in polycystic ovarian syndrome by quantitating the joint synchrony of leptin and LH release in adolescents with this syndrome and eumenorrheic controls. To this end, hyperandrogenemic oligo- or anovulatory patients with polycystic ovarian syndrome (n = 11) and healthy girls (n = 9) underwent overnight blood sampling every 20 min for 12 h to monitor simultaneous secretion of leptin (immuno-radiometric assay), LH (immunofluorometry), and androstenedione and T (RIA). Synchronicity of paired leptin-LH, leptin-androstenedione, and leptin-T profiles was appraised by two independent bivariate statistics; viz., lag-specific cross-correlation analysis and pattern-sensitive cross-approximate entropy. The study groups were comparable in chronological and postmenarchal age, body mass index, fasting plasma insulin/glucose ratios, and serum E2 concentrations. Overnight mean (+/- SEM) serum leptin concentrations were not distinguishable in the two study groups at 30 +/- 4.8 (polycystic ovarian syndrome) and 32 +/- 7.4 microg/liter (control). Serum LH concentrations were elevated at 9.5 +/- 1.4 in girls with polycystic ovarian syndrome vs. 2.8 +/- 0.36 IU/liter in healthy subjects (P = 0.0015), androstenedione at 2.8 +/- 0.30 (polycystic ovarian syndrome) vs. 1.2 +/- 0.11 ng/ml (control) (P = 0.0002), and T at 1.56 +/- 0.29 (polycystic ovarian syndrome) vs. 0.42 +/- 0.06 ng/ml (P < 0.0001). Cross-correlation analysis shows that healthy adolescents maintained a positive relationship between leptin and LH release, wherein the latter lagged by 20 min (P < 0.01). No such association emerged in girls with polycystic ovarian syndrome. In eumenorrheic volunteers, leptin and androstenedione concentrations also covaried in a lag-specific manner (0.0001 < P < 0.01), but this linkage was disrupted in patients with polycystic ovarian syndrome. Anovulatory adolescents further failed to sustain normal time-lagged coupling between leptin and T (P < 0.01). Approximate entropy calculations revealed erosion of orderly patterns of leptin release in polycystic ovarian syndrome (P = 0.012 vs. control). Cross-entropy analysis of two-hormone pattern regularity disclosed marked disruption of leptin and LH (P = 0.0099), androstenedione and leptin (P = 0.0075) and T-leptin (P = 0.019) synchrony in girls with polycystic ovarian syndrome. In summary, hyperandrogenemic nonobese adolescents with oligo- or anovulatory polycystic ovarian syndrome manifest: 1) abrogation of the regularity of monohormonal leptin secretory patterns, despite normal mean serum leptin concentrations; 2) loss of the bihormonal synchrony between leptin and LH release; and 3) attenuation of coordinate leptin and androstenedione as well as leptin and T output. In ensemble, polycystic ovarian syndrome pathophysiology in lean adolescents is marked by vivid impairment of the synchronous outflow of leptin, LH and androgens. Whether analogous disruption of leptin-gonadal axis integration is ameliorated by therapy and/or persists into adulthood is not known.     

Innate immunity modulates adipokines in humans

CONTEXT: Chronic inflammation converges in type 2 diabetes and atherosclerosis. Modulation of adipokine signaling by innate immunity in humans is of considerable interest given the role of adipokines in insulin resistance and atherosclerosis. OBJECTIVE: The aim of the study was to examine effects of low-grade endotoxemia, a model of human inflammation, on adipokines in vivo. DESIGN/SETTING: An open-label, placebo-controlled, fixed-sequence clinical study was conducted at a General Clinical Research Center. PATIENTS: There were 20 healthy male (50%) and female volunteers aged 18-40 yr. INTERVENTION: Serial blood sampling and adipose biopsies were performed for 24 h before and after iv bolus endotoxin [lipopolysaccharide (LPS), 3 ng/kg]. MAIN OUTCOME MEASURES: We measured plasma leptin, adiponectin, resistin, soluble leptin receptor, cytokines, insulin, and glucose; distribution of adiponectin among multimeric complexes; whole blood, monocyte and adipose mRNA for adipokines and their receptors. RESULTS: LPS induced fever, blood, and adipose TNF and IL-6 and increased homeostasis model assessment of insulin resistance. These were associated with increases in plasma leptin (from 4.1 +/- 1.1 to 6.1 +/- 1.9 ng/ml in men; 21.1 +/- 4.4 to 27.4 +/- 4.7 ng/ml in women; P < 0.005), doubling of the leptin:soluble leptin receptor ratio, and marked induction of whole blood resistin mRNA (13.7 +/- 7.3-fold; P < 0.001) and plasma resistin (8.5 +/- 2.75 to 43.2 +/- 15.3 ng/ml; P < 0.001). Although total adiponectin levels and low and high molecular weight adiponectin complexes were unaltered by LPS treatment, whole blood mRNA for adiponectin receptors 1 (49%; P < 0.005) and 2 (65%; P < 0.001) was suppressed. CONCLUSIONS: Modulation of adipokine signaling may contribute to the insulin resistant, atherogenic state associated with human inflammatory syndromes. Targeting of individual adipokines or their upstream regulation may prove effective in preventing acute and chronic inflammation-related metabolic complications.     

Elevated serum leptin concentrations in women with components of multiple risk factor clustering syndrome

This cross sectional study was undertaken to determine whether serum leptin levels were associated with multiple risk factor (MRF) clustering syndrome. We examined the relationship between serum leptin concentrations and blood pressure (BP), serum lipids levels, calculated insulin resistance (HOMA-ratio) and adiposity among 581 Japanese adult women. The serum leptin was increased in female subjects with systolic (> or =160 mmHg) and diastolic > or =90 mmHg) hypertension compared with the normotensive females (mean+/-SE; 9.3+/-0.5 vs 7.7+/-0.3; 10.2+/-0.6 vs 7.1+/-0.3 ng/ml, both p<0.001). Serum leptin was elevated in those with hyper-cholesterolemia (C; > or =220 mg/dl) and triglyceridemia (TG; > or =150 mg/dl) compared with the normolipidemia (9.4+/-0.4 vs 7.8+/-0.3; 11.7+/-0.6 vs 7.5+/-0.2 ng/ml, both p <0.001). Serum leptin was also elevated in those with adiposity (BMI > or =26.4 kg/m2) and insulin resistance (HOMA-ratio > or =2.5) compared with the normal females (14.8+/-0.7 vs 5.2+/-0.2; 11.3+/-1.1 vs 7.1+/-0.4ng/ml, both p<0.001). Even after adjusting for BMI or percent body fat mass (BFM), leptin levels remained to be elevated significantly in all these diseases. There was a positive correlation between serum leptin and systolic, diastolic BP, TC, TG, BMI, BFM, IRI and HOMA-ratio (r=0.12, p=0.005; r=0.24, p<0.0001; r=0.19, p<0.0001; r=0.35, p<0.0001; r=0.72, p<0.0001; r=0.73, p<0.0001; r=0.47, p< 0.0001; r=0.44, p<0.0001), and a negative correlation with HDL-C levels (r= -0.20, p< 0.0001). These correlations were also observed in leptin levels after adjusting for the BMI or BFM. Multiple regression analysis showed that BFM, HOMA-ratio and TG were significant determinants of leptin concentration before (t=12.6, p<0.0001; t=3.33, p=0.001; t=3.22, p=0.001) and after adjusting for BMI or BFM.These results suggest that because serum leptin levels were elevated in components of MRF clustering syndrome, leptin may have a pathophysiological role in MRF clustering syndrome.     

Regulation of expression of 11beta-hydroxysteroid dehydrogenase type 1 in adipose tissue: tissue-specific induction by cytokines

Patients with glucocorticoid excess develop central obesity, yet in simple obesity, circulating glucocorticoid levels are normal. We have suggested that the increased activity and expression of the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) generating active cortisol from cortisone within adipose tissue may be crucial in the pathogenesis of obesity. In this study primary cultures of human hepatocytes and adipose stromal cells (ASC) were used as in vitro models to investigate the tissue-specific regulation of 11betaHSD1 expression and activity. Treatment with tumor necrosis factor-alpha (TNFalpha) caused a dose-dependent increase in 11betaHSD1 activity in primary cultures of both sc [1743.1 +/- 1015.4% (TNFalpha, 10 ng/ml); P < 0.05 vs. control (100%)] and omental [375.8 +/- 57.0% (TNFalpha, 10 ng/ml); P < 0.01 vs. control (100%)] ASC, but had no effect on activity in human hepatocytes [90.2 +/- 2.8% (TNFalpha, 10 ng/ml); P = NS vs. control (100%)]. Insulin-like growth factor I (IGF-I) caused a dose-dependent inhibition of 11betaHSD1 activity in sc [49.7 +/- 15.0% (IGF-I, 100 ng/ml]; P < 0.05 vs. control (100%)] and omental [71.6 +/- 7.5 (IGF-I, 100 ng/ml); P < 0.01 vs. control (100%)] stromal cells, but not in human hepatocytes [101.8 +/- 15.7% (IGF-I, 100 ng/ml); P = NS vs. control (100%)]. Leptin treatment did not alter 11betaHSD1 activity in human hepatocytes, but increased activity in omental ASC [135.8 +/- 14.1% (leptin, 100 ng/ml); P = 0.08 vs. control (100%)]. Treatment with interleukin-1beta induced 11betaHSD1 activity and expression in sc and omental ASC in a time- and dose-dependent manner. 15-Deoxy-12,14-PGJ2, the putative endogenous ligand of the orphan nuclear receptor peroxisome proliferator-gamma, significantly increased 11betaHSD1 activity in omental cells [179.7 +/- 29.6% (1 microM); P < 0.05 vs. control (100%)] and sc [185.3 +/- 12.6% (1 microM); P < 0.01 vs. control (100%)] ASC, and it is possible that expression of this ligand may ensure continued cortisol generation to permit adipocyte differentiation. Protease inhibitors used in the treatment of human immunodeficiency virus infection are known to cause a lipodystrophic syndrome and central obesity, but saquinavir, indinavir, and neflinavir caused a dose-dependent inhibition of 11betaHSD1 activity in primary cultures of human omental ASC. 11betaHSD1 expression is increased in human adipose tissue by TNFalpha, interleukin-1beta, leptin, and orphan nuclear receptor peroxisome proliferator-gamma agonists, but is inhibited by IGF-I. This autocrine and/or paracrine regulation is tissue specific and explains recent clinical data and animal studies evaluating cortisol metabolism in obesity. Tissue-specific 11betaHSD1 regulation offers the potential for selective enzyme inhibition within adipose tissue as a novel therapy for visceral obesity.     

Leptin modulates inflammatory cytokine and neuroendocrine responses to endotoxin in the primate

Leptin, which plays a crucial role in regulating energy balance, can also modulate the inflammatory response. Although leptin-deficient rodents are more sensitive to the toxic effects of bacterial endotoxin, it is unknown if leptin can modulate inflammatory cytokine or neuroendocrine responses to inflammation in a primate model. We have therefore studied the effects of leptin on plasma cytokine and hypothalamic-pituitary-adrenal responses to endotoxin (5 microg iv) in nine ovariectomized rhesus monkeys. Human leptin (50 microg/h) or saline was infused iv for 16 h before and 4 h after endotoxin injection; mean plasma leptin increased from 3.6 +/- 1.0 ng/ml to 18 +/- 1.7 ng/ml (P < 0.001). Leptin infusion had no effect on baseline plasma cytokine and hormone levels before endotoxin injection. As expected, endotoxin stimulated TNF-alpha, IL-6, IL-1 receptor antagonist (IL-1ra), ACTH, and cortisol in the saline-infused animals (P < 0.001). There was a significant attenuation of the IL-6 (P < 0.005) and cortisol (P < 0.001) responses (repeated measures ANOVA) to endotoxin in the leptin-infused animals. There was a significant reduction (by paired analysis) in the responses of the leptin compared with saline-treated animals: 47% for TNF-alpha, 48% for IL-6, 30% for IL1ra, 42% for ACTH, and 22% for cortisol (P < 0.05). We conclude that an increase in circulating leptin, within the physiological range of our monkey colony, can blunt the inflammatory cytokine and hypothalamic-pituitary-adrenal responses to an inflammatory challenge. These results, coupled with our recent finding that endotoxin stimulates leptin release in the monkey, demonstrate that leptin can be both released in response to inflammatory cytokines and act to attenuate the responses to these cytokines.     

Plasma soluble tumor necrosis factor-alpha receptors circulate in proportion to leptin levels during the menstrual cycle in lean but not in obese women

OBJECTIVE: In recent studies serum leptin levels were significantly higher in the luteal phase than in the follicular phase, but the mechanism of changing leptin levels are unknown. Several research lines indicate a potential role for tumor necrosis factor (TNF-alpha) in ovulation and reproductive events. As TNF-alpha appears to regulate leptin secretion, we speculated that TNF-alpha might be involved in leptin variations during the menstrual cycle. DESIGN AND METHODS: Nine healthy never obese and ten overweight normally cycling women were studied. TNF-alpha action - through the plasma levels of the soluble fraction of the tumor necrosis factor receptors 1 and 2 (sTNFR1 and sTNFR2) - and leptin concentrations were measured in the follicular (F), peri-ovulatory (PO) and luteal phases (L) of their menstrual cycles. RESULTS: Circulating leptin levels were significantly associated with the stage of the menstrual cycle (P<0.001), being higher in PO and L phases. However, only three of ten overweight subjects vs eight of nine lean women (Chi square P=0.014 after Fisher's exact test) showed significantly higher leptin levels in the PO and L than in the F phase (95% confidence interval (95% CI) of the differences, 3.7 to 10.2 ng/ml, paired t-test P=0.001). In these women (group 1), the changes in leptin levels parallelled the variations observed in plasma sTNFR1 (2.50+/-0.1 vs 2.11+/-0.05 ng/ml, P<0.0001, 95% CI, 0.21 to 0.56) and sTNFR2 levels (5.19+/-0.28 vs 4.55+/-0.25 ng/ml, P<0.0001, 95% CI, 0. 47 to 0.81). In the remaining women (group 2), leptin (95% CI, -1 to 9.2 ng/ml, P=not significant (NS)), sTNFR1 (95% CI, -0.3 to 0.14 ng/ml, P=NS) and sTNFR2 levels (95% CI, -0.95 to 0.39 ng/ml, P=NS) were essentially unaltered throughout the menstrual cycle. Group 2 women were similar in age (36.1+/-2.9 vs 37.3+/-1.4 years) and significantly overweight (body mass index 31+/-2.9 vs 23.9+/-1. 2 kg/m(2)) compared with group 1 women. A negative correlation was observed between leptin levels in the follicular phase and the change in plasma leptin from F to L phase in all subjects (r=-0.67, P=0.002). CONCLUSIONS: Circulating leptin and sTNFRs levels change significantly during the menstrual cycle of most lean women. In contrast, the levels of these molecules remain essentially unaltered during the F, PO and L phases in the majority of overweight women. Obesity might be associated not only with blunted diurnal excursions and dampened pulsatility, but also with blunted excursions during the menstrual cycle.