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Fasting Serum Levels of Adiponectin, Ghrelin, and Leptin in Men With Spinal Cord Injury
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ORIGINAL ARTICLE
Fasting Serum Levels of Adiponectin, Ghrelin, and Leptin in Men With Spinal Cord Injury Yen-Ho Wang, MD, Tien-Shang Huang, MD, Huey-Wen Liang, MD, MPH, Ta-Chen Su, MD, Ssu-Yuan Chen, MD, PhD, Tzung-Dau Wang, MD, PhD ABSTRACT. Wang Y-H, Huang T-S, Liang H-W, Su T-C, Chen S-Y, Wang T-D. Fasting serum levels of adiponectin, ghrelin, and leptin in men with spinal cord injury. Arch Phys Med Rehabil 2005;86:1964-8. Objectives: To measure serum levels of adiponectin, ghrelin, and leptin in men with spinal cord injury (SCI) and to investigate possible correlations between these serum levels and various factors, such as body mass index (BMI), age, injury level, and duration of injury. Design: Cross-sectional. Setting: A university hospital that is a tertiary referral center. Participants: Eighty-nine men with traumatic neurologically complete SCI (30 with tetraplegia, 59 with paraplegia) and 37 age- and BMI-matched male controls. Subjects with SCI were injured at the mean age ⫾ standard error of 28.5⫾1.0 years (range, 14.7–59.1y) and the mean injury duration was 10.8⫾0.7 years (range, 1.1–27.7y). Interventions: Not applicable. Main Outcome Measures: Serum levels of adiponectin, ghrelin, and leptin and BMI. Results: Serum leptin levels in subjects with SCI (mean, 7.0⫾0.5ng/mL) were significantly higher than those in ablebodied controls (mean, 4.7⫾0.6ng/mL) (P⬍.01). The group with tetraplegia had higher serum leptin levels than the group with paraplegia, but this did not reach a statistically significant level (8.2⫾1.1ng/mL vs 6.4⫾0.5ng/mL, P⫽.097). There were significant differences in serum leptin levels among the 3 groups by 1-way analysis of variance (P⫽.008). Serum adiponectin levels in subjects with SCI (7.1⫾0.5ng/mL) were higher than those in able-bodied controls (5.6⫾0.5ng/mL), but this was not statistically significant (P⫽.08). In contrast, serum levels of ghrelin in subjects with SCI (302.0⫾17.5pg/mL) were similar to those in the controls (264.0⫾27.0pg/mL) (P⫽.24). Serum leptin levels correlated positively with BMI (SCI, r⫽.698, P⬍.001; controls, r⫽.782, P⬍.001), whereas serum adiponectin (SCI, r⫽–.527, P⬍.001; controls, r⫽–.315, P⫽.057) and ghrelin (SCI, r⫽–.368, P⬍.001; controls, r⫽–.447, P⫽.006) levels correlated negatively with the BMI in both subjects with SCI and controls. Conclusions: Men with SCI have significantly higher serum leptin levels than able-bodied controls, and serum leptin levels
From the Departments of Physical Medicine and Rehabilitation (Wang, Liang, Chen) and Internal Medicine (Huang, Su, Wang), National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan. Supported by the National Science Council of the Republic of China (grant no. NSC 92-2314-B002-196). No commercial party having a direct financial interest in the results of the research supporting this article has or will confer a benefit upon the authors or upon any organization with which the authors are associated. Reprint requests to Tien-Shang Huang, MD, Dept of Internal Medicine, National Taiwan University Hospital, No 7 Chung-Shan S Rd, Taipei 100, Taiwan, e-mail: [email protected]. 0003-9993/05/8610-9734$30.00/0 doi:10.1016/j.apmr.2005.04.017
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correlated with the degree of neurologic deficit. Men with SCI had a tendency toward higher serum adiponectin level than able-bodied controls. Serum levels of ghrelin in men with SCI were similar to those of controls. Key Words: Adipocytes; Leptin; Rehabilitation; Spinal cord injuries. © 2005 by the American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and Rehabilitation EPTIN, THE PRODUCT OF the OB gene, is a 16-kd L protein secreted by adipocytes that signals the size of adipose tissue. Leptin acts on the hypothalamus to suppress 1-3
food intake by inhibiting appetite and to stimulate energy expenditure by increasing sympathetic nervous system (SNS) activity,4,5 and thus it represents one of the defense mechanisms against the development of obesity. However, the satiety effect of leptin is less convincing in humans than in rodents. Leptin also regulates the neuroendocrine response to fasting6 and has been suggested to regulate thyroid, gonad, and adrenal function.7-9 There are complex interactions between leptin and insulin.10 Subjects with spinal cord injury (SCI) have higher serum levels of leptin compared with able-bodied controls.11,12 Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor,13 functions as an orexigenic signal from the stomach when an increase in metabolic efficiency is needed.14 In the hypothalamus, ghrelin acts via neuropeptide Y and agouti-related protein in an antagonistic manner to leptin.15,16 Both ghrelin and leptin receptors are found in the stomach, and endocrine and vagal pathways are involved in their gastrointestinal actions.17-19 Whether ghrelin secretion is altered in subjects with SCI is unclear. Adiponectin is a 30-kd protein produced primarily by adipocytes.20-23 In contrast to leptin levels, adiponectin levels are decreased in obesity.24 Because people with SCI have higher adiposity, we expect that people with SCI would have lower serum adiponectin levels compared with able-bodied controls. There is evidence that adiponectin is an important regulator of insulin sensitivity.25,26 Various in vitro and in vivo studies27-29 suggest that adiponectin has anti-inflammatory and antiatherogenic activities. It is suggested that adiponectin is an important factor in obesity-associated comorbidities.30 People with SCI have an increased prevalence of insulin resistance.31,32 A recent epidemiologic study33 showed an increased cardiovascular mortality in subjects with SCI. Earlier studies12 had shown that serum leptin correlates positively with body mass index (BMI) and adiposity but correlates negatively with serum cortisol level in subjects with SCI and able-bodied controls. However, there is no information on levels of adiponectin and ghrelin in subjects with SCI. In this study, we measured serum levels of adiponectin, ghrelin, and leptin in 89 men with SCI and investigated the correlations between these serum levels and various factors, such as BMI, age, injury level, and duration of injury.
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ADIPONECTIN, GHRELIN, AND LEPTIN IN SCI, Wang Table 1: Comparison of Demographic Data of 126 Subjects Control (n⫽37)
SCI (n⫽89)
P (t test)*
38.1⫾1.4 (23.8–57.6) 68.2⫾2.0 (46–96) 170.8⫾1.1 (159–186) 23.3⫾0.6 (17.3–29.9)
39.3⫾1.1 (21.4–68.3) 65.1⫾1.2 (46–93) 170.8⫾0.6 (159–183) 22.3⫾0.40 (15.0–30.1)
.54
Characteristic
Age (y) Body weight (kg) Body height (cm) BMI (kg/m2)
.18 .98 .21
Paraplegic (n⫽59)
Tetraplegic (n⫽30)
P (ANOVA)†
T6 and Above (n⫽49)
T7 and Below (n⫽40)
P (ANOVA)‡
39.5⫾1.4 (21.4–68.3) 65.9⫾1.5 (46–93) 171.2⫾0.8 (155–183) 22.4⫾0.5 (15.0–29.6)
38.9⫾2.0 (21.5–62.0) 64.3⫾2.1 (46–87) 170.2⫾0.9 (162–180) 22.2⫾0.8 (15.7–30.1)
.80
40.6⫾1.7 (21.5–68.3) 67.1⫾1.7 (46–93) 170.4⫾0.7 (157–183) 23.1⫾0.6 (15.7–30.1)
37.6⫾1.5 (21.4–62.7) 62.8⫾1.7 (46–83) 171.3⫾1.0 (155–183) 21.4⫾0.6 (15.0–28.3)
.31
.36 .76 .45
.09 .76 .05
NOTE. Values are mean ⫾ SE (range). *Comparison between controls and SCI. † Comparison among controls, paraplegics, and tetraplegics. ‡ Comparison among controls, injury at T6 and above, and injury at T7 and below.
METHODS Participants Eighty-nine men with traumatic SCI (59 with paraplegia, 30 with tetraplegia) were recruited from the SCI clinic of the National Taiwan University Hospital. Their SCIs were neurologically complete, as defined by the American Spinal Injury Association.34 The injury levels were from C3 to L1. Those with injury levels at or above C8 were classified as tetraplegics and the others as paraplegics. Subjects had been injured at the mean age ⫾ standard error (SE) of 28.5⫾1.0 years (range, 14.7–59.1y), and the mean injury duration was 10.8⫾0.7 years (range, 1.1–27.7y). Subjects with paraplegia had longer injury duration than those with tetraplegia (11.8⫾1.9y vs 8.6⫾1.0y, P⫽.042). BMI was defined as weight divided by height squared (in kg/m2). The control group consisted of 37 age- and BMI-matched healthy men. No subjects had known diabetes or endocrine disorders. Except for a few patients who were taking stool softeners (magnesium oxide) or low-dose antispastic agents (baclofen), none were taking drugs regularly. All subjects voluntarily agreed to participate in the study. They all gave written informed consent as approved by the Research Ethics Committee of National Taiwan University Hospital. Radioimmunoassays Blood samples were taken from all subjects between 8:00 and 9:00 AM after overnight fasting, and serum was prepared and stored frozen at –70°C until assayed. Leptin and adiponectin levels were assayed using Linco radioimmunoassay (RIA) kits,a which had been previously used in our laboratory with interassay and intra-assay variations of less than 10%. Ghrelin levels were measured using RIA kits obtained from Phoenix Pharmaceuticals.b
Statistical Analyses Analyses were performed using SPSS, version 10.0,c for Windows. The continuous results are reported as the mean ⫾ SE. The differences of demographic characteristics (age, body weight, body height, BMI), serum leptin, adiponectin, and ghrelin among subjects with SCI and able-bodied controls were compared by t tests. The differences of the above variables among paraplegics, tetraplegics, and able-bodied controls were compared with 1-way analysis of variance (ANOVA) with post hoc Bonferroni tests. The correlation between BMI and serum levels of leptin, adiponectin, or ghrelin was examined using the Pearson correlation coefficient. Multiple linear regression analysis was performed using serum levels of leptin, adiponectin, and ghrelin as dependent variables and BMI, age, injury classification, and injury duration as independent variables. Stepwise methods were used to determine the model. Both linear and nonlinear regression models were used. A P value less than .05 was considered statistically significant. RESULTS Table 1 shows the demographic data for the 30 subjects with tetraplegia, 59 subjects with paraplegia, and 37 controls. The serum levels of leptin, ghrelin, and adiponectin were in a fairly normal distribution curve (table 2). The SCI group had a higher mean serum leptin level than the able-bodied controls (7.0⫾0.5ng/mL vs 4.7⫾0.6ng/mL, P⫽.01) by t test. Serum leptin levels differed significantly among the control, tetraplegic, and paraplegic groups by 1-way ANOVA (P⫽.008). Based on post hoc Bonferroni testing, the serum leptin levels of tetraplegics were significantly higher than those of able-bodied controls. The tetraplegic group had higher— but not statisti-
Table 2: Comparison of Serum Leptin, Adiponectin, and Ghrelin Levels of 126 Subjects Protein
Leptin (ng/mL) Adiponectin (ng/mL) Ghrelin (pg/mL)
Control (n⫽37)
4.7⫾0.6 (1.1–19.1) 5.6⫾0.5 (1.3–15.1) 264.0⫾27.0 (16–687)
SCI (n⫽89)
7.0⫾0.5 (0.6–22.3) 7.1⫾0.5 (1.3–26) 302.0⫾17.5 (53–989)
P (t test)*
.010 .080 .240
Paraplegic (n⫽59)
Tetraplegic (n⫽30)
6.4⫾0.5 (0.6–16.2) 6.8⫾0.5 (1.3–15.3) 318.2⫾21.4 (63–768)
8.2⫾1.1 (1.3–22.3) 7.7⫾1.1 (2.1–26.0) 270.2⫾30.0 (53–989)
P (ANOVA)†
.008 .130 .220
T6 and Above (n⫽49)
T7 and Below (n⫽40)
7.9⫾0.7 (1.3–22.3) 7.3⫾0.8 (1.4–26.0) 267.8⫾20.3 (53–989)
5.9⫾0.7 (0.6–16.6) 6.9⫾0.6 (1.3–14.6) 344.0⫾28.7 (63–768)
P (ANOVA)‡
.004 .200 .050
NOTE. Values are mean ⫾ SE (range). *Comparison between controls and SCI. † Comparison among controls, paraplegics, and quadriplegics. ‡ Comparison among controls, injury at T6 and above, and injury at T7 and below.
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cally significant—serum leptin levels than the paraplegic group (8.2⫾1.1ng/mL vs 6.4⫾0.5ng/mL, P⫽.097) by Bonferroni adjustment. However, subjects with SCI who had a neurologic injury level of T6 and above had significantly higher serum leptin levels than subjects with SCI who had a neurologic injury level of T7 and below (7.9⫾0.7ng/mL vs 5.9⫾0.7ng/ mL, P⫽.049). Serum adiponectin levels in subjects with SCI (7.1⫾0.5ng/mL) were higher than those in able-bodied controls (5.6⫾0.5ng/mL) (P⫽.08), but this was not statistically significant. In contrast, serum levels of ghrelin in subjects with SCI (302.0⫾17.5pg/mL) were similar to those in the controls (264.0⫾27.0pg/mL) (P⫽.24). Although the serum level of adiponectin between subjects with SCI who had injuries above or below T6 were similar (7.3⫾0.8ng/mL vs 6.9⫾0.6ng/mL, P⫽.682), the serum levels of ghrelin in subjects with SCI who had a neurologic injury level of T6 and above were significantly lower than those in subjects with SCI who had a neurologic injury level of T7 and below (267.8⫾20.3pg/mL vs 344.0⫾28.7pg/mL, P⫽.029). Using the Pearson correlation coefficient, we found serum leptin levels to correlate positively with BMI, whereas serum adiponectin and ghrelin levels correlated negatively with BMI in both subjects with SCI and controls (tables 3, 4). Multiple regression analysis showed that serum leptin levels correlated not only with the BMI but also with the degree of neurologic deficit (r⫽.752, P⬍.001):
关leptin兴⫽⫺15.519⫹0.870⫻关BMI兴⫹4.373⫻关GP1兴
⫹2.438⫻关GP2兴
where GP1 is the group effect for tetraplegia and GP2 is the group effect for paraplegia. Serum adiponectin levels correlated negatively with serum leptin levels in subjects with SCI, but the correlation was less significant in controls (see tables 3, 4). The influence of SCI was not found for serum adiponectin by stepwise multiple linear regression analysis. Both BMI and age were found to be influencing factors for adiponectin levels by stepwise multiple regression analysis (r⫽.595, P⬍.001):
关adiponectin兴⫽17.752⫺0.684⫻关BMI兴⫹0.123⫻关age兴. There was also a negative correlation between serum ghrelin levels and leptin levels in both subjects with SCI and controls (see tables 3, 4). Meanwhile, only BMI was a significant influencing factor for ghrelin levels by stepwise multiple linear regression analysis (r⫽.368, P⬍.001):
关ghrelin兴⫽647.867⫺15.475⫻关BMI兴. Table 3: Pearson Correlation Coefficients and P Values for BMI Compared With Serum Levels of Leptin, Adiponectin, or Ghrelin in Normative Controls
Age Leptin Adiponectin Ghrelin
BMI
Age
Leptin
Adiponectin
r⫽.078 P⫽.648 r⫽.782 P⬍.001 r⫽–.315 P⫽.057 r⫽–.447 P⫽.006
—
—
—
r⫽.182 P⫽.280 r⫽.023 P⫽.890 r⫽–.189 P⫽.269
—
—
r⫽–.278 P⫽.096 r⫽–.336 P⫽.045
—
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r⫽.114 P⫽.508
Table 4: Pearson Correlation Coefficients and P Value for BMI Compared With Serum Levels of Leptin, Adiponectin, or Ghrelin in the Subjects With SCI
Age Leptin Adiponectin Ghrelin
BMI
Age
Leptin
Adiponectin
r⫽.156 P⫽.143 r⫽.698 P⬍.001 r⫽–.527 P⬍.001 r⫽–.368 P⬍.001
—
—
—
r⫽.113 P⫽.292 r⫽.190 P⫽.074 r⫽–.141 P⫽.187
—
—
r⫽–.363 P⬍.001 r⫽–.260 P⫽.014
— r⫽.169 P⫽.114
DISCUSSION In this study, men with SCI had significantly higher serum leptin levels than the able-bodied group. This finding is consistent with the results of previous studies.11,12 Furthermore, subjects with tetraplegia had higher serum leptin levels than those with paraplegia, although the difference was not significant. However, when subjects with SCI were grouped into those with an injury above or below T6, leptin levels were significantly higher in the former group. T6 appears to be the lowest level of injury in most patients with SCI to develop autonomic dysreflexia.35 With SCIs above the level of T6, there is reduced SNS outflow and supraspinal control to the splanchnic outflow and the lower-extremity blood vessels.36 Multiple regression analysis showed that serum leptin levels in men with SCI correlated not only with BMI but also with the neurologic deficit. This finding supports the notion that decentralization of sympathetic nervous activity relieves its inhibitory tone on leptin secretion,37,38 because subjects with tetraplegia have a more severe deficit of sympathetic nervous activity. Jeon et al39 reported that the leptin response to short-term fasting and its effects on resting metabolic rate are impaired in subjects with SCI. Spungen et al40 reported that subjects with SCI were 13% fatter per unit of BMI compared with able-bodied controls. However, subjects with SCI had high serum leptin levels out of proportion with their adiposity—that is, a nearly 50% increase in this study and the Bauman et al study11and a 100% increase in another study.12 This implies that there are other contributing factors in hyperleptinemia in subjects with SCI. There is a leptin-SNS-leptin feedback loop.37,41 Leptin produces widespread sympathetic activation in various tissues and organs, including brown adipose tissue, kidney, hind limb, and adrenal glands,42,43 and activation of the SNS modulates leptin expression and secretion in rodents and humans in vitro and in vivo.44 People with SCI show decentralization of the SNS after the injury. This interruption blocks not only the inhibitory effect of the SNS on leptin expression and secretion41 but also the stimulatory effect of leptin on energy expenditure.39 Subjects with SCI therefore develop hyperleptinemia, a decreased resting metabolic rate, and increased adiposity. In this study, serum adiponectin levels in men with SCI were higher than those in the able-bodied group, and there was no significant difference in serum adiponectin levels between subjects with paraplegia and tetraplegia. Serum adiponectin levels are reported to correlate negatively with BMI in able-bodied men.24 In our study, they were shown to correlate negatively with the BMI in both the controls and subjects with SCI. Because subjects with SCI have a higher degree of adiposity and prevalence of insulin resistance, we expected that they would have higher serum leptin levels and lower serum adiponectin levels than controls. There are several possible expla-
ADIPONECTIN, GHRELIN, AND LEPTIN IN SCI, Wang
nations for the high serum adiponectin levels in subjects with SCI. First, RIA measures various forms of adiponectin, only some of which are biologically active.20,22,45 Second, adipose tissue in subjects with SCI may secrete different forms of adiponectin that are biologically inactive20,22; Delporte et al46 have reported the presence of a different form of adiponectin in -agonist–stimulated adipocytes and that administration of a 3 agonist to mice reduces adiponectin messenger RNA levels in subcutaneous and visceral adipose tissue and adiponectin levels in the plasma. An inhibitory effect of a  agonist on adiponectin messenger RNA levels was also shown in human adipocyte cultures.46 SCI results in decreased sympathetic activity; therefore, SCI may relieve its inhibitory effect on adiponectin secretion by adipose tissue. Third, adiponectin is not the only determining factor in insulin resistance, because other factors are known to be involved.47,48 In this study, serum ghrelin levels in men with SCI were similar to those in able-bodied controls. In humans, ghrelin secretion is under cholinergic control.49 Relative preservation of vagal parasympathetic function has been noted in subjects with SCI.50 In our study, we found a negative correlation between serum ghrelin levels and BMI or serum leptin levels in both the men with SCI and the normative controls. However, multiple regression analysis showed that BMI was the only significant factor for serum ghrelin levels. This finding is consistent with early studies51,52 and suggests that the stomach is part of the neuroendocrine system for energy homeostasis.53 BMI is a surrogate marker for fat mass, and it has been correlated with adipocytokines in study. However, BMI does not distinguish the anatomic site of fat mass. Actually not only subjects with SCI as a group but also subjects with paraplegia and tetraplegia have different body compositions as compared with able-bodied controls. Despite the above limitations, BMI is still a good surrogate measure for adiposity within the same neurologic-deficit group. One must be careful in using BMI to compare between groups. Thus, the definition of obesity should be redefined with a lower level in subjects with SCI. CONCLUSIONS Men with SCI have significantly higher serum leptin levels than able-bodied controls, and the serum leptin levels correlate with the degree of neurologic deficit. In addition to higher serum leptin levels, men with SCI also have higher serum levels of adiponectin but similar serum ghrelin levels compared with controls. The roles of these 3 hormones in the pathogenesis of insulin resistance in subjects with SCI require further investigation. References 1. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman SM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:425-32. 2. Halaas JL, Gajiwala KS, Maffei M, et al. Weight-reducing effect of the plasma protein encoded by the obese gene. Science 1995; 269:543-6. 3. Mantzoros CS. The role of leptin in human obesity and disease: a review of current evidence. Ann Intern Med 1999;130:671-80. 4. Scarpace PJ, Matheny M, Pollock BH, Tumer N. Leptin increases uncoupling protein expression and energy expenditure. Am J Physiol 1997;273:E226-30. 5. Tang-Christensen M, Havel PJ, Jacobs RR, Larsen PJ, Cameron JL. Central administration of leptin inhibits food intake and activates the sympathetic nervous system in rhesus macaques. J Clin Endocrinol Metab 1999;84:711-7. 6. Ahima RS, Prabakaran D, Mantzoros C, et al. Role of leptin in the neuroendocrine response to fasting. Nature 1996;382:250-2.
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28. Ouchi N, Kihara S, Arita Y, et al. Adiponectin, an adipocytederived plasma protein, inhibits endothelial NF-kappaB signaling through a cAMP-dependent pathway. Circulation 2000;102:1296301. 29. Kubota N, Terauchi Y, Yamauchi T, et al. Disruption of adiponectin causes insulin resistance and neointimal formation. J Biol Chem 2002;277:25863-6. 30. Ukkola O, Santaniemi M. Adiponectin: a link between excess adiposity and associated comorbidities? J Mol Med 2002;80:696702. 31. Duckworth WC, Solomon SS, Jallepalli P, Heckemeyer C, Finnern J, Powers A. Glucose intolerance due to insulin resistance in patients with spinal cord injuries. Diabetes 1980;29:906-10. 32. Bauman WA, Adkins RH, Spungen AM, Waters RL. The effect of residual neurological deficit on oral glucose tolerance in persons with chronic spinal cord injury. Spinal Cord 1999;37:765-71. 33. Whiteneck GG, Charlifue SW, Frankel HL, et al. Mortality, morbidity, and psychosocial outcomes of persons spinal cord injured more than 20 years ago. Paraplegia 1992;30:617-30. 34. Maynard FM Jr, Bracken MB, Creasey G, et al. International Standards for Neurological and Functional Classification of Spinal Cord Injury. American Spinal Injury Association. Spinal Cord 1997;35:266-74. 35. Erickson RP. Autonomic hyperreflexia: pathophysiology and medical management. Arch Phys Med Rehabil 1980;61:431-40. 36. Teasell RW, Arnold JM, Krassioukov A, Delaney GA. Cardiovascular consequences of loss of supraspinal control of the sympathetic nervous system after spinal cord injury. Arch Phys Med Rehabil 2000;81:506-16. 37. Trayhurn P, Duncan JS, Rayner DV. Acute cold-induced suppression of ob (obese) gene expression in white adipose tissue of mice: mediation by the sympathetic system. Biochem J 1995;311:72933. 38. Rayner DV, Simon E, Duncan JS, Trayhurn P. Hyperleptinaemia in mice induced by administration of the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine. FEBS Lett 1998;429:395-8. 39. Jeon JY, Harber VJ, Steadward RD. Leptin response to short-term fasting in sympathectomized men: role of the SNS. Am J Physiol Endocrinol Metab 2003;284:E634-40. 40. Spungen AM, Adkins RH, Stewart CA, et al. Factors influencing body composition in persons with spinal cord injury: a crosssectional study. J Appl Physiol 2003;95:2398-407. 41. Rayner DV, Trayhurn P. Regulation of leptin production: sympathetic nervous system interactions. J Mol Med 2001;79:8-20.
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42. Haynes WG, Sivitz WI, Morgan DA, Walsh SA, Mark AL. Sympathetic and cardiorenal actions of leptin. Hypertension 1997; 30:619-23. 43. Dunbar JC, Hu Y, Lu H. Intracerebroventricular leptin increases lumbar and renal sympathetic nerve activity and blood pressure in normal rats. Diabetes 1997;46:2040-3. 44. Mark AL, Rahmouni K, Correia M, Haynes WG. A leptinsympathetic-leptin feedback loop: potential implications for regulation of arterial pressure and body fat. Acta Physiol Scand 2003;177:345-9. 45. Sato C, Yasukawa Z, Honda N, Matsuda T, Kitajima K. Identification and adipocyte differentiation-dependent expression of the unique disialic acid residue in an adipose tissue-specific glycoprotein, adipo Q. J Biol Chem 2001;276:28849-56. 46. Delporte ML, Funahashi T, Takahashi M, Matsuzawa Y, Brichard SM. Pre- and post-translational negative effect of betaadrenoceptor agonists on adiponectin secretion: in vitro and in vivo studies. Biochem J 2002;367:677-85. 47. Fruhbeck G, Gomez-Ambrosi J, Muruzabal FJ, Burrell MA. The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation. Am J Physiol Endocrinol Metab 2001;280:E827-47. 48. Havel PJ. Control of energy homeostasis and insulin action by adipocyte hormones: leptin, acylation stimulating protein, and adiponectin. Curr Opin Lipidol 2002;13:51-9. 49. Broglio F, Gottero C, Van Koetsveld P, et al. Acetylcholine regulates ghrelin secretion in humans. J Clin Endocrinol Metab 2004;89:2429-33. 50. Wang YH, Huang TS, Lin JL, et al. Decreased autonomic nervous system activity as assessed by heart rate variability in patients with chronic tetraplegia. Arch Phys Med Rehabil 2000;81:1181-4. 51. Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000;407:908-13. 52. Tschop M, Weyer C, Tataranni PA, Devanarayan V, Ravussin E, Heiman ML. Circulating ghrelin levels are decreased in human obesity. Diabetes 2001;50:707-9. 53. Inui A, Asakawa A, Bowers CY, et al. Ghrelin, appetite, and gastric motility: the emerging role of the stomach as an endocrine organ. FASEB J 2004;18:439-56. Suppliers a. Linco Research, 6 Research Park Dr, St Charles, MO 63304. b. Phoenix Pharmaceuticals Inc, 530 Harbor Blvd, Belmont, CA 94002. c. SPSS Inc, 233 S Wacker Dr, 11th Fl, Chicago, IL 60606.
ORIGINAL ARTICLE
Fasting Serum Levels of Adiponectin, Ghrelin, and Leptin in Men With Spinal Cord Injury Yen-Ho Wang, MD, Tien-Shang Huang, MD, Huey-Wen Liang, MD, MPH, Ta-Chen Su, MD, Ssu-Yuan Chen, MD, PhD, Tzung-Dau Wang, MD, PhD ABSTRACT. Wang Y-H, Huang T-S, Liang H-W, Su T-C, Chen S-Y, Wang T-D. Fasting serum levels of adiponectin, ghrelin, and leptin in men with spinal cord injury. Arch Phys Med Rehabil 2005;86:1964-8. Objectives: To measure serum levels of adiponectin, ghrelin, and leptin in men with spinal cord injury (SCI) and to investigate possible correlations between these serum levels and various factors, such as body mass index (BMI), age, injury level, and duration of injury. Design: Cross-sectional. Setting: A university hospital that is a tertiary referral center. Participants: Eighty-nine men with traumatic neurologically complete SCI (30 with tetraplegia, 59 with paraplegia) and 37 age- and BMI-matched male controls. Subjects with SCI were injured at the mean age ⫾ standard error of 28.5⫾1.0 years (range, 14.7–59.1y) and the mean injury duration was 10.8⫾0.7 years (range, 1.1–27.7y). Interventions: Not applicable. Main Outcome Measures: Serum levels of adiponectin, ghrelin, and leptin and BMI. Results: Serum leptin levels in subjects with SCI (mean, 7.0⫾0.5ng/mL) were significantly higher than those in ablebodied controls (mean, 4.7⫾0.6ng/mL) (P⬍.01). The group with tetraplegia had higher serum leptin levels than the group with paraplegia, but this did not reach a statistically significant level (8.2⫾1.1ng/mL vs 6.4⫾0.5ng/mL, P⫽.097). There were significant differences in serum leptin levels among the 3 groups by 1-way analysis of variance (P⫽.008). Serum adiponectin levels in subjects with SCI (7.1⫾0.5ng/mL) were higher than those in able-bodied controls (5.6⫾0.5ng/mL), but this was not statistically significant (P⫽.08). In contrast, serum levels of ghrelin in subjects with SCI (302.0⫾17.5pg/mL) were similar to those in the controls (264.0⫾27.0pg/mL) (P⫽.24). Serum leptin levels correlated positively with BMI (SCI, r⫽.698, P⬍.001; controls, r⫽.782, P⬍.001), whereas serum adiponectin (SCI, r⫽–.527, P⬍.001; controls, r⫽–.315, P⫽.057) and ghrelin (SCI, r⫽–.368, P⬍.001; controls, r⫽–.447, P⫽.006) levels correlated negatively with the BMI in both subjects with SCI and controls. Conclusions: Men with SCI have significantly higher serum leptin levels than able-bodied controls, and serum leptin levels
From the Departments of Physical Medicine and Rehabilitation (Wang, Liang, Chen) and Internal Medicine (Huang, Su, Wang), National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan. Supported by the National Science Council of the Republic of China (grant no. NSC 92-2314-B002-196). No commercial party having a direct financial interest in the results of the research supporting this article has or will confer a benefit upon the authors or upon any organization with which the authors are associated. Reprint requests to Tien-Shang Huang, MD, Dept of Internal Medicine, National Taiwan University Hospital, No 7 Chung-Shan S Rd, Taipei 100, Taiwan, e-mail: [email protected]. 0003-9993/05/8610-9734$30.00/0 doi:10.1016/j.apmr.2005.04.017
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correlated with the degree of neurologic deficit. Men with SCI had a tendency toward higher serum adiponectin level than able-bodied controls. Serum levels of ghrelin in men with SCI were similar to those of controls. Key Words: Adipocytes; Leptin; Rehabilitation; Spinal cord injuries. © 2005 by the American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and Rehabilitation EPTIN, THE PRODUCT OF the OB gene, is a 16-kd L protein secreted by adipocytes that signals the size of adipose tissue. Leptin acts on the hypothalamus to suppress 1-3
food intake by inhibiting appetite and to stimulate energy expenditure by increasing sympathetic nervous system (SNS) activity,4,5 and thus it represents one of the defense mechanisms against the development of obesity. However, the satiety effect of leptin is less convincing in humans than in rodents. Leptin also regulates the neuroendocrine response to fasting6 and has been suggested to regulate thyroid, gonad, and adrenal function.7-9 There are complex interactions between leptin and insulin.10 Subjects with spinal cord injury (SCI) have higher serum levels of leptin compared with able-bodied controls.11,12 Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor,13 functions as an orexigenic signal from the stomach when an increase in metabolic efficiency is needed.14 In the hypothalamus, ghrelin acts via neuropeptide Y and agouti-related protein in an antagonistic manner to leptin.15,16 Both ghrelin and leptin receptors are found in the stomach, and endocrine and vagal pathways are involved in their gastrointestinal actions.17-19 Whether ghrelin secretion is altered in subjects with SCI is unclear. Adiponectin is a 30-kd protein produced primarily by adipocytes.20-23 In contrast to leptin levels, adiponectin levels are decreased in obesity.24 Because people with SCI have higher adiposity, we expect that people with SCI would have lower serum adiponectin levels compared with able-bodied controls. There is evidence that adiponectin is an important regulator of insulin sensitivity.25,26 Various in vitro and in vivo studies27-29 suggest that adiponectin has anti-inflammatory and antiatherogenic activities. It is suggested that adiponectin is an important factor in obesity-associated comorbidities.30 People with SCI have an increased prevalence of insulin resistance.31,32 A recent epidemiologic study33 showed an increased cardiovascular mortality in subjects with SCI. Earlier studies12 had shown that serum leptin correlates positively with body mass index (BMI) and adiposity but correlates negatively with serum cortisol level in subjects with SCI and able-bodied controls. However, there is no information on levels of adiponectin and ghrelin in subjects with SCI. In this study, we measured serum levels of adiponectin, ghrelin, and leptin in 89 men with SCI and investigated the correlations between these serum levels and various factors, such as BMI, age, injury level, and duration of injury.
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ADIPONECTIN, GHRELIN, AND LEPTIN IN SCI, Wang Table 1: Comparison of Demographic Data of 126 Subjects Control (n⫽37)
SCI (n⫽89)
P (t test)*
38.1⫾1.4 (23.8–57.6) 68.2⫾2.0 (46–96) 170.8⫾1.1 (159–186) 23.3⫾0.6 (17.3–29.9)
39.3⫾1.1 (21.4–68.3) 65.1⫾1.2 (46–93) 170.8⫾0.6 (159–183) 22.3⫾0.40 (15.0–30.1)
.54
Characteristic
Age (y) Body weight (kg) Body height (cm) BMI (kg/m2)
.18 .98 .21
Paraplegic (n⫽59)
Tetraplegic (n⫽30)
P (ANOVA)†
T6 and Above (n⫽49)
T7 and Below (n⫽40)
P (ANOVA)‡
39.5⫾1.4 (21.4–68.3) 65.9⫾1.5 (46–93) 171.2⫾0.8 (155–183) 22.4⫾0.5 (15.0–29.6)
38.9⫾2.0 (21.5–62.0) 64.3⫾2.1 (46–87) 170.2⫾0.9 (162–180) 22.2⫾0.8 (15.7–30.1)
.80
40.6⫾1.7 (21.5–68.3) 67.1⫾1.7 (46–93) 170.4⫾0.7 (157–183) 23.1⫾0.6 (15.7–30.1)
37.6⫾1.5 (21.4–62.7) 62.8⫾1.7 (46–83) 171.3⫾1.0 (155–183) 21.4⫾0.6 (15.0–28.3)
.31
.36 .76 .45
.09 .76 .05
NOTE. Values are mean ⫾ SE (range). *Comparison between controls and SCI. † Comparison among controls, paraplegics, and tetraplegics. ‡ Comparison among controls, injury at T6 and above, and injury at T7 and below.
METHODS Participants Eighty-nine men with traumatic SCI (59 with paraplegia, 30 with tetraplegia) were recruited from the SCI clinic of the National Taiwan University Hospital. Their SCIs were neurologically complete, as defined by the American Spinal Injury Association.34 The injury levels were from C3 to L1. Those with injury levels at or above C8 were classified as tetraplegics and the others as paraplegics. Subjects had been injured at the mean age ⫾ standard error (SE) of 28.5⫾1.0 years (range, 14.7–59.1y), and the mean injury duration was 10.8⫾0.7 years (range, 1.1–27.7y). Subjects with paraplegia had longer injury duration than those with tetraplegia (11.8⫾1.9y vs 8.6⫾1.0y, P⫽.042). BMI was defined as weight divided by height squared (in kg/m2). The control group consisted of 37 age- and BMI-matched healthy men. No subjects had known diabetes or endocrine disorders. Except for a few patients who were taking stool softeners (magnesium oxide) or low-dose antispastic agents (baclofen), none were taking drugs regularly. All subjects voluntarily agreed to participate in the study. They all gave written informed consent as approved by the Research Ethics Committee of National Taiwan University Hospital. Radioimmunoassays Blood samples were taken from all subjects between 8:00 and 9:00 AM after overnight fasting, and serum was prepared and stored frozen at –70°C until assayed. Leptin and adiponectin levels were assayed using Linco radioimmunoassay (RIA) kits,a which had been previously used in our laboratory with interassay and intra-assay variations of less than 10%. Ghrelin levels were measured using RIA kits obtained from Phoenix Pharmaceuticals.b
Statistical Analyses Analyses were performed using SPSS, version 10.0,c for Windows. The continuous results are reported as the mean ⫾ SE. The differences of demographic characteristics (age, body weight, body height, BMI), serum leptin, adiponectin, and ghrelin among subjects with SCI and able-bodied controls were compared by t tests. The differences of the above variables among paraplegics, tetraplegics, and able-bodied controls were compared with 1-way analysis of variance (ANOVA) with post hoc Bonferroni tests. The correlation between BMI and serum levels of leptin, adiponectin, or ghrelin was examined using the Pearson correlation coefficient. Multiple linear regression analysis was performed using serum levels of leptin, adiponectin, and ghrelin as dependent variables and BMI, age, injury classification, and injury duration as independent variables. Stepwise methods were used to determine the model. Both linear and nonlinear regression models were used. A P value less than .05 was considered statistically significant. RESULTS Table 1 shows the demographic data for the 30 subjects with tetraplegia, 59 subjects with paraplegia, and 37 controls. The serum levels of leptin, ghrelin, and adiponectin were in a fairly normal distribution curve (table 2). The SCI group had a higher mean serum leptin level than the able-bodied controls (7.0⫾0.5ng/mL vs 4.7⫾0.6ng/mL, P⫽.01) by t test. Serum leptin levels differed significantly among the control, tetraplegic, and paraplegic groups by 1-way ANOVA (P⫽.008). Based on post hoc Bonferroni testing, the serum leptin levels of tetraplegics were significantly higher than those of able-bodied controls. The tetraplegic group had higher— but not statisti-
Table 2: Comparison of Serum Leptin, Adiponectin, and Ghrelin Levels of 126 Subjects Protein
Leptin (ng/mL) Adiponectin (ng/mL) Ghrelin (pg/mL)
Control (n⫽37)
4.7⫾0.6 (1.1–19.1) 5.6⫾0.5 (1.3–15.1) 264.0⫾27.0 (16–687)
SCI (n⫽89)
7.0⫾0.5 (0.6–22.3) 7.1⫾0.5 (1.3–26) 302.0⫾17.5 (53–989)
P (t test)*
.010 .080 .240
Paraplegic (n⫽59)
Tetraplegic (n⫽30)
6.4⫾0.5 (0.6–16.2) 6.8⫾0.5 (1.3–15.3) 318.2⫾21.4 (63–768)
8.2⫾1.1 (1.3–22.3) 7.7⫾1.1 (2.1–26.0) 270.2⫾30.0 (53–989)
P (ANOVA)†
.008 .130 .220
T6 and Above (n⫽49)
T7 and Below (n⫽40)
7.9⫾0.7 (1.3–22.3) 7.3⫾0.8 (1.4–26.0) 267.8⫾20.3 (53–989)
5.9⫾0.7 (0.6–16.6) 6.9⫾0.6 (1.3–14.6) 344.0⫾28.7 (63–768)
P (ANOVA)‡
.004 .200 .050
NOTE. Values are mean ⫾ SE (range). *Comparison between controls and SCI. † Comparison among controls, paraplegics, and quadriplegics. ‡ Comparison among controls, injury at T6 and above, and injury at T7 and below.
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cally significant—serum leptin levels than the paraplegic group (8.2⫾1.1ng/mL vs 6.4⫾0.5ng/mL, P⫽.097) by Bonferroni adjustment. However, subjects with SCI who had a neurologic injury level of T6 and above had significantly higher serum leptin levels than subjects with SCI who had a neurologic injury level of T7 and below (7.9⫾0.7ng/mL vs 5.9⫾0.7ng/ mL, P⫽.049). Serum adiponectin levels in subjects with SCI (7.1⫾0.5ng/mL) were higher than those in able-bodied controls (5.6⫾0.5ng/mL) (P⫽.08), but this was not statistically significant. In contrast, serum levels of ghrelin in subjects with SCI (302.0⫾17.5pg/mL) were similar to those in the controls (264.0⫾27.0pg/mL) (P⫽.24). Although the serum level of adiponectin between subjects with SCI who had injuries above or below T6 were similar (7.3⫾0.8ng/mL vs 6.9⫾0.6ng/mL, P⫽.682), the serum levels of ghrelin in subjects with SCI who had a neurologic injury level of T6 and above were significantly lower than those in subjects with SCI who had a neurologic injury level of T7 and below (267.8⫾20.3pg/mL vs 344.0⫾28.7pg/mL, P⫽.029). Using the Pearson correlation coefficient, we found serum leptin levels to correlate positively with BMI, whereas serum adiponectin and ghrelin levels correlated negatively with BMI in both subjects with SCI and controls (tables 3, 4). Multiple regression analysis showed that serum leptin levels correlated not only with the BMI but also with the degree of neurologic deficit (r⫽.752, P⬍.001):
关leptin兴⫽⫺15.519⫹0.870⫻关BMI兴⫹4.373⫻关GP1兴
⫹2.438⫻关GP2兴
where GP1 is the group effect for tetraplegia and GP2 is the group effect for paraplegia. Serum adiponectin levels correlated negatively with serum leptin levels in subjects with SCI, but the correlation was less significant in controls (see tables 3, 4). The influence of SCI was not found for serum adiponectin by stepwise multiple linear regression analysis. Both BMI and age were found to be influencing factors for adiponectin levels by stepwise multiple regression analysis (r⫽.595, P⬍.001):
关adiponectin兴⫽17.752⫺0.684⫻关BMI兴⫹0.123⫻关age兴. There was also a negative correlation between serum ghrelin levels and leptin levels in both subjects with SCI and controls (see tables 3, 4). Meanwhile, only BMI was a significant influencing factor for ghrelin levels by stepwise multiple linear regression analysis (r⫽.368, P⬍.001):
关ghrelin兴⫽647.867⫺15.475⫻关BMI兴. Table 3: Pearson Correlation Coefficients and P Values for BMI Compared With Serum Levels of Leptin, Adiponectin, or Ghrelin in Normative Controls
Age Leptin Adiponectin Ghrelin
BMI
Age
Leptin
Adiponectin
r⫽.078 P⫽.648 r⫽.782 P⬍.001 r⫽–.315 P⫽.057 r⫽–.447 P⫽.006
—
—
—
r⫽.182 P⫽.280 r⫽.023 P⫽.890 r⫽–.189 P⫽.269
—
—
r⫽–.278 P⫽.096 r⫽–.336 P⫽.045
—
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r⫽.114 P⫽.508
Table 4: Pearson Correlation Coefficients and P Value for BMI Compared With Serum Levels of Leptin, Adiponectin, or Ghrelin in the Subjects With SCI
Age Leptin Adiponectin Ghrelin
BMI
Age
Leptin
Adiponectin
r⫽.156 P⫽.143 r⫽.698 P⬍.001 r⫽–.527 P⬍.001 r⫽–.368 P⬍.001
—
—
—
r⫽.113 P⫽.292 r⫽.190 P⫽.074 r⫽–.141 P⫽.187
—
—
r⫽–.363 P⬍.001 r⫽–.260 P⫽.014
— r⫽.169 P⫽.114
DISCUSSION In this study, men with SCI had significantly higher serum leptin levels than the able-bodied group. This finding is consistent with the results of previous studies.11,12 Furthermore, subjects with tetraplegia had higher serum leptin levels than those with paraplegia, although the difference was not significant. However, when subjects with SCI were grouped into those with an injury above or below T6, leptin levels were significantly higher in the former group. T6 appears to be the lowest level of injury in most patients with SCI to develop autonomic dysreflexia.35 With SCIs above the level of T6, there is reduced SNS outflow and supraspinal control to the splanchnic outflow and the lower-extremity blood vessels.36 Multiple regression analysis showed that serum leptin levels in men with SCI correlated not only with BMI but also with the neurologic deficit. This finding supports the notion that decentralization of sympathetic nervous activity relieves its inhibitory tone on leptin secretion,37,38 because subjects with tetraplegia have a more severe deficit of sympathetic nervous activity. Jeon et al39 reported that the leptin response to short-term fasting and its effects on resting metabolic rate are impaired in subjects with SCI. Spungen et al40 reported that subjects with SCI were 13% fatter per unit of BMI compared with able-bodied controls. However, subjects with SCI had high serum leptin levels out of proportion with their adiposity—that is, a nearly 50% increase in this study and the Bauman et al study11and a 100% increase in another study.12 This implies that there are other contributing factors in hyperleptinemia in subjects with SCI. There is a leptin-SNS-leptin feedback loop.37,41 Leptin produces widespread sympathetic activation in various tissues and organs, including brown adipose tissue, kidney, hind limb, and adrenal glands,42,43 and activation of the SNS modulates leptin expression and secretion in rodents and humans in vitro and in vivo.44 People with SCI show decentralization of the SNS after the injury. This interruption blocks not only the inhibitory effect of the SNS on leptin expression and secretion41 but also the stimulatory effect of leptin on energy expenditure.39 Subjects with SCI therefore develop hyperleptinemia, a decreased resting metabolic rate, and increased adiposity. In this study, serum adiponectin levels in men with SCI were higher than those in the able-bodied group, and there was no significant difference in serum adiponectin levels between subjects with paraplegia and tetraplegia. Serum adiponectin levels are reported to correlate negatively with BMI in able-bodied men.24 In our study, they were shown to correlate negatively with the BMI in both the controls and subjects with SCI. Because subjects with SCI have a higher degree of adiposity and prevalence of insulin resistance, we expected that they would have higher serum leptin levels and lower serum adiponectin levels than controls. There are several possible expla-
ADIPONECTIN, GHRELIN, AND LEPTIN IN SCI, Wang
nations for the high serum adiponectin levels in subjects with SCI. First, RIA measures various forms of adiponectin, only some of which are biologically active.20,22,45 Second, adipose tissue in subjects with SCI may secrete different forms of adiponectin that are biologically inactive20,22; Delporte et al46 have reported the presence of a different form of adiponectin in -agonist–stimulated adipocytes and that administration of a 3 agonist to mice reduces adiponectin messenger RNA levels in subcutaneous and visceral adipose tissue and adiponectin levels in the plasma. An inhibitory effect of a  agonist on adiponectin messenger RNA levels was also shown in human adipocyte cultures.46 SCI results in decreased sympathetic activity; therefore, SCI may relieve its inhibitory effect on adiponectin secretion by adipose tissue. Third, adiponectin is not the only determining factor in insulin resistance, because other factors are known to be involved.47,48 In this study, serum ghrelin levels in men with SCI were similar to those in able-bodied controls. In humans, ghrelin secretion is under cholinergic control.49 Relative preservation of vagal parasympathetic function has been noted in subjects with SCI.50 In our study, we found a negative correlation between serum ghrelin levels and BMI or serum leptin levels in both the men with SCI and the normative controls. However, multiple regression analysis showed that BMI was the only significant factor for serum ghrelin levels. This finding is consistent with early studies51,52 and suggests that the stomach is part of the neuroendocrine system for energy homeostasis.53 BMI is a surrogate marker for fat mass, and it has been correlated with adipocytokines in study. However, BMI does not distinguish the anatomic site of fat mass. Actually not only subjects with SCI as a group but also subjects with paraplegia and tetraplegia have different body compositions as compared with able-bodied controls. Despite the above limitations, BMI is still a good surrogate measure for adiposity within the same neurologic-deficit group. One must be careful in using BMI to compare between groups. Thus, the definition of obesity should be redefined with a lower level in subjects with SCI. CONCLUSIONS Men with SCI have significantly higher serum leptin levels than able-bodied controls, and the serum leptin levels correlate with the degree of neurologic deficit. In addition to higher serum leptin levels, men with SCI also have higher serum levels of adiponectin but similar serum ghrelin levels compared with controls. The roles of these 3 hormones in the pathogenesis of insulin resistance in subjects with SCI require further investigation. References 1. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman SM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:425-32. 2. Halaas JL, Gajiwala KS, Maffei M, et al. Weight-reducing effect of the plasma protein encoded by the obese gene. Science 1995; 269:543-6. 3. Mantzoros CS. The role of leptin in human obesity and disease: a review of current evidence. Ann Intern Med 1999;130:671-80. 4. Scarpace PJ, Matheny M, Pollock BH, Tumer N. Leptin increases uncoupling protein expression and energy expenditure. Am J Physiol 1997;273:E226-30. 5. Tang-Christensen M, Havel PJ, Jacobs RR, Larsen PJ, Cameron JL. Central administration of leptin inhibits food intake and activates the sympathetic nervous system in rhesus macaques. J Clin Endocrinol Metab 1999;84:711-7. 6. Ahima RS, Prabakaran D, Mantzoros C, et al. Role of leptin in the neuroendocrine response to fasting. Nature 1996;382:250-2.
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