| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
University of Warwick (K.K., A.L.H., N.F.d.S., J.P.O., P.G.M., S.K.), Unit for Diabetes and Metabolism, Clinical Sciences Research Institute, Coventry CV2 2DX, United Kingdom; Department of Forensic Medicine and Department of Clinical Pathology (A.T., G.C.), Varna Medical University, Varna 9002, Bulgaria; Department of Histopathology (S.J., D.R.S.), University Hospital of Coventry and Warwickshire National Health Service (NHS) Trust, Coventry CV2 2DX, United Kingdom; and Department of Anaesthetics (B.H.), Birmingham Heartlands Hospital, Heart of England NHS Foundation Trust, Birmingham B9 5SS, United Kingdom
Address all correspondence and requests for reprints to: Professor Sudhesh Kumar, University of Warwick, Clinical Sciences Research Institute, University Hospital Coventry and Warwickshire, Clifford Bridge Road, Coventry CV2 2DX, United Kingdom. E-mail: Sudhesh.Kumar{at}warwick.ac.uk.
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Objective: The objective was to establish the presence of adiponectin or resistin in human cerebrospinal fluid (CSF) and to compare their distribution with leptin. Furthermore, we wished to examine the expression of the adiponectin receptors 1 and 2 (AdipR1, AdipR2) in the human hypothalamus.
Methods: For this purpose, serum and CSF samples were collected from 20 men and 19 women matched for age [men, 69.8 ± 8.6 yr (mean ± SD); women, 69.4 ± 4.3 yr] and BMI (men, 29.4 ± 3.4 kg/m2; women, 27.3 ± 4.8 kg/m2) undergoing elective surgery under spinal anesthesia.
Results: Adiponectin was identified in CSF with levels 1000-fold less than serum, whereas resistin and leptin levels were 100-fold less. Unlike their serum levels, adiponectin CSF levels showed no gender difference or correlation with insulin resistance, which is similar to resistin CSF levels. The adiponectin and leptin CSF/serum ratios in our study exhibit the same pattern of gender-specific BMI association with inverse correlation in women (r = –0.61; P = 0.02) and no correlation in men (r = 0.026; P = not significant). Furthermore, immunostaining established AdipR1 and -2 in the hypothalamus and increased AdipR2 expression in the paraventricular nucleus, which is involved in energy regulation.
Conclusion: In summary, our findings show both the presence of adiponectin and resistin in human CSF, with no effect of insulin resistance on CSF levels. The CSF entry of adiponectin and leptin in women appears to be impaired in obesity.
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
The aim of this study was to examine the potential central role of resistin and adiponectin in humans. We examined matched paired serum and human cerebrospinal fluid (CSF) from male and female patients to explore the relationship between CSF and the corresponding serum levels and to determine the CSF/serum ratio as a measure of efficient uptake into the CSF. This study also considered possible correlations between adiponectin, resistin, and leptin in CSF with age, gender, BMI, and homeostasis assessment model (HOMA) index. Finally, we determined the expression and distribution of adiponectin receptors in the human hypothalamus. These issues have become particularly important in the light of recent reports that adiponectin is not present in human CSF (10) and exogenous adiponectin does not enter the BBB by endocytosis of rat brain endothelium 4 cerebral microvessel endothelial cells (11).
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Serum- and CSF-matched samples were obtained from age- and BMI-matched subjects [men, age, 69.8 ± 8.6 yr (mean ± SD); BMI, 29.4 ± 3.4 kg/m2, n = 20; women, age, 69.4 ± 4.3 yr; BMI, 27.3 ± 4.8 kg/m2, n = 19] undergoing elective surgery, under spinal anesthesia, fasted for at least 8 h (Table 1
). Exclusion criteria were malignancy, acute or chronic renal or liver disease, neurological disorders, and/or the use of immunosuppressants, current or recent use of systemic high-dose corticosteroids, antibiotics, or weight-modifying medication. Patients with C-reactive protein (CRP) levels above 10 mg/dl were also excluded. All samples were collected with the approval of the local ethics committees and with the informed written consent of study subjects. A fasting blood sample (5 ml) was taken at the time of venopuncture, and a clear CSF sample (2 ml) was collected before the spinal anesthetic agent was injected. Six patients had T2DM (two on insulin treatment), and three had impaired glucose tolerance. The T2DM subjects were on glucose/insulin infusion at the time of cannulation and could not be considered for HOMA calculation. Serum and plasma EDTA samples were immediately centrifuged, snap-frozen in liquid nitrogen, and stored at –80 C. CSF samples were passed through a 0.2-µm syringe filter (Schleicher & Schuell, Middlesex, UK), flash-frozen, and stored at –80 C.
|
The serum samples were analyzed for the determination of adiponectin (1:500 dilution; Linco Research Inc., St. Charles, MO) with the assay limit of 0.78 ng/ml; intraassay percent coefficient of variation (%CV) was 7.4%, and interassay variability was 2.4–8.4%. Serum resistin levels were analyzed by ELISA (1:5 dilution; Quantikine, R&D Systems, Abingdon, UK). Assay limits were noted between 0.01 and 0.055 ng/ml, with intraassay %CV noted as between 3.8 and 5.3% and interassay variability being 7.8–9.2%. We have previously assessed the human resistin R&D Systems ELISA to examine resistin binding specificity. The recovery of resistin in serum and cross-reactivity with resistin-like molecules was assessed as previously described (13).
The leptin ELISA (1:100 dilution; Quantikine, R&D Systems) has a minimum detectable value of 7.8 pg/ml with an intraassay precision of 3–3.3% CV and interassay variability of 3.5–5.4% CV. The high-sensitive CRP ELISA (Life Diagnostics Inc., West Chester, PA) has a functional sensitivity of 0.1 mg/dl, intraassay precision of 2–7.5% CV, and interassay variability of 2.5–4.1% CV. An insulin ELISA (Linco Research Inc.) was used with the sensitivity of 2 µU/ml, intraassay %CV of 5.96, and interassay variability of 10.3% CV, in accordance with the manufacturer’s protocol. Glucose levels were analyzed using the YSI-2300 STAT PLUS (YSI, Inc., Yellow Springs, OH), according to the manufacturer’s instructions.
Assessment of CSF levels
CSF samples were used undiluted for assessment of adiponectin (20 µl), resistin (100 µl), and (10 µl) leptin. The serial dilution of CSF detected adiponectin levels up to a 1-in-4 dilution (86% of value expected 1-in-2 dilution and 80% of value expected 1-in-4 dilution). A similar serial dilution was documented for resistin, with serial dilution ranging from undiluted to 1 in 8. The use of neat CSF sample produced results with a recovery of 100%; therefore, they were deemed most acceptable in this system.
We further assessed potential sources of cross-reactivity in the adiponectin ELISA with C1q (Abcam, Cambridge, UK), collagen type III (Sigma, Poole, UK), and collagen type IV (Sigma). For this recombinant human (rh) C1q, rh collagen type III and rh type IV were analyzed at two concentrations (10 and 100 ng/ml) either in the presence of PBS pH 7.6 (Sigma) or in combination with CSF. No detectable cross-reactivity occurred between rh C1q, rh collagen type III, and rh type IV alone or in combination with CSF samples and adiponectin in this ELISA. The interassay and intraassay coefficients were evaluated for samples ranging from 12.1–15.9 ng/ml CSF adiponectin. The within percentage CV was 2.5%, and the between CVs were 6.7, 2.4, and 5.3%. The means between variations were calculated as described in the manufacturer’s instructions.
Spiking and recovery of human adiponectin in CSF was further examined. rh Adiponectin (20 and 50 ng/ml) was added to known concentrations of adiponectin in pooled CSF samples (12.3 and 15.9 ng/ml, respectively), and adiponectin content was determined in three separate assays. The percentage of recovery was determined in accordance with manufacturer’s instructions. We observed that recovery ranged from 93–97% in CSF, which was within a comparable range as noted for serum according to the manufacturer’s detailed assessments.
Spiking and recovery of human resistin in CSF was also addressed. For these studies, two concentrations of rh resistin (0.5 and 2 ng/ml) were added to known concentrations of resistin in pooled CSF samples (0.03 and 0.08 ng/ml, respectively), and resistin concentration was determined in three separate ELISA assays. The percentage of resistin recovery ranged from 81–99%, with a mean of 88% in CSF; a similar recovery was noted for serum findings by the manufacturer as well as in our previous publications (13, 14).
Blood contamination was excluded with use of the micropore filter described. The cell count in CSF samples with the hemocytometer did not show any nucleus-bearing cells. Further screening of the CSF samples with the hemoccult test (Hema-screen; Immunostics Inc., Ocean, NJ) was negative, yet the test remained positive with spiking of full blood at a concentration of 1:10,000.
HOMA index analysis
The HOMA for insulin resistance (HOMA-IR) index (12) was calculated using the HOMA calculator available at www.dtu.ox.ac.uk/index.php?maindoc=/homa/index.php.
Immunohistochemistry
Human adipose tissue was obtained from Medical Solutions (Peterborough, UK). Human hypothalamic postautopsy sections were obtained with the approval of the Varna Medical Research Ethics Committee from Varna University, Bulgaria. Tissue was incubated with primary polyclonal adiponectin receptor (AdipR)1 and AdipR2 antibody (Phoenix Pharmaceuticals, Burlingame, CA) in a dilution of 1:2500 and 1:200, respectively, and dual-stained with neuropeptide Y (NPY) in a dilution of 1:400. Sections were developed using peroxidase substrate kit VIP (Vector Laboratories Ltd., Peterborough, UK) for AdipR1 and -2 and diaminobenzidine (BioGenex, San Ramon, CA) for NPY. To demonstrate specific binding, the primary antibody was omitted for negative control for both AdipR antibodies, independently.
Statistics
Statistics were performed using SPSS version 12 (SPSS UK, Surrey, UK). P values of less than 0.05 were considered as statistically significant. Analysis of normally distributed variables was by linear regression analysis and two-tailed Student’s t test. For nonparametric distributions, e.g. HOMA, the Spearman correlation was used. Values are expressed as mean ± SD, unless otherwise stated.
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Serum adiponectin was significantly higher in women than in men (31.5 ± 1.2 vs. 22.2 ± 1.1 µg/ml; P = 0.016). Adiponectin correlated positively with age in men (r = 0.56; P = 0.01) but not significantly in women [r = 0.36; P = not significant (NS)]. Serum adiponectin showed a negative association with BMI in males (r = –0.46; P = 0.04), whereas only a trend was noted in women (r = –0.27; P = NS). In agreement with previous studies, the HOMA index and serum adiponectin were inversely correlated for the total study population (r = –0.48; P < 0.001), and the adiponectin levels were significantly lower in the T2DM and insulin-resistant subjects compared with nondiabetic subjects (19.2 ± 3 µg/ml, n = 9, vs. 28.8 ± 2.9 µg/ml, n = 30; P = 0.020).
Mean CSF adiponectin was 16.3 ± 6 ng/ml, with a range of 7.2–41.6 ng/ml; therefore, the CSF levels were approximately 1000-fold lower than the serum adiponectin levels (Fig. 1). Unlike the serum, CSF adiponectin did not show sexual dimorphism (men, 16.7 ± 8.3 ng/ml; women, 15.9 ± 3.6 ng/ml; P = 0.7) (Table 1
). In the total population, a positive age correlation was noted (r = 0.36; P = 0.036; n = 35), which was maintained when men and women were analyzed individually (r = 0.55, P = 0.035; r = 0.5, P = 0.036, respectively). CSF adiponectin was inversely correlated with BMI in men (r = –0.62; P = 0.01) but not in women (r = –0.26; P = NS). HOMA-IR and CSF adiponectin levels showed no correlation, and no change was noted due to diabetic status.
|
. The CSF levels failed to rise with high serum levels and demonstrate a threshold effect, suggesting a saturated transport. CSF/serum adiponectin ratios were significantly higher in men [1.0 ± 0.11 (*10–3)] than women [0.65 ± 0.06 (*10–3)] (P < 0.01). The CSF/serum ratio was inversely correlated with BMI in women (r = –0.61, P = 0.02) and did not show a correlation in men (r = 0.026; P = NS) (Fig. 3A).
|
|
Mean serum resistin levels were 14.8 ± 8 ng/ml, with slightly higher levels shown in women than in men (15.8 vs. 14.97 ng/ml) (Table 1), which did not attain significance (P = 0.080). Resistin levels correlated positively with increasing age (r = 0.45; P < 0.01); however, no correlation was observed with BMI (r = –0.21; P = NS) or HOMA-IR (r = –0.32; P = NS) index.
The mean CSF resistin level was measured as 0.20 ± 0.13 ng/ml, with a range of 0.01–0.63 ng/ml. CSF resistin levels were not influenced by gender (P = 0.65) (Table 1). No relationship between CSF resistin levels and serum was noted (Fig. 2B). Furthermore, CSF resistin levels did not correlate with age (r = 0.09; P = NS) or HOMA-IR index (r = 0.24; P = NS) and remained unaltered by diabetic status. The CSF/serum ratio did not differ significantly between men and women and bore no relationship with BMI (Fig. 3B).
Serum and CSF leptin
Mean serum leptin levels were 17.4 ± 2.5 ng/ml, with significantly higher levels in women (22.5 ± 3.7 ng/ml) vs. men (13.0 ± 3.0 ng/ml; P = 0.03) (Table 1). Serum leptin levels were inversely correlated with BMI (r = 0.52, P < 0.001; men, r = 0.75, P = 0.001; women, r = 0.82; P < 0.001) but showed no correlation with age.
The CSF levels showed a tendency toward higher leptin levels in women than men but did not attain significance (women, 79.0 ± 9.0 pg/ml vs. men, 56.0 ± 9.0 pg/ml; P = NS). CSF leptin was positively correlated with BMI in men and women (r = 0.42; P = 0.028) but not with age. The correlation of CSF with serum levels was best described by a logarithmic curve fit (r = 0.65; P < 0.001), similar to previous observations (15) (Fig. 2C). The CSF/serum ratio was inversely correlated with BMI in women (r = –0.81; P = 0.001) but showed no correlation with BMI in men (r = –0.17; P = NS) (Fig. 3C). Serum leptin was higher in T2DM than nondiabetic subjects (28.9 ± 5.8 ng/ml vs. 12.5 ± 8.7 ng/ml; P = 0.02), whereas CSF leptin was not significantly different in the T2DM subjects (T2DM subjects, 105 ± 2 pg/ml vs. nondiabetic subjects, 62 ± 7 pg/ml; P = NS).
Expression of adiponectin receptors in the human hypothalamus
Immunohistochemistry showed the expression of AdipR1 and AdipR2 in the neuronal cells in the human hypothalamus. AdipR1 was diffusely expressed in both the anterior and posterior hypothalamus, whereas AdipR2 was concentrated in the paraventricular nucleus (PVN) along with neurons that were dual-stained with NPY. Human adipose human tissue was also observed to express AdipR1 (Fig. 4).
|
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Although there is some confirmation of the role of adiponectin in CSF through receptor expression studies in the brain and cFos activity with adiponectin (1), other studies have failed to establish the presence of adiponectin in human CSF (10) or to verify the passage of adiponectin through the BBB (11). This may be due to the need for high-sensitivity assays to be used for its detection. Furthermore, entry of additional adiponectin, when administered as radioactive-labeled iv adiponectin, may not lead to observable differences due to potentially already saturated levels of adiponectin in the CSF.
Leptin is a known potent central appetite regulator (21), and its serum levels directly correlate with fat mass (21, 22). Comparison of leptin CSF levels within our and other studies (15, 23, 24, 25) has highlighted variation (15). The average leptin CSF levels in our findings were relatively low in comparison with some previously reported data (15, 23) but similar to levels identified in other studies (21, 24). In comparison, mean CSF fasting levels of leptin are much smaller than those of adiponectin and resistin, suggesting that their levels are within the range to activate central nervous system effects. Much lower concentrations than in the periphery are sufficient for activation of neural pathways and signaling in the hypothalamic nuclei for proteins like leptin (25, 26).
AdipR1 and AdipR2 can be activated by both globular and full-length adiponectin (3, 5). AdipR1 shows a high affinity for globular adiponectin, but data for binding affinities only exist for the muscle receptors in mice (5) and not for neuronal cells in humans. Adiponectin receptors mediate action through signaling molecules such as peroxisome proliferated activated receptor-
, MAPK, and AMP kinase (27, 28). AMP kinase pathways that can be activated by globular adiponectin, as well as trimers, are involved in hormonal and nutrient signaling in the hypothalamus, including the regulation of food intake by leptin and insulin (8, 28). Hexamers and higher molecular weight (HMW) isoforms of adiponectin can also bind to T-cadherin (29) with unclear biological function. Given that HMW isoforms do not induce AMP kinase activation but mediate their action through nuclear factor-
B in the periphery (30), T-cadherin is unlikely to play a role in central appetite regulation.
In concordance with previous observations, serum circulating levels of adiponectin were found at higher levels in women (31). Similar to other studies, serum and CSF adiponectin were inversely correlated with BMI in men but not in women (32). Although the serum adiponectin levels showed sexual dimorphism, intrathecal adiponectin levels showed no gender difference. Higher circulating serum levels in women and the lack of a CSF gender difference have previously been observed with leptin levels (33) and reaffirmed within our study population.
Our current study does not allow us to determine which isoforms of adiponectin enter the CSF directly. However, we note that higher serum HMW adiponectin was reported in women (31), but we found no gender difference in CSF. Therefore, it is likely that HMW structures do not enter the CSF. Additionally, due to the size of the HMW structures (>500 kDa), these are unlikely to pass through the BBB.
Resistin appears to have a role in obesity-associated inflammation and insulin resistance, which remains controversial in the human context (13, 14, 34). In this study, raised circulating resistin levels do not correspond with higher CSF levels, suggesting limited uptake into the CSF. The wide variation in resistin concentration requires further studies to examine entry of resistin across the BBB. No correlation between serum and CSF resistin levels was observed with BMI, and neither of the adipokines’ CSF levels were noted to be affected by insulin resistance. T2DM is also associated with reduced HMW isoforms (27) and impaired multimerization of adiponectin (35). Thus adiponectin CSF levels may predominantly be made up of LMW isoforms for reasons explained above and, as such, may not be affected by diabetes.
In summary, adiponectin and resistin are present in human CSF with levels unaffected by gender. AdipR1 and AdipR2 are present in the human hypothalamus, especially in the PVN. These findings highlight the potential involvement of adipokines in central energy homeostasis in humans. Based on our data, we postulate an active transport mechanism for both adiponectin and resistin across the BBB.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
First Published Online January 9, 2007
Abbreviations: AdipR, Adiponectin receptor; BBB, blood-brain barrier; BMI, body mass index; CSF, cerebrospinal fluid; CRP, C-reactive protein; CV, coefficient of variation; HMW, higher molecular weight; HOMA, homeostasis assessment model; HOMA-IR, HOMA for insulin resistance; LWM, lower molecular weight; NPY, neuropeptide Y; NS, not significant; PVN, paraventricular nucleus; rh, recombinant human; T2DM, type 2 diabetes.
Received August 22, 2006.
Accepted December 28, 2006.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
-MSH in obese women and their relationship to negative energy balance. J Clin Endocrinol Metab 86:4849–4853This article has been cited by other articles:
 |
|
 |
|
  M. Pini, M. E. Gove, J. A. Sennello, J. W. P. M. van Baal, L. Chan, and G. Fantuzzi Role and Regulation of Adipokines during Zymosan-Induced Peritoneal Inflammation in Mice Endocrinology, August 1, 2008; 149(8): 4080 - 4085. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Lu, Q. Tang, J. M. Olefsky, P. L. Mellon, and N. J. G. Webster Adiponectin Activates Adenosine Monophosphate-Activated Protein Kinase and Decreases Luteinizing Hormone Secretion in L{beta}T2 Gonadotropes Mol. Endocrinol., March 1, 2008; 22(3): 760 - 771. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. S. Singhal, M. A. Lazar, and R. S. Ahima Central Resistin Induces Hepatic Insulin Resistance via Neuropeptide Y J. Neurosci., November 21, 2007; 27(47): 12924 - 12932. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kos, A. L. Harte, S. James, D. R. Snead, J. P. O'Hare, P. G. McTernan, and S. Kumar Secretion of neuropeptide Y in human adipose tissue and its role in maintenance of adipose tissue mass Am J Physiol Endocrinol Metab, November 1, 2007; 293(5): E1335 - E1340. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Neumeier, J. Weigert, R. Buettner, J. Wanninger, A. Schaffler, A. M. Muller, S. Killian, S. Sauerbruch, F. Schlachetzki, A. Steinbrecher, et al. Detection of adiponectin in cerebrospinal fluid in humans Am J Physiol Endocrinol Metab, October 1, 2007; 293(4): E965 - E969. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
