| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
University of Cambridge Metabolic Research Laboratories (X.P., Y.C.L.T., I.S.F., S.O'R., A.P.C.), Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge CB2 0QQ, United Kingdom; and Department of Biochemistry (J.L.G.), University of Cambridge, Cambridge CB2 1QW, United Kingdom
Address all correspondence and requests for reprints to: Stephen O'Rahilly or Anthony P. Coll, University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, Level 4, Addenbrooke’s Hospital, Box 289, Cambridge CB2 OQQ, United Kingdom. E-mail: so104{at}medschl.cam.ac.uk or apc36{at}cam.ac.uk.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(skeletal muscle) to an extent not replicated by pair feeding. Leptin had profound effects on peripheral lipid metabolism, but the majority were explained by its effects on food intake. Leptin had additional centrally mediated effects to increase the expression of a limited number of genes concerned with fatty acid oxidation. Whereas we cannot exclude direct peripheral effects of leptin on certain aspects of lipid metabolism, we were unable to detect any such effects on the parameters measured in this study. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Compelling data indicate that leptin acts through the central nervous system to regulate food intake and energy expenditure (reviewed in (4, 5).
Leptin has also been reported to have profound effects on carbohydrate and lipid metabolism that may, at least in part, be independent of its centrally mediated effects on energy balance (reviewed in Refs. 6 , 7). Indeed, in the decade since the first report of a specific lipid depleting effect of leptin (8), there has been a wealth of experiments attempting to discern the extent and nature of the direct effects of leptin on peripheral tissues (9, 10, 11, 12).
However, many of these studies have used in vitro or ex vivo preparations in a hormonal milieu somewhat removed from that seen in vivo, making it difficult to place the data within a proper physiological context.
In this study we used the well-established abnormalities of peripheral lipid metabolism in the ob/ob mouse as an experimental tool to more rigorously examine two questions. First, to what extent are the beneficial effects of leptin on the lipid metabolism in ob/ob mice mediated through central vs. peripheral mechanisms? Second, if acting through the central nervous system, what are the relative contributions of effects on suppression of food intake vs. food intake-independent mechanisms in the mediation of these effects? To address these questions, we compared the effects of 3 d of leptin administered ip with a much smaller dose administered centrally [intracerebroventricular (icv)], both doses resulting in equivalent degrees of suppression of food intake. We also studied a sham injected group of ob/ob mice that were pair fed to the leptin-treated group. We compared plasma lipid and lipoprotein levels, hepatic fatty acid composition measured by gas chromatography coupled to mass spectrometry (GC-MS), and the expression of lipogenic and lipolytic genes in liver, muscle, and three different white adipose tissue depots.
Leptin therapy is increasingly being used in human disorders of congenital and acquired leptin deficiency, with, in some cases, dramatic therapeutic impact on dyslipidemia. To be able to fully understand and potentially develop these findings further, there is a need to have a clear and precise understanding of this aspect of the biology of leptin.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To allow icv injection, on day 0 all mice underwent stereotaxic surgery to place an indwelling guide cannula into the lateral ventricle. Mice were anesthetized with a mix of inhaled isoflurane and oxygen and a 26-gauge steel guide cannula (internal diameter 0.24 mm, outer diameter 0.46 mm, length 2 mm; Semat International, Hertfordshire, UK) was implanted into the right lateral ventricle using the following coordinates: 1.0 mm lateral from bregma, 0.5 mm posterior to bregma. The guide cannula was secured to the skull using quick-drying cyanoacrylate glue and a dental cement (Associated Dental Products, Wiltshire, UK), and a dummy cannula was inserted. All animals received analgesia (Rimadyl, 5 mg/kg; Pfizer Animal Health, Kent, UK) and antibiotic (Teramycin LA, 60 mg/kg, Pfizer Animal Health) before being returned to their home cage.
After surgery, food intake and body weight were measured daily. Only mice that maintained their presurgery body weight were studied further.
Leptin administration
On d 7, mice were divided into four experimental groups (n = 6 per group). All mice received a 2-µl injection icv plus a 200-µl injection ip injection at the same times (every 12 h, first injection started at the onset of light cycle on d 7). The injection scheme for each groups were as follow: icv group, icv 1 µg leptin, ip saline; ip group, icv saline, ip 1 mg/kg body weight leptin; leptin-treated (PF) group, icv saline, ip saline; control group, icv saline, ip saline. Recombinant murine leptin was obtained from Amgen (Thousand Oaks, CA).
The pair-feeding group was fed with the average food intake of the icv and ip group that was equal to 4.2 and 2.5 g, respectively, on d 7 and 8.
After the fifth injection (d 9), all food was withdrawn from all mice. The mice were killed 4 h later. Blood was collected by cardiac puncture, allowed to clot, and then spun for 10 min. Serum was frozen and stored until further analyzed. Tissues were dissected (skeletal muscle = quadriceps), weighed, frozen on dry ice, and then stored at –80 C until analyzed.
Plasma lipid analyses
Enzymatic assay kits were used for determination of plasma free fatty acids (Roche Diagnostics, West Sussex, UK) and total triglycerides (Sigma-Aldrich, St. Louis, MO). Plasma lipoproteins were fractionated by gel filtration on two Superose 6 columns (GE Healthcare, Little Chalfont, Buckinghamshire, UK) connected in series, and cholesterol level was evaluated in each individual fraction by using a enzymatic assay for cholesterol measurement kit (BioMerieux, Marcy l’Etoile, France).
Fatty acid profile analysis by GC-MS
Frozen liver (100 mg wet weight) was ground in liquid nitrogen, and lipids were extracted using methanol and chloroform in a 2:1 ratio. After vortexing and sonication (15 min), 200 µl chloroform and 200 µl water were added to the sample. After centrifugation (1 min at 10,000 rpm), the lower layer (lipid layer) was collected and evaporated. The lipid pellet was resuspended in 250 µl methanol-chloroform (1:1) and 125 µl of BF3. The mixture was heated for 90 min at 90 C and then cooled down. The lower phase was dried and analyzed by GC-MS.
The derivatized organic metabolites were injected onto a ZB-WAX column (30 m x 0.25 mm inner diameter x 0.25 µm df; 100% polyethylene glycol). The initial column temperature was 60 C; this was held for 2 min and then increased by 10 C/min–1 to 150 C and then by 4 C/min–1 up to a temperature of 230 C at which it was held for 7 min.
The column eluent was introduced into a Trace DSQ quadrupole mass spectrometer (Thermo) (transfer line temperature = 310 C for aqueous metabolites and 240 C for lipid metabolites, ion source temperature = 250 C, electron beam = 70 eV). The detector was turned on after a solvent delay of 240 sec, and data were collected in full scan mode using three scans per second across a mass range of 50–650 m/z.
GC-MS chromatograms were analyzed using Xcalibur (version 2.0. Thermo Fisher Corp., Waltham, MA), integrating each peak individually. Deconvolution of overlapping peaks was achieved by generating traces of selected ions. A 0.1-min threshold window was used for the deviation of peaks away from the predicted retention time across the data set. Structures were assigned using both the National Institute of Standards and Technology database of mass spectra and analysis of standard compounds (e.g. FAME standard from Sepulcho; Sigma-Aldrich, Gillingham, Dorset, UK).
RNA analysis
Total RNA was purified using STAT60 (Iso-Text Diagnostics, Friendswood, TX) according to the manufacturer’s instructions. Real-time quantitative PCR was used to analyze RNA from tissues. Total RNA (500 ng) was reverse transcribed and real-time quantitative PCR performed on TaqMan 7900 sequence detection system (Applied Biosystems, Warrington, UK). Primers were designed using the Primer Express 2.0 software (sequence available on request). Cyclophilin mRNA was used as control to normalize gene expression.
Statistics
All data are reported as mean ± SEM. All data sets were analyzed for statistical significance using Student’s t test (Excel; Microsoft, UK, Thames Valley Park, Reading, Berkshire, UK) (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Both central and peripheral leptin treatment led to a significant but comparable reduction on food consumption compared with saline controls (ip: 2.5 ± 1.2 g; icv: 1.5 ± 0.8 g; control: 6.0 ± 0.8 g/d, P < 0.001; Fig. 1
A). A third group of ob/ob mice was food restricted to the level that leptin-treated mice consumed (pair fed). The reduction in percentage of body weight seen was similar across the three groups (7.0 ± 3.0 vs. 18.0 ± 2.5 vs. 8.0 ± 1.8%, ip vs. icv vs. PF, respectively, P < 0.001 compared with control; Fig. 1B).
|
). Compared with sham-treated controls, all three intervention groups showed a significant and similar reduction in TG (2.54 ± 0.6 vs. 1.12 ± 0.2 vs. 1.28 ± 0.3 vs. 1.40 ± 0.3 mg/dl, control vs. icv vs. ip vs. PF, respectively) and FFAs (1.51 ± 0.2 vs. 0.94 ± 0.34 vs. 1.02 ± 0.12 vs. 1.06 ± 0.20 µmol/liter, icv vs. ip vs. PF, respectively). These equate to a halving in TG concentration and a reduction in FFA by more than 3-fold.
|
). Cholesterol levels in the LDL/HDL1 fraction were reduced by about 50% in leptin-treated groups (icv and ip) compared with sham-treated control ob/ob mice. Interestingly, a similar effect was observed after food restriction in PF group. Very low-density lipoprotein cholesterol levels were detectable in control mice but were rendered undetectable in the icv, ip, and PF groups.
Effects of central and peripheral leptin administration on hepatic lipid metabolism
Liver weight (Fig. 3A) was decreased significantly by all three treatments compared with control [(liver weight/total body weight) x 100: 6.3 ± 0.43 vs. 7.2 ± 0.50 vs. 7.3 ± 0.47 vs. 8.3 ± 0.6 mg/g, icv vs. ip vs. PF vs. control, respectively, P < 0.01 (icv), P < 0.05 (ip and PF)]. Of note, the effect seen within the icv group was significant greater than that seen in the ip and PF groups.
|
). No change was seen in myristic (C14) or oleic (C18:1) acid concentration. Central and peripheral leptin treatments as well as food restriction had similar effects to markedly reduce eicosanoic (C20:1) and slightly reduce palmitic (C16) concentrations. Concentrations of docohexanoic (C22:6), arachidonic (C20:4), linoleic (C18:2), and, to a lesser extent, stearic (C18) acids were increased equally in all three treatment groups.
|
). mRNA levels for acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) were markedly reduced (60% reduction for ACC, 80% reduction for FAS) in all three treatment groups with no differences between groups. Similarly, mRNA levels of stearoyl-coenzyme A desaturase 1 (SDC-1) was decreased by central and peripheral leptin administration and pair fed to a similar degree in comparison with sham saline-treated controls (icv: 75%, ip: 65%, PF: 75%). In contrast, we did not detect any significant effect of any treatment on sterol receptor binding protein 1c (SREBP1c) mRNA levels, a key transcription factor in the regulation of the lipogenesis pathway.
Because leptin has been shown to regulate β-oxidation in the liver, we also assessed the effect of leptin treatment on the expression of genes implicated in β-oxidation (Fig. 3C). mRNA levels encoding acetyl-coenzyme A acetyltransferase 1 (ACAT1; an enzyme that catalyzes the reversible formation of acetoacetyl-coenzyme A from two molecules of acetyl-coenzyme A) and carnitine palmitoyl transferase 1 (CPT1; the transporter that allows the entrance of fatty acids into the mitochondria) were increased equally in icv, ip, and PF groups. The expression of peroxisomal proliferator-activated receptor (PPAR) and medium-chain acyl-coenzyme A dehydrogenase (MCAD; which catalyzes one step of β-oxidation pathway) was not modified by leptin treatment (data not shown). Notably mRNA levels encoding both acyl-CoA oxidase (ACOX) isoforms, a key enzyme involved in fatty acid catabolism, were significantly increased (50% increase for the peroxisomal isoform and 65% increase for the mitochondrial isoform) after both icv and ip leptin treatment but were not significantly increased by food restriction.
Finally, we analyzed the expression of some of the key enzymes in cholesterol metabolism (Fig. 3D). The expression of 3-hydroxy-3-methylglutaryl-coenzyme A (the key enzyme involved in the de novo synthesis of cholesterol) reductase was down-regulated (60% decrease, P < 0.001) by both leptin treatment and food restriction. We measured apolipoprotein A1 mRNA, the main component of HDL and a key player in cholesterol efflux, and found that central and peripheral leptin treatment increased apolipoprotein A1 expression by 2-fold. Comparable changes were seen in the PF group.
Effects of central and peripheral leptin administration on gene expression in skeletal muscle
Previous data have indicated that leptin may have a direct effect on β-oxidation in muscle. In each of our treatment groups, we therefore also measured the expression levels of several genes involved in β-oxidation: the muscle-specific isoform of CPT1 (CPT1muscle), ACOX, and MCAD (Fig. 4A). None were significantly regulated by leptin treatment. However, in skeletal muscle, the expression of PPAR, a nuclear receptor that plays a key role in regulation of β-oxidation, was increased by 80% with both central and peripheral leptin treatment compared with sham-treated controls (P < 0.05). This effect was not seen in the PF, suggesting that the effect of leptin on PPAR expression in skeletal muscle was independent of food intake.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The vast majority of the effects on parameters of plasma, hepatic, muscle, and white adipose tissue lipid metabolism after peripheral administration of leptin were identical with those seen after central leptin treatment. In addition, changes seen with leptin treatment were mostly indistinguishable from those seen in sham-treated animals pair fed to mice treated with leptin, indicating that many of the changes seen with leptin treatment over 3 d occur through a centrally mediated reduction in food intake. In liver, muscle, and white adipose tissue, leptin acted primarily to repress lipogenesis with a smaller increase in the expression of enzymes involved in β-oxidation.
Several previous animal (1, 2, 3) and human studies (15) have reported the effects of leptin upon lipid metabolism. In this current study, we also demonstrate that leptin treatment, either centrally or peripherally, is sufficient to decrease plasma TG and FFA levels by approximately 50% and suppress the characteristic high LDL/HDL1-cholesterol profile. Because this effect was also seen in PF ob/ob mice, this effect seems to be largely dependent on the anorectic effects of leptin. This finding contrasts with a recent study in lean rats (16) in which a reduced plasma TG was observed after 7 d of central leptin treatment but not in pair-fed rats. This difference may reflect species-specific effects, differences in time course, or difference in endogenous leptin levels between the two models.
Liver weights of the ob/ob mice were significantly decreased with leptin administered either centrally or peripherally (17), consistent with previously reports. Previous data have also suggested that leptin can act directly on the liver to increase lipid oxidative metabolism (12) with the acute effects of systemically delivered leptin on liver TG levels not replicated by icv leptin (18). However, in our study, we clearly observed that central leptin administration reproduced all of the effects on TG metabolism seen after ip treatment.
The liver gene expression profiles of our intervention groups (both leptin treated and pair fed) indicated that the major effect was the repression of de novo lipogenesis, with an 80% decrease of FAS gene expression. This is further supported by the lipidemic data in which a trend for a reduction in levels of palmitic acid (C16) is in keeping with reduced de novo lipogenesis.
In our current study, we also found SCD-1 mRNA expression was strongly decreased (60–70%) in the liver, not only after central and peripheral leptin treatment but also to a similar extent in the PF group. Furthermore, the reduction of hepatic eicosanoic acid (C20:1) levels, a product of SCD-1, indicates that in each intervention group there was a reduction in function SCD-1 levels.
SCD-1 has been previously identified by microarray analysis as a specific leptin target in the liver, down-regulated by leptin treatment in the liver of the ob/ob mouse but to a much lesser extent by pair feeding (19). The different findings in these studies could potentially be attributable to differences in the mode of leptin administration (ip injection vs. minipump infusion).
Leptin has been suggested to activate the β-oxidation pathway (20, 21) and, consistent with this, we found an increase in mRNA expression of genes involved in hepatic fatty acid oxidation. In the liver, ACOX, CPT1, and ACAT-1 mRNA levels were increased by leptin treatment but also by pair feeding, indicating these changes were driven by the reduction in food intake. Importantly, there were also some changes in enzyme expression, which appeared to be independent of the anorexigenic effects of leptin. The expression of two genes involved in the fatty acid oxidation-hepatic ACOX (both mitochondrial and peroxisomal isoforms) was increased by leptin treatment independently of food restriction.
Leptin has also been reported to have a direct action in skeletal muscle. However, because of the concentrations of leptin used and the highly atypical conditions within which some of the ex vivo analyses took place the physiological relevance of these reports is uncertain. For example, one study reporting a direct effect of leptin on human skeletal muscle lipid metabolism (22) used a leptin concentration of 10,000 ng/ml, a value several log scales higher than that seen naturally. In our current study, the expression of skeletal muscle PPAR was increased similarly by icv and ip leptin treatment but not by food restriction, suggesting a central leptin effect independent of its anorexigenic effect rather than a direct action. This is in keeping with previous data supporting a centrally mediated effect of leptin on β-oxidation in skeletal muscle (21).
Previous data have indicated leptin may directly activate metabolic pathways within adipose tissue (23, 24). In the three different adipose depots we studied, the expression of lipogenic genes was similarly decreased in leptin-treated and PF animals. The only exception was SREBP1c in the gonadal fat in which expression levels were significantly decreased only by leptin treatment. It is of course possible that because of the limited range of genes that we studied some direct effects of leptin on adipocyte gene expression were overlooked in our study. However, our findings support the idea that the action of leptin on lipogenesis in WAT is mainly mediated through its central anorexigenic effects.
There are a number of limitations to our present study. A change in mRNA expression is not necessarily translated into a change in functional protein level. However, the transcript data are not presented in isolation but together with data on circulating lipids and hepatic lipid composition, which all support our assertions. We administered leptin for 3 d, whereas other studies have used longer regimens, which may have uncovered a difference between the treatment groups. For example, Levin et al. (17) administered leptin to ob/ob mice for 12 d. The weight loss over the first 6 d was equivalent in both leptin-treated and pair-fed group. However, after 6 d a plateau in weight loss was observed in pair-fed mice, whereas ob/ob mice receiving leptin continued to lose weight. This raises the possibility that with time, the central action of leptin to promote lipid β-oxidation persists, whereas the purely anorexigenic effect become muted, although our current study was not designed to address this. We chose to study ob/ob mice because we wanted to examine the physiological effects of hormonal repletion in a hormone naïve animal, a classical paradigm for the understanding of endocrine physiology. However, we appreciate that the effects of leptin supplementation in animals who already have some endogenous leptin may be different in extent and conceivably in kind.
In conclusion, although several studies support a direct effect of leptin on peripheral aspects of lipid metabolism, in our model we have not been able to detect such effects. Taken together, our data suggest that over the short term of leptin administration used in the current study, the majority of leptin’s effects on lipid metabolism appears to be centrally mediated. In addition, our results suggest that, except for some centrally mediated effects on the expression of a subset of genes involved in β-oxidation, the bulk of leptin’s effects on peripheral lipid metabolism can be explained by its anorectic action.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Disclosure Statement: The authors have nothing to disclose.
First Published Online July 17, 2008
Abbreviations: ACAT1, Acetyl-coenzyme A acetyltransferase 1; ACC, acetyl-CoA carboxylase; ACOX, acyl-coenzyme A oxidase; CPT1, carnitine palmitoyl transferase 1; FAS, fatty acid synthase; FFA, free fatty acid; GC-MS, gas chromatography coupled to mass spectrometry; HDL, high-density lipoprotein; icv, intracerebroventricular; LDL, low-density lipoprotein; PF, pair-fed to the leptin-treated mice; PPAR, peroxisomal proliferator-activated receptor ; SDC-1, stearoyl-coenzyme A desaturase 1; TG, triglyceride; WAT, white adipose tissue.
Received April 9, 2008.
Accepted for publication July 8, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  J. German, F. Kim, G. J. Schwartz, P. J. Havel, C. J. Rhodes, M. W. Schwartz, and G. J. Morton Hypothalamic Leptin Signaling Regulates Hepatic Insulin Sensitivity via a Neurocircuit Involving the Vagus Nerve Endocrinology, October 1, 2009; 150(10): 4502 - 4511. [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 |
