help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0583
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Danaher, R. N.
Right arrow Articles by Cooper, G. J. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Danaher, R. N.
Right arrow Articles by Cooper, G. J. S.
Endocrinology Vol. 149, No. 1 154-160
Copyright © 2008 by The Endocrine Society

Evidence that {alpha}-Calcitonin Gene-Related Peptide Is a Neurohormone that Controls Systemic Lipid Availability and Utilization

Rachel N. Danaher, Kerry M. Loomes, Bridget L. Leonard, Lynda Whiting, Debbie L. Hay, Lance Yi Xu, Edward W. Kraegen, Anthony R. J. Phillips and Garth J. S. Cooper

School of Biological Sciences and Maurice Wilkins Centre of Research Excellence for Molecular Biodiscovery (R.N.D., K.M.L., B.L.L., L.W., D.L.H., L.Y.X., A.R.J.P., G.J.S.C.), University of Auckland, Auckland 1142, New Zealand; Diabetes and Obesity Research Program (E.W.K.), Garvan Institute of Medical Research, Darlinghurst, New South Wales 2010, Australia; and Medical Research Council Immunochemistry Unit (G.J.S.C.), Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom

Address all correspondence and requests for reprints to: Garth J. S. Cooper, School of Biological Sciences, University of Auckland, Private Bag 92 019, Auckland, New Zealand. E-mail: g.cooper{at}auckland.ac.nz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
{alpha}-Calcitonin gene-related peptide ({alpha}CGRP) is released mainly from sensory and motor nerves in response to physiological stimuli. Despite well-documented pharmacological effects, its primary physiological role has thus far remained obscure. Increased lipid content, particularly in skeletal muscle and liver, is strongly implicated in the pathogenesis of insulin resistance, but the physiological regulation of organ lipid is imperfectly understood. Here we report our systematic investigations of the effects of {alpha}CGRP on in vitro and in vivo indices of lipid metabolism. In rodents, levels of {alpha}CGRP similar to those in the blood markedly stimulated fatty acid β-oxidation and evoked concomitant mobilization of muscle lipid via receptor-mediated activation of muscle lipolysis. {alpha}CGRP exerted potent in vivo effects on lipid metabolism in muscle, liver, and the blood via receptor-mediated pathways. Studies with receptor antagonists were consistent with tonic regulation of lipid metabolism by an endogenous CGRP agonist. These data reveal that {alpha}CGRP is a newly recognized regulator of lipid availability and utilization in key tissues and that it may elevate the availability of intramyocellular free fatty acids to meet muscle energy requirements generated by contraction by evoking their release from endogenous triglyceride.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
THE WORLDWIDE PREVALENCE of type 2 diabetes is projected to rise to 300 million by 2025, with the majority of this increase likely to occur in the developing world (1). Central to the pathophysiology of the disease are insulin resistance in the liver, skeletal muscle, and adipose tissue and impaired insulin secretion from the β-cell. Insulin resistance is the strongest predictor of type 2 diabetes, and historically its development has been attributed to altered regulation of carbohydrate metabolism. There is growing evidence that disordered lipid metabolism is fundamental to the pathology of this condition (2). Skeletal muscle plays a critical role in the maintenance of whole-body energy balance. It can normally use both lipid and carbohydrate as fuels and switches readily between the two; however, this switching becomes impaired in insulin-resistant states, apparently in relation to pathological accumulation of intramyocellular triglyceride (TG) (3, 4). There is, therefore, significant interest in biological processes that stimulate intramyocellular TG breakdown and fatty acid oxidation.

The two calcitonin gene-related peptides, {alpha}-calcitonin gene-related peptide (CGRP) and βCGRP, and amylin are closely related 37-amino acid peptides. {alpha}CGRP, the more widely distributed of the two CGRPs, has pleiotropic biological activities in processes that mediate vasodilatation, nociception, and cardioprotection (5, 6, 7) and various metabolic effects such as modulation of insulin sensitivity in skeletal muscle and liver (8). It has thus been postulated as a regulator of the circulation and a neurotransmitter. Pharmacological {alpha}CGRP doses evoke insulin resistance in rodents, mainly through effects in muscle and liver (8, 9, 10, 11). However, mice with specific deletion of {alpha}CGRP displayed no evident phenotypic abnormalities (12), and the primary physiological role of CGRP is still uncertain.

{alpha}CGRP is coreleased into the neuromuscular junction with the neurotransmitter acetylcholine in response to physiological stimuli (13) and is structurally similar to amylin, an endocrine metabolic regulator (6). We hypothesized that it might play a role in energy use and to investigate this possibility studied its effects on lipid metabolism. The data demonstrate that {alpha}CGRP increases fatty acid availability and use in key tissues including skeletal muscle, perhaps to meet the energy requirements generated by contraction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Animals
Male Wistar rats, weighing 200–250 g, were housed in a constant 12-h light, 12-h dark cycle and received standard rat chow or an in-house-derived high-fat diet [standard rat chow to which 30% (wt/wt) of lard had been added] and water ad libitum. All protocols were approved by the University of Auckland Animal Ethics Committee.

Peptides
Rat amylin, rat {alpha}CGRP, rat amylin-(8–37), and human βCGRP and CGRP-(8–37) were all purchased from Bachem (Bubendorf, Switzerland). Full-length mouse adiponectin was produced by recombinant expression and characterized according to our previously published methods (14).

In vitro experiments
Animals were anesthetized using halothane; soleus muscles of both legs were excised and the tissue immersed in Krebs-Henseleit buffer (KHB) that was gassed continuously with 95% O2-5% CO2. Muscles were separated into two equal parts and rested for 20 min. They were then incubated in the various treatments (concentrations as indicated) for stated times. For acetylcholine experiments, incubation concentrations were 100 µM and 100 nM. Muscles were frozen in liquid nitrogen and stored for later analysis. For β-oxidation pulse-chase studies, the muscles were incubated in KHB containing 4% fatty acid-free BSA (ICP Bio, Auckland, New Zealand), 2 mM pyruvate, and 0.5 mM palmitate with 2 µCi [9,10-3H] palmitate (GE Healthcare, Buckinghamshire, UK) and incubations performed as described (15), with minor modifications. In the short incubation study, muscles were stimulated with 1 µM {alpha}CGRP for 15 min, washed, and placed into fresh CGRP-free buffer, and fatty acid oxidation was then assessed at 60 min. Sixty-minute treated and control muscles were similarly treated but replaced into CGRP containing or KHB buffer as appropriate.

Lipid analysis
Total lipids were extracted from organs and tissues using a modified version of the method of Bligh and Dyer as described (16). Tissue TG levels were quantified using a GPO reagent set (Pointe Scientific, Canton, MI). Tissue free fatty acids (FFAs) were separated using bonded phase columns as described (17) and quantified using a colorimetric method (Boehringer, Ingelheim, Germany) adapted for microanalyses. For determination of lipid content by gas chromatography/mass spectrometry, separated lipids were processed for derivatization of methyl esters by the boron trifluoride-methanol method (18) and detected using a gas chromatograph/mass spectrometer (Hewlett Packard, Portland, OR).

cAMP analysis
Muscle strips were incubated for 10 min with peptide as indicated and then freeze dried and homogenized in 0.5 M HClO4, heated, centrifuged, and the supernatant removed. The supernatant was then neutralized with NH4OH, the samples refrigerated for 2 h, and centrifuged; finally cAMP content was determined using a commercial radioreceptor assay (Amersham, Buckinghamshire, UK), and values are expressed relative to dry tissue weight.

Western blot analysis of AMP-activated protein kinase (AMPK)
Phosphorylation and protein levels of AMPK were determined using anti-p-AMPK and anti-{alpha}AMPK antibodies (Cell Signaling, Beverly, MA) with detection via Qdot 605 Goat F(ab')2 antirabbit IgG conjugate (Invitrogen, Carlsbad, CA) per the manufacturer’s instructions. Bands were quantified by Gel Doc XR (Bio-Rad, Hercules, CA) and Quantity One software using glyceraldehyde-3-phosphate dehydrogenase (Abcam, Cambridge, UK) as a loading control.

In vivo studies
Male Wistar rats were randomly assigned to experimental groups. Induction and maintenance of surgical anesthesia were by 3–5% halothane and 2 liters/min oxygen. The trachea was cannulated and the animal ventilated at 60–70 breaths/min with air supplemented with O2. The respiratory rate and end-tidal pressure (10–15 cmH2O) were adjusted to maintain end-tidal CO2 at 35–40 mm Hg. Body temperature was maintained at 37 C throughout by a heating pad. The carotid artery and jugular vein were cannulated with saline-filled PE 50 catheters. Fluids (saline or peptide dissolved in saline) were delivered to the jugular vein. Infusion of saline or CGRP antagonist, as appropriate, was commenced 30 min before the start of {alpha}CGRP or saline infusion. Blood samples were taken at time zero (baseline reading), 30 min, and thereafter every 20 min until the end of the experiment at 90 min. The carotid artery line was connected to a blood pressure transducer. After 90 min a terminal blood sample was taken, and organs and tissues were removed and frozen until further analysis.

Plasma metabolites and hormones
Lactate and blood gas parameters were measured in heparinized whole blood using an arterial blood gas analyzer. The remaining blood was centrifuged for 15 min, 10 C at 3000 rpm, and the serum stored at –80 C. TG and FFAs were measured in serum as described above. Insulin, amylin, and {alpha}CGRP (Bachem) were analyzed with specific RIAs and catecholamines measured using an HPLC-based method (Endolab, Christchurch, New Zealand).

Calculations and statistical analysis
All results are presented as mean ± SEM except for EC50 values, which are presented as means (95% confidence intervals). Nonlinear curves were fitted according to a four-parameter logistic fit using Prism GraphPad 3.0 (GraphPad, San Diego, CA). The quantity of palmitate oxidized (nanomoles) was calculated from the specific activity of the incubation medium [radiolabeled palmitate (disintegrations per minute)/total palmitate (nanomoles)]. Data were analyzed by Student t tests or one-way ANOVA followed by Tukey’s post hoc test, as appropriate. Differences whose corresponding P values were less than 0.05 were considered statistically significant.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Exogenous {alpha}CGRP dose-dependently elevates muscle FFA content
We first measured the effects of exogenous {alpha}CGRP on muscle FFA content after 1 h incubation of isolated rat soleus muscle strips. This revealed that {alpha}CGRP evoked a biphasic, concentration-dependent increase in muscle FFA content (Fig. 1AGo), for which the half-maximal response of the high-potency component was in the subpicomolar range, with an EC50 of 0.25 pM (0.13–0.50; mean ± 95% confidence interval). FFA content in skeletal muscle was also analyzed by gas liquid chromatography, with identification of individual FFAs by mass spectrometry (data not shown). We thereby verified that the FFA pool was accurately measured using our enzymatic method and that the quantities reported here are consistent with other literature reports (16, 19). Additionally, we incubated soleus muscle with physiological and supraphysiological acetylcholine concentrations and determined that this coreleased neurotransmitter did not itself evoke any changes in lipid content (data not shown). The calcitonin receptor-like-receptor/receptor activity-modifying protein (RAMP)-1 receptor complex shows the highest affinity to {alpha}CGRP of all the receptor combinations known. However, the binding characteristics for this receptor combination lie within the 0.5–1 nM range (20, 21), suggesting that the responses observed in our study are the result of a novel receptor complex.


Figure 1
View larger version (23K):
[in this window]
[in a new window]

  		FIG. 1. Concentration-dependent effects of peptides on muscle lipid. FFA content in isolated rat soleus muscles from normal-fed Wistar rats were incubated with rat {alpha}CGRP (A); human βCGRP (B); or rat amylin (C). D, Comparison of the lipid levels in soleus muscles from animals fed a standard or high-fat diet. Effect of rat {alpha}CGRP on TG (E) and FFA (F) content in soleus muscles from high-fat-fed animals are shown. Each point represents the mean ± SEM (n = 10–16 individual muscle strips).

 
By contrast, the half-maximal response for {alpha}CGRP (Fig. 1AGo) in the low-potency component of the biphasic curve was in the low nanomolar range [EC50 = 42 nM (8.5–90)], similar to EC50 values derived for βCGRP, (Fig. 1BGo) [EC50 = 4.8 nM (2.1–11.1)] and amylin (Fig. 1CGo) (EC50 = 7.8 nM (4.1–14.7)]. These similar concentration dependencies are consistent with the known and comparable actions of both CGRP and amylin as noncompetitive antagonists of insulin-stimulated glycogen synthesis in muscle (8). Additionally, this response closely parallels published potency orders for complexes of the calcitonin receptor and RAMPs (RAMP1 or -3), which display similar affinity for the CGRPs and amylin (22).

We next determined whether {alpha}CGRP-mediated increases in muscle FFAs were due to mobilization of intrinsic lipid stores by measuring muscle TG content after incubation with {alpha}CGRP. In soleus muscle isolated from standard chow-fed animals, there was no significant change in TG content in response to {alpha}CGRP, although a significant decrease in muscle glycerol content did occur (P < 0.05) (data not shown); this effect is most likely due to its conversion to glycerol 3-phosphate (23). Skeletal muscle from animals fed a 30% lard diet for 40 d showed increases in basal TG and FFA contents of 1.8- and 2-fold, respectively (Fig. 1DGo). Incubation of soleus muscle strips from these animals with {alpha}CGRP for 1 h revealed marked dose-dependent decreases in TG content to a maximal value of 50% (Fig. 1EGo), and concomitant increases in FFA content (Fig. 1FGo). Half-maximal responses were in the subpicomolar range [TG: EC50 = 0.25 pM (0.03–1.96), FFA: EC50 = 0.7 pM (0.4–1)], showing that these actions were of very high potency.

{alpha}CGRP-mediated muscle lipid effects are mediated via CGRP receptors
To further confirm the specificity of this newly described action of {alpha}CGRP, we investigated its specific blockade. We incubated soleus muscles with 100-fold molar excess concentrations of the NH2-terminally truncated peptides of CGRP [CGRP-(8–37)] and amylin [amylin-(8–37)]. Both peptide fragments effectively blocked the effects of exogenous {alpha}CGRP on TG and FFA content (Fig. 2Go, A and B). This result is interesting because much higher concentrations of amylin-(8–37) were required to block cAMP responses in COS-7 cells transfected with CGRP1 (calcitonin receptor-like-receptor/RAMP1) (24) or amylin (1a) [calcitonin (a)/RAMP] (22) receptors, further suggesting the presence of a novel receptor phenotype.


Figure 2
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 2. {alpha}CGRP can evoke increases in cAMP and can be inhibited by NH2-terminally truncated CGRP and amylin peptide fragments. TG (A) or FFA (B) content in high-fat-fed soleus muscle after treatment with specific receptor antagonists are indicated. Effects of masoprocol on TG (C) and FFA (D) content in soleus muscle from high-fat-fed animals are also shown. cAMP content in soleus from high-fat-fed (E) and normal-fed (F) animals are indicated. *, Significant difference, compared with untreated (P < 0.05); **, P < 0.01; *** P < 0.001; ++, significant difference, compared with 100 nM {alpha}CGRP (P < 0.01), +++, P < 0.001; #, significant difference, compared with 1 pM {alpha}CGRP (P < 0.05); ##, P < 0.01; ###, P < 0.001. Each bar represents the mean ± SEM (n = 10–16 individual muscle strips).

 
It is currently unclear what the components of the high potency receptor complex might be, and it is therefore possible that these receptors might be differentially sensitive to the effects of antagonists, thus explaining why the effects of {alpha}CGRP on muscle lipid are not completely blocked as observed in Fig. 2BGo.

Additionally, the effects of {alpha}CGRP were inhibited by coincubation of soleus muscles with the lipoxygenase inhibitor, masoprocol (nordihydroguaiaretic acid) (Fig. 2Go, C and D), a compound reported to lower isoproterenol-induced lipolysis by decreasing the phosphorylation of hormone-sensitive lipase (25).

Evidence that {alpha}CGRP effects on muscle lipid are evoked via receptor-mediated increases in cAMP
We next investigated {alpha}CGRP signaling in soleus muscle at concentrations corresponding to the two components of the biphasic dose-response curve. {alpha}CGRP (100 nM) significantly increased cAMP content in muscle from both high-fat-fed (Fig. 2EGo) and standard chow-fed rats (Fig. 2FGo), whereas 1 pM {alpha}CGRP significantly increased cAMP only in soleus muscle from high-fat-fed animals. Thus, high-fat-feeding appears to sensitize the {alpha}CGRP-mediated cAMP response and increase TG breakdown (Fig. 1EGo) and FFA availability (Fig. 1FGo). This may reveal a role of endogenously released {alpha}CGRP in the sensitization of muscle lipid metabolism in the context of conditions of high-fat feeding.

Increases in cAMP content were inhibited by coincubation with 100-fold molar excesses of either of the peptide fragments used above, consistent with a previous report that {alpha}CGRP-mediated cAMP stimulation in skeletal muscle is CGRP receptor-mediated (26).

Interestingly, we observed that high-fat feeding induced an increase in basal muscle cAMP content (5.7 ± 0.2 vs. 3.9 ± 0.2 pmol/mg, P < 0.01). This finding is novel to our knowledge and complements our previous experiments, which showed that high-fat feeding elevated the phosphorylation of P20, a common intracellular target for insulin and several of its antagonists [Fig. 5 in Wang et al. (27)]. Together the increases in cAMP and changes in P20 could contribute to the insulin resistance observed after high-fat feeding.

{alpha}CGRP stimulates β-oxidation in vitro
To further investigate the regulatory role of {alpha}CGRP in muscle energy use, we used pulse-chase methodologies to measure fatty acid β-oxidation. {alpha}CGRP-mediated increases in muscle FFA content were significantly lower than expected from concomitant disappearance of muscle TG (data not shown). This finding suggested that muscle FFAs were undergoing further metabolism once released from TG by hormone action. Theoretically, FFAs can undergo one or more of three possible fates in muscle once freed from TG: release via the cell membrane; reesterification into TG; or β-oxidation in mitochondria. In this study, FFA concentrations were below the detection limit of the assay in muscle incubation medium (~10–6 M), indicating that measurable amounts of FFAs were not released from the muscle after {alpha}CGRP-evoked stimulation. Furthermore, because im TG concentrations decreased after hormone treatment, it is unlikely that re-esterification was taking place. Administration of 1 µM {alpha}CGRP for 1 h stimulated fatty acid oxidation in vitro (Fig. 3AGo) to a degree similar to that induced by full-length mouse adiponectin (89 nM), an established activator of fat oxidation (14, 28). Additionally, treatment of isolated soleus muscle with 100 nM {alpha}CGRP for 2 h significantly increased rates of lipid oxidation (Fig. 3BGo). We also compared the effects of 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR; 2 mM), a commonly used experimental stimulator of fatty acid oxidation, with that of {alpha}CGRP and found that the response was again similar [control, 1.95 ± 0.13; {alpha}CGRP, 2.79 ± 0.21 (P = 0.0031 vs. control); AICAR, 2.95 ± 0.28 (P = 0.0051 vs. control] (data not shown). Interestingly, we also found that {alpha}CGRP increased fatty acid oxidation when applied to skeletal muscle for 15 min (Fig. 3CGo).


Figure 3
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 3. {alpha}CGRP increases fatty-acid oxidation and AMPK signaling in skeletal muscle. Fatty-acid oxidation was determined in isolated soleus muscles in the presence of 1 µM rat {alpha}CGRP, 89 nM mouse full-length adiponectin, or modified KHB buffer (control) (A) and 1 µM rat {alpha}CGRP, 100 nM rat {alpha}CGRP, or control (B); and modified KHB buffer or 1 µM rat {alpha}CGRP (C). Each bar represents the mean ± SEM (n = 12 individual muscle strips). Representative immunoblots of AMPK Thr172 phosphorylation (pAMPK) and total protein content (D) and densitometric quantification show increased pAMPK levels (D and E). Each bar represents the mean ± SEM (n = 6 individual muscle strips). *, Significant difference, compared with untreated (P < 0.05), **, P < 0.01.

 
{alpha}CGRP stimulates phosphorylation of 5'-AMPK-activated protein kinase
The protein kinase, AMPK, acutely stimulates fatty acid oxidation via inhibitory phosphorylation of acetyl-CoA carboxylase (29). We hypothesized that {alpha}CGRP-mediated stimulation of fatty acid oxidation is mediated through AMPK. Using quantitative Western blotting, we determined that 1 h exposure of skeletal muscle to {alpha}CGRP (1 µM) stimulated phosphorylation in the {alpha}-subunit of AMPK, similar to that induced by 2 mM AICAR (Fig. 3DGo). There was no difference in the total amount of nonstimulated {alpha}AMPK protein between treated and untreated muscle. We next determined the effect of {alpha}CGRP on skeletal muscle AMPK phosphorylation using two lower concentrations, the first (100 nM) sufficient to stimulate maximally both the low- and high-potency phases and the second (50 pM) sufficient to maximally stimulate only the high-potency phase. Figure 3EGo shows that 100 nM {alpha}CGRP significantly increased phosphorylation of AMPK, whereas phosphorylation after 1 h stimulation with 50 pM {alpha}CGRP was not significantly different to control.

These findings show that the effects on skeletal muscle lipid at higher concentrations of {alpha}CGRP occur via pathways involving both cAMP and AMPK. In contrast, effects on muscle lipolysis at {alpha}CGRP concentrations within the high-potency phase apparently occur via a potentially novel mechanism independent of cAMP and AMPK.

{alpha}CGRP evokes specific effects on in vivo lipid metabolism
We also examined the tissue specificity of {alpha}CGRP treatment on in vivo organ lipid content after its infusion into anesthetized male Wistar rats in the presence or absence of a 100-fold molar excess of the {alpha}CGRP receptor antagonist, CGRP-(8–37) (Table 1Go). At an infusion rate of 100 pmol/kg·min, plasma [{alpha}CGRP] was significantly elevated after 1 h, compared with saline infusion (325 ± 75 vs. 6.3 ± 0.8 pM, P < 0.001), whereas plasma concentrations of insulin, epinephrine, norepinephrine, and amylin were unchanged (data not shown). Analysis of tissue-lipid content revealed 3-fold increases in FFA (P < 0.01) and 2-fold decreases in TG (P < 0.01) content in soleus muscle but not in heart, epididymal adipose tissue, or kidney. Additional acute effects on lipid content were observed in liver, in which FFAs increased 7-fold (P < 0.01) and TG decreased 2.5-fold (P < 0.05); plasma TG underwent a concomitant 7-fold decrease (P < 0.01) and serum FFAs a 4-fold increase (P < 0.01) (Table 1Go). These effects were eliminated by coinfusion of CGRP-(8–37). In addition to its use via lipolysis and β-oxidation, TG may also be exported in the form of very low-density lipoprotein and used elsewhere in the body. Interestingly, infusion of CGRP-(8–37) alone caused changes in the reverse direction in muscle and liver TG content (both P < 0.05), consistent with blockade of an endogenous CGRP agonist, presumably {alpha}CGRP.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Tissue FFA and TG content and plasma lipid profile after {alpha}CGRP infusion

 
These current studies show that, at least in vitro, {alpha}CGRP at concentrations sufficient to stimulate the low potency phase elicits skeletal muscle TG breakdown via a pathway that is apparently coupled to cAMP production. This pathway can be blocked by both peptide fragments of CGRP and amylin, and an inhibitor of hormone-sensitive lipase-mediated lipolysis. In addition to TG breakdown, {alpha}CGRP increased FFA partitioning toward β-oxidation and activated AMPK in muscle. Interestingly, recent evidence shows that AMPK can be activated by increased FFA supply within muscle cells and that this is independent of changes in the energy status of the cell (30).

There are several other endogenous compounds that have been demonstrated to increase lipid use, including adiponectin and other adipokines such as leptin. These, along with exercise and the antidiabetic drug metformin, all activate AMPK, and it is now clear that this enzyme plays a crucial role in the regulation of energy expenditure (31). The physiological significance of the low-potency phase in fatty acid oxidation is unclear because {alpha}CGRP concentrations at the neuromuscular junction after stimulation are unknown. Nevertheless, the maximal {alpha}CGRP effect is similar to that of leptin- and AICAR-induced activation of AMPK, which reach significance 60 min after administration (32, 33). Compounds that activate the AMPK pathway, as {alpha}CGRP appears to, could improve the lipid profile of patients with obesity and type 2 diabetes.

At {alpha}CGRP concentrations sufficient to stimulate the high-potency phase, the breakdown of TG occurs via a mechanism that, at least in skeletal muscle, is not coupled to cAMP production or mediated via the AMPK pathway. Further studies will be required to fully elucidate the signaling pathways of {alpha}CGRP on muscle lipid metabolism, particularly at these lower concentrations. Further studies to determine the dose responsiveness of the {alpha}CGRP-induced effects on lipid in vivo and determine whether {alpha}CGRP alters lipid metabolism after stimulation of the neural supply to muscle would provide further support for the physiological significance of this mechanism. As mentioned, there is a lack of obvious phenotype in the {alpha}CGRP knockout mouse model. However, this knockout model has yet to be characterized in sufficient depth because there are no available reports pertaining to the effects of high-fat feeding nor have lipid measurements been reported. Consequently, determination of the effects of a high-fat dietary challenge on the lipid profile of these knockout mice will be of significant interest.

In summary, the present findings provide the first evidence that {alpha}CGRP, released from motor neurons, could act as a neurohormone that coordinates the availability of intramyocellular lipid to meet muscle energy requirements created by concomitant activation of contraction by acetylcholine.


   Acknowledgments
 
We thank P. J. Scott for discussions and encouragement.


   Footnotes
 
This work was supported by grants from the Endocore Research Trust; the Maurice and Phyllis Paykel Trust; Lottery Health (New Zealand); the University of Auckland; the Department of Education (New Zealand) through a grant to the Maurice Wilkins Centre of Excellence for Molecular Biodiscovery; program grants from the Foundation for Research Science and Technology, New Zealand (to G.J.C.); the Health Research Council of New Zealand; and a grant from Protemix Corp., Auckland, New Zealand.

Disclosure Summary: R.N.D., K.M.L., L.W., L.Y.X., and A.P. have been employed by Protemix Ltd. R.D., K.L., B.L.L., D.L.Y.H., L.W., L.X., A.R.J.P., and G.J.S.C. received grant support from Protemix Ltd. D.L.H. has share options in Protemix Ltd. E.W.K. and G.J.S.C. received consulting fees from and have equity interests in Protemix Ltd.

Competing Interest Statement: The authors declare association with Protemix Corp., Auckland, New Zealand.

First Published Online October 11, 2007

Abbreviations: AICAR, 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside; AMPK, AMP-activated protein kinase; CGRP, calcitonin gene-related peptide; FFA, free fatty acid; KHB, Krebs-Henseleit buffer; RAMP, receptor activity-modifying protein; TG, triglyceride.

Received May 2, 2007.

Accepted for publication September 25, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. King H, Aubert RE, Herman WH 1998 Global burden of diabetes, 1995–2025: prevalence, numerical estimates, and projections. Diabetes Care 21:1414–1431[Abstract]
  2. McGarry JD 1994 Disordered metabolism in diabetes: have we underemphasized the fat component? J Cell Biochem 55(Suppl):29–38
  3. Oakes ND, Cooney GJ, Camilleri S, Chisholm DJ, Kraegen EW 1997 Mechanisms of liver and muscle insulin resistance induced by chronic high-fat feeding. Diabetes 46:1768–1774[Abstract]
  4. Furler SM, Oakes ND, Watkinson AL, Kraegen EW 1997 A high-fat diet influences insulin-stimulated posttransport muscle glucose metabolism in rats. Metabolism 46:1101–1106[CrossRef][Medline]
  5. Brain SD, Grant AD 2004 Vascular actions of calcitonin gene-related peptide and adrenomedullin. Physiol Rev 84:903–934[Abstract/Free Full Text]
  6. Cooper GJ 1994 Amylin compared with calcitonin gene-related peptide: structure, biology, and relevance to metabolic disease. Endocr Rev 15:163–201[Abstract/Free Full Text]
  7. Uddman R, Edvinsson L, Ekblad E, Hakanson R, Sundler F 1986 Calcitonin gene-related peptide (CGRP): perivascular distribution and vasodilatory effects. Regul Pept 15:1–23[CrossRef][Medline]
  8. Leighton B, Cooper GJ 1988 Pancreatic amylin and calcitonin gene-related peptide cause resistance to insulin in skeletal muscle in vitro. Nature 335:632–635[CrossRef][Medline]
  9. Rossetti L, Farrace S, Choi SB, Giaccari A, Sloan L, Frontoni S, Katz MS 1993 Multiple metabolic effects of CGRP in conscious rats: role of glycogen synthase and phosphorylase. Am J Physiol 264:E1–E10
  10. Choi SB, Frontoni S, Rossetti L 1991 Mechanism by which calcitonin gene-related peptide antagonizes insulin action in vivo. Am J Physiol 260:E321–E325
  11. Ye JM, Lim-Fraser M, Cooney GJ, Cooper GJ, Iglesias MA, Watson DG, Choong B, Kraegen EW 2001 Evidence that amylin stimulates lipolysis in vivo: a possible mediator of induced insulin resistance. Am J Physiol Endocrinol Metab 280:E562–E569
  12. Lu JT, Son YJ, Lee J, Jetton TL, Shiota M, Moscoso L, Niswender KD, Loewy AD, Magnuson MA, Sanes JR, Emeson RB 1999 Mice lacking {alpha}-calcitonin gene-related peptide exhibit normal cardiovascular regulation and neuromuscular development. Mol Cell Neurosci 14:99–120[CrossRef][Medline]
  13. Hokfelt T, Holets VR, Staines W, Meister B, Melander T, Schalling M, Schultzberg M, Freedman J, Bjorklund H, Olson L, Lindh B, Elfvin LG, Lundberg JM, Lindgren JA, Samuelsson B, Pernow B, Terenius L, Post C, Everitt B, Goldstein M 1986 Coexistence of neuronal messengers—an overview. Prog Brain Res 68:33–70[Medline]
  14. Xu A, Wang Y, Keshaw H, Xu LY, Lam KS, Cooper GJ 2003 The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice. J Clin Invest 112:91–100[CrossRef][Medline]
  15. Bruce CR, Dyck DJ 2004 Cytokine regulation of skeletal muscle fatty acid metabolism: effect of interleukin-6 and tumor necrosis factor-{alpha}. Am J Physiol Endocrinol Metab 287:E616–E621
  16. Gorski J, Nawrocki A, Murthy M 1998 Characterization of free and glyceride-esterified long chain fatty acids in different skeletal muscle types of the rat. Mol Cell Biochem 178:113–118[CrossRef][Medline]
  17. Kaluzny MA, Duncan LA, Merritt MV, Epps DE 1985 Rapid separation of lipid classes in high yield and purity using bonded phase columns. J Lipid Res 26:135–140[Abstract]
  18. Coalson RE, Schwartz MJ 1970 Boron trifluoride-methanol for histochemical methylation. Acta Histochem 36:227–237[Medline]
  19. Molaparast-Saless F, Shrago E, Spennetta TL, Donatello S, Kneeland LM, Nellis SH, Liedtke AJ 1988 Determination of individual long-chain fatty acyl-CoA esters in heart and skeletal muscle. Lipids 23:490–492[Medline]
  20. Poyner DR, Sexton PM, Marshall I, Smith DM, Quirion R, Born W, Muff R, Fischer JA, Foord SM 2002 International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors. Pharmacol Rev 54:233–246[Abstract/Free Full Text]
  21. Mallee JJ, Salvatore CA, LeBourdelles B, Oliver KR, Longmore J, Koblan KS, Kane SA 2002 Receptor activity-modifying protein 1 determines the species selectivity of non-peptide CGRP receptor antagonists. J Biol Chem 277:14294–14298[Abstract/Free Full Text]
  22. Hay DL, Christopoulos G, Christopoulos A, Poyner DR, Sexton PM 2005 Pharmacological discrimination of calcitonin receptor: receptor activity-modifying protein complexes. Mol Pharmacol 67:1655–1665[Abstract/Free Full Text]
  23. Marks DB, Marks A, Smith C 1996 Basic medical biochemistry: a clinical approach. Baltimore: Williams, Wilkins
  24. Bailey RJ, Hay DL 2006 Pharmacology of the human CGRP1 receptor in Cos 7 cells. Peptides 27:1367–1375[CrossRef][Medline]
  25. Gowri MS, Azhar RK, Kraemer FB, Reaven GM, Azhar S 2000 Masoprocol decreases rat lipolytic activity by decreasing the phosphorylation of HSL. Am J Physiol Endocrinol Metab 279:E593–E600
  26. Pittner RA, Wolfe-Lopez D, Young AA, Beaumont K 1996 Different pharmacological characteristics in L6 and C2C12 muscle cells and intact rat skeletal muscle for amylin, CGRP and calcitonin. Br J Pharmacol 117:847–852[Medline]
  27. Wang Y, Xu A, Ye J, Kraegen EW, Tse CA, Cooper GJ 2001 Alteration in phosphorylation of P20 is associated with insulin resistance. Diabetes 50:1821–1827[Abstract/Free Full Text]
  28. Tomas E, Tsao TS, Saha AK, Murrey HE, Zhang CCC, Itani SI, Lodish HF, Ruderman NB 2002 Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation. Proc Natl Acad Sci USA 99:16309–16313[Abstract/Free Full Text]
  29. Jorgensen SB, Richter EA, Wojtaszewski JF 2006 Role of AMPK in skeletal muscle metabolic regulation and adaptation in relation to exercise. J Physiol 574:17–31[Abstract/Free Full Text]
  30. Watt MJ, Steinberg GR, Chen ZP, Kemp BE, Febbraio MA 2006 Fatty acids stimulate AMP-activated protein kinase and enhance fatty acid oxidation in L6 myotubes. J Physiol 574:139–147[Abstract/Free Full Text]
  31. Musi N 2006 AMP-activated protein kinase and type 2 diabetes. Curr Med Chem 13:583–589[CrossRef][Medline]
  32. Minokoshi Y, Kim YB, Peroni OD, Fryer LG, Muller C, Carling D, Kahn BB 2002 Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415:339–343[CrossRef][Medline]
  33. Smith AC, Bruce CR, Dyck DJ 2005 AMP kinase activation with AICAR simultaneously increases fatty acid and glucose oxidation in resting rat soleus muscle. J Physiol 565:537–546[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Danaher, R. N.
Right arrow Articles by Cooper, G. J. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Danaher, R. N.
Right arrow Articles by Cooper, G. J. S.

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