Globular and full-length forms of adiponectin mediate specific changes in glucose and fatty acid uptake and metabolism in cardiomyocytes
- Rengasamy Palanivela,
- Xiangping Fanga,
- Min Parka,
- Megumi Eguchia,
- Shelley Pallana,
- Sabrina De Girolamoa,
- Ying Liua,
- Yu Wangb,
- Aimin Xuc and
- Gary Sweeneya,*
- aDepartment of Biology, York University, Toronto, Ontario, Canada M3J 1P3
- bGenome Research Center, University of Hong Kong, China
- cDepartment of Medicine, University of Hong Kong, China
- * Corresponding author. Tel: +1 416 736 2100x66635; fax: +1 416 736 5698. gsweeney{at}yorku.ca
- Received February 11, 2007.
- Revision received March 21, 2007.
- Accepted April 16, 2007.
Abstract
Objective Our aim was to investigate the regulation of glucose and fatty acid metabolism in cardiomyocytes by the globular (gAd) and full-length (fAd) forms of adiponectin.
Methods We produced fAd (consisting of high, medium and low molecular weight oligomers) in a mammalian expression system and gAd in bacteria. These were used to treat primary neonatal rat cardiomyocytes (up to 48 h), and we employed 3H- or 14C-labeled substrates to monitor glucose uptake and subsequent metabolism via oxidation, glycogen synthesis or lactate production and fatty acid uptake and oxidation. Enzymatic assay for acetyl CoA carboxylase activity was employed, and protein phosphorylation and expression was determined by immunoblotting cell lysates. The role of adiponectin receptor (AdipoR) isoforms was determined via siRNA-mediated knockdown.
Results There was an initial (1 h) increase in glucose uptake and oxidation in response to gAd or fAd. Fatty acid uptake was stimulated by gAd or fAd, and by 24 h a decrease in acetyl CoA carboxylase activity and elevated fatty acid oxidation were observed. After 48 h increased fatty acid oxidation correlated with decreased glucose oxidation and pyruvate dehydrogenase activity, while glycogen synthesis and lactate production increased. Both gAd and fAd elicited phosphorylation of AMP kinase, insulin receptor substrate-1, Akt and glycogen synthase kinase-3β. Knockdown of AdipoR1 or AdipoR2 attenuated the effect of both gAd and fAd on fatty acid uptake and oxidation. Only AdipoR1 knockdown prevented the ability of gAd (1 h) to increase glucose uptake and oxidation; however, reducing either AdipoR1 or AdipoR2 expression attenuated the long-term (24 h) effects of gAd.
Conclusions These results clearly demonstrate that gAd and fAd mediate distinct and time-dependent effects on cardiomyocyte energy metabolism via AdipoR1 and AdipoR2.
Key words
Time for primary review 25 days
1 Introduction
There is currently great interest in the mechanisms via which obesity can impact upon cardiac structure and function [1]. In this study we focus on the myocardial metabolic effects of the adipose-derived hormone adiponectin [2, 3], whose plasma levels show a negative correlation with obesity [4]. Many studies have demonstrated a clear correlation between adiponectin and various aspects of cardiovascular disease [5–7]. In particular, several studies in the last year have focused on defining the role of adiponectin in heart failure and myocardial remodeling [8–11]. It is now believed that adiponectin is a key metabolic regulator and that decreased levels or function of adiponectin confer increased risk for cardiovascular disease [1,5].
Adiponectin exists as a full-length protein of 30 kDa (fAd) which circulates in trimeric, hexameric and higher order complexes [12]. A fragment containing the globular domain of adiponectin (gAd) has also been shown to exhibit potent metabolic effects in various tissues [13–18]. Importantly, gAd and fAd have been shown to mediate tissue-specific effects as well as to regulate distinct signaling pathways and end-point responses in the same tissue [14,19,20]. Thus, rather than examining total adiponectin levels, it is now imperative to measure and understand the distinct effects of the various adiponectin forms [21]. Understanding this specificity was aided by the recent identification of two adiponectin receptor (AdipoR) isoforms [22]. AdipoR1 is expressed primarily in skeletal muscle and exhibits a high binding affinity for gAd and weak affinity for fAd. Conversely, AdipoR2 predominates in liver and has a high affinity for binding to fAd and an intermediate affinity for gAd [22]. Detailed analysis of the effects of gAd and fAd on cardiomyocyte metabolism and the role of each receptor isoform in mediating these effects has not been investigated.
Altered myocardial metabolism of long chain fatty acids and glucose may play a role in the development of compromised ventricular performance and progressive left ventricular remodeling [23,24]. The metabolic effects of adiponectin in skeletal muscle [13–16], liver [25] and adipocytes [18] have been extensively characterized but little is known regarding regulation of cardiomyocyte metabolism. Recently it was shown that gAd increased fatty acid oxidation in newborn rabbit hearts [17] and that adiponectin increased glucose and fatty acid uptake in cultured cardiomyocytes [26]. In this study we perform a detailed analysis of fatty acid and glucose metabolism in response to gAd or fAd and examine the mechanisms utilized to mediate these effects in neonatal rat cardiomyocytes.
2 Methods
2.1 Materials
Dulbecco's modified eagle medium (DMEM/F-12 medium) was from Gibco (Grand Island, NY). Gentamicin sulfate was obtained from Mediatech Inc. (Herndon, VA), penicillin/ streptomycin from Wisent Inc. (Quebec), Primaria TM Easy GripTM (surface modified polystyrene, nonpyrogenic) tissue culture dishes and plates were from Becton Dickinson (Franklin, NJ). Bromodeoxyuridine was from Sigma (St. Louis, MO). [3H] palmitate, D-[U-14C] glucose, 2-deoxy-D-[3H] glucose, [1-14C] pyruvate and [14C] sodium bicarbonate were from Amersham (Quebec). FATP1, FATP4, FAT/CD36, AMPK-α1 and AMPK-α2 antibodies were obtained from Santa Cruz (Santa Cruz, CA). ACC, phospho-ACC (Ser-79), phospho-AMPK (Thr-172), phospho-Akt Thr-308 and Ser-473 primary antibodies, and HRP-conjugated anti-rabbit secondary antibody were purchased from Cell Signaling (Beverly, MA). Phospho-IRS-1 (Tyr-612) and phospho-GSK-3β (Ser-9) antibodies were obtained from Biosource (Montreal). Enhanced chemiluminescence reagent was purchased from PerkinElmer (Burlington).
2.2 Production of globular and full-length adiponectin
We used recombinant gAd produced by subcloning murine gAd cDNA (a kind gift from Dr. Philipp Scherer, Albert Einstein College of Medicine, NYC) into the pTrisEx expression vector (Novagen) as we described previously [14]. Since biological activity of fAd depends upon post-translational modifications [25], we produced in a mammalian expression system as we described previously [14,25] which produced high, medium and low molecular weight oligomers in a ratio of ∼35:40:25%, respectively. The concentrations of adiponectin used in this study were based upon preliminary studies at a range of concentrations and correlate with those used previously by ourselves and others [13,14,22].
2.3 Isolation and culture of neonatal ventricular myocytes
Primary cultures of cardiomyocytes were prepared from 1- to 3-day-old Sprague–Dawley rats and conducted in accordance with protocol approved by the York University Animal Care Committee and which conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Briefly, neonatal hearts were removed and immediately put into ice-cold calcium and bicarbonate-free Hank's to HEPES buffer and then cut into pieces. Cells in suspension were collected after several rounds of trypsin digestion of heart pieces and for selective enrichment of cardiomyocytes cells were pre-plated into several 100×20 mm culture dishes and incubated for 1 h. The suspension containing unattached cardiomyocytes was then collected and seeded at a density of 1×106 cells/ml in culture media (DMEM/F-12 with 10% fetal bovine serum, 0.1 mM 5-bromodeoxyuridine, 50 mg/ml gentamycin, 100 U/ml penicillin and 100 mg/ml streptomycin). More than 90% of the cells were myocytes, as evaluated by indirect immunofluorescence staining with an antibody to myosin heavy chain (MF20, a kind gift from Dr. J.C. McDermott, York University, Toronto). After incubation at 37 °C in humid air (5% CO2 and 95% O2) and serum-free medium for 24 h, the cardiomyocytes were then used for experimental treatment as detailed below.
2.4 siRNA-mediated knockdown of AdipoR1 and AdipoR2 expression and analysis of adiponectin receptor expression by real-time quantitative PCR
One hour prior to transfection with siRNA, cells were incubated with serum-reduced medium. Several 21-nucleotide small interfering RNA (siRNA) sequences (Ambion, Inc. Austin, TX) designed to knockdown rat AdipoR1 or AdipoR2 were tested in these cardiomyocytes and the sequence of siRNAs ultimately used here for providing optimal efficiency were: AdipoR1, GCUCAUGUUGAGAUUUACtt; AdipoR2, GAGACACCUGUUUGUUCUUtt. 100nM of AdipoR1 and AdipoR2 siRNAs were transfected into myocytes using the TransIT-TKO reagent (Mirus Bio Corporation, Madison WI), precisely according to instructions provided by the manufacturer. After 24 h of incubation, the medium was replaced by serum-free medium and cells were then treated with adiponectin as described in each experimental method section. Isolation of RNA, cDNA synthesis from 1 μg of total RNA, cycling conditions and primers used to amplify AdipoR1 and AdipoR2 products as well as β-actin as housekeeping control were as we published previously [14].
2.5 Measurement of long chain fatty acid uptake and oxidation
To determine fatty acid uptake we used [3H]-palmitate as previously described [27] and fatty acid oxidation was measured by the production of 14CO2 from [1-14C] palmitate as previously described [13,28]. ACC activity was assayed in isolated cardiomyocytes by the [14C] bicarbonate fixation assay [29].
2.6 Analysis of glucose uptake and metabolism
To determine glucose uptake cardiomyocytes were seeded in 24-well plates and pretreated with or without gAd (2.5 g/ml) or fAd (2.5 μg/ml) for periods of 1 h or 24 h. Where indicated insulin was used at 100 nM for 20 min. Subsequently, glucose transport was assayed essentially as we previously described [14] and results are calculated as pmol of glucose uptake per min per mg protein. Glucose oxidation was measured by the production of 14CO2 from d-[U-14C] glucose essentially as we previously described [13,30]. Glycogen synthesis was measured by the incorporation of d-[U-14C] glucose to glycogen as we described previously [13,30]. Lactate content in culture medium was determined using a lactate assay kit from Trinity Biotech (St. Louis, MO). PDH activity was estimated according to the method described previously [31] based on [1-14C] pyruvate turnover.
2.7 Analysis of protein phosphorylation and expression by Western blot analysis
Myocytes were seeded on 35×10 mm culture dishes and treated with gAd (2.5 μg/ml) and fAd (2.5 μg/ml) as indicated in figures. Lysates were prepared exactly as we previously described [14,30]. Primary antibodies were used as follows: phospho-IRS (Y612) at 1:3000, GLUT4 and GLUT1 at 1:5000 dilution and all other antibodies at 1:1000 dilution in combination with appropriate horseradish peroxidase-conjugated secondary antibodies (anti-rabbit at 1:10,000 dilution and goat 1:3,000) were used in each case and detected by the enhanced chemiluminescence method. Equal loading of protein is routinely ensured via protein assay of lysates, ponceau S staining of PVDF membranes and by Western blotting of the same membranes stripped and reprobed for analysis of β-actin expression. The degree of protein phosphorylation or expression was determined by scanning densitometry analysis as we described previously [13,14].
2.8 Statistical analysis
Data are expressed as mean values±SEM with number of repeats (n) stated in each case. Statistical analysis was undertaken using one-way ANOVA or the paired Student's t-test where appropriate. Differences between groups were considered statistically significant when p<0.05 and are indicated by symbols as described in figure legends.
3 Results
We first investigated the effect of short- and long-term treatment of neonatal rat cardiomyocytes with either gAd or fAd (both 2.5 μg/ml) on 2-deoxyglucose uptake. Fig. 1A shows that gAd and fAd caused a statistically significant increase in glucose uptake (basal uptake was calculated as 19.8±2.1 pmol/min/mg, values in graph are expressed as fold relative to this) after both 1 h and 24 h. This initially correlated with increased glucose oxidation and PDH activity. These increases were transient and in the case of gAd had dropped to basal levels by 24 h or 48 h with fAd treatment (Fig. 1B and C). Whereas glucose oxidation increased rapidly in response to gAd or fAd, metabolism of glucose via glycogen synthesis or lactate production only increased after 24 h (Fig. 1D and E), suggesting that the route of glucose metabolism dictated by gAd or fAd was time dependent.
Fig. 1
Regulation of glucose uptake and metabolism by adiponectins. We examined the effect of gAd (2.5 μg/ml) or fAd (2.5 μg/ml) for 1 and 24 h on glucose uptake (A) and subsequent metabolism. Glucose oxidation (B) was also determined up to 48 h together with PDH activity (C). Lactate production (D), glycogen synthesis (E) and GSK3β (S9) phosphorylation (0–30 min) are also shown (F). The results presented are mean±SEM of n≥3 where *indicates significance of p<0.05 with respect to control.
We found that both gAd and fAd (0–30 min) could stimulate phosphorylation of AMPK on T172, IRS-1 on Y612 and Akt on T308 and S473 (Fig. 2A), suggesting that gAd and fAd activated insulin-like and insulin-independent signaling. After 24 h treatment, there was no change in phosphorylation of IRS-1, Akt or GSK-3β but AMPK phosphorylation increased relative to control (24 h gAd: 1.33±0.07 or fAd 1.35±0.05). We also found no change in IRS-1, Akt, GSK-3β, AMPKα1 or AMPKα2 protein expression when cells were treated with gAd or fAd for 24 h (data not shown). Neonatal cardiomyocytes express mainly GLUT1 with low levels of GLUT4 [32,33] and here 24 h treatment with gAd increased the levels of GLUT1 protein and to a lesser extent GLUT4. No change occurred after 24 h exposure to fAd (Fig. 2B).
Fig. 2
Mechanisms regulating glucose uptake by adiponectin. The effect of gAd (2.5 μg/ml, 0–30 min) or fAd (2.5 μg/ml, 0–30 min) on phosphorylation of AMPK (T172), IRS1 (Y612) and Akt (T308 or S473) is shown in A. Part B shows the effect of gAd (2.5 μg/ml, 24 h) or fAd (2.5 μg/ml, 24 h) on glucose transporter protein (GLUT)1 and GLUT4 expression. Representative immunoblots together with graphs showing quantitative analysis of n≥4 experiments are shown in all cases. Results presented are mean±SEM of n≥3 where * indicates significance of p<0.05 with respect to control.
To examine long chain fatty acid uptake and metabolism we used palmitate, a preferred substrate for heart. Fig. 3A highlights the ability of both gAd and fAd at 1 h or 24 h to significantly increase palmitate uptake in neonatal rat cardiomyocytes. We examined expression of fatty acid transporters after prolonged exposure (24 h) of cells to adiponectins and found selective regulation of these transporters by different forms of adiponectin. Only fAd selectively increased expression of FATP1 after as little as 1 h whereas gAd had no effect on FATP1 expression (Fig. 3B). Neither FATP4 nor CD36 expression was altered in response to gAd or fAd (Fig. 3B). No significant change in the oxidation of palmitate was observed in response to gAd or fAd within 1 h treatment but by 24 h, and particularly 48 h treatment of both gAd and fAd there was a significant increase palmitate oxidation observed (Fig. 4A). The increase in palmitate oxidation after 24 h treatment with gAd or fAd correlated with attenuation of ACC activity under these conditions (Fig. 4B) and also with decreased oxidation of glucose (Fig. 1B). ACC (280 kDa) phosphorylation was increased by both gAd or fAd at both acute (1 h) and prolonged (24 h) treatment times (Fig. 4C) while the total expression level of ACC was unaffected (data not shown). PDH activity in response to fAd was elevated at 1 and 24 h but returned to basal by 48 h and again this correlated with decreased glucose oxidation only after 48 h (Fig. 1B and C). We analyzed expression of PDK4 at both 24 and 48 h in response to gAd or fAd and found no significant difference (data not shown).
Fig. 4
Regulation of fatty acid oxidation and ACC activity and phosphorylation. The effect of gAd and fAd (2.5 μg/ml, 1 h, 24 h and 48 h), on fatty acid oxidation measured by production of 14CO2 from [1-14C] palmitate is shown in A. We also measured the effect of gAd and fAd (2.5 μg/ml, 1 h and 24 h) on incorporation of [14C]HCO3− into malonyl CoA from [14C]bicarbonate as a measure of ACC activity (B) and ACC (S79) phosphorylation (C). Values shown in A and B are expressed as mean±SEM of n≥3 and * indicates p<0.05 with respect to control. A representative immunoblot together with quantitative analysis of n≥3 experiments are shown for ACC phosphorylation.
Fig. 3
Regulation of fatty acid uptake and fatty acid transporter expression. Uptake of [3H] palmitate in response to gAd (2.5 μg/ml, 1 h and 24 h) or fAd (2.5 μg/ml, 1 h and 24 h) is shown in A. We also examined the effect of similar treatment conditions on fatty acid transporter protein (FATP1, FATP4 and CD36) expression as shown in B and representative immunoblots together with graphs showing quantitative analysis of n≥3 experiments are shown in all cases. Data represent mean±SEM of n≥4 where * indicates significance of p<0.05 with respect to control.
We determined using quantitative real-time PCR that primary neonatal rat cardiomyocytes express approximately 7-fold more AdipoR1 than AdipoR2 mRNA (data not shown). The use of siRNA designed to target AdipoR1 or AdipoR2 significantly reduced the mRNA expression of these receptor isoforms (51±4% and 66±3%, respectively). The expression of AdipoR1 or AdipoR2 mRNA was unaffected by siRNA for the other receptor isoform or by a scrambled siRNA sequence (data not shown). The effects of gAd on glucose uptake and oxidation after 1 h were significantly attenuated only after AdipoR1 siRNA treatment and were unaffected by AdipoR2 siRNA (Fig. 5A). However, stimulation of glucose uptake and metabolism by gAd after 24 h was attenuated upon knockdown of either AdipoR1 or AdipoR2 (Fig. 5B and C). The ability of fAd to stimulate glucose uptake and oxidation after 1 h was significantly attenuated only after AdipoR2 siRNA treatment (Fig. 5D). There was a small but significant attenuation of the stimulation of glucose uptake and metabolism by fAd after 24 h upon knockdown of AdipoR1 or AdipoR2 (Fig. 5E and F). Importantly, siRNA treatments did not significantly alter basal glucose and fatty acid metabolism without adiponectin (data not shown). siRNA to AdipoR1 or AdipoR2 attenuated the short- and long-term effects of both gAd or fAd on fatty acid uptake and metabolism, although inhibition of some responses did not reach statistical significance (Fig. 6).
Fig. 6
Effect of AdipoR1 or AdipoR2 knockdown on regulation of fatty acid uptake and metabolism by adiponectin. Here again we used siRNA to selectively knockdown AdipoR1 or AdipoR2 expression then determined functional effects of gAd or fAd on fatty acid metabolism. The effects of gAd and fAd on palmitate uptake are shown in A and C, respectively, while B and D show the consequences of AdipoR knockdown on palmitate oxidation and ACC activity after only 24 h treatment with gAd or fAd since neither had any effect after 1 h (see Fig. 4). The results shown are expressed as % of maximum responses which are highlighted in Fig. 4 to clearly indicate the responses attenuated by AdipoR1 or AdipoR2 siRNA and represent mean±SEM of n≥6 where *indicates significance of p<0.05 with respect to gAd or fAd alone where appropriate. R1 and R2 indicate use of AdipoR1 or AdipoR2 siRNA, respectively.
Fig. 5
Effect of siRNA-mediated knockdown of AdipoR1 or AdipoR2 on regulation of glucose uptake and metabolism by gAd or fAd. Primary neonatal cardiomyocytes were treated with siRNA for to effectively reduce AdipoR1 or AdipoR2 expression and consequently treated as shown in Fig. 1 with gAd (2.5 μg/ml) or fAd (2.5 μg/ml) for 1 h and 24 h. We established previously that gAd or fAd only altered glucose uptake and oxidation after 1 h and the effect of AdipoR1 or AdipoR2 siRNA on these effects are shown in A and D, respectively. After 24 h gAd or fAd altered glucose uptake and oxidation, glycogen synthesis and lactate production and the effect of AdipoR1 or AdipoR2 siRNA on these are shown in B and C or E and F, respectively. The results shown are expressed as % of maximum responses which are highlighted in Fig. 1 to clearly indicate the responses attenuated by AdipoR1 or AdipoR2 siRNA and represent mean±SEM of n≥6 where *indicates significance of p<0.05 with respect to gAd or fAd alone where appropriate. R1 and R2 indicate use of AdipoR1 or AdipoR2 siRNA, respectively.
4 Discussion
Cardiac energy metabolism plays a vital role in optimal cardiac performance and an imbalance can contribute to heart failure [1,23]. In the healthy heart under aerobic conditions, the majority of energy required for contractile performance is derived from fatty acids while the remainder (∼30%) is principally obtained via metabolism of glucose [23]. Well-controlled fatty acid metabolism is also important to prevent triglyceride accumulation as this can lead to lipotoxic effects such as apoptosis or insulin resistance [34]. In ischemic heart failure it is beneficial to switch from the prevailing excessive fatty acid metabolism toward glucose oxidation [23,24]. Many studies have established that altered circulating levels of adiponectin correlated with various aspects of cardiovascular disease [5–7], yet little is known to date on the metabolic effects of adiponectin in cardiomyocytes.
In our study, both gAd and fAd stimulated glucose uptake in cardiomyocytes and the magnitude of this increase is in keeping with the only previous report which demonstrated that fAd (10 μg/ml, 1 h) increased glucose uptake almost 1.5-fold in rat cardiomyocytes [26]. AMPK has been shown to play an important role in mediating glucose uptake in cardiomyocytes [35] and in response to adiponectin in skeletal muscle [15] and AMPK has previously been proposed to mediate the effects of gAd and fAd on cardiac hypertrophy in cardiomyocytes [9,10]. Here we show that both gAd and fAd increase AMPK phosphorylation in neonatal rat cardiomyocytes. Increased glucose uptake also correlated with increased insulin-like signaling, including phosphorylation of IRS-1 and Akt. Thus, it appears that adiponectin can stimulate both insulin-mimetic and insulin-independent mechanisms [32], which lead to glucose uptake in cardiomyocytes.
Beyond simply increasing glucose uptake, adiponectins mediated time-dependent effects on glucose metabolism. Initially, gAd directs glucose entering the cell for oxidation but by 24 h of treatment there is a lower level of oxidation, which is accompanied by enhanced glycogen synthesis and lactate production. Since we observed that fatty acid oxidation is increased by gAd after 24 and 48 h, we speculated that this may promote lower levels of glucose oxidation via inhibiting PDH [23]. Indeed, we confirmed that as fatty acid oxidation increased there was a decrease in PDH activity and subsequently in glucose oxidation. We observed that as glucose oxidation returns to basal levels in the face of increased fatty acid oxidation and increase in glycogen and lactate production is observed. In the normal aerobic heart lactate oxidation and glycolysis contribute equally to pyruvate production [23]; however, under various parameters associated with heart failure, for example ischemia and diabetes, pyruvate oxidation is diminished and lactate production becomes significant [23]. Lactate accumulation may exert detrimental effects on cardiac performance, or in the worst case scenario lead to sudden death [36]. Based on our data, transient but not prolonged administration of adiponectin would appear to be most effective in improving the myocardial metabolic profile, particularly glucose oxidation, in cardiomyocytes.
Little is known regarding the regulation of fatty acid uptake by adiponectin in any tissue and here we examined expression of fatty acid transporters FATP1, FATP4 and CD36, which have been shown to mediate fatty acid uptake in cardiomyocytes [37,38]. Interestingly fAd selectively increased expression of FATP1, suggesting that the chronic (24 h) effects of fAd on fatty acid uptake may be mediated by selectively increasing expression of this fatty acid transporter protein isoform. Although we found no change in total CD36 expression this does not rule out a contribution of this transporter via translocation and enhanced plasma membrane content [37]. The regulation of ACC activity and fatty acid oxidation by gAd and fAd was rather similar and our results are also in keeping with a previous report showing increased fatty acid uptake in an immortalized murine atrial myocyte cell line in response to 10 μg/ml fAd (30 min and 2 h) [26]. A recent study showed that gAd (1.5 μg/ml, 10 min) but not fAd (10 μg/ml, 10 min) increased fatty acid oxidation in 1-day-old rabbit hearts [17]. The reason why this study did not observe an increase in response to fAd whereas we did may be simply accounted for by the time difference 10 min versus 24 h or the fact that the fAd used in that study appears to be recombinant bacterial protein which has previously been well characterized as lacking potent biological activity [25].
Cloning of adiponectin receptors represented an important advance in allowing an understanding of the molecular mechanisms of adiponectin action [2,22]. gAd binds to AdipoR1 with high affinity and to AdipoR2 with intermediate affinity whereas fAd binds with intermediate affinity to AdipoR2 and with low affinity to AdipoR1 [22]. In this study we found using quantitative RT-PCR that primary neonatal rat cardiomyocytes express approximately 7-fold more AdipoR1 mRNA than AdipoR2. It was recently hypothesized that gAd, but not fAd, mediates fatty acid oxidation in rabbit hearts by binding a high affinity receptor isoform [17]. Indeed, we observed that the acute effects of gAd on glucose uptake and oxidation were significantly attenuated by AdipoR1, but not AdipoR2, siRNA. The ability of fAd to stimulate glucose uptake and oxidation after 1 h was significantly attenuated only after AdipoR2 siRNA treatment which is similar to observations made in liver where metabolism is regulated by fAd via AdipoR2 [22]. The possibility that prolonged adiponectin treatment of cells may downregulate its own isoform(s) expression should be borne in mind [39]. Based on our studies we believe that acute signaling by AdipoR1 and 2 may diverge and allow activation of different pathways and thus distinct metabolic effects, but that chronic stimulation of either receptor leads to activation of a common pathway which leads to changes in gene expression which correlate with the common metabolic effects we observe.
In summary, here we characterize the metabolic effects of gAd and fAd in cardiomyocytes and the role played by each AdipoR isoform in mediating these effects. The main conclusions are that both gAd and fAd possess the ability to regulate glucose and fatty acid uptake and oxidation in primary neonatal rat cardiomyocytes. It is also important to emphasize the temporal nature of these effects since the initial increase in glucose oxidation elicited by both adiponectins decreased at later time points when fatty acid oxidation was elevated and can inhibit PDH.
Acknowledgments
Funding was provided by a Grant-in-aid from the Heart and Stroke Foundation of Canada (Ontario) to GS, the Canadian Institutes of Health Research via a New Investigator Award to GS, and the Canadian Diabetes Association via a scholarship award to GS in honor of the late Mary A. Bodington. RP and XF acknowledge postdoctoral fellowship and studentship support, respectively, from Heart and Stroke Foundation of Canada. XF is also supported by a Doctoral Student Research Award from Canadian Diabetes Association.
- Copyright © 2007, European Society of Cardiology
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