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Endocrinology Vol. 148, No. 9 4411-4419
Copyright © 2007 by The Endocrine Society

Coculture with Primary Visceral Rat Adipocytes from Control But Not Streptozotocin-Induced Diabetic Animals Increases Glucose Uptake in Rat Skeletal Muscle Cells: Role of Adiponectin

Vivian Vu, Wi Kim, Xiangping Fang, Yuan-Tao Liu, Aimin Xu and Gary Sweeney

Department of Biology (V.V., W.K., X.F., Y.-T.L., G.S.), York University, Toronto, Ontario, Canada M3J 1P3; and Department of Medicine (A.X.), University of Hong Kong, Hong Kong

Address all correspondence and requests for reprints to: Gary Sweeney, Department of Biology, York University, Toronto M3J 1P3, Ontario, Canada. E-mail: gsweeney{at}yorku.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We developed a coculture system comprising primary rat adipocytes and L6 rat skeletal muscle cells to allow investigation of the effects of physiologically relevant mixtures of adipokines. We observed that coculture, or adipocyte-conditioned media, increased glucose uptake in muscle cells. An adipokine that could potentially mediate this effect is adiponectin, and we demonstrated that small interfering RNA-mediated knockdown of adiponectin receptor-2 in muscle cells reduced the uptake of glucose upon coculture with primary rat adipocytes. Analysis of coculture media by ELISA indicated total adiponectin concentration of up to 1 µg/ml, and Western blotting and gel filtration analysis demonstrated that the adipokine profile was hexamer greater than high molecular weight much greater than trimer. We used the streptozotocin-induced rat model of diabetes and found that high-molecular-weight adiponectin levels decreased in comparison with control animals and this correlated with the fact that diabetic rat-derived primary adipocytes in coculture did not stimulate glucose uptake to the same extent as control adipocytes. Coculture induced phosphorylation of AMP-activated protein kinase (T172) and interestingly also insulin receptor substrate-1 (Y612) and Akt (T308 & S473), which could be attenuated after adiponectin receptor-2-small interfering RNA treatment. In summary, we believe that this coculture system represents an excellent model to study the effects of primary adipocyte-derived adipokine mixtures on skeletal muscle metabolism, and here we have established that in the context of physiologically relevant mixtures of adipokines, adiponectin may be an important determinant of positive cross talk between adipocytes and skeletal muscle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADIPOSE TISSUE, ONCE considered simply a lipid storage depot, is now known to be a dynamic endocrine organ that plays an important role in the regulation of whole-body carbohydrate and lipid metabolism (1). Adipocytes release various factors (adipokines) that can affect insulin action and have been implicated in the development of insulin resistance and type 2 diabetes (2). It is now well established that insulin action is markedly impaired in individuals with visceral obesity (3, 4) or lipoatrophy (5). Overall, an extensive amount of data strongly suggests changes in the expression and/or secretion of adipokines as putative mechanisms by which visceral fat may modulate whole-body glucose and fatty acid metabolism (6). Adiponectin (7, 8) is one of the most abundant plasma proteins, but mRNA expression and circulating levels are reduced in diabetic obese (9, 10, 11, 12, 13, 14) and lipoatrophic (15, 16) humans and rodent models. Adiponectin has insulin-sensitizing and insulin-mimetic properties in liver and muscle and consequently mediates antidiabetic effects. This is evidenced by observations such as that adiponectin administration in rodents decreased resting blood glucose levels (17, 18), prevented insulin resistance induced by diet-induced obesity (19), protected ob/ob mice from diabetes (17), and helped reverse insulin resistance associated with lipoatrophy (15).

Regulation of physiological responses to adiponectin is complicated and occurs at many levels, including formation of oligomers, enzymatic cleavage, and availability of its receptor isoforms. Full-length adiponectin (fAd) can form trimers (low molecular weight), hexamers (middle molecular weight), or larger [high molecular weight (HMW)] oligomeric forms (20). Cleavage of fAd liberates the globular C-terminal domain (gAd), which also mediates metabolic effects. The fAd and gAd forms have different affinities for two recently characterized adiponectin receptor (AdipoR) isoforms (21). Importantly, the oligomerization or cleavage of adiponectin, not total amount of adiponectin, is now thought to be more relevant to understanding the pathogenesis of type 2 diabetes, the metabolic syndrome, and vascular disease (8). Previous in vitro studies by ourselves and others (15, 18, 19, 21, 22, 23) investigated the direct effects of adiponectin by adding recombinant protein as a single stimulus to cells. Whereas it is vital to delineate the effects of adiponectin acting in isolation, we believe it is important to stress that the ultimate in vivo effect of adiponectin is likely to be dictated by the prevailing intra- and extracellular milieu at any given time. Specifically, cross talk with signaling pathways induced by other adipokines or growth factors may prime a cell for adiponectin action or may render it in a state that may be described as adiponectin resistant. Therefore, here we established a coculture system that would allow us to develop a better understanding of the effect of more physiologically relevant combinations of adipokines on skeletal muscle glucose metabolism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Cytochalasin B, BSA, collagenase type II, streptozotocin (STZ), and enhanced avian reverse transcriptase kit were purchased from Sigma (St. Louis, MO). Human insulin (Humulin R) was obtained from Eli Lilly (Toronto, Ontario, Canada) and 2-deoxy-D-[3H]glucose from Amersham (Baie d’Urfe, Québec, Canada). The cell culture medium ({alpha}-MEM; 5 mM glucose), fetal bovine serum (FBS), and antibiotic/antimycotic solution were purchased from Wisent (St. Foy, Québec, Canada). TRIzol reagent and platinum SYBR green qPCR SuperMix UDG kit were from Invitrogen Life Technologies (Burlington, Ontario, Canada). TransIT-TKO reagent was from Mirus Bio Corp. (Madison, WI). Polyclonal phosphospecific antibodies to insulin receptor substrate (IRS)-1 (Y612), Akt (T308 and S473) and AMP-activated protein kinase (AMPK) (T172), and horseradish peroxidase (HRP)-conjugated antirabbit-IgG were from Cell Signaling Technology (Beverly, MA). Polyvinylidene difluoride (PVDF) membrane were from Bio-Rad (Burlington, Ontario, Canada) and chemiluminescence reagent plus from PerkinElmer LAS (Boston, MA). All other reagents used were of the highest purity available.

Cell culture
Rat L6 skeletal muscle cells were grown in {alpha}-MEM containing 2 or 10% (vol/vol) FBS growth medium and 1% (vol/vol) antibiotic/antimycotic (100 U/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml amphotericin B) in a humidified atmosphere of 95% air-5% CO2 at 37 C. We used wild-type L6 cells or these same cells stably transfected to overexpress myc-tagged glucose transporter (GLUT)-4 (a kind gift from Dr. Amira Klip, Hospital for Sick Children, Toronto, Ontario, Canada). Wild-type cells were differentiated in 2% FBS {alpha}-MEM media for 5–7 d, whereas L6-GLUT4myc cells grown in 10% FBS {alpha}-MEM media could be used as myoblasts when fully confluent.

Experimental animals
Male Wistar rats (Charles River, Montreal, Québec, Canada) were housed in temperature-controlled conditions on a 12-h light, 12-h dark cycle and had free access to food and tap water. Animals were used at 6–8 wk (250–300 g) and were either used directly for adipocyte isolation (wild type) or diabetes was induced with STZ before adipocyte isolation. All experimentation described in the submitted manuscript was conducted in accord with accepted standards of humane animal care and governed by York University Animal Care Committee.

Induction of diabetes using STZ
Diabetes was induced in 6- to 8-wk-old male Wistar rats with a single ip injection of STZ [in 50 mM citrate buffer (pH 4.5)] at a dose of 100 mg/kg body weight. Blood glucose levels were measured with the OneTouch Ultra Meter glucometer (Lifescan; Burnaby, British Columbia, Canada) upon removal of blood from the tail vein. The glucose tolerance test (2 g glucose per kilogram body weight) was performed 3 or 6 d after STZ injection to confirm the induction of diabetes. Blood samples from the greater saphenous vein were collected daily for up to 14 days after STZ injection to monitor adiponectin levels. Approximately 100 µl of blood were collected, centrifuged at 1000 rpm at 4 C, and serum collected for analysis of adiponectin levels.

Isolation of epididymal adipocytes and coculture with skeletal muscle cells
Adipocytes were isolated as previously described (24) during which epididymal adipose tissue was removed from 6- to 8-wk-old male Wistar rats, always between 1000 and 1200 h to avoid diurnal variations in adipokine profiles, and chopped with scissors into 2 ml Krebs-Ringer HEPES buffer (131.5 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.25 mM MgSO4, 2.5 mM NaH2PO4, 10.0 mM HEPES) supplemented with 1% BSA. Tissues were digested with collagenase type II (1 mg/ml) for 1 h at 37 C in a 250-rpm shaker. After 1 h digestion, infranatant was removed and adipocytes were washed with fresh Krebs-Ringer HEPES buffer. It is established that using this method of isolation effectively removed macrophages. The number of adipocytes was counted and diluted to 1 x 106 cells/ml with 10% FBS {alpha}-MEM. Adipocytes were stabilized in a 25 cm2 cell suspension flask for 3 h in humidified atmosphere (95% air and 5% CO2) at 37 C. After stabilization, media were removed from adipocytes and changed to serum-free {alpha}-MEM media and then used either directly in coculture or to prepare conditioned media. L6 rat skeletal muscle myotubes or L6-GLUT4myc myoblasts were used for glucose uptake studies. In coculture experiments, the muscle cells grew attached to the surface of the plate, whereas isolated adipocytes floated in the medium allowing for straightforward coculture (i.e. without need for semipermeable membrane devices). Conditioned media were collected from incubation of 1 x 106 adipocytes per milliliter for 2 h in serum-free {alpha}-MEM and used to treat muscle cells for times indicated in the figure legends. The adipocytes were then aspirated, the wells subsequently rinsed, and functional analyses conducted in the muscle cells.

Glucose transport in L6 rat skeletal muscle cells
L6 cells grown in 24-well plates were serum starved for 2 h before experimental treatments. Cells were then treated with 1 x 106 cells per milliliter of wild-type or STZ diabetic adipocytes or adipocyte-conditioned media for various time points; 100 nM insulin for 20 min served as a positive control but was not present in coculture samples. After treatments, glucose uptake was determined as previously described (22). Briefly, cells were incubated in transport solution [140 mM NaCl, 20 mM HEPES-Na, 2.5 mM MgSO4, 1 mM CaCl2, 5 mM KCl, and 0.5 µCi/ml 2-deoxy-D-[3H]glucose (pH 7.4)] for 5 min at room temperature. Nonspecific uptake was measured in the presence of cytochalasin B (10 µM). Cells were then lysed with 1 M KOH, and aliquots were transferred to scintillation vials for 3H radioactivity counting and results calculated as picomoles per minute per milligram protein and expressed as fold increase over control.

Use of small interfering RNA (siRNA) to knock down expression of AdipoR1 and AdipoR2
L6-GLUT4myc myoblasts were cultured in 24-well tissue culture plates. Several siRNA sequences (Ambion, Inc., Austin, TX) were tested to knock down rat AdipoR1 or AdipoR2. The sequence of the 21-nucleotide siRNAs ultimately used for best effect in L6 rat skeletal muscle cells were as follows: AdipoR1, CCAUGCCAUGGAGAAGAUGtt; AdipoR2, GAGACACCUGUUUGUUCUUtt. Before coculture experiments, L6-GLUT4myc myoblasts were transfected by treating for 24 h with 100 nM AdipoR1, AdipoR2, or unrelated siRNAs by using the TransIT-TKO reagent following manufacturer’s protocol.

Analysis of adiponectin receptor expression by RT-PCR
After siRNA treatment for 24 h, total RNA was isolated from L6-GLUT4myc myoblasts using the TRIzol reagent. cDNAs were synthesized by reverse transcription using the enhanced avian reverse transcriptase kit with 1 µg of total RNA and 1 U of reverse transcriptase according to the manufacturer’s recommendations. Primers for real-time quantitative PCR were based on sequences of adiponectin receptors in L6-GLUT4myc myoblasts. Primer sequences and their respective PCR fragment length were as follows: AdipoR1 (158 bp), forward 5'-GCTGGCCTTTATGCTGCTCG-3', reverse 5'-TCTAGGCCGTAACGGAATTC-3'; AdioR2 (415 bp), forward 5'-CCCTCTGCAAGAGAAAGTGG-3', reverse 5'-TAGCCAGCCTATCTGCCCTA-3'; ß-actin (133 bp), forward 5'-CTGTGCCCATCTATGAGGGT-3', reverse 5'-CTCTCAGCTTGGTGGTGAA-3'. AdipoR1 and AdipoR2 mRNA levels in L6 cells were determined by using platinum SYBR Green qPCR SuperMix UDG kit.

Determination of adipokine content in conditioned medium and plasma
Plasma samples and conditioned media obtained by culturing adipocytes (1 x 106 cells/ml for various times) were analyzed for adiponectin content. ELISA, immunoblotting, and gel filtration were used to analyze the profile of adiponectin forms in plasma and conditioned media. For immunoblotting, 20 µl of cultured media or 1 µl of plasma were diluted with Laemmli sample buffer (LSB) [conditioned media in 5x LSB in a ratio of 4:1 (vol/vol), plasma in 1x LSB 1:1 (vol/vol)]. LSB consisted of 62.5 mM Tris-HCl (pH 6.8), 2% (wt/vol) sodium dodecyl sulfate, 50 mM dithiothreitol, 0.01% (wt/vol) bromophenol blue, and phosphatase inhibitors (1 µM Na3VO4, 1 µM leupeptin, 1 µM pepstatin, 1 µM okadaic acid, and 1 µM phenylmethylsulfonyl fluoride). Aliquots of diluted 20 µl conditioned media and 1 µl plasma were resolved by 6 and 15% SDS-PAGE under nonreducing and nondenaturing conditions and then immunoblotted onto PVDF membrane using methods as we described previously (25). Membranes were blocked with 3% BSA dissolved in 1x wash buffer solution for 1 h and incubated overnight with antiadiponectin antibody (2 µg/ml). Membranes were then incubated for 1 h with HRP-coupled antirabbit antibody (1:10,000) and proteins visualized using enhanced chemiluminescence. Gel filtration and ELISA studies were conducted as previously described (25). Briefly, PBS-diluted, 1-ml samples were loaded onto an AKTA Explorer fast protein chromatography system, fractionated through a HiLoad 16/60 Superdex 200 column (GE Healthcare, Piscataway, NJ), and eluted with PBS at a flow rate of 1 ml/min. One milliliter-fractions were collected and subjected to in-house ELISA analysis for adiponectin.

Immunoblotting to analyze phosphorylation of IRS1, Akt and AMPK
Western blotting was conducted as described by us previously (23) where, after coculture or treatment with conditioned media, L6 cells were lysed in 1x LSB sample buffer containing 10% (vol/vol) ß-mercaptoethanol, passed through a syringe several times, and heated (65 C, 5 min). Cell lysates were then centrifuged for 5 min in a bench top microfuge (13,000 rpm), and approximately 30 µg protein were resolved by 10% SDS-PAGE and immunoblotted onto PVDF membrane. Membranes were blocked with 3% BSA dissolved in 1x wash buffer solution of 50 mM Tris-base, 150 mM NaCl, 1% Triton X-100, and 1% Nonidet P-40 for 1 h. Membranes were incubated overnight with primary antibodies at the following dilutions: phospho-AMPK (T172), -IRS1 (Y612), and -Akt (T308 and S473) (all 1:500). Membranes were then washed four times in 1x wash buffer for 15 min each at room temperature and incubated with appropriate HRP-coupled secondary antibody (1:10,000) for 1 h. Membranes were washed five times in 1x wash buffer for 10 min each and proteins visualized using enhanced chemiluminescence.

Statistical analysis
Data are expressed as means ± SEM. Statistical analysis was undertaken using paired Student’s t test. Differences between groups were considered statistically significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We first tested whether coculture of primary rat adipocytes with L6 rat skeletal muscle cells had any effect on glucose uptake by L6 cells. We observed that coculture of rat adipocytes for times from 30 min up to 3 h significantly enhanced uptake of 2-deoxyglucose into L6-GLUT4myc myoblasts (Fig. 1AGo). The magnitude of this response (~2-fold) was similar to that induced by insulin as positive control. The use of L6-GLUT4myc cells myoblasts was validated by similar experiments using differentiated wild-type L6 myotubes (data not shown). Because the majority of adipokines are thought to have attenuating effects on insulin action (1), the most likely candidates for mediating this effect may be adiponectin or visfatin. The latter would be expected to elevate insulin receptor phosphorylation (26), but we did not detect such a response on coculture (data not shown). Hence, to further investigate the role played by adiponectin, we used the strategy of knocking down its receptor expression in L6 cells to nullify its effects. We used siRNA to AdipoR1 and AdipoR2 (achieving up to 90% knockdown of relevant mRNA; Fig 1BGo) and found that the ability of adipocyte coculture to increase glucose uptake in L6 cells was attenuated on AdipoR2, but not AdipoR1, knockdown (Fig. 1Go, C and D).


Figure 1
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  		FIG. 1. Effect of coculture with primary rat adipocytes on glucose uptake in L6 skeletal muscle cells and role of adiponectin. A, Uptake of 2-deoxyglucose was measured in the presence (black bars) or absence (white bars) of adipocyte coculture (1 x 106 adipocytes/ml) for 0.5–3 h. Control is assigned a value of 1 and values represent mean ± SEM (n ≥ 3). *, P < 0.05 vs. respective time controls. B, Expression of AdipoR1 and AdipoR2 isoform mRNA in L6 cells upon knockdown with AdipoR1, AdipoR2, and unrelated siRNA. Results represent mean ± SEM (n = 3), and in each case the level of AdipoR expression was assigned an arbitrary value of 1, and content after siRNA treatment was expressed relative to this value. *, P < 0.05 vs. control in absence of siRNA. C and D, 2-Deoxyglucose uptake was measured after siRNA knockdown of AdipoRs in the presence (black bars) or absence (white bars) of adipocyte coculture. Unrelated siRNA control was set to 1 and values represent mean ± SEM (n ≥ 3). *, P < 0.05 vs. control; # P < 0.05 vs. coculture in the absence of AdipoR2 siRNA (D).

 
We also used the STZ-induced diabetic rat model and examined the amount and profile of adiponectin secreted from adipocytes of these and control animals. We first examined total circulating levels of adiponectin and found a significant drop after only 2 d after STZ injection which was further decreased at days 4 and 6 post-injection (Fig. 2AGo). When the profile of various forms of adiponectin found in circulation of control or diabetic animals was analyzed by Western blotting over the course of 14 d after STZ injection, we observed relatively constant levels of HMW and hexameric adiponectin in control animals (Fig. 2BGo). A clear decrease, particularly in the HMW form, was observed in diabetic animals as soon as just a few days after STZ injection (Fig. 2BGo). We did not detect significant changes in levels of trimeric adiponectin in plasma by Western blotting (data not shown). The decrease in HMW adiponectin in STZ-treated animals was verified upon separation of various oligomeric forms of adiponectin in serum (0–6 d after injection) by gel filtration (Fig. 2Go, C and D). The accumulation of total adiponectin over time (up to 12 h) in rat adipocyte-conditioned media was also significantly less when adipocytes derived from diabetic animals were used (Fig. 3AGo). Upon analysis of oligomeric forms by Western blotting, it is apparent that lower amounts of total, but particularly HMW adiponectin, are secreted by adipocytes derived from diabetic rats (Fig. 3BGo).


Figure 2
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  		FIG. 2. Total adiponectin and adiponectin multimeric complex distribution in wild-type and STZ diabetic rat serum. Total adiponectin in rat serum was quantified by ELISA after STZ injection (A). Values represent means ± SEM (n ≥ 3). *, P < 0.05 vs. d 0 levels. B, SDS-PAGE separation of adiponectin multimeric complexes from wild-type (top) and diabetic (bottom) rat serum over the 14-d period after STZ injection under nonreducing and nondenaturing conditions, followed by immunoblotting for adiponectin. Representative elution profiles of serum adiponectin from wild-type (C) and STZ diabetic (D) rats are shown. Individual gel filtration chromatography fractions were quantified by ELISA for adiponectin levels. D (inset), Quantitative analysis of HMW composition expressed as a percentage of total adiponectin.

 

Figure 3
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  		FIG. 3. Total adiponectin and adiponectin multimeric complex distribution in wild-type and STZ diabetic rat adipocyte conditioned media. Total adiponectin in wild-type (white bars) and STZ diabetic rat (black bars) adipocyte-conditioned medium was quantified by ELISA (A). Values represent mean ± SEM (n ≥ 3). B, Representative SDS-PAGE separation of adiponectin multimeric complexes from adipocyte conditioned media under nonreducing and nonheating conditions followed by immunoblotting for adiponectin.

 
To investigate the metabolic consequences of the altered adiponectin profile secreted by diabetic rat adipocytes, we then measured the effects of coculturing normal or diabetic rat adipocytes with L6 muscle cells. We used adipocytes derived from animals 3 and 6 d after injection. Although an apparent decrease from the level of glucose uptake stimulated by normal rat adipocytes at each time point (0.5, 1, 2, and 3 h) was apparent when adipocytes from 3-d diabetic rats were used, these changes were only statistically significant at 2 and 3 h (Fig. 4AGo). When 6-d diabetic rat adipocytes were used, a significant decrease was observed at all time points (Fig. 4BGo). Due to the potential that the stimulatory effects we observed on glucose uptake in L6 cells on coculture with adipocytes may have been due to factors such as adipocytes remaining in wells, even after rinsing, we have been careful to repeat the above experiments using adipocyte-conditioned media. In these experiments conditioned media, in which primary adipocytes have been cultured for 2 h, alone was used to treat muscle cells. Upon using this approach, we found the same observations (Fig. 5Go, A and B) to those presented in Fig. 4Go.


Figure 4
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  		FIG. 4. Effect of wild-type and STZ diabetic rat adipocyte coculture on glucose uptake in L6 myoblasts. L6-GLUT4myc myoblasts were cocultured with adipocytes isolated from wild-type (white bars) or diabetic rats (black bars) 3 d (A) and 6 d (B) after STZ injection. The data represent mean ± SEM (n = 3). Control represents basal levels (light gray bars) and 100 nM insulin for 20 min served as a positive control (dark gray bars). *, P < 0.05 vs. control under basal conditions; #, P < 0.05 vs. paired wild-type adipocyte coculture response.

 

Figure 5
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  		FIG. 5. Effect of wild-type and STZ diabetic rat adipocyte conditioned media on glucose uptake in L6 myoblasts. Uptake of 2-deoxyglucose in L6-GLUT4myc myoblasts was measured under basal conditions (light gray bars), in response to 100 nM insulin (20 min, dark gray bars), and on treatment with wild-type (white bars) or STZ diabetic rat adipocyte (black bars) conditioned medium: 3 d (A) and 6 d (B) after STZ injection. Values are means ± SEM (n = 3).*, P < 0.05 vs. control; #, P < 0.05 vs. paired wild-type conditioned medium treatment.

 
We examined the ability of adipocyte-conditioned media to regulate signaling pathways known to control glucose uptake in L6 cells. The representative Western blots together with quantitative analysis of several experiments shown in Fig. 6Go, A–D, highlight the fact that media prepared using adipocytes from normal rats (added for 5–30 min) caused increases in IRS1 (Y612) phosphorylation and enhanced stimulation of Akt (T308 and S473) and AMPK (T172). In each case the degree of phosphorylation observed was lower when adipocytes derived from STZ-diabetic rats were used (Fig. 6Go, A–D). Whereas the ability of coculture media containing adiponectin to phosphorylate AMPK might be expected (27), the phosphorylation of IRS-1 and Akt is a more novel observation in this study. We again used siRNA-mediated knockdown of AdipoR2 (Fig. 1Go) and found that this approach attenuated the phosphorylation of IRS1 (Fig. 6EGo) and Akt (Fig. 6Go, F and G) in the coculture system.


Figure 6
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  		FIG. 6. Effect of wild-type and STZ diabetic rat adipocyte-conditioned media on phosphorylation of IRS1, Akt, and AMPK and the effect of siRNA-mediated knockdown of AdipoR2. Representative immunoblots for phosphorylated IRS1 (Y612) (A), Akt (T308) (B), Akt (S473) (C), and AMPK (T172) (D) on exposure to coculture media collected from wild-type or diabetic rat adipocytes are shown in the panels above graphs showing data from quantitative analysis. Also shown is quantitative analysis of IRS (E), Akt (T308) (F), and Akt (S473) (G) phosphorylation after siRNA-mediated knockdown of AdipoR2 and exposure to coculture media collected from wild-type adipocytes. In all cases graphs represent n = 3 experiments and data are expressed as mean ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To analyze the metabolic effects of adipokines in vitro, the majority of studies to date have used the strategy of simply adding recombinant protein. However, this approach inherently neglects many factors which occur in vivo, such as the extensive cross talk between signaling pathways regulated by various adipokines. Similarly, in vivo studies are often complicated by additional inputs (e.g. via nervous system). We established the use of a coculture system to allow analysis of more physiologically relevant combinations of adipokines acting in concert with one another. Several previous studies also used a coculture approach. First, primary human skeletal muscle cells were cocultured with human fat cells or incubated with adipocyte-conditioned medium in the presence or absence of gAd to analyze its capacity to restore normal insulin signaling in muscle cells (28). In this study adipocyte-conditioned medium caused impaired Akt and glycogen synthase kinase-3 phosphorylation and GLUT4 translocation in response to insulin, all of which were corrected by adding gAd to the medium. The observations made in our experiments and in this study differ somewhat. However, many differences exist between this system and our own coculture model including: 1) cells derived from human rectus abdominus vs. L6 cells, 2) preadipocytes isolated from mammary fat tissue of normal and overweight women and then differentiated to adipocytes over approximately 18 d before use vs. visceral fat from normal male rats, 3) insulin (1 pmol/liter) present throughout coculture vs. insulin added only before metabolic assays where indicated as positive control, 4) conditioned medium generated by culturing adipocytes for 48 h, lyophilization, reconstitution and addition to myotubes vs. various time points of coculture followed by direct addition to myoblasts, and 5) no analysis of adiponectin forms present in coculture system was performed in the paper by Dietze-Schroeder et al. (28). Blunted insulin signaling and regulation of glucose metabolism was recently observed in primary hepatocytes on coculture with differentiated 3T3-L1 adipocytes (29). We believe our system is more likely to accurately reflect the effects of physiological combinations of adipokines because it uses freshly isolated primary adipocytes from visceral adipose tissue and target cells from the same species. Another recent study has also documented the use of a coculture system as a source of bioactive adipokines. Human adipocytes were cocultured with rat cardiomyocytes and the cardioactive substances secreted by adipocytes induced a decrease in inotropic activity of cardiomyocytes (30).

Our results clearly demonstrated that the mixture of adipokines derived from primary rat visceral adipocytes increased glucose uptake in L6 cells. Although a common perception is that adipokines are detrimental to glucose homeostasis, this is largely derived from consideration of obese individuals or animal models. However, the development of insulin resistance and diabetes in lipoatrophy clearly suggests a need for a normal level of adipose tissue that contributes to glucose homeostasis (9, 10, 11, 12, 13, 14, 15, 16). The important beneficial role played by adipocytes was further exemplified by studies in which induction of caspase-8 was used to selectively ablate adipocytes in adult mice (31). These mice quickly became glucose intolerant and had very low circulating levels of adipokines, including adiponectin. These changes were reversible on cessation of caspase-8 activation and recovery of adipose tissue mass (31). Furthermore, transgenic overexpression of adiponectin in the context of an ob/ob mouse background resulted in an even greater degree of obesity but an essentially normal metabolic profile (43). As with our study, this suggests an important beneficial role played by adiponectin.

Based on the above and our knowledge of metabolic effects of other adipokines, only a few candidates exist that may be responsible for mediating the enhanced glucose uptake we observe on coculture. Whether leptin exerts positive or negative influence on glucose uptake in skeletal muscle is somewhat controversial (32). Hence, the most likely adipokine to elicit an increase in glucose uptake is adiponectin, recombinant forms of which have been well documented to increase glucose uptake in skeletal muscle in a large number of previous studies by ourselves and others (15, 18, 21, 22, 23). Analysis of adiponectin action is complicated by the fact that monomers of the adiponectin gene product can form trimeric or higher-order complexes that are found in circulation. Furthermore, cleavage of fAd (33) liberates the C-terminal globular domain (gAd), which exerts potent metabolic effects in skeletal muscle (19, 21, 22, 23).

Each form of adiponectin may have different potency for, and end point responses in, different tissues (23, 34, 35, 36). For example, it has been demonstrated that only trimeric and not hexameric or oligomeric adiponectin could phosphorylate AMPK in extensor digitorum longus (36). This may be at least partly explained by the discovery of two AdipoR isoforms that have different binding affinity for adiponectin multimers and gAd (21). Thus, to build on the inference from our work to date and clearly delineate the role of adiponectin in our coculture system, we used the approach of knocking down L6 cell AdipoR expression using siRNA. We have previously shown that L6 cells contain approximately 7-fold more AdipoR1 than AdipoR2 (23), yet in this study knockdown of AdipoR2 attenuated the ability of coculture to enhance glucose uptake, IRS1 and Akt phosphorylation. This is not entirely surprising because, although many studies have shown that gAd mediates potent metabolic effects via AdipoR1, it is known that AdipoR2 has a higher affinity for fAd (21), and we have shown that enhancing AdipoR2 expression enhances the metabolic effects of fAd in L6 cells (23). When we analyzed the adiponectin composition in coculture media, we observed primarily hexamer and HMW with only very low levels of low molecular weight. This would suggest that multimers of fAd, acting via AdipoR2, can rapidly increase glucose uptake in skeletal muscle cells in the context of this coculture system.

We used the STZ-induced diabetic rat model to manipulate the levels of adipokines in coculture system. It has been suggested that adiponectin levels change in type 1 diabetic patients, although these studies are often confounded by patients receiving insulin therapy (37, 38, 39). First, we examined the change in total adiponectin in circulation after injection of STZ and found a significant decrease after as little as 2 d. When we examined the profile of each form of adiponectin in plasma up to 14 d after STZ injection or secreted by primary adipocytes in culture, we observed that the main change was a decrease in the HMW form of adiponectin. Importantly, when we examinied the functional significance of this alteration, we observed that coculture using adipocytes from diabetic rats elicited a significantly smaller increase in glucose uptake compared with that observed with adipocytes from normal rats. Similar results were observed using conditioned media prepared using diabetic rat adipocytes. When both insulin-like (IRS1 and Akt phosphorylation) and insulin-independent (AMPK phosphorylation) signaling was examined, a lower response was observed when adipocytes from diabetic rats were used. Therefore, our results suggest that reducing the content of oligomeric forms of adiponectin in the coculture system can attenuate signaling and the uptake of glucose normally seen in skeletal muscle cells. Importantly, it must also be borne in mind that the profile of certain deleterious peptides secreted by primary adipocytes may also change in diabetic rats and partly contribute to the observed effects. Although it is tempting to speculate on their role, it is beyond the scope of this single article to analyze all. Indeed, we believe that our coculture system is of great value because it allows direct analysis of this adipokine mixture on muscle cells rather than at the whole-body level in vivo in which interpretation of direct cross talk between fat and muscle may be confounded by influences such as centrally mediated effects (40).

When comparing our results with those published previously using recombinant forms of adiponectin, some interesting observations arise. We have shown here that in the presence of physiological mixtures of adipokines, fAd is capable of enhancing glucose uptake at 30 min to 3 h exposure times. Previous in vitro studies using recombinant protein by ourselves (22, 23) and others (21) using as low as 0.3 µg/ml fAd, similar to those found in our coculture system, found no acute effect and suggest longer exposure times (7 h and higher) are necessary. However, another study has demonstrated that 2.5 µg/ml fAd increased glucose uptake in C2C12 cells after only 30 min. As far as we are aware, although phosphorylation of AMPK has been observed, an ability of fAd to stimulate Akt phosphorylation in skeletal muscle has not been observed. However, gAd increased Akt phosphorylation in endothelial cells (41). Our results suggest that adipokines in the coculture mixture may enhance cross talk between adiponectin and insulin-like signaling (42). Other adipokines in coculture may mediate direct effects but as discussed previously, few would be expected to increase Akt phosphorylation.

In summary, the studies described here establish a coculture system to examine metabolic effects of adipokines secreted by primary rat visceral adipocytes on skeletal muscle cells. We believe that this system is useful to study the effects of naturally secreted mixtures of adiponectin oligomers in the context of physiologically relevant mixtures of adipokines, and it may be applied to examination of primary rat adipocyte-derived adipokine mixtures on a variety of target cells. Interfacing previous studies using animal models or recombinant adiponectin in vitro, we believe our observations in this study further suggest that in the context of physiologically relevant mixtures of adipokines, adiponectin acts as an important regulator of cross talk between adipocytes and skeletal muscle cells with respect to control of glucose uptake.


   Footnotes
 
This work was supported by Canadian Institutes of Health Research via an operating grant and New Investigator Award (to G.S.). X.F. was supported by doctoral student research awards from Heart and Stroke Foundation of Canada and the Canadian Diabetes Association.

First Published Online June 14, 2007

Abbreviations: AdipoR, Adiponectin receptor; AMPK, AMP-activated protein kinase; fAd, full-length adiponectin; FBS, fetal bovine serum; gAd, globular C-terminal domain of adiponectin; GLUT, glucose transporter; HMW, high molecular weight; IRS, insulin receptor substrate; LSB, Laemmli sample buffer; PVDF, polyvinylidene difluoride; siRNA, small interfering RNA; STZ, streptozotocin.

Received January 8, 2007.

Accepted for publication June 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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