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Department of Human Nutrition, Ohio State University, Columbus, OH 43210
Published, JLR Papers in Press, October 18, 2006.
1 A. A. Wendel and L-F. Liu contributed equally to this work. 
2 To whom correspondence should be addressed. e-mail: belury.1{at}osu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESEARCH DESIGN AND METHODSRESULTSDISCUSSIONREFERENCES |
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Supplementary key words hepatic steatosis • adipokines • hepatic steatosis • rosiglitazone
Abbreviations: AOX, acetyl-coenzyme A oxidase; CLA, conjugated linoleic acid; c9t11, cis9,trans11; CON, control; FBG, fasting blood glucose; IL-6, interleukin-6; PPAR
, peroxisome proliferator-activated receptor ; ROSI, rosiglitazone; TG, triglyceride; TNF-
, tumor necrosis factor-
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESEARCH DESIGN AND METHODSRESULTSDISCUSSIONREFERENCES |
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Conjugated linoleic acid (CLA) consists of positional and geometric isomers of octadecadienoate that are naturally found in foods such as beef, lamb, milk, and other dairy products (11). It is well established that cis9,trans11 (c9t11)-CLA and trans 10,cis 12 (t10c12-CLA) have unique effects on lipid metabolism (12), and it is the t10c12-CLA isomer that is mainly associated with decreases in body fat in experimental rodent models (13–16) as well as in some but not all human studies (17–19), independently of energy intake (20).
Feeding CLA to mice is associated with lipodystrophy and worsening of insulin sensitivity (16, 21–24). These effects have been attributed to a rapid and significant reduction of adipose tissue and a sharp decline in insulin-sensitizing adipokines such as adiponectin and leptin (23). Although feeding dietary CLA has been shown previously to deplete adipokines and cause hyperinsulinemia (23), it is unknown whether removing CLA from the diet can restore the level of adipokines and attenuate insulin resistance, showing that there is, in fact, a causal link between adipokine depletion by feeding dietary CLA and the development of hepatic steatosis and insulin resistance in mice. Furthermore, dietary CLA has been shown to induce insulin resistance in ob/ob mice, which lack functional leptin (16), raising the possibility that adipokines other than leptin may be more important in CLA-mediated insulin resistance in mice. Thus, we further investigated the role of adiponectin in insulin resistance mediated by CLA in leptin-deficient ob/ob mice. To this end, we used the peroxisome proliferator-activated receptor (PPAR) agonist rosiglitazone (ROSI) in conjunction with CLA and hypothesized that maintaining serum adiponectin in ob/ob mice, which lack functional leptin, attenuates the effects of CLA on insulin resistance and hyperglycemia.
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RESEARCH DESIGN AND METHODS |
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TOPABSTRACTINTRODUCTIONRESEARCH DESIGN AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
Eleven week old male C57BL/6 mice and 6 week old male ob/ob mice were purchased from Harlan (Indianapolis, IN) and Charles River Laboratories, Inc. (Wilmington, MA), respectively. Mice were housed four per cage at 22 ± 0.5°C on a 12 h day/night cycle. Mice received standard chow for 1 week while adjusting to their new environment. All procedures were in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of Ohio State University.
Study 1: depletion-repletion of adipokines
To determine the role of adipokine depletion by CLA in the development of insulin resistance and hepatic steatosis, 12 week old male C57BL/6 mice (n = 10) were consecutively fed two different experimental diets. For the first 4 weeks, mice were fed a 1.5% CLA experimental diet, which contained 5% soybean oil plus 1.5% CLA-TG mix by weight, for a total of 6.5% fat. This dose of CLA provided 
0.6% (by weight) each of the c9t11-CLA and t10c12-CLA isomers. Supplementation with either 0.5% purified t10c12-CLA isomer or 1% CLA mixture has been shown to effectively reduce adipose tissue in mice and produce liver steatosis (15, 23, 24). At the end of the first 4 week diet period, half of the mice were euthanized, and the remaining mice were switched to a diet without CLA (chow diet containing 4–5% total fat). All mice had free access to food and water. Body weights were measured at the indicated time points.
Study 2: sustenance of adiponectin
Six week old male ob/ob mice were randomized by body weight and fed experimental diets containing 6.5% total fat for 4 weeks. The diets contained 6.5% soybean oil [control (CON) diet; n = 8] by weight. Additionally, 10 mice were maintained on the CON diet for the first 2 weeks, after which 6 mice were switched to the CLA diet and 4 mice were continued on the CON diet for the last 2 weeks of the study period. During the last 2 weeks, mice received daily intraperitoneal injections of PBS (vehicle control: 10% DMSO and 90% PBS solution) or 10 mg/kg body weight/day ROSI (25, 26). Body weights were measured weekly.
Insulin tolerance test and fasting blood glucose
Insulin sensitivity was determined for studies 1 and 2 using an insulin tolerance test at the indicated times. Mice were fasted overnight and received intraperitoneal injections of insulin [Humulin R (Eli Lily, Inc.)] at doses of 0.75 U/kg body weight for C57BL/6 mice and 1.5 U/kg body weight for ob/ob mice. Insulin-stimulated glucose clearance was determined by tail vein bleeding at 0, 15, 30, 45, 60, 90, and 120 min after insulin injection. Insulin sensitivity was determined by calculating the areas under the curves, and individual baselines were used to normalize data. Fasting blood glucose (FBG) levels were measured at baseline, 2 weeks, and 4 weeks for study 2. Mice were fasted overnight for 12 h, and tail vein blood was used to analyze FBG using a One Touch Basic glucose analyzer (Lifescan, Milpitas, CA).
Necropsy (studies 1 and 2)
To avoid the effects of injected insulin on gene expression, mice were anesthetized by isoflurane in the fasted state 2 days after the insulin tolerance test. Blood was collected by heart puncture, centrifuged at 1,500 g at 4°C to isolate serum, and stored at –80°C for hormone and metabolite analyses. Liver, epididymal adipose, and gastrocnemius muscle tissues were weighed, snap-frozen in liquid nitrogen, and stored at –80°C for further analysis.
Serum hormone and metabolite determination
Time course depletion-repletion of adipokines from study 1 was determined using 4 h fasted serum from retro-orbital eye bleeds at the indicated time points. Mice were anesthetized using isoflurane. Additionally, fasted serum insulin, adiponectin, and resistin levels from study 2 were determined using ELISAs (LINCO Research, St. Charles, MO). Fasting serum TGs and NEFAs from study 2 were measured using spectrophotometric assays from Sigma (St. Louis, MO) and Wako Chemicals (Richmond, VA), respectively.
Tissue TG analysis (studies 1 and 2)
Liver and muscle tissues were homogenized and lysed in 10x Tris (w/v) buffer containing 20 mM trizma base, 1% Triton X-100, 50 mM NaCl, 250 mM sucrose, 50 mM NaF, 5 mM Na4P2O7·10H2O, and protease inhibitors. TGs were extracted with 2:1 (v/v) chloroform-methanol, final extracts were dissolved in 3:1:1 (v/v/v) tert-butanol-methanol-Triton X-100 (27), and TGs were quantitatively measured with an enzymatic colorimetric kit (Sigma). Values are expressed as percentage tissue weight.
Real-time RT-PCR (study 2)
Sections from liver and muscle tissue were homogenized in Trizol reagent (Invitrogen, Carlsbad, CA), and RNA was isolated using the manufacturer's protocol. RNA from adipose tissue was isolated using the RNeasy lipid extraction kit (Qiagen, Valencia, CA). RNA was diluted in RNase-free water and quantified by spectrophotometry. RNA integrity was assessed by electrophoresis using agarose gel and ethidium bromide staining. The first transcripts were reverse-transcribed using reverse transcriptase (Invitrogen), and cDNA was amplified using real-time PCR with FAM-labeled TaqMan gene expression assays (Applied Biosystems, Foster City, CA). In short, 5 ng of the reverse transcription reaction was amplified in a total reaction volume of 25 µl using predesigned and validated primers for liver fatty acid synthase (FAS), the fatty acid transporter CD36, acetyl-coenzyme A oxidase (AOX), and tumor necrosis factor- (TNF-) using universal cycling conditions. Target gene expression was normalized to Vic-labeled 18S, which was used as an endogenous control and amplified in the same reaction as the target gene.
Statistical analysis
All data are presented as means ± SEM. Data were analyzed using MINITAB (version 14). Data from study 2 were analyzed by one-way ANOVA. Posthoc analysis was performed using Tukey's test. Other comparisons were analyzed by Student's t-test as appropriate. Weight gain and serum adipokine concentrations over time were analyzed by repeated-measures ANOVA using SAS (version 9.1; Cary, NC). Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESEARCH DESIGN AND METHODSRESULTSDISCUSSIONREFERENCES |
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Study 2
Body weights and organ weights
ROSI administration for 2 weeks prevented weight loss in male ob/ob mice fed dietary CLA, and weight gain was comparable to that in CON-PBS mice in both ROSI-treated groups (Fig. 1B). Epididymal adipose mass was also not significantly different in the CLA-ROSI group compared with the CON-ROSI and CON-PBS groups (Table 2
).
Serum metabolites Two weeks of treatment with ROSI not only prevented increases in both glucose and insulin in mice fed dietary CLA (CLA-ROSI vs. CON-ROSI and CON-PBS) but also significantly decreased FBG levels compared with CON-PBS mice (Table 2). ROSI administration significantly increased adiponectin levels in mice fed the CON or CLA diet compared with mice fed the CON-PBS diet (Table 2). Serum resistin levels were significantly higher in CON-PBS mice compared with CON-ROSI mice; however, ROSI administration did not have an effect on resistin levels in mice fed CLA. Furthermore, ROSI administration significantly decreased serum levels of TG and NEFA in both CON- and CLA-fed mice compared with the CON-PBS group (Table 2).
Insulin tolerance Compared with the CON-PBS group, ROSI significantly improved insulin sensitivity in both CON- and CLA-fed ob/ob mice, and CLA-ROSI-fed mice had insulin sensitivity comparable to CON-ROSI mice (area under the curve) (Table 2, Fig. 3B).
Liver and muscle TG Although ROSI treatment did not decrease hepatic lipids in CON-fed mice, interestingly, there was a significant reduction in liver TG in the CLA-ROSI group compared with the CON-PBS and CON-ROSI groups after 2 weeks of ROSI treatment (Table 2). There was no effect of diet or treatment on muscle TG in ob/ob mice (data not shown).
Liver mRNA expression
Because the combination of CLA with ROSI led to significantly lower liver TG, we measured mRNA levels of genes indicative of lipid oxidation and lipid synthesis in the liver. ROSI treatment significantly increased mRNA levels of liver AOX in both CON- and CLA-fed mice compared with CON-PBS mice (Fig. 4A
). There were no significant differences in the mRNA levels of PPAR and carnitine palmitoyl transferase between the groups (data not shown). Although ROSI treatment significantly increased mRNA levels of FAS in the CON-fed mice, interestingly, the combination of CLA with ROSI led to significantly lower levels of FAS mRNA comparable to CON-PBS mice (Fig. 4B).
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Adipose mRNA expression
ROSI administration significantly decreased TNF- mRNA levels in both CON- and CLA-fed mice compared with PBS-injected mice fed the CON diet (Fig. 4E).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESEARCH DESIGN AND METHODSRESULTSDISCUSSIONREFERENCES |
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and interleukin-6 (IL-6)] were also not significantly different between the CON and CLA groups after 4 weeks (data not shown). In this study, we show that CLA causes rapid changes in weight and adiposity in mice. The depletion in adipose tissue is accompanied by worsened insulin sensitivity and the development of hepatomegaly (likely attributable, in part, to increased hepatic lipids). These results are in accordance with other studies using both mixed isomers of CLA and the t10c12 isomer alone (16, 21–24). Previously, it was shown that CLA causes a time course depletion of adiponectin and leptin and is associated with the development of insulin resistance in female C57BL/6J mice (23). Here, we show for the first time that removing CLA from the diet results in the reversal of depressed adipokines, insulin resistance, and hepatic steatosis.
In study 1, the level of adiponectin was depleted significantly at 6 days in male C57BL/6 mice supplemented with dietary CLA. These results are in accordance with a study conducted by Poirier et al. (23). However, in contrast to their study, in this study, using comparable levels of dietary CLA, leptin levels were not significantly lower than baseline. These differences may be attributable to differences in gender in the two studies. Associated with the depletion of adiponectin, there was a significant decrease in the phosphorylation of key mediators of glucose and lipid metabolism pathways, such as AMP-activated protein kinase and acetyl-CoA carboxylase, in the livers of C57BL/6 mice, and levels of these phosphoproteins were restored by removing CLA from the diet (data not shown). The observed decrease in phosphorylation state of AMP-activated protein kinase and acetyl-CoA carboxylase may be indicative of decreased lipid oxidation in the liver and impaired insulin responsiveness (9, 28–30). Switching mice to the diet without CLA resulted in a significant improvement in serum adiponectin levels as early as 7 days. Of interest here, although insulin resistance and hepatic TG were significantly attenuated when CLA was removed from the diet, adiponectin levels were only partially restored, suggesting that the presence of leptin may compensate for insufficient adiponectin levels and that the overall adipokine status may influence the response to dietary CLA in different animal models. The concomitant repletion of adipokines and reversal of insulin resistance by removing CLA from the diets is novel and further supports a strong association between the dysregulation of adipokine synthesis by dietary CLA and the worsening of insulin sensitivity in mice.
Previously, it was demonstrated that leptin infusion into C57BL/6 mice reverses insulin resistance and hepatic steatosis when adipose stores are not completely ablated (21). However, leptin has no effect when infused into A-ZIP/F-1 fatless mice (31). These data suggest that leptin insufficiency alone is not the principal cause of lipodystrophy-associated insulin resistance caused by CLA (31). The effects of CLA on insulin resistance and hepatic steatosis have been demonstrated previously in ob/ob mice by Roche et al. (16). Preliminary findings from our study are in agreement with the study conducted by Roche et al. (16). In addition, we measured serum adiponectin levels over time in female ob/ob mice fed CLA and found that levels were significantly and maximally depressed by 2 weeks (data not shown). Furthermore, mice developed significant hepatic steatosis and insulin resistance along with the depletion of adiponectin. Because ob/ob mice lack functional leptin, these data in conjunction with our data from study 1 suggest that the worsening of insulin sensitivity caused by dietary CLA may be more strongly associated with the depletion of adiponectin or overall adipokine status than leptin alone. To further examine the role of adiponectin in CLA-induced insulin resistance, we injected leptin-deficient male ob/ob mice with an adiponectin enhancer, ROSI, for 2 weeks.
ROSI treatment significantly increased serum concentrations of adiponectin in mice fed either the CON or CLA diet compared with CON-PBS-treated mice. These effects of ROSI on serum adiponectin are consistent with other studies (10, 32, 33). Because higher circulating levels of serum resistin are often associated with insulin resistance and diabetes in rodents (32), we measured serum levels of resistin. Preliminary data from male ob/ob mice fed dietary CLA for 4 weeks did not show significantly increased resistin levels compared with CON-PBS mice (data not shown). Furthermore, ROSI treatment did not have an effect on serum resistin levels in ob/ob mice fed dietary CLA. The lack of effect of ROSI on serum resistin levels in CLA-fed mice further demonstrates that CLA-mediated insulin resistance is related more to the depletion of adiponectin. The increase in serum adiponectin in the CLA-fed mice was accompanied by significantly lower levels of fasting glucose, insulin, insulin sensitivity, serum TG, and NEFA, similar to those in CON-ROSI-treated mice. Cotreatment of CLA with ROSI for 2 weeks prevented lipodystrophy and the associated increases in serum levels of glucose and insulin that are usually associated with the supplementation of dietary CLA in mice (21–24). Additionally, ROSI treatment also prevented significant body weight loss, which is often reported with treatment of CLA in mice. This may be attributable to the maintenance of adipose mass, as ROSI, a PPAR agonist, often increases adipose mass (25, 26). These data suggest that when adipose mass and adiponectin levels are maintained, the lipodystrophic effects associated with dietary CLA supplementation are attenuated.
In this study, increased adiponectin levels were associated with higher levels of hepatic AOX mRNA in CON- and CLA-fed mice, but only the CLA-ROSI group had significantly lower hepatic FAS mRNA levels and hepatic TG. To our knowledge, the interactive effect of the combination of CLA and ROSI on hepatic TG is novel and suggests a complementary effect of these two agents for restoring normal lipid levels in the liver. Furthermore, there were significant increases in the levels of the lipid transporter CD36 and the lipid oxidative enzyme AOX in the muscle of the CLA-ROSI group. Administration of adiponectin has similar effects on CD36 and AOX mRNA in mice fed high-fat diets (10), suggesting that at least part of the effects of ROSI are mediated through adiponectin. Although it is unclear why the combination of CLA with ROSI has additional effects in liver and muscle, it may be attributable to increased adiponectin levels with ROSI administration. It is possible that in the presence of adequate adiponectin, CLA increases lipid utilization and decreases lipid accumulation in tissues, similar to observations from rat studies (33–35).
It has been shown previously that supplementation with dietary CLA significantly decreases adipokines and induces hyperinsulinemia in female C57BL/6J mice by 6 days (23). In this study, ROSI was administered for 2 weeks. In this short-term treatment, ROSI prevented CLA-induced insulin resistance by maintaining adequate adiponectin levels in ob/ob mice. Although we cannot speculate on the effects of ROSI if treatment had been prolonged in this model, these results are in accordance with a recent study conducted in C57BL/6 mice (M. A. Belury, unpublished data) that were administered the combination of CLA-ROSI for 6 weeks.
Because other adipokines secreted from the adipose tissue, such as TNF-, IL-6, and resistin, are known to modulate insulin sensitivity in addition to leptin and adiponectin, the overall adipokine status and not any particular adipokine alone may influence CLA's effects on insulin resistance and hepatic steatosis. It was recently shown that short-term t10c12-CLA administration induced adipose IL-6 and TNF- mRNA without affecting serum levels (36). In contrast to these findings, preliminary data from male ob/ob mice fed mixed isomers of CLA for 4 weeks did not show significantly higher mRNA levels of TNF- and IL-6 compared with CON mice (data not shown). Thus, whereas in this study, adiponectin alone seems to be more important than other adipokines in insulin resistance mediated by CLA, future studies using adiponectin-deficient mice are necessary to make conclusions regarding the relative importance of this adipokine. ROSI treatment significantly decreased adipose tissue TNF- mRNA levels in both CON- and CLA-fed mice compared with CON-PBS-treated mice. These data are in accordance with previous reports (37, 38). Because ROSI is a thiazolidinedione known to improve insulin sensitivity, possibly through multiple pathways, the increased synthesis of adiponectin is not the only possible explanation for our findings. In fact, ROSI may have additional effects in the adipose tissue and may decrease insulin resistance mediated by CLA by directly or indirectly modulating inflammatory cytokines such as TNF- and IL-6.
In conclusion, we show that removing CLA from the diet of mice reverses insulin resistance and restores adiponectin, establishing a link between depletion of this adipokine and the development of hepatic steatosis and insulin resistance by dietary CLA in mice. We further show that in the absence of significant changes in leptin levels (male C57BL/6 mice fed dietary CLA) or in mice lacking functional leptin (male ob/ob mice), adiponectin depletion alone results in worsened insulin sensitivity by CLA. In addition, restoration of adiponectin (by either removal of CLA from the diet or administration of ROSI) is sufficient for the reversal of these effects. Thus, in this study, we show that adiponectin is an important factor responsible for insulin resistance caused by CLA provided as mixed isomer oil at 1.5% of the diet. We based our dose (1.5% CLA equaling approximately 0.6% t10c12-CLA) on studies by others showing this dose to be effective for adipose suppression in mice. We observed that C57Bl/6 mice weighing an average 30 g, ate 2.0 g diet per day (and ob/ob mice weighing 45 g, consumed 5.0 g diet per day). If this were translated to a human of 55 kg, our dose of CLA could equal 44 g CLA (or 21.2 g of t10-CLA). With this estimate, it seems an unrealistic choice for humans to use and achieve such a rapid loss of body fat with foods at this time. However, there are other factors to consider between species (mice versus humans) including rate of metabolism of CLA isomers, rapidity of adipose tissue metabolism, and specific to this design, differences in adipose catabolism. Nevertheless, we consider these preclinical studies to be important for improving our understanding of mechanisms of anti-adipose effects of CLA.
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ACKNOWLEDGMENTS |
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Manuscript received August 31, 2006 and in revised form October 3, 2006 and in re-revised form October 5, 2006.
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REFERENCES |
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P < 0.05 versus the last time point on the diet containing CLA. B: Male ob/ob mice were fed either control (CON) or CLA-supplemented (CLA) diets and received either PBS or rosiglitazone (ROSI) for 2 weeks by intraperitoneal injection daily. Closed circles, CON-PBS (n = 8); open circles, CON-ROSI (n = 4); closed triangles, CLA-ROSI (n = 6). Data shown are means ± SEM.
