Effects of Postweaning Administration of Conjugated Linoleic Acid on

Article pubs.acs.org/JAFC

Effects of Postweaning Administration of Conjugated Linoleic Acid on Development of Obesity in Nescient Basic Helix−Loop−Helix 2 Knockout Mice Yoo Kim,† Daeyoung Kim,‡ Deborah J. Good,§ and Yeonhwa Park*,† †

Department of Food Science and ‡Department of Mathematics and Statistics, University of Massachusetts, Amherst, Massachusetts 01003, United States § Department of Human Nutrition, Foods and Exercise, Virginia Tech, Blacksburg, Virginia 24061, United States ABSTRACT: Conjugated linoleic acid (CLA) has been reported to prevent body weight gain and fat accumulation in part by improving physical activity in mice. However, the effects of postweaning administration of CLA on the development of obesity later in life have not yet been demonstrated. The current study investigated the role of postweaning CLA treatment on skeletal muscle energy metabolism in genetically induced inactive adult-onset obese model, nescient basic helix−loop−helix 2 knockout (N2KO) mice. Four-week-old male N2KO and wild type mice were fed either control or a CLA-containing diet (0.5%) for 4 weeks, and then CLA was withdrawn and control diet provided to all mice for the following 8 weeks. Postweaning CLA supplementation in wild type animals, but not N2KO mice, may activate AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-δ (PPARδ) as well as promote desensitization of phosphatase and tensin homologue (PTEN) and sensitization of protein kinase B (AKT) at threonine 308 in gastrocnemius skeletal muscle, improving voluntary activity and glucose homeostasis. We suggest that postweaning administration of CLA may in part stimulate the underlying molecular targets involved in muscle energy metabolism to reduce weight gain in normal animals, but not in the genetically induced inactive adultonset animal model. KEYWORDS: conjugated linoleic acid, childhood obesity, voluntary activity, glucose homeostasis, AMP-activated protein kinase

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INTRODUCTION Since 1980, the prevalence of childhood obesity has almost tripled.1,2 Along with overnutrition, lack of physical activity during the childhood period is one of the significant contributing factors to the development of childhood obesity. Similar to adult obesity, childhood obesity is known to be associated with the increased risk of chronic metabolism diseases.3,4 Even though early childhood has been considered to be critical for health later in life, most studies in humans and animals have mainly addressed the effects of dietary and exercise regimens rather than focusing on early-onset interventions during critical developmental periods.5 Conjugated linoleic acid (CLA) is a term describing a mixture of 18-carbon unsaturated fatty acid isomers with conjugated double bonds. The name, CLA, was first used when its anticancer activities were reported in 1987.6 Since then, other biological activities of CLA have been reported, including its effects on prevention of atherosclerosis development, modulation of immune responses, promotion of young animal growth, and modulation of body composition, particularly reduction of body fat and improvement of lean and bone masses.7 It has been suggested that CLA reduces body fat by multiple biochemical mechanisms, including increased energy expenditure, modulated adipogenesis, and/or enhanced fatty acid β-oxidation.7,8 The beneficial effects of CLA supplementation during the obesity onset period in nescient basic helix−loop−helix 2 (Nhlh2) knockout mice (N2KO) have been previously reported.9 Targeted deletion of the Nhlh2 transcription factor expressed in the hypothalamus precedes adult-onset obesity in © XXXX American Chemical Society

mice, resulting from a disruption of the hypothalamic−pituitary axis. While N2KO mice have normal levels of food intake, their spontaneous voluntary movement is decreased by more than 50% in both genders.10 It was suggested that CLA supplementation in young N2KO animals prevented excessive weight gain by improving voluntary activity.9 However, it is not clear if supplementation of CLA during the preobesity stage would benefit later in life, as was reported in nonobese animals.11 Thus, the purpose of this study was to determine the effects of CLA supplementation during the early growth period in N2KO animals on the development of obesity and symptoms of type 2 diabetes later in life.

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MATERIALS AND METHODS

Materials. CLA was provided by Natural Lipids Ltd. AS (Hovdebygda, Norway). The CLA used consisted of 80.7% CLA (37.8% cis-9,trans-11, 37.6% trans-10,cis-12, and 5.3% other CLA isomers) and 13.7% oleic acid, 3.2% stearic acid, 0.4% palmitic acid, 0.2% linoleic acid, and 1.8% unknown. Semipurified powdered diet (TD07518) was obtained from Harlan Laboratories (Madison, WI). Serum triacylglyceride (TG), glucose, and total cholesterol assay kits were purchased from Genzyme Diagnostics (Charlottetown, PE, Canada). A DC protein assay kit (Bio-Rad, Hercules, CA) was used for protein quantitation. Rabbit antibodies for phosphorylated AMPactivated protein kinase α (p-AMPKα), AMPKα, phosphorylated

Received: February 12, 2015 Revised: May 13, 2015 Accepted: May 15, 2015

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DOI: 10.1021/acs.jafc.5b00840 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Composition of Experimental Diet ingredient

amount (g/kg)

casein, “vitamin-free” tested L-cystine sucrose cornstarch maltodextrin cellulose soybean oil CLA or soybean oil mineral mix, AIN-93M-MX (TD 94049) vitamin mix, AIN-93-VX (TD 94047) choline bitartrate tert-butylhydroquinone total

169.1 2.2 100 288.5 132 50 195 5 42.8 12.4 3 0.04 1000

phosphatase and tensin homologue (p-PTEN), phosphorylated phosphoinositide-dependent kinase (p-PDK), phosphorylated protein kinase B at threonine 308 (p-Akt Thr308) and serine 473 (p-Akt Ser473), Akt, and superoxide dismutase 2 (SOD2) were obtained from Cell Signaling (Berberly, MA). Rabbit antibodies for peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor-δ (PPARδ), nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (Tfam), cytochrome c (Cyt C), cytochrome c oxidase (COX IV), insulin receptor substrate 1 (IRS-1), glucose transporter type 4 (GLUT4), and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidase-conjugated goat antirabbit IgG antibody was obtained from Boston Bioproducts Inc. (Ashland, MA). Other chemicals used were purchased from either Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). Animals and Diet. All animal work was conducted in compliance with the Institutional Animal Care and Use Committee at the University of Massachusetts. As described in a previous report,10 129 Sv/J male offspring mice were obtained through inbreeding between heterozygous male and female breeders. Litters were housed together with dams until weaning. At 3 weeks old, all male mice were biopsied (0.5 cm tail) for genotyping. Ten wild type and eight N2KO mice were selected for this study and then placed in individual wire-bottomed cages on a 12 h:12 h light:dark cycle in a temperature- and humidity-controlled room. During a 1 week acclimatization period, all mice were fed a semipurified powdered control diet (20 w/w % fat) to induce obesity in rodents9,12,13 and were subjected to a baseline test for voluntary movement and glucose tolerance in serum. On the basis of body weight, all animals in different genotype groups (wild type and N2KO) were divided into two diet groups and fed either control or 0.5% CLA containing diet for 4 weeks. Then, all animals received the control diet for 8 weeks. Previously it was determined that feeding CLA for 4 weeks in mice was enough to result in changes in body composition as well as changes in activity levels.14,15 Moreover, it was reported that most CLA remaining in the tissues (liver, adipose tissue, and muscle) returned to levels comparable to that of control animals at 8 weeks postwithdrawal.14 Thus, we selected 8 weeks post CLA supplementation in the current study. The dose of CLA was determined on the basis of previous reports that supplementing 0.5% CLA for 2−4 weeks in rats led to a serum level range of 23−120 μM and serum levels of CLA after supplementation of 0.8−3.2 g per day for 2 months in humans ranged from 23 to 200 μM.16,17 The compositions of diets are shown in Table 1. Diet and water were available ad libitum throughout the experiment and were provided freshly twice per week. Body weight and food intake were monitored weekly. At the end of the study, mice were fasted for 6 h and sacrificed by CO2 asphyxiation. Blood was immediately collected by cardiac puncture, and the internal organs (liver, heart, kidney, spleen, and white adipose tissues including epididymal, retroperitoneal, and mesenteric fat pads) were weighed at sacrifice.

Figure 1. Effect of postweaning administration of conjugated linoleic acid (CLA) on body weight (A) and food intake (B) in wild type and nescient helix−loop−helix knockout (N2KO) mice. Mice were treated with either control or 0.5% CLA diets until 4 weeks and then switched diets from CLA supplementation to control for the following 8 weeks. Values represent means ± SE (n = 3−5). n.s.: not significant. Means with different letters at the same time points are significantly different (P < 0.05). P values in the table are from two-way repeated measures ANOVA with the slice option in the Least Square Means statement to compare the group differences at each week. Voluntary Movement Measurement (Nonexercise Physical Activity Test). Nonexercise physical activity (voluntary movement) was recorded using LoliTrack Quatro Video Tracking Software Version 1.0 (Loligo Systems, Tjele, Denmark) with a high-resolution infrared camera under dark conditions. This tracking system simultaneously monitored four mice placed in individual cages (30 × 46 × 40 cm) during the dark phase (5:00 pm−5:00 am) once a month with free access to diet and water (provided as HydroGel, Clear H2O, Portland, ME). Movement data for 8 h (7:00 pm−3:00 am) excluding the first 2 h (5:00 pm−7:00 pm) during the early phase for adapting to environmental change and 2 h (3:00 am−5:00 am) during the late phase due to lack of movement were analyzed. Serum Parameters. The levels of glucose, total cholesterol, and TG in serum separated by centrifugation at 3000g for 20 min at 4 °C were B


Article

Journal of Agricultural and Food Chemistry Table 2. Organ and Adipose Tissue Weightsa body weight (%) wild type control

N2KO CLA

epididymal fat retroperitoneal fat mesenteric fat total

6.89 ± 0.37ab 1.47 ± 0.18 4.12 ± 0.29 12.5 ± 0.30ab

4.56 ± 1.12b 1.05 ± 0.18 3.82 ± 0.42 9.43 ± 1.68b

liver heart kidney spleen

3.47 ± 0.24bc 0.37 ± 0.02 1.13 ± 0.08ab 0.26 ± 0.03

4.37 ± 0.22a 0.41 ± 0.02 1.38 ± 0.08a 0.23 ± 0.01

control Adipose Tissue 9.39 ± 0.92a 1.57 ± 0.16 4.74 ± 0.86 15.7 ± 1.61a Organ 2.80 ± 0.11c 0.34 ± 0.01 0.89 ± 0.05b 0.21 ± 0.03

effect (P value) CLA

genotype

CLA

interaction

7.89 ± 0.69ab 1.39 ± 0.07 5.25 ± 0.23 14.5 ± 0.51a

0.007 n.s. 0.0448 0.007

(0.0545) n.s. n.s. n.s.

n.s. n.s. n.s. n.s.

4.21 ± 0.22ab 0.46 ± 0.04 1.16 ± 0.11ab 0.24 ± 0.04

n.s. n.s. 0.0261 n.s.

0.0002 0.0234 0.0118 n.s.

n.s. n.s. n.s. n.s.

a Values represent means ± SE (n = 3−5). Means with different superscripts within the same row are significantly different (P < 0.05). P values in the table are from two-way ANOVA. Abbreviations: N2KO, nescient helix−loop−helix knockout mice; CLA, conjugated linoleic acid; n.s., not significant.

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determined using commercial kits as specified by the manufacturer’s instructions. Glucose Tolerance. As described previously,18 intraperitoneal glucose tolerance tests (IGTT) were conducted every 4 weeks. All mice underwent 6 h fasting and then were subjected to baseline measurement of blood glucose levels at 0 min. Blood was collected from the tail vein using a blood glucose meter, Advocate Redi-Code+ (Advocate Meters Inc., Dorado, Puerto Rico). Subsequently, 30% glucose solution (2.0 g of glucose/kg of body weight) was administered through intraperitoneal injection and then blood glucose levels were monitored at 15, 30, 60, 90, and 120 min. Collected data were calculated as the areas under the curve (AUC) with SigmaPlot 11.0 (Systat Software, Inc., San Jose, CA). Western Blot Analysis. The frozen gastrocnemius skeletal muscle tissue was ground, and 50 mg aliquots were prepared and stored at −80 °C until analysis. Each sample was homogenized in premixed lysis radioimmune precipitation assay (RIPA; 50 mM Tris-HCl, 10 mM NaCl, 5 mM MgCl2, and 0.5% NP-40, pH 8.0) buffer (Boston Bioproducts Inc., Ashland, MA), protease inhibitor cocktail (SigmaAldrich, St. Louis, MO), and phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL) and then lysed on ice for 2 h and centrifuged at 12000g for 20 min at 4 °C. The concentration of protein from tissue lysate was determined using the DC protein assay kit. The normalized lysates (37.5 μg/mL of protein) were separated by an 8−15% (w/v) sodium dodecyl sulfate−polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). After incubation with the appropriate primary rabbit antibodies, membranes were treated with horseradish peroxidase-conjugated goat antirabbit IgG antibody for 1 h at room temperature. Detection was performed using Enhanced Chemiluminescence solution (Bio-Rad, Hercules, CA) with an Image Station 4000MM instrument (Carestream Health, New Heaven, CT). The band density was normalized by β-actin intensity as an internal control. The results were quantified using ImageJ software (U.S. National Institutes of Health). Statistical Analyses. All data analyses were performed by two-way analysis of variance (ANOVA) followed by the PROC MIXED procedure with Least Squares (LS) Means statement of the SAS software (Version 9.3, SAS Institute Inc., Cary, NC). For data on body weight, food intake, voluntary movement, and glucose tolerance test, two-way repeated measures ANOVA and the slice option in the LS means statement to examine the group differences at each time point with PROC MIXED were used. For internal organ, adipose tissue weight, and serum parameter data, two-way ANOVA with PROC MIXED was conducted. The experimental groups were compared by the multiple comparison basic Tukey−Kramer method in SAS. The Dunnett test in SAS was used for the relative protein expression level. Data are shown as the means ± SE; P values less than 0.05 are reported as statistically significant.

RESULTS Body Weights and Food Intake. There was significant genotype effect on weight gain; N2KO mice had significantly greater body weight gain over wild type mice (P = 0.0001, Figure 1A). CLA supplementation during the first 4 weeks resulted in no significant different effects on weight gain overall, although the wild type CLA-fed group had the least body weight gain among all treatment groups during the whole experimental period (Figure 1A). Overall N2KO mice consumed more food than wild type animals during the experimental period (P = 0.0252). No significant differences in food intake were observed between controls and CLA treatment during the preobese period, although the N2KO CLA group temporarily consumed more food right after withdrawal of CLA supplementation for 2 weeks (N2KO control; 50.5 ± 5.6 g vs N2KO CLA; 63.0 ± 2.3 g, the mean ± SE). Although three-way interaction among genotype, CLA, and time was observed, there was no significant two-way interaction in overall food intake (Figure 1B). Tissue and Organ Weights. Total adipose tissue weights, including epididymal, mesenteric, and retroperitoneal adipose tissue, and internal organ weights (liver, heart, kidney, and spleen) are shown in Table 2. Epididymal, mesenteric, and total adipose tissue weights were significantly increased in N2KO animals in comparison to wild types (all P values were less than 0.05), but no significant effects of genotypes was observed for retroperitoneal fat weights. No significant effects of CLA supplementation for the first 4 weeks were observed for adipose tissue weights. No differences of genotype effects were observed in the liver, heart, and spleen weights, while significant genotype effects were observed for the kidney weights (P = 0.0261). CLA-fed animals had significantly increased liver, heart, and kidney weights in comparison to control animals. No significant interaction between genotype and CLA was observed in any tissue or organ weight. Voluntary Movement (Nonexercise Physical Activity). To evaluate the effects of postweaning administration of CLA on physical activity, voluntary movement (nonexercise physical activity) was measured every 4 weeks. Travel distance as total (19:00−03:00) and two time periods, early (19:00−23:00) and late (23:00−03:00), are shown in Figure 2A−C. The changes in total travel distance over time are shown in Figure 2D. C


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Journal of Agricultural and Food Chemistry

Figure 2. Effect of postweaning conjugated linoleic acid (CLA) treatment on voluntary movement (nonexercise physical activity) in wild type and nescient helix−loop−helix knockout (N2KO) mice. Mice were fed control or 0.5% CLA diets for 4 weeks (from 4 to 8 weeks old) and then changed diets from CLA-containing to control for the next 8 weeks (from 9 to 17 weeks old). Voluntary movement was recorded during 17:00−05:00 every 4 weeks for 12 weeks (A−C). The data show different time points (total, 19:00−23:00 and 23:00−03:00). Total stands for movement measure during 19:00−03:00, divided into 4 h segments (19:00−23:00 and 23:00−03:00) representing the early or late phase of the dark cycle, respectively. (D) Trend for total travel distances (19:00−03:00). Values represent means ± SE (n = 3−5). n.s.: not significant. Means with different letters at the same time points are significantly different (P < 0.05).P values in the tables are from two-way repeated measures ANOVA with the Least Square Means statement to compare the group differences at each time point.

Consistent with the previous reports,9,19 N2KO mice showed a significantly decreasing tendency of activity in comparison to wild type animals (P = 0.0492). It was evident that all experimental groups except the wild type CLA group showed declining activity over time (Figure 2D), with overall CLA effects being significant (P = 0.0444), particularly during the late period (P = 0.0234). No difference in activity between N2KO groups was observed, while CLA-fed wild types, but not N2KO animals, maintained activity levels throughout the experiment. This suggests that supplementation of CLA during the preobese state prevented reduction of voluntary movement over time in wild types but not in N2KO animals. Glucose Tolerance Test. To determine the effects of postweaning CLA treatment on glucose homeostasis, glucose tolerance tests were conducted every 4 weeks (Figure 3A−D). N2KO mice showed glucose intolerance in comparison to wild type animals (P = 0.0061). In addition, there was a significant

overall time effect (P = 0.0148), suggesting these animals develop glucose intolerance with aging along with the high-fat diet as used in this study. No significant CLA effect was observed; however, the CLA-treated wild type animals showed improved glucose tolerance at week 12 in comparison to wild-type controls, even after CLA withdrawal for 8 weeks (Figure 3E, P < 0.0001 for the interaction effect between CLA and time). This suggests that postweaning CLA administration may improve glucose tolerance in wild types but not in N2KOs. Serum Parameters. The serum levels of TG, glucose, and total cholesterol were analyzed and are shown in Table 3. There were no genotype or CLA effects on these markers in this study, except glucose levels for CLA (P = 0.036). No interactions between genotype and CLA were observed in any of these parameters. Effects on Muscle Energy Metabolism. To assess whether postweaning administration of CLA contributes to improved D


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Figure 3. Effect of time-specific postweaning conjugated linoleic acid (CLA) treatment on glucose levels (A−D), the area under the curve (AUC) change (E), and the overall trend of AUC change (F) in wild type and nescient helix−loop−helix knockout (N2KO) mice. Glucose tolerance was measured every 4 weeks during the whole experimental period. Blood was collected from the tail vein, and glucose levels were determined at 0 min for a baseline. Then glucose solution was administrated by intraperitoneal injection and measured at five different time points (15, 30, 60, 90, and 120 min). The data (E) are changed values of AUC and main effect analysis of AUC change dependent on different time points in the table. Values represent means ± SE (n = 3−5). n.s.: not significant. Means with different letters at the same time points are significantly different (P < 0.05). P values in the table are from two-way repeated measures ANOVA with the slice option in the Least Square Means statement to compare the group differences at each time point.

interaction between them was observed (P = 0.0471) (Figure 4B). For phosphorylated AMPKα (the active form of AMPKα), N2KO animals had significantly lower levels of p-AMPKα in comparison to wild type animals (P = 0.0399). The mice supplemented CLA for the first 4 weeks showed significantly higher expression of phosphorylation of AMPKα in comparison

skeletal muscle energy metabolism, we investigated the AMPK signaling pathway, a master sensor to maintain energy homeostasis (Figure 4). Total AMPKα was measured, as it is known that two subunits (α1 and α2) are regulated differentially and overall AMPKα activity is more important.20 There were no overall effects of any genotype or CLA effects on AMPKα expression, but E


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Journal of Agricultural and Food Chemistry Table 3. Serum Parametersa concentration (mg/dL) wild type triglyceride glucose total cholesterol

N2KO

effect (P value)

control

CLA

control

CLA

genotype

CLA

interaction

120.3 ± 18.3 189.8 ± 11.2 267.4 ± 35.0

99.2 ± 10.6 169.8 ± 15.9 328.5 ± 25.4

112.9 ± 2.0 286.8 ± 55.3 261.9 ± 18.9

128.4 ± 20.8 168.5 ± 35.3 232.1 ± 18.2

n.s. n.s. n.s.

n.s. 0.036 n.s.

n.s. n.s. n.s.

Values represent means ± SE (n = 3−5). P values in the table are from two-way ANOVA. Abbreviations: N2KO, nescient helix−loop−helix knockout mice; CLA, conjugated linoleic acid; n.s., not significant.

a

Figure 4. Effect of postweaning treatment of conjugated linoleic acid (CLA) on protein expression of total and phosphorylated AMP-activated protein kinase (AMPK), SIRT1, and PGC-1α from the gastrocnemius muscle. Pictures show representative results (A). The graph expresses the relative expression levels of total AMPKα (B), phosphorylated AMPKα at Threonine 172 (p-AMPKα; C), a ratio of phosphorylated AMPKα to total AMPK expression (D), sirtuin1 (SIRT1; E) and peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α; F). All data were normalized by β-actin. Values represent means ± SE (n = 3−5). # and $ denote significant overall genotype and CLA effect, respectively (P < 0.05). * denotes a significant difference in comparison to the wild type or N2KO control group (P < 0.05). N2KO: nescient helix−loop−helix knockout. F


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Figure 5. Effect of postweaning treatment of conjugated linoleic acid (CLA) on mitochondrial biogenesis-related factors from the gastrocnemius muscle. Pictures represent results of immunoblotting (A). The relative expression levels of peroxisome proliferator-activated receptor δ (PPARδ; B), nuclear respiratory factor 1 (NRF-1; C), and mitochondrial transcription factor A (Tfam; D) were determined and normalized using β-actin as the internal control. Values represent means ± SE (n = 3−5). ## denotes a significant overall genotype effect (P < 0.01). * denotes a significant difference in comparison to the wild type or N2KO control group (*, P < 0.05; **, P < 0.01). N2KO: nescient helix−loop−helix knockout.

to the control groups (Figure 4C, P = 0.0264); in particular, the wild type CLA group in comparison to the respective control (Figure 4C). In addition, an increased ratio of phosphorylated AMPKα to AMPKα by CLA was observed (Figure 4D, P = 0.0471). Along with AMPKα, SIRT1 (a NAD-dependent deacetylase) is a key activator of PGC-1α (one major downstream marker for AMPKα). There were no significant effects of genotype and CLA on SIRT1 observed, whereas the wild type CLA group showed increased SIRT1 expression level in comparison to the N2KO control (Figure 4E). The expression of PGC-1α, a master regulator of mitochondrial biogenesis, was also not influenced by genotype or CLA (Figure 4F). However, it was evident that CLA treatment had a greater effect on PGC-1α in comparison to the control diet group in wild type animals. Effects on Mitochondrial Biogenesis-Related Factors. We further determined the effect of postweaning CLA supplementation on mitochondrial biogenesis in the gastrocnemius muscle. Mitochondria are important organelles in determining oxidative capacity and fatigue resistance, resulting in prolonged endurance capacity.21−23 For mitochondrial biogenesis several nuclear encoded precursor proteins are involved in this metabolic process;24−26 among them, we selected PPARδ, NRF-1, and Tfam. PPARδ is a marker involved in mitochondrial biogenesis as well as muscle type transformation from glycolytic to oxidative fiber.27 No significant overall effects of genotypes or CLA were

observed on PPARδ; however, wild type CLA-fed animals showed significantly greater expression levels of PPARδ in comparison to both control diet groups (Figure 5B). No significant difference in NRF-1, a regulator to mediate nuclearencoding mitochondrial proteins, was observed in any experimental group (Figure 5C). The expression of Tfam, which is the key regulator for mitochondrial DNA replication and transcription, was significantly downregulated in N2KO mice in comparison to wild type mice (P = 0.0062). The CLA supplemented group in wild type animals showed significantly greater Tfam expression over the control group in N2KO mice (Figure 5D). To estimate overall mitochondrial relative content, we used an indirect method to measure mitochondrial proteins, including cytochrome c and COX IV, located in the inner membrane of mitochondria.28,29 Additionally, we tested another mitochondrial protein, superoxide dismutase 2 (SOD2) in the inner mitochondrial matrix, to assess mitochondrial function.30 The expression levels of cytochrome c were significantly decreased in N2KO mice in comparison to wild type animals (P = 0.0496), but no CLA effects were observed (Figure 6B). No genotype or CLA effects were observed in COX IV (Figure 6C). Overall SOD2 protein levels were significantly reduced in N2KO mice (P = 0.0224), while no effects of CLA were observed (Figure 6D). Effects on Markers of Glucose Metabolism. Along with assessment of postweaning administration of CLA on G


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Figure 6. Effect of postweaning treatment of conjugated linoleic acid (CLA) on mitochondrial content and function from the gastrocnemius muscle. Pictures are representative results (A). The relative expression levels of cytochrome c (Cyt C, B), cytochrome c oxidase (COX IV, C), and superoxide dismutase 2 (SOD2, D) were detected. β-Actin was used as the loading control. Data are means ± SE (n = 3−5). # denotes a significant overall genotype effect (P < 0.05). N2KO: nescient helix−loop−helix knockout. Means with different letters are significantly different (P < 0.05).

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DISCUSSION It was previously reported that supplementation of CLA prevents weight gain and body fat accumulation both during the growth period and in aged N2KO mice.9,31,32 In particular, CLA increased voluntary activity when supplemented during the growth period.9,31 Results from the current study suggest that supplementation of CLA in the preobese state may reduce weight gain in normal animals but not in the genetically induced inactive adult-onset animal model, although the stimulation of voluntary activity and improved glucose tolerance remain even after CLA withdrawal in this model. Previously CLA was reported to be linked with reduced food intake in a number of animal models, although it was not consistent.14,33,34 Moreover, it was determined that the reduction of food intake by CLA was independent of its effects on body fat reduction.14,35 Others suggested that CLA reduces food intake by decreasing neuropeptides Y (NPY) and agouti-related protein (AgRP), both of which are known to be related to increased appetite36 and inducing the storage of glycogen in muscle and liver, which may subsequently serve as satiety signals.37,38 In the current study, we did not observe any significant differences in food intake during CLA supplementation periods in both genotypes. However, when CLA was withdrawn, apparent compensation of food intake occurred in N2KO animals fed CLA but not in wild type animals, coinciding with greater weight gain during this period (weeks 4−6 in Figure 1A). It is not clear how CLA influenced food intake in these animals; however, these

mitochondrial biogenesis-related factors, we determined the effects of CLA supplementation during the first 4 weeks on markers of glucose metabolism in the gastrocnemius muscle. The expression levels of IRS-1, a member of the IRS family regulating insulin signaling, were significantly decreased in N2KO animals over wild types, while no CLA effects were observed (Figure 7B, overall genotype effect, P = 0.0335). A downstream biomarker of IRS (PTEN, attributed to negatively regulate the AKT signaling pathway) significantly reduced phosphorylation (inactive form of PTEN) in N2KO mice in comparison to wild type animals (P = 0.0094). No significant effect of postweaning administration of CLA was observed on PTEN expression in N2KO mice. However, CLA-treated wild type animals significantly upregulated phosphorylated PTEN in comparison to the respective control, while no differences were observed in N2KO animals (Figure 7C). No difference in protein expression of phosphorylated PDK, a downstream marker of PTEN, was observed for either genotype or CLA (Figure 7D). There were no significant effects of genotypes and CLA on total AKT expressions and phosphorylated AKT (Serine 473) and GLUT4, one of the insulinregulated glucose transporters (Figure 7E−H). However, we observed marginally increased responses from wild type animals fed CLA on phosphorylated AKT (threonine 308) in comparison to the respective controls (P = 0.0547 vs wild-type control and P = 0.0528 vs N2KO control, respectively). H


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Figure 7. Effect of postweaning treatment of conjugated linoleic acid (CLA) on glucose metabolism from the gastrocnemius muscle. Pictures represent results of Western blotting (A). The graph represents the relative expression levels of insulin receptor substrate 1 (IRS-1; B), phosphorylated phosphatase and tensin homologue (p-PTEN; C), phosphorylated phosphoinositide-dependent kinase (p-PDK; D), protein kinase B (AKT; E), phosphorylated AKT at threonine 308 (p-AKT Thr308; F) and serine 473 (p-AKT Ser473; G), and glucose transporter type 4 (GLUT4; H). All data were normalized by β-actin. Values represent means ± SE (n = 3−5). # denotes a significant overall genotype effect (#, P < 0.05; ##, P < 0.01). * denotes a significant difference in comparison to the wild type or N2KO control group (*, P < 0.05). N2KO: nescient helix−loop−helix knockout. P values are from two-way ANOVA. I


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the liver and adipose tissues, increased hepatic lipogenesis, and decreased fat accumulation in adipose tissues.8,60−62 It is unlikely that an enlarged liver caused by CLA is due to increased glycogen content, as it was previously shown that CLA had no effects on enzyme activities involved in gluconeogenesis.57 In addition, previous studies reported no pathologically significant observations after 18 months of CLA supplementation or no changes in hepatic gene expressions in lipogenesis from N2KO animals.9,32,63 Although we provided CLA only during the preobese state and then withdrew CLA, the significant effect of CLA on liver weight still remains. This is consistent with the previous study that withdrawal of CLA did not completely reverse liver enlargement at the end of a 4 week recovery period.64 Our study might not have enough adaptive time to complete this reversal of CLA’s effect. Thus, additional investigation of the relationship between short-term CLA supplementation and changes in hepatic genes should be considered to determine if the liver enlargement caused by CLA is completely reversible or not. Clinical trials with CLA reported no changes or increased markers for liver function, and all were within the normal ranges. In addition, it was reported that a high dose of CLA (14.6 g of cis-9,trans-11 and 4.7 g of trans-10,cis-12 CLA per day) for 3 weeks had no changes on markers of liver function in healthy subjects. Thus, it is possible that rodents are more sensitive to CLA’s effect in comparison to humans. In conclusion, CLA supplementation during the preobese state may attenuate reduction of voluntary activity and glucose intolerance after CLA withdrawal in this mouse model. However, it is not sufficient per se to maintain health benefits such as prevention of weight gain and fat accumulation until later life in genetically obese mice. Wild type animals fed CLA may activate AMPKα and PPARδ as well as promote desensitization of PTEN and sensitization of AKT at threonine 308 in gastrocnemius skeletal muscle, improving voluntary activity and glucose homeostasis. We suggest that postweaning administration of CLA may partially stimulate the underlying molecular targets involved in muscle energy metabolism in normal animals but not in the genetically induced inactive adult-onset animal model. The present study should provide a scientific foundation for future trials of targeted application of postweaning CLA intervention in modulating long-term obesity. If further research on genetically predisposed obese mice discovered an appropriate intervention or combination with an additional regime such as exercise, it may be possible to more effectively use it to prevent childhood obesity.

results suggest that CLA may influence food intake differently in obese animals, which has been previously reported in other animal models.14,33,34,39 We observed that N2KO mice had significantly reduced physical activity, consistent with previous reports for N2KO animals.9,10,40 Overall, postweaning CLA supplementation significantly prevented reduction of voluntary activity levels in comparison to controls, particularly in wild type animals. Intriguingly, our results showed that the effects of CLA on activity during the late phase (23:00−03:00) might remain even after termination of CLA treatment up to 8 weeks, whereas during the early phase (19:00−23:00) there was no significant CLA effect in all experimental groups. Among various molecular mechanisms to affect increased or maintained physical activity levels, the AMPK signaling pathway has drawn significant attention since the 1990s.41 An upregulated AMPK signaling pathway is involved in skeletal muscle energy metabolism as well as mitochondrial biogenesis, resulting in enhanced endurance capacity and attenuated exercise intolerance.42−45 Previously, our group demonstrated the effect of CLA on stimulation of mitochondrial biogenesis through an AMPK signaling pathway.46 In the same context, our data supported that postweaning administration of CLA might stimulate activation of AMPKα and related factors in wild type but not in N2KO animals. Wild type animals fed CLA for the first 4 weeks showed improved glucose tolerance over time even after no benefits on weight were observed in comparison to the respective control, suggesting that CLA may have lasting effects on glucose metabolism later in life. On the basis of this observation, we assessed the effects of CLA treatment during the growth period on glucose metabolism through the insulin signaling pathway. Among several measured markers involved in insulin signaling cascades, phosphorylated PTEN and phosphorylated AKT at threonine 308 partially provided evidence regarding how to moderate glucose tolerance in the current study. PTEN and AKT are important effectors of insulin responses mediating glucose uptake and glycogen synthesis in skeletal muscle.47−50 Our data are consistent with early studies that CLA-specific isomers mediate AKT sensitization.51,52 The current results suggest potential benefits of postweaning CLA administration to reduce health risk, as suggested previously.19,31 This is important, since one potential health concern associated with CLA supplementation is glucose intolerance. We observed a nonsignificant increase in glucose intolerance following CLA supplementation in wild type mice (Figure 3B, week 4); however, when CLA was withdrawn, glucose tolerance improved in the treated groups (week 12 in Figure 3D). As suggested before, this may be due in part to a significant shift of energy metabolism by CLA, such as increased fatty acid β-oxidation associated with reduced glucose utilization.53,54 However, further studies such as HOMA-IR to better understand glucose and insulin metabolism are needed to determine how postweaning administration of CLA changes energy sources. As seen in previous studies,15,55,56 we observed a significant increase in liver weight, as fatty liver, after CLA supplementation. It is unlikely that CLA is directly accumulated in the liver, as levels of CLA in the liver and muscle reached a maximum 1 week for the liver and 2 weeks for muscle after supplementation in mice.57 Previously it was reported that CLA-fed animals accumulate TG in the liver.58,59 This is believed to be associated with CLA’s dramatic effects on lipid metabolism, particularly in

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AUTHOR INFORMATION

Corresponding Author

*Y.P.: tel, (413) 545-1018; fax, (413) 545-1262; e-mail, ypark@ foodsci.umass.edu. Funding

This material is based upon work supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, the Massachusetts Agricultural Experimental Station, and the Department of Food Science under Project No. MAS00998. Y.K. was supported in part by the Charm Sciences scholarship from the Department of Food Science, University of Massachusetts, Amherst. Notes

The authors declare no competing financial interest. J


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ACKNOWLEDGMENTS The authors thank Ms. Jayne M. Storkson for help in preparing this paper and Mr. Hyungtaek Cho and Ms. Sunnye Bang for assistance with the experiments. Y.P. is one of the inventors of CLA use patents that are assigned to the Wisconsin Alumni Research Foundation.

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ABBREVIATIONS USED Akt, protein kinase B; AMPK, AMP-activated protein kinase; AUC, areas under the curve; CLA, conjugated linoleic acid; COX IV, cytochrome c oxidase subunit IV; Cyt C, cytochrome c; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLUT4, glucose transporter type 4; IRS-1, insulin receptor substrate 1; Nhlh2, nescient basic helix−loop−helix 2; NRF, nuclear respiratory factor; N2KO, nescient basic helix−loop−helix 2 knockout; PDK, phosphorylated phosphoinositide-dependent kinase; PGC-1α, peroxisome proliferator-activated receptor γ coactivator 1α; PPARδ, peroxisome proliferator-activated receptor δ; PTEN, phosphatase and tensin homologue; SIRT1, sirtuin 1; SOD2, superoxide dismutase 2; Tfam, mitochondrial transcription factor A; TG, triacylglyceride

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Effects of Postweaning Administration of Conjugated Linoleic Acid on

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