- Email: [email protected]
Gene expression related to lipid and glucose metabolism in white adipose tissue
ORCP-449; No. of Pages 9
ARTICLE IN PRESS
Obesity Research & Clinical Practice (2015) xxx, xxx—xxx
ORIGINAL ARTICLE
Gene expression related to lipid and glucose metabolism in white adipose tissue Yoshito Kadota ∗, Takashige Kawakami, Satoshi Takasaki 1, Masao Sato, Shinya Suzuki Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima 770-8514, Japan Received 25 July 2014 ; received in revised form 13 April 2015; accepted 23 April 2015
KEYWORDS Gene expression; Obesity; White adipose tissue; Glucose transporter; Adipokine
∗ 1
Summary Problem: A number of endogenous and external factors influence the development of obesity. However, the factors responsible for these differences in obesity pathogenesis between males and females are largely unknown. Methods: We investigated the expression of 35 genes related to lipid and glucose metabolism and to receptors for insulin signaling in white adipose tissue (WAT) of 8-week-old 129/Sv mice and mice fed standard diet (STD) or high fat diet (HFD) for 35 weeks in males and females. Results: At 8 weeks, the expression levels of two genes for fatty acid synthesis, Acaca and Fasn, were higher in females than in males. Female mice fed a STD for 35 weeks also had higher expression levels of an additional four genes related to glucose transporters (Slc2a1 and Slc2a4) and adipokines (Adipoq and Nampt). The expression levels of these six genes were also higher in females than in males fed a HFD for 35 weeks. At 43 weeks old, the female-to-male expression ratio of these six genes was similar for the STD and HFD groups. Furthermore, glucose tolerance testing showed that the half-life for the elimination of elevated blood glucose was shorter in females than males, although blood glucose parameters were generally similar between females and males.
Corresponding author. Tel.: +81 88 602 8457; fax: +81 88 655 3051. E-mail address: [email protected] (Y. Kadota). Present address: Department of Pharmacy, Ohira Medical Care Hospital, Kitakyusyu, Fukuoka 807-0083, Japan.
http://dx.doi.org/10.1016/j.orcp.2015.04.009 1871-403X/© 2015 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
ORCP-449; No. of Pages 9
ARTICLE IN PRESS
2
Y. Kadota et al. Conclusions: These findings suggest that sex and aging may cause diet-independent differences in gene expression levels in female and male mice, and that higher expression of these genes in females could contribute to higher metabolic activity and resistance to obesity compared with males. © 2015 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
Introduction
Materials and methods
Obesity is characterized by an increase in adipose tissue mass and excess lipid deposition, and is associated with enlarged adipocytes. In developed countries, it is one of the most notable causes of accumulative lipid-related diseases such as type 2 diabetes, cardiovascular disease, and cancer [1]. There is a variety of genes associated with the development of obesity [2], and susceptibility to obesity is thought to be dependent on certain genetic factors. For example, leptin-receptor mutant rats [3], as well as interleukin-6-deficient [4] and interleukin-18-deficient mice [5], exhibit obesity. These gene deficiencies induce obesity even when their rodents are fed a standard diet (STD). A number of factors influence the development of obesity. Endogenous and external factors such as various stressors, aging, and low physical activity contribute to obesity and may change patterns of gene expression. Some changes are unlikely to be able to induce obesity independently, but rather promote diet-induced obesity. In addition, the phenotypes of obesity in men and women are characterized by certain differences: men have more visceral adipose tissue, whereas women have more peripheral and subcutaneous adipose tissue [6]. However, the factors responsible for these differences in obesity pathogenesis between males and females are largely unknown. Since there are at least 400 genes associated with obesity [2], it is necessary to narrow the number of genes on which to focus. We selected 35 genes that are particularly important for lipid and glucose metabolism. First, we studied the expression of these genes in young male and female mice. Second, since aging may contribute to excess accumulation of lipids, we compared the expression of these genes in older males and females fed either a STD or a high fat diet (HFD) for 35 weeks. Third, since sex differences may be another important factor for the development of obesity, we compared the expression of these genes and glucose metabolic activity between males and females.
Animals 129/SvCPJ mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Eight-week-old mice were fed a STD (343 kcal/100 g, crude fat 3.8%) or HFD (508 kcal/100 g, crude fat 32%) (CE-2, CLEA Japan, Osaka, Japan) for 35 weeks (seven males and nine females for each diet group). All experimental procedures were approved by the Animal Care and Use Committee of Tokushima Bunri University. They also conformed to the guidelines established by the Japanese Ministry of Education, Culture, Sports, Science, and Technology.
Animal treatment The body weight of each animal was measured weekly. We used 8-week-old mice (five males and four females) and 43-week-old mice (seven males and nine females for each diet group). The mice fasted for 6 h before sampling to reduce the effects of dietary intake on plasma constituents. The animals received an overdose of pentobarbital anesthesia, after which dissection was performed to collect their organs and plasma. White adipose tissue (WAT) surrounding the ovaries, testes, kidneys, and posterior belly lining was weighed. Plasma glucose levels were determined using a NIPRO FreeStyle Sensor (NIPRO, Osaka, Japan). Plasma insulin and leptin were measured with an Ultra-Sensitive Mouse Insulin ELISA Kit and a Mouse Leptin ELISA Kit (Morinaga Institute of Biological Science, Kanagawa, Japan), respectively. Plasma adiponectin was determined with a mouse/rat adiponectin ELISA kit (Otsuka Pharmaceuticals, Tokyo, Japan).
Intraperitoneal glucose tolerance test Glucose metabolism was evaluated by intraperitoneal glucose tolerance test (IPGTT) as follows. Mice at 8 weeks (n = 4 per each sex) and 21—24 weeks (n = 4 per each diet and sex group) were
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
ORCP-449; No. of Pages 9
ARTICLE IN PRESS
Gene expression related to lipid and glucose metabolism in white adipose tissue subjected to fasting for 12 h, and then an intraperitoneal inoculation of glucose solution (2 g/10 ml/kg body weight). Blood was sampled from the tail vein at 0, 15, 30, 60, 120, and 180 min post-injection before they were sacrificed. The area under the curve (AUC) for elevated blood glucose during the IPGTT was calculated by the trapezoidal rule. The half-life (t1/2 ) of elimination of elevated blood glucose was calculated using first-order elimination kinetics.
RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from frozen mouse tissue with RNAiso Plus (TaKaRa Bio, Otsu, Japan) using a Polytron homogenizer, and then were subjected to reverse transcription reactions using a highcapacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. The primer sets and PCR conditions employed are summarized in Table 1. The PCR products were separated by electrophoresis on 2% agarose gels stained with ethidium bromide. The signal intensity of the amplified DNA was quantified by densitometry using an ImageQuant LAS 4000 (Fujifilm, Tokyo, Japan) and a CS Analyzer version 3.0 (ATTO, Tokyo, Japan). The densities of the PCR products were normalized to those of the internal control genes, 36b4 for adipose tissue. The density ratios of the products were used for comparison.
Statistical analysis Ekuseru-Toukei 2010 for Windows (Social Survey Research Information, Tokyo, Japan) was used for statistical analysis. Results are presented as mean ± standard error of the mean (SEM). Significant differences in means were determined by Student’s t-tests and one-way ANOVA. Significance was established at P < 0.05.
Results Fundamental parameters of 129/Sv mice Table 2 shows various measurements of 8-weekold 129/Sv mice before the diet intervention. Male mice were constitutionally bigger than female mice, and liver weight and perirenal WAT were greater in male mice than in female mice.
3
Perigonadal and subcutaneous WAT were similar between males and females. Mice were fed a STD or HFD ad libitum starting at 8 weeks of age for 35 weeks. At this time (43 weeks), mice reach their maximum body weight (data not shown), so we considered this time-point as optimal for comparisons. Table 3 compares various parameters in 43-week-old male and female mice. For mice fed a STD, the body weight and liver weight were greater in males than in females. Body weight gain and liver weight per gram of body weight were similar between male and female mice. Amounts of perigonadal, perirenal, and subcutaneous WAT were also similar. Taken together, these results indicate that consuming a STD for 35 weeks did not induce obesity in either female or male mice. The body weight and weight gain in male mice were significantly greater for those fed a HFD for 35 weeks compared with STD-fed males, whereas these differences were not remarkable for the corresponding female groups. In addition, the weight of perigonadal, perirenal, and subcutaneous WAT, as well as the ratio of WAT weight to body weight were markedly increased in the HFD-fed male mice compared with STD-fed mice (Table 3).
Gene expression levels in WAT of 129/Sv mice We investigated 35 genes related to glucose and lipid metabolism in WAT. Fig. 1 indicates that mRNA products such as Acaca (acetyl-CoA carboxylase 1 (ACC1)-encoding gene), and Fasn (fatty acid synthase (FAS)-encoding gene) in WAT were more highly expressed in young females than in males (Fig. 1A and B) at 8 weeks. We then investigated these genes in the WAT of 43-week-old 129/Sv mice fed STD (i.e., under non-obese conditions). Fig. 2 displays the mRNA products that were more highly expressed in females than in males. Representative bands of amplified PCR products are shown in Fig. 2H. These genes were Acaca, Fasn, Slc2a1 (glucose transporter (GLUT) 1-encoding gene), and Slc2a4 (GLUT4-encoding gene) in perigonadal WAT (Fig. 2A—D). In addition, expression levels of the adiponectin-encoding gene (Adipoq) and the nicotinamide phosphoribosyl-transferase gene (Nampt, visfatin-encoding gene) were higher in females (Fig. 2E and F). The expression levels of these genes in the WAT of male mice were 40—60% of those in female mice. However, expression levels of four genes — Slc2a1, Slc2a4, Adipoq, and Nampt — were similar between male and female mice at 8 weeks (Fig. 1C—F). On the other hand, the expression levels of the other 33 genes, which are involved
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
Primer information.
Acaca Acsl1 Adipoq Ar Cd36 Cebpa Cebpb Cebpd Esr1 Fabp4 Fasn Fsp27 G6pc Insr Irs1 Irs2 Ldlr Lep Lipe Lpl Mest Nampt Pck1 Pparg2 Ppargc1a Retn Scd1 Serpine1 Slc2a1 Slc2a2 Slc2a4 Slc27a1 Tnf Ucp2 Vldlr 36b4 actb
Forward 5 -CAAAATGCAACTGGACAACCC-3 5 -ACGTTCGCAGTGGCATCG-3 5 -CAGGATGCTACTGTTGCAAGC-3 5 -GTGAAATGGGACCTTGGATG-3 5 -AGCCATCTTTGAGCCTTCAC-3 5 -AAGCCAAGAAGTCGGTGGA-3 5 -ATCCGGATCAAACGTGGCT-3 5 -GGAACACGGGAAAGCATGA-3 5 -TTGACAAGAACCGGAGGAAG-3 5 -GCTTTGCCACAAGGAAAGTG-3 5 -GTGGTGGGTTTGGTGAATTGT-3 5 -GCACAATCGTGGAGACAGAA-3 5 -GCTGAAACTTTCAGCCACATCC-3 5 -ATGGACATCCGGAACAACCT-3 5 -CCCACAGCAGATCATTAACC-3 5 -GGCCTCTGTGGAAAATGTCTC-3 5 -TCCTACGACACCATCATCAGTG-3 5 -ATGTGCTGGAGACCCCTGTGTC-3 5 -CTTCCAGTTCACACCTGCCAT-3 5 -GAG AAGCCATCCGTGTGATT-3 5 -AACCGCAGAATCAACCTGCT-3 5 -AGTGGCCACAAATTCCAGAG-3 5 -GTGTTTACTGGGAAGGCATCGA-3 5 -GGTGAAACTCTGGGAGATTC-3 5 -ATGTGTCGCCTTCTTGCTCT-3 5 -CCACGTACCCACGGGATGAA-3 5 -ACCTGCCTCTTCGGGATTTT-3 5 -GTTCACTTTACCCCTCCGAGAA-3 5 -CGGCCTCTTTGTTAATCGCT-3 5 -GGCTAATTTCAGGACTGGTT-3 5 -TGAGAAACGGAAGTTGGAG-3 5 -CAAGGTCAATGAGGACACGA-3 5 -CTCTTCTCATTCCTGCTTGTGG-3 5 -CCTACAAGACCATTGCACGA-3 5 -TCAGAAGCTGTTTTGGGCTG-3 5 -GAGATTCGGGATATGCTGTTGG -3 5 -GACCCTGAAGTACCCCATTGAA-3
Primer sets used in RT-PCR assays
Reverse 5 -CGTAGTGGCCGTTCTGAAACT-3 5 -TTAGATCTTGATGGTGGCGT-3 5 -TGCAGTCAGTTGGTATCATGG-3 5 -TCGTTTCTGCTGGCACATAG-3 5 -GGAACCAAACTGAGGAATGG-3 5 -CAGTTCACGGCTCAGCTGTT-3 5 -TGCTCGAAACGGAAAAGGTT-3 5 -GGGTTAAGCCCGCAAACATTA-3 5 -AAGGACAAGGCAGGGCTATT-3 5 -ATTTCCATCCAGGCCTCTTC-3 5 -AGGGTACATCCCAGAGGAAGTCA-3 5 -TTGCAGCATCTTCAGACAGG-3 5 -ATGGGAGCAACTTGCTGAGTTC-3 5 -TTGATGACAGTGGCAGGACA-3 5 -AGAGACGAAGATGCTGGTGC-3 5 -CTGTGGCTTCCTTCAAGTGATG-3 5 -GGAATCAACCCAATAGAGACGG-3 5 -TCAGCATTCAGGGCTAACATCCAA-3 5 -TGGCATCTCAAAGGCCTCA-3 5 -CCATCCTCAGTCCCAGAAAA-3 5 -CGAAGAAATTCATGAGCCTGG-3 5 -TATGGTACTGTGCTCTGCTGCT-3 5 -TTGCCATCTTTGTCCTTCCG-3 5 -TAATAAGGTGGAGATGCAGG-3 5 -ATCCCTCTTGAGCCTTTCGT-3 5 -TCAGGAAGCGACCTGCAGCTTACA-3 5 -GTTTTCCGCCCTTCTCTTTGA-3 5 -CAAAGATGGCATCCGCAGT-3 5 -CAAGTCTGCATTGCCCATG-3 5 -TTTCTTTGCCCTGACTTCCT-3 5 -GAGACATAGCTCATGGCTGGAA-3 5 -ATCTGAAGGTGCCTGTGGT-3 5 -CTTTGAGATCCATGCCGTTG-3 5 -GAAGGCATGAACCCCTTGTA-3 5 -GCATGTGCAACTTGGAATCC-3 5 -GTTGTCAAACACCTGCTGGATG-3 5 -GCTTCTCTTTGATGTCACGCAC-3
Product size (bp)
Cycles
Anneal. temp. (◦ C)
GenBank at NCBI accession #
572 899 753 238 216 189 254 274 244 347 556 452 307 494 446 464 350 504 332 328 223 271 472 344 341 360 301 302 307 278 369 531 268 343 430 297 452
25 30 25 30 26 28 30 30 31 25 27 35 28 30 33 35 33 30 30 34 28 36 28 28 33 28 30 30 32 28 35 37 39 38 33 24 23
58 60 60 58 60 58 60 60 58 58 58 60 58 60 60 60 60 60 58 58 58 60 58 58 60 58 60 60 58 58 62 60 58 58 60 58 60
[NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM
133360] 007981] 009605] 013476] 001159558] 007678] 009883] 007679] 007956] 024406] 007988] 178373] 008061] 010568] 010570] 001081212] 010700] 008493] 010719] 008509] 008590] 021524] 011044] 011146] 027710] 022984] 009127] 008871] 011400] 031197] 009204] 011977] 013693] 011671] 013703] 007475] 007393]
ARTICLE IN PRESS
Primer sequence
Y. Kadota et al.
Gene
ORCP-449; No. of Pages 9
4
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
Table 1
ORCP-449; No. of Pages 9
ARTICLE IN PRESS
Gene expression related to lipid and glucose metabolism in white adipose tissue Table 2
5
Fundamental parameters of 8-week-old male and female mice. Sex
Female
Weight
Body weight (g) Perigonadal WAT (g) Perirenal WAT (g) Subcutaneous WAT (g) Liver (g)
18.58 0.16 0.04 0.23 0.56
Blood/plasma
Glucose (mg/dl) Insulin (ng/ml) Adiponectin (g/ml)
IPGTT
C0 (mg/dl) Cmax (mg/dl) t1/2 (min) AUC0-2 h ((mg/dl) h)
± ± ± ± ±
Male 0.65 0.03 0.00 0.01 0.02
22.45 0.26 0.08 0.26 0.80
47 ± 5 0.13 ± 0.06 29.2 ± 1.2 50 167 44 51
± ± ± ±
± ± ± ± ±
0.35** 0.03 0.01** 0.02 0.01**
57 ± 2 0.18 ± 0.10 21.1 ± 0.9**
5 14 5 3
35 187 30 79
± ± ± ±
4 14 4 6**
Body, WAT, and liver weights in 8-week-old mice were measured. Blood glucose and plasma insulin, adiponectin, and leptin levels were measured after 6 h of fasting (male mice, n = 5; female mice, n = 4). Parameters of glucose kinetics during an IPGTT were measured after 12 h of fasting (n = 4 per each sex group). C0 , concentration of blood glucose at 0 min; Cmax , maximum glucose concentration; t1/2 , half-life of the elimination of elevated blood glucose; AUC0—2 h , area under the curve (AUC) for elevated blood glucose during the IPGTT. Values are mean ± SEM. ** P < 0.01 compared with female mice.
Table 3
Comparison of various parameters of 43-week-old male and female mice.
Diet
Standard diet
High fat diet
Sex
Female
Male
Female
Male
Body weight (g) Body weight gain (g)
25.98 ± 0.32 8.22 ± 0.20
29.27 ± 0.64** 7.76 ± 0.61
27.45 ± 0.75 9.99 ± 0.63#
41.80 ± 2.16**, 20.36 ± 1.94**,
Perigonadal WAT (g) per gram of body weight (mg/g)
0.44 ± 0.02 15.95 ± 0.88
0.55 ± 0.07 18.68 ± 2.26
0.81 ± 0.11## 29.41 ± 3.96##
1.97 ± 0.37*, 47.57 ± 6.56*,
Perirenal WAT (g) Per gram of body weight (mg/g)
0.11 ± 0.01 4.08 ± 0.25
0.14 ± 0.02 4.84 ± 0.67
Subcutaneous WAT (g) Per gram of body weight (mg/g)
0.25 ± 0.02 9.57 ± 0.75
0.35 ± 0.06 11.79 ± 2.04
Liver (g) Per gram of body weight (mg/g)
1.04 ± 0.03 40.02 ± 1.34
1.19 ± 0.05* 40.69 ± 1.45
Blood/plasma Glucose (mg/dl) Insulin (ng/ml) Adiponectin (g/ml) Leptin (ng/ml)
122 0.84 38.6 4.0
± ± ± ±
10 0.11 1.7 0.6
109 1.34 17.4 5.6
± ± ± ±
5 0.18* 0.9** 0.7
0.17 ± 0.03# 6.18 + 0.79#
## ## ## ##
0.59 ± 0.10**, 14.14 ± 1.86**,
##
0.45 ± 0.06## 16.37 ± 1.97##
1.41 ± 0.27**, 34.04 ± 4.74**,
##
1.04 ± 0.04 38.08 ± 1.41
1.34 ± 0.05** 34.88 ± 1.38
122 0.71 33.5 6.8
± ± ± ±
7 0.15 4.1 0.5#
103 1.30 18.4 39.8
± ± ± ±
5 0.30 1.3** 8.6**,
##
##
##
Body weight gain was measured as the difference in body weight between 8- and 43-week-old mice. Blood glucose and plasma insulin, adiponectin, and leptin levels were measured in 43-week-old mice after 6 h of fasting. Values are mean ± SEM (male mice, n = 7; female mice, n = 9). * P < 0.05 compared with female mice fed the corresponding diet. ** P < 0.01 compared with female mice fed the corresponding diet. # P < 0.05 compared with corresponding sex-matched mice fed a standard diet. ## P < 0.01 compared with corresponding sex-matched mice fed a standard diet.
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
ARTICLE IN PRESS
ORCP-449; No. of Pages 9
6
Y. Kadota et al. A. Acaca
B. Fasn
80
**
60 40 20 female
120
100
100
**
80 60 40 20 0
male
female
D. Slc2a4
40 20 0
female
male
40 20
100
male
F. Nampt
100
80 60 40 20 0
female
120 relative mRNA expression
relative mRNA expression
relative mRNA expression
60
60
0
male
120
80
80
E. Adipoq
120 100
relative mRNA expression
100
0
C. Slc2a1
120 relative mRNA expression
relative mRNA expression
120
female
male
80 60 40 20 0
female
male
Figure 1 mRNA levels in perigonadal WAT of 8-week-old mice. RT-PCR was performed for (A) Acaca (ACC1-encoding gene), (B) Fasn (FAS-encoding gene), (C) Slc2a1 (GLUT1-encoding gene), (D) Slc2a4 (GLUT4-encoding gene), (E) Adipoq (adiponectin-encoding gene), and (F) Nampt (visfatin-encoding gene) in perigonadal WAT of 8-week-old mice. Data are presented as mean ± SEM (male mice, n = 5: female mice, n = 4). ** P < 0.01 compared with female mice.
in adipocyte differentiation, fatty acid synthesis, adipocyte enlargement, glucose metabolism, glucose uptake, insulin signaling, or lipid accumulation (listed in Table 1), were similar in the WAT of STDfed males and females (data not shown). We then compared gene expression between female and male mice fed a HFD for 35 weeks. Tellingly, the expression levels of the six genes identified above — Acaca, Fasn, Slc2a1, Slc2a4, Adipoq, and Nampt — in perigonadal WAT (Fig. 2A—F) were also higher in female mice than in male mice under HFD conditions.
Glucose metabolism capacity in 129/Sv mice In 8-week-old mice, blood glucose and plasma insulin levels were similar between males and females (Table 2). In 43-week-old mice, the insulin level, but not glucose level, became to be higher in males than females (Table 3). The t1/2 and maximum concentration (Cmax ) of glucose as evaluated by IPGTT showed no differences between females and males. However, the AUC0—2 h was slightly
lower in female mice than in male mice, suggesting that the glucose metabolism capacity is marginally greater in females than in males (Table 2). Even by the halfway point of their 43-week lifespan, 129/Sv male mice were already exhibiting HFD-induced obesity (data not shown). We accordingly hypothesized that changes in glucose metabolism would also be observed in mice fed a STD even at 21—24-weeks-old. The half-life for elimination of elevated blood glucose (t1/2 ) indeed was longer in male mice than in female mice, indicating that glucose absorption and metabolism were faster in female mice fed a STD (Table 4 and Fig. 3). In HFD-fed mice, the AUC0—3 h was significantly elevated in both 21—24-week-old males and females, though it was lower in females than in males (Table 4). Thus, t1/2 may correlate with age, and HFD-induced obesity at 24 weeks (data not shown) may affect AUC0—3 h .
Other parameters in 129/Sv mice Plasma adiponectin was slightly but significantly higher in female mice than in male mice fed a STD at
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
ARTICLE IN PRESS
ORCP-449; No. of Pages 9
Gene expression related to lipid and glucose metabolism in white adipose tissue
80 60 40 20
relative mRNA expression
120
*
100
0
80 60 40 20 0
STD
60 40 20
STD
120 relative mRNA expression
**
STD
HFD
80 60 40 20 0
**
**
HFD
80 60 40 20
40 20
female male female male STD
H
**
250
HFD
Female
Male
Acaca Fasn
200
Slc2a1
150
Slc2a4 100
Adipoq
50
Nampt
0 female male female male STD
60
HFD
300
0 female male female male
80
G. lep
100
**
100
0
F. Nampt
100
STD
80
**
120
female male female male
relative mRNA expression
*
*
0
E. Adipoq 120
D. Slc2a4
100
female male female male
HFD
*
120
*
100
female male female male
relative mRNA expression
C. Slc2a1
relative mRNA expression
**
120 relative mRNA expression
B. Fasn
**
relative mRNA expression
A. Acaca
7
female male female male
HFD
STD
HFD
36B4
Figure 2 mRNA levels in perigonadal WAT of mice fed a STD or a HFD for 35 weeks. RT-PCR was performed for (A) Acaca, (B) Fasn, (C) Slc2a1, (D) Slc2a4, (E) Adipoq, (F) Nampt, and (G) lep (leptin-encoding gene) in perigonadal WAT for mice fed a STD or a HFD for 35 weeks (n = 4 per group). (H) Representative bands of amplified PCR products. The gene expression levels were shown as percentages of value of STD-fed female mice. Data are presented as mean ± SEM for n = 4 mice per set. * P < 0.05 and ** P < 0.01 compared with female mice.
8 weeks old (Table 2). We also noted expression levels of Lep mRNA (Fig. 2G) and plasma leptin levels (Table 3) were up-regulated in HFD-fed mice. Since we have previously shown that plasma leptin correlates with WAT weight [7], we hypothesize lipid accumulation might mediate this up-regulation. In contrast, levels of blood glucose, plasma insulin, and adiponectin remained unchanged despite the consumption of a HFD for 35 weeks (Table 3). Plasma adiponectin was elevated in female mice but reduced in male mice fed a STD after 35 weeks (Tables 2 and 3). Table 4
Discussion Men have been observed to exhibit a higher prevalence of diabetes and early abnormalities in glucose metabolism than women do [8]. However, the factors responsible for the differences in the development of insulin resistance are largely unknown. In order to assign relative responsibilities to these factors, the present study compared factors associated with lipid accumulation and glucose metabolism in the WAT and blood of non-obese female and male mice fed a STD.
Parameters of glucose kinetics during an IPGTT in 21—24-week-old male and female mice.
Diet
Standard diet
Sex
Female
IPGTT C0 (mg/dl) Cmax (mg/dl) t1/2 (min) AUC0—3 h ((mg/dl) h)
63 157 50 64
± ± ± ±
3 9 4 23
High fat diet Male 60 130 110 73
± ± ± ±
Female 11 16 18* 25
64 243 78 190
± ± ± ±
2 17 2 24
Male 73 258 115 331
± ± ± ±
8 13 5** 51*
Parameters of glucose kinetics during an IPGTT were measured in 21—24-week-old mice after 12 h of fasting (n = 4 per group). Values are presented as the mean ± SEM. * P < 0.05 compared with diet-matched female mice. ** P < 0.01 compared with diet-matched female mice.
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
ORCP-449; No. of Pages 9
ARTICLE IN PRESS
8
Y. Kadota et al.
Figure 3 IPGTT for 21—24 week-old 129/Sv mice after 12 h fasting. IPGTTs were performed after 12 h of fasting on STD-fed female mice (open circles), STD-fed male mice (filled circles), HFD-fed female mice (open square) and HFD-fed male mice (filled square) (n = 4 per group). A glucose solution (2 g/10 ml/kg body weight) was administered intraperitoneally and blood glucose levels (mg/dl) were measured in venous blood at 0, 15, 30, 60, 120, and 180 min post-injection. Values are presented as mean ± SEM for n = 4 mice per set. ** P < 0.01 compared with HFD-fed female mice.
Rankinen et al. suggested that over 400 genes could affect obesity [2], and so it is impractical to analyze them all in one setting. From this large number, we picked 35 genes associated with lipid and glucose metabolism in WAT. We focused on gene expression changes in the WAT of 8-week-old 129/Sv mice and mice fed a STD or HFD for 35 weeks. The expression levels of four genes — Slc2a1, Slc2a4, Adipoq, and Nampt — were greater in the WAT of 43-week-old female mice than in corresponding male mice (Fig. 2), suggesting that females might have a greater ability to metabolize glucose in WAT regardless of diet fat content. However, expression levels of these four genes were similar in male and female mice at 8 weeks old (Fig. 1). Aging might affect also gene expression levels independently of diet. The expression of genes mediating glucose uptake by cells, such as Slc2a1 and Slc2a4, in WAT (Fig. 2C and D) was also greater in female mice than in male mice at 43 weeks regardless of diet fat content. GLUT1 is a ubiquitously expressed glucose transporter and GLUT4 is found in insulin-responsive tissues such as adipose tissue, skeletal muscle, and the heart [9]. GLUT4 proteins migrate to the plasma membrane in response to insulin stimulation, and play a role in cellular glucose absorption. The up-regulation of
both GLUT1 and GLUT4 gene expression levels are insulin-dependent [10]. Gene expression levels for Acaca and Fasn were higher in females than in males at both 8 weeks (Fig. 1A and B) and 43 weeks (Fig. 2A and B). Thus, the differences in expression of these genes between male and female mice appear at an earlier stage as compared with other genes. These gene expression levels may be associated with sex but not aging. ACC1 and FAS are rate-limiting enzymes in fatty acid synthesis, which creates triglycerides (TGs), meaning they contribute to the control of lipid homeostasis. A decrease in tissue TG levels may induce protein synthesis of ACC1 and FAS, which would lead to TG accumulation. We performed IPGTTs to measure the mice’s ability to metabolize glucose. The value of t1/2 was not significantly different between female and male mice at 8 weeks (Table 2). At 43 weeks, t1/2 was lower in females than in males fed a STD for 35 weeks (Table 4), suggesting that the capacity to metabolize glucose may change with age in a sexdependent manner. The insulin level of 43-week old mice fed a STD was higher in males than in females. This might also alter glucose metabolism of male mice as they grow older. In addition, consuming a HFD for 35 weeks significantly increased AUC0—3 h in male and female mice compared with mice fed a STD; moreover, the elevated AUC0—3 h was greater in male mice than in female mice (Table 4). Furthermore, t1/2 was greater in male mice compared with female mice fed a HFD (Table 4). These results demonstrate that a HFD may cause a decrease in the ability to metabolize glucose. Although further studies are required to elucidate the contributions of higher expression levels of GLUT1 and GLUT4 to the AUC0—3 h and t1/2 of female versus male mice (Fig. 2C and D), these results demonstrate that the ability of female mice to absorb and metabolize glucose is superior to that of male mice, even when fed a STD (Table 4). Male mice did not become obese after consuming a STD, but their glucose metabolism was nonetheless slower, and as a result, the males may potentially be vulnerable to weight gain. Glucose tolerance test results further emphasized the effects of diet on glucose metabolism for females compared with males. Genetic and other factors that contribute to such differences in glucose metabolism between males and females should be investigated further. In conclusion, we focused on the expression of 35 genes related to lipid metabolism, receptors for insulin signaling, and glucose metabolism out of over 400 obesity-related genes in the WAT of male and female mice. Expression of Acaca and Fasn in 8-week-old mice and four additional
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
ORCP-449; No. of Pages 9
ARTICLE IN PRESS
Gene expression related to lipid and glucose metabolism in white adipose tissue genes — Slc2a1, Slc2a4, Adipoq and Nampt — in 43-week-old mice was higher in females compared with males. The relative gene expression ratios of female to male mice were similar between the STD and HFD groups at 43 weeks. The data suggest that sex and aging may cause differences in gene expression levels in females and males independently of diet. Since male and female mice fed a STD for 43 weeks did not become obese, obesity cannot be attributed to these observed gene expression changes alone. Nonetheless, the changes may be responsible for regulating sensitivity to HFD-induced obesity. Further studies on the mechanisms of candidate gene expression are required to elucidate factors involved in obesity development in males and females.
Acknowledgment This work was supported in part by JSPS KAKENHI Grant Numbers 24790133 to Y.K., 24590771 to T.K., and 21590144 to M.S.
References [1] van Kruijsdijk RC, van der Wall E, Visseren FL. Obesity and cancer: the role of dysfunctional adipose tissue. Cancer Epidemiol Biomarkers Prev 2009;18:2569—78.
9
[2] Rankinen T, Zuberi A, Chagnon YC, Weisnagel SJ, Argyropoulos G, Walts B, et al. The human obesity gene map: the 2005 update. Obesity (Silver Spring) 2006;14: 529—644. [3] Moralejo DH, Hansen CT, Treuting P, Hessner MJ, Fuller JM, Van Yserloo B, et al. Differential effects of leptin receptor mutation on male and female BBDR Gimap5−/Gimap5− spontaneously diabetic rats. Physiol Genomics 2010;41:9—20. [4] Wallenius V, Wallenius K, Ahren B, Rudling M, Carlsten H, Dickson SL, et al. Interleukin-6-deficient mice develop mature-onset obesity. Nat Med 2002;8:75—9. [5] Netea MG, Joosten LA, Lewis E, Jensen DR, Voshol PJ, Kullberg BJ, et al. Deficiency of interleukin-18 in mice leads to hyperphagia, obesity and insulin resistance. Nat Med 2006;12:650—6. [6] Geer EB, Shen W. Gender differences in insulin resistance, body composition, and energy balance. Gend Med 2009;6(Suppl. 1):60—75. [7] Sato M, Kawakami T, Kondoh M, Takiguchi M, Kadota Y, Himeno S, et al. Development of high-fat-diet-induced obesity in female metallothionein-null mice. FASEB J 2010;24:2375—84. [8] Kuhl J, Hilding A, Ostenson CG, Grill V, Efendic S, Bavenholm P. Characterisation of subjects with early abnormalities of glucose tolerance in the Stockholm Diabetes Prevention Programme: the impact of sex and type 2 diabetes heredity. Diabetologia 2005;48:35—40. [9] Wood IS, Trayhurn P. Glucose transporters (GLUT and SGLT): expanded families of sugar transport proteins. Br J Nutr 2003;89:3—9. [10] McGowan KM, Long SD, Pekala PH. Glucose transporter gene expression: regulation of transcription and mRNA stability. Pharmacol Ther 1995;66:465—505.
Available online at www.sciencedirect.com
ScienceDirect
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
ARTICLE IN PRESS
Obesity Research & Clinical Practice (2015) xxx, xxx—xxx
ORIGINAL ARTICLE
Gene expression related to lipid and glucose metabolism in white adipose tissue Yoshito Kadota ∗, Takashige Kawakami, Satoshi Takasaki 1, Masao Sato, Shinya Suzuki Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima 770-8514, Japan Received 25 July 2014 ; received in revised form 13 April 2015; accepted 23 April 2015
KEYWORDS Gene expression; Obesity; White adipose tissue; Glucose transporter; Adipokine
∗ 1
Summary Problem: A number of endogenous and external factors influence the development of obesity. However, the factors responsible for these differences in obesity pathogenesis between males and females are largely unknown. Methods: We investigated the expression of 35 genes related to lipid and glucose metabolism and to receptors for insulin signaling in white adipose tissue (WAT) of 8-week-old 129/Sv mice and mice fed standard diet (STD) or high fat diet (HFD) for 35 weeks in males and females. Results: At 8 weeks, the expression levels of two genes for fatty acid synthesis, Acaca and Fasn, were higher in females than in males. Female mice fed a STD for 35 weeks also had higher expression levels of an additional four genes related to glucose transporters (Slc2a1 and Slc2a4) and adipokines (Adipoq and Nampt). The expression levels of these six genes were also higher in females than in males fed a HFD for 35 weeks. At 43 weeks old, the female-to-male expression ratio of these six genes was similar for the STD and HFD groups. Furthermore, glucose tolerance testing showed that the half-life for the elimination of elevated blood glucose was shorter in females than males, although blood glucose parameters were generally similar between females and males.
Corresponding author. Tel.: +81 88 602 8457; fax: +81 88 655 3051. E-mail address: [email protected] (Y. Kadota). Present address: Department of Pharmacy, Ohira Medical Care Hospital, Kitakyusyu, Fukuoka 807-0083, Japan.
http://dx.doi.org/10.1016/j.orcp.2015.04.009 1871-403X/© 2015 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
ORCP-449; No. of Pages 9
ARTICLE IN PRESS
2
Y. Kadota et al. Conclusions: These findings suggest that sex and aging may cause diet-independent differences in gene expression levels in female and male mice, and that higher expression of these genes in females could contribute to higher metabolic activity and resistance to obesity compared with males. © 2015 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
Introduction
Materials and methods
Obesity is characterized by an increase in adipose tissue mass and excess lipid deposition, and is associated with enlarged adipocytes. In developed countries, it is one of the most notable causes of accumulative lipid-related diseases such as type 2 diabetes, cardiovascular disease, and cancer [1]. There is a variety of genes associated with the development of obesity [2], and susceptibility to obesity is thought to be dependent on certain genetic factors. For example, leptin-receptor mutant rats [3], as well as interleukin-6-deficient [4] and interleukin-18-deficient mice [5], exhibit obesity. These gene deficiencies induce obesity even when their rodents are fed a standard diet (STD). A number of factors influence the development of obesity. Endogenous and external factors such as various stressors, aging, and low physical activity contribute to obesity and may change patterns of gene expression. Some changes are unlikely to be able to induce obesity independently, but rather promote diet-induced obesity. In addition, the phenotypes of obesity in men and women are characterized by certain differences: men have more visceral adipose tissue, whereas women have more peripheral and subcutaneous adipose tissue [6]. However, the factors responsible for these differences in obesity pathogenesis between males and females are largely unknown. Since there are at least 400 genes associated with obesity [2], it is necessary to narrow the number of genes on which to focus. We selected 35 genes that are particularly important for lipid and glucose metabolism. First, we studied the expression of these genes in young male and female mice. Second, since aging may contribute to excess accumulation of lipids, we compared the expression of these genes in older males and females fed either a STD or a high fat diet (HFD) for 35 weeks. Third, since sex differences may be another important factor for the development of obesity, we compared the expression of these genes and glucose metabolic activity between males and females.
Animals 129/SvCPJ mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Eight-week-old mice were fed a STD (343 kcal/100 g, crude fat 3.8%) or HFD (508 kcal/100 g, crude fat 32%) (CE-2, CLEA Japan, Osaka, Japan) for 35 weeks (seven males and nine females for each diet group). All experimental procedures were approved by the Animal Care and Use Committee of Tokushima Bunri University. They also conformed to the guidelines established by the Japanese Ministry of Education, Culture, Sports, Science, and Technology.
Animal treatment The body weight of each animal was measured weekly. We used 8-week-old mice (five males and four females) and 43-week-old mice (seven males and nine females for each diet group). The mice fasted for 6 h before sampling to reduce the effects of dietary intake on plasma constituents. The animals received an overdose of pentobarbital anesthesia, after which dissection was performed to collect their organs and plasma. White adipose tissue (WAT) surrounding the ovaries, testes, kidneys, and posterior belly lining was weighed. Plasma glucose levels were determined using a NIPRO FreeStyle Sensor (NIPRO, Osaka, Japan). Plasma insulin and leptin were measured with an Ultra-Sensitive Mouse Insulin ELISA Kit and a Mouse Leptin ELISA Kit (Morinaga Institute of Biological Science, Kanagawa, Japan), respectively. Plasma adiponectin was determined with a mouse/rat adiponectin ELISA kit (Otsuka Pharmaceuticals, Tokyo, Japan).
Intraperitoneal glucose tolerance test Glucose metabolism was evaluated by intraperitoneal glucose tolerance test (IPGTT) as follows. Mice at 8 weeks (n = 4 per each sex) and 21—24 weeks (n = 4 per each diet and sex group) were
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
ORCP-449; No. of Pages 9
ARTICLE IN PRESS
Gene expression related to lipid and glucose metabolism in white adipose tissue subjected to fasting for 12 h, and then an intraperitoneal inoculation of glucose solution (2 g/10 ml/kg body weight). Blood was sampled from the tail vein at 0, 15, 30, 60, 120, and 180 min post-injection before they were sacrificed. The area under the curve (AUC) for elevated blood glucose during the IPGTT was calculated by the trapezoidal rule. The half-life (t1/2 ) of elimination of elevated blood glucose was calculated using first-order elimination kinetics.
RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from frozen mouse tissue with RNAiso Plus (TaKaRa Bio, Otsu, Japan) using a Polytron homogenizer, and then were subjected to reverse transcription reactions using a highcapacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. The primer sets and PCR conditions employed are summarized in Table 1. The PCR products were separated by electrophoresis on 2% agarose gels stained with ethidium bromide. The signal intensity of the amplified DNA was quantified by densitometry using an ImageQuant LAS 4000 (Fujifilm, Tokyo, Japan) and a CS Analyzer version 3.0 (ATTO, Tokyo, Japan). The densities of the PCR products were normalized to those of the internal control genes, 36b4 for adipose tissue. The density ratios of the products were used for comparison.
Statistical analysis Ekuseru-Toukei 2010 for Windows (Social Survey Research Information, Tokyo, Japan) was used for statistical analysis. Results are presented as mean ± standard error of the mean (SEM). Significant differences in means were determined by Student’s t-tests and one-way ANOVA. Significance was established at P < 0.05.
Results Fundamental parameters of 129/Sv mice Table 2 shows various measurements of 8-weekold 129/Sv mice before the diet intervention. Male mice were constitutionally bigger than female mice, and liver weight and perirenal WAT were greater in male mice than in female mice.
3
Perigonadal and subcutaneous WAT were similar between males and females. Mice were fed a STD or HFD ad libitum starting at 8 weeks of age for 35 weeks. At this time (43 weeks), mice reach their maximum body weight (data not shown), so we considered this time-point as optimal for comparisons. Table 3 compares various parameters in 43-week-old male and female mice. For mice fed a STD, the body weight and liver weight were greater in males than in females. Body weight gain and liver weight per gram of body weight were similar between male and female mice. Amounts of perigonadal, perirenal, and subcutaneous WAT were also similar. Taken together, these results indicate that consuming a STD for 35 weeks did not induce obesity in either female or male mice. The body weight and weight gain in male mice were significantly greater for those fed a HFD for 35 weeks compared with STD-fed males, whereas these differences were not remarkable for the corresponding female groups. In addition, the weight of perigonadal, perirenal, and subcutaneous WAT, as well as the ratio of WAT weight to body weight were markedly increased in the HFD-fed male mice compared with STD-fed mice (Table 3).
Gene expression levels in WAT of 129/Sv mice We investigated 35 genes related to glucose and lipid metabolism in WAT. Fig. 1 indicates that mRNA products such as Acaca (acetyl-CoA carboxylase 1 (ACC1)-encoding gene), and Fasn (fatty acid synthase (FAS)-encoding gene) in WAT were more highly expressed in young females than in males (Fig. 1A and B) at 8 weeks. We then investigated these genes in the WAT of 43-week-old 129/Sv mice fed STD (i.e., under non-obese conditions). Fig. 2 displays the mRNA products that were more highly expressed in females than in males. Representative bands of amplified PCR products are shown in Fig. 2H. These genes were Acaca, Fasn, Slc2a1 (glucose transporter (GLUT) 1-encoding gene), and Slc2a4 (GLUT4-encoding gene) in perigonadal WAT (Fig. 2A—D). In addition, expression levels of the adiponectin-encoding gene (Adipoq) and the nicotinamide phosphoribosyl-transferase gene (Nampt, visfatin-encoding gene) were higher in females (Fig. 2E and F). The expression levels of these genes in the WAT of male mice were 40—60% of those in female mice. However, expression levels of four genes — Slc2a1, Slc2a4, Adipoq, and Nampt — were similar between male and female mice at 8 weeks (Fig. 1C—F). On the other hand, the expression levels of the other 33 genes, which are involved
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
Primer information.
Acaca Acsl1 Adipoq Ar Cd36 Cebpa Cebpb Cebpd Esr1 Fabp4 Fasn Fsp27 G6pc Insr Irs1 Irs2 Ldlr Lep Lipe Lpl Mest Nampt Pck1 Pparg2 Ppargc1a Retn Scd1 Serpine1 Slc2a1 Slc2a2 Slc2a4 Slc27a1 Tnf Ucp2 Vldlr 36b4 actb
Forward 5 -CAAAATGCAACTGGACAACCC-3 5 -ACGTTCGCAGTGGCATCG-3 5 -CAGGATGCTACTGTTGCAAGC-3 5 -GTGAAATGGGACCTTGGATG-3 5 -AGCCATCTTTGAGCCTTCAC-3 5 -AAGCCAAGAAGTCGGTGGA-3 5 -ATCCGGATCAAACGTGGCT-3 5 -GGAACACGGGAAAGCATGA-3 5 -TTGACAAGAACCGGAGGAAG-3 5 -GCTTTGCCACAAGGAAAGTG-3 5 -GTGGTGGGTTTGGTGAATTGT-3 5 -GCACAATCGTGGAGACAGAA-3 5 -GCTGAAACTTTCAGCCACATCC-3 5 -ATGGACATCCGGAACAACCT-3 5 -CCCACAGCAGATCATTAACC-3 5 -GGCCTCTGTGGAAAATGTCTC-3 5 -TCCTACGACACCATCATCAGTG-3 5 -ATGTGCTGGAGACCCCTGTGTC-3 5 -CTTCCAGTTCACACCTGCCAT-3 5 -GAG AAGCCATCCGTGTGATT-3 5 -AACCGCAGAATCAACCTGCT-3 5 -AGTGGCCACAAATTCCAGAG-3 5 -GTGTTTACTGGGAAGGCATCGA-3 5 -GGTGAAACTCTGGGAGATTC-3 5 -ATGTGTCGCCTTCTTGCTCT-3 5 -CCACGTACCCACGGGATGAA-3 5 -ACCTGCCTCTTCGGGATTTT-3 5 -GTTCACTTTACCCCTCCGAGAA-3 5 -CGGCCTCTTTGTTAATCGCT-3 5 -GGCTAATTTCAGGACTGGTT-3 5 -TGAGAAACGGAAGTTGGAG-3 5 -CAAGGTCAATGAGGACACGA-3 5 -CTCTTCTCATTCCTGCTTGTGG-3 5 -CCTACAAGACCATTGCACGA-3 5 -TCAGAAGCTGTTTTGGGCTG-3 5 -GAGATTCGGGATATGCTGTTGG -3 5 -GACCCTGAAGTACCCCATTGAA-3
Primer sets used in RT-PCR assays
Reverse 5 -CGTAGTGGCCGTTCTGAAACT-3 5 -TTAGATCTTGATGGTGGCGT-3 5 -TGCAGTCAGTTGGTATCATGG-3 5 -TCGTTTCTGCTGGCACATAG-3 5 -GGAACCAAACTGAGGAATGG-3 5 -CAGTTCACGGCTCAGCTGTT-3 5 -TGCTCGAAACGGAAAAGGTT-3 5 -GGGTTAAGCCCGCAAACATTA-3 5 -AAGGACAAGGCAGGGCTATT-3 5 -ATTTCCATCCAGGCCTCTTC-3 5 -AGGGTACATCCCAGAGGAAGTCA-3 5 -TTGCAGCATCTTCAGACAGG-3 5 -ATGGGAGCAACTTGCTGAGTTC-3 5 -TTGATGACAGTGGCAGGACA-3 5 -AGAGACGAAGATGCTGGTGC-3 5 -CTGTGGCTTCCTTCAAGTGATG-3 5 -GGAATCAACCCAATAGAGACGG-3 5 -TCAGCATTCAGGGCTAACATCCAA-3 5 -TGGCATCTCAAAGGCCTCA-3 5 -CCATCCTCAGTCCCAGAAAA-3 5 -CGAAGAAATTCATGAGCCTGG-3 5 -TATGGTACTGTGCTCTGCTGCT-3 5 -TTGCCATCTTTGTCCTTCCG-3 5 -TAATAAGGTGGAGATGCAGG-3 5 -ATCCCTCTTGAGCCTTTCGT-3 5 -TCAGGAAGCGACCTGCAGCTTACA-3 5 -GTTTTCCGCCCTTCTCTTTGA-3 5 -CAAAGATGGCATCCGCAGT-3 5 -CAAGTCTGCATTGCCCATG-3 5 -TTTCTTTGCCCTGACTTCCT-3 5 -GAGACATAGCTCATGGCTGGAA-3 5 -ATCTGAAGGTGCCTGTGGT-3 5 -CTTTGAGATCCATGCCGTTG-3 5 -GAAGGCATGAACCCCTTGTA-3 5 -GCATGTGCAACTTGGAATCC-3 5 -GTTGTCAAACACCTGCTGGATG-3 5 -GCTTCTCTTTGATGTCACGCAC-3
Product size (bp)
Cycles
Anneal. temp. (◦ C)
GenBank at NCBI accession #
572 899 753 238 216 189 254 274 244 347 556 452 307 494 446 464 350 504 332 328 223 271 472 344 341 360 301 302 307 278 369 531 268 343 430 297 452
25 30 25 30 26 28 30 30 31 25 27 35 28 30 33 35 33 30 30 34 28 36 28 28 33 28 30 30 32 28 35 37 39 38 33 24 23
58 60 60 58 60 58 60 60 58 58 58 60 58 60 60 60 60 60 58 58 58 60 58 58 60 58 60 60 58 58 62 60 58 58 60 58 60
[NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM [NM
133360] 007981] 009605] 013476] 001159558] 007678] 009883] 007679] 007956] 024406] 007988] 178373] 008061] 010568] 010570] 001081212] 010700] 008493] 010719] 008509] 008590] 021524] 011044] 011146] 027710] 022984] 009127] 008871] 011400] 031197] 009204] 011977] 013693] 011671] 013703] 007475] 007393]
ARTICLE IN PRESS
Primer sequence
Y. Kadota et al.
Gene
ORCP-449; No. of Pages 9
4
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
Table 1
ORCP-449; No. of Pages 9
ARTICLE IN PRESS
Gene expression related to lipid and glucose metabolism in white adipose tissue Table 2
5
Fundamental parameters of 8-week-old male and female mice. Sex
Female
Weight
Body weight (g) Perigonadal WAT (g) Perirenal WAT (g) Subcutaneous WAT (g) Liver (g)
18.58 0.16 0.04 0.23 0.56
Blood/plasma
Glucose (mg/dl) Insulin (ng/ml) Adiponectin (g/ml)
IPGTT
C0 (mg/dl) Cmax (mg/dl) t1/2 (min) AUC0-2 h ((mg/dl) h)
± ± ± ± ±
Male 0.65 0.03 0.00 0.01 0.02
22.45 0.26 0.08 0.26 0.80
47 ± 5 0.13 ± 0.06 29.2 ± 1.2 50 167 44 51
± ± ± ±
± ± ± ± ±
0.35** 0.03 0.01** 0.02 0.01**
57 ± 2 0.18 ± 0.10 21.1 ± 0.9**
5 14 5 3
35 187 30 79
± ± ± ±
4 14 4 6**
Body, WAT, and liver weights in 8-week-old mice were measured. Blood glucose and plasma insulin, adiponectin, and leptin levels were measured after 6 h of fasting (male mice, n = 5; female mice, n = 4). Parameters of glucose kinetics during an IPGTT were measured after 12 h of fasting (n = 4 per each sex group). C0 , concentration of blood glucose at 0 min; Cmax , maximum glucose concentration; t1/2 , half-life of the elimination of elevated blood glucose; AUC0—2 h , area under the curve (AUC) for elevated blood glucose during the IPGTT. Values are mean ± SEM. ** P < 0.01 compared with female mice.
Table 3
Comparison of various parameters of 43-week-old male and female mice.
Diet
Standard diet
High fat diet
Sex
Female
Male
Female
Male
Body weight (g) Body weight gain (g)
25.98 ± 0.32 8.22 ± 0.20
29.27 ± 0.64** 7.76 ± 0.61
27.45 ± 0.75 9.99 ± 0.63#
41.80 ± 2.16**, 20.36 ± 1.94**,
Perigonadal WAT (g) per gram of body weight (mg/g)
0.44 ± 0.02 15.95 ± 0.88
0.55 ± 0.07 18.68 ± 2.26
0.81 ± 0.11## 29.41 ± 3.96##
1.97 ± 0.37*, 47.57 ± 6.56*,
Perirenal WAT (g) Per gram of body weight (mg/g)
0.11 ± 0.01 4.08 ± 0.25
0.14 ± 0.02 4.84 ± 0.67
Subcutaneous WAT (g) Per gram of body weight (mg/g)
0.25 ± 0.02 9.57 ± 0.75
0.35 ± 0.06 11.79 ± 2.04
Liver (g) Per gram of body weight (mg/g)
1.04 ± 0.03 40.02 ± 1.34
1.19 ± 0.05* 40.69 ± 1.45
Blood/plasma Glucose (mg/dl) Insulin (ng/ml) Adiponectin (g/ml) Leptin (ng/ml)
122 0.84 38.6 4.0
± ± ± ±
10 0.11 1.7 0.6
109 1.34 17.4 5.6
± ± ± ±
5 0.18* 0.9** 0.7
0.17 ± 0.03# 6.18 + 0.79#
## ## ## ##
0.59 ± 0.10**, 14.14 ± 1.86**,
##
0.45 ± 0.06## 16.37 ± 1.97##
1.41 ± 0.27**, 34.04 ± 4.74**,
##
1.04 ± 0.04 38.08 ± 1.41
1.34 ± 0.05** 34.88 ± 1.38
122 0.71 33.5 6.8
± ± ± ±
7 0.15 4.1 0.5#
103 1.30 18.4 39.8
± ± ± ±
5 0.30 1.3** 8.6**,
##
##
##
Body weight gain was measured as the difference in body weight between 8- and 43-week-old mice. Blood glucose and plasma insulin, adiponectin, and leptin levels were measured in 43-week-old mice after 6 h of fasting. Values are mean ± SEM (male mice, n = 7; female mice, n = 9). * P < 0.05 compared with female mice fed the corresponding diet. ** P < 0.01 compared with female mice fed the corresponding diet. # P < 0.05 compared with corresponding sex-matched mice fed a standard diet. ## P < 0.01 compared with corresponding sex-matched mice fed a standard diet.
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
ARTICLE IN PRESS
ORCP-449; No. of Pages 9
6
Y. Kadota et al. A. Acaca
B. Fasn
80
**
60 40 20 female
120
100
100
**
80 60 40 20 0
male
female
D. Slc2a4
40 20 0
female
male
40 20
100
male
F. Nampt
100
80 60 40 20 0
female
120 relative mRNA expression
relative mRNA expression
relative mRNA expression
60
60
0
male
120
80
80
E. Adipoq
120 100
relative mRNA expression
100
0
C. Slc2a1
120 relative mRNA expression
relative mRNA expression
120
female
male
80 60 40 20 0
female
male
Figure 1 mRNA levels in perigonadal WAT of 8-week-old mice. RT-PCR was performed for (A) Acaca (ACC1-encoding gene), (B) Fasn (FAS-encoding gene), (C) Slc2a1 (GLUT1-encoding gene), (D) Slc2a4 (GLUT4-encoding gene), (E) Adipoq (adiponectin-encoding gene), and (F) Nampt (visfatin-encoding gene) in perigonadal WAT of 8-week-old mice. Data are presented as mean ± SEM (male mice, n = 5: female mice, n = 4). ** P < 0.01 compared with female mice.
in adipocyte differentiation, fatty acid synthesis, adipocyte enlargement, glucose metabolism, glucose uptake, insulin signaling, or lipid accumulation (listed in Table 1), were similar in the WAT of STDfed males and females (data not shown). We then compared gene expression between female and male mice fed a HFD for 35 weeks. Tellingly, the expression levels of the six genes identified above — Acaca, Fasn, Slc2a1, Slc2a4, Adipoq, and Nampt — in perigonadal WAT (Fig. 2A—F) were also higher in female mice than in male mice under HFD conditions.
Glucose metabolism capacity in 129/Sv mice In 8-week-old mice, blood glucose and plasma insulin levels were similar between males and females (Table 2). In 43-week-old mice, the insulin level, but not glucose level, became to be higher in males than females (Table 3). The t1/2 and maximum concentration (Cmax ) of glucose as evaluated by IPGTT showed no differences between females and males. However, the AUC0—2 h was slightly
lower in female mice than in male mice, suggesting that the glucose metabolism capacity is marginally greater in females than in males (Table 2). Even by the halfway point of their 43-week lifespan, 129/Sv male mice were already exhibiting HFD-induced obesity (data not shown). We accordingly hypothesized that changes in glucose metabolism would also be observed in mice fed a STD even at 21—24-weeks-old. The half-life for elimination of elevated blood glucose (t1/2 ) indeed was longer in male mice than in female mice, indicating that glucose absorption and metabolism were faster in female mice fed a STD (Table 4 and Fig. 3). In HFD-fed mice, the AUC0—3 h was significantly elevated in both 21—24-week-old males and females, though it was lower in females than in males (Table 4). Thus, t1/2 may correlate with age, and HFD-induced obesity at 24 weeks (data not shown) may affect AUC0—3 h .
Other parameters in 129/Sv mice Plasma adiponectin was slightly but significantly higher in female mice than in male mice fed a STD at
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
ARTICLE IN PRESS
ORCP-449; No. of Pages 9
Gene expression related to lipid and glucose metabolism in white adipose tissue
80 60 40 20
relative mRNA expression
120
*
100
0
80 60 40 20 0
STD
60 40 20
STD
120 relative mRNA expression
**
STD
HFD
80 60 40 20 0
**
**
HFD
80 60 40 20
40 20
female male female male STD
H
**
250
HFD
Female
Male
Acaca Fasn
200
Slc2a1
150
Slc2a4 100
Adipoq
50
Nampt
0 female male female male STD
60
HFD
300
0 female male female male
80
G. lep
100
**
100
0
F. Nampt
100
STD
80
**
120
female male female male
relative mRNA expression
*
*
0
E. Adipoq 120
D. Slc2a4
100
female male female male
HFD
*
120
*
100
female male female male
relative mRNA expression
C. Slc2a1
relative mRNA expression
**
120 relative mRNA expression
B. Fasn
**
relative mRNA expression
A. Acaca
7
female male female male
HFD
STD
HFD
36B4
Figure 2 mRNA levels in perigonadal WAT of mice fed a STD or a HFD for 35 weeks. RT-PCR was performed for (A) Acaca, (B) Fasn, (C) Slc2a1, (D) Slc2a4, (E) Adipoq, (F) Nampt, and (G) lep (leptin-encoding gene) in perigonadal WAT for mice fed a STD or a HFD for 35 weeks (n = 4 per group). (H) Representative bands of amplified PCR products. The gene expression levels were shown as percentages of value of STD-fed female mice. Data are presented as mean ± SEM for n = 4 mice per set. * P < 0.05 and ** P < 0.01 compared with female mice.
8 weeks old (Table 2). We also noted expression levels of Lep mRNA (Fig. 2G) and plasma leptin levels (Table 3) were up-regulated in HFD-fed mice. Since we have previously shown that plasma leptin correlates with WAT weight [7], we hypothesize lipid accumulation might mediate this up-regulation. In contrast, levels of blood glucose, plasma insulin, and adiponectin remained unchanged despite the consumption of a HFD for 35 weeks (Table 3). Plasma adiponectin was elevated in female mice but reduced in male mice fed a STD after 35 weeks (Tables 2 and 3). Table 4
Discussion Men have been observed to exhibit a higher prevalence of diabetes and early abnormalities in glucose metabolism than women do [8]. However, the factors responsible for the differences in the development of insulin resistance are largely unknown. In order to assign relative responsibilities to these factors, the present study compared factors associated with lipid accumulation and glucose metabolism in the WAT and blood of non-obese female and male mice fed a STD.
Parameters of glucose kinetics during an IPGTT in 21—24-week-old male and female mice.
Diet
Standard diet
Sex
Female
IPGTT C0 (mg/dl) Cmax (mg/dl) t1/2 (min) AUC0—3 h ((mg/dl) h)
63 157 50 64
± ± ± ±
3 9 4 23
High fat diet Male 60 130 110 73
± ± ± ±
Female 11 16 18* 25
64 243 78 190
± ± ± ±
2 17 2 24
Male 73 258 115 331
± ± ± ±
8 13 5** 51*
Parameters of glucose kinetics during an IPGTT were measured in 21—24-week-old mice after 12 h of fasting (n = 4 per group). Values are presented as the mean ± SEM. * P < 0.05 compared with diet-matched female mice. ** P < 0.01 compared with diet-matched female mice.
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
ORCP-449; No. of Pages 9
ARTICLE IN PRESS
8
Y. Kadota et al.
Figure 3 IPGTT for 21—24 week-old 129/Sv mice after 12 h fasting. IPGTTs were performed after 12 h of fasting on STD-fed female mice (open circles), STD-fed male mice (filled circles), HFD-fed female mice (open square) and HFD-fed male mice (filled square) (n = 4 per group). A glucose solution (2 g/10 ml/kg body weight) was administered intraperitoneally and blood glucose levels (mg/dl) were measured in venous blood at 0, 15, 30, 60, 120, and 180 min post-injection. Values are presented as mean ± SEM for n = 4 mice per set. ** P < 0.01 compared with HFD-fed female mice.
Rankinen et al. suggested that over 400 genes could affect obesity [2], and so it is impractical to analyze them all in one setting. From this large number, we picked 35 genes associated with lipid and glucose metabolism in WAT. We focused on gene expression changes in the WAT of 8-week-old 129/Sv mice and mice fed a STD or HFD for 35 weeks. The expression levels of four genes — Slc2a1, Slc2a4, Adipoq, and Nampt — were greater in the WAT of 43-week-old female mice than in corresponding male mice (Fig. 2), suggesting that females might have a greater ability to metabolize glucose in WAT regardless of diet fat content. However, expression levels of these four genes were similar in male and female mice at 8 weeks old (Fig. 1). Aging might affect also gene expression levels independently of diet. The expression of genes mediating glucose uptake by cells, such as Slc2a1 and Slc2a4, in WAT (Fig. 2C and D) was also greater in female mice than in male mice at 43 weeks regardless of diet fat content. GLUT1 is a ubiquitously expressed glucose transporter and GLUT4 is found in insulin-responsive tissues such as adipose tissue, skeletal muscle, and the heart [9]. GLUT4 proteins migrate to the plasma membrane in response to insulin stimulation, and play a role in cellular glucose absorption. The up-regulation of
both GLUT1 and GLUT4 gene expression levels are insulin-dependent [10]. Gene expression levels for Acaca and Fasn were higher in females than in males at both 8 weeks (Fig. 1A and B) and 43 weeks (Fig. 2A and B). Thus, the differences in expression of these genes between male and female mice appear at an earlier stage as compared with other genes. These gene expression levels may be associated with sex but not aging. ACC1 and FAS are rate-limiting enzymes in fatty acid synthesis, which creates triglycerides (TGs), meaning they contribute to the control of lipid homeostasis. A decrease in tissue TG levels may induce protein synthesis of ACC1 and FAS, which would lead to TG accumulation. We performed IPGTTs to measure the mice’s ability to metabolize glucose. The value of t1/2 was not significantly different between female and male mice at 8 weeks (Table 2). At 43 weeks, t1/2 was lower in females than in males fed a STD for 35 weeks (Table 4), suggesting that the capacity to metabolize glucose may change with age in a sexdependent manner. The insulin level of 43-week old mice fed a STD was higher in males than in females. This might also alter glucose metabolism of male mice as they grow older. In addition, consuming a HFD for 35 weeks significantly increased AUC0—3 h in male and female mice compared with mice fed a STD; moreover, the elevated AUC0—3 h was greater in male mice than in female mice (Table 4). Furthermore, t1/2 was greater in male mice compared with female mice fed a HFD (Table 4). These results demonstrate that a HFD may cause a decrease in the ability to metabolize glucose. Although further studies are required to elucidate the contributions of higher expression levels of GLUT1 and GLUT4 to the AUC0—3 h and t1/2 of female versus male mice (Fig. 2C and D), these results demonstrate that the ability of female mice to absorb and metabolize glucose is superior to that of male mice, even when fed a STD (Table 4). Male mice did not become obese after consuming a STD, but their glucose metabolism was nonetheless slower, and as a result, the males may potentially be vulnerable to weight gain. Glucose tolerance test results further emphasized the effects of diet on glucose metabolism for females compared with males. Genetic and other factors that contribute to such differences in glucose metabolism between males and females should be investigated further. In conclusion, we focused on the expression of 35 genes related to lipid metabolism, receptors for insulin signaling, and glucose metabolism out of over 400 obesity-related genes in the WAT of male and female mice. Expression of Acaca and Fasn in 8-week-old mice and four additional
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009
ORCP-449; No. of Pages 9
ARTICLE IN PRESS
Gene expression related to lipid and glucose metabolism in white adipose tissue genes — Slc2a1, Slc2a4, Adipoq and Nampt — in 43-week-old mice was higher in females compared with males. The relative gene expression ratios of female to male mice were similar between the STD and HFD groups at 43 weeks. The data suggest that sex and aging may cause differences in gene expression levels in females and males independently of diet. Since male and female mice fed a STD for 43 weeks did not become obese, obesity cannot be attributed to these observed gene expression changes alone. Nonetheless, the changes may be responsible for regulating sensitivity to HFD-induced obesity. Further studies on the mechanisms of candidate gene expression are required to elucidate factors involved in obesity development in males and females.
Acknowledgment This work was supported in part by JSPS KAKENHI Grant Numbers 24790133 to Y.K., 24590771 to T.K., and 21590144 to M.S.
References [1] van Kruijsdijk RC, van der Wall E, Visseren FL. Obesity and cancer: the role of dysfunctional adipose tissue. Cancer Epidemiol Biomarkers Prev 2009;18:2569—78.
9
[2] Rankinen T, Zuberi A, Chagnon YC, Weisnagel SJ, Argyropoulos G, Walts B, et al. The human obesity gene map: the 2005 update. Obesity (Silver Spring) 2006;14: 529—644. [3] Moralejo DH, Hansen CT, Treuting P, Hessner MJ, Fuller JM, Van Yserloo B, et al. Differential effects of leptin receptor mutation on male and female BBDR Gimap5−/Gimap5− spontaneously diabetic rats. Physiol Genomics 2010;41:9—20. [4] Wallenius V, Wallenius K, Ahren B, Rudling M, Carlsten H, Dickson SL, et al. Interleukin-6-deficient mice develop mature-onset obesity. Nat Med 2002;8:75—9. [5] Netea MG, Joosten LA, Lewis E, Jensen DR, Voshol PJ, Kullberg BJ, et al. Deficiency of interleukin-18 in mice leads to hyperphagia, obesity and insulin resistance. Nat Med 2006;12:650—6. [6] Geer EB, Shen W. Gender differences in insulin resistance, body composition, and energy balance. Gend Med 2009;6(Suppl. 1):60—75. [7] Sato M, Kawakami T, Kondoh M, Takiguchi M, Kadota Y, Himeno S, et al. Development of high-fat-diet-induced obesity in female metallothionein-null mice. FASEB J 2010;24:2375—84. [8] Kuhl J, Hilding A, Ostenson CG, Grill V, Efendic S, Bavenholm P. Characterisation of subjects with early abnormalities of glucose tolerance in the Stockholm Diabetes Prevention Programme: the impact of sex and type 2 diabetes heredity. Diabetologia 2005;48:35—40. [9] Wood IS, Trayhurn P. Glucose transporters (GLUT and SGLT): expanded families of sugar transport proteins. Br J Nutr 2003;89:3—9. [10] McGowan KM, Long SD, Pekala PH. Glucose transporter gene expression: regulation of transcription and mRNA stability. Pharmacol Ther 1995;66:465—505.
Available online at www.sciencedirect.com
ScienceDirect
Please cite this article in press as: Kadota Y, et al. Gene expression related to lipid and glucose metabolism in white adipose tissue. Obes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.orcp.2015.04.009