Just Accepted by International Journal of Neuroscience
Long Term Metabolic Alterations in a Febrile Seizure Model Duanhe Heng, Zhongcheng Wang, Yuanteng Fan, Liang Li, Jian Fang, Song Han, Jun Yin, Biwen Peng, Wanhong Liu, Xiaohua He doi:10.3109/00207454.2015.1018385
Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
Abstract Objective: Febrile seizures (FS) are the most common neurological disease in infancy and early childhood, it can lead to metabolic changes and have long-term health implications. Aim of this study was to investigate the long-term effects of FS on metabolism. Methods: We measured certain metabolic parameters in hyperthermia-prone (HP) rats, which were developed using a selective breeding process and showed a lower seizure threshold than wild-type (WT) rats. Body weight, body length, abdominal circumference and the levels of fasting blood glucose, serum triglyceride and total cholesterol concentrations were analyzed. The mRNA expression of genes involved in glucose and lipid metabolism was determined by qPCR and the histone methylation level in the liver was determined by western blot. Results: We found that the body weight of the HP rats was significantly lower than that of the WT rats. Similarly, the fasting blood glucose and serum triglyceride levels were lower in the HP group compared with the WT group. These changes were accompanied by increased mRNA expression of genes such as phosphoenolpyruvate carboxykinase (PEPCK) and carnitine palmitoyl transferase-1 (CPT-1), but not peroxisome proliferator-activated receptor α (PPARα). We also found tri-methylation of histone 3 at Lys9 and Lys27 was decreased in the HP group. Conclusions: These data may suggest an underlying mechanism by which FS have a long-term effect on energy metabolism via histone methylation.
© 2015 Informa Healthcare USA, Inc. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
Long Term Metabolic Alterations in a Febrile Seizure Model Duanhe Heng1#,Zhongcheng Wang1#, Yuanteng Fan1 , Liang Li1, Jian Fang1,
1
TE D
Song Han1, Jun Yin1, Biwen Peng2, Wanhong Liu2, Xiaohua He1,2§
Department of Pathophysiology, School of Basic Medical Sciences, Wuhan
2
Hubei Provincial Key Laboratory of Developmentally Originated Disease,
§
C EP
School of Basic Medical Sciences, Wuhan University, Wuhan, China
Corresponding authors. Dr. Xiaohua He: School of Basic Medical Sciences,
AC
Wuhan University, Donghu Road No. 185, Wuchang, Wuhan 430071, China. Tel.:86-27-68759985. Fax: 86-27-68759991. E-mail: [email protected].
These authors contributed equally to this work.
ST
#
Abstract
JU
Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
University, Wuhan, China
Objective: Febrile seizures (FS) are the most common neurological
disease in infancy and early childhood, it can lead to metabolic changes and have long-term health implications. Aim of this study was to investigate the long-term effects of FS on metabolism. Methods: We measured certain metabolic parameters in hyperthermia-prone (HP) rats, which were developed using a selective breeding process and showed a 1
lower seizure threshold than wild-type (WT) rats. Body weight, body length, abdominal circumference and the levels of fasting blood glucose, serum triglyceride and total cholesterol concentrations were analyzed.
TE D
The mRNA expression of genes involved in glucose and lipid metabolism was determined by qPCR and the histone methylation level in the liver
of the HP rats was significantly lower than that of the WT rats. Similarly,
C EP
the fasting blood glucose and serum triglyceride levels were lower in the HP group compared with the WT group. These changes were
accompanied by increased mRNA expression of genes such as
AC
phosphoenolpyruvate carboxykinase (PEPCK) and carnitine palmitoyl transferase-1 (CPT-1), but not peroxisome proliferator-activated receptor α (PPARα). We also found tri-methylation of histone 3 at Lys9 and Lys27
ST
was decreased in the HP group. Conclusions: These data may suggest an underlying mechanism by which FS have a long-term effect on energy metabolism via histone methylation.
JU
Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
was determined by western blot. Results: We found that the body weight
Key
words:
Febrile
seizures,
metabolism,
methylation
2
epigenetics,
histone
Introduction Febrile seizures (FS) are the most common disease of the nervous system in the pediatric population [1], affecting approximately 2-5% of
TE D
all children and usually occurring between 3 months and 5 years of age
[2]. During FS, the central nervous system appears to be overexcited, and
C EP
Meanwhile, the rates of glucose metabolism and neuronal oxygen
consumption increase [4]. FS can also cause malfunctions in the brain and result in various metabolic disorders in peripheral tissue [5]. For example, FS are frequently associated with stress hyperglycemia in children, which
AC
is associated with a high risk for the development of type I diabetes [6, 7]. The coexistence of seizures or epilepsy and type 1 diabetes has been observed in epidemiological surveys [8]. Furthermore, decreased blood
ST
glucose levels were detected in the kainite-induced epilepsy model, and blood glucose levels modulate seizure sensitivity [9]. Other research showed that an increasing seizure duration might be associated with
JU
Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
the sensitivity of brain cells to internal and external stimuli increases [3].
aberrant glucose levels [10]. Other studies have suggested that the serum sodium, calcium and blood glucose levels in children with FS are generally abnormal [11]. However, there have been few studies on the long-term effects of FS on metabolism. Altered phenotypes and metabolism can involve changes in the epigenome [12], including altered DNA methylation and histone 3
modification [13]. For example, histone methyltransferases and demethylases, which correlate with metabolic abnormalities in certain metabolic disorders such as type-2 diabetes and obesity, can connect
TE D
metabolism and transcription [14, 15]. PEPCK and CPT-1 are the rate-limiting enzymes in gluconeogenesis and fatty acid oxygenolysis,
triglyceride metabolism. PEPCK is transcriptionally regulated by histone
C EP
H3 lysine 27 trimethylation (H3K27me3) [16]. The mRNA expression of
hepatic PPARα and CPT-1, which play important roles in glucose and lipid metabolism, is regulated by DNA methyltransferase activity [17].
processes.
AC
Therefore, epigenetic modifications potentially regulate metabolic
We previously developed a rat model of FS with a lower seizure
ST
threshold, the hyperthermia-prone (HP) rat [18, 19]. Using this rat model, we identified numerous FS susceptibility genes related to metabolism [20]. In this study, we measured particular metabolic parameters and the
JU
Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
respectively. Therefore, they are critical for fasting glucose and
expression of key genes that are related to metabolism in HP and wild-type (WT) rats. We aimed to determine whether the differences between the two groups were induced by epigenetic changes that altered specific metabolites.
4
Materials and Methods Ethics Statement All the protocols complied with the recommendations in the Guide for
TE D
the Care and Use of Laboratory Animals, Biosafety Level 3 (ABSL-3), and were approved by the Animal Ethics Committee of Wuhan University
C EP
suffering. Animals and Sample Collection
WT Sprague-Dawley rats (n=12) were obtained from the ABSL-3
AC
Laboratory. Unless otherwise noted, 14-day-old HP and WT rats were used for the experiments. Rats were housed individually on a 12-h light-dark cycle with ad libitum access to food and water. The HP pups
ST
(n=12) were randomly selected from the HP rat strains we built previously [19]. The body weight of the two groups was measured daily from PN14 to PN23. From 4th week to 16th week, the body weight, body
JU
Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
(Permit Number: SCXK 2008-0004). All efforts were made to minimize
length and abdominal circumference of each rat were measured each week. Blood samples were collected from the 24 rats via the tail vein to measure metabolism markers at 1 month and 4 months of age. After 4 months, all the animals were sacrificed after anesthesia with chloral hydrate. The liver was dissected and frozen in liquid nitrogen.
5
Analysis of Metabolic Parameters In all the groups, tail vein blood was collected to analyze the metabolic parameters. The tail vein blood of the rats was collected after a
TE D
night of fasting subjects. Fasting blood glucose levels were immediately measured using a Roche glucometer (Roche Diagnostics GmbH,
C EP
ice and centrifuged (3000 rpm, 15 min) to obtain serum. Serum triglyceride and total cholesterol levels were then measured using commercial assay kits (Mingdian, Shanghai, China). The catalog numbers
Real-Time PCR
AC
are triglyceride (T010M0120) and total cholesterol (C011M0120).
Total RNA was isolated from livers, and 1 μg was used as a template to prepare cDNA. cDNA was amplified using real-time PCR primers using a
ST
RevertAidTM First Strand cDNA Synthesis Kit (Thermo Scientific, Pittsburgh, USA). The following primers were used to selectively amplify regions of the β-actin, PEPCK, CPT-1 or PPARα promoters: β-actin,
JU
Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
Mannheim, Germany), and the remaining blood samples were stored on
5’-CACGATGGAGGGGCCGGACTCATC-3’
and
5’-TAAAGACCTCTATGCCAACACAGT-3’;
PEPCK,
5’-AGCTGCATAATGGTCTGG-3’
and
5’-GAACCTGGCGTTGAATGC-3’;
CPT-1,
5’-ACCACTGGCCGAATGTCAAG-3’ 5’-AGCGAGTAGCGCATGGTCAT-3’; 6
and and
PPARα,
and
5’-CGGGTCATACTCGCAGGAAAG-3’
5’-TGGCAGCAGTGGAAGAATCG-3’. All primers were designed by Primer Premier 5.0 software. Using a Real Master mix (SYBR Green) Kit
TE D
(GeneCopoeia, China) and an iQ5 Real Time PCR Detection System (Bio-Rad, US), qRT-PCR was performed according to the manufacturer’s
analysis. Rat β-actin was always amplified in parallel with the
C EP
experimental genes. The relative expression ratio was determined by the 2-△△Ct method. Western Blot
AC
After anesthesia, freshly isolated liver tissues were collected, and the protein content was determined using a BCA Protein Quantitation Kit (Thermo Scientific, Pittsburgh, USA). Twenty micrograms of protein was
ST
loaded per lane. After separation in SDS-PAGE gel, the protein was transferred to nitrocellulose paper, and nonspecific protein binding sites were blocked with 5% bovine serum albumin. The blots were incubated
JU
Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
protocols. Single PCR products were further verified by melt curve
with primary antibodies overnight at 4 ℃ . All primary antibodies (H3K4me3(ab8580), H3K9me3(ab8898), H3K27me3(mab6002) and β-actin(mab8226)) were purchased from Abcam. The primary antibody dilution buffer was 3% bovine serum albumin. The blots were subsequently incubated for 2 h with a horseradish peroxidase-conjugated secondary antibody (1:20,000; Abmart). Immunoreactivity was visualized 7
using an ECL detection system (Thermo Scientific, Pittsburgh, USA). Data were quantified by using the Quantity One software. Statistical Methods
TE D
The data were analyzed with SPSS 17.0 software to evaluate the
differences between the two groups. The data were presented as the mean
C EP
weight, body length and abdominal circumference comparisons between the groups were analyzed by repeated measures ANOVA. Fasting blood glucose, serum triglyceride and total cholesterol levels as well as the
Results
AC
real-time PCR results were analyzed using a t test.
Body Weight, Body Length, and Abdominal Circumference
ST
The body weight of infant rats was lower in the HP group (n=12) than in the WT group from PN14 to PN23 (P
Long Term Metabolic Alterations in a Febrile Seizure Model Duanhe Heng, Zhongcheng Wang, Yuanteng Fan, Liang Li, Jian Fang, Song Han, Jun Yin, Biwen Peng, Wanhong Liu, Xiaohua He doi:10.3109/00207454.2015.1018385
Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
Abstract Objective: Febrile seizures (FS) are the most common neurological disease in infancy and early childhood, it can lead to metabolic changes and have long-term health implications. Aim of this study was to investigate the long-term effects of FS on metabolism. Methods: We measured certain metabolic parameters in hyperthermia-prone (HP) rats, which were developed using a selective breeding process and showed a lower seizure threshold than wild-type (WT) rats. Body weight, body length, abdominal circumference and the levels of fasting blood glucose, serum triglyceride and total cholesterol concentrations were analyzed. The mRNA expression of genes involved in glucose and lipid metabolism was determined by qPCR and the histone methylation level in the liver was determined by western blot. Results: We found that the body weight of the HP rats was significantly lower than that of the WT rats. Similarly, the fasting blood glucose and serum triglyceride levels were lower in the HP group compared with the WT group. These changes were accompanied by increased mRNA expression of genes such as phosphoenolpyruvate carboxykinase (PEPCK) and carnitine palmitoyl transferase-1 (CPT-1), but not peroxisome proliferator-activated receptor α (PPARα). We also found tri-methylation of histone 3 at Lys9 and Lys27 was decreased in the HP group. Conclusions: These data may suggest an underlying mechanism by which FS have a long-term effect on energy metabolism via histone methylation.
© 2015 Informa Healthcare USA, Inc. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
Long Term Metabolic Alterations in a Febrile Seizure Model Duanhe Heng1#,Zhongcheng Wang1#, Yuanteng Fan1 , Liang Li1, Jian Fang1,
1
TE D
Song Han1, Jun Yin1, Biwen Peng2, Wanhong Liu2, Xiaohua He1,2§
Department of Pathophysiology, School of Basic Medical Sciences, Wuhan
2
Hubei Provincial Key Laboratory of Developmentally Originated Disease,
§
C EP
School of Basic Medical Sciences, Wuhan University, Wuhan, China
Corresponding authors. Dr. Xiaohua He: School of Basic Medical Sciences,
AC
Wuhan University, Donghu Road No. 185, Wuchang, Wuhan 430071, China. Tel.:86-27-68759985. Fax: 86-27-68759991. E-mail: [email protected].
These authors contributed equally to this work.
ST
#
Abstract
JU
Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
University, Wuhan, China
Objective: Febrile seizures (FS) are the most common neurological
disease in infancy and early childhood, it can lead to metabolic changes and have long-term health implications. Aim of this study was to investigate the long-term effects of FS on metabolism. Methods: We measured certain metabolic parameters in hyperthermia-prone (HP) rats, which were developed using a selective breeding process and showed a 1
lower seizure threshold than wild-type (WT) rats. Body weight, body length, abdominal circumference and the levels of fasting blood glucose, serum triglyceride and total cholesterol concentrations were analyzed.
TE D
The mRNA expression of genes involved in glucose and lipid metabolism was determined by qPCR and the histone methylation level in the liver
of the HP rats was significantly lower than that of the WT rats. Similarly,
C EP
the fasting blood glucose and serum triglyceride levels were lower in the HP group compared with the WT group. These changes were
accompanied by increased mRNA expression of genes such as
AC
phosphoenolpyruvate carboxykinase (PEPCK) and carnitine palmitoyl transferase-1 (CPT-1), but not peroxisome proliferator-activated receptor α (PPARα). We also found tri-methylation of histone 3 at Lys9 and Lys27
ST
was decreased in the HP group. Conclusions: These data may suggest an underlying mechanism by which FS have a long-term effect on energy metabolism via histone methylation.
JU
Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
was determined by western blot. Results: We found that the body weight
Key
words:
Febrile
seizures,
metabolism,
methylation
2
epigenetics,
histone
Introduction Febrile seizures (FS) are the most common disease of the nervous system in the pediatric population [1], affecting approximately 2-5% of
TE D
all children and usually occurring between 3 months and 5 years of age
[2]. During FS, the central nervous system appears to be overexcited, and
C EP
Meanwhile, the rates of glucose metabolism and neuronal oxygen
consumption increase [4]. FS can also cause malfunctions in the brain and result in various metabolic disorders in peripheral tissue [5]. For example, FS are frequently associated with stress hyperglycemia in children, which
AC
is associated with a high risk for the development of type I diabetes [6, 7]. The coexistence of seizures or epilepsy and type 1 diabetes has been observed in epidemiological surveys [8]. Furthermore, decreased blood
ST
glucose levels were detected in the kainite-induced epilepsy model, and blood glucose levels modulate seizure sensitivity [9]. Other research showed that an increasing seizure duration might be associated with
JU
Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
the sensitivity of brain cells to internal and external stimuli increases [3].
aberrant glucose levels [10]. Other studies have suggested that the serum sodium, calcium and blood glucose levels in children with FS are generally abnormal [11]. However, there have been few studies on the long-term effects of FS on metabolism. Altered phenotypes and metabolism can involve changes in the epigenome [12], including altered DNA methylation and histone 3
modification [13]. For example, histone methyltransferases and demethylases, which correlate with metabolic abnormalities in certain metabolic disorders such as type-2 diabetes and obesity, can connect
TE D
metabolism and transcription [14, 15]. PEPCK and CPT-1 are the rate-limiting enzymes in gluconeogenesis and fatty acid oxygenolysis,
triglyceride metabolism. PEPCK is transcriptionally regulated by histone
C EP
H3 lysine 27 trimethylation (H3K27me3) [16]. The mRNA expression of
hepatic PPARα and CPT-1, which play important roles in glucose and lipid metabolism, is regulated by DNA methyltransferase activity [17].
processes.
AC
Therefore, epigenetic modifications potentially regulate metabolic
We previously developed a rat model of FS with a lower seizure
ST
threshold, the hyperthermia-prone (HP) rat [18, 19]. Using this rat model, we identified numerous FS susceptibility genes related to metabolism [20]. In this study, we measured particular metabolic parameters and the
JU
Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
respectively. Therefore, they are critical for fasting glucose and
expression of key genes that are related to metabolism in HP and wild-type (WT) rats. We aimed to determine whether the differences between the two groups were induced by epigenetic changes that altered specific metabolites.
4
Materials and Methods Ethics Statement All the protocols complied with the recommendations in the Guide for
TE D
the Care and Use of Laboratory Animals, Biosafety Level 3 (ABSL-3), and were approved by the Animal Ethics Committee of Wuhan University
C EP
suffering. Animals and Sample Collection
WT Sprague-Dawley rats (n=12) were obtained from the ABSL-3
AC
Laboratory. Unless otherwise noted, 14-day-old HP and WT rats were used for the experiments. Rats were housed individually on a 12-h light-dark cycle with ad libitum access to food and water. The HP pups
ST
(n=12) were randomly selected from the HP rat strains we built previously [19]. The body weight of the two groups was measured daily from PN14 to PN23. From 4th week to 16th week, the body weight, body
JU
Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
(Permit Number: SCXK 2008-0004). All efforts were made to minimize
length and abdominal circumference of each rat were measured each week. Blood samples were collected from the 24 rats via the tail vein to measure metabolism markers at 1 month and 4 months of age. After 4 months, all the animals were sacrificed after anesthesia with chloral hydrate. The liver was dissected and frozen in liquid nitrogen.
5
Analysis of Metabolic Parameters In all the groups, tail vein blood was collected to analyze the metabolic parameters. The tail vein blood of the rats was collected after a
TE D
night of fasting subjects. Fasting blood glucose levels were immediately measured using a Roche glucometer (Roche Diagnostics GmbH,
C EP
ice and centrifuged (3000 rpm, 15 min) to obtain serum. Serum triglyceride and total cholesterol levels were then measured using commercial assay kits (Mingdian, Shanghai, China). The catalog numbers
Real-Time PCR
AC
are triglyceride (T010M0120) and total cholesterol (C011M0120).
Total RNA was isolated from livers, and 1 μg was used as a template to prepare cDNA. cDNA was amplified using real-time PCR primers using a
ST
RevertAidTM First Strand cDNA Synthesis Kit (Thermo Scientific, Pittsburgh, USA). The following primers were used to selectively amplify regions of the β-actin, PEPCK, CPT-1 or PPARα promoters: β-actin,
JU
Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
Mannheim, Germany), and the remaining blood samples were stored on
5’-CACGATGGAGGGGCCGGACTCATC-3’
and
5’-TAAAGACCTCTATGCCAACACAGT-3’;
PEPCK,
5’-AGCTGCATAATGGTCTGG-3’
and
5’-GAACCTGGCGTTGAATGC-3’;
CPT-1,
5’-ACCACTGGCCGAATGTCAAG-3’ 5’-AGCGAGTAGCGCATGGTCAT-3’; 6
and and
PPARα,
and
5’-CGGGTCATACTCGCAGGAAAG-3’
5’-TGGCAGCAGTGGAAGAATCG-3’. All primers were designed by Primer Premier 5.0 software. Using a Real Master mix (SYBR Green) Kit
TE D
(GeneCopoeia, China) and an iQ5 Real Time PCR Detection System (Bio-Rad, US), qRT-PCR was performed according to the manufacturer’s
analysis. Rat β-actin was always amplified in parallel with the
C EP
experimental genes. The relative expression ratio was determined by the 2-△△Ct method. Western Blot
AC
After anesthesia, freshly isolated liver tissues were collected, and the protein content was determined using a BCA Protein Quantitation Kit (Thermo Scientific, Pittsburgh, USA). Twenty micrograms of protein was
ST
loaded per lane. After separation in SDS-PAGE gel, the protein was transferred to nitrocellulose paper, and nonspecific protein binding sites were blocked with 5% bovine serum albumin. The blots were incubated
JU
Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
protocols. Single PCR products were further verified by melt curve
with primary antibodies overnight at 4 ℃ . All primary antibodies (H3K4me3(ab8580), H3K9me3(ab8898), H3K27me3(mab6002) and β-actin(mab8226)) were purchased from Abcam. The primary antibody dilution buffer was 3% bovine serum albumin. The blots were subsequently incubated for 2 h with a horseradish peroxidase-conjugated secondary antibody (1:20,000; Abmart). Immunoreactivity was visualized 7
using an ECL detection system (Thermo Scientific, Pittsburgh, USA). Data were quantified by using the Quantity One software. Statistical Methods
TE D
The data were analyzed with SPSS 17.0 software to evaluate the
differences between the two groups. The data were presented as the mean
C EP
weight, body length and abdominal circumference comparisons between the groups were analyzed by repeated measures ANOVA. Fasting blood glucose, serum triglyceride and total cholesterol levels as well as the
Results
AC
real-time PCR results were analyzed using a t test.
Body Weight, Body Length, and Abdominal Circumference
ST
The body weight of infant rats was lower in the HP group (n=12) than in the WT group from PN14 to PN23 (P
