proteomic changes in the hypothalamus and retroperitoneal fat from male f344 rats subjected to...

RESEARCH ARTICLE

Proteomic changes in the hypothalamus and

retroperitoneal fat from male F344 rats subjected

to repeated light–dark shifts

Archana Mishra1, Chung-Hsien Cheng2, Wen-Chien Lee2 and Ling-Ling Tsai1

1 Department of Psychology, National Chung Cheng University, Chia-yi, Taiwan, ROC2 Department of Chemical Engineering, National Chung Cheng University, Chia-yi, Taiwan, ROC

Received: October 17, 2008

Revised: April 29, 2009

Accepted: May 20, 2009

Chronic circadian desynchronization induced by repeated 12 h light–dark cycle shifts conducted

twice weekly resulted in elevated food intake, body weight gain, and retroperitoneal fat mass in

male F344 rats. Using a proteomic approach, we found that repeated light–dark shifts caused

changes in expression levels of five hypothalamic (four upregulated) and 22 retroperitoneal fat

(13 upregulated) 2-DE protein spots. Proteins involved in carbohydrate metabolism and in the citric

acid cycle were upregulated, indicating a positive energy balance status. In addition, the hypotha-

lamic gamma-amino butyric acid (GABA) aminotransferase was upregulated, thus suggesting a

connection between the brain GABAeric system and the modulation of food intake. Furthermore,

the upregulation of fatty acid-binding protein 4 and the downregulation of 78 kDa glucose-regulated

protein in the fat implicated the development of insulin resistance. We observed the upregulation of

two antioxidant enzymes that might serve as protection against insulin dysfunction associated with

oxidative stress. Finally, the downregulation of hypothalamic voltage-dependent anion-selective

channel protein 1 and fat ATP synthase suggested a reduction in synthesis of mitochondrial ATP.

These findings are in partial agreement with those of studies of obesity induced by genotype and a

high-fat diet.

Keywords:

Animal proteomics / Circadian rhythms / Energy intake / Fat / Obese / Shift work

1 Introduction

Many workers regularly or irregularly work a shift other than a

normal daytime shift. Although there has been little change in

the proportion of those working alternate shifts over the last

three decades, i.e. 15–17% from 1973–1978 [1], 16% in 1985 [2],

17% in 1997 [3], and 18% in 2004 [4] in the US, the number of

shift workers increased to ten million from 1985 to 2004, when

the population of full-time workers is considered. The percen-

tage of shift workers in Europe is 11–23% [5] and is probably

quite similar among other industrialized countries. In contrast

to the advantages that work shift systems provide to employers

and to people who need round-the-clock services, the practice

has repeatedly been reported to have adverse effects on workers

and their families. Obesity is one of the most widespread public

health problems today and most of the survey studies suggest

that shift work is associated with metabolic changes. These

include increased weight gain [6], body mass index [7–13], waist-

to-hip ratio [12–14], and prevalence of overweight [15] and

obesity [7, 16]. The increased rate of these measures in meta-

bolism is also correlated with the duration of shift work status

[9, 11, 13, 15].

The circadian rhythm system can be entrained

(synchronized) to a regular schedule of the Zeitgeber

Abbreviations: FABP4, fatty acid-binding protein 4; GABA,

gamma-aminobutyric acid; GAPDH, glyceraldehyde-3-phos-

phate dehydrogenase; GRP78, 78 kDa glucose-regulated protein;

HT, hypothalamus; LC, control group; LD, light–dark; LS,

repeated light–dark shift group; SCN, suprachiasmatic nucleus;

VDAC1, voltage-dependent anion-selective channel protein 1

Correspondence: Dr. Ling-Ling Tsai, Department of Psychology,

National Chung Cheng University, 168 University Road,

Minhsiung Township, Chiayi County 62102, Taiwan, ROC

E-mail: [email protected]

Fax: 1886-5-2420857

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Proteomics 2009, 9, 4017–4028 4017DOI 10.1002/pmic.200800813

(time cue, e.g. light–dark (LD) cycle, working/activity time,

meal time, etc.) and become reentrained after several days

when a sudden shift of the Zeitgeber’s phase occurs [17, 18].

In addition, the speed of reentrainment to a new LD cycle is

different between the central circadian pacemaker located in

the suprachiasmatic nucleus (SCN) of the hypothalamus

(HT) and behavioral and physiological rhythms supposedly

regulated by the SCN [19], between different behavioral/

physiological rhythms [20], and even between SCN subdi-

visions [21]. In response to a new feeding cycle, circadian

expression of clock genes such as Per1 in peripheral circa-

dian oscillators also takes several periods to be synchronized

with different speeds in different organs [22, 23]. If the

mealtime is in conflict with the LD cycle, e.g. when food

availability is restricted in the middle of the light period (the

rest period in nocturnal mice and rats), two uncoupled

pacemakers occur: one entrained to mealtime and the other

to the LD cycle. The clock gene expression rhythm in the

liver and other organs is reset gradually and stabilizes

eventually in response to a new mealtime; whereas, the

circadian phase in the SCN is not affected [22–24]. Conco-

mitantly, wheel running activity shows two split rhythms:

one with an intense activity bout before mealtime and the

other with activity mainly occurring in the dark period [25].

Most shift work schedules involve chronic night shifts

and/or shift rotations. Thus, continual practice of shift work

may give rise to disturbances in circadian rhythm systems,

i.e. desynchronization, as long as permanent reentrainment

is not achieved, due to a fast speed of shift rotation or phase

conflicts between different Zeitgebers, e.g. the LD cycle and

mealtime, particularly when workers work at unusual hours

such as nighttime. Experimental evidence supporting the

causal connection between chronic circadian desynchroni-

zation and the consequence of metabolic changes is

presented by two animal paradigms of circadian desyn-

chronization, induced by genetic mutations in the central

clock genes and by artificial manipulations of the Zeitge-

ber’s phase. A mutation in the clock gene Clock or loss of

another clock gene, Bmal1, in mice results in impaired

entrainment of activity rhythm to the LD cycle [26, 27] and

metabolic changes [28–31]. On the other hand, the shifting

of the LD cycle by several hours once or more weekly,

concomitant with ad lib feeding, fixed mealtime, or shifted

mealtime can induce desynchronization in activity and

temperature rhythms in laboratory animals [32–34].

Furthermore, an activity schedule regularly enforced in the

light period or shifting 12 h twice weekly can also induce

disturbances in circadian rhythms [35, 36]. Chronic

manipulations in lighting, feeding, and/or activity sche-

dules, as described above, result in changes in metabolic

measures such as weight gain, food intake, and activity

levels [32–38].

In our previous study [33], we found that repeated 12 h LD

cycle shifts twice weekly resulted in elevated body weight gain

and food intake, and reduced activity levels in male F344 rats.

In this study, we aimed to identify protein molecules in the

HT and retroperitoneal fat specifically related to chronic

circadian desynchronization in the same rat strain under-

going the same shifting schedule of the LD cycle as that

reported in the previous study [33]. The retroperitoneal fat is

one of major abdominal fat depots and composed mainly by

white adipose tissue. The HT is a major homeostatic regu-

latory center involved in body energy balance in the brain. It

receives and integrates the information of energy status from

different sensor mechanisms in response to external cues of

food availability and/or internal metabolic sensory cues of

energy stores and then activates effector mechanisms to

adjust energy intake and expenditure in order to maintain

body energy homeostasis (reviewed in [39, 40]). In addition,

glucose-sensing neurons are found in the lateral and

ventromedial nuclei of the HT; they also respond to fatty

acids and hormones from the periphery (reviewed in [41]). On

the other hand, the adipose tissue is known as a major energy

store, and an endocrine and secretory organ; it releases the

adiposity signal leptin that runs through the circulation

system and interacts with the neurons within the arcuate

nucleus and other HT nuclei associated with energy regula-

tion [39, 40, 42, 43]. The action of leptin in the HT results in

reduced food intake, increased energy expenditure, and

increased lipolytic action in adipose tissues. The brain action

of ghrelin, synthesized primarily in the stomach, and several

hypothalamic neuropeptides, such as neuropeptide Y,

expressed in the arcuate nucleus can also modulate lipid

metabolism and glucose utilization in the fat (reviewed in

[43]). In light of such an intimate connection between the HT

and adipose tissue regarding the regulation of energy

homeostasis, a proteomic approach was applied to reveal the

protein profiles in the HT and adipose tissue that varied with

metabolic changes induced by repeated LD shifts.

2 Material and methods

2.1 Animals

The experimental (LS) and control (LC) rat pairs were

matched on age and body weight recorded at the baseline.

The procedure of LD shifts has previously been described in

detail [33]. Briefly, specific pathogen-free, male inbred rats

(F344/NNarl) aged 6–8 wk were purchased from the

National Laboratory Animal Center, Taipei, Taiwan, ROC.

When the rats were 11–12 wk old, they received aseptic

surgery of sham or temperature/activity transponders (E-

Mitter, Mini-Mitter, Bend, OR, USA) implantation in the

abdomen under isoflurane anesthesia. After 1 wk, when all

of the rats showed a body weight close to the pre-surgical

level, they were subjected to weekly measurements of body

weight, food intake on a regular diet (3.25 kcal/g), and water

intake throughout the experimental period. This included a

1 wk baseline with light exposure from 02:00–14:00, 13 wk of

12 h shifts twice a week in the LD cycle for the LS rats, and

13 wk of a fixed LD cycle except the lights were extended to

4018 A. Mishra et al. Proteomics 2009, 9, 4017–4028


24 h every Monday for the LC rats in order to maintain a

weekly light exposure duration equal to that received by the

LS rats (Fig. 1). Both LC and LS rats then underwent 1–3 wk

of a recovery period with the same lighting schedule as that

at the baseline until the daily activity/temperature rhythms

in the LS rats showed reentrainment to the LD cycle

(see Fig. 1). Subsequently, they were killed at 09:00 under

isoflurane without undergoing fasting (for brain samples) or

with 19 h of fasting (for fat samples). The retroperitoneal fat

depot was dissected out from each side of the body. The

kidney and adrenal gland connected to the retroperitoneal

depot were carefully removed prior to weighing the depot.

All brain and fat samples were stored at �701C. All animal

facilities and care followed the guidelines provided by the

Guild for the Care and Use of Laboratory Animals (National

Academy Press, Washington, DC, 1996), and all procedures

and experimental protocols were approved by the National

Chung Cheng University Animal Care and Use Committee.

2.2 Tissue preparation

The frozen rat brain was placed on its dorsal surface on an

aluminum foil paper kept cold on powdered dry ice and a

frontal section was made by the use of a surgical blade. The

HT block was bounded anteriorly at the optic chaism and

posteriorly at the mammillary body and extended dorsally to

the columns of the fornix (anterior) or the mammilotha-

lamic tracts (posterior) and laterally to the telencephalic–

diencephalic junction. The HT samples obtained from six LS

and LC rat pairs and the retroperitoneal fat samples obtained

from eight LS and LC rat pairs were each crushed in a

mortar in the presence of liquid nitrogen. The tissue powder

was then loaded in a microcentrifuge tube and homogenized

using a pestle in 200mL (for HT) or a 1:2 w/v ratio (for

adipose tissue) of lysis buffer containing urea (7 M), thiourea

(2 M), CHAPS (4% w/v), DTT (50 mM), and Bio-Lyte

ampholyte, pH 3–10, (0.2%). The HT suspension was then

centrifuged at 21 000� g at 121C for 72.0 min and the

supernatant (mean protein concentration, 5.472.0 mg/mL,

was collected. For the fat sample, the tissue suspension was

centrifuged at 21 000� g at 181C for 30 min and the interface

between the low-density lipid layer and the insoluble pellet

was carefully collected and spun down again [44]. Total

protein content was quantified based on the method of

Bradford: Protein Assay Dye Reagent Concentrate (BioRad)

was added to the tissue protein solution, it was measured at

595 nm with a spectrophotometer, and the results were

compared with a linear BSA (Sigma-Aldrich) standard curve.

Protein extracts were stored at �801C. When not specified,

the chemical reagents used in each buffer solution were

purchased from Merck or Sigma-Aldrich.

2.3 2-DE

For each 2-DE experiment, a pair of protein samples of the

same tissues randomly selected from one LS and one LC rat

were performed in parallel. Thus, there were six and eight

pairs of 2-DE gels run for the HT and fat, respectively. A

350 mL aliquot of rehydration buffer containing protein

extracts (100 mg), urea (7 M), thiourea (2 M), CHAPS

(2% w/v), DTT (50 mM), and Bio-Lyte ampholyte, pH 3–10,

(0.2%) was applied to each of 18 cm nonlinear pH 3–10 IPG

strip (GE Healthcare) settled in the slot of a strip holder

(BioRad) and rehydrated at 50 V for 16 h at 201C. For IEF,

the BioRad Protean IEF cell was used with the current limit

set at 50mA/IPG strip and with the following conditions at

201C: 500 V for 500 Vh, 1000 V for 1000 Vh, and 8000 V for

57 000 Vh. After IEF, the strips were equilibrated for 15 min

in an equilibration buffer containing urea (6 M), glycerol

(30%), SDS (0.2%), Tris-HCl (50 mM), pH 8.8, and DTT

A

B

Mon Tue Wed Thu Fri Sat Sun

Mon Tue Wed Thu Fri Sat Sun

Fixed Light-Dark Cycle

Shifted Light-Dark Cycle

02:00 08:00 14:00 20:00 02:00 08:00 14:00 20:00 02:00

Rec

ove

ryB

asel

ine

Sh

ifte

d L

igh

t-D

ark

Cyc

le

02:00 08:00 14:00 20:00 02:00 08:00 14:00 20:00 02:00

Figure 1. Weekly lighting schedules over the 13 wk treatment

period (A) and an example of double-plotted body temperature

in one experimental (LS) rat throughout the experimental period

(B). Open and filled horizontal bars represent the light and dark

periods, respectively. Each temperature data point indicates a

mean body temperature difference in 30 s from daily mean body

temperature and is plotted only when it is larger than 0. Vertical

lines on the left side of the temperature graph represent the

period with lights on at 14:00.

Proteomics 2009, 9, 4017–4028 4019


(2%), and a further 15 min in the same buffer except DTT

was substituted by iodoacetamide (2.5%). For the second

dimension, equilibrated IPG strips were placed on the top of

SDS-polyacrylamide (12%) gels, and sealed with agarose

(0.5%) containing a trace amount of bromophenol blue.

Electrophoresis was performed at 201C and at 10 mA/gel for

1 h followed by 40 mA/gel until the bromophenol blue

reached the bottom of the gel. Protein spots on the 2-DE gel

were visualized using MS compatible silver stain [45].

2.4 Image analysis

The developed 2-DE gels were scanned using an image

scanner (Amersham Biosciences) at 300 dpi resolution with

a blue filter. Quantitative analysis of protein spot intensity

from 2-DE gel images was performed using Imagemaster

2D Platinum software (version 5.0, Amersham Biosciences).

The relative spot volume was defined as the value of the

intensity integration over the feature area of one spot divi-

ded by the total intensity integration over all of the spots in

the whole gel image.

2.5 Protein digestion

Preparative 2-DE gels loaded with 600–800mg protein

extracts pooled from the same tissues of different animals

were performed following the same conditions described

above except that the protein spots on the 2-DE gel were

stained with Coomassie blue [46]. Selected protein spots

showing statistically significant (po0.05, independent t-test)

rat group differences in relative spot intensity were manu-

ally excised from the preparative 2-DE gel and subjected to

in-gel digestion following the procedure as described in [47]

and with minor modifications. The excised gel plug was

washed twice with 50 mM ammonium acetate and 30%

ACN for 20 min for destaining. The protein in the gel plug

was reduced with 10 mM DTT for 45 min and alkylated with

55 mM iodoacetamide for 45 min in the dark. The gel plug

was then washed twice again with 50 mM ammonium

acetate and 30% ACN as described above, air dried after the

addition of ACN, and digested with 12.5 ng/mL sequencing

grade-modified trypsin (Promega) in 50 mM ammonium

acetate for 18 h at 371C. At the end of digestion, the gel

sample was centrifuged and the supernatant was filtered

through a 0.2 mM nylon filter syringe (Titans, Sun Sri),

dried using a SpeedVac concentrator, and stored at �801C

until MS/MS analysis.

2.6 Protein identification by LC-MS/MS

The tryptic digests were mixed with 10 mL 0.1% formic acid

and filtered through a 0.22 mm nylon syringe filter, and 2mL

of the solution was loaded onto a Bio-Basic capillary C18

column (150 mm� 75 mm, 5mm, 100 A, Thermo Electron,

Bremen, Germany). The mobile phases A and B contained

ACN (5 and 80%, respectively) in 0.1% formic acid. The

peptides were eluted over a linear solvent gradient of 5–60%

over 50 min at a flow rate of 300 nL/min. MS/MS analysis of

the eluted peptides were performed using an LTQ linear ion

trap MS (Thermo Electron) fitted with a nanospray ioniza-

tion source coupled online to an X’TremeSimple nanoflow

LC system (Microtech, Orange, CA, USA). MS/MS spectra

were obtained at a heated capillary temperature of 2001C

with a capillary voltage of 1.2–1.4 kV. MS/MS spectral data

were searched against the National Centre for Biotechnology

Information protein database with Rattus norvegicus speci-

fied and analyzed using the TurboSEQUEST algorithm in

the BioWorks 3.2 software package (Thermo Electron). The

search parameters were as follows: allowing two missed

cleavages; peptide mass tolerance 2 Da; fragment ion toler-

ance 1 Da; permitted modifications including carbamido-

methylated cysteine (157 Da) and oxidized cysteine

(148 Da) and methionine (116 Da); cross-correlation score

(Xcorr) Z1.5 for singly charged ions, Z2.0 for doubly

charged ions, and Z2.5 for triply charged ions; and peptide

probability o0.05. The matched proteins that passed the

above criteria and ranked within the top five peptide prob-

ability scores were further subjected to manual inspection.

The peptide probability indicates the probability that the

peptide is a random match to the spectral data. The best

peptide probability value was transformed into a probability

score (�10 log(peptide probability)). The protein candidate

that had a theoretical Mr within the range of the experi-

mental Mr720% and the highest total number of peptide

matches (more than one match) was finally selected.

2.7 Western blot analysis

The retroperitoneal fat powder was homogenized in 0.25 M

sucrose and then centrifuged at 14 000� g at 181C for

10 min and the interface between the low-density lipid layer

and the insoluble pellet was collected. Total protein content

was quantified using the Protein Assay Rapid Kit (Wako).

Protein samples (15 mg) were separated by electrophoresis

on a 15% polyacrylamide/0.1% SDS gel at 196 V for 3 h and

then electrotransferred to a PolyScreens PVDF membrane

(PerkinElmer) at 41C and 60 V for 15 h. The membrane was

blocked in 5% milk in TBS-Tween 20 (0.05% v/v), incubated

at room temperature for 1 h with a goat polyclonal antibody

(Santa Cruz Biotechnology, Santa Cruz, CA, USA) to fatty

acid-binding protein 4 (FABP4; 1:20,000), and then washed

and incubated at room temperature for 1 h with HRP-

conjugated donkey anti-goat IgG (1:10 000; Santa Cruz

Biotechnology). Bands were detected using the Western

Lightning Plus-ECL detection kit (PerkinElmer) and visua-

lized on Kodak X-Omat Blue XB-1 imaging film. Auto-

radiograms were scanned on a GS-800 calibrated

densitometer (BioRad). Signals were quantified using



Imagemaster 2D Platinum software. We also performed the

western blot analysis on the protein samples prepared for

the 2-DE experiment and followed the same blotting

procedure as described above. The two independent western

blot analyses were named Run1 and Run2, respectively.

2.8 Statistical analysis

Rat group differences were evaluated using independent

t-tests. The degree of association between the relative

protein spot volume and body weight and fat mass was

analyzed by calculating Pearson’s r. All statistical analyses

were performed using SYSTATs 7.0 for Windowss. The

statistical significance level was set at 0.05.

3 Results

Consistent with previous findings [33], the LS rats showed

continual phase shifts, either advanced or delayed, in daily

activity and body temperature (see Fig. 1) rhythms in

response to repeated shifts in the LD cycle. The LS and LC

rats in either tissue sample group maintained comparable

values in the mean food intake and body weight at the

baseline. However, the LS rats ate and gained more weight

than the LC rats during the 13 wk treatment period,

consisting of two 12 h LD shifts per week (Table 1). The

mean retroperitoneal fat mass in absolute weight or relative

weight to body weight was significantly greater in the LS rats

than in the LC rats (Table 1).

There were 519 (SD 5 100) and 679 (129) mean total

protein spots shown in the hypothalamic 2-DE gels of the LS

and LC rats, respectively. When one missing sample was

permitted, there were 203 spots overlapped among all the

gels; among these, five spots showed significant rat group

differences in the relative spot volume (Table 2 and Fig. 2).

The final body weight was not significantly correlated with

the relative spot volume of any of the five protein spots, but

the retroperitoneal fat mass was significantly correlated with

the relative volume of spots 114 (r 5 0.581, po0.05) and 937

(r 5 0.644, po0.05). All five spots were unambiguously

identified (Table S1 of Supporting Information and Table 2),

and they are metabolism-related enzymes or porins. We did

not perform western blot analyses on the HT sample to

validate the proteomic results because of the shortage of the

tissue protein.

There were 632 (71) and 651 (93) mean total protein spots

shown in the retroperitoneal fat 2-DE gels of the LS and LC

rats, respectively. When a maximum of two missing

samples was permitted, there were 337 spots overlapped

among all the gels; among these, 22 spots showed signifi-

cant rat group differences in the relative spot volume

(Table 3 and Fig 3). The final body weight was significantly

correlated with the relative volume of spots 4684 (r 5 0.694,

po0.01), 5335 (r 5 0.688, po0.01), 6159 (r 5 0.543,

po0.05), and 6200 (r 5�0.566, po0.05), and the retro-

peritoneal fat mass was correlated with spots 5335

Table 1. Effects of repeated LD shifts on metabolic variables

HT group Retroperitoneal fat group

LC (n 5 6) LS (n 5 6) LC (n 5 8) LS (n 5 8)

Food intake (g)a)

Baseline 16.9 (1.0) 17.3 (1.3) 19.1 (1.6) 18.8 (0.7)M1 17.0 (1.3) 19.1 (1.1)� 18.5 (1.1) 18.9 (0.5)M2 17.3 (1.8) 18.7 (1.8) 16.8 (1.4) 16.9 (0.4)M3 17.6 (1.7) 18.9 (1.7) 16.2 (1.2) 17.7 (0.9)�

Recovery 18.1 (2.1) 20.1 (1.7) 16.7 (1.9) 19.4 (1.6)��

Body weight (g)b)

Baseline 277 (15) 290 (12) 307 (19) 310 (17)M1 314 (13) 341 (15)�� 345 (15) 359 (15)M2 342 (17) 369 (16)� 356 (19) 370 (13)M3 369 (15) 396 (17)� 384 (19) 410 (16)��

Recovery 375 (21) 398 (17) 383 (20) 410 (17)�

Sacrifice 356 (26) 373 (21) 369 (20) 394 (16)�

Retroperitoneal fat

Absolute weight (g) 5.7 (0.8) 7.6 (1.2)�� 7.3 (1.5) 8.8 (0.8)�

Relative weight (%) 1.59 (0.13)_ 2.04 (0.25)�� 1.97 (0.31) 2.23 (0.20)

a) Daily food intake data were averaged over the days in the baseline, successive months (M1–M3) during LD shifts and recovery,respectively. The rats in the HT sample group underwent a longer recovery period (2–3 wk) than those of the fat sample group becausethose in the HT sample group received glucose and insulin tests during recovery (data not reported). Data are means (SD). Significantdifferences between experimental (LS) and control (LC) rats in the same tissue sample group (�po0.05, ��po0.01, and ��po0.005,independent t-test).

b) Body weight was measured on the last day of baseline, of each successive month during LD shifts and of the first week in recovery andon the day of sacrifice.

Proteomics 2009, 9, 4017–4028 4021


(r 5 0.698, po0.01), 6159 (r 5 0.743, po0.01), and 6200

(r 5�0.582, po0.05). Among the 22 spots showing signif-

icant group differences, 17 spots were unambiguously

identified (Table S2 of Supporting Information and Table 3).

Most of the identified proteins are metabolism-related

enzymes. Two proteins (FABP4; and 78 kDa glucose-regu-

lated protein, GRP78) appeared at multiple spots, suggest-

ing that they might be posttranscriptionally and/or

posttranslationally modified. The Western blot analysis on

the fat tissue protein with two independent repetitions

showed that the protein level of FABP4 tended to be higher

in the LS group than in the LC group (Fig. 4).

4 Discussion

Consistent with our previous findings [33], this study

showed that repeated LD shifts increased food intake and

body weight gain in male F344 rats. In addition, this study

found that the LS rats had a greater retroperitoneal fat mass

than the LC rats. A previous study in diet-induced obesity

showed that after 10-wk-old male F344 rats were fed a high-

fat diet (4.47 kcal/g) for 3 wk, their food intake, weight gain,

and retroperitoneal fat mass were 10, 41, and 93%, respec-

tively, greater than those of the control rats fed a low-fat diet

(3.3 kcal/g) [48]. Comparable to the effect of a high-fat diet,

while rats were fed a regular diet (3.25 kcal/g), repeated LD

shifts increased food intake and weight gain to 13 and 35%,

respectively, in the first month (calculated using the data of

the HT sample group). Although repeated LD shifts also

increased body fat accumulation (33% in the HT sample

group and 21% in the fat group), the rate of accumulation

appeared to be lesser efficient than that of a high-fat diet.

Concomitant with changes in food intake, weight gain,

and fat mass, repeated LD shifts resulted in changes in

expression levels of five hypothalamic and 22 retroperitoneal

fat 2-DE protein spots. Among these spots, the expression of

four protein spots in the HT sample and 13 protein spots in

the fat sample was upregulated. Furthermore, the expres-

sion level of two protein spots in the HT and three in the fat

were correlated with the retroperitoneal fat mass; the

expression level of four protein spots in the fat was corre-

lated with body weight. All the five HT protein spots and 17

of the 22 adipose protein spots that were differentially

expressed in response to repeated LD shifts were identified

by MS (see Tables 2 and 3). These proteomic results are

confined in several aspects: (i) Direct comparisons across

2-DE gels may be limited due to inter-gel variation. (ii) The

proteome of whole HT may confound the dynamic changes

in specific HT nuclei involved in different physiological and

behavioral mechanisms. (iii) There is lack of additional

confirmative data, e.g. from immunoassays, for all the

protein spots identified by MS. An exception is FABP4. The

Western blot analysis on FABP4 showed a tendency of rat

group differences consistent with the finding of the

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To our knowledge, this is the first study applying a

proteome analysis approach to identify and quantify hypo-

thalamic proteins related to positive energy balance, i.e.elevated body weight/fat mass setpoint. Most of the five HT

proteins that were differentially expressed in response to

repeated LD shifts are known to be involved in food intake

and/or energy regulation (see Table 2). First, the expression

of the glycolytic enzyme fructose-bisphosphate aldolase A

and the citric acid cycle enzyme aconitase 2 were upregu-

lated in the LS rat group, suggesting a state of elevated

energy supply related to chronic circadian desyncronization.

Upregulation of aldolase and aconitase might also reflect a

central mechanism in the regulation of fat mass since the

expression of the two enzymes was correlated with the

retroperitoneal fat mass. Second, the increased expression of

gamma-amino butyric acid (GABA) aminotransferase

corresponds to the role of the brain GABA mechanism in

the control of food intake in rats. Elevations of brain GABA

induced by intracisternal injection of the GABA amino-

transferase inhibitor produced dose-dependent anorexia in

both normal and obese rats [49–51]. Thus, upregulation of

GABA aminotransferase in the HT suggests attenuated

brain GABA levels, and thus, an enhanced appetite; this

corresponds to increased food intake in the LS rat group.

Aldehyde dehydrogenase family 1 member B1 belongs to a

group of enzymes that play a major role in the oxidation of a

wide range of endogenous aldehydes formed during the

metabolism of carbohydrates, lipids, amino acids, and

exogenous aldehydes such as alcohol [52]. However, the role

of upregulated aldehyde dehydrogenase family 1 member

B1 in the HT is not clear. Last, the only downregulated

protein in the HT, voltage-dependent anion-selective chan-

nel protein 1 (VDAC1), lies in the outer mitochondrial

membrane and plays a crucial role in the regulation of

mitochondrial function [53, 54] and in mitochondrial

apoptosis [55]. Cells with low levels of VDAC1 show a

reduced ATP-synthesis capacity and growth rate. It seems

odd that upregulated aldolase and aconitase, representing

an elevated energy supply, coexists with downregulated

VDAC1, representing a reduced energy supply. How such a

deviation in intracellular energy regulation is involved in

metabolic changes related to circadian desynchronization

requires clarification.

In the retroperitoneal fat, most of the identified proteins

showing differential expression in response to repeated LD

shifts are metabolism-related enzymes (see Table 3).

Consistent with the findings of elevations in all enzymes of

glycolysis, of the pentose phosphate pathway, and of the

citric acid cycle in the adipose tissue derived from geneti-

cally obese Zucker rats (fa/fa) [56], we found that the

expression of the glycolytic enzymes triosephosphate

isomerase, glyceraldehyde-3-phosphate dehydrogenase

(GAPDH), and glycerol-3-phosphate dehydrogenase, the

pentose phosphate pathway enzyme transketolase, and the

citric acid cycle enzyme isocitrate dehydrogenase was higher

in the LS rat group than in the LC rat group. Among these

five upregulated enzymes, GAPDH was the only one

showing expression levels correlated with both body weight

and the retroperitoneal fat mass. Increased GAPDH activity,

specifically in the adipose tissue, has been shown to be

concomitant with a slight but significant increase in

subcutaneous inguinal fat-pad weight detected as early as 7

days of age in obese Zucker pups as compared with lean

heterozygous pups [57]. Thus, a fatty genotype and circadian

desynchronization appear to have a common mechanism in

increasing fat mass involved in an enhanced GAPDH

activity, through which glycolytic flux and supply of

substrates for lipogenesis in adipose tissue is elevated. In

contrast, previous proteomic studies in high-fat-diet-induced

obesity fail to identify any glycolytic enzymes upregulated in

pI

1701309572

55

43

34

26

17

11

kDa

4.0 4.5 5.0 5.5 6.0 6.5 7.0 8.0 9.0 10.0

1701309572

55

43

34

26

17

11

kDa

pI4.0 4.5 5.0 5.5 6.0 6.5 7.0 8.0 9.0 10.0

Figure 2. Hypothalamic 2-DE gels from one control (LC, upper)

and one experimental (LS, lower) rat. Circles indicate protein

spots that showed a higher mean of the relative volume for the

LS group than the LC group.

Proteomics 2009, 9, 4017–4028 4023


Tab

le3.

Lis

to

fth

ep

rote

insp

ots

dif

fere

nti

all

yexp

ress

ed

inth

era

tre

tro

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aft

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13

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rep

eate

dLD

shif

ts

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ot

no

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ldch

an

ge

a)

pV

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eb

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Pro

tein

nam

eP

ep

tid

es

matc

hed

%C

overa

ge

c)

Sco

red

)

3800

(nLC

5n

LS

57)

1.4

70.0

27

gi|62647962

Tri

ose

ph

osp

hate

iso

mera

se(E

C5.3

.1.1

)3

14.1

654

6159

1.3

20.0

19

gi|62647964

GA

PD

H(E

C1.2

.1.1

2)

36.3

190

3284

1.8

90.0

04

gi|57527919

Gly

cero

l-3-p

ho

sph

ate

deh

yd

rog

en

ase

[NA

D1

],cy

top

lasm

ic(E

C1.1

.1.8

)10

30.6

667

1548

(nLS

57)

1.5

40.0

45

gi|12018252

Tra

nsk

eto

lase

(EC

2.2

.1.1

)12

23.6

6125

2619

1.6

60.0

38

gi|16758446

Iso

citr

ate

deh

yd

rog

en

ase

[NA

D1

]su

bu

nit

alp

ha,

mit

och

on

dri

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(EC

1.1

.1.4

1)

822.9

580

5335

1.9

80.0

22

gi|62643089

FA

BP

42

21.2

182

5647

1.4

20.0

41

gi|62643089

FA

BP

48

52.2

787

5702

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80.0

07

gi|62643089

FA

BP

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3398

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gi|62652539

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se(E

C2.8

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170

3918

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38

gi|34855391

Bil

iverd

inre

du

ctase

B(E

C1.3

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4)

426.2

1160

3458

1.7

50.0

10

3872

2.4

10.0

11

4684

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40.0

44

2252

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60.0

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gi|40538742

AT

Psy

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ase

sub

un

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mit

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48.1

081

2091

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10

gi|6980956

Glu

tam

ate

deh

yd

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ase

1,

mit

och

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(EC

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8.0

674

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(nLC

57)e

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90.0

06

gi|42476287

Tra

nsg

luta

min

ase

2,

Cp

oly

pep

tid

e(E

C2.3

.2.1

3)

14

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2104

1167

0.7

30.0

47

gi|16758748

Dyn

ein

,cy

top

lasm

ic,

inte

rmed

iate

po

lyp

ep

tid

e2

611.4

4141

1882

0.6

40.0

39

gi|51036655

Alp

ha-1

-an

tip

rote

inase

12

28.2

295

6200

(nLS

56)

0.4

40.0

28

gi|25742763

GR

P78

24

38.6

9121

6201

(nLC

56)

0.5

20.0

11

gi|25742763

GR

P78

20

39.1

4126

1776

0.6

60.0

25

1787

(nLC

57)

0.6

70.0

10

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the fat in obese rats [44] or mice but have identified one

downregulated in obese mice [58]. Downregulation in

glycolytic enzymes is assumed to reflect that at elevated

plasma fatty acids concentrations resulting from a high-fat

diet, fatty acids compete with glucose as an oxidative fuel

source.

However, diet-induced obese rats show upregulation of a

cytosolic fatty acid chaperone, FABP4, also known as

adipocyte-fatty-acid-binding protein, in the epidydimal fat; it

is reverted by treatment with the antiobesity agent tungstate

[44], which decreases body weight gain and adiposity with-

out modifying food intake in obese rats [59]. The LS rats in

this study also showed upregulation of FABP4 in the

retroperitoneal fat. One of the multiple protein spots iden-

tified as FABP4 (5335) showed that expression levels were

correlated positively with both body weight and the retro-

peritoneal fat mass. Recently, plasma FABP4 released from

adipocytes was found to be higher in overweight/obese

individuals than in lean persons and correlated positively

with obesity and metabolic syndrome [60]. However, mice

deficient in FABP4 are not protected from genetic or high-

fat-diet-induced obesity and, when fed a regular diet, have

higher elevated plasma fatty acids than wild-type mice.

Although increased plasma fatty acids are positively corre-

lated with the development of obesity and insulin resistance

in general, mice deficient in FABP4 are more insulin

sensitive (reviewed in [61]). If the upregulation of FABP4 is

a critical link between obesity and insulin resistance, the

development of insulin resistance is predicted for the LS rats

in the long run. Furthermore, we have found multiple

protein spots identified as FABP4 and all of them were

consistently upregulated in the LS rat group. Whether

these multiple protein spots represent specific protein

0

2

4

6

8

10

12

Run1 Run2

FA

BP

4 co

nten

t (a

rbit

rary

uni

ts)

LC LS

15kDa

21_CL10_SL LC_02 LS_11

Figure 4. Representative Western blot analyses from two control

(LC_02, LC_12) and two experimental (LS_01, LS_11) rats and

mean densitometric results (the error bar depicts SEM) of FABP4

in the retroperitoneal fat tissue. A two-factor (two-rat groups two

runs) analysis of variance showed a tendency of rat group

difference to be significant (F1,13=4.03, p=0.066; n=7 in the LC

group due to one tissue sample unavailable for Western blot

analyses).

4.0 4.5 5.0 5.5 6.0 6.5 7.0 8.0 9.0 10.0pI

170

130

95

72

55

43

34

26

17

kDa

4.0 4.5 5.0 5.5 6.0 6.5 7.0 8.0 9.0 10.0pI

170

130

95

72

55

43

34

26

17

kDa

Figure 3. Retroperitoneal fat 2-DE gels from one control (LC,

upper) and one experimental (LS, lower) rat. Circles indicate

protein spots that showed a higher mean of the relative volume

for the LS group than the LC group.

Proteomics 2009, 9, 4017–4028 4025


modifications requires further study. For example, it has

been shown that high-fat-diet-induced obesity results in an

increase in the carbonylation of a number of adipose-regu-

latory proteins including FABP4 [62]. Protein carbonylation

is mediated by reactive aldehydes in response to oxidative

stress. It is postulated that increased protein carbonylation

and oxidative stress are components linking obesity to

insulin resistance. In contrast, the upregulation of two

antioxidant enzymes, 3-mercaptopyruvate sulfurtransferase

and biliverdin reductase B, in the LS rat group might serve

as a compensatory mechanism in response to increased

reactive oxygen species resulting from increased metabolic

processes, and thereby, might protect the body from

(excessive) oxidative stress. To what extent upregulation in

antioxidant enzymes can protect the body from insulin

resistance in LS rats could not be predicted by this study.

In contrast to the proteome of high-fat-diet-induced

obesity that shows upregulation in ATP synthase in fat [44],

the LS rats had a lower expression level of ATP synthase.

ATP synthase has been found in both the mitochondrial and

plasma membrane. Mitochondrial ATP synthase is the

central enzymatic complex of mitochondrial oxidative

phosphorylation and the cell-surface ATP synthase complex

contributes to the regulation of signal transduction. The

expression of the cell-surface ATP synthase is markedly

increased during adipogenesis and inhibition of ATP

synthase activity by ATP synthase inhibitors or by antibodies

against ATP synthase inhibits lipid accumulation in adipo-

cytes [63]. Thus, it appears paradoxical that the LS rats had a

greater fat mass and a lower expression level of ATP

synthase than the LC rats. However, a reduced expression

level of mitochondrial ATP synthase might represent an

adaptation mechanism to elevated energy supply. Reduction

in mitochondrial ATP synthase may increase the uncou-

pling of ATP generation from respiratory chain oxidation,

which leads to increased proton leakage, resulting in more

energy dissipated as heat.

Glutamate dehydrogenase, a key regulator of amino acid

and ammonia metabolism, was downregulated in the LS

rats. Glutamate dehydrogenase activity in brown adipose

tissues is lower in diet-induced obese rats than in control

lean rats [64], and this, accompanied by reductions in the

activity of other amino acid metabolism-related enzymes in

adipose tissues, represents an adaptive mechanism of

depressed amino acid utilization as fuel in obese rats. The

other four downregulated proteins in the LS rats were tissue

glue protein transglutaminase 2, cellular structure protein

dynein, plasma protein alpha1 antiproteinase, and chaperon

GRP78. The role of the first three above-mentioned proteins

in metabolism and obesity is not clear. The expression of

GRP78 does not change in the fat of diet-induced obese rats

but is upregulated after tungstate treatment [44]. In addi-

tion, mice with a fat-specific insulin receptor knock-out

show an elevated expression level of GRP78 in adipose

tissues accompanied by reduced fat mass and protection

against age-related and hypothalamic lesion-induced obesity

and obesity-related glucose intolerance [65, 66]. In this

study, it was found that the expression level of GRP78 was

negatively correlated with both body weight and the retro-

peritoneal fat mass. Thus, downregulation of GRP78 in the

LS rats might be related to the development of obesity and

insulin resistance.

In summary, rats undergoing chronic circadian desyn-

chronization induced by repeated LD cycle shifting consis-

tently showed increased food intake, weight gain, and fat

mass, accompanied by expression changes in several

proteins in the HT and fat. Those identified proteins

involved in carbohydrate metabolism and citric acid cycle

were upregulated in the HT and fat, which might reflect a

positive energy balance status. On the other hand, upregu-

lation of the GABA metabolic enzyme in the HT suggested

that a suppressed GABAeric system might be responsible

for enhanced food intake induced by repeated LD shifts.

Furthermore, upregulation of FABP4 and downregulation

of GRP78 predicted the development of insulin resistance.

However, upregulation of antioxidant enzymes might serve

as protection against oxidative stress-related insulin

dysfunction. Thus, how insulin function responds to repe-

ated LD shifts awaits future studies.

This work was supported by the National Science Council(NSC), Taiwan R. O. C., Grant NSC95-2413-H-194-029-MY2.AM was supported by the NSC Postdoctoral Research FellowGrants NSC95-2811-H-194-002 and NSC96-2811-H-194-001.We thank Yu-Che Tsai and Kuang-Jen Huang for their assis-tance in tissue dissection, Wan-Chen Wang for her participationin the proteome experiment, Wen-Peng Lin and Yu-Chin Chenfor their work on the LC-MS/MS analysis performed in theDepartment of Chemistry and Biochemistry at the NationalChung Cheng University, and Ya-Wen Liu, Fan-Ci Hsiao, Yu-Shan Huang, and Chun-Yu Chen for their work on the westernblot analysis performed at Dr. Hsien-Bin Huang’s laboratory inthe Department of Life Sciences at the National Chung ChengUniversity.

The authors have declared no conflict of interest.

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