baburski2016

Circadian rhythm of the leydig cells endocrine function is attenuated during
aging
Aleksandar Z. Baburski, Srdjan J. Sokanovic, Maja M. Bjelic, Sava M.
Radovic, Silvana A. Andric, Tatjana S. Kostic
PII: S0531-5565(15)30080-2
DOI: doi: 10.1016/j.exger.2015.11.002
Reference: EXG 9729
To appear in: Experimental Gerontology
Received date: 23 May 2015
Revised date: 11 October 2015
Accepted date: 3 November 2015
Please cite this article as: Baburski, Aleksandar Z., Sokanovic, Srdjan J., Bjelic, Maja M.,
Radovic, Sava M., Andric, Silvana A., Kostic, Tatjana S., Circadian rhythm of the leydig
cells endocrine function is attenuated during aging, Experimental Gerontology (2015), doi:
10.1016/j.exger.2015.11.002
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Title:
Circadian rhythm of the Leydig cells endocrine function is attenuated during aging
Aleksandar Z Baburski, Srdjan J Sokanovic, Maja M Bjelic, Sava M Radovic, Silvana A Andric, Tatjana S Kostic
LaRES, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Novi Sad, Serbia
Running title: Aging & Leydig cell circadian rhythm
Key words: Aging, Leydig cell, clock, testosterone, 24-hour rhythm
All correspondence to:
Tatjana S. Kostic,
Department of Biology and Ecology
Faculty of Sciences,
University of Novi Sad,
Dositeja Obradovica Sq 2
21000 Novi Sad
Serbia
Fax: [phone]
E-mail: tatjana.ko[email protected]ns.ac.rs

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ABBREVIATIONS:
Bmal1, brain-muscle Arnt-like protein 1;
BSA, bovine serum albumin;
cAMP, cyclic adenosine monophosphate;
Ck1, gene for casein kinase 1;
Clock/CLOCK, gene/protein for circadian locomotor output cycles kaput;
CRE, cAMP-responsive element;
Creb/CREB, gene/protein for cAMP response element-binding protein;
Cry/CRY, gene/protein for cryptochrome;
Cyp11a/CYP11a, gene/protein for cytochrome P450 side chain cleavage enzyme;
Cyp17a-hydroxylase/C17-20 lyase;
DBP, D site of albumin promote bind protein;
DHT, dihydrotestosterone;
DMEM/F12, Dulbecco's modified eagle medium;
Gapdh, gene for glyceraldehyde 3-phosphate dehydrogenase;
HDL, high density lipoprotein;
Hmgcr, gene for 3-hydroxy-3-methyl-glutaryl-CoA reductase;
Hsd17b
Hsd3b, 
Insl3/INSL3, gene/protein for insulin-like 3;
LDL, low density lipoprotein;
LH, luteinizing hormone;
Lhr/LHR, gene/protein for luteinizing hormone receptor;
Lipe, gene for Hormone sensitive lipase (HSL);
mo, month;
Nampt/NAMPT, Nicotinamide phosphoribosyltransferase;
Npas2, gene for Neuronal PAS domain-containing protein 2;
Nur77, gene for nerve growth factor;
Per1, gene for period circadian protein 1;
Per2, gene for period circadian protein 2;
Per3, gene for period circadian protein 3;
PKA, cAMP dependent protein kinase;
RIA, radioimmunoassay;
Rev-erba/b / REV-ERBA/B, gene/protein for reverse viral erythroblastis oncogene product alpha/beta;
Ror/ROR, gene/protein for retinoid acid related orphan receptor;
RQ-PCR, relative quantification polymerase chain reaction;
Scarb1, gene for Scavenger Receptor Class B, Member 1;
Sf1, gene for steroidogenic factor 1;
Sirt1/SIRT1, gene/protein for NAD-dependent protein deacetylase sirtuin-1;
Soat1, gene for Sterol O-acyltransferase (acyl-Coenzyme A: cholesterol acyltransferase) 1;
Soat2, gene for Sterol O-acyltransferase (acyl-Coenzyme A: cholesterol acyltransferase) 2;
Star/StAR, gene/protein for steroidogenic acute regulatory protein;

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T, testosterone;
Tspo, gene for translocator protein;
VLDL, very low density lipoprotein ;
ZT, Ze

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Abstract
Although age-related hypofunction of Leydig cells is well illustrated across species, its circadian
nature has not been analyzed. Here we describe changes in circadian behavior in Leydig cells isolated from
adult (3-month) and aged (18- and 24-month) rats. The results showed reduced circadian pattern of
testosterone secretion in both groups of aged rats despite unchanged LH circadian secretion. Although
arrhythmic, the expression of Insl3, another secretory product of Leydig cells, was decreased in both groups.
Intracellular cAMP and most important steroidogenic genes (Star, Cyp11a1 and Cyp17a1), together with
positive steroidogenic regulator (Nur77), showed preserved circadian rhythm in aging although rhythm
robustness and expression level were attenuated in both aged groups. Aging compromised cholesterol
mobilization and uptake by Leydig cells: the oscillatory transcription pattern of genes encoding HDL-receptor
(Scarb1), hormone sensitive lipase (Lipe, enzyme that converts cholesterol esters from lipid droplets into
free cholesterol) and protein responsible for forming the cholesterol esters (Soat2) were flattened in 24-
month group. The majority of examined clock genes displayed circadian behavior in expression but only a
few of them (Bmal1, Per1, Per2, Per3 and Rev-Erba) were reduced in 24-month-old group. Furthermore,
aging reduced oscillatory expression pattern of Sirt1 and Nampt, genes encoding key enzymes that connect
cellular metabolism and circadian network. Altogether circadian amplitude of Leydig cell`s endocrine function
decreased during aging. The results suggest that clock genes are more resistant to aging than genes involved
in steroidogenesis supporting the hypothesis about peripheral clock involvement in rhythm maintenance
during aging.