Journal of Life Sciences Volume 6 Number (1)
J LS
Journal of Life Sciences
Volume 6, Number 10, October 2012 (Serial Number 54)
Contents
Biochemical and Molecular Biology
1077 The Study of Cholesterol Content in Synbiotic Fermented Dairy Products Ilze Beitane and Inga Ciprovica 1082 Metabolic and Endocrine Responses of Desert-Adapted Mice Reproductive System to Increased
Salinity
Elena Bukovetzky, Fuad Fares and Abraham Haim 1094 Influence of Abiotic Elicitors on Accumulation of Thymol in Callus Cultures of Origanum vulgare L.
Abedaljasim M. Aljibouri, Ashwaq S. Abd, Duha M. Majeed and Eman N. Ismail 10 Low Peptone Dose as Inductor of Alkaline Protease Promoter Used for Invertase Gene Expression
in Yarrowia lipolytica
Łukasz Śnieżewski, Ewa Walczak, Zbigniew Lazar and Małgorzata Robak
Biomedicine
1109 In Vitro Study on Virulence Potentials of Burkholderia pseudomallei Isolated from
Immunocompromised Patients
Hadeel Tawfiq Al-Hadithi and Rana Muhammad Abdulnabi 1117 The Control of Malaria among PLWHA in Calabar, Cross River State, Nigeria
Patience Edoho Samson-Akpan, Olaide Bamidele Edet, Ekaette Francis Asuquo, Mary Achi Mgbekem and Idang Neji Ojong
1124 Spatiotemporal Distribution of Phlebotomine Sand Flies (Diptera: Psychodidae) in a Focus of
Cutaneous Leishmaniasis in Foum Jamâa (Azilal, Atlas of Morocco)
Hassan Arroub, Abdelaaziz Alaoui, Hicham El Miri, Meryem Lemrani and Khalid Habbari
13 Determination of Fungal Colonization in the Oral Cavity of College Students Floridia Ricardo, Rodriguez Graciela, Ampuero Verónica and Gonzalez Luis 1142 Preliminary Results of Crayfish Distribution and Diseases in Latvia Inese Briede
Interdisciplinary Researches
1145 The Effects of Simultaneous Application of Different Organic and Biological Fertilizers on
Quantitative and Qualitative Characteristics of Cucurbita pepo L.
Mohsen Jahan, Alireza Koocheki, Mohammad-Kazem Tahami, Mohammad-Behzad Amiri and Mahdi Nassiri-Mahallati
1150 The Impact of Deforestation in Anambra State: The Ekwusigo Example Joel Ekwutosi Umeuduji and Chukwuma Onyebueke Egbuonu 1158 Contribution of Study Bioecology of the Fauna Chamaerops humilis in the Region of Tlemcen
(Algeria)
Damerdji Amina 1167 The Floristic Diversity of the Tlemcen Southern Slope Scrublands (Western Algeria) Belhacini Fatima and Bouazza Mohammed 1174 Tools for Protein Structure Prediction at the bri-shur.com Web Portal
Sergey Feranchuk, Ulyana Potapova, Vladimir Potapov, Dmitry Mukha, Vladimir Nikolaev and Sergei Belikov
1180 The Elaboration of Horse Meat Products Technology Аbzhanova Sholpan, Kizatova Мaigul, Мukhtarkhanova Rauan, Тarakbaeva Raushan and
Аbilmazhinova Nazum
Journal of Life Sciences 6 (2012) 1077-1081
The Study of Cholesterol Content in Synbiotic Fermented Dairy Products
Ilze Beitane and Inga Ciprovica Faculty of Food Technology, Latvia University of Agriculture, Jelgava LV-3001, Latvia
Received: April 11, 2012 / Accepted: June 22, 2012 / Published: October 30, 2012.
Abstract: The influence of prebiotics as lactulose as well inulin on the ability of Bifidobacterium lactis to reduce cholesterol in milk was studied during milk fermentation. Pasteurized milk, freeze-dried starter culture Bb-12 (Bifidobacterium lactis, Chr. Hansen, Denmark), inulin—RAFTILINE ® HP (ORAFI, Belgium), syrup of lactulose (Duphalac ® , the Netherlands) in following concentrations: 0, 1%, 2%, 3%, 4% and 5% were used for experiments. The fermentation process was realized at 37 °C for 16 h. The content of cholesterol was determined according to AOAC Official Method 976.26A. The results showed that it is possible considerable to lower the level of cholesterol in fermented milk using B. lactis. The ability of B. lactis to decrease the level of cholesterol in milk can be influenced with addition of prebiotics. The lower concentration of cholesterol was determined in fermented samples with 4% of lactulose (9.5 mg/100g) and with 1% of inulin (10.4 mg/100g). Evaluating the influence of prebiotics on cholesterol content in fermented milk samples, it is obvious that the influence depends on the type of prebiotics (P > 0.05) and their concentrations (P < 0.05).
Key words: Cholesterol, B. lactis, lactulose, inulin, fermented milk.
1. Introduction have been recommended as dietary supplements because of their hypocholesterolemic effect in
Elevated total cholesterol and LDL (low-density humans [7]. Evaluating the relationship between lipoprotein) cholesterol levels are widely LAB (lactic acid bacteria) and the serum cholesterol, represented as a contributory risk factor for the it has found that lactobacilli or bifidobacteria can development of artherosclerosis, coronary heart exhibit hypocholesterolemic properties in humans disease and stroke [1-3]. It has been reported that [8-10]. The possible mechanism it could be, that hypercholesterolemia promotes to 45% of heart LAB with active bile salt hydrolase or products attacks in Western Europe and 35% in Central and containing them have been suggested to lower Eastern Europe [4]. In addition, it is known that high cholesterol levels through interaction with host bile cholesterol levels and mortality are close related [5]. salt metabolism [11]. Bifidobacteria are one of the Manson et al. [6] have pointed out that even a 1% most important probiotics associated with human reduction in serum cholesterol could reduce the risk health. They have varied positive influence on of coronary heart disease by 2-3%. Therefore it is human health, inter alia, the lowering of serum important to control the cholesterol intake by food cholesterol in blood [10]. Xiao et al. [10] observed and to use the products with the ability to lowering that consumption of Bifidobacterium milk leads to a the blood cholesterol level. Fermented milk products meaningful reduction in triglyceride, low-density
Inga Ciprovica, Ph.D., Prof., research field: dairy science lipid and total cholesterol. Therefore it is important and technology. E-mail: [email protected].
Corresponding authors: Ilze Beitane, Ph.D., Assist. Prof., to produce products with low cholesterol content or research field: functional dairy products. E-mail: products which compounds should be reduced
The Study of Cholesterol Content in Synbiotic Fermented Dairy Products
cholesterol level in blood by regular intake. One of
2 mL of milk suspension (10 6 cfu/mL) and cultured at the possibilities is to add probiotics and prebiotics in
37 °C for 16 h. The control sample was prepared dairy products. The numerous studies indicate that
without the prebiotics for comparing with the obtained bifidobacteria have the ability to assimilate results. cholesterol [12-14] which should be promoted with
The level of cholesterol was determined according adding prebiotics. There are limited studies [12]
to AOAC Official Method 976.26A. about the influence of prebiotics on the ability of
The differences at the level of cholesterol were bifidobacteria to reduce the level of cholesterol in
analyzed using the analysis of variance (ANOVA). fermented milk. Therefore the task of the research
t -test was applied to compare the mean values, and was to investigate the influence of lactulose and
P -value at 0.05 was used to determine the significant inulin on the ability of Bifidobacterium lactis to
differences. Experiments were carried out in triplicate. reduce cholesterol level in fermented dairy product
3. Results and Analysis
during milk fermentation. Cholesterol is included in the membrane of fat
2. Materials and Methods
globules, and it makes up to 95% of the total sterol Pasteurized milk with fat content 2.5% and the
content [15], the others 5% are cholesteryl esters. The strain of Bifidobacterium lactis (Bb-12, Chr.Hansen,
cholesterol content in milk is within the range from Denmark) was used for experiments. During the
0.09 g/L to 0.22 g/L, on average 0.16 g/L [16]. The experiments, the culture was maintained at -18 °C. As
cholesterol content in milk is possible to decrease by prebiotics were used inulin RAFTILINE ® HP (ORAFI,
different techniques as cholesterol distillation, complexes Belgium) with polymerization degree ≥ 5 and degree
with cyclodextrins as well microorganisms [13]. of purity 99.5% and syrup of lactulose (Duphalac ® ,
Consequently, ability of Bifidobacterium lactis to the Netherlands) with following composition (%):
decrease the cholesterol level in fermented milk, as lactulose—no less than 67, lactose—less than 6,
well as the content of decreased cholesterol level galactose—less than 10.
influenced by added prebiotics was studied. Different lactulose and inulin concentrations (1%;
The level of cholesterol in milk, control and in 2%; 3%; 4% and 5%) were added individually to 100
fermented milk samples with different concentrations
g of milk. Bifidobacterium lactis was inoculated with of lactulose and inulin is presented in Fig. 1.
Fig. 1 The level of cholesterol in milk, control and in fermented milk samples with different concentrations of lactulose and inulin.
The Study of Cholesterol Content in Synbiotic Fermented Dairy Products
The research results showed that it is considerable Summarizing the research results it should be possible to lower the level of cholesterol in milk using
induced that the considerable decrease of cholesterol
B. lactis . The ability of B. lactis to decrease the level content should be reached up to 25% during milk of cholesterol in milk can be induced with adding the
fermentation. This tendency is possible to facilitate by prebiotics. The lower content of cholesterol was
using appropriate prebiotics.
determined in fermented milk samples with 4% of
4. Discussion
lactulose (9.5 mg/100g) and with 1% of inulin (10.4 mg/100g). It confirms the conclusions of Palframan et
In scientific articles there are achievements that al. [17] that a greater bifidogenic effect of inulin was
consumption of fermented dairy products significantly obtained in 1% concentration. It indicates on the
decreases the cholesterol level in blood serum [22, 23] relationship between the multiply of B. lactis and the
and bifidobacteria have the ability to lower serum assimilation of cholesterol in milk. In preliminary
cholesterol level in humans [14]. Manning et al. [24] studies were established the ability of inulin and
have indicated the ability of lactic acid bacteria to lactulose to promote the growth of B. lactis in milk
decrease the total and LDL cholesterol level in blood. (Table 1).
Whereas Kiessling et al. [25] in study with human The results showed that the highest amount of B.
reported about increase of high-density lipoprotein lactis did not provide the lowest level of cholesterol in
(HDL) level but no reduction in total cholesterol (P = fermented milk with prebiotics. It is linked with the
0.001) in subjects fed yoghurt with Lactobacillus activity of cholesterol esterase to catalyze the acidophilus and B. longum. Contradictory data in hydrolysis of cholesteryl esters [19], thereby it should
literature show the necessity to continue the research
be decreased the level of cholesterol in fermented milk. in this field, because the mechanism how it happens is The beneficial influence of lactulose on cholesterol
not quite clear yet. The production of bile salt level in fermented milk can be explained with the
hydrolase has been suggested as one of possible ability of lactulose to promote the growth of
mechanisms [26].
bifidobacteria comparing with other prebiotics [20, 21]. The effect of lactic acid bacteria is inconsistent; Evaluating the research data it should be concluded
there is possible the significant decrease of cholesterol that influence of different prebiotics is not significant
content and also unchangeable cholesterol content. It (P > 0.05). The type of prebiotics has not significant
depends mainly on the bacteria species used for influence on the content of cholesterol in milk. The
fermentation [27]. Pereira’s [12] research has significant decrease was determined in fermented milk
confirmed that depending on the species of bacteria
samples with different concentration of prebiotics (P < the decrease of the cholesterol content is possible from 0.05). There is established between control sample
0.4% to 47% in the selective culture mediums. Zhao and fermented milk sample with 4% of lactulose.
et al. [28] reported that Lactobacillus acidophilus was
Table 1 The influence of the concentrations of lactulose and inulin on the growth rate of B. lactis in fermented milk samples, lg cfu·mL -1 [18].
Concentrations(%) Lactulose Inulin
a: no disparity (P > 0.05) compared to control; b: a disparity (P < 0.05) compared to control.
The Study of Cholesterol Content in Synbiotic Fermented Dairy Products
effective in reducing cholesterol level in MRS
Acknowledgments
medium. Whereas Ziarno et al. [13] have indicated Publication and disamination of research results has that Lactobacillus acidophilus and Bifidobacterium been made due to the funding of the ERAF Project spp. in fermented milk are able to assimilate “Promotion of scientific activities of LLU”, Contract cholesterol from 18% to 38%. Nr. 2010/0198/2DP/2.1.1.2.0/10/APIA/VIAA/020. The results of the research have shown that it is
possible to achieve a considerable decrease of the
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Journal of Life Sciences 6 (2012) 1082-1093
Metabolic and Endocrine Responses of Desert-Adapted Mice Reproductive System to Increased Salinity
1 1 Elena Bukovetzky 1, 2 , Fuad Fares and Abraham Haim 1. Department of Evolutionary and Environmental Biology, University of Haifa, Mount Carmel, Haifa 31905, Israel
2. The Israeli Center for Interdisciplinary Studies in Chronobiology, University of Haifa, Mount Carmel, Haifa 31905, Israel
Received: March 12, 2012 / Accepted: April 27, 2012 / Published: October 30, 2012.
Abstract: From an evolutionary point of view, reproduction timing is an important adaptation which enables the transfer of genetic properties, thus enabling species continuation. Rodents inhabiting arid environments need reliable cues for triggering their reproduction. Results of previous studies showed that increased dietary salinity plays an important role as an ultimate regulator for desert adapted rodents’ reproductive system. The authors aimed discovering pathways by which high salinity can affect the reproductive system and metabolic status of desert adapted common spiny mice, Acomys cahirinus. Mice were challenged with osmotic stress, water source salinity increased gradually from 0.9% - 5% NaCl under short days (SD) and long days (LD). The authors assessed leptin and free fatty acid (FFA) levels using ELISA while, SYBR green technology was used for relative receptor expression (RQ) of target genes. Results revealed that serum levels of the hormone leptin were significantly (P < 0.05) reduced in salinity treated (ST) mice. Levels of FFA were significantly (P < 0.05) increased in LD- and SD-ST-males. In ST-SD females a significant increase (P < 0.05) in expression levels of leptin (Ob-Rt) mRNA receptor gene, in ovaries was noted. Aldosteron (Nr3c2) and vasopressin (AVP) mRNA receptor expression genes levels were significantly (P < 0.05) increased in both LD- and SD- ST- males.
Key words: Acomys cahirinus, salinity, desert-adapted, AVP, Nr3c2, Ob-Rt receptor genes, leptin, FFA.
1. Introduction The past management of agricultural land and climatic changes, alone or acting together, have led to
Mammals are known to use various environmental the accumulation of excessive salts in land, water and cues to forecast the occurrence of the optimal season vegetation—a phenomenon named salination. At for reproduction. One such cue most commonly used sufficient levels, salinity has negative impacts on by them is day length changes (photoperiod) [1, 2], human and natural assets such as plants, animals, which is a common cue in predictable ecosystems aquatic ecosystems, infrastructure, water supplies and with minimal year to year changes in climatic agricultural land [6]. Salt known as sodium chloride conditions. Desert regions are unpredictable and (NaCl) holds a unique position in the annals of human therefore, additional cues for successful breeding are and animal history and in health and disease research. needed. Because of unpredictable climatic changes, Both the level of dietary NaCl and the background desert species could be “misled” if they entirely rely diet are important in generating a hypertensive only on photoperiod. However, photoperiod is used as phenotype in the rat [7]. But in the last years, an initial cue for breeding regulation of desert-adapted increased salinity levels in plants attracted researchers rodents [3-5]. with regards to possible effects on reproduction,
especially in desert adapted rodents [4, 5, 8]. Due to Corresponding author: Elena Bukovetzky, Ph.D. candidate, research fields: environmental biology and endocrinology.
increased evaporation under desert conditions, particle E-mail: [email protected].
Metabolic and Endocrine Responses of Desert-Adapted Mice
Reproductive System to Increased Salinity
concentration in plants elevate as the dry season as an endocrine and immunological organ incorporates progresses since the last water input into the
cross-talk and co-stimulation of a multitude of ecosystem. It was suggested that increased dietary
hormonal signals [13]. It is possible that localized salinity could be used as an ultimate signal for
hormonal positive and negative feedback loops reproductive system regulation of the desert adapted
interact with the tissue itself and/or through additional golden spiny mouse Acomys russatus [4]. The results
pathways.
of previous studies showed a strong negative effect of By the identification of leptin, the high salt diet on the reproductive status of adipocyte-derived hormone, and the leptin gene (Lep desert-adapted males and females of A. russatus but
Ob), followed by its cognate receptor Ob-Rt located not on a mesic population of common spiny mice
within the ventral medial hypothalamus (VMH), Acomys cahirinus [5]. Sheep females, kept on high
paved the way for this area of research. Leptin is a 167 salt intake diet during pregnancy showed hormonal
amino acid residue hormone almost exclusively changes, which can negatively affect the offspring via
derived from WAT that shares structural similarities
a mechanism called fetal programming [6]. High with cytokines. The primary effects of leptin involve salinity gradually treated rodents showed a significant
the mobilization of lipolytic pathways of energy
expenditure as it signals to the hypothalamus that suggested that high salt consumption decrease food
decrease in body mass (W b ). It has previously been
energy sufficiency has been met [14]. intake in sheep [9].
In rodents it is well established that leptin gene Another mechanism by which the increased dietary
expression and plasma levels are proportional to total salinity affects reproduction in rodents could be
body WAT stores [15]. Recently, it has been reported associated with the hormones vasopressin [4, 5] and
that leptin has a role in the early onset of the hormone leptin, the latter produced by adypocites,
reproduction [16, 17]. In mice it was noted that leptin which regulates W b , metabolism and reproduction [10, 11].
serves as a permissive signal for the reproductive Salinity treatment to desert adapted A. russatus,
system [18]. Results of studies on bats have shown
that serum leptin levels are positively correlated with reproductive hiatus (decreasing number of estrous
revealed a significant decrease in W b , progressive
WAT mass and negatively correlated with the cycles and relative decreased testis mass), which was
reproductive function [19, 20]. Leptin has been shown coupled with a massive (about 80%) reduction of
to promote sexual maturation in various rodent species white adipose tissue (WAT) mass, compared with
and its role in reproduction has been investigated at their control group [5].
various sites within the hypothalamo-pituitary-gonadal The primary role of WAT commonly referred to as
axis [21]. It was suggested that the pituitary-derived “fat” in mammals [12], is to store free fatty acids
leptin acts on the gonads and many other organs (FFA) as triglycerides (triacylglycerol) during periods
equipped with leptin receptor through endocrine of caloric excess (lipogenesis) and to mobilize these
function in addition to paracrine action with pituitary reserves and release free fatty acids for ATP synthesis
hormone secretory cells [22].
via the Kreb’s Cycle during periods when expenditure By using a transgenic animal model, it was shown of calories exceeds the intake (lipolysis) [12]. WAT is
that brown adipose tissue (BAT) expresses now recognised as a multifunctional organ; in addition
mineralocorticoid receptor (MR), which can be to the central role of lipid storage, it has a major
activated by aldosterone [23]. Little is known about endocrine function secreting several hormones, the potential involvement of the mineralocorticoid notably leptin. Clearly, the emerging picture of WAT
system in WAT development. The results of previous
Metabolic and Endocrine Responses of Desert-Adapted Mice
Reproductive System to Increased Salinity
pharmacological studies have suggested that avoid bias results related to W b , both SD and LD aldosterone may promote adipogenesis [24, 25]. It was
acclimated groups had a similar average W b (34-36 ± 3 noted that chronic exposure to aldosterone induces
g) at the beginning of experiments. Ethical clearance morphological, biochemical and molecular markers of
for the use of animals was provided by The Ethical adipose conversion by stimulating the adipogenic
Committee, University of Haifa [8]. transcriptional program [26].
2.1 Acclimation to Increasing Salinity under Different The results of a study carried out on obese prone
Photoperiod Regimes
(OP) rats [27], revealed that high-salt intake induced an increase in the size of WAT but a reduction in
36 individuals (18 females and 18 males) were number of adipocytes, accompanied by twofold
acclimated to short days (SD, 8L:16D, lights on from increase in circulating leptin. It was suggested that
07:00 a.m.) while 26 individuals (12 females and 14 high sodium chloride content diet could modulate
males) were acclimated to long days (LD, 16L:8D, leptin levels, independently of obesity. In accordance
lights on from 07:00 a.m.) inside a controlled with this recently conceived concept regarding leptin
environmental cabinet (158 × 77 × 74 cm; Meditest physiology [28], rodents treated with a high salt (HS)
600/1300, Austria) for three weeks. As these
individuals were used also in for measuring other and adipose mass-reducing [29, 30], but leptin’s
diet were resistant to metabolic effects of leptin: as W b ,
variables for further information on acclimation sympatho-excitatory actions still remains intact. The
groups [8].
accumulated knowledge so far brings us to test the
2.2 Sacrificing Animals and Collecting Samples follow hypothesis: if leptin, vasopressin and
aldosterone are involved in breeding regulation of At the end of acclimation to the highest dietary desert-adapted rodents than the three hormones will
salinity (5% NaCl) mice were, anesthetized by have a direct effect through their receptors on the
injecting a cocktail of Ketamin (10 mg/kg) and gonads or indirect effect of these hormones via WAT.
Rampoone (100 mg/kg), and then sacrificed by In order to establish the metabolic status of the tested
decapitation. All animals were sacrificed between mice we also detected serum levels of free fatty acid
10:00 and 12:00 as reported earlier for our studied (FFA) and leptin in males and females of a desert
individuals [8]. (For both photoperiod groups three adapted population of common spiny mice, A.
hours after lights were on). Blood samples were cahirinus acclimated to short day (SD) or long day
collected and centrifuged at 3,000 rpm for 10 min. (LD) photoperiods.
Serum was collected and stored at -20 °C for analyzing serum leptin hormone level and FFA.
2. Materials and Methods
WAT and gonads were removed and weighed. The Studied mice were recruited from a desert adapted
different organs mass was determined by using an laboratory colony of A. cahirinus, maintained at
analytical scale (1907 MP8 Sartorius, Germany) and Oranim campus, University of Haifa. Mice in the
calculated as percent of W b . Organs were stored at colony are descendants of individuals originally
-20 °C in RNA later (Beit Haemek, Israel) for RNA captured on the western Dead Sea shores. All tested
stability.
mice, at the beginning of acclimation, were adults,
2.3 Determination of Serum Leptin Levels aged four months. In the colony, mice were
maintained at an ambient temperature of 26 ± 2 °C Serum leptin levels were measured by using a under a photoperiod regime of 12L:12D. In order to
commercial ELISA (R&D System, USA) kit
Metabolic and Endocrine Responses of Desert-Adapted Mice
Reproductive System to Increased Salinity
according to manufacturer’s instructions. This assay For reference we used NCBI sequence NM_016847.2) employs the quantitative sandwich enzyme receptor genes. The expression of leptin receptor was immunoassay technique. The product of these measured only in gonads (Ob-Rt: For reference we enzymatic reactions determined used NCBI sequence NM_146146.2). For reference spectrophotometrically, by ELISA reader (Power
housekeeping gene GAPDH (glyceraldehyde-3- Wave XS, BioTek Gen 5) at a wavelength of 450 nm.
phosphate dehydrogenase: NM_008084.2 as reference sequence) was used.
2.4 Determination of Serum FFA Levels
2.7 Primer Design
In the assay used, fatty acids are converted to their CoA derivates, which are subsequently oxidized with
All primer sets on exon-exon junction site, were the concomitant generation of color/fluorescence. C-8
designed to have a T m of approximately 60 °C, to (octanoate) and longer fatty acids can be easily
have a GC content of approximately 50%, and to quantified by colorimetric (570 nm) method with
generate a PCR amplicone less than 150 bps. Finally, palmitic acid employed as a standard (Free Fatty Acid
BLAST searchers were performed on primer pair Quantification Kit, BioVision, USA) according to
sequences using the NCBI database to check for manufacturer’s instructions.
uniqueness. Primer sets and identifiers are provided (Appendix 1).
2.5 Total RNA Extraction and Reverse Transcription
2.8 qRT-PCR Amplification
The reverse transcription-polymerase chain reaction (RT-PCR) has become a standard tool in
qRT-PCR (quantitative real-time reverse quantification of gene expression analysis studies [31].
transcription-PCR) has become the benchmark for the Total RNA was extracted from mice testis using
detection and quantification of RNA targets. RNeasy Mini Kit (Qiagen, Germany). For mRNA
Quantitative real-time PCR (qRT-PCR) was purification from testis and RNA extraction from
performed using SYBR Green technology [31]; WAT the authors used EZ-RNA kit (Biological
primers were designed by Sigma, Israel. For the Industries, Israel, Beit Haemek Ltd.). Isolated total
real-time PCR procedure, gene-specific primers and RNA was quantified photometrically at a SYBR Green Real-Time PCR Master Mix (T Applied wavelength of 260 nm. Quality of RNA was verified
Biosystems, CA, USA) were used. Relative gene by loading 1 μL of total RNA onto a RNA 6,000
expression was detected by the ABI Prism 7000 Nano Chip using the Nano Assay 600 Bioanalyzer
Genetic Analyzer (Applied Biosystems, CA, USA). (Agilent, Waldbronn, Germany) following the The relative expression (relative quantification: RQ) manufacturer’s instructions. Single stranded cDNA
of each target gene (Ob-Rt; AVP; Nr3c2 genes), was was generated out of 0.5 μg total RNA using a High
normalized to the amount of GAPDH as housekeeping Capacity cDNA RT Kit (Applied Biosystems, UK).
gene transcript in the same cDNA. RQ relates the The obtained cDNA was stored (-20 °C) for further
PCR signal of the target transcript in a treatment analysis.
group to that of another sample such as an untreated control. A melting curve analysis was performed to
2.6 Receptors Detection verify that a single PCR product was generated.
Testis and WAT were tested for the expression of Negative controls, performed by omitting reverse aldosteron (Nr3c2: For reference we used NCBI
transcriptase from the RT step, remained consistently sequence NM_001083906.1) and vasopressin (AVPr:
negative.
Metabolic and Endocrine Responses of Desert-Adapted Mice
Reproductive System to Increased Salinity
2.9 Statistical Analysis Because of massive reduction in W b , and WAT mass as result of salinity treatment, WAT could only
All values are given as mean ± SEM, measured
be collected from LD and SD control groups mRNA values of control and experimental groups were receptor expression using our primers only compared using two-tailed independent t test on SPSS Aldosterone, but not AVP was detected. However, no
15.0.1 for Windows. For group comparison one way significant differences between the two photoperiod ANOVA was used. Post-Hoc test was conducted
acclimated groups were noted.
using LSD. Difference in mean values were considered significant when P < 0.05. Actual
3.1 Female Responses to Photoperiod Manipulations probability values were given for each comparison.
and ST Treatment
FFA levels were significantly elevated in The metabolic status of SD- and LD-acclimated
3. Results and Analysis
ST-LD-acclimated group, compared with the males and females (control) as well as ST-males and
ST-SD-mice (P < 0.05; F 1,11 , = 4.589). ST caused a females was estimated from serum FFA and leptin
decrease in serum leptin levels in both, LD- and concentrations. The relation between WAT as SD-acclimated females, compared with their control metabolic and endocrine tissue is assessed from the
groups (P < 0.05). There was a trend to increase leptin correlation between FFA and leptin (Figs. 1 and 2).
levels (~ 25%) in SD-acclimated females, compared In order to assess these relations with the
with the LD-females (Table 1A). However, FFA reproductive system, the response to increased salinity
levels in LD- and SD-acclimated (control) females in diet is evaluated from the mRNA receptors
were similar. There was a strong positive correlation expression of Nr3c2, AVP and Ob-Rt in the gonads 2 (R = 0.48, P < 0.05) between leptin and FFA serum
(Figs. 3 and 4). levels in SD-acclimated females (Fig. 1). In addition, the role of photoperiod effect on
As ovaries of ST-LD-acclimated females were metabolic and reproduction molecular response was
atrophied we could only compare between ovaries of highlighted, in both sexes of desert-adapted A.
SD- and ST-SD-females. Expression of Nr3c2 cahirinus population by studying receptors activation
mRNA was not affected by ST in SD-acclimated in the gonads.
females while, a strong effect of ST was noted in
Fig. 1A Correlations between serum FFA levels (nmol/µl) and leptin levels (pg/mL) of control long day acclimated (LD control) and salinity treated (LD-ST) desert-adapted female common spiny mice Acomys cahirinus. (n = 6 in each group). R 2 = 0.26 for LD control group; R 2 = 0.03 for LD-ST group.
Metabolic and Endocrine Responses of Desert-Adapted Mice
Reproductive System to Increased Salinity
Fig. 1B Correlations between serum FFA levels (nmol/µL) and leptin levels (pg/mL) of control short day acclimated (SD control) and salinity treated (SD-ST) desert-adapted female common spiny mice Acomys cahirinus. R 2 = 0.48, *P < 0.05 (Pearson) for SD-control group, R 2 = 0.27 for SD-ST group.
Fig. 2A Correlations between serum FFA levels (nmol/µL) and leptin levels (pg/mL) of control long day acclimated (LD control) and salinity treated (LD-ST) desert-adapted male common spiny mice Acomys cahirinus. R 2 = 0.63; *P < 0.01 (Pearson) for LD-control group, R 2 = 0.51, *P < 0.05 (Pearson) for LD-ST group.
Fig. 2B Correlations between serum FFA levels (nmol/µL) and leptin levels (pg/mL) of control short day acclimated (SD control) and salinity treated (SD-ST) desert-adapted male common spiny mice Acomys cahirinus. R 2 = 0.16 under SD-acclimation. R 2 = 0.65, *P < 0.01 (Pearson) for SD-ST group.
Metabolic and Endocrine Responses of Desert-Adapted Mice
Reproductive System to Increased Salinity
RQ
Fig. 3 Receptors mRNA expression (relative quantification—RQ) in ovaries of short day (SD control) acclimated and salinity treated (SD-ST) desert adapted female common spiny mice A. cahirinus. Nr3c2: aldosterone receptor gene expression; AVP: vasopressine receptor gene expression; Ob-Rt: leptin receptor gene expression. *P < 0.05 in SD-ST females, compared with SD-control ones.
RQ
Fig. 4 Receptors mRNA expression (Relative quantification—RQ) in testis of short and long day (SD, LD-control) acclimated and salinity treated (SD-ST, LD-ST) desert adapted female common spiny mice A. cahirinus. Nr3c2: aldosterone receptor gene expression; AVP: vasopressine receptor gene expression; Ob-Rt: leptin receptor gene expression. *P < 0.05 in treated groups, compared with their controls.
Table 1A Leptin and FFA concentration in serum (± SEM) of short and long day acclimated (SD, LD-control) and salinity treated (SD-ST, LD-ST) desert-adapted female common spiny mice Acomys cahirinus.
FFA levels (nmol/µL) LD female
Treatment
Leptin levels (pg/mL)
LD-ST female
SD female
SD-ST female
Leptin (pg/mL) and FFA (nmol/ μL) concentration (n = 9 in each SD group) and (n = 6 in each LD group) . Leptin levels in LD-ST and SD-ST females compared with their control *P < 0.05. FFA serum levels in LD-ST females compared with their control, *P <
0.05. FFA serum levels in SD-ST females compared with the LD-ST ones * P < 0.05, (F 1,12 = 1.34).
Table 1B Leptin and FFA serum levels (± SEM) of short and long day acclimated (SD, LD control), SD and LD salinity treated (SD-ST, LD-ST) desert adapted males of common spiny mice Acomys cahirinus.
Treatment
FFA levels (nmol/µL) LD male
Leptin levels (pg/mL)
LD-ST male
SD male
SD-ST male
Leptin (pg/mL) and FFA (nmol/ μL) concentration (n = 9 in each SD group) and LD (n = 6 in each LD group). Leptin levels in LD-ST and SD-ST males compared with their control, *P < 0.05. FFA serum levels in LD-ST and SD-ST males compared with their control *P < 0.05.
Metabolic and Endocrine Responses of Desert-Adapted Mice
Reproductive System to Increased Salinity
mRNA receptors expression genes of AVP and Ob-Rt, reported earlier [8] for A. cahirinus, individuals used which were significantly (P < 0.05) increased in
in the present study. Some other previous studies ST-SD females compared with their controls (Fig. 3).
showed that dietary sodium chronic restriction has been related to increased WAT mass in rats [32].
3.2 Male Responses to Photoperiod Manipulations WAT development depends on a balance between and ST Treatment food consumption and energy expenditure [33]. It was
No effect of photoperiod was noted, as under both also revealed that high salt dietary consumption LD and SD acclimation FFA serum levels were
significantly decreased feed intake in sheep [9, 34]. similar. However, ST caused a significant (P < 0.05)
The results of our study, in addition to the progressive increase in serum FFA and a significant decrease (P <
decrease in W b , revealed an almost complete
0.05) in leptin levels, in both, LD and SD group abolishment of WAT in ST-mice as reported earlier [8]. compared with their control groups (Table 1B). The
WAT reduction and W b values decrease in our study correlations between leptin and FFA serum levels
could not be explained by reduced energy intake, as were significant and negative for both, ST-LD (R 2 =
ST mice consumed the same amount of food as the
0.51, P < 0.05) and ST-SD (R 2 = 0.65, P < 0.01) mice. control mice throughout the experimental period [8]. The same correlation was noted as positive and
We tested adipocytes lipid mobilization resulted from significant (R 2 = 0.63, P < 0.01) in LD-acclimated ST compared with their control. This process known
mice (Fig. 2). as lipolysis, consists of Triacylglycerole (TAG) The mRNA receptor expression of Nr3c2 was affected
hydrolysis, FFA and glycerol release, which represent by ST in both, LD- and SD- males. A significant (P <
an important mechanism for controlling WAT mass
0.05) increase in mRNA receptor expression genes and metabolism [35]. ST males showed an increase in was noted in both Nr3c2 and AVP under both
FFA release, during both, LD and SD acclimation compared with their controls. ST-LD-acclimated
photoperiod regimes. However, Ob-Rt mRNA receptor expression genes increased significantly (P <
females showed increased FFA release compared with ST-SD females (Table 1A). Chronic salt loading may
0.05) by ST only under LD-acclimation. ST under increase dysfunction of fat cells in lean and obese
SD-acclimation had no effect on testis Ob-Rt mRNA alike [35]. A significant linear positive relationship
receptor expression genes (Fig. 4). between leptin levels and WAT was noted in previous
4. Discussion
study where lower serum leptin levels in cold acclimated animals could act as a starvation signal [36].
4.1 Metabolic Response to ST It was also shown that ST induced hyperleptinemia
According to the results of previous studies, ST and it may stimulate the lipolytic process by a direct
induces a dramatically W b decrease [4, 5] in
action [37].
desert-adapted A.russatus. A massive reduction in W b High salt (3% NaCl) treated rats exhibited higher and WAT mass was noted in ST-SD males where W b plasma leptin levels compared with those of controls [38]. decreased significantly (P < 0.001) by 26% ± 0.1%,
Higher plasma leptin levels were also reported for rats while in ST-LD males the loss was only of 13% ±
kept on a 4% NaCl diet for 10 weeks compared with
those kept on 0.8% NaCl [27]. In our study, ST caused only 17% ± 0.03% but still significant (P < 0.01),
0.03% (P < 0.01). In SD-ST females a W b loss was of
hypoleptinemia in both, males and females, under while for LD-ST females a 3% ± 0.2% significant (P
both, LD and SD-conditions compared with their
< 0.01) decrease in W b from their initial values as
controls. In addition, the correlations between leptin
Metabolic and Endocrine Responses of Desert-Adapted Mice
Reproductive System to Increased Salinity
and FFA levels were significant, in both, males and osmoregulation [40] on the one hand and they affect females (Figs. 1 and 2). These correlations were
the reproductive ability of desert adapted rodents on positive in LD and SD-acclimated groups (controls),
the other [8]. The AVP gene is now known to be but negative, in ST groups. Combining our results
expressed in a number of peripheral organs such as the with Ref. [35], we suggest that ST may alter (directly
adrenal glands, ovaries, and testes [41]. Ivell [42] or indirectly) the secretion function and metabolic
reported that AVP mRNA is detectable in the rat testis. activity of WAT.
It is now well documented that aldosterone receptor are also expressed in non-epithelial tissues, including
4.2 Reproductive Response to ST the cardiovascular and central nervous systems as well
Previous studies on leptin receptors (leptin-R) in as on the white adipose tissue [43]. rodents have demonstrated the expression of leptin-R
The results of our study show that a part of the gene in the hypothalamus, ovary, uterus, testis and
involvement of these two hormones in osmoregulation, pituitary by reverse transcriptase polymerase chain
they are also possibly involved in inhibiting reaction (RT-PCR) [39]. In our study using the same
reproduction under osmolarity stress as there serum method we detected a significant increase in levels increased also mRNA receptor expression genes expression levels of mRNA receptors genes for
increase. These increases suggest an involvement of aldosterone (Nr3c2) in testis of ST-males under the
molecular mechanism in the gonads as a response to two photoperiods (P < 0.05, Fig 4). ST caused a
increased osmolarity stress.
significant increase (P < 0.05, Fig. 4) in expression Human WAT cells secrete mineralocorticoid- level of vasopressin (AVP) mRNA receptor genes in
releasing factors [44]. The AVP receptors (V1b and testis under both photoperiods while for females only
V2) were attributed in mice to lipid metabolism; SD-ST could be measured (P < 0.05, Fig 3). High
expression of the two genes was also noted in the gene expression levels of leptin (Ob-Rt) mRNA
heart, liver, kidney, skeletal muscle, BAT, and WAT. receptors were noted in SD-ST females (P < 0.05,
The V1a receptor was expressed in all tissues Fig 3) while in males only in LD-ST individuals (P <
examined, but the V1b receptor was expressed only in
0.05, Fig. 4), compared with their controls. We WAT, while the V2 receptor in mice was expressed suggest that ST caused a significant decrease of leptin
only in the kidney [45]. We searched for AVP and levels in serum and for this reason; leptin receptors
Nr3c2 mRNA receptor genes expression in WAT. For sites expressed on gonads were not occupied by the
expression of receptors we had compared only SD- hormone.
and LD-acclimated control males, as WAT was The authors have no data on receptors gene
abolished due to ST. There was no detectable AVP expression in ovaries of ST-LD females, as there
mRNA receptor gene expression, but only of Nr3c2. ovaries were atrophied. Yet the authors can suggest
There was no significant difference in expression that ST had an effect on metabolism and reproductive
levels between LD- and SD-acclimated groups. ability of ST-LD-acclimated females, as leptin levels
5. Conclusions
were decreased under both photoperiod acclimations regimes. It is important to note that ingestion of