Testicular Metabolic Reprogramming in Neonatal Streptozotocin-Induced Type 2 Diabetic Rats ...

13 Pages·2015·1.33 MB·English

Hindawi Publishing Corporation
Journal of Diabetes Research
Volume 2015, Article ID 973142, 13 pages
http://dx.doi.org/10.1155/2015/973142
Research Article
Testicular Metabolic Reprogramming in Neonatal
Streptozotocin-Induced Type 2 Diabetic Rats Impairs
Glycolytic Flux and Promotes Glycogen Synthesis
L.Rato,1M.G.Alves,1,2T.R.Dias,1J.E.Cavaco,1andPedroF.Oliveira1,3,4
1HealthSciencesResearchCentre(CICS),FacultyofHealthSciences,UniversityofBeiraInterior(UBI),Covilha˜,Portugal
2DepartmentofLifeSciences,FacultyofSciencesandTechnologyandCenterforNeurosciencesandCellBiology(CNC),
UniversityofCoimbra,Portugal
3DepartmentofMicroscopy,LaboratoryofCellBiology,AbelSalazarInstituteofBiomedicalSciences(ICBAS),
UniversityofPorto,Portugal
4UnitforMultidisciplinaryResearchinBiomedicine(UMIB),AbelSalazarInstituteofBiomedicalSciences(ICBAS),
UniversityofPorto(UP),Portugal
CorrespondenceshouldbeaddressedtoPedroF.Oliveira;[email protected]
Received26November2014;Revised22April2015;Accepted23April2015
AcademicEditor:MariaPiaFrancescato
Copyright©2015L.Ratoetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Defectsintesticularmetabolismaredirectlyimplicatedwithmaleinfertility,butmostofthemechanismsassociatedwithtype
2diabetes-(T2DM)inducedmaleinfertilityremainunknown.WeaimedtoevaluatetheeffectsofT2DMontesticularglucose
metabolism by using a neonatal-streptozotocin- (n-STZ) T2DM animal model. Plasma and testicular hormonal levels were
evaluatedusingspecifickits.mRNAandproteinexpressionlevelswereassessedbyreal-timePCRandWesternBlot,respectively.
1
Testicularmetabolicprofilewasassessedby H-NMRspectroscopy.T2DMratsshowedincreasedglycemiclevels,impairedglucose
toleranceandhyperinsulinemia.Bothtesticularandserumtestosteronelevelsweredecreased,whereasthoseof17𝛽-estradiolwere
notaltered.TesticularglycolyticfluxwasnotfavoredintesticlesofT2DMrats,since,despitetheincreasedexpressionofbothglucose
transporters1and3andtheenzymephosphofructokinase1,lactatedehydrogenaseactivitywasseverelydecreasedcontributingto
lowertesticularlactatecontent.However,T2DMenhancedtesticularglycogenaccumulation,bymodulatingtheavailabilityof
theprecursorsforitssynthesis.T2DMalsoaffectedthereproductivespermparameters.Takentogethertheseresultsindicatethat
T2DMisabletoreprogramtesticularmetabolismbyenhancingalternativemetabolicpathways,particularlyglycogensynthesis,
andsuchalterationsareassociatedwithimpairedspermparameters.
1.Introduction prevalenceofmalesubfertility/infertility[3–6]anddecreased
birthrates[7,8].
Diabetesmellitus(DM)ischaracterizedbyhyperglycaemia, Recently our team reported that diet-induced predia-
resulting from defects in insulin secretion, insulin action, betes, which is a prodromal stage of T2DM, compromises
or both. The most prevalent form of DM is type 2 diabetes sperm quality and promotes marked alterations in overall
mellitus(T2DM),comprisingupto95%ofalldiagnoseddia- testicular metabolism and ionic homeostasis [9, 10]. The
beticindividualsindevelopedcountries[1].T2DMinduces testiclescompriseaheterogeneouscellpopulationconsisting
metabolicalterations,disruptingtheendocrinesystem,with ofbothsomaticandgermcells.Testicularcellspresentunique
a subsequent dysfunction of the hypothalamus-pituitary metaboliccharacteristics,inpartduetotheexistenceofthe
testicles (HPT) axis [2], that may end up in impairment of blood-testis barrier. The Sertoli cells (SCs) are responsible
themalereproductivehealth.Althoughnotalldiabeticmen fortheproductionofmetabolicprecursorsessentialforgerm
are infertile, evidences strongly associate T2DM with high cellsdevelopment[11,12].Thesecellsshowa“Warburg-like
2 JournalofDiabetesResearch
metabolism”[13],usingtheexternalglucosetoproducelac- wateradlibituminaconstantroomtemperature(20±2∘C)
tate,whichisthepreferredsubstrateofdevelopinggermcells ona12-hourcycleofartificiallighting.Allexperimentswere
[14–17].TesticularcellsinparticularSCstakeupextracellular performed according to the Guide for the Care and Use of
glucose via specific glucose transporters (GLUTs), which is LaboratoryAnimalspublishedbytheUSNationalInstitutes
thenoxidizedtopyruvateandpromptlyreducedtolactateby of Health (NIH Publication number 85–23, revised 1996)
lactatedehydrogenase(LDH),withaconcomitantoxidation and the European directives for the care and handling of
of NADH. Once produced, lactate is exported through the laboratoryanimals(Directive86/609/EEC).
monocarboxylate transporter 4 (MCT4) to the intratubular
fluidtobecomeavailableforthedevelopinggermcells.Thus, 2.2. Experimental Design. Animals were randomly distri-
SCs and germ cells establish a tight metabolic cooperation buted in a control and neonatal-streptozotocin- (n-STZ-)
that is highly dependent on glucose uptake and lactate induced T2DM group. Animals from T2DM group were
production[9,17].Fromthephysiologicalpointofviewthe injected with a low dose of STZ to achieve a T2DM
metabolism of carbohydrates, specially glucose, is vital for model, according to the method described by Iwase and
male reproductive health, so the maintenance of testicular collaborators [26]. In brief, two-day-old male Wistar rats
glucose metabolism homeodynamics is of particular rele- wereintraperitoneallyinjectedwithSTZ(40mg/kg)freshly
vance;otherwisespermatogenesisisarrested[18,19].Hence, dilutedincitratebuffer(0.1M,sodiumcitrate,pH4.5).The
thefullenlightenmentoftesticularglucosemetabolismand controlgroupreceivedthevehiclesolutioninanequivalent
the molecular mechanisms that control it is of extreme volume.Allanimalswerefedwithstandardchowdiet(4RF21
importance,sincealterationsinthesemechanismsmaybeon certificate, Mucedola, Italy) and water. Animals’ glycaemias
thebasisofmaleinfertility[9,20,21].Aroleforalternative were weekly monitored between the 30th and 90th days of
substrates during diabetic conditions has been suggested ageusingaglucometer(OneTouchUltraLifescan-Johnson,
(forreviewsee[18]).Theuseofthesealternativesubstrates, Milpitas,CA,USA).Aftertreatment,animalswerekilledby
suchasglycogen,mayactasacompensatorymechanismfor decapitation. Blood was collected in heparinized tubes for
fluctuationsininsulinandglucoseconcentrations.Glycogen further analysis and testicles were removed, weighed, and
is a readily mobilized fuel store, which provides the body storedat−80∘C.Thelevelsofglycatedhemoglobin(HbA1c)
with a readily available source of energy if blood glucose werealsodeterminedusingA1cNow+meter(BayerDiabetes
availabilitydecreases.Thepresenceofglycogenandglycogen Care,USA).
metabolism-related enzymes in testicular tissue has already
been reported [22–24]. In fact, glycogen seems to play a
2.3. Glucose and Insulin Tolerance Test. At 3 months of
pivotalrolewithinthetesticles,particularlyduringtesticular
age, animals were submitted to a glucose tolerance test, as
development [24], where it acts as modulator of germ cell
describedbyRatoandcollaborators[9].Inbrief,14hbefore
survival[24].However,theroleofglycogenwithintesticular
the test, food was removed and the animals were kept in
milieu has been overlooked, particularly under abnormal
fast. An intraperitoneal (IP) injection with 6mL of glucose
physiologicalconditionssuchasT2DM.Apartfromastudy
30%(w/v)perkgofbodyweightwasgiventoeachanimal.
showing that diabetic animals present increased testicular
Bloodsampleswereobtainedfromthetailandglucoselevels
levelsofuridinediphosphoglucose(UDP-glucose),aglycosyl
measuredevery30minduring2h.Theareaunderthecurve
donorfortheinitialstepofglycogensynthesis[25],noother
fortheglucosetolerancetest(AUCGTT)wascalculatedusing
studies were performed to evaluate the impact of T2DM
thetrapezoidalrule,asdescribedpreviously[20].
on testicular glycogen. Herein we aimed to evaluate the
The animals were also subjected to an insulin tolerance
effects of T2DM on testicular metabolism, with particular
testasdescribedbyHolmesandcollaborators[27].Inbrief,
emphasisonglycolyticandglycogenmetabolism.Wehypoth-
4h before the test, food was removed and animals were
esized that T2DM may impair male fertility by acting in
kept in fast. An IP injection with 0.75U insulin per kg of
keyglycolytic-associatedenzymesandtransporters,altering
body weight was givento eachanimal.Bloodsampleswere
testicular metabolism. We also hypothesized that T2DM
obtained from the tail and glucose levels measured every
inducescrucialchangesintesticularglycogenmetabolism.
30minduring2h.Theareaunderthecurvefortheinsulin
tolerancetest(AUCITT)wascalculatedusingthetrapezoidal
2.MaterialsandMethods rule,asdescribedpreviously[20].
All chemicals were purchased from Sigma-Aldrich (St. 2.4. Testosterone, 17𝛽-Estradiol, and Insulin Measurement.
Louis,MO,USA)unlessspecificallystated.MoloneyMurine
Hormonal levels were measured as described by Rato and
LeukemiaVirusReverseTranscriptaseanddeoxynucleotides collaborators[9].Inbrief,testosterone(T),17𝛽-estradiol(E2),
triphosphates were purchased to NZYTech (Lisbon, Portu-
and insulin levels were determined using commercial rat
gal).
EIA kits according to manufacturer instructions. T and E2
EIAKitswerepurchasedfromCaymanChemicalCompany
2.1.Animals. Twelve-three-month-oldmaleWistarrats(Rat- (AnnArbor,MI,USA).InsulinELISAmeasurementkitwas
tus norvegicus) (Charles River Laboratories, Barcelona, purchased from Mercodia (Uppsala, Sweden). The EIA kits
Spain)wereusedinthisstudy.Theanimalswerehousedin usedhaddetectionlimitsofapproximatelyof40𝜇U/mL(for
ouraccreditedanimalcolonyandmaintainedwithfoodand insulin),6pg/mL(forT),and20pg/mL(forE2).
JournalofDiabetesResearch 3
Table1:OligonucleotidesandcyclingconditionsforPCRamplificationofGLUT1,GLUT2,GLUT3,PFK1,LDHA,MCT4,ALT2,GYS1,
PYGL,and𝛽2MG.
󸀠 󸀠 ∘
Gene Sequence(5-3) AT(C) Accessionnumber
Sense:TCCGGCGGGAGACGCATAGT
GLUT1 61 NM 138827.1
Antisense:CCCGCATCATCTGCCGACCC
Sense:GGGTTCCTTCCAGTTCGGAT
GLUT2 60 NM 012879.2
Antisense:TCGTATGTGCTGGTGTGACT
Sense:GCGCAGCCCTTCCGTTTTGC
GLUT3 63 NM 017102.2
Antisense:CCCCTCGAAGGCCCGGGTAA
Sense:GAGTGCTGACAAGCGGCGGT
PFK1 61 NM 013190.4
Antisense:GTGGCCCAGCACGGTCACTC
Sense:GCGCAGCCCTTCCGTTTTGC
LDHA 63 NM 017025.1
Antisense:CCCCTCGAAGGCCCGGGTAA
Sense:TGAGGTAATCCGAGCCAACA
ALT2 60 NM 001012057.1
Antisense:CACGTCCTCTCGGATACAGT
Sense:ATGTGGGCATGGCGTGTGCC
MCT4 66 NM 001013913.1
Antisense:CCCAGCCATGGCAGCTCGAA
Sense:CAGCTATGGGACACAGCCAA
GYS1 60 NM 001109615.1
Antisense:TTCGTCGGATGGTGGTCAAG
Sense:CTCCCAATCAGCCAGACCTC
PYGL 60 NM 022268.1
Antisense:GGAAGGCTCCATGTTCCAGA
𝛽2MG Sense:ATGAGTATGCCTGCCGTGTG 60 NM 012512.2
Antisense:CAAACCTCCATGATGCTGCTTAC
Legend: AT: annealing temperature; ALT2: alanine aminotransferase 2; GLUT1: glucose transporter 1; GLUT2: glucose transporter 2; GLUT3: glucose
transporter3;GYS1:muscleglycogensynthase;LDHA:lactatedehydrogenaseA;MCT4:monocarboxylatetransporter4;PFK1:phosphofructokinase1;PYGL:
liver-typeglycogenphosphorylase;𝛽2MG:beta2microglobulin.
2.5.NMRSpectroscopy. Acombinedextractionofpolarand as internal control to normalize gene expression (Table 1).
nonpolar metabolites was performed as described by Rato qPCRwascarriedoutinaniQ5system(Bio-Rad,Hercules,
and collaborators [9]. The aqueous phase containing the CA, USA). Efficiency of the amplification was determined
1
water-solublemetaboliteswaslyophilized. H-NMRspectra for all primer sets using serial dilutions of cDNA (1, 1:5
were acquired as previously described by Rato and col- and 1:25). PCR conditions were previously optimized and
laborators [9]. Sodium fumarate was used as an internal specificity of the amplicons was determined by melting
reference𝛿H [2H2O]6.5[s,2xCH]toquantifythefollowing curves. qPCR amplifications were performed with 1𝜇L of
malaentainbeoli1t.e4s5: [lda,ctJat−e14𝛿.H36[H2Hz,23OC]H13.]3,3an[dd,UJD6P.9-gHluzc,o3sCeH7.39]7, sMynaxthimesaizSeYdBRcDGNreAeni/nFluaor2e0sc𝜇eLinrqePaCctRioMnacsotenrtMainixin(gBio10ra𝜇dL)
[d,J8.1Hz,CH].Therelativeareasof1H-NMRresonances and 300nM of sense and antisense primers for each gene.
werequantifiedusingthecurve-fittingroutinesuppliedwith Amplification conditions comprised 5min denaturation at
∘ ∘
the NUTSpro NMR spectral analysis program (Acorn, CA, 95 C, followed by 40 cycles at 95 C for 10sec, a specific
annealing temperature for each gene (Table 1) for 30sec,
USA).
∘
and 72 C for 10sec. Samples were run in triplicate in each
PCR assay. Normalized expression values were calculated
2.6.TesticularGlycogenContent. Testicularglycogencontent
following the mathematical model proposed by Pfaffl using
wasdeterminedbyusingacommercialkit(AbnovaKA0861,
theformula2−ΔΔCt[28].
CA, USA) and following the manufacturer’s instructions.
Glycogen content was expressed as nanomoles of glycogen
permilligramoftissue(wetweight). 2.8.WesternBlot. WesternBlotprocedurewasperformedas
previously described by Simo˜es and collaborators [29]. The
2.7.QuantitativeReal-TimePCR. Quantitativereal-timePCR resultingmembraneswereincubatedwithrabbitanti-GLUT1
(qPCR) was performed to determine glucose transporter (1:500, SC-7903, Santa Cruz Biotechnology, Heidelberg,
1 (GLUT1), glucose transporter 2 (GLUT2), glucose trans- Germany),rabbitanti-GLUT2(1:1000,SC-9117,SantaCruz
porter 3 (GLUT3), phosphofructokinase 1 (PFK1), lac- Biotechnology, Heidelberg, Germany), rabbit anti-GLUT3
tate dehydrogenase A (LDHA), alanine aminotransferase 2 (1:1000,ab41525,Abcam,Cambridge,MA,USA),rabbitanti-
(ALT2),MCT4,muscleglycogensynthase(GYS1),andliver- PFK1 (1:500, SC-67028, Santa Cruz Biotechnology, Heidel-
type glycogen phosphorylase (PGYL) mRNA expression berg,Germany),rabbitanti-MCT4(1:1000,SC-50329,Santa
levels. Specific primers were designed for the amplification Cruz Biotechnology, Heidelberg, Germany), rabbit anti-
of target genes and for 𝛽2-microglobulin which was used LDH (1:10000, ab52488, Abcam, Cambridge, MA, USA),
4 JournalofDiabetesResearch
rabbit anti-ALT (1:500, SC-99088, Santa Cruz Biotechnol- Table2:Averagevaluesoftheanimalsweight,glycaemia,HbA1c,
ogy, Heidelberg, Germany), rabbit anti-GYS1 (1:100, SC- andinsulinlevelsmeasuredinanimalsfromthecontrol(CTR)and
81173,SantaCruzBiotechnology,Heidelberg,Germany),and T2DMgroupafterthreemonthsofage.
rabbitanti-PYG(1:500,SC-66913,SantaCruzBiotechnology,
Parameters CTR T2DM
Heidelberg,Germany).Mouseanti-tubulin(1:5000,T9026,
Bodyweight(g) 347±8 363±11
Sigma-Aldrich, Ro¨dermark, Germany) was used as protein
loading control for testicular tissue. The immunoreactive AverageGlycaemia(mg/dL) 99±1 126±1∗
proteinswereseparatelydetectedwithgoatanti-rabbitIgG- HbA1c(%) 4.80±0.02 5.60±0.07∗
AP (1:5000, Sc-2007, Santa Cruz Biotechnology, Heidel- Insulin(𝜇U/mL) 4.3±0.3 5.2±0.4∗
berg, Germany) or goat anti-mouse IgG-AP (1:5000, Sc- Legend:CTR:control;HbA1c:glycatedhemoglobin;T2DM:type2diabetes
2008, Santa Cruz Biotechnology, Heidelberg, Germany). mellitus.Resultsareexpressedasmean±SEM(𝑛 = 6foreachcondition).
Membranes were reacted with enhanced chemifluorescence ∗Significantlydifferentrelativetocontrol(𝑃<0.05).
detection system (GE Healthcare, Webling, Germany). The
densitiesfromeachbandwereobtainedusingtheQuantity n-STZ-treated animals (126.0 ± 1mg/dL), when compared
OneSoftware(Bio-Rad,HemelHempstead,UK),dividedby to control group (99.0 ± 1mg/dL; Table 2). Blood HbA1c
the respective tubulin band densities and then normalized
levels were also significantly increased (by 17%) in n-STZ-
againsttherespectivecontrolanimals. treated animals (5.60 ± 0.07%) when compared to control
group (4.80 ± 0.02%; Table 2). Together, these results pre-
2.9. Enzymatic Assays. LDH activity was determined using
figure a prolonged state of hyperglycaemia and subsequent
a commercial assay kit (Promega, Madison, USA) and fol-
impaired glucose metabolism. Indeed, the results attained
lowing the manufacturer’s instructions. PFK activity was
for the glucose tolerance test show that blood glycaemia
determined as previously described by Alves and collabo-
of n-STZ-treated animals increased during the 120min of
rators [30]. ALT was determined by a colorimetric assay as
thetest(Figure1(a)),indicatingthedevelopmentofglucose
previouslydescribedbyMohunandCook[31].Theattained
intolerance.Thiscanbeseenbythesignificantlyincreased(by
activitieswereexpressedasfoldvariationversusthecontrol 29%)AUCGTTvaluesinn-STZ-treatedanimals(23364±2231
group.
arbitrary units (a.u.)) when compared to animals from the
controlgroup(18153±735a.u.)(Figure1(b)).Theseresults
2.10. Epididymal Sperm Parameters. Evaluation of epididy- ledustoinvestigatetheinsulinresponsivenessstatus,sowe
malspermparameterswasperformedaspreviouslydescribed performed an IP insulin tolerance test. Our results showed
byDiasandcollaborators[32].Inbrief,caudaepididymiswas thatn-STZ-treatedanimalsdidnotrespondtoinsulin(Fig-
∘
isolated and placed in prewarmed (37 C) Hank’s Balanced ure 1(c)) as observed by the significant increase of AUCITT
Salt Solution (pH 7.4), minced with a scalpel blade and the (by30%)inn-STZ-treatedanimals(10570±1054a.u.),when
suspension incubated for 5 minutes (37∘C). Sperm motility comparedwithratsfromthecontrolgroup(7420±657a.u.)
was evaluated by assessing the percentage of motile sperm (Figure 1(d)), illustrating that these rats developed insulin
in 10 random fields, and the average value was used as the resistance. The higher levels of fasting insulin (increased
totalspermmotility.Spermviabilitywasassessedexamining by 21%) observed in n-STZ-treated animals (Table 2) are
eosin-nigrosinstainedepididymalspermsmears.Epididymal consistent with these results, corroborating that these rats
sperm concentration was determined using a Neubauer developedinsulinresistance.Altogetherthesecharacteristics
countingchamber.Fortheassessmentofspermmorphology clearlyillustratethatthen-STZ-treatedratsdevelopedtype2
we used standard methods [33]. Sperm morphology was diabetes(T2DMgroup).
evaluated using Diff-Quick (Baxter Dale Diagnostics AG,
Dubinger,Switzerland)stainedsmearsaccordingtotheman- 3.2.T2DMDecreasesSerumandTesticularTestosteroneLevels.
ufacturer’s instructions. To be classified as normal, a sperm We evaluated the levels of the sex steroid hormones (T
cell must have a hook-shaped head and no defects of head, and E2) in serum and testicular environment, since they
neck,ortail.Otherwise,spermwereconsideredabnormal. are pivotal for several events that control spermatogenesis,
including testicular metabolism [17]. T serum concentra-
2.11. Statistical Analysis. The statistical significances of dif- tion was significantly decreased (by 95%) in T2DM group
ferences of all experimental data were assessed by Student’s (1.06±0.09nmol/L)whencomparedwiththecontrolgroup
𝑡-test (Graph Pad Software 6.0, San Diego, CA, USA). All (23.16 ± 9.76nmol/L) (Figure 1(e)). On the other hand,
experimentaldataareshownasmean±standarderrorofthe serum E2 levels were not altered in the animals that devel-
mean;𝑃<0.05wasconsideredsignificant. oped T2DM (272 ± 87pmol/L) when compared to control
(127 ± 20pmol/L) (Figure 1(f)). Testicular cells are bathed
by testicular interstitial fluid (TIF) and the establishment
3.Results
of an appropriate fluid is crucial for an adequate hormonal
3.1. Streptozotocin-Treated Rats Developed Type 2 Diabetes control of spermatogenesis. In this fluid, sex steroid levels
MellitusExhibitingMildHyperglycaemia,GlucoseIntolerance, are100-to1000-foldhigherthaninserum,asobservedina
and Insulin Resistance. After three months of age average previousstudy[9].TlevelspresentinTIFweresignificantly
glycaemic values were significantly increased (by 26%) in decreasedby348%inT2DMgroupanimals(7±2nmol/L)
JournalofDiabetesResearch 5
300 30000
∗
∗
dL) ∗ ∗
mia (mg/ 200 (a.u.)T 20000
ae GT
d glyc 100 AUC 10000
o
o
Bl
0 0
0 30 60 90 120 CTR T2DM
Time (min)
(a) (b)
150 15000
∗
L)
a (mg/d 100 ∗ ∗ ∗ a.u.) 10000
mi (
T
e T
ca CI
d gly 50 AU 5000
o
o
Bl
0 0
0 30 60 90 120 CTR T2DM
Time (min)
(c) (d)
40 4000 400 3000
Serum T (nmol/L) 123000 ∗ 2235000000 Intratesticular T (nmol/L) SerumE(pmol/L)2 123000000 12000000 IntratesticularE(pmol/L)2
∗
0 0 0 0
CTR T2DM CTR T2DM CTR T2DM CTR T2DM
Serum TIF Serum TIF
(e) (f)
Figure1:Type2diabetesmellitus(T2DM)induces(aandb)glucoseintoleranceand(candd)insulinresistanceandsignificantlyalters
both(e)serumandtesticulartestosteronelevels.(a)Bloodglucoselevelsofthecontrolgroup(󳵳)andT2DMgroup(e)measuredduring
intraperitoneal glucose tolerance test. (b) Area under the curve for the intraperitoneal glucose tolerance test (AUCGTT) performed in
controlandT2DMgrouprats.(c)Bloodglucoselevelsofthecontrolgroup(󳵳)andT2DMgroup(e)measuredduringintraperitoneal
insulintolerancetest.(d)Areaunderthecurvefortheintraperitonealinsulintolerancetest(AUCITT)performedincontrolandT2DM
grouprats.(e)TestosteronelevelsinintratesticularinterstitialfluidandserumofcontrolandT2DMgrouprats.(f)17𝛽-Estradiollevels
inintratesticularinterstitialfluidandserumofcontrolandT2DMgrouprats.Resultsarepresentedasmean±SEMofsixindependent
experiments,correspondingtosixanimals/group.*Significantlydifferentrelativetocontrol(𝑃<0.05).
6 JournalofDiabetesResearch
NA expression (fold variation to control) 0112....5050 ∗ ein expression (fold variation to control) 0112....5050 ∗ ∗
mR 0.0 Prot 0.0
GLUT1 GLUT2 GLUT3 PFK1 GLUT1 GLUT2 GLUT3 PFK1
(a) (b)
ol) 2.0
ntr
co ∗
n to 1.5
o
ati
ari
d v 1.0
ol
G55LUkDTa1 CTR T2DM 60G–L6U2kTD2a CTR T2DM G48LUkDTa3 CTR T2DM ctivity (f 0.5 ∗
a
e
m
PFK1 TUB zy
85kDa 50kDa En 0.0
CTR T2DM CTR T2DM PFK LDH ALT
(c) (d)
Figure 2: Type 2 diabetes mellitus (T2DM) modulates the (a) mRNA, (b) protein levels, and (d) activity of glycolytic enzymes and
transporters.(a)Pooleddataofindependentexperiments,indicatingthefoldvariationofglucosetransporters(GLUT1,GLUT2,andGLUT3)
andofphosphofructokinase1(PFK1)mRNAlevelsfoundintesticlesofT2DMratswhencomparedwiththecontrolrats(dashedline).(b)
Pooleddataofindependentexperiments,indicatingthefoldvariationofGLUT1,GLUT2,GLUT3,andPFK1proteinlevelsfoundintesticles
ofT2DMratswhencomparedwiththecontrolrats(dashedline).(c)IllustrativeWesternBlotexperiment.(d)Pooleddataofindependent
experiments,indicatingthefoldvariationofPFK1,lactatedehydrogenase(LDH),andalaninetransaminase(ALT)enzymaticactivitiesin
testiclesofT2DMratswhencomparedwiththecontrolrats(dashedline).Resultsareexpressedasmean±SEM(𝑛=6foreachcondition).
*Significantlydifferentrelativetocontrol(𝑃<0.05).
whencomparedwithanimalsfromthecontrolgroup(2457± controlgroup(Figure2(b)).ConcerningGLUT2expression,
583nmol/L)(Figure1(e)).Contrastingly,T2DManimalsdid no alteration was observed in both transcript and protein
not exhibited significantly altered E2 concentrations in TIF levels of T2DM animals when compared with the control
(2585 ± 255pmol/L) when compared to control (2192 ± group(Figures2(a)and2(b)).GLUT3mRNAexpressionwas
239pmol/L)(Figure1(f)). significantlyincreasedby40%inT2DMgroup(1.40±0.17-
foldvariationtocontrol)(Figure2(a))andwasfollowedby
3.3.T2DMIncreasesGLUT1/GLUT3LevelsandPFKActivity a 31% increase in protein levels (1.31 ± 0.13-fold variation
inRatTesticles. Glucosemetabolismispivotalforthenormal tocontrol)(Figure2(b)).Afterbeinginternalized,glucoseis
occurrenceofspermatogenesis.Itsuptakefromtheextracel- metabolizedviaglycolysis,inwhichtheirreversibleconver-
lularmediumisarate-limitingstepforglucosemetabolism. sionoffructose6-phosphatetofructose1,6-bisphosphateby
HenceweevaluatedtheeffectsofT2DMontheexpressionof PFKisakeycontrolpoint.WeevaluatedtheeffectsofT2DM
themostrelevantglucosemembranetransportersintesticles in PFK1 levels (widely expressed in testicular tissue) and
(GLUT1, GLUT2, and GLUT3). No significant differences observednoalterationinbothtranscriptsandproteinlevels
wereobservedintesticularGLUT1transcriptlevelsbetween ofPFK1.Nevertheless,whenweassessedtheactivityofPFK
animalsfrombothgroups(Figure2(a)),whileGLUT1protein itwassignificantlyincreasedby44%inthetesticulartissueof
inanimalsfromT2DMgroupwassignificantlyincreasedin T2DMrats(1.44±0.14-foldvariation),whencomparedwith
13% (1.13 ± 0.07-fold variation), when compared with the testiculartissueofratsfromthecontrolgroup(Figure2(d)).
JournalofDiabetesResearch 7
Table3:Metabolitecontentoftesticulartissueinanimalsfromthe WethenassessedthemRNAlevelsofthemaintesticular
control(CTR)andT2DMgroup. ALTisoform(ALT2).Ourresultsshowasignificantincrease
(by35%)inT2DMrats(1.35±0.19-foldvariationtocontrol)
Metabolites(nmol/mgtissue) CTR T2DM
(Figure3(a)).Additionally,weobservedthattheoveralltes-
Lactate 2.90±0.60 1.45±0.30∗
ticularproteinlevelsofALTwerealsosignificantlyincreased
Alanine 0.70±0.20 0.73±0.20 by21%inT2DMrats(1.21±0.05-foldvariationtocontrol)
Glycogen 0.25±0.03 0.41±0.10∗
(Figure3(b)).However,whenweevaluatedtheactivitylevels
UDP-Glucose 0.48±0.01 0.54±0.01∗
ofALTintestiswefoundnosignificantdifferencesbetween
Lactate/Alanineratio 3.70±0.30 2.70±0.40∗ both groups (Figure 2(d)). Importantly, the lower levels of
Legend:CTR:control;T2DM:type2diabetesmellitus.Resultsareexpressed lactatetogetherwiththeslightincreaseinthealaninelevels
asmean±SEM(𝑛 = 6foreachcondition).∗Significantlydifferentrelative detected in the testicular tissue of T2DM rats illustrate a
tocontrol(𝑃<0.05). decrease of the lactate/alanine ratio (Table 3) (2.70 ± 0.40),
ascomparedwiththecontrolgroup(3.70±0.30).
3.4. T2DM Causes a Reduction of Testicular Lactate Levels
byDecreasingLDHActivity. Intesticulartissuethemajority 3.6. T2DM Enhances Testicular Glycogen Deposition, Mod-
of glucose is converted into lactate, which is not a “waste” ulating the Expression of Glycogen-Associated Enzymes. We
end-product of glycolysis but a critical “fuel” for germ cells evaluatedthetesticularcontentofglycogenandofitsprecur-
development.Thus,weevaluatedtheeffectsofT2DMonthe sormonomersUDP-glucose.Weobservedthatglycogenlev-
testicularglycolyticmetabolicprofile.Ourresultsshowthat elsinthetesticlesofT2DMratsweresignificantlyincreased
testicularlactatecontentwassignificantlydecreasedby50% by26%(0.41±0.10nmol/mgtissue),ascomparedwiththe
inratsfromT2DMgroup(1.45±0.30nmol/mgtissue),when control group (0.25 ± 0.03nmol/mg tissue; Table 3). We
comparedtoratsfromthecontrolgroup(2.90±0.60nmol/mg alsoobservedthatUDP-glucosetesticularcontentinT2DM
tissue)(Table3). rats was significantly increased by 12.5% when compared
with the control group, from 0.48±0.01nmol/mg tissue to
Sincetesticularlactatecontentwassignificantlyreduced,
0.54 ± 0.01nmol/mg tissue (Table 3). We further evaluated
we evaluated the effects of T2DM on the expression and
the effects of T2DM on testicular glycogen metabolism by
activity of LDH. When we assessed the LDHA transcript
assessing the expression levels of key rate-limiting enzymes
levels (which is highly expressed in the lactate-producing
involvedintesticularglycogenmetabolism.ThemRNAlevels
SCs), we observed a significant reduction (by 19%) in the
testicles of rats from the T2DM group (0.81 ± 0.07-fold of GYS1 (the main GYS isoform expressed in the testicles)
weresignificantlydecreasedby20%inthetesticlesofT2DM
variation)whencomparedwithratsfromthecontrolgroup rats(0.80±0.01-foldvariation)whencomparedtoratsofthe
(Figure 3(a)). However, the overall protein levels were not
controlgroup,whileitsproteinlevelswerenotsignificantly
alteredinthetesticlesofratsfrombothgroups(Figure3(b)).
altered(Figures4(a)and4(b)).
WefurtherevaluatedLDHactivityonthetesticulartissueof
On the other hand, when we evaluated the testicular
animalsfrombothgroupsandobservedasignificantdecrease
(by 48%) in the testicles from T2DM rats (0.52 ± 0.05- expression levels of glycogen phosphorylase (PYG), a key
enzymeresponsibleforglycogendegradation,wefoundthat
foldvariation;Figure2(d)),ascomparedwithratsfromthe
theseweresignificantlyalteredbyT2DM.WeassessedPYGL
controlgroup.
transcriptlevels(theisoformpreferentiallyexpressedinthe
Onceproduced,inorderforlactatetoreachthedevelop-
testicles) and overall PYG protein levels. Our results show
ing germ cells it must be exported from SCs MCT4. When
thatPYGLmRNAlevelsweresignificantlyincreasedby44%
weevaluatedthetesticularexpressionofMCT4,weobserved
in T2DM rats (1.44 ± 0.20-fold variation) when compared
no differences between animals from T2DM and control
with rats from the control group (Figure 4(a)). The overall
groups concerning mRNA levels (Figure 3(a)). However,
PYGproteinlevelswerealsosignificantlyincreasedby48%
MCT4 protein levels were significantly increased (72%) in
thetesticulartissueofT2DMrats(1.72±0.35-foldvariation) in the testicles of T2DM rats (1.48 ± 0.06-fold variation to
control)(Figure4(b)).
whencomparedwithratsfromcontrolgroup(Figure3(b)).
3.7. T2DM Affects Epididymal Sperm Quality. T2DM is
3.5. T2DM Slightly Increases the Testicular Content of Ala-
known to affect sperm quality parameters, such as concen-
nine,AlteringtheLactate/AlanineRatio. Lactateisconverted
tration, motility, viability, and morphology [5]. As could
from pyruvate, which is at a crossroad of several metabolic
pathways. Pyruvate is an intermediary metabolite that can be expected, our results showed that T2DM rats presented
be reversibly converted either to lactate (by LDH) or to alterations in specific epididymal sperm parameters when
alanine(byalanineaminotransferase(ALT)).Followingthe compared with rats from the control group. Although the
observeddecreaseintesticularlactatecontentofT2DMrats, sperm concentration in control rats was 4.10 ± 0.40 ×
we further evaluated testicular alanine levels. We found it 107cell/mL,whichwasnotsignificantlydifferentfromT2DM
tobeslightly(butnotsignificantly)increasedinT2DMrats rats(4.70±0.40×107cell/mL),spermatozoatotalmotilityof
(0.73±0.20nmol/mgtissue;Table3),ascomparedwithrats T2DMratswassignificantlylower(73.2±1.0%)relativelyto
fromthecontrolgroup(0.70±0.20nmol/mgtissue). controlrats(78.2±0.9%).Furthermore,whenweevaluated
8 JournalofDiabetesResearch
ntrol) 2.0 ntrol) 2.5
n to co 1.5 ∗ n to co 2.0 ∗
o o
n (fold variati 1.0 ∗ n (fold variati 11..05 ∗
o o
A expressi 0.5 n expressi 0.5
N ei
mR 0.0 Prot 0.0
LDHA ALT2 MCT4 LDH ALT MCT4
(a) (b)
LDH AALLTT
37kDa 4488kkDDaa
CCTTRR TT22DDMM CCTTRR TT22DDMM
MCT4 TTUUBB
43kDa 5500kkDDaa
CTR T2DM CTR T2DM
(c)
Figure3:Type2diabetesmellitus(T2DM)modulatestheexpressionoflactateproduction-relatedenzymesandtransporter.(a)Pooled
data of independent experiments, indicating the fold variation of lactate dehydrogenase (LDH), alanine transaminase 2 (ALT2), and
monocarboxylatetransporter4(MCT4)mRNAlevelsfoundintesticlesofT2DMratswhencomparedwiththecontrolrats(dashedline).
(b)Pooleddataofindependentexperiments,indicatingthefoldvariationofLDH,ALT,andMCT4proteinlevelsfoundintesticlesofT2DM
ratswhencomparedwiththecontrolrats(dashedline).(c)IllustrativeWesternBlotexperiment.Resultsareexpressedasmean±SEM(𝑛=6
foreachcondition).*Significantlydifferentrelativetocontrol(𝑃<0.05).
ol) 2.0 ol) 2.0
ntr ntr
o co ∗ o co ∗
n t 1.5 n t 1.5
o o
old variati 1.0 ∗ old variati 1.0
n (f n (f
o o
pressi 0.5 pressi 0.5
A ex n ex
N ei
mR 0.0 Prot 0.0
GYS1 PYGL GYS1 PYG
(a) (b)
GYS1 PPYYGG
86kDa 9977kkDDaa
CCTTRR TT22DDMM CCTTRR TT22DDMM
TTUUBB
5500kkDDaa
CTR T2DM
(c)
Figure 4: Type 2 diabetes mellitus (T2DM) modulates the expression of glycogen metabolism-related enzymes. (a) Pooled data of
independentexperiments,indicatingthefoldvariationofglycogensynthase1(GYS1)andglycogenphosphorylaseL(PYGL)mRNAlevels
foundintesticlesofT2DMratswhencomparedwiththecontrolrats(dashedline).(b)Pooleddataofindependentexperiments,indicating
thefoldvariationofGYS1andPYGproteinlevelsfoundintesticlesofT2DMratswhencomparedwiththecontrolrats(dashedline).(c)
IllustrativeWesternBlotexperiment.Resultsareexpressedasmean±SEM(𝑛 = 6foreachcondition).*Significantlydifferentrelativeto
control(𝑃<0.05).
JournalofDiabetesResearch 9
Table 4: Epididymal sperm concentration, motility, viability, and isessentialforthenormalfunctionofthereproductiveaxis.
morphologyinanimalsfromthecontrol(CTR)andT2DMgroup. Deficiencyinthishormonehasbeendirectlyassociatedwith
a decrease in T secretion by Leydig cells [39, 40]. In this
Spermparameters CTR T2DM
context, we evaluated sex steroids levels and observed a
C(×o1n0c7ecnetllr⋅amtiLo−n1) 4.10±0.40 4.70±0.40 significantdecreaseinbothtesticularandserumTlevelsin
T2DM rats. Undoubtedly, insulin intolerance compromises
Motility(%) 78.20±0.90 73.20±1.00∗ testicular T production and consequently whole body T
Viability(%) 74.00±2.00 51.00±1.00∗ levels, as previously observed in insulin resistant individ-
Morphology uals [39]. Concerning the E2 levels, we did not observe
(%abnormal 38.00±3.00 49.00±3.00∗ significant differences at both testicular and serum levels
spermatozoa) between groups, which is in accordance with other reports
Legend:CTR:control;T2DM:type2diabetesmellitus.Resultsareexpressed [41]. Unaltered E2 levels may be explained not only by
asmean±SEM(𝑛 = 6foreachcondition).∗Significantlydifferentrelative the unchanged levels and/or enzyme activity of aromatase
tocontrol(𝑃<0.05). observed in early stages of DM [41], but also by the contri-
butionofperipheraltissues,suchasadiposetissue,bone,and
skin[42].ThereforetheT2DMpromotedbySTZmightnot
epididymalspermatozoaviability,controlratsshowedasig-
nificantlyhigherviability(74.0±0.2%)thanT2DMrats(51.0± be sufficiently advanced to induce significant differences in
1.0%; Table 4). When assessing sperm morphology, our E2levelsthatareknowntooccurinmoreseverestagesofthe
disease[38,43].
resultsshowedthatthepercentageofabnormalspermatozoa
incontrolgroupwas38.0±3.0%andT2DMratspresented The endocrine disruption observed in T2DM rats is
reflected in whole body metabolism and may also disturb
a significantly higher percentage of abnormal spermatozoa
(49.0±3.0%;Table4). testicularenergymetabolismandmalereproductivefunction
[17, 44, 45]. Testicular tissue consists of a heterogeneous
population of somatic and germ cells, where germ cells are
4.Discussion
dependent on the nutritional support provided by SCs and
DMaltersthismetaboliccooperation.Ithasbeendiscussed
T2DM is the most prevalent form of DM, characterized by
thatthealteredmetabolismwithintesticularenvironmentis
insulinresistanceandimpairmentofwholebodymetabolism
closely associated with decreased male fertility (for review
[1].Severalanimalmodelshavebeendevelopedtostudythe
see [46]). Previous studies from our team showed that,
mechanismsinvolvedinthepathophysiologyofthevarious
in a prodromal stage of T2DM, known as prediabetes,
comorbiditiesassociatedwithT2DM[34].Inthisstudy,we
testicularcellsareabletoadapttheirmetabolismtopromote
usedan-STZ-inducedT2DManimalmodelthatdisplaysthe
an adequate environment for germ cell development [9].
typicalcharacteristicsoftheearlystagesofT2DM.STZisa
Glucoseuptakeisenhancedintesticulartissueofprediabetic
widely used diabetogenic agent, which produces a selective
animals, as well as PFK activity, favoring a high glycolytic
toxic effect on pancreatic 𝛽-cells [35]. When administrated
flux[9].Moreover,thetesticularexpressionlevelsofproteins
to rats at low doses during postnatal age they are reported
involvedinlactateproductionandtransportwerealsofound
to develop T2DM [26]. In our study the n-STZ-treated
to be enhanced in prediabetic animals, resulting in higher
animalsshowedmildhyperglycaemiaandimpairedglucose
amounts of testicular lactate [9]. Like prediabetic rats, our
tolerance, as observed by the significant increase in the
T2DMratspresentedincreasedtesticularexpressionlevelsof
AUCGTTvalues.Infact,whensubjectedtoaglucosetolerance GLUT1 and GLUT3, favoring the glucose uptake. This was
test,theirglycaemialevelsremainedwithintheinterval140–
accompanied by increased activity of PFK1, thus showing
200mg/dL2hoursafterglucoseload.Thisisconsistentwith
thatthetworate-limitingstepsofglycolysisarenotaffected
theobservedincreaseofHbA1cvaluesinT2DMrats,which
byT2DMwhichillustratesthatthehightesticularglycolytic
wereinaccordancewithothers[26,36].HbA1cisamarker
fluxobservedinthetesticlesofprediabeticanimalsmayalso
of cumulative glycaemic exposure over a preceding of 8 to
occurinthetesticlesofT2DM.Insum,testiclesmetabolize
12 weeks and a strong predictor of the development of DM
glucose at high rates not only in prodromal stages but also
[1].Insulinintolerance(resistance)isalsocloselyassociated
when T2DM is already established. However, we observed
withT2DM[1]andwasalsoobservedinouranimalmodel.
that testicular lactate production is reduced by T2DM. In
Furthermore,plasmainsulinlevelswerealsoincreased.This
the present study, the main contributor to the lower lactate
may be explained as a compensatory mechanism in order
content detected in testicles of T2DM rats seems to be the
to maintain normoglycemia to face the detected insulin
decrease of LDH activity. Moreover, lactate export is not
intoleranceinT2DMrats[37].Hyperinsulinemiapresented
impaired by T2DM, since MCT4 levels were increased in
by T2DM rats is also concomitant with the marked insulin T2DMrats.Concurrentresultshavebeendescribedinapre-
intolerance observed in these animals. Altogether, the dis- viousstudyusingaspontaneousT2DManimalmodel[47].
playedcharacteristicsofSTZ-treatedanimalsclearlyindicate Thoseauthorsreportedthat3-month-oldGoto-Kakizakirats,
thataT2DMconditionwasattainedintheserats. which constitute an important model to study the initial
T2DM rats presented insulin intolerance that has been eventsofDMdevelopment(sinceatearlyagetheseanimals
associated with disruption of the HPT axis, leading to an do not exhibit the severe complications associated with the
imbalanceinthelevelsofsexsteroidhormones[38].Insulin disease)[48],showedreducedtesticularlactateproduction,
10 JournalofDiabetesResearch
illustratingtheexistence oftesticularmetabolicadaptations presentseveralconditionsthatfavorglycogenesissuchas(1)
accordingtothedegreeofseverityofT2DM. elevatedlevelsofinsulin,whichstimulateglycogensynthesis
Thepresentdataillustratesthattesticularglycolyticflux, through inactivation of glycogen synthase kinase 3 (GSK-
which ends up in lactate production, is compromised in 3) that is known to maintain GYS1 in an activated form;
T2DM rats. Glucose taken up by testicular cells is not (2) hyperglycaemia, which increases the blood-to-testicles
converted into lactate as efficiently, being most probably glucoseavailability,exertinganallostericactivationonGYS1
redirected to other metabolic pathways. Pyruvate, the end- via glucose-6-phosphate (accelerating glycogen synthesis)
productofglycolysis,canbeusedasaprecursormetabolite [55, 56]; (3) increased testicular levels of UDP-glucose, an
for several metabolic pathways. Nevertheless, within the active intermediate in the synthesis of glycogen [25]. As
testicular milieu it is preferentially converted to lactate by glycogen synthase is the rate-limiting enzyme in glycogen
LDH or alanine by ALT [17]. We found that alanine levels synthesis, we evaluated testicular GYS1 levels. We found
wereslightly(butnotsignificantly)increasedinT2DMrats, no significant alterations in GYS1 protein levels of T2DM
whichwasassociatedwithaslight(nonsignificant)increase rats, although a significant decrease was observed in GYS1
ofALTactivity.Thisslightincreaseinalaninelevelstogether mRNA levels. These divergent results concerning mRNA
withthelowerlevelsoflactateledtoadecreaseintesticular and protein levels may be due to factors associated with
lactate/alanineratioinT2DMrats.Thislactate/alanineratio regulatory mechanisms in gene expression, such as mRNA
is often used as an index of the redox state of tissues retention in the nucleus, processing, stability, and half-life
+
[49], because it reflects the NAD /NADH ratio, since the time of mRNA molecule [57]. The simultaneous increase
conversionofpyruvatetoalanineiscoupledwithreoxidation intesticularglycogenamountandoverallPYGlevelsseems
+ +
of NADH into NAD . NAD /NADH is directly implicated to be somewhat contradictory. A possible explanation may
withenergymetabolismconstitutingametabolicnodewell rely on the increased levels of sugar nucleotides, such as
suited for integration of energy metabolism. The decreased UDP-glucose,whichistheimmediateprecursoringlycogen
testicularlactate/alanineratioillustratesthat,inthepresent synthase reaction. Increased levels of UDP-glucose were
conditions, the feed-forward pathway in which LDH fuels detectedinthetesticlesofT2DMratsillustratingtheability
+
glyceraldehyde 3-phosphate dehydrogenase with NAD is of testicular tissue to synthesize glycogen. This increase of
not favored, which may eventually end up in compromised UDP-glucose and glycogen content in tissues promoted by
glycolyticpathway. T2Dwasalsoreportedintheheart[58,59].Theregulationof
Kimandcollaborators[50]havedescribedanassociation glycogenmetabolismiscomplexandseveralenzymestaking
between alterations in glucose metabolism and decreased part in this process allosterically respond to metabolites
sperm quality in diabetic mice models. Although those and/or hormones. It has been proposed that the increased
authors used a distinct type 1 diabetes rodent model, they cardiacglycogensynthesisobservedinSTZ-induceddiabetic
observedadecreaseinspermparametersthattheycorrelated rats [60] is due to a metabolic reprogramming of cardiac
with an abnormal metabolic activity, which could also be tissue,inwhichtheincreasedglucoseinfluxtogetherwiththe
exacerbatedbythelocalautoimmunemechanismknownto reduced glucose oxidation augments the cardiac content of
occur in this pathological state [5, 51]. In that study, Kim UDP-glucose [61]. This effect with the combined activation
andcollaborators[50]reportedanalterationofexpressionof ofglycogensynthaseincreasescardiacglycogenstorage.Our
specificGLUTsinvolvedeitherinthesupplyofsubstratesto results support that this phenomenon may also occur in
thepentosephosphatepathwayknowntooccurinthesperm testicles. The increased GLUT1 and GLUT3 expression, in
head(forreviewsee[52])orintheuptakeoffructosefrom opposition to the low lactate production and together with
the seminal plasma (see review [53]), pointing to possible thehigherlevelsofUDP-glucoseinT2DMrats,illustratethat
impairment in the maturation and fertilization events and T2DMstimulatesglycogenaccumulation.
not in spermatogenesis [50]. No information was given Despite the ability of testicles to adopt alternative path-
concerningtheotherrate-limitingstepsoftheglycolyticflux ways,thealteredglucoseutilizationandtheglycogenaccu-
andproductionoflactate. mulation observed in testicles of T2DM rats were concur-
Still, our results demonstrate that T2DM condition rent with the impairment of sperm parameters. In fact,
altered testicular GLUT’s expression, favoring the glucose although no changes were observed in sperm concentra-
uptake,whiletheproductionoflactate,thekeysubstratefor tion of T2DM rats, the percentage of abnormal sperm was
developing germ cells, was compromised. These evidences significantly increased. A side effect of hyperglycaemia and
suggestedthat,inouranimalmodel,T2DMinducedatestic- altered testicular bioenergetics is the increase of oxidative
ular metabolic reprogramming, promoting distinct glucose stress and reactive oxygen species overproduction within
metabolic pathways and/or substrate preferences. Bearing testicularenvironment[20],whichcontributestoabnormal
in mind that glycogen metabolism plays a preponderant sperm morphology. Thus, our results are consistent with
role in T2DM, contributing to high glucose disposal (for previousstudies[47,62],illustratingthattesticularmetabolic
review see [9, 54]) and that testicles readily synthetize and alterations induced by T2DM are closely associated with a
store glycogen [24] we hypothesized that, under T2DM decreaseinmalefertility.However,onecannotdiscardthat
conditions, testicular glycogen may represent an alternative subnormal androgenic status prevailing in T2DM animals
metabolic pathway and contribute to adaptive metabolic also contributes to the impairment of secretory activity of
mechanisms. We evaluated testicular glycogen content and epididymalepitheliumwhichmaycontributetotheobserved
observedasignificantincreaseinT2DMrats.T2DManimals declineofspermparameters[63].