The Effect of Progesterone and Pregnenolone on Diabetes Status in Chinese Rural Population: a Dose–Response Analysis from Henan Rural Cohort

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J Jiang and others Progestogen and diabetes 181:6 603–614 Clinical Study status

The effect of progesterone and pregnenolone on diabetes status in Chinese rural population: a dose–response analysis from Henan Rural Cohort

Jingjing Jiang1,2, Xue Liu1, Xiaotian Liu1, Zhongyan Tian1, Haiqing Zhang1, Xinling Qian1, Zhicheng Luo1, Dandan Wei1, Shuna Jin3, Chongjian Wang1 and Zhenxing Mao1

1Department of Epidemiology and Biostatistics, College of Public Health, Zhengzhou University, Zhengzhou, Henan, People’s Republic of China, 2Medical Insurance Office, Liuzhou General Hospital, Liuzhou, Guangxi, People’s Republic Correspondence of China, and 3Key Laboratory of Environment and Health, Ministry of Education & Ministry of Environmental should be addressed Protection, and State Key Laboratory of Environmental Health, School of Public Health, Tongji Medical College, to Z Mao Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China Email [email protected]

Abstract

Objective: Previous studies have uncovered a progestin-only contraceptive association with an increased risk of diabetes, but limited studies have explored the relationship of endogenous progesterone and pregnenolone levels with diabetes status. A case–control study was conducted in Henan Rural Cohort (register number: ChiCTR- OOC-15006699) to evaluate the dose–response independent and interactive relationship of progesterone and pregnenolone levels with prediabetes and type 2 diabetes mellitus (T2DM) in Chinese rural population. Design: A case-control study. Methods: A total of 798 T2DM patients, 779 prediabetes patients, and 782 individuals with normal fasting plasma glucose were included in this study. Serum progesterone and pregnenolone were detected by liquid chromatography-

European Journal of Endocrinology tandem mass spectrometry. Logistic regression and restricted cubic splines were used to assess the independent effects of progesterone and pregnenolone on prediabetes and T2DM. Interactive plots were employed to examine the interaction effects of progesterone and pregnenolone. Results: Progesterone in the fourth versus first quartile was positively associated with prediabetes (odds ratio (OR) (95% CI): 2.66 (1.99–3.55)) and T2DM (OR (95% CI): 6.41 (4.57–8.98)), whereas pregnenolone in the fourth versus first quartile was inversely related to prediabetes (OR (95% CI): 0.23 (0.16–0.33)) and T2DM (OR (95% CI): 0.44 (0.31–0.62)). Additionally, the nonlinear dose–response associations between progesterone and pregnenolone with prediabetes and T2DM were found. Interactive effects of progesterone and pregnenolone on prediabetes and T2DM were observed, and these significant associations remained in gender-stratified analysis. Conclusions: Prediabetes and T2DM were positively linked to serum concentration of progesterone and negatively related to pregnenolone in a dose–response manner in Chinese rural population.

European Journal of Endocrinology (2019) 181, 603–614

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-19-0352 European Journal of Endocrinology https://eje.bioscientifica.com based studyhasbeencarried out toexploretherelationship found ( the riskofdiabetes,butno significantassociationwas association ofserumconcentration ofprogesteronewith opposite-sex twinsinaSwedishpopulation,assessedthe only oneepidemiologicalstudy, whichfocused onelderly progestogen withprediabetesordiabetes.Uptodate, is littleinformationontherelationshipofendogenous risk ofgestationaldiabetesmellitus( 17-OH progesteronecaproatewererelatedtoanelevated analysis indicatedthatwomentakingsupplemental ( may playacrucialroleinthedevelopmentofdiabetes demonstrated that progestogen-only contraceptives the secondtrimester( generally presentswithhighlevelsofprogesteronein level ofinsulin( and alsoaprogestogen,isassociatedwiththeblood Pregnenolone, theimmediateprecursorofprogesterone release andapoptosisofpancreatic glucose, increasedinsulinresistance,elevated glucose metabolism,includingahighlevelofplasma concentration ofprogesteroneisrelatedtoabnormal T2DM ( and increasedbloodpressure,allofwhicharerelatedto is associatedwithobesity, abnormalbloodlipidlevels, T2DM ( hormones areassociatedwiththedevelopmentof and T2DM. the factorsthatinfluencedevelopmentofprediabetes diabetes ( accounts for approximately 90% of the individuals with population inChina( escalated morerapidlyinruralpopulationthanurban alarmingly ( be 10.9and35.7%inChina2013haveincreased The ratesofdiabetesandprediabeteswereestimatedto developing diabetes, and can even be reverted to normal. individuals with prediabetes can be prevented from diabetes andcardiovasculardisease( and diabeticstatus,isassociatedwithanincreasedriskof glucose levelsandintermediatestatusbetweennormal Moreover, prediabetes, characterized by elevated blood org/key-messages.html 425 millionindividualsworldwide( mortality and disability ( Diabetes mellitus is one of the most common causes of Introduction 18 Clinical Study , Previous researches haveindicated thatendocrine 19 , 22 10 8 20 , 1 ). To the best of our knowledge, no population- , , ). Additionally, afindingfromrecentmeta- 9 11 2 6 ). Progesterone,theprincipalprogestogen, ). Hence,itisurgentandcrucialtoidentify ). Notably, the prevalence of diabetes has , 12 ). Severalstudieshaveshownthathigh 17 ). Interestingly, gestationaldiabetes 7 14 , accessed11September2018)( ). Type 2diabetesmellitus(T2DM) ). Moreover, severalstudieshave 1 , 2 , 3 J Jiangandothers ), affecting more than β 5 cells( ). Mostimportantly, 21 http://diabetesatlas. ). However, there 13 , 14 , 15 , 16 4 ). ). and the WHO criteria (1999) ( Diabetes Association(ADA)diagnosticcriteria(2009)( Chinese ruralpopulation( potential riskfactorsofcommonchronicdiseasesin an ongoingprospectivestudydesignedtoexplorethe Rural Cohort(registernumber:ChiCTR-OOC-15006699), data (fromJuly2015toSeptember2017)oftheHenan Data forthecurrentstudywereextractedfrombaseline Study designandparticipants Subjects andmethods population. with theriskofprediabetesandT2DMinChineserural of endogenous(serum)progesteroneandpregnenolone dose–response independentandinteractiveassociations with theriskofprediabetes. of endogenousprogesteroneandpregnenolonelevels University LifeScienceEthics Committeeandconducted Ethic approvalcode:(2015) MEC (S128). informed consentwasobtained fromallparticipants. University LifeScienceEthics Committee’,andwritten Ethics approvalwasobtainedfromthe‘Zhengzhou Ethics approvalandconsenttoparticipate on progestogenlevels. were alsoexcludedbecauseofthepotentialimpactHRT Participants withhormonereplacementtherapy(HRT) serum samplestodetectprogestogenwereruledout. blood levels.Individualswithmissingorinsufficient the significanteffectofmenstrualcycleonprogestogen study. Premenopausalwomenwerenotincludeddueto women agedfrom18to79yearswereincludedinthis (3-year groups) and gender. Menandpostmenopausal matched toparticipantswithT2DMaccordingage individuals weresampledrandomlyandfrequency 798 controlindividuals.Prediabetespatientsand 782 individualswithT2DM,779prediabetespatients,and and FPG prediabetes was determined as individuals without T2DM ≥ during the past fortnight, or fasting plasma glucose (FPG) by physician and taking antidiabetic drugs or insulin T2DM wasdefinedassubjectswithdiagnosisof diabetes mellitus,andcausedbyotherreasons, status Progestogen anddiabetes 7.0 Therefore, thisstudywasconductedtoexplorethe The protocolofthis study wasapprovedbyZhengzhou A totalof2359participantswereincludedinthisstudy: mmol/L, orglycosylatedhemoglobin(HbA1c) ≥ 6.1 mmol/LorHbA1c Downloaded fromBioscientifica.com at09/28/202101:23:55PM 23 ). OnthebasisofAmerican 24 ≥ ), after excluding type 1 5.7%. 181 :6 ≥ 6.5%; 604 via freeaccess 5 ) European Journal of Endocrinology system, Waters), blinded toT2DM,prediabetes,orcontrol method (Watersspectrometry e2695,Waters XEVOTQ-S was determinedbyliquidchromatography-tandem mass dtu.ox.ac.uk/homacalculator/ indexes, namelyHOMA2-IRandHOMA2- resistance and homeostasis model(HOMA2)wasusedtoestimateinsulin data Table(Supplementary 1,seesection on measurements than 3%forabove-mentionedlaboratory and inter-assaycoefficientsofvariation(CVs)wereless using HPLCmethod(Bio-RadVARIANT II).Theintra- lipoprotein cholesterol(LDL-C).HbA1cwasmeasured density lipoproteincholesterol(HDL-C),andlow-density of FPG, triglycerides (TG), total cholesterol (TC), high- Cobas C501)wasusedtomeasuretheconcentration at centrifugation at1409 participants were collected. Serum was separated after After anovernightfasting,venousbloodsamplesof Laboratory measurements weight (kg)dividedbyheight(m)squared. measured bytrainedinvestigators.BMIwascalculatedas ofdiabetes.Heightandbodyweightwere family history ofdiabeteswereconsideredtobewitha with ahistory mediate and high level. Parents or siblings of participants ( on InternationalPhysicalActivityQuestionnaire(IPAQ) to provideinformationontheirmenopausalstatus.Based structured questionnaire. Women were further requested activity, witha werecollectedbywell-trainedinterviewers habit (currentdrinker, neverorformer),andphysical (current smoker, never or former), alcohol drinking usage ofHRT, behavioralhabitsincludingsmokinghabit ( school, middleschoolorabove),averagemonthlyincome school,primary education level(belowthanprimary cohabitating andunmarried/divorced/widowed), including age, gender, marital status (married/ Information onsociodemographiccharacteristics, Covariates procedures used. after theyweretoldthepurposeandnatureofall All participantsprovidedtheinformedwrittenconsents according totheguidelinesofDeclarationHelsinki. 25 < Clinical Study 500, 500~,1000 ), physicalactivitywasdividedintothreegroups:low, − The serumlevelofprogesterone andpregnenolone givenattheendofthisarticle).Theupdated 80°C. Anautomatic biochemical analyzer(Roche β -cell functionwiththecorresponding ~ RMB), family history ofdiabetes, RMB), familyhistory g in4°Cfor10minandstored ) ( 26 J Jiangandothers ). β supplementary supplementary ( https://www. Because oftheskeweddistributionsallcontinuous Statistical analysis highest quartile). (Q3, thethirdquartile),andQuartile4(Q4,fourth/ quartile), Quartile2(Q2,thesecond3 as thereferencegroups:Quartile1(Q1,first/lowest classified intofourgroupswiththelowestquartile the control group, progesterone and pregnenolone were Tables 2and3.Accordingtogender-specificquartilesof of thedetectionmethodsispresentedinSupplementary were 0.02 the detectionlimitsofprogesteroneandpregnenolone progesterone andpregnenolonerespectively. Moreover, limits ofquantificationwere0.05 pregnenolone were 9.78and 11.68% respectively. The 1.48% forprogesterone,andthecorrespondingvalues status ( order toimprovenormalization. To furtherexaminethe homeostasis markerswerenatural log-transformed(ln-)in ruled out,andprogesterone, pregnenolone,andglucose taking hypoglycemicdrugs duringpast14dayswere and HOMA2- including FPG,HbA1c,fastinginsulin,HOMA2-IR, pregnenolone levelswithglucosehomeostasismarkers, to furtherexploretherelationshipofprogesterone and on theirdistribution. Linear regression wasperformed reference valuesforprogesteroneandpregnenolonebased 90th percentiles; 0.5 and T2DM,withthreeknotsplacedat30th,60th, progesterone andpregnenolonewiththeriskofprediabetes estimate thedose–responseassociationsofcontinuous variables. Restricted cubicsplines were conducted to response relationships by including quartiles as continuous Furthermore, trendtestswereusedtoassessthedose– ofdiabetes,TC,TG,HDL-C,andLDL-C. family history habit, alcoholdrinkingphysicalactivity, BMI, education level,averagemonthlyincome,smoking in adjustedmodelincludedage,gender, maritalstatus, quartiles ofprogesteroneandpregnenolone.Covariates ratios (ORs)with95%CIsforprediabetesandT2DMby presented asnumbers(percentages). in categoricalvariableswerecomparedusing medians (interquartilerange(IQR)),whereasdifferences compared usingMann–Whitney and controlindividualsincontinuousvariableswere all continuous covariates), differences between case covariates (Kolmogorov–Smirnov test: status Progestogen anddiabetes Logistic regressionwasemployedtoestimateodds 27 , ng/mL and0.2 28 ); theintra-andinter-dayCVswere1.12 β . Inlinearregressionmodel, individuals ng/mL and1.0 Downloaded fromBioscientifica.com at09/28/202101:23:55PM ng/mL. Adetaileddescription https://eje.bioscientifica.com ng/mL and0.4 U testsandexpressedas ng/mL weresetasthe 181 :6 P

χ < 2 .5 for 0.05 ng/mL for testsand 605 via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com prediabetes (OR(95%CI): Q3 vsQ1:0.34(0.25–0.45); T2DM persistedafterstratification bysex. associations betweenprogesterone withprediabetesand progesterone levels and T2DM. The significantly positive positively dose–responserelationshipwasfoundbetween further adjustmentforpotentialconfounders.Similarly, a and thetendencywasnotsignificantlyattenuatedafter 95% CI:1.28–2.33;Q4:OR < ( across progesteronequartileswasobserved in Estimated ORsforprediabetesandT2DMarepresented prediabetes andT2DM Effects ofprogesteroneandpregnenoloneon and postmenopausalwomen. the controlgroup.Asimilardistributionwasseeninmen the prediabetesandT2DMgroupwaslowerthanthatin groups, whereas the concentration of pregnenolone in progesterone was higher in the prediabetes and T2DM Compared withthecontrolgroup,concentrationof prediabetes weremorelikelytohaveahigherLDL-C. of diabetes, and participants with have a family history with controls.Diabeticindividualsweremorelikelyto higher HbA1C,TC,andTGcompared tended tohavehigherBMI,FPG,insulin, shown in of participants according to diabetes status by gender are 62.00 (12.00)wereincludedinthisstudy. Characteristics A total of 2359 individuals with a median age (IQR) of Characteristics ofparticipants Results (two-tailed) wasdefinedasstatisticalsignificance. Inc.) andRLanguagesoftware(version3.4.4). was alsotested. the research. Theinteractionofprogestogenandgender ( generalized linearmodelandvisualizedinContourplot pregnenolone onprediabetesandT2DMweretestedby Additionally, the interaction effects of progesterone and separately addedtoadjustedlogisticregressionmodel. and T2DM, HOMA2-IR, HOMA2- independent associationofprogestogenwithprediabetes 29 0.001; Q2:OR Clinical Study ). Gender-stratifiedanalysiswasemployedthroughout Table 2 Analyses wereperformedusingSAS9.1(SASInstitute However, pregnenolone was inversely associated with Table 1 . A graded increment in ORs of prediabetes = . Individuals with prediabetes and T2DM 1.30, 95%CI:0.95–1.77;Q3:OR = 2.84, 95%CI:2.15–3.76), β J Jiangandothers , and fasting insulin were P P trend =

< 1.73, 0.05

association in men,andpostmenopausalwomen (all serum concentration of progesterone in all participants, increased graduallywithanincrementalincreaseinthe stratified analysis. in gender- of prediabetes and T2DM was also observed negative relationshipbetweenpregnenolonewiththerisk controlling forpotentialconfounders( with theriskofprediabetesandT2DMbeforeafter relationships werefoundbetweenpregnenolonelevels after adjustingforpotentialconfounders.Dose-response relationships wereslightlyalteredbutstillremainedeven vs Q1:0.44(0.33–0.59);Q4Q1:0.39(0.29–0.53));these Q4 vsQ1:0.24(0.17–0.33))andT2DM(OR(95%CI):Q3 increment of pregnenolone in all participants, and the In contrast,ORsofprediabetesweredecreasedwiththe relationship of progesterone with prediabetes and T2DM. indicating asignificantnonlineardose–response weaker inmen. slightly stronger in postmenopausal women and slightly stratified analysisindicated thattherelationshipwas (95% CI: 15.1~26.6%) lower ln-HOMA2- (95% CI:5.3~9.0%)higherln-HbA1c,whereasa20.9% mmol/L(95% CI:0.082~0.137)higherln-FPG,and7.2% quartile of ln-progesterone was associated with 0.110 with thelowestquartileofln-progesterone,highest adjusting forpotentialconfounders.Similarly, compared and 9.2%(95%CI:6.3~12.1%)lowerln-HOMA2- higher ln-FPG,3.6%(95%CI:2.6~4.5%)ln-HbA1c, was relatedtoa0.052mmol/L(95%CI:0.037~0.066) TableSupplementary 4.A100% increaseinln-progesterone homeostasis markersaredisplayedin ln-progesterone andln-pregnenolonewithglucose Linear regressionresultsoftherelationshipbetween glucose homeostasismarkers Effects ofprogesteroneandpregnenoloneon P postmenopausal women( P was enhancedinmen( Gender-stratified analysisshowedthattherelationship for nonlinearity the totalpopulation( < (all relationship remainedevenaftergenderstratification status Progestogen anddiabetes o nonlinearity =0.360). for fornonlinearity 0.001). AsimilarassociationwasobtainedinT2DM P As shownin foroverallassociation < 0.001 andall = 0.015), although the trend was weaker. Fig. 1 P = foroverallassociation , ORsofprediabetesandT2DM 0.005), butdisappearedin Downloaded fromBioscientifica.com at09/28/202101:23:55PM P P foroverallassociation foroverallassociation < P 0.001, all fornonlinearity 181 P :6 fornonlinearity P

< Fig. 2 .0) The 0.001). P β foroverall . Gender- = < 0.050, = = 0.001), β 0.018, 0.639, and after 606 via freeaccess P

European Journal of Endocrinology Clinical Study

Table 1 Characteristics of participants according to diabetes status by gender.

Total Men Postmenopausal women Variable Control Prediabetes T2DM Control Prediabetes T2DM Control Prediabetes T2DM Subjects (n) 798 779 782 352 352 352 446 427 430 Age (years) 62.00 (12.00) 62.00 (12.00) 62.00 (11.00) 61.00 (12.00) 61.00 (12.00) 61.00 (12.00) 63.00 (9.00) 64.00 (8.00) 64.00 (8.00) Male, n (%) 352 (44.11) 352 (45.19) 352 (45.01) – – – – – – Married/cohabitating, n (%) 702 (87.97) 677 (86.91) 685 (87.60) 315 (89.49) 313 (88.92) 311 (88.35) 387 (86.77) 364 (85.25) 374 (86.98) Education level, n (%) J Jiangandothers

Continues variables are expressed as median (interquartile range) and compared with Mann–Whitney U tests; categorical variables are presented as numbers (percentages) and compared with χ2 tests. 181 # $

*P < 0.05 when comparing with controls in total population. P < 0.05 when comparing with controls in men. P < 0.05 when comparing with controls in postmenopausal women. :6 FPG, fasting plasma glucose; HbA1c, glycosylated hemoglobin; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; T2DM, type 2 diabetes mellitus; TC, total cholesterol; TG, triglycerides. 607 via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com relationship vanishedaftergender stratification. to thelowestquartileofpregnenolone. However, this (95% CI:0.4~13.5%)higherln-HOMA2- quartile ofpregnenolonewasassociatedwitha6.9% men andpostmenopausalwomen.Whereasthehighest in lower ln-HbA1c,andasimilarassociationwasobserved quartile wasassociatedwitha4.3%(95%CI:2.3~6.4%) diabetes mellitus. 95% CI,CI;OR,oddsratio;Q1,thefirst/lowestquartile;Q2,second quartile;Q3,thethirdQ4,fourth/highestT2DM,type2 lipoprotein cholesterol,HDL-C.*Significant men), alcoholdrinkinghabit(intotalpopulationandinphysicalactivity, BMI,andfamilyhistoryofT2DM,totalcholesterol,triglycerides,low-density † Q4 Q3 Q2 Q1 Postmenopausal women Pregnenolone Q4 Q3 Q2 Q1 Men Q4 Q3 Q2 Q1 Total Pregnenolone Q4 Q3 Q2 Q1 Postmenopausal women Q4 Q3 Q2 Q1 Men Q4 Q3 Q2 Q1 Total Progesterone Variable Table 2 Adjusted forage,gender(onlyintotalpopulation),maritalstatus,education level,averagemonthlyincome,smokinghabit(intotalpopulationandin Clinical Study P P P P P P Ln-pregnenolone inthehighestquartilevslowest fortrend fortrend fortrend fortrend fortrend fortrend Odds ratios(95%confidenceintervals)ofprediabetes/T2DM. Unadjusted ORs(95%CIs) 0.25 (0.16–0.39)*** 0.32 (0.21–0.48)*** 0.22 (0.13–0.35)*** 0.36 (0.23–0.56)*** 0.24 (0.17–0.33)*** 0.34 (0.25–0.45)*** 3.54 (2.42–5.17)*** 2.16 (1.43–3.28)*** 2.84 (2.15–3.76)*** 1.73 (1.28–2.33)*** 1.92 (1.28–2.87)** 1.03 (0.74–1.45) 0.98 (0.67–1.44) 1.01 (0.79–1.30) 1.48 (0.97–2.26) 1.51 (0.96–2.36) 1.09 (0.68–1.75) 1.30 (0.95–1.77) < < < < < < J Jiangandothers P 1.00 1.00 1.00 1.00 1.00 1.00 0.001 0.001 0.001 0.001 0.001 0.001

< 0.05, **Significant Prediabetes β compared Adjusted ORs(95%CIs) P 3.21 (2.17–4.75)*** 2.66 (1.99–3.55)*** 0.25 (0.16–0.39)*** 0.30 (0.20–0.46)*** 0.21 (0.13–0.36)*** 0.36 (0.23–0.58)*** 0.23 (0.16–0.33)*** 0.33 (0.25–0.45)*** 2.13 (1.37–3.31)** 1.58 (1.16–2.15)**

1.70 (1.12–2.57)* < 1.39 (0.90–2.14) 1.46 (0.91–2.35) 1.12 (0.69–1.84) 1.26 (0.92–1.74) 1.04 (0.73–1.47) 1.01 (0.68–1.50) 1.02 (0.79–1.32) 0.01, ***Significant < < < < < < 1.00 1.00 1.00 1.00 1.00 1.00 0.001 0.001 0.001 0.001 0.001 0.001 (Supplementary Table(Supplementary 5). for HOMA2-IR,HOMA2- attenuated butstillremainedafterfurtheradjustment progestogen toprediabetesandT2DMwasmoderately Logistic regressionshowedthattherelationship of fasting insulin The modifiedeffectsofHOMA2-IR,HOMA2- status Progestogen anddiabetes † P

< 0.001. Unadjusted ORs(95%CIs) 9.21 (6.03–14.06)*** 7.34 (5.39–10.01)*** 2.52 (1.58–4.02)*** 5.54 (3.51–8.75)*** 2.21 (1.56–3.11)*** 0.28 (0.18–0.45)*** 0.33 (0.21–0.52)*** 0.39 (0.29–0.53)*** 0.44 (0.33–0.59)*** 0.52 (0.34–0.78)** 0.55 (0.37–0.81)** 1.86 (1.11–3.10)* 0.91 (0.52–1.60) 0.92 (0.52–1.63) 0.93 (0.63–1.39) 1.39 (0.98–1.97) 1.02 (0.70–1.49) 1.20 (0.93–1.55) < < < < < < 1.00 1.00 1.00 1.00 1.00 1.00 0.001 0.001 0.001 0.001 0.001 0.001 Downloaded fromBioscientifica.com at09/28/202101:23:55PM β , andfastinginsulin T2DM 181 8.41 (5.34–13.24)*** Adjusted ORs(95%CIs) 4.47 (2.67–7.47)*** 6.41 (4.57–8.98)*** 1.95 (1.34–2.84)*** 0.32 (0.19–0.54)*** 0.38 (0.23–0.63)*** 0.44 (0.31–0.62)*** 0.48 (0.35–0.66)*** 2.00 (1.21–3.32)** 0.57 (0.37–0.87)** 1.84 (1.03–3.27)* 0.56 (0.36–0.89)* 0.81 (0.44–1.48) 0.87 (0.46–1.64) 0.88 (0.57–1.35) 1.41 (0.96–2.05) 0.89 (0.58–1.37) 1.16 (0.88–1.54) :6 < < < < < < 1.00 1.00 1.00 1.00 1.00 1.00 0.001 0.001 0.001 0.001 0.001 0.001 β , and 608 † via freeaccess

European Journal of Endocrinology with thehighestprogesterone andlowestpregnenolone on prediabetesandT2DMare shownin The interactioneffectsofprogesterone andpregnenolone Interaction effects 95% confidenceinterval. low-density lipoproteincholesterol,high-densitycholesterol; T2DM,type2diabetesmellitus;OR,oddsratio;95%CI, population andinmen),physicalactivity,bodymassindex, familyhistoryofT2DM,totalcholesterol,triglycerides, education level,averagemonthlyincome,smokinghabit(intotal populationandinmen),alcoholdrinkinghabit(intotal and 1 ng/mLforpregnenoloneaccordingtothedistribution. Adjusted forage,gender(onlyintotalpopulation),maritalstatus, cubic splines.Threeknotsweresetatthe30th,60th,and90th percentileswiththereferencevalueat0.5 ng/mLforprogesterone The dose–responseassociationsbetweenprogesteroneand pregnenolone withprediabetesandT2DMestimatedbyrestricted Figure 1 Clinical Study J Jiangandothers Fig. 3 . Individuals value ofprogesteroneon prediabeteswasobviously with theincreaseofpregnenolone, theestimatedeffect risk ofprediabetes( progesterone andhighestpregnenolone wereatlowest were athighestriskofprediabetes;thosewiththelowest status Progestogen anddiabetes P forinteraction Downloaded fromBioscientifica.com at09/28/202101:23:55PM https://eje.bioscientifica.com < 181 0.001). Furthermore, :6

609 via freeaccess

European Journal of Endocrinology https://eje.bioscientifica.com Clinical Study J Jiangandothers status Progestogen anddiabetes T2DM, type2diabetesmellitus. high-density lipoproteincholesterol. low-density lipoproteincholesterol, T2DM, totalcholesterol,triglycerides, body massindex,andfamilyhistoryof population andinmen),physicalactivity, men), alcoholdrinkinghabit(intotal smoking habit(intotalpopulationandin education level,averagemonthlyincome, (only intotalpopulation),maritalstatus, predictors wereadjustedforage,gender blue (lowrisk)topink(highrisk).Linear exhibited indifferentcolors,rangingfrom progesterone andpregnenolonewere function fordifferentconcentrationsof The linearpredictorsofthehazard linear predictorofthehazardfunction. generalized linearmodelshowedthe T2DM. Contourplotsestimatedby and pregnenoloneonprediabetes The interactioneffectsofprogesterone Figure 3 model (HOMA2)wasusedtoestimate normalization. Theupdatedhomeostasis transformed (ln)inordertoimprove pregnenolone werenaturallog- HOMA2-IR, HOMA2- cholesterol. FPG,HbA1c,fastinginsulin, cholesterol, high-densitylipoprotein triglycerides, low-densitylipoprotein history ofT2DM,totalcholesterol, activity, bodymassindex,andfamily total populationandinmen),physical and inmen),alcoholdrinkinghabit(in income, smokinghabit(intotalpopulation status, educationlevel,averagemonthly gender (onlyintotalpopulation),marital homeostasis markers.Adjustedforage, pregnenolone (D,E,F)withglucose Association ofprogesterone(A,B,C)and Figure 2 glucose; HbA1c,glycosylatedhemoglobin. confidence interval;FPG,fastingplasma HOMA2-IR andHOMA2- the correspondingindexes,namely insulin resistanceand Downloaded fromBioscientifica.com at09/28/202101:23:55PM β 181 , progesterone,and β -cell functionwith :6 β . 95%CI, 610 via freeaccess European Journal of Endocrinology 19 insulin sensitivity, andelevatedriskofdiabetes( associated with increased insulin resistance, decreased that theuseofaprogestogen-only contraceptivewas power todetectanassociation. Severalstudiesshowed of theSwedishstudymayresultinalimitedstatistical the different conclusions. Further, the limited sample size ethnicity, aswelldiverseeconomicstatus mayexplain ranged from71to80years.Differentagerange,varied targeted elderlySwedishopposite-sextwinswhoseage toourfindings.Theprior researchwhich wascontrary progesterone and diabetes was not significant ( individuals indicatedthattheassociationbetween prediabetes andT2DMwasdiminished. of pregnenolone,thepositiveeffectprogesteroneon inthisstudy:witheachincrement T2DM wereobserved progesterone andpregnenoloneonprediabetes HbA1c. Furthermore,significantinteractionsbetween nonlinear dose–responsefashionandwaslinkedtolower was negativelyassociatedwithprediabetesandT2DMina HOMA2- was relatedtohigherFPG,HbA1c,aswelllower and T2DM in a nonlinear dose–response manner, and also progesterone waspositivelyassociatedwithprediabetes status in Chinese rural population.The serum level of endogenous progesteroneandpregnenolonetodiabetes first studytoexplorethedifferentialrelationshipsof To thebestofourknowledge,thisresearch isthe Discussion not foundinthisstudy(all < after stratificationaccordingtogender( diminished but still existed. A similar association was seen the impactofprogesteroneonT2DMwasslightly < progesterone andhighestpregnenolone( whereas thelowestriskofT2DMwasfoundin highest progesteroneandlowestpregnenolonelevels, an increaseofpregnenolone( was diminished and eventually disappeared in men with < was similarinpostmenopausalwomen( Gender-stratified analysisshowedthattherelationship decreased, but the statistical significance remained. 0.001 inmenandpostmenopausalwomen). 0.001). Additionally, withanincrement of pregnenolone 0.001), whereastheeffectofprogesteroneonprediabetes Clinical Study , 20 Previous research conducted in 425 elderly Swedish Interactions betweenprogestogenandgenderwere withthe The highestriskofT2DMwasobserved , 30 β . In contrast, the serum level of pregnenolone , 31 , 32 , 33 , 34 ), whichpartlysupportedour P

> P 0.05). o interaction =0.004). for J Jiangandothers P P P forinteraction forinteraction forinteraction 22 18 ), , lager pancreaticisletsinprogesterone receptor-knockout increased insulinrelease,improved glucosetolerance,and receptor antagonist( improved aftertheadministration ofaprogesterone mice, whereasthebloodglucose levelwassignificantly elevated progesteroneleadstodiabetesinfemale animal experimentconductedbyPicard ( diabetes. Itcontributestothedecreaseofinsulinrelease an importantroleinsomephysiologicalprocesses of are severalpotentialexplanations.Progesteroneplays with diabetesstatushasnotbeenelucidated,butthere our findings. normal bloodglucose( but lowerconcentrationofHDL-Cthanindividualswith tended to have a higher concentration of TC and TG, reported that individuals with prediabetes andT2DM Moreover, astudyconductedinChinesepopulation in reported that an increase in progesterone was observed of progesterone. Similar to our study, a previous study study tended to be obese and with higher serum levels in postmenopausalwomenandmen( to thesimilarsource ofprogestogen production(adrenal) the findings inall participants, which partly may be due The resultsofgenderstratificationanalysisweresimilarto reported thattheeffectofahighdoseglibenclamide- T2DM. Additionally, anexperimentalresearch with rats is neededtoevaluatetheeffectofprogestogenon results. Thus, a study conducted in thegeneral population pregnant women,whichmayresultincontradictory postmenopausal women, whileother studies aimedat outcomes. Thecurrentstudywasfocusedonmenand results mayduetothedifferentethnicitiesanddisease T2DM inChineseruralpopulation.Thedifferencethe cell function,alongwithhigherriskofprediabetesand with higherFPG,HbA1c,anddecreasedbeta- study showedthatserumprogesteronewasassociated did not( gestational diabetesmellitus,whilenaturalprogesterone higher prevalenceofabnormalglucosemetabolismand progesterone: syntheticprogestogenmaycontributetoa that synthetic progestogen was different from natural biological activity. However, prior research indicated findings, asprogesteronewasthemainprogestogenwith consistence withthepresentstudytosomedegree( than glibenclamideorglibenclamide-OH,whichwasin pregnenolone onanti-hyperglycemicactivitywasbetter status Progestogen anddiabetes 16 Db , The mechanismbywhichprogestogenisassociated Individuals withprediabetesandT2DMinthis 43 / db , 44 mice with obesity and high blood glucose ( 35 ) andinsulin-secretingcellproliferation( , 36 , 37 ). However, thefindingsofcurrent 16 42 ). Thefurtherexperimentshowed Downloaded fromBioscientifica.com at09/28/202101:23:55PM ), whichwasinaccordancewith https://eje.bioscientifica.com 181 39 et al :6 , 40 . showedthat ). 13 Db 611 ). An 41 38 / via freeaccess db ). ).

European Journal of Endocrinology https://eje.bioscientifica.com status, as well asT2DM; added new findings to these glucose homeostasismarkers, modifiableprediabetes relationships ofprogesterone andpregnenolonewith progesterone andpregnenolone ondiabetesstatus. may explainthe opposite direction of the impact of resulting inanaccumulation of pregnenolone, which pregnenolone toprogesteronelikelywouldbedecreased, the activityof3BHSDwasinhibited,conversion of (including insulinresistanceandCpeptide)( accompanied byanimprovementinglucosemetabolism research notedthattheinhibitionof3BHSDwas beta hydroxysteroiddehydrogenase(3BHSD).Previous from positionC-5toC-4.Thisprocessiscatalyzedby 3 to aketogroupandthenthedoublebondwastransferred two steps of pregnenolone,itcanbeconvertedintoprogesteronein noradrenaline) whichelevatebloodglucose( accelerates thegenerationofotherincretion(forexample, the actualrelationship.Additionally, progesteronealso which couldleadto a weakerdetected association than lowing medicinewereruledoutinlinearregression, to detectsuchassociation.Participantstakingglucose- Henan RuralCohort,whichmayresultinaweakability data ofthisstudywasderivedfromthebaseline inthepresentstudy.resistance wasnotobserved The positive association between progesterone and insulin but remainedaftergenderstratification.Nevertheless,a current study, andtheserelationshipswereslightlyaltered of progesteroneonFPGandHbA1cwerefoundinthe and insulinresistance( obesity maymediatetheassociationbetweenprogesterone the relationshipandsuggestedthatadiponectin glucose intake( leading toincreasedinsulinresistanceanddecreased may suppressdifferentstepsofthePI3kinasepathway, muscle aswelladiposetissue.Inaddition,progesterone increased insulinresistance( the expressionofglucosetransporter4( studies also revealed that progesterone could suppress which wasconsistentwithpriorresearch. Previous was relatedtohigherFPGandimpairedbeta-cellfunction, of thisstudyalsodemonstratedthathigherprogesterone reactive oxygenspeciesandoxidativestress.Thefindings acting throughamechanismrelatedtothegenerationof induced apoptoticdeathofinsulin-producingcellsby from Nunes release, whichmayeventuallyleadtodiabetes.Evidence impact ontheproliferationofislet mice. Thesefindingssuggestedthatprogesteronehadan Clinical Study The currentstudyevaluated theopposing in vivo t al et 47 , wherethe3-hydroxylgroupisoxidized ). Kin-ChuenLeungfurthervalidated . ( 13 15 ) suggested that progesterone ). Significantlypositiveeffects 46 ), particularlyinskeletal J Jiangandothers β -cells andinsulin 45 ), leadingto 48 ). Interms 49 ). As was observed, themechanismisstillunclear.was observed, Fifth,the of progesteroneandpregnenoloneondiabetesstatus cannot beruledout.Fourth,althoughoppositeeffect adjusted, thepossibilityofotherpotentialconfounders many confoundingfactorswereconsideredand may notapplytourbanpopulations.Third,although were allfromtheHenanruralarea,soconclusions drawn in this study. Second, participants in this study Cohort; therefore,thecausalassociationscannotbe study was basedon the baseline data of Henan Rural of this study should be noted. First, this case–control potential confounders.Nevertheless,severallimitations guaranteeingcontroloverawiderangeof interviewers, information throughastandardizedprotocolbytrained on ageneralpopulationinruralregionandcollected derived datafromalargeongoingcohortstudybased in theChinesepopulation.Furthermore,thisstudy inindividualsathighriskofdiabetes and intervention relationships; andprovidedevidencefortheclassification receptor functiononT2DM. studies areneededtoexplore theeffectofprogestogen T2DM remains to be elucidated. Additionally, further potential mechanismbywhich progestogeninfluence be validated in prospective multicenter studies, and the for publichealth.However, theserelationshipsremainto diabetes, ourfindingsmayhaveanimportantimplication of T2DM.Giventhehighprevalenceandhugeburden of pregnenolone mayplaypivotalrolesinthedevelopment Ourfindingsindicatethatprogesterone and were observed. progesterone and pregnenolone onprediabetes and T2DM postmenopausal women. In addition, interactive effects of to serumpregnenolonelevelsinChineseruralmenand concentration ofprogesterone,andnegativelyrelated prediabetes andT2DMarepositivelylinkedtotheserum In conclusion,the results ofthisstudy suggest that Conclusions in diabetes. our abilitytoestablishthemechanismofprogestogen function wasnotanalyzedinthisstudy, furtherlimiting prediabetes andT2DM.Finally, progestogenreceptor oral glucose tolerance test was not employed to diagnose were consistentwiththefindingsofthisstudy. Sixth,an results associatedwithprogestogen-onlycontraceptives in participantswithoutpregnancy. However, similar relationship of endogenous progesterone and diabetes best ofourknowledge,onlyonestudyevaluatedthe evidence frompreclinicalstudywaslimited.To the status Progestogen anddiabetes Downloaded fromBioscientifica.com at09/28/202101:23:55PM 181 :6 612 via freeaccess

European Journal of Endocrinology 4 References their suggestionshelpustoimprovethecurrentpaper. and reviewers, anonymous from advice and comments valuable the for Thanks measurement. laboratory and collection data the to contributed who team research the for Thanks study. this in participants all to Thanks Acknowledgements participants. Ethicapprovalcode:(2015)MEC(S128). all from obtained was consent informed written and Committee’, Ethics Science Life University ‘Zhengzhou the from obtained was approval Ethics Ethics approvalandconsenttoparticipate decision topublish,orpreparationofthemanuscript. analysis, and collection data design, 2016M602264). study the in role no had number funders The (grant numbers Foundation by Science funded Postdoctoral (grant was China project and China 81602925); 81573243, 21607136, of (grant numbers China of Initiative’ Foundation Science Natural National Medicine 2016YFC0900803); ‘Precision Program Development and Research Key National the by supported was work This Funding be could that interest of conflict perceived asprejudicingtheimpartialityofthisstudy. no is there that declare authors The Declaration ofinterest EJE-19-0352 at paper the of version online the to linked is This Supplementary data 8 7 6 5 3 2 1 Clinical Study Joseph JJ, Echouffo-Tcheugui JB, Kalyani RR, Yeh HC, Bertoni AG, Liu X, Li Y, Li L,Zhang L,Ren Y, Zhou H,Cui L,Mao Z,Hu D& Wang L, Gao P, Zhang M,Huang Z,Zhang D,Deng Q,Li Y, Zhao Z, American DiabetesAssociation.Diagnosisandclassificationof IncidenceandPrevalence GBD 2016DiseaseandInjury Stumvoll M, Goldstein BJ&vanHaeften TW. Type 2diabetes: Zimmet P, Alberti KG&Shaw J.Globalandsocietalimplications Effoe VS, Casanova R,Sims M,Correa A, Wu WC srep31426) study. mellitus andriskfactorsinChinese rural population:theRuralDiab Wang C. Prevalence, awareness,treatment,controloftype2diabetes doi.org/10.1001/jama.2017.7596) prediabetes inChina2013. Qin X, Jin D (https://doi.org/10.2337/dc09-S062) diabetes mellitus. September 2018. http://diabetesatlas.org/key-messages.html org/10.1016/S0140-6736(17)32154-2) of DiseaseStudy2016. countries, 1990–2016:asystematicanalysisfortheGlobalBurden and yearslivedwithdisabilityfor328diseasesinjuries195 Collaborators. Global,regional,andnationalincidence,prevalence, (https://doi.org/10.1016/S0140-6736(05)61032-X) principles ofpathogenesisandtherapy. org/10.1038/414782a) of thediabetesepidemic. Scientific Reports . et al . Prevalenceandethnicpatternofdiabetes Diabetes Care 2016 Lancet Nature 6 2017 31426. JAMA 2009 2001 390 2017 32 (https://doi.org/10.1038/ J Jiangandothers 414 1211–1259. Lancet (Supplement 1)S62–S67. 317 782–787. . Accessedon11 https://doi.org/10.1530/ 2005 2515–2523. et al 365 (https://doi. (https://doi. . Aldosterone, 1333–1346. (https://

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