Regulation of Glucokinase As Lslets Adapt to Pregnancy

Home , Human placental lactogen

Matschinsky FM, Magnuson MA (eds): Glucokinase and Glycemic Disease: From Basics to Novel Therapeutics. Front Diabetes. Basel, Karger, 2004, vol 16, pp 222-239

Regulationof Glucokinaseas lsletsAdapt to Pregnancy

RobertL. Sorenson,Anthony J. Weinhaus,T. Clark Brelje

Department of Genetics Cell Biology and Development, University of Minnesota Medical School, Minneapolis,Minn., USA

Pregnancyis an occasionin the life history of the B-cell where there is an increasedneed for insulinthat'occurs over a relativelyshort period of time. This amountsto daysin rodentsand monthsin humans.The needfor enhanced islet function emergesas a consequenceof an increasein peripheralinsulin resistanceat the sametime as the placenta,a major targetorgan for insulin, develops.To accommodatethis increasein insulin demand,the islet must undergochanges that lead to increasedinsulin secretionunder normal glucose conditions. The primary short-termregulation of insulin secretionis achievedby ele- vating the glucoseconcentration. However, if this were the primary adaptive mechanismsduring pregnancy,there would be a need for persistenthyper- glycemia- a conditiondeleterious to the developingembryo, fetus and mother. Thus, in the face of this increaseddemand for insulin, islets must undergo structuraland functional changes.The outcomeof this long term upregulation of isletsmust be enhancedinsulin secretion at normalglucose levels. Failure of this long-termadaptive process can lead to gestationaldiabetes. Evidence for functional changes in islets during pregnancy first appearedin the 1960sshortly after the developmentof a sensitiveradioimm- unoassayfor insulin. Spellacyand coworkers[1 3] reportedthat there was a progressiveincrease in both fasting and glucose-stimulatedinsulin secretion throughoutthe courseof pregnancy.These and subsequentstudies led to the charucterizationof pregnancyas a condition of elevatedserum insulin levels, slightly lower blood glucose levels and peripheralinsulin resistance(see reviews[4, 5]). 300 E E' zso .a 'fr E zoo E G ; 300 .9 o X r

0 6 1012141820 0 6 101214171A20 0 610 12 14't820 Days pregnancy Days pregnancy Days pregnancy

Fig. 1. F,ffect ofpregnancy on BrdU labeling ofislet nuclei (center panel) and extent ofglucose stimulated insulin secretionin rats (right panel) [9]. The increasein islet cell proliferation and insulin secretion correlateswith the onset of placental lactogen secretion (left panel). The two peaks of serum lactogen activity correspond to the secretion of PL-I and PLJI during rat pregnancy.

lslet p-Cell Proliferation and Hypertrophy during Pregnancy

Islets can respondto an increaseddemand for insulin by increasingislet' massand the functionality of the B-cells.In fact, suggestionsthat isletsundergo changesduring pregnancywas suggestedas early as the 1930swhen it was first reportedthat there was an increasein total islet volume. Subsequently,a number of studieshave shownthat the increasein islet massis due to both hypertrophy and hyperplasia[see review, 6]. When we examinedDNA content/islet on day 20 andprotein contenton days l5 and 20 ofpregnancy,there wasa25o/o increasein DNA and a 50 and 100%increase in protein, respectively. Islet cell proliferationduring pregnancyin rats hasbeen examined by triti- atedthymidine incorporation and bromodeoxyuridine (BrdLf labeling.Tritiated thymidineincorporation was increased,2-to 3-fold on day 12-14 of pregnancy and then approachedcontrol levelsby day 19 [7, 8]. Similarly,there was a progressiveincrease in BrdUlabeled nuclei from day l0 to 14 of pregnancyat which the labelingwas I 0-fold greaterthan in controls(fig. I ). Subsequently,the numberof labelednuclei/islet declined to control levelsby day l8 of pregnancy [9]. Theserates of BrdU labelingare consistent with a 30% increasein cell mass during pregnancy and correspond with the 25o/oincrease in DNA that was

Glucokinase during Pregnancy 223 7

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0 2 4 6 8 1012 2 4 6 8 1012 2 4 6 8 1012 Glucose (nM)

12 c €roo o o .EB J -c .E o6 o E o c p4 u

2.8 5.6 8.3 11.1 61012151920 Glucose (n^,f) Days pregnancy

Sorenson/Weinhaus/Brelie observed.A study designedto determinethe numberof islets and islet volume during pregnancyindicated that the increasedislet volume resultsfrom growth of pre-existingislets and not from neo-formationof islets [10]. Thus, there is uniform agreementthat pregnancyresults in an increasein total islet mass.This growth is due to both B-cell hyperplasiaand hypertrophy. The increasein B-cell proliferation is first observedaround day 10 and corre- spondsto the observedincrease in placentallactogen [9]. The B-cell proliferation peaksaround day 14 ofpregnancyand then returnsto control levelsby the end of pregnancy.From this dataone can surmisethat the capacityfor insulin secretion,based on islet mass,is increasedabout 2-fold during pregnancy.

Insulin Secretion during Pregnancy

During pregnancyin rodents,fasting serum insulin levels are increased aboutT5o/oand glucose levels are decreased by about25%ll l-131. Glucosetol- erancetests are normal, but showenhanced insulin secretion[11]. A similar sit- uationis observedin humansduring pregnancy [-5]. Therehave been relatively few studiesexamining insulin secretionfrom islets isolatedduring pregnancy. These studies showedthat pregnancyresults in enhancedglucose-stimulated insulin secretionfrom theseislets [8, ll, l4]. Greenet al. |4, 15] alsonoted that there was a leftward shift in the glucoseresponse curves for insulin secretion and insulin synthesis.This was the first report indicatingthat the thresholdfor glucose-stimulatedinsulin secretioncould be loweredbelow that observedunder normal, non-pregnantconditions. That is, an increasein the islet's sensitivityto glucosestimulation. Although these studies indicated that there was an increasein insulin secretionduring pregnancy,there were no studiesexamining the correlationof secretionparameters with changesin placentallactogen (PL) secretionthrough- out pregnancy.To investigatethe temporalprofile of changesin islets during pregnancy,we examinedinsulin secretionduring gestationin rats (fig. 2) I9l. Differencesin both the thresholdof glucose-stimulatedinsulin secretionand the amountof insulin releasedabove this thresholdcould be detectedbv dav l0

Fig. 2. The top six panels show glucose-dependentinsulin secretion from perfused pancreasesduring the course ofpregnancy in comparison to the controls [9]. The shift in the glucose-stimulationthreshold and abovethreshold insulin secretionis first detectedon day I 0. The differences increase until day 15 and then return to control levels by day 20 ofpregnancy.The bottom left panel showsthe ratio ofinsulin secretionduring pregnancy compared to controls for each glucose concentration.The bottom right panel showsthe fold increasein insulin secretioncompared to control at a normal blood glucose concentration of 5.6mM.

Glucokinaseduring Pregnancy 225 (fig.2). By day l2,the thresholdwaslowered from5.7mM glucoseIo3.3mM, remainedat this level throughday 15, and returnedtowards normal by day 20. concomitant with the increasedsensitivity of B-cellsto glucose,the amountof insulin releasedabove the thresholdwas increased by day 12,peakedonday 15, and returned to control levels by day 20. This lowering of the thresholdfor glucose-stimulatedinsulin secretionis an important feature of islets as they adaptto pregnancy.It is only by this maneuverthat a large increasein insulin secretioncan be achievedat normal blood glucoselevels. The magnitudeof this effect canbe seenby comparingthe ratesof insulin secretionat 5.6mMglucose from pregnantanimals versus controls (fig. 2). The onsetofthe changesin islet cell proliferationand insulin secretioncor- relate with the appearanceof circulating PL [9]. However,pL remains elevated until the end of pregnancywhen islet cell proliferation and insulin secretionhave returnedto control levels.Since islets culturedin the presenceof pRL or pL show persistentincreases in islet cell dMsion and insulin secretion,it is likely that the increasein steroidsor other effectorsduring the later stagesofpregrrancy are coun- teracting the effects of PL. This hypothesisis supportedby experimentsin vitro whereislets are cultured with bothPRL andprogesterone [16]. In this case,insulin secretionand Brdu labeling increasedduring the first 4 days, but subsequently returnedto control levelsby day 8. This temporalpattern of changesin islet function closely mimics that observed in islets during pregnancy [9]. Similarly, increasedplasma glucocorticoid levels during the later part of pregnancycould effectively reversethe lactogen-inducedupregulation of islet function by inhibit- ing insulin secretionand cell proliferationwhile increasingapoptosis [17]. The summaryof the evidencethat the adaptationof islets to pregnancyis mediatedby way of lactogenicactivity (i.e. pRL or pL) is shownin table l. In addition,the magnitudeof the effectsobserved with the homologouspRL or pL in vitro is comparableto thoseobserved during pregnancy. It should also be mentioned that the physiological relevance of pL secretion has been questionedbecause placentas of many speciesdo not producea distinct PL (e.g. rabbits,pigs, dogs, and cats) [18]. In addition, the occurrenceof normal pregnanciesin womenwith undetectableserum concentrations of human PL, becauseof a gene deletion,has beenreported Ug-211. unfortunately, insulin secretionduring pregnancyhas not been examinedin either of these conditions.An elevation in insulin secretionmay still occur becauseother mechanismsmay compensatefor an absenceof a distinct pL. First, the decreasedcapacity for glucosedisposal from the induction ofperiph- eral insulin resistancemay be sufficient to indirectly stimulatean increasein islet function. Second,an increasein serumlevels of pRL may compensatefor the absenceof a distinct PL during pregnancy.An increasein serum levels of PRL during pregnancy has been shown in dogs l22l and,humans 123151.

Sorenson/Weinhaus/Brelj e Table 1. Comparison of the effects of pregnancy with species homologous lactogenic hormones (prolactin and or placental lactogen)

Islet structureand function Pregnancy PRL/PL

B-Ce1lproliferation +++ +++ Islet volume +++ +++ Glucose-stimulatedinsulin +++ +++ secretion Lower threshold for insulin +++ +++ secretion Insulin synthesis +++ +++ Insulin content +++ +++ B-Cell junctional coupling +++ +++ Glucose utilization +++ +++ Glucose oxidation +++ +++ Glucokinase activity +++ +++ Glucokinase protein/DNA +++ +++ Glucose transporter 2 leveis +++ +++ c-AMP metabolism +++ +++

Furthermore,the extent of the PRL receptorexpression in target tissuesmay also be importantbecause the binding of PRL to the mammarygland is much greaterin specieswithout a distinct PL U8l. Until additionalevidence is available, thesestudies should not be interpretedas disprovingthat an increasein islet function is an essentialcomponent of the alterationsof maternalmetabo- lism during pregnancyin mammalianspecies.

Role of Lactogensin the Regulationof lslets during Pregnancy

The long form of the PRL receptoris presentin islets [26-31]. Within islets,the PRL receptoris only presenton B-cellsand not a-cells or 6-cells [32] (fig. 3). The intensityof immunohistochemicalstaining in control isletsis quite variableamong B-cells, with many showinga low level of PRL receptorexpression. By day 14 of pregnancyin rats,this cellular heterogeneityfor PRL receptors is markedlydecreased with nearlyall cells havinga uniformly high level of staining [31]. Also, an increasein PRL receptormRNA has been reportedin islets during pregnancyand after treatmentwith PRL in vitro [33]. Thesestud- ies suggestthat lactogensinduce the expressionofthe PRL receptorand this likely hasa role in the islet'sadaptation to pregnancy.The long form of the GH

Glucokinase during Pregnancy Fig. 3. Immunohistochemical detection of STAT5 in isolated islets (top panels). The left panel is a control islet and the right panel shows cytoplasm to nuclear translocation of STAI5 after 30min of prolactin ffeatment.The bottom panel is an immunohistochemical demonstrationofprolactin receptorsin islet B-cells.Note the absenceofprolactin receptors in the mantel containing ct- and 6-cells. receptoris alsopresent in isletsl2l ,291.However, in contrastto thepRL receptor, GH hasno effecton the expressionof the GH receptor[33]. PRL and GH receptorsbelong to the cytokine superfamilyof receptors, which do not haveintrinsic tyrosine kinase activity but interactwith membersof the JAK (Januskinase) family of tyrosine kinases [34]. The activatedJAK kinases phosphorylatea member of the srAr (Signal Transduction and Activatorsof Transcription)family of transcriptionfactors, which then dimerize andtranslocate to the nucleusto regulatethe expressionof specificgenes [35]. The principalcomponents involved in PRL andGH receptorsignaling are JAK-2 and STAI5, althoughothers may be involved.In recentimmunohistochemical studies,we have identified JAK2 in rat islets and INS-I B-cell line and have observedthe translocationof srAr5 to the nucleusafter treatmentwith pRL [32,361.This translocationof srAr5 is a rapid event,detectable within a few minutes,and is maximalwithin 30min (fig. 3). Interestingly,the dose/response relationshipbetween PRL concentrationand STAI5 activation(hanslocation from cytoplasmto nucleus)parallels the doseresponse relationship between pRL and glucose-stimulatedinsulin secretion and BrdU labelingof islet p-cerls[32].

Sorenson/Weinlaus/Bre1je 228 Mechanismsof lncreasedGlucose-Stimulated InsulinSecretion during Pregnancy

With the demonstrationthat lactogensinduce the changesin islets during pregnancy,it was important to identify the cellular mechanismsinvolved. In particular,the loweringof the thresholdof glucose-stimulatedinsulin secretion is an essentialfeature ofthe successfuladaptation of isletsto pregnancy.Although conditions have been identified with an increasein the glucose-stimulation threshol4for examplesomatostatin, fasting and reduced glucokinase expression 131-391,few studieshave examined insulin secretionwhen there is a lowering of the threshold[14, 40,411.Therefore, we undertooka studyto investigate whetherthe thresholdcan changein vivo after PRL or GH infusion,glucose loading or fasting, or in vitro after treatmentwith forskolin, glucagon,chole- cystokinin,carbamylcholine or gastricinhibitory peptide[42]. Unexpectedly, all treatmentsthat enhanceinsulin secretionlowered the thresholdof glucose stimulationinto the rangeof 3.0 4.0mMglucose.This suggeststhat the metabolism of approximately3.0mM glucoseactivates all of the pathwaysnecessary for glucose-stimulatedinsulin secretion.Further increases in glucosemetabolism would increasethe activity of thesepathways and/or stimulateadditional regulatorypathways. This modelof insulinsecretion is supportedby the obser- vation that most secretagoguesrequire stimulatoryor near stimulatoryconcen- trationsof glucoseto havean effect on insulin secretion. Glucosemetabolism is centralto regulatedinsulin secretionand glucokinase is regardedas the critical glucosesensor. Moreover, a reduction in glucokinaseactivity increases the threshold of glucose-stimulatedinsulin secretion [37].Therefore, we examinedwhether changes in glucosemetabolism, glucokinaseactivity and expression,and the expressionof the glucosetransporter Glut2 correlatedwith the changesin insulin secretionobserved in islets during pregnancyor after culture with PRL in vitro [43]. The ratesof glucoseoxidation andutilization in isletswere elevated during pregnancy.This enhancedglu- cosesensitivity results in a shift of the glucosemetabolism profiles to lower concentrations.A similar increasein glucosemetabolism was found in islets treatedwith PRL in vitro. This increasein glucosemetabolism correlates with a 50o/oincrease in glucokinaseactivity observed in theseislets. This changealso correlateswith the increasein glucokinaseprotein (fig. a). The glucosetransporter Glut2 expressionwas also increasedmore than 2-fold in theseislets. Thesechanges support the hypothesisthat the upregulationofkey components in glucosemetabolism has a primaryrole in the long-termadaptation of islets to pregnancy.The bestcandidate for a centralrole in this processis glucokinase, which is the rate limiting stepin islet glucosemetabolism [aa]. The observed 50oloincrease in glucokinaseactivity is close to the predicted amount [45]

Glucokinaseduring Pregnancy 229 Glucoseutilization Glucoseoxidation I c""t';il 2.0 F 1A <7 lrDaylsl z z .^ o o t.o go6 9 rn 'Fc c E A 1.0 b o oc 3 o.e ov l f ^^ E u.o o- o.o E Eo o1 o.2 0 0.0 2.8 5.6 8.4 11.2 2.8 5.6 8.4 11.2 Glucose (mM) Enzymeexpression Enzymeactivity (Westernanalysis) 180 180

160 160

140 140 E tzo E tzo c c 8 roo 8 roo o 580 880 I 860 &uo 40 40 20 20 0 0 Pregnant Pregnant

Fig. y'. Glucose oxidation, utilization, glucokinase and hexokinaseduring pregnancyin rat islets. On day l5 ofpregnancy, when glucose stimulated insulin secretion is at its great- est, there is an increase in glucose metabolism as demonstrated by measuring glucose utilization and glucose oxidation. At this time there is also an increase in glucokinase enzyme activity and protein as demonstratedby Western analysis. needed to lower the glucose-stimulation threshold to that observed during pregnancy and in islets treated with PRL in vitro. Since hexokinase could also potentiallycontribute to changesin the glucose-stimulatedinsulin secretion threshold it was also examined.In mid-pregnancy,when the greatestchange in insulin secretion occurs, there were no changesin hexokinaseactivity or expressionlevels. Also, in the in vitro experimentsusing PRl-treated islets, hexokinaseis barely detectableand again no changeswere observed.Thus, it

Sorenson/Weinhaus/Brelje Culture2 days Stimulatet hr ------> Assay and Western in G-10 mM glucose in 8.2mM glucose

lnsulinsecretion Glucokinaseexpression* 0.5 4.O

3.5 0.4 en c E - 9 nr o 2.5 = E gl = 2.O c c nt o t.c f o a c '1.0 0.1 u.c

0.0 6 10 o2 Glucoseconcentration during culture (mM)

Frg 5. Effect of glucose and prolactin treatment on subsequentglucose-stimulated insulin secretionand expressionof glucokinasein isolated islets. Culturing in increasing concentrat'ionsof glucose for 2 days leads to a subsequentincrease in glucose-stimulated insulin secretionand glucokinaseexpression. At all concentrationsofglucose therewas an incremental increasein insulin secretionand slucokinase exoressionin the islets treatedwith prolactin. appears that hexokinase is not required for the changes in insulin secretion that occur during pregnancy. The above studies strongly implicated glucokinase in the changes in insulin secretionthat occur during pregnancy.Therefore, additional studieswere done to further characterizeits role and the mechanismsby which this occurs. Initial experiments were done using INS-I cells examining the effect of PRL on glucose metabolism, insulin secretion and glucokinase expression by Western blots. These experimentsshowed glucose-dependent increases in each of these parameters with an additional increase with PRL treatment at all glucose concentrations.Similar resultswere observedwith isolated islets (fig. 5). These experimentsdemonstrate that both glucoseand PRL can regulatethe expression of glucokinase in islets. Furthermore, this increasein glucokinase expression resulted in a lowering of the threshold for glucose-stimulatedinsulin secretion and an overall increasein the extent of insulin release. Because these changeswith PRL were observed even in the absenceof glucose,PRL appearedto be able to alter glucokinaseexpression independent of glucosemetabolism. Consequently, additional experimentswere done with islets cultured in the absenceof slucose and then examined for slucose-stimulated

Glucokinase during Pregnancy 23l Culture2 days Stimulatet hr ------) Assay and Western in 0mM glucose in 4-8mM glucose Westem blot analysis Insulinsecretion Glucokinase PRL receptor 0.40 c""tr;il 0.9 2.0 F 1.8 F o.ss IrPRL I 0.8 t.o # o.so o.7 1.4 - 0.6 - o.zs o t.z E FC E o.zo =6 "" O rn p @ oA 6 0.15 o 6 o.a o o a 0.6 .E 0. 10 = 0.2 0.4 C U.UC 0.1 o.2 0.00 0.0 0.0 468 Glucose (mtl,/)

Fig.6. Effect of prolactin treatment on islets cultured for two days in the absenceof glucose. PRL treatment resulted in an increaseglucokinase expression(middle panel) and a coordinateincrease in glucosestimulated insulin secretion(left panel).An increasein prolactin receptor expressionwas also observed (right panel). insulin secretionand glucokinase expression. PRL receptorexpression was also examinedas this had previouslybeen demonstrated as being underthe direct regulation by the PRL/JAK/STAIS pathway [30]. These results demonstrate that glucokinaseexpression could be inducedby PRL treatmentand that it did not requireglucose or its metabolismfor the effect to be observed(fig. 6). Theseexperiments also show that the increasein glucokinaseinduced by PRL in the absenceof glucoseis sufficientto lower the thresholdof glucose-stimulated insulin secretion.This raisedthe possibilitythat glucokinase,like the PRL receptor,may be directly regulatedby a STAI5 mediatedmechanism. STAI5 bindsto DNA by way of a GAS relatedsequence (GRE) which con- sists of the consensusmotif TTC(n : l 4)GAA. Six of theseputative STAT5 binding sitescan be found in the rat B-cell glucokinasepromoter [46]. To exam- ine the possibilitythat glucokinaseis regulatedby a STAT5mechanism. experiments were done to characterizethe putative interactionbetween STAI5 and GRE containing oligonucleotidesfrom the glucokinasepromoter. The initial experimentswere southwesternanalysis and consistedof treating INS-I cells with PRL. The nuclei were extractedand electrophoresisand first examinedby Westernblots, which demonstratedthe presenceof activated(phosphorylated) STI{I5. Probingwith a GAS containingoligonucleotide from the glucokinase promoterrevealed binding to STAI5. Similar resultswere obtained when examining GRE containingoligonucleotide probes from the PRL receptorpromoter

Sorenson/Weinhaus/Brelie 232 PRL-inducednuclear extract binding to glucokinasepromoter (GRE containing sequence) 3.0

2.5

2.O - o E o t.c .=q ()c o 1.0

0.5

0.0 Nuclearextract:Control Prolactintreated ---l Probe:F- cK13-cRE cK13-Mutated 10 x cold competition

Fig. 7. Effect of prolactin treatment on nuclear extract protein binding to a GRE containing oligonucleotidesequence (GKl3-GRE) from the glucokinasepromoter. The EMSA shows that prolactin treatmentinduces a protein which is capableof binding GKl3-GRE, but not when its STAT5 binding motif is mutated (CKl3-mutated). The putative STAT5 binding of labeled GKl3-GRE is competitivelyinhibited by incubatingwith a l0-fold excessof unlabeledprobe. and the insulin fromoter. To further charucteizethe interaction betweenSTI{I5 andthe glucokinasepromoter, electromobility shift assays(EMSA) weredone. In theseexperiments, a GRE-containingoligonucleotide probe of the glucokinase promoterwas incubatedwith nuclearextracts from NS-l cells that weretreated with or without PRL for 30min and then separatedby electrophoresis.Nuclear extractprotein that bound to the probe was identified as a retardedband. The retardedband from the PRL treatedcells had a density2.5-fold greaterthan that observedfor the controlcells. The banddensity was reduced to controllevels by competitionwith a cold probe. Similarly a mutatedprobe was unableto bind greaterthan that observedfor the control(fig.7). Evidencethat the retardedband wasSTAI5 wasdemonstrated in supershiftexperiments. A supershiftedband was producedby the bindingof anti-STAT5b,but not anti-STAI5a(fig. 8). Thesedata demonstratethat PRl-treatment induces the binding of STAI5b to GRE sequencesin the glucokinasepromoter and is evidencethat glucokinaselevels are increasedin PRL treatedislet cellsbv wav of a STAI5b mediatedmechanism.

Glucokinaseduring Pregnancy Antibody: STAT5b STAT5a Supershift w

Bound probe

Freeprobe

Nuclearextract: None Ctrl. Prolactin Treated Probe:------GK13-GRE---

Fig. 8. Supershift experiment of prolactin induced binding of the GRE containingoligonucleotide sequence (GKl3-GRE) from the glucokinasepromoter. Inclusion of anti-STATb,but not anti-STATa,in the incubationmedia results in a supershiftof the bound probe and is evidencethat STAT5b contributesto protein retardationin the EMSA expenments.

Previousstudies have also reportedan increasein c-AMP metabolism during pregnancyand proposedthat it was responsiblefor the increasein glucose-stimulatedinsulin secretion [40,41]. An increasein c-AMP levelshas beenshown to potentiateinsulin secretion and lower the thresholdfor glucose- stimulatedinsulin secretionin controlislets [42, 4149]. We re-examinedthis issueby studyingc-AMP metabolismin isletsduring pregnancy and after treatment with PRL in vitro [50]. This study indicatedthat thesechanges c-AMP metabolismare most likely a consequenceof the increasedglucose metabolism and not a primary eventin alteringthe patternof insulin secretion.Support for this idea is that underall conditionsstudie{ whetherpregnancy, PRL treatedor

Sorenson/Weinhaus/Brelje c-AMP versus secretion c-AMP versusoxidation o.12 100

^ 0.10 1.6 o o t i i E l o ? 0.08 F E e ; 7 1-2 .F E60 6 0.06 @ 6 6 6 o ne c o 8+o .s 0.04 =.g c f @ a (I c E o.+ o.o2 8.4 >20 o

0.00 0 0 20 40 60 80 100 120 0 5 1015202530 0 5 1015202530 c-AMP (fmol/islet) Glucose oxidation (pmol/islet) Glucoseoxidation (pmol/islet)

Fig. 9. Summary of data on the relationships among PRL treatment and glucose metabolism, c-AMP metabolism and insulin secretion.Under all treatment conditions, with and without PRL and with different concentrations of glucose, the levels of c-AMP are comparablefor similar ratesof glucosemetabolism. The resultssuggest that the changesin c-AMP metabolismare most likely a consequenceof the increasedglucose metabolism and not a primary event in pregnancy or PRL induced alterations of insulin secretion. with different glucoseconcentrations, the levels of c-AMP are comparablefor similarrates of glucosemetabolism (fig. 9). Although the amount of insulin secretionin pregnancyor PRl-treated isletsis higherthan that in controlislets at the sameglucose concentration, it is appropriatefor the amountof glucoseoxidation observed in controlislets at a higher glucoseconcentration [50]. This is strongevidence that the increased insulin secretionseen in pregnancyor PRl-treatedislets in vitro is primarily dueto an increasein glucoseoxidation because ofthe increasein glucokinase activity. Thus, it is expectedthat in addition to c-AMP, all pathwaysnormally influencedby glucosemetabolism in isletswill be upregulatedin parallelto the enhancedglucose metabolism that occursas a consequenceof the increasein glucokinaseprotein and its activity.

Model of the Mechanismsby which PRL and PL InduceChanges in p-Cellsduring Pregnancy

Based on the available information, we have developed a model for lactogen regulation of islets (fig. l0). During pregnancy,islets undergo a number of

Glucokinase during Pregnancy 235 Placentallactogen/prolactin

Fig. 10. A model of the mechanismswhereby prolactin/placental lactogen bring about changesin islets characteristicofthose observedduring the adaptationofislets to pregnancy. changesto adapt to the increaseddemand for insulin. These changesoccur under direct influenceoflactogenic hormonesand undernoffnal blood glucose conditions.This is not an acuteshort-term regulatory process, but ratheroccurs overan extendedperiod of time.The most significant outcomes are an increase in isletmass and enhanced insulin secretion with a loweringof the thresholdof glucose-stimulatedinsulin secretion.These are two quite diverse cellular processes,one which is commonto many cells (mitosis)and one which is highly differentiated(insulin secretion). Binding of PL or PRL to the PRL receptoron B-cells leadsto the activation of tyrosinekinase JAK2 andthe transcriptionfactor STAI5. With translo- cationofactivated STAI5 to thenucleus, an increase in theexpression ofseveral genesrequired for the upregulationof islet function occurs.An increasein the expressionof the PRL receptorleads to an increasedsensitivity to lactogens.

Sorenson/Weinhaus/Brelje ] The increasedexpression of glucokinaseresults in enhancedglucose metabolism. This increasein glucosemetabolism is the primary eventresponsible for the loweringof the thresholdof glucose-stimulatedinsulin secretion. As a consequenceof this increasein glucosemetabolism, all otherregulatory pathways, such as cAMP and intracellular calcium concentration142, 51, 52], are activated at higher levels than normally observedfor a comparableconcentration of glucosein normal(i.e., non-pregnant) islets. The combinationof this lowering of the thresholdand increasedactivity of regulatorypathways results in a markedenhancement of insulin secretionat normalblood glucoselevels. An increasein the expressionof the cell cycle regulatorcyclin D2 shouldlead to an increasein B-cellproliferation [53, 54].This effectwill be furtherincreased dueto the stimulationof B-cellproliferation by the enhancedglucose metabolism. Althoughthis increasein islet masscontributes to the increasedinsulin secretionduring pregnancy,it is only by the lowering of the threshold that islets can secretelarge amountsof insulin without the need for prolonged hyperglycemia.

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Robert L. Sorenson.PhD Dcpartment of Genetics Cell Biology and Development University of Mimesota Medical School 6 160 JacksonHall, 321 Church St. S.8., Minneapolis,MN 55455 (JSA) Tel. + I 612 624 6414,Fax + I 612 624 8118, E-Mail sorenO0l(rlumn.edu

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