The effects of metabolic acidosis on glucose metabolism

Transcription

1 Yale University EliSchlar A Digital Platfrm fr Schlarly Publishing at Yale Yale Medicine Thesis Digital Library Schl f Medicine 1977 The effects f metablic acidsis n glucse metablism Alan Dennis Beckles Yale University Fllw this and additinal wrks at: Recmmended Citatin Beckles, Alan Dennis, "The effects f metablic acidsis n glucse metablism" (1977). Yale Medicine Thesis Digital Library This pen Access Thesis is brught t yu fr free and pen access by the Schl f Medicine at EliSchlar A Digital Platfrm fr Schlarly Publishing at Yale. It has been accepted fr inclusin in Yale Medicine Thesis Digital Library by an authrized administratr f EliSchlar A Digital Platfrm fr Schlarly Publishing at Yale. Fr mre infrmatin, please cntact elischlar@yale.edu.

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5 Digitized by the Internet Archive in 2017 with funding frm The Natinal Endwment fr the Humanities and the Arcadia Fund

6 THE EFFECTS F METABLIC ACIDSIS N GLUCSE METABLISM Alan Dennis Beckles B.S. Clumbia University, 1971 M.S. Clumbia University, 1973 Presented t The Faculty f Yale University Schl f Medicine In Candidacy fr the Degree f Dctr f Medicine 1977

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8 The authr wishes t thank Mrs. Lis Mishwiec fr her patience, gd humr and technical assistance, Mr. Ralph Jacb fr his technical assistance and Ms. Creen Bentham fr tlerating cnstantly changing deadlines.

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10 TABLE F CNTENTS I. INTRDUCTIN II. METHDS A) Subjects B) Hyperglycemic Clamp C) Euglycemic Clamp D) Calculatins E) Analytical Prcedures III. RESULTS A) Hyperglycemic Clamp B) Euglycemic Clamp IV. DISCUSSIN V. SUMMARY VI. REFERENCES VII. LEGENDS T FIGURES VIII. FIGURES IX. TABLES

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12 INTRDUCTIN Impaired carbhydrate tlerance is a cmmn accmpaniment f diabetic ketacidsis (1) and chrnic renal failure (2). is the presence f metablic acidsis. Cmmn t bth these states Althugh several previus animal (3-5) and in vitr (6-8) studies have suggested that acidsis per se may result in an impairment f glucse metablism, such an effect has nt been well studied in man and the mechanism(s) by which acidsis impairs glucse metablism have nt been defined. Haldane et.al. (9) were the first t examine the effect f metablic acidsis n glucse metablism in man. Fllwing the injestin f ammnium chlride they fund an impairment f glucse tlerance which persisted fr sme time fllwing the return f bld ph t nrmal. Walker et.al. (6) nted decreased sensitivity t exgenusly administered insulin in six diabetics with ketacidsis (bld ph <7.20) and imprvement in insulin sensitivity fllwing crrectin f the acidsis. Hwever, these authrs culd nt exclude ther changes in the diabetic state as respnsible fr the imprvement in insulin sensitivity. In the nly ther study in man, Baird (10) was unable t demnstrate any adverse effect f ammnium chlride induced acidsis n the intravenus glucse tlerance test. Hwever, all f his subjects were treated with quinalbarbitne prir t study and this culd have bscured any differences between the cntrl and ammnium chlride treated grups. Mackler et.al. examined the effect f ammnium chlride induced metablic acidsis (mean bld ph and serum bicarbnate cncentratin = 7.06 and 6 meq/l) in dgs (3-5). They fund a cnsistent increase in fasting glucse cncentra tin, a blunted respnse t exgenus insulin, and impairment in the intra venus glucse tlerance test. They als bserved a failure f the nrmal

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14 decline in serum ptassium and phsphate cncentratin (11-14) in respnse t bth insulin and glucse, again suggesting impaired sensitivity t the actin f insulin. Hwever, the interpretatin f the abve studies is bscured by the fact that the dgs were als fasted ver the three days during which the metablic acidsis was induced. Thus, it is difficult t differentiate an effect f acidsis versus fasting n the decline in glucse tlerance. In the nly ther study perfrmed in dgs, Deuel and Gulick were unable t demnstrate an effect f ammnium chlride-induced acidsis n carbhydrate tlerance (15). In vitr studies emplyed the rat epididymal fat pad (6), the rat hemidiaphragm (6,8), and circulating erythrcytes and leukcytes (5,7) have als suggested that acidsis inhibitsglucse uptake and xidatin. T examine the effect f metablic acidsis n glucse metablism and t define the relative cntributins f impaired insulin resistance, imparied beta-cell secretin f insulin, and augmented hepatic qlucse rductin t the carbhydrate intlerance, we studied 12 subjects with the glucse clamp technique befre and after the inductin f chrnic metablic with ammnium chlride. METHDS A. Subjects: (Table I). frm 22 t 31 were studied. Twelve healthy vlunteers ranging in age They were evenly divided between males and females and all were within 20% f their ideal bdy weight. The middle f the weight range fr subjects f medium frame frm the 1959 Metrplitan Life Insurance Cmpany table fr desirable weight was used t determine ideal bdy weiqht. All subjects were cnsuming a weight maintainina diet cntaining at least 200 gms f carbhydrate per day fr 3 days prir t study. ther than subjec

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16 M.K., M.D. and M.S., wh were n ral cntraceptives, subjects cnsumed n ther medicatins fr at least tw weeks prir t study. n family histry f diabetes. the 12 subjects. There was A ttal f 24 studies were perfrmed n All subjects were studied at 8 a.m. fllwing a twelve hur vernight fast. Fllwing the initial study each subject received ammnium chlride in a dse f either 0.1 r 0.15 gm/kg f bdy weight per day fr three days and the study was repeated n the mrning f the furth day. Each subject thereby served as his wn cntrl. The ammnium chlride was administered in divided dses every 6 hurs with the last dse being given at 11 p.m. n the evening prir t study. Althugh tw subjects cmplained f mild abnrmal discmfrt, there was n vmiting and all cntinued n the diet as stated abve. Prir t each study a plyethylene catheter was inserted int an anticubital vein under lcal xylcaine anesthesia fr administratin f all test substances. sampling. A secnd catheter was inserted int a hand vein fr bld The hand was then inserted int a heated chamber in which the air temperature was maintained at 70±2 degrees centigrade. This was dne t insure arterilizatin f venus bld (16). B. Hyperglycemic Clamp: After a cntrl perid f at least thirty minutes a priming infusin f glucse designed t rapidly raise the bld glucse cncentratin t 125 gm/dl abve the fasting level was administered in a lgarithmically falling manner ver 15 minutes. Arterilized venus bld was sampled every tw minutes fr the first ten minutes t quantitate the early releasable insulin peak. every five minutes fr 110 minutes. Thereafter, bld samples were drawn The plasma glucse cncentratin was rapidly determined (analytical delay was less than tw minutes) n each sample and the infusin rate f a 20% glucse slutin was adjusted t maintain the bld

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18 glucse cncentratin cnstant at 125 mg/dl abve the basal level. The frmula fr the serv-crrectin is based upn the negative feedback principle. Thus, if the actual plasma glucse cncentratin is greater than the gal, the glucse infusin rate is decreased and vice-versa. f pertinence t this paper is the fact that the actual plasma glucse cncentratin was maintained within narrw limits f the specified gal (see Results). Under these steady-state cnditins f cnstant hyperglycemia the amunt f glucse infused (mg/kg f bdy weight/min) minus urinary lsses (mg/kg f bdy weight/min) is a measure f the glucse metablized, M. Althugh the clamp methd des essentially eliminate variance frm subject t subject in bld glucse cncentratin, the plasma insulin levels are nt the same in all subjects. In the euglycemic clamp (see belw), insulin levels vary nly as a functin f its metablic clearance rate (MCR). In the hyperglycemic clamp insulin levels vary nt nly as a functin f MCR, but als n a functin f the sensitivity f the beta cell t hyperglycemia. The fact that insulin levels vary makes it essential t crrect the value fr M in sme manner t take this variance int accunt. The simplest technique is t cmpute the rati f M t the plasma insulin level, I, that is, t cmpute M/I. The underlying mathematical assumptins f such a cmputatin are that M is linearly related t I and that there is n intercept f M n I. If either f these tw assumptins is incrrect, then a mre cmplex frmulatin wuld be required. might be justified. Fr example, cmputatin f an M/lg I We have chsen t crrect fr the insulin cncentratin by the simplest pssible technique, M/I. The mean insulin cncentratin in the 24 euglycemic and hyperglycemic clamp studies is 104 uu/ml (Tables 2 and 3) with a standard deviatin f ±32. Within these reasnably narrw limits it is dubtful whether mre cmplex frmulatins wuld significantly change the cnclusins presented.

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20 C. Euglycemic Clamp: A prime plus cntinuus infusin f crystalline prcine insulin (Eli Lilly C., Indianaplis, In.) was administered t btain cnstant hyperinsulinemia (17). The primary dse was administered in a lgarithmically falling manner ver ten minutes at which time the cntinuus insulin infusin was begun. The ttal amunt f insulin infused during the priming perid was twice that infused during subsequent ten minute intervals. The cntinuus infusin (40 mu/m 110 minutes. p surface area per min) was maintained fr In rder t prevent insulin adsrptin t glassware and t the plastic infusin apparatus, infusates were prepared with the additin f 2 ml f the subject's whle bld per 50 ml f infusate. Hypglycemia with its attendant cunter-regulatry respnses was prevented by the glucse clamp technique (see under hyperglycemic clamp) which was used t maintain each subject at his wn basal bld glucse cncentratin during insulin administratin. The initial glucse infusin rate was 2.0 mg/kg bdy weight/ min and was started fur minutes after the insulin infusin was begun. At ten minutes the glucse infusin was increased t 3.0 mg/kg/min; these dses were empirically determined. minutes. The first serv-crrectin was made at 14 Under the cnditin f cnstant euglycemia all f the glucse infused (M) is metablized and is thus a measure f the bdy's sensitivity t insulin. During the euglycemic clamp the effect f insulin n hepatic glucse prductin was examined with tritiated glucse labeled in the 3 psitin ([%]glucse). Fr 3 hurs prir t beginning the insulin infusin, each subject's glucse pl was labeled by a primed cntinuus infusin f [^T] glucse. The labeled glucse was administered as an initial intravenus priming dse fllwed immediately by a cntinuus intravenus infusin at a rate f abut 0.25 uci/min. Plasma samples fr determinatin f glucse specific activity were taken at 30 minute intervals fr the first tw hurs and at 15 minute intervals fr the subsequent hur. A steady state plateau

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22 glucse specific acticity was achieved in all subjects during the third hur f [3t] glucse infusin and this plateau value was used t calculate basal hepatic glucse prductin. After 3 hurs f cntinuus [^T] glucse infusin, the euglycemic clamp was begun as previusly described and the cntinuus infusin f [~T] glucse was cntinued at the same rate. During the clamp study plasma samples fr glucse specific activity were drawn every 15 minutes. Three cntrl samples fr amin acids and glucagn were btained prir t beginning bth the euglycemic and heperglycemic clamp studies; subsequent samples were drawn at minute intervals. D. Calculatins: Hyperglycemic Clamp: The fasting plasma insulin cncentratin, the early (0-10 minute), late ( min), and ttal plasma insulin (I) respnses, the amunt f glucse metablized ( minute time perid) and the M/I rati (a measure f tissue sensitivity t insulin) were cmpared befre and after NH^l lading. The basal glucagn and amin acid levels as well as the change in plasma glucagn and amin acid cncentratins fllwing glucse were als cmpared befre and after NH4C1 lading. Euglycemic Clamp: Tissue sensitivity t insulin (M/I rati during time = minutes) and the metablic clearance rate (MCR) f insulin were cmpared befre and after ammnium chlride lading. The MCR f insulin was calculated by dividing the insulin infusin rate by the steady state plasma insulin cncentratin during the minute time perid minus the basal insulin cncentratin. Plasma glucagn and amin acid cncentratins were analyzed as described abve fr the hyperglycemic clamp. Basal hepatic glucse prductin befre and after ammnium chlride lading was determined 3 by dividing the [ T] glucse infusin rate (cunts/min) by the steady state plateau f [ T] glucse specific activity during the last hur f the cntrl perid. Fllwing insulin-glucse administratin (euglycemic clamp) a nn steady state cnditin in glucse specific activity exists and hepatic glucse

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24 prductin was apprximately using the Steele equatins (equatins 4A and 5A, reference #18) average values fr the rates f glucse turnver fr the intervals between tw cnservative sampling times. All data are presented as the mean - SEM. Statistical significance between the difference f tw means was calculated by paired T-test analysis. E. Analytical Prcedures: Plasma glucse analyses were perfrmed in duplicate n arterized venus samples using the glucse xidase methd (Glucstat, Beckman Instruments Crp., Fullertn, Ca ). Insulin was determined by radiimmunassay with talc t separate bund frm free insulin (19). Plasma glucagn was analyzed by radiimmunassay with Unger antibdy 30K (20). Fr determinatin f amin acids, plasma prteins were remved by precipitatin with sulfsalicylic acid (21). Amin acids were then determined by autmated in exchange chrmatgraphy (22) with a Beckman 120C amin acid analyzer (Beckman Instruments, Inc., Spinc Div., Pal Alt, Ca.) mdified fr single clumn analysis f basic as well as acidic and neutral amin acids (23). Fr determinatin f [^T] glucse specific activity, plasma samples were centrifuged immediately and plasma deprteinized using ZnS04 and Ba (0H)2 (24). The filtrates were analyzed fr glucse cntent by the glucse xidase methd as previusly described. Aliquts (0.5 ml) f plasma filtrates were then evaprated t dryness in cunting vials in a partial vacuum at 70 C t eliminate metablic T2. The dried residues were redisslved in ne ml f water, t which 10 ml f aquasl was added fr cunting (Packard Mdel 3003 Tri-Carb Liquid Scintillatin Spectrmeter, 78% efficiency, with internal quenching).

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26 RESULTS Hyperglycemic clamp: Prir t NH^Cl the fasting bld glucse cncentratin averaged 94-3 mg/dl. Durin the perid f sustained hyperglycemia the mean glucse cncentratin f the five subjects was ma/dl. Fllwing NH^Cl the fastina bld glucse and steady state hyperglycemic levels averaged 92-1 mq/dl and mg/dl respectively. The stability f the bld glucse cncentratin during the perid f hyperglycemia is shwn by the cefficient f variatin which averaged during the cntrl studies and % pst NH4CI. The amunt f glucse metablized (M) during the hyperalvcemic clamp decreased frm 9.09 ma/kg bdy wt./min. t 7.43 mg/kq bdy wt./min. fllwina NH4CI (p<0.10. Table 2). The fasting plasma insulin cncentratin in the 12 subjects fllwing NH4CI (17-1 yu/ml) was significantly hiqher than during the cntrl study (13-1 yu/ml, p 0.02). Fllwing the creatin f a square wave plateau f hyperglycemia, the plasma insulin respnse was biphasic with an early burst f insulin within the first six minutes fllwed by a gradually increasing phase f insulin release that lasted until the end f the study. There was n difference in the early insulin respnse (0-10 min.) between the cntrl and NH4C1 studies (Table 2, Figure 1). Hwever, the late insulin respnse ( min.) tended t be greater fllwing the inductin f metablic acidsis and the plasma insulin cncentratin was statistically significantly higher during the minute perid. Tissue sensitivity t insulin (M/I rati) pst NH^Cl decreased in all five subjects by an average f 32-6% (Table II, p<0.02). The fastina plasma alucaan cncentratin in the twelve subjects fllwing NH4CI was significantly higher than during the cntrl study

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28 (110 ± 18 pa/ml versus pg/ml, p<0.01). Fllwinq the creatin f a steady state plateau f hyperglycemia, plasma lucagn cncentratin was suppressed similarly in bth the pre and pst NH4CI studies (Figure 2). Euglycemic Clamp (Table 3). The prime-cntinuus insulin infusin resulted in a steady state plasma insulin level ( minutes) that averaed yli/ml during the cntrl study and yll/ml fllwing the inductin f metablic acidsis with NH4CI (Fiqure 3). The stability f the plasma insulin cncentratin is shv/n by the cefficient f variatin which averaed 9-1% durina the cntrl study and 10-1% fllwinq NF^Cl. There was n difference in the metablic clearance rate f insulin (insulin infusin rate divided by the increase abve basal in the minute insulin cncentratin) between the cntrl ( ml/min./m2) and acidtic ( ml/min/m^) studies. The fastina plasma glucse cncentratins during the cntrl and NH4CI studies were 91-2 mg/dl and 88-1 m/dl. During the perid f hyperinsulinemia, the plasma alucse cncentratin was held cnstant at the basal level with a mean cefficient f variatin f % and % respectively. The amunt f glucse metablized fllwing NH4CI decreased frm t m/kg bdy wt/min (p=0.07) and the M/I rati declined frm t ^5 in units f (mg/kq/min per yu/ml) X 100 (p<0.10). Fllwinq the infusin f insulin and lucse, plasma lucan suppressed nrmally during the cntrl study (p<0.01, figure 4).

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30 Fllwing the inductin f metablic acidsis with NH^Cl, the suppressin f glucagn by insulin and glucse was less cmplete, becming statistically significantly different at nly the 105 and 120 minute values. Plasma amin acid cncentratins were measured in subjects M.K., A.P., and M.S. N difference in fasting levels were bserved between the cntrl and NH4CI studies. Fllwing the creatin f hyperinsulinemia, a decrease in the levels f valine, leucine, isleucine, phenylalanine, threnine, tyrsine, methinine, and serine was bserved. DISCUSSIN Previus studies examining the effect f metablic acidsis n glucse metablism have yielded cnflicting results. Several reprts have suggested that ammnium chlride-induced metablic acidsis results in an impairment f the intravenus glucse tlerance test and decreased tissue sensitivity t exgenus insulin (3-6). Hwever, several investigatrs have failed t find a decrease in glucse tlerance (10,15) and the plasma insulin respnse t glucse fllwing metablic acidsis has nt been reprted. In the present study we emplyed the glucse clamp technique t examine the relative cntributins f impaired beta cell secretin f insulin versus tissue insensitivity t insulin in the genesis f the glucse intlerance fllwing chrnic ammnium chlride induced metablic acidsis. This technique ffers several advantages ver the usual techniques emplyed t assess glucse metablism: (1) During the hyperglycemic clamp the glucse stimulus presented t the beta cell is cntrlled; thus, repeat studies f beta cell sensitivity can be accurately interpreted and cmpared befre and after inductin f

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32 metablic acissis. Furthermre, since the glucse stimulus is presented as a square wave ne can examine the early and late phases f insulin secretin independently. (2) In bth the hyperglycemic and euglycemic clamps, the plasma glucse cncentratin is held cnstant. Cnsequently, the amunt f glucse infused must equal the amunt f glucse metablized by all the tissues f the bdy (minus urinary glucse lsses) and thus allws quantitatin f the glucse metablized. (3) The euglycemic clamp ffers majr advantages ver the insulin tlerance test in assessing insulin sensitivity, since the basal glucse cncentratin is maintained, thereby aviding the cmplex neur-endcrine respnse t hypglycemia. The test thus prvides a purer estimate f sensitivity t insulin. (4) Tissue insensitivity t insulin culd reside in peripheral tissues (muscle, adipse) r in the liver. By perfrming the euglycemic clamp in cmbinatin with [^T] glucse the cntributin f increased hepatic glucse prductin t the bserved insulin resistance can be determined. (5) The hyperglycemic and euglycemic clamps prvide independent tests f the sensitivity f the bdy t endgenusly-released and t exgenusly-administered insulin. The results f the hyperglycemic and euglycemic clamps clearly demnstrate that metablic acidsis results in the impairment f glucse tlerance and that the effect f acidsis is mediated via decreased sensitivity t insulin. During the hyperglycemic clamp, a decline in glucse metablism was bserved despite a 26% increase in the plasma insulin respnse. Cnsequently, the M/I rati (a measure f tissue sensitivity

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34 t insulin) fell significantly in all five subjects by an average f 32% (p<0.02). The presence f insulin resistance is als suggested by the elevated fasting insulin levels in the face f nrmal glucse cncentratins. Seven subjects als received a euglycemic clamp study t prvide an independent measure f tissue sensitivity t insulin. Tissue sensitivity t insulin declined in 6 f the 7 by an averaae f 1 2%. When the results f the euglycemic clamp are cmbined with thse f the hyperglycemic clamp a hiahly significant decline (p<0.01) in tissue sensitivity t insulin fllwing metablic acidsis is bserved. These results indicate that mild metablic acidsis (mean decline in bld ph and serum bicarbnate cncentratin = 0.04 and 4 me q/l respectively) results in decreased glucse tlerance which is mediated via tissue insensitivity t insulin. Beta cell sensitivity t lucse is nt affected and insulin secretin is augmented in an attempt t vercme this insulin resistance. It is likely that mre severe dearees f metablic acidsis which are maintained fr lnger perids f time will result in even areater insulin resistance. Attempts t administer ammnium chlride lads nreater than 0.15 grams/kg bdy wt/day in human subjects were prlv tlerated and the effect f mre severe acidsis n lucse metablism culd nt be examined. The studies with [^T]-qlucse suest that the insulin resistance resides in the periphery rather than with the liver. Prir t the administratin f NH^Cl, basal hepatic glucse prductin averaaed 1.85 mg/kg/min. Fllwing NH^Cl basal glucse prductin was unchanged.(table V). Thus, unlike ranal glucneaenesis which is stimulated bv metablic acidsis (25), hepatic glucse prductin appears t be unaffected. Althugh these results exclude augmented basal hepatic lucse prductin

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36 as the cause f the bserved insulin resistance, it is still pssible that incmplete suppressin f glycgenlysis and/r glucnegenesis by insulin is respnsible fr the decline in glucse tlerance. The answer t this questin awaits final cmpletin f the pt] glucse data. Althugh basal plasma glucagn levels were mildly elevated fllwing NH4CI, it is unlikely that such a small increase culd have any significant effect in augmenting hepatic glucse prductin n a sustained basis (26). Furthermre, bth hyperinsulinemia and hyperglycemia resulted in nrmal glucagn suppressin in the presence f systemic acidsis. It is well knwn that insulin lwers the systemic cncentratin f certain amin acids, mst ntably valine, leucine, isleucine, methinine, tyrsine, and phenylalanine (27). This effect f insulin appears t be the result f net inhibitin f amin acid utput frm muscle (28). T examine whether the insulin resistance bserved with glucse metablism als extends t amin acid metablism, we measured the fall in circulating amin acids during the euglycemic clamp studies in three subjects. Fllwing the administratin f insulin a prmpt decline in plasma valine, leucine, isleucine, methinine, tyrsine, phenylalanine, and serine was bserved in after NH4Cl.*Thus, all subjects bth befre and it appears that the insulin resistance bserved fllwing the inductin f metablic acidsis is specific fr carbhydrate metablism. * TABLE IV

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38 SUMMARY: The effect f metablic acidsis n glucse metablism was examined in 12 healthy vlunteers emplying tw types f glucse clamp experiments. Each subject was studied befre and after the ingestin f 0.1 gm f ammnium chlride per kg bdy wt. per day fr three days. Fr all subjects, the fasting insulin cncentratin rse significantly withut change in the fasting glucse cncentratin. Hyperglycemic clamp: The plasma glucse cncentratin is acutely raised and maintained at 125 mg/dl abve the fasting level fr tw hurs by the peridic adjustment f a variable glucse infusin. Since the glucse cncentratin is held cnstant, the glucse infusin rate is n index f glucse metablism (M). Fllwing ammnium chlride M declined frm 9.09 t 7.43 mg/kg bdy wt/min (p=0.10) despite a rise in plasma insulin respnse. Cnsequently, the M/I rati, an index f tissue sensitivity t endgenus insulin, declined in all subjects by 35% (p<0.005). Euglycemic clamp: apprximately 100 The plasma insulin cncentratin is acutely raised U/ml abve basal levels and is maintained by a prime- cntinuus insulin infusin. The bld glucse cncentratin is held cnstant by a variable glucse infusin. M/I is again a measure f time sensitivity t exgenus insulin and fell by 10% (p=0.09). A nrmal decline in plasma amin acids value, leucine, isleucine, methinine, tyrsine, phenylalanine, and serine was bserved bth befre and after the inductin f metablic acidsis. Bth the hyperglycemic and euglycemic clamp techniques demnstrate tissue insensitivity t insulin fllwing NH4CI induced metablic acidsis. The tissue resistance t insulin is specific fr glucse metablism and des nt extend t amin acid metablism.

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40 REFERENCES 1. Field, J.B., bservatins Cncerning a Hrmnal Insulin Antagnists During Diabetic Ketsis. Diabetes 1_: (1958). 2. DeFrnz, RA, Andres, R, Edgar, P, and Walker, WG. Metablism in Uremia: A Review. Medicine 52_: 3. Mackler, B, Lichtenstein, H, and Guest, GM. Carbhydrate (1973). Effects f Ammnium Chlride Acidsis n the Actin f Insulin in Dgs. Physil. 166: (1951). 4. Mackler, B, Lichtenstein, H, and Guest, GM. Effects f Ammnium Chlride Acidsis n Glucse Tlerance in Dgs. 168: Amer. J. Amer. J. Physil (1952). 5. Guest, GM, Mackler, B, and Knwles, HC. Effects f Acidsis n Insulin Actin and n Carbhydrate and Mineral Metablism. Diabetes U (1952). 6. Walker, BG, Phear, DN, Martin, FIR, and Baird, CW. Insulin Actin by Acidsis. Lancet 2_: 7. Graubarth, H, Mackler, B, and Guest, GM. Inhibitin f (1973). Effects f Acidsis n Utilizatin f Glucse in Erythrcytes and Leukcytes. Physil. V72: 8. Rgers, TA. Amer. J (1953) Inhibitin f Glucse Uptake by Acidsis in vitr. Prc. Sc. Exp. Bil, and Med. 97: (1958). 9. Haldane, J.B.S., Wiggleswrth, U.B., Wdcw, C.E. Effects f Reactin Change n Human Carbhydrate and xygen Metablism. Prc.Ry.Sc. 96; 15 (1924). 10. Baird, CW. Subjects. Glucse Tlerance in Metablic Acidsis: Med. J. Australia. 47 : (1960). Nndiabetic

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42 11. Fenn, W. The Depsitin f Ptassium and Phsphate with Glycgen in Rat Livers J. Bi. Chem. 128: (1939). Levine, R, Lube, SD, and Weisberg, HF. Nature f the Actin f Insulin n The Level f Serum Inrganic Phsphate. Am.J. Physil. 159: (1949). DeFrnz, RA, Cke, CR, Andres, R. Falna, GR, and Davis, PJ. The Effect f Insulin n Renal Handling f Sdium, Ptassium, Calcium, and Phsphate in Man. J. Clin. Invest. 55_: (1975). 14. Hiatt, N, Yamakutwa, T, and Davidsn, MB. Necessity fr Insulin in Transfer f Excess Infused K t Intracellular Fluid. 15. Metablism Deuel, H.J., Gulick, M. Tlerance in the Dg * (1974). The Relatin Between Alkali Deficit and Glucse J. Bil. Chem. 89: (1930). McGuire, EAH, Helderman, JH, Tbin, JD, Andres, R, and Berman, M. Effects f Arterial Verus Venus Sampling An Analysis f Glucse Kinetics in Man. J. App. Physil. 41_: 17. Sherwin, RS, Kramer, KJ, Tbin, JD, Insel, PA, Liljenquist, JE, Berman, M. and Andres, R. Man. 18. J. Clin. Invest. 53: A Mdel f the Kinetics f Insulin in (1974). Strele, R. Influence f Glucse Lading and f Infected Insulin n Hepatic Glucse utput (1976). Ann. N.Y. Acad. Sci. 82: (1959). Rsselin, GR, Assan, R, Yalw, RS, and Bersn, SA. Separatin f Antibdy-bund and Unbund Peptide Hrmnes Labelled with Idine-131 By Talcum Pwder and Precipitated Silica. Nature (Lnd.) 212: (1966)

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44 20. Aguilar-Parada, E, Eisentraut, AM, and Unger, RH. Pancreatic Glucagn Secretin in Nrmal and Diabetic Subjects. Med. Sci : Amer. J (1969). Blck, WD, Markvs, ME, Steele, BF. Cmparisn Between Free Amin Acid Levels in Plasma Deprteinated With Picric Acid and With Sulfsalicylic Acid. Prc. Sc. Exp. Bil. Med. 122: (1966). 22. Spckman, DH, Stein, WH, and Mre, S. Autmatic Recrding Apparatus Fr Use in the Chrmatgraphy f Amin Acids. Anal. Chem. 30.: (1958). 23. Kedenburg, CP. A Lithium Buffer System fr Accelerated Single-Clumn Amin Acid Analysis in Physilgical Fluids. 40: 24. Anal. Bichem (1971). Smgyi, M. Determinatin f Bld Sugar. J. Bil. Chem. 60: (1945). 25. Steiner, AL, Gdman, AD, Treble, DH. n Renal Glucnegenesis in Viv. Effect f Metablic Acidsis Amer. J. Physil. 215: (1968). 26. Shelwin, R.S., Fisher, M. Dendler, R. and Felig, P. Hyperglucagnemia and Blc Glucse Regulatin in Nrmal, bese and Diabetic Subjects. NEJM 294: (li 27. Felig, P, Marliss, E, and Cahill, GF. Insulin Secretin in besity. Plasma Amin-acid Levels and N. Engl. J. Med. 281: (1969). 28. Pzefsky, T, Felig, P, Tbin, J, Seldner, JS, and Cahill, GF, J. Clin. Invest. 48: (1971).

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46 Legend t Figures Figure 1. The time curse f the mean plasma insulin cncentratin during the hyperglycemic clamp in nrmal subjects pre ( and pst ( represent the Figure 2. ) ammnium chlride. ) The height f the bars EM.*=P <0.05; **=P <0.02. The time curse f the mean (-SEM) plasma glucagn cncentratin during the hyperglycemic clamp in nrmal subjects pre and pst ammnium chlride. Figure 3. *=P< 0.005; **=P <0.05. The time curse f the mean (*SEM) plasma insulin cncentratin during the euglycemic clamp in nrmal subjects pre arid pst ammnium chlride. Figure 4. *=P< 0.05; **=P <0.02. The time curse f the mean (-SEM) plasma glucagn cncentratin during the euglycemic clamp in nrmal subjects pre and pst ammnium chlride. *=P <0.02; **=P <0,05.

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48 PLASMA INSULIN CNCENTRATIN UU/ml ) iv) ) r & l TIME (minutes) Figure I

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50 PLASMA GLUCAGN CNCENTRATIN (pg/ml) r (D cd r 140 r I Figure 2 100

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52 PLASMA INSULIN CNCENTRATIN (//,U/ml) r n ^ TIME ( m inutes ) Figure 3

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54 PLASMA GLUCAGN CNCENTRATIN (pg/ml) r 4^ > p ^ Figure 4 100

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56 SERUM HC03 (meg/l) PRE NH4CL PST NH4CL 0 CD cd c r-** D d- CD BLD ph PRENH4CI PST NH4C1,_ C LD +1 LD C LD +1,_ CD c CD r- LD 0 C C,_ LD LD c c00 r^cricr>r^^--)r^^ c^-ccrcc r*-. r- r^r^r^r^r^r^r^r^ r CD c +1 Lf) r «d* r *=3* CTYC^T C C BESITY INDEX FBS (mg/dl) r^r^.r^r>.r^r^.r^.r^ c C C c 1 cd cd C +i D»* CD Y CD CD C C CD +1 C»xj- C CD C (T> C C cd '* r_ +1 LD +1 LD 00 <3- CD C C CDCCCDCr^')Ln cm cd t ^ c c 1 cd 1 cd cd +1 cr^uc^ra)r^cd+i CD CD C I r CD cm CD CD D- GLr^rcD'^rcMcD0^ l^'tr-r-^cj) c 1 ld CD +1 CD r^. LD r-. +1 CD r* +1 AGE SEX HEIGHT(cm) WEIGHT(kg) CD cr> C D CD CD C SUBJECTS UJ CD ' CL. lu s: Q.< >- _1 iu CD >- Q_ CQ z: Q Z CL 2 1 DC 21 ' es; LU z: LU -h CD C ZD I LU Cl. Q zc = DC <X. LU Q. +1

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58 Glucse Metablism (M), Plasma Insulin Respnse, and Tissue sensitivityt Insulin During the Hyperglycemic Clamp Pre and Pst NH-C1 ^3" C X UJ C»4-> r * c -r.j E ca: h- <*\J ea sz < ZI zc cn C c < c E rn -f- LU h- J < r I I s < QC E c 03

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60 SUBJECTS M( min) (mg/kg/min) Plasma Insulin Cne. ( min) (nu/ml) Change in Insulin Sensitivity (M/I) Metablic Clearance Rate (ml/min/m^) CD + *5* CD r- *3" C r C CTi l r CD CD C CD E CD c_ s CD CD c + D2: CD QJ 4J +-> C r <D i_ C r _Q E </> 03

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62 c c 00 C DURING THE EUGLYCEMIC CLAMP *= C <c «^C c J LU l *=0 CT> C» t s- Ci

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66 ) 1 BASAL GLUCSE PRDUCTIN

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70 YALE MEDICAL LIBRARY Manuscript Theses Unpublished theses submitted fr the Master s and Dctr s degrees and depsited in the Yale Medical Library are t he used nly vith due regard t the rights f the authrs. Bibligraphical references may re nted, but passages must nt he cpied withut permissin f the authrs, and withut prper credit being given in subsequent written r published wrk. This thesis by has been used by the fllwing persns, whse signatures attest their acceptance f the abve restrictins. DATE

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