. 11.11111 ‘4 . 3.. “ L4""""‘""" .41 ‘I‘Ifiqt' ..R'I;I~uu;qm_q~m .I41"1: 1‘..V “4 .441 " (I'm! ‘4 “RI?“ .1. :;. "”"""""1":"" 1. '1'2',4,;' “1.1.2:? 41 V 40:.4 ’ 4' WI I:'! -L V... 4.13 11.4 .44 ' 412141314312 1'1I. “3'1 I. 3‘4“"; ~14H1'I‘4" V“ I "‘.4"4H 4.14 g: E‘ ('V . , 1.4...V ' E. - '4‘, . ':w;4'141'4 I‘II‘H'V'w ' " " H .'v‘:" '1'1'4 4 " I??? ' ‘ I‘l'V'U ' V "4"} ‘ '4II'IV3314 '1,le 441333" 4., 4").‘111' 41'I4‘4IJ "3' 41'4:,';1"1-1v :'1‘:| "'th't" I‘I‘I‘W IF;2'IE"1' ',' 14: 'I'Vfi'11"'l’,1'."':~‘ '1'L'A’ “EVER. ' V.‘!.L' LL‘I 'fix’I'H" 4 . 4 1,-1.1. 5: . 44.44“ 13,1,”‘11'x’1'nv £4,431. 1 '. 1‘1'4'1'44 .. ‘I‘L’E‘Ij I" I,’ 9'1"}: 1,.1‘. ’V ‘ #4141 1'13'" “""£-’I U. 1'? 7114“} 3113‘) " E. 114:" 412' . ,. .I1"4{3~" Lu‘ ''1' '.I‘1:‘4}."1:4v('1'l ,. 41.1\ix:‘\ ":23 ' ”HUI .. .' . .. ' ‘.'~ .4'1'4'4‘414,4;4,414 .41 4.4“”? I: '3'II'H‘9‘15'3‘E'3'3'L'E'g'e'I‘ " 1"!" "3"“ “11111114144, 1|,I .1. 1:533 ‘11?” 211.11.“ ‘ '1“ ‘ :E'QIL'I"I':1.'""'I'I}I" [I IL.1'3"1.~’”.4.4}I."L;-4:{"4$:14,fix’t' ”411411 I'M 14"C‘41'111‘ 44 4 4114144 4‘4. I 1 11 I" I, . 11:45:11. ‘11}. 1.42.: .4; 64.14;. 1.1.]; “$534, {"45" 4.1%.; 41134141 2.4.. n: w.‘ ' 4 .4 L 4.444'II.::1'.'.I11.1I.41’11~. i1.1444111241'41111114.I, . f . - I; 1 4 '4- ".V' 4,1I $13.11", ‘47 ‘13, 112111. 9:41;, 4. 4 “ 44444I'1V121‘1‘1fl4nwlW1V4M4'I'I'I": ..I‘3"1'V1§‘; l I IE.“ ‘ '1‘4,‘ ‘ L’3. 4"?! .. .IU I1:1‘. :_141|1414;N ”.1 1 1 ”III [11” :hsql 1 [$21 :‘1:1' 'zsfizrkvyl ’9’4‘1' I 541.1(- 4 1.1.41.4 444.5 . "4'; 444-1. 14.414131444- : 4.1.211 “I" . 4.1 1 '4""I. 444""4’“ " VIH'V'W‘I' "" 14.21:,"4).‘411‘1.,!;‘1I111:414121‘1H 4V-4V4 "qr'gI'b'V ’4 X'If'tflhé‘g" {33:4, "n’q‘ 9‘1“ 1' I L‘4'1’Ifl‘l'“51'31'4‘h4i's4'h‘fip‘ 4 Inn C"1“~:“‘44‘1‘4,Ib.:4",}:1 'Iktfiv'v'x'i' i???" ' 1. 4 H 1 44.}.{18141‘1”11413111411151V.V.41:S1I\‘-'44:4:4,u.11.:§.“€ 1.1M}?_\{1,l1k‘1’¢1;~1‘1,,4:1‘1§::k1-.§ng‘ .. .'. .‘3' 44'“ “Iv-.54 51321: “3‘ 1, . . "L, 2 1 .I 3. 11:34 .. .444 ..- _ 4 HI I V".;'.'4‘I"I'I V', SWIM 3‘51 3 " 1:41:4er :2 » 4 V 1.1.4.41414111114144441 . “V4314." 44"»ny 1.143.115?» ‘“ W. ‘4‘ '.“44'4"','1 14 ;" II: I'I'I'k‘,1 :4'I’V4I 'h' "‘ "$151?! 'IS"'"";' "'3' ‘ .I‘Q ‘? "" l| :1 I 1.4. 11111.1 .113" I111 1131111” 4% 11:; 1:? ”2:! . 410:: '13:?“ . :‘I: M!‘ '2‘?" 1| I'I‘gzé'fi‘ HEM}, R‘a‘x. 332?: .V L ’3 44 4 4 1'41H‘111‘14J444. “5"?“ 4 4'4‘11'13 In I 5.. .. 4 . Vn. :4 . . 4,41. 4"4" .11.. ‘1" 41:1 .4' V3 1444’ '454I‘VV-i1'zu-IVis'4IV-t."IV ”6444?": “W :- IN 414'.“ '4’4’44'H' 'I'I“"‘4’4'V'LV"'""I‘I’I’I‘N'I 1211121511111E,11:11:11.1,1121‘L'11'EIEJHI... ”#5:: "KI-"53.: 4 1’4" 1.4;. 4.3.: "... .44 11.1.4;.1411111111414”.4441 4"“1114... .1... "1;" 1V 1 4411 . V 4.541143%“ 44.44;. 41:34:41 'I'VI V. 44'44'! ." 44H“ ,"14I1VIV'. \1-34 "4‘ ' " ' '11:}: V14.4I‘I.4‘I:4'|‘.’4’4’4” "4L'4'"""’I""‘!1'I"VI"'IF‘EI' \ Mk V" 13.21V’33‘ “$31.31; 1144 VI“.'.V .4‘4'441"..44I44Vu4' 5.334444 V-Q‘.V4!’I"4‘ 'IV41Q414141114. '431 4‘4. V. 31113.} 3.1" 241' 4'4" «1'14“: 11.14VV444‘4 ‘4'41.'.! 41"" 144'4'4‘I44‘4 414.1111"4’11EI4'444:I "‘44I‘I' 'IS "'4’1’4, "‘ 31%. 41211111114141444411111141141'11‘1114WH11'4I 44411,.“ [1 1.1111141Ih1'g 41111111111} ”11"!“ “In 13,103.11“! 1 41 41:11: ‘. 'I I..I1‘"'I4 .‘ $444 "' V" LN” 4H. 41”,” .. 111 ':':;1 IV “ """' "V" """"'V"' I‘I' !.'4V"I‘<" ‘Ei? """"'""' "' I'm V4}'.'III‘"'.‘I'I‘¢I'I§11“I'4‘11‘V'IV.V4'E4'4'. 44.444441] 44 444"”! 464111.114111'11‘1 ’uV'IV‘V1'1I‘1'lVI 4: {54411,1 I414. 41' Hg. 1 1411"” 4II'H.III1I1V1'1:1"44II11 1” I {‘4 71" fl“! I11I. 9:114, R‘QIT'PEV? 1.14,].44'3'11 1 4 '1’141'41I1Imu'4 41'144’4'1V14141'4I'I II11$IIVI11eV1H§IVI§EIJI I'fi “113:? I.I,I 12A ’111'4511‘5'1,‘ 1 4. I 75!. r '44 V1 ‘4 ""' I“ II ""V 4"‘I1':"VV4VIV'1'I"" "4" . 14.1414'I111{:3111.r."4""4' IV "I' 5:2? __ ,A, -H~.~:-_:-: v'xgxzz ’52: 1,41 “MEI. 'l .. , . ~4.’ 1 4’4“ 444 44 4 “fig 14') I'"§"I:’ 4'4 . ERIE. ”‘11-: ""{II "'"Ii 11";'!1j.1."..!‘:4"“"' 1.5119‘5' 114' 4' 414.1% .141‘1‘:VV114“:'I1"'1"‘:1:1“ “VIE 44:11112‘4‘1."""'1§'!I1"I1'§"4"4$.';E 4% £33 WI‘I‘ 1,:{352Liféfi'fi ,."";' ’VV" "”1"! 4VI41V4‘44 V‘4'I4' ".‘-' '4VI’4‘I‘I "I'V' " V""'4I'I" 4"“' 1’1.V-4411‘.4"""IV4‘4 :' 'E' ' ~ {I' ' 4.V.1'4‘44V.V..'4v4.'l.11-,1'1 """ 4'3“ «V'hI V' I:I.I"4:"I:4 I'I'I‘II" II”! I431 '1i "I'I'E’I'L ":4 «a: 4.V"..:.4:‘:V4.3..4‘ V. 4141 ”44.4444 4"‘"'4I""' "'4 4.4444444 JV" $44.1 V" "”‘4 ”.4124... . "1' V"??? " " _ 1'14'r4'll',‘! I? 41l11'IIIV' 444'1’1 11:I"11I.:""1I V111 11114111111 11'I'I'I11I IS'IE'HN'II'? 4'31“ [4119‘ """"§ """'" "h‘ 43%; 4’3“ H‘nfiékflk'figzéa 1‘" "‘4: . . 1.1.1.44141111 111.14.411.11 $441111 V1111": "'"I'I 41I4 {INA i‘I 11E 14,414} '1111 4:4.41'4'} 4111 14?": 2.111331%” 14.41% 4112.4..1.;41;41 .,: "I‘I'EI' .14, 4.11,}, 1 1 .1 .. 41114114114 44:4:411411.4.11}111441411{41 V4 41E"144 41.14141411EII 111114111414 14141411, I 1.14 4:141’If"4144121'12'i441‘1‘41-14154132” .11.4§.’E1: Egg- 3 V3: " 1‘ «4D ...'1,;5..1..1.,. . V'""1 "1"" I'L'V" 4"" “"V"I. I 44'" ”114'. "L4 44442544?" .4. . 14. '13 41141441,. “4.4.II'423'4‘4414E3I‘4111‘4119H.321 7.1111,». 33125.1, (1'43. 62': 111.11-1111114‘1‘4311‘14 V2I13.1'I'I L141 14 ['1 I.“ 1:41” (”1.10"I14'1’138133III‘1FIL1314I11‘ II “21%;! E ”511%“4' 11'1“???" #211113”? 'I 1:)“ I‘iqé'ka'éi‘fi‘t'i‘g“ 'i “43:4“; #1 t "V " V' 1" “"‘V"I‘ V'VV‘V 1.11:1. "'"' " 44 4'4" 4'44 V4; 4"" ‘.'.'.'4,."'VEIV""1.1.1,.IV'EVVVIVV'V‘VEViIE'I'E’EI ‘ 4 1.1.114...“ .1 VI ' ',.....V.""" |"‘4 ‘4‘ 4‘1‘4 '1'” IIIEI 'IE1 4 4 ‘4") 444.4 la". 41 5";‘4‘4414 NI“ 4". ‘4“. 11‘... 'g ' 111' 11:411I'1'14'1.,114\I1‘ 1I1I“1'4?“sz11111111I1'III4'I1I'1'I'E9III3’1121V”? 'I'E'I'" 111111116111331 Q‘E’IIEEP""'III'"I"':-"I'I: [14' 4.9%41‘). $4151“: - r"! . v 1.1 1, ”‘3‘ I "Ch? 4 . I. I4; 11111111111114.» V1}('V1I'-\4‘11"’4‘i144”11 'VV‘E E 'I'l' 411'1'1?41 I44 ’4‘: 1'I‘I 151461214 """I""}'I1’""'% .‘ Rafi" ' fi'fik‘fifl". ' .31. .4111131:4111.111§111§?E'111111: 151111 1 “1‘14‘111144IE1'114V4E31E14‘11;11:14:12111‘14441114144 11111111.: 1 4114141 11 111111 114%}? a]: #411111111 11“ ‘41:"? E 1w.44144w44444.mw.14 '.§I:VE1 1-mE4» m4"E'4§1 4. 44‘ 4 ’ 444114.111.11.4141.444.44.41.“‘4H1431;44.143V4114.1,1.1V4‘43'11 44'V4EVIE'I1. '4 4' VV'VEE‘EVCVI'V "'5' " '7’ "E354 "2'45 ‘4' ‘-V 111... '11, '. ‘ 1 ‘.,,,,.’4‘ ' I .I‘ u 5'51“ I"? .‘ «114;.1411I'1111141111414 111E11111111E11 11111 ”1.11.44 "4’I " HI 1% 14441411'I'4""V"4':I:"I.:1I'44.'111’II£411141':1§111151V11§"E111 ' kg??? _ if}? iggfiEkfifififi '1 (44'1“. 1313.. 1141‘. 4' '44 414"V ["4414 411? 44'1:1E1111"414:;'14111111I§'1"1I?1I4E1E1 11' 1; ‘4. ,1 44,414,341“ 2‘4 4 3'13 ‘ 11 I 5'4 ""'1'"41’ 4.4.EH44II1441EII114114111,"1I1‘441 '11.; 41'11‘31‘5:§'§§1y4;";lfi 4": 14:4" " 4“" 141114.344“ 4V 'VVI"4VV44VII'V'4'IVV134E‘IVV'V'V‘V‘V: I I' I? 4 VVV'I V4410 4"I'V EIL'V4E41'4V44 "5' 5'4"} ' " ""-“’""‘"V""‘é‘“ '4"". ""ufiVV'V'V' " ""‘ "’I 'I'I’4'I‘ "II4I'1"1"4'44 "14'I-VI'1H V"""4 'I"4"' 4. 'b" "’1"V'\44"I"“9‘I"I"‘I '4' V‘." , "n ' .'. ""' "'2' ' 1.11.. 111411.111 4121 1411114! ‘11. 1'11"" 4 4141!1 III1 111'1II11I“ ‘41th MI 14111; “1‘ IfiIa‘EE1E1-Elf%§ 4. 4- $5113: ‘iV4" . ' 41" I 4 ‘1 . ' ' '144'4114141. 41444" E44} 4E4" "' VVVV'V V4: .4" V‘VV4IV '44VV'V" " ~ ' E "4I:1I 144114111 11 1 1414'!" 4. 1 1 11 411%}? 'IE'E E41411": 1141:"! 1 1 44 1. 1414 _..'.."'I'{3,441 .1141 21 :41 .1 4.411141414411114. 4.4 ‘4'! ., “V4434. 111.4111 41411134 11%,4114'1'51‘4 114,4 .4 .1 "'1 411144114, 1! ‘11 111.4 . . 4 31444. 414‘ 114.5 .1,;4;11$=;.1..1.11 1'3 4'14' 4 I'VVIV V " ’4414414"V '1434’ 'E'"‘" ' ‘ 'iV 1"" " ""' 'II" " "' "’4?- 'm' "a 11:11:. 11.411 '444. 1,111.11 1 11111111511111 141'1114 4, 11:4??? 14114411'11'1141IH-1 1 1‘ 11 1 . '1 "V"! " "2 "2‘4' '4’V‘V‘44 " .V‘V 1' 4 E" “EVEN 4'. V' ‘4‘ . 1 .4 . 4 "' :V 11' ' 111:1 'I‘I'. ‘ 'V'IIII'EI1"JVV4,11414{4.V1JV'1'I'I111 11"II'12I . V ' 4'414I' .1Ii' 4.21;”??4, I 11 ..V4 V“4""" V"';'V 4...,11 4 '4 I! ‘ ' V" ~ .444» V'VV'V 44 «44.4 . 4.1311111..I.II':41I4111 11 III'. 'I" 1‘ 41 1.1' {111 "".VVI"'V"'V.VV' VV'-'V'VVV"'"'4 4'4'4'4IIVIII'IVI' "' "' "" ' RI"? 4i" " ' . I .,. .. 4 . . ' .1. 4"“4' 5"V'4'VV4 ‘I4V4 4‘ ' “1"? '4'? " . 4 1. . . . 1 1 . 4 4V4.«:V4441411‘V114444E 444:. = V . ‘VV. 1. ' EI"'1I ‘ I ' {I .4... 44444 14 4 I ‘ I 'II' V - '41 '4o"'I.'“’ 'I 4 {,4 111. I 312-233" mat... ’3‘ 44! .m‘ -c- 3%“;qu “Ag-h . .1. ‘3‘ J m :;r £2“ -~....='1.- . ,~. “A‘ctx. —:_.- r-':-'r 3—5:? zz‘ " A 41" 344%?! 1 4 “W H" ’I “2'19: ,. ‘2’ Y «— z.- 1'55 . .15?“ .3: “ 1:5,. .4.- at“ 4: VV .4‘I'I4'4 EH“ 2, x. LIBRARY Michigan State Uni'VCfSitY This is to certify that the thesis entitled In Vivo and In Vitro Studies of Glucose Metabolism in the Chicken presented by Linda Jean Brady has been accepted towards fulfillment of the requirements for PhD . Department degree in of Food Science and Human Nutrition Major professor Date 7' (2/-73 0-7 639 IN VIVO AND IN VITRO STUDIES OF GLUCOSE METABOLISM IN THE CHICKEN BY Linda Jean Brady A DISSERTATION Submitted to Michigan State University in partial fulfillments of the requirements for the degree of Doctor of Philosophy Department of Food Science and Human Nutrition bf) ABSTRACT IN VIVO AND IN VITRO STUDIES OF GLUCOSE METABOLISM IN THE CHICKEN BY Linda Jean Brady Glucose replacement rate, percent recycling, mean transit time, and glucose mass were examined using various double- labelled glucose tracers. Estimates of replacement rate were greatest for 2-3H glucose (2T), with 3T, 4T, ST and 6T glucose all having similar values. Calculated glucose mass based on all tritiated tracers agreed closely with the direct deter- mination of body glucose (734-1086 mg/kg body weight vs. 969 mg/kg). Reincorporation of tritium from 3H 0 into glucose did 2 not occur to any significant degree. The young chick was found to have a rapid rate of,glucose turnover and high percent re— cycling compared to mammals. When chickens were fasted for l, 4, or 8 days, significant decreases occurred in total body protein and fat with fasting, the greatest energy loss from fat. Glucose production remained remained constant with fasting at 10-13 mg/min/kg body weight. Blood lactate and glycerol were unchanged with fasting, while pyruvate increased and plateaued. Plasma alanine, serine and glycine levels were high compared to values in fasted mammals. Linda Jean Brady Blood B-hydroxybutyrate increased dramatically with fasting (350-3500 nmol/ml), while acetoacetate remained constant.The hepatic lactate:pyruvate ratio was unchanged with fasting, while the B-hydroxybutyrate:acetoacetate ratio increased. These ratios may influence phosphoenolpyruvate and glucose production in mammals. Hepatic and renal phosphoenolpyruvate carboxykinase levels remained constant, while hepatic lactate dehydrogenase increased with fasting. B—hydroxybutyrate dehy- drogenase levels were very low at all times. The results indicated that little glucose sparing adaptation per kg occurs in the chicken with fasting. Glucose production was studied in the isolated hepatocyte. The highest rate of glucose production was obtained from lac— tate, followed by dihydroxyacetone, glyceraldehyde and fructose. Alanine was converted to glucose at only 4% the rate of lac- tate, in contrast to the rat and guinea pig, where it is con- verted at 40-50% the rate of lactate. Pyruvate was utilized to form glucose at 20—30% the rate of lactate, again in con— tast to rat and guinea pig, where lactate and pyruvate are utilized equally well. Addition of lOmM sorbitol, xylitol, or ethanol increased glucose production from pyruvate 25—40%, while glycerol addition increased it only 9%. Addition of B- hydroxybutyrate had no effect on glucose production from lac- tate or pyruvate. Addition of octanoate had no effect on glu- cose production from pyruvate, but depressed it from lactate at 5mM. Differences in the formation of glucose from various subatrates suggest some basic differences in the mode of Linda Jean Brady glucose production between the chick, and the rat and guinea pig. Lactate and alanine turnover and percent glucose arising from lactate and alanine were calculated in vivo. Lactate turnover was 12.1 umole/min/kg, while alanine turnover was 6.9 umole/min/kg. The percent of glucose derived from lac- tate was 47%, while that of alanine was 8.5%. The results also indicated a significant conversion of 140 alanine to luc lactate. The estimate of alanine conversion to glucose in vivo is higher than in Vitro and may indicate that organs other than the liver contribute to alanine conversion to glucose. Lactate conversion to glucose was higher than in other species. ACKNOWLEDGEMENTS I wish to thank the National Institutes of Health for supporting me throughout my program and allowing me funds for traveling to meetings and laboratory supplies. I also wish to thank the members of my committee: Dr. Gilbert Leveille, Dr. Maurice Bennink, Dr. Duane Ullrey, and Dr. Werner Bergen. My deepest thanks are to Dr. Dale Romsos, my advisor, who accepted my failures without losing faith, and my success- es without claiming credit. To Paul and Gillian, just THANKS. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . Tracer Methodology and Glucose Turnover . . . . Lactate and Alanine Turnover . . . . . . . . . . The Use of Isolated Hepatocytes . . . . . . . . The Role of Redox State and Phosphoenol Pyruvate kinase Distribution . . . . . . . . . Carboxy— Page .12 GLUCOSE TURNOVER IN THE YOUNG CHICK USING VARIOUSLY LABELLED (3H,U-1"C) GLUCOSE TRACERS . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . .24 .25 .28 .32 THE EFFECTS OF FASTING ON BODY COMPOSITION, GLUCOSE TURNOVER, ....2 METABOLITES, AND ENZYMES IN THE CHICKEN . . . . . Introduction . . . . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . 42 43 46 .57 GLUCONEOGENESIS IN ISOLATED CHICKEN Introduction . . . . . . . . . . Materials and Methods . . . . . . Results and Discussion . . . . . LACTATE AND ALANINE TURNOVER IN THE Introduction . . . . . . . . . . Materials and Methods 0 o v c v 0 Results . . . . . . . . . . . . . Discussion . . . . . . . . . . GENERAL CONCLUSIONS . . . . . . . LITERATURE CITED . . . . . . . . . HEPATOCYTES O :I: H O N L11 2 Page . .67 . ~87 Table 10 ll 12 13 14 15 16 LIST OF TABLES Page Glucose replacement rates in chicks, mg/min/kg . . .29 Glucose recycling in chicks . . . . . . . . . . . .30 Mean transit times of variously labelled glucoses in chicks, minutes . . . . . . . . . . . . . . . . .33 Glucose mass in chicks, mg/kg . . . . . . . . . . .34 3H incorporation into plasma glucose from 3H20 . . .36 Body weight and composition of chickens fasted for O, l, 4, or 8 days (experiment 1) . . . . . . . . .47 Glucose metabolism in fasted chickens (experiment 2) . . . . . . . . . . . . . . . . . . . . . . . . .49 Circulating levels of metabolites in fed and fasted chickens (experiment 3) . . . . . . . . . . . . . .51 Amino acid and uric acid levels in the fasted chicken (experiment 3) . . . . . . . . . . . . . . . . . . .53 Liver and kidney metabolites in fasted chickens . .55 Enzyme activities in fasted chickens . . . . . . . .56 Conversion of various substrates (lOmM) to glucose .70 Pyruvate conversion to glucose in the presence of glcerol, xylitol, sorbitol, or ethanol . . . . . . .74 Glucose, lactate, and pyruvate accumulation from various substrates (lOmM) . . . . . . . . . . . . .75 Effect of B—hydroxybutyrate on glucose production from lactate and pyruvate . . . . . . . . . . . . .78 Effect of octanoic acid on glucose production in isolated chick hepatocytes . . . . . . . . . . . . .79 LIST OF FIGURES Figure Page 1 3H/“C ratios in plasma glucose of chicks . . . . 31 2 Conversion of lactate, pyruvate, and fructose 69 to glucose at various substrate concentrations . vi REVIEW OF LITERATURE Introduction The use of experimental animals to detail intermed- iary metabolism and its regulation is a generally accept— ed research technique to gain specific information which might be applicable to human metabolism. The laboratory rat is usually the animal of choice, for obvious reasons. How— ever, it has become clear that the rat is not the "ideal" human model. The only truly ideal human model is the human himself. But with increasing restrictions on human exper— imentation, another approach may be valid; that is, to use a variety of species for experimentation, choosing a partic- ular species for study based on how closely it resembles the human in that particular situation. The chick may represent such a model when control and maintenance of blood glucose levels in humans is considered. Even though several features of avian carbohydrate metabolism appear applicable to various pathological human disorders. very little study of avian intermediary carbohydrate metabo- lism has occurred. Possessing blood glucose levels of 150— 300 mg/100 ml, the chick is an excellent model for hyperglyc- emia. In addition, glucagon, not insulin is the primary blood glucose regulatory hormone, in contrast to mammals. but similar to diabetes where insulin exerts little effect on blood glucose levels. The chick also appears able to resist severe fasting hypoglycemia, a capability important in humans and sometimes lacking. Thus, the chick may serve as a model for abnormal human glucose metabolism, especially abnormalities in glucose production. The importance of an adequate ability to regulate gluc- ose metabolism begins at birth in humans, when the infant's blood glucose supply from the mother is severed. From this point, the neonate's blood glucose level depends on a bal- ance between his tissues' utilization, the availability of his liver glycogen, and his ability to make glucose from lac- tate, glycerol, and amino acids. The enzyme phosphoenolpyr- uvate carboxykinase (PEPCK), glucagon, and catecholamines are usually elevated at this time and the infant's blood glucose levels are maintained. If enzyme, substrates. or hormones' effects are not existent at this time, neonatal hypoglycemia occurs. with concomitant problems in nervous function. Thus, adequate maintenance of blood glucose is of utmost importance in proper development of the human neonate. In contrast is the diabetic human with severe hypergly- cemia. In these people, a 100 g glucose load may result in 50-75 g of glucose escaping into the peripheral circulation, while in normal subjects only 30-40 g of glucose escape hepatic uptake (Felig,l975). Therefore, postprandially, diabetics are found to have higher peripheral blood glucose levels. As the diabetes progresses, hyperglycemia also characterizes the fasting state. In this condition, the liver loses its sensitivity to rising blood glucose levels and continues to produce glucose. In normal subjects, ris- ing blood glucose levels would signal the liver to decrease glucose output. Splanchnic uptake of glucose precursors 3 is also elevated in the fasted diabetic. This ensures that sufficient substrate is available to the liver for continued glucose production. The consistently high blood glucose levels in the chick lead to speculation on the control of blood glucose in this species and perhaps to a partial answer to glucose overprod- uction in the human diabetic. Thus, it was of interest to examine avian glucose metabolism in some detail. The re- search is divided into four areas: tracer methodology to meas- ure glucose production, tracer methodology for measuring ala- nine and lactate conversion to glucose, the use of isolated hepatocytes , and the role of redox state. Tracer Methodology for Glucose Turnover Basic to the study of carbon flux in animals in vivo is the determination of glucose production (utilization) and turnover rate. Katz et al (1974 a,b) defined some important concepts in establishing methods of evaluation of glucose carbon flux. The labelled glucose tracer may be given by a single pulse or a continuous intravenous infusion, the math- ematical treatment being slightly different in each case. The plasma sampling pool is assumed to equilibrate with the tracer immediately and may be treated as one of a series of pools or by non-compartmental analysis (Katz et al.1974 a,b). The organism is also assumed to be in a metabolic steady state (postabsorptive). In this case, the rate of disappear- ance of tracer glucose from the plasma (rate of phosphor— ylation throughout the body) is equal to the rate of 4 production (release by liver or kidney)(Katz et al, 1974a; 1974b). Many studies have used 140 glucose to estimate the above parameters. However, this label usually leads to an under- estimate of glucose utilization because of the recycling of .labelled carbon from tricarbon units or 002 back into églucose (Katz et al, 1974a; 1974b; Brockman et al, 1975; EShipley and Gibbon, 1975). Labelled 14C-lactate from extra- liepatic tissues and any lac-lactate derived from hepatic Inetabolism may be recycled to 14C-glucose in the liver and Iceleased into the blood. Thus, the rate of disappearance of lblood 14C-glucose would be less than if no recycling occur- need. The search for an ideal label, which would not recycle loack into glucose, resulted in the proposal of 3H-glucose ‘tracers (Katz and Dunn, 1967; Dunn et al, 1967). The 3H is J.ost irreversibly to water during extrahepatic glucose cata- Ioolism (Katz et al, 1974a; 1974b). Originally, all 3H glucose tracers were considered Eagppropriate except l-3H glucose (Katz et al, 1974a; 1974b). FVIore recently, questions have arisen of the meaning of data cinerived with several of the 3H tracers. The 2—3H glucose is JExresumed to lose 3H at the isomerization of glucose-6- I?Qnosphate and fructose-6—phosphate (Katz and Rognstad, 1976). lilqproximately 50% of 3H in position 2 of glucose-6-phos- IPfuate and position 1 of fructose-6—phosphate is lost in each 5 isomerization (Katz and Rognstad,l976). The hexose isomerase has been reported to possess high enough activity to shuttle the compounds back and forth several times before further metabolism (Katz and Rognstad,l976). Immediately obvious is a problem with 2-3H—glucose tracers. If glucose-6—phos- phate can be rapidly converted to fructose-é-phosphate and vice versa, glucose-6—phosphate could then return to glucose, without retaining the 3H label, since gluconeogenic flow will never be completely nil (Clark et al,l974). Thus, through "futile cycling" glucose will lose 3H without metabolism to tricarbon units. The label of 6—3H-glucose is presumed lost from the meth- yl group of pyruvate in carboxylation to oxaloacetate. If oxaloacetate, malate, and fumarate equilibrate. the methyl label may be further diluted (Katz and Rognstad,l976). Since the 3H loss occurs late in glucose metabolism. the 6—3H—gluc- ose has been proposed as truly indicative of glucose utiliza— tion (production). In most species, the rate of loss of 6-3H is much lower than 2-3H, and slightly lower than 3-3H, 4-3H, or 5—3H. The former three labels are lost at the triose stage of metabolism (Katz and Rognstad,l976). Futile cycling is also possible with 5—3H—glucose, due to an excess of al— dolase and triose phosphate isomerase resulting in rapid 3H loss from fructose—1,6—diphosphate (Clark et al,l974). However, this has been disputed by Rognstad et al (1975). They found significant retention of 3H during fructose conver— sion to glucose via fructokinase (fructose is metabolized 6 by conversion to tricarbon units after phosphorylation, then converted to glucose, C02, or lactate). Thus, even though all 3H tracers should provide valid estimates of glucose production, 6-3H-glucose has been found most satisfactory (Dunn et al,l976; Katz et al,l976; Anwer et al,l976; Belo et al,l976). Both 2—3H and 5-3H glucoses have been used for estimation of futile cycling at their res— pective stages of glycolysis. Bloxham et a1 (1973) and Clark et a1 (1973) have shown increased futile cycling in the flight muscle of bumblebees exposed to cold. They have postulated that futile cycles are a method of fine regulation of metab- olism. The futile cycling in bumblebee muscle which produces heat has been found necessary to allow the muscle to warm enough to prepare for flight. Thus, all tracers have potential use. The studies rep- orted here make use of several of the methods mentioned above. Lactate and Alanine Turnover The rate of glucose appearance indicates the total gluc- ose production from all substrates, but does not indicate the quantitative importance of the various glucose precur— sors. It is possible to measure not only glucose turnover, but also the turnover of key substrates related to glucose metabolism such as lactate and alanine. It is also possible to measure the interconversion of glucose with either lactate or alanine by several methods. Although it is feasible to use a single pulse injection to measure lactate or alanine turnover, Forbath et al (1967) 7 compared this technique to that of constant infusion for the estimate of lactate turnover. The constant infusion method estimated lower rates of lactate production than obtained with the single injection technique. The authors attrib- uted the discrepancy to a high estimate of the lactate pool and apparent distribution space. The value is derived from the intercept of the terminal portion of the specific act- ivity versus time curve. They postulated that the problem inherent in the method was the metabolism of tracer before equilibration between a rapidly mixing pool (blood) and peripheral compartments. In a multicompartmental system, the overestimate of pool size by this technique occurs if the substrate turns over at a fast rate. The primed infus- ion estimates rates in the dynamic steady state without ref— erence to the amount of tracer intermixing. However, it should be noted that the single injection can be used sucess— fully. Belo et al (1977) found the rates of lactate, ala- nine, and serine turnover in dogs on various diets with the single injection method. The rates for lactate turnover were similar to those obtained by Forbath et a1 (1967) with the constant infusion technique. The rates of interconversion of metabolites have been measured by several methods. Depocas and DeFreitas (1970) proposed a model for glucose—lactate or glucose-glycerol interconversion. Their model involves injecting tracer amounts of m1 (glucose) and m2 (lactate) into two different animals. According to their theory, the rate of total net formation 8 of each compound from all sources can be measured, as well as the rate that m1 is formed from m2 and the rate at which ml is formed from all other precursors. The same calcula- tions can be made for m2. Kreisberg et al (1970) used essentially the same technique, but injected each tracer into lean and obese subjects on successive days, rather than pairing subjects. The method of sampling in metabolite turnover studies has also been shown to be of importance (Chiasson et al, 1977; Reilly and Chandrasena,1977). This includes differ— ences between whole blood and plasma and also arterial vs. venous sampling. Chiasson et a1 (1977) discussed methodol— ogical approaches for in vivo tracer work. It was noted that specific activity of alanine in the plasma of fasting sub— jects differed greatly from that in whole blood following constant infusion. They estimated that 90% of the alanine extracted from the Splanchnic bed was from the plasma com— partment. This is an important distinction because it is most correct to use the actual specific activity to which the liver is exposed in the calculation of precursor conversion to glucose. However, this is difficult as the liver's blood supply is complicated, as dicussed below. Bergman and Heitman (1978) also found that plasma and red blood cell amino acid content did not rapidly equilibrate in sheep. Reilly and Chandrasena (1977) assessed the errors in lactate turnover in sheep which resulted from sampling 9 jugular vein rather than carotid artery blood. The main problem in the sampling of the venous blood appears to be a dilution of tracer by lactate (or alanine) released from the perfused tissues into the venous system. This decreases the specific activity of the metabolite and leads to increased estimates of turnover. Reilly and Chandrasena (1977) found a mean difference in lactate concentration between the art— erial and venous system to be 30%. Thus, it appears to be quite necessary to measure the A—V difference across the tis- sues in consideration before applying a continuous infusion technique for measurement of metabolite turnover. In the liver, the situation is much complicated by the fact that the portal vein supplies as much as 70% of its perfusion and the arterial system only about 30%. Thus, the most correct measure of specific activity presented to the liver would be a mixture of portal and arterial blood specific activity (Chiasson et al, 1977). In the case of alanine, this differ— ence is important since the gut releases alanine to the por— tal vein; arterial blood would present quite a different specific activity. Ruderman et a1 (1976) have stated that the major gluco— neogenic precursors in man are amino acids, lactate and gly- cerol. 0f the amino acids, alanine has received major at— tention as it accounts for approximately 50% of amino acid release from muscle and an equivalent percent of amino acids extracted by the Splanchnic bed. Felig (1975) has calcul- ated that the alanine contribution to the glucose pool in 10 postabsorptive man is approximately 50% of that observed for lactate. The effects of fasting and diet on lactate and alanine metabolismhave been studied in rats, dogs and man. In 24 hr fasted man, Kreisberg et al (1970) found a 30% reduction in glucose pool size, turnover, and oxidation; lactate turn- over was unchanged, despite decreased conversion of glucose to lactate. The conversion of lactate to glucose increased 33% and represented 36% of lactate turnover. The fraction of glucose from lactate increased 2-fold. They postulated that the combination of decreased glucose conversion to lactate and increased lactate to glucose with unchanged blood lactate concentration and turnover means that lactate was increasing- ly synthesized from substrates other than glucose. Freminet et a1 (l975a,b; 1976a,b) studied the effects of feeding and fasting on various parameters of lactate and glucose interrelationships in rats. Lactate turnover decreased progressively with fasting from 24-72 hr, then returned to fed contrdlvalues with refeeding as did the % lactate turn- over oxidized. When they used the Depocas-DeFreitas model in fed rats, they found that 17% of glucose turnover comes from lactate, while 28% of lactate turnover goes to glucose. They also found that the Cori cycle represented only 20-30% of glucose recycling, measured as the difference between 3H and 14C glucose turnover in rats. They also studied the effect of fasting on the Cori cycle. Fractionally, lactate to glucose almost doubled, glucose to lactate remained the 11 same, and lactate and glucose oxidized decreased. The Cori cycle increased from 10-20% in fed to 25—30% in fasted animals. Several studies have been performed with dogs. Belo et a1 (1977) studied the effects of different diets on lactate and alanine turnover. While alanine turnover and pool size increased with the high protein diet, lactate pool was not influenced by diet or fasting. Dogs fed the high carbohydrate diet had low lactate to glucose in the fed state, which increased after fasting. The high protein diet decreased lactate to glucose but increased alanine to glucose. Forbath and Hetenyi (1969) showed that 14-20% of lactate comes from glucose in normal fasted dogs, while 11-18% comes from glucose in diabetic dogs. Alanine has been shown to be an important glucose pre- cursor in vivo in the dog (Belo et al,l977), man (Felig and Wahren,l974;Felig,l975; Cahill,l976), and lamb (Prior and Christensen,1977). In vivo turnover studies have not been done in the chicken; however, Veiga et a1 (1975,1977) have studied U-luc-alanine incorporation into glucose after a single injection to both chickens and black vultures. The vulture, a carnivorous bird, showed higher rates of incorporation (umoles/g liver) than the chicken. Fasted vultures had decreased incorporation compared to fed vul- tures, while fasted chickens had higher rates than fed chickens. This type of response is somewhat similar to that of dogs found by Belo et a1 (1977), where plasma 12 alanine was higher in animals on the high protein diet, as was alanine turnover. Bergman and Heitman (1978) recognized that there are two approaChes for studying gluconeogenesis from amino acids: 1) using 14C tracers as a minimum estimate for whole . . 14 body converSion Since some C can cross over to the TCA cycle from oxaloacetate 2) arterial—venous differences, a maximum estimate, since the liver uses amino acids for pro- tein synthesis also. The present work utilizes the first approach. The Use of Isolated Hepatocytes The use of isolated liver cells for the study of met- abolic processes is advantageous for several reasons: 1) the response of the parenchymal cells can be separated from that of structural cells 2) the substrate and hormone additions can be made independently to test the effect of single addit- ions, or added in physiological proportions. Howard et a1 (1967), Howard and Pesch (1968), and Berry and Friend (1969) pioneered work with isolated rat hepatocytes. and since that time many modifications in technique have been made. Wagle and Ingebretsen (1975) reported detailed isolation and metabolic studies of rat hep- atocytes. They studied net glucose production from various substrates in cells from fed and fasted rats. The rates of glucose production from highest to lowest were: fruc- tose, lactate, pyruvate, glycerol, galactose, alanine, and succinate. Compared to the perfused liver, they assessed 13 the isolated cells to produce twice as much glucose from lactate, and slightly more from alanine, pyruvate, and galactose. These authors and Hems et a1 (1966) suggested that a stringent test of metabolic integrity of cells is the production of glucose from lactate, since this process involves integration of control between the mitochondria and cytosol and also the interchange of metabolites between them. Wagle and Ingebretsen (1975) also calculated that their isolated hepatocytes produced glucose at 50-90% the in vivo rate with 10 mM lactate or fructose. Jeejeebhoy and Phillips (1976) have reviewed the entire technique of hepatocyte isolation and suggested the criteria for assessing normal hepatocyte function after isolation. They cited morphological integrity, functional membrane barrier, and normal protein synthesis and glucose production as essential for "normal" liver cells. The specific cri- teria included: spherical shape of cells, representation of all liver lobes, exclusion of trypan blue dye, minimal leak— age of cell enzymes, and normal RNA and ATP levels. They also compared various buffers, ion concentrations, oxygen— ation, and incubation methods for short and long term cul- tures. Isolated cells have been used successfullly to study gluconeogenesis by various investigators. Arinze and Row- ley (1975) studied gluconeogenesis from guinea pig hepat- ocytes and the effect of redox state on glucose production in this species. The values obtained were similar to those 14 obtained for rats. Allan and Sneyd (1975) have used isol- ated rat hepatocytes to study the effects of glucagon on various gluconeogenic steps, while Veneziale et al (1976 a,b) have used this system to study the effects of hormones on gluconeogenic and glycolytic enzymes. Story et a1 (1976) have used isolated rat cells to determine glucose and ketone production from substrates with added oleate or octanoate in the fed and fasted state. Clark et a1 (1976) have studied basic glucose production rates in lamb hepatocytes and the .51. effects of glucagon and butyrate on that process, while Siess et a1 (1977) have used hepatocytes to determine the effects of glucagon on compartmentation and gluconeogenesis from lactate in the rat. Zaleski and Bryla (1977) have used cells to determine the effects of oleate, palmitate, and octanoate in rabbits. The use of the chicken isolated hepatocyte has not been as extensive as that of the rat, but Capuzzi et a1 (1971) successfully isolated adult chicken hepatocytes by initial perfusion and subsequent incubation with collagenase and found them metabolically comparable to rat cells. Good- ridge (1973) isolated embryonic chick hepatocytes by a simpler procedure and found them to be a useful model in the study of avian fatty acid synthesis. However, Dickson and Langslow (1975), Anderson and Langslow (1975), and Dickson and Langslow (1977) first reported studies of glu- cose production in isolated chicken hepatocytes. They found that damaged cells accounted for 25% of cells in fed and 34% 15 in fasted chickens. In 24 hr fasted chickens, endogenous glucose production was low, as was ATP concentration. How- ever, the cells' ability to synthesize glucose and sensitiv- ity to hormones suggested that they were physiologically com— petent. The percent of competent cells decreased with time based on trypan blue dye exclusion, and correlated with in- creases of cellular enzymes in the media. They also found that lactate was the best glucose precursor in chick hepato- cytes, followed by pyruvate, alanine, and glycerol. Cells from starved birds responded to glucagon by increasing the conversion of lactate to glucose. Dickson and Langslow (1977) further studied glucose production in the chick. Sev- eral additional substrates were characterized-~aspartate, dihydroxyacetone, glyceraldehyde, malate, and succinate. They also supplied cytosolic reducing equivalents in the case of pyruvate by adding sorbitol, xylitol, ethanol, or glycerol. They noted increased glucose production over that from pyruvate alone. The work presented in this thesis examines some additional substrates and the effect of supply— ing mitochondrial reducing equivalents, as well as the above mentioned parameters. The Role of Redox State and Phosphoenolpyruvate Carbokainasg Distribution Gumaa et al (1971) have provided an excellent review of the theory and methods for redox state calculations through metabolite measurement. The basis for the evaluation of compartmental metabolite concentrations is the knowledge 16 that total cell concentrations of metabolites may be mis— leading. The concentration of effectors of enzymes should be known in close proximity to the enzyme, or at the very least, in the same compartment. The most applied method for metabolite measurement is the "freeze clamp" pro- cedure (Hess,l974), which fixes cell constituents in a state close to the "in vivo" state, but does not allow the separation of organelle constituents. Metabolite concen- trations in freeze clamped whole cells may be used to derive compartmental redox state estimates via the calculation of the nicotinamide nucleotide redox states from related de- hydrogenases and their reactants (Gumaa et al,l97l). Nicotinamide adenine dinucleotide (NAD) is mostly ox— idized in the cell, while nicotinamide adenine dinucleotide phosphate (NADP) is largely reduced (Gumaa et al,l97l). Since the inner mitochondrial membrane is impermeable to nicotinamide nucleotides, a spatial compartmentation of these metabolites with distinct redox states was proposed (Gumaa et al,l97l)- These same investigators have also presented the common criteria for calculation of nicotinamide nucleotide redox states through the "redox metabolite indicator method"; 1) the NAD pool and the metabolite pair should be related by a dehydrogenase located in a particular cellular compartment 2) the dehydrogenase activity should be high in relation to hydrogen flux such that reactants are at or near equilibrium 3) the reactants should be either uniformly distributed in the cell or confined to the compartment of the enzyme. 17 In establishing the cytosolic NAD+/NADH ratio, sev- eral dehydrogenases and their metabolite pairs have been proposed to meet these criteria--1actate dehydrogenase (EC 1.1.1.27), glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), and malate dehydrogenase (EC 1.1.1.37). The redox ratios calculated from these agree in certain metabolic states, but discrepancies have been found (Willms et al,l970). Since glycerol—3-phosphate dehydrogenase is not always sufficient— ly active and malate dehydrogenase is both cytosolic and mitochondrial, the lactate/pyruvate rationediated by lactate dehydrogenase is usually chosen for the estimation of cyto- solic redox state. The NAD+/NADH ratio can be calculated from the lactate/pyruvate ratio by the equation: NADVNADH = Pyr/Lact x l/KLDH assuming that lactate and pyruvate are evenly distributed in the cell. The mitochondrial redox ratio may be calculated by the B-hydroxybutyrate/acetoacetate ratio or the glutamate/ a-ketoglutarate, NH3 ratio. Most commonly the B-hydroxy- butyrate/acetoacetate ratio is used, although this calcula- tion may be incorrect in certain circumstances (Sylvia and Miller,l973). Redox state and compartmentation of metabolites are es— pecially important in studies of gluconeogenesis, as the pro- cess is both mitochondrial and cytosolic. Gluconeogenesis from many three carbon intermediates begins with conversion to pyruvate, mitochondrial entry, and carboxylation to 18 oxaloacetate via pyruvate carboxylase (EC 6.4.1.1). In all species studied, this enzyme is mitochondrial. Alternative- ly, some substrates may be converted directly to oxaloacetate in the mitochondria. The disposition of oxaloacetate de— pends, in a species specific way, on the distribution of phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) and metabolite flux in the cell. Oxaloacetate itself is highly impermeable to the inner mitochondrial membrane. The roles of PEPCK distribution, oxaloacetate dispos- ition, transmitochondrial flux, and redox state are of par— amount importance in discussing various species responses in gluconeogenesis. PEPCK is an enzyme which converts oxalo— acetate (0AA) to phosphoenolpyruvate (PEP), a high energy intermediate in glucose production. It is a key regulatory enzyme in the gluconeogenic pathway (Garber and Hanson, 1971 a,b; Hanson,l974). PEPCK is almost completely cytosolic in the rat,mouse, and hamster; almost entirely mitochondrial in the chicken, pigeon, and rabbit; and equally divided in the guinea pig, cat, and human (Hanson,l974). As the perm- eability of the inner mitochondrial membrane is metabolite specific (Meijer and VanDam,1975), this distribution has important metabolic consequences. Metabolite concentrations affecting enzymes may thus themselves be regulated by specific permeability and com- partmental redox states. Both cytosolic and mitochondrial redox state and PEPCK distribution are important in regul— ating gluconeogenesis and in creating species differences 19 in that regulation (Garber and Hanson,l97la,b; Arinze et al,l973; Willms et al,l970). Once mitochondrial 0AA is formed, its fate varies by PEPCK distribution and the redox state of the mitochondria. In the rat, PEPCK is mainly cytosolic. Intramitochondrial 0AA is unable to traverse the inner membrane as such, thus is removed from a key enzyme in its metabolism. The prob- lem is solved by intramitochondrial conversion of 0AA to aspartate or malate. Both transfer carbon into the cytosol in the rat (Parilla and Parilla,1976). In addition, malate also carries reducing equivalents necessary for the reduction of 1,3 diphosphoglycerate in the reversal of glycolysis. Which metabolite is the transfer form of 0AA depends on the gluconeogenic substrate. When lactate is the substrate, its conversion to pyruvate produces NADH sufficient for glycolytic reversal (Hanson,l974). Hence, aspartate trans- fers only carbons to the cytosol. In the case of pyruvate, no NADH is produced in cytosolic metabolism, so malate trans— fers both carbon and reducing equivalents into the cytosol. How does the redox state of the mitochondria affect the process? The addition of fatty acids or B-hydroxybutyrate to liver preparations has been used to promote intramito— chondrial NADH production and thus alter redox state (Will- iamson et al,l969; Arinze et al,l973). Soling et a1 (1970) perfused livers of 48 hr fasted rats with 20 mM lactate and 2 mM oleate and found significantly increased glucose prod- uction in the presence of oleate. The rat livers produced 20 significant amounts of ketones concurrently. With lactate as a glucose precursor, sufficient cytosolic NADH should be available. Therefore, it is likely that fatty acid stimulation in rat liver occurs between pyruvate and PEP. A more reduced redox state in the rat liver is therefore favorable to increased glucose production. Octanoate (0.2 mM) also increases glucose production from lactate, pyruvate, and alanine in rat liver (Arinze et al,l973). Again, mito- chondrial redox state was more reduced. The mechanism of increased glucose production in rats under these conditions has been ascribed to acetyl CoA dep— endent activation of pyruvate carboxylase, increased mito- chondrial reducing equivalents, or both (Arinze et al,l973). but redox shifts appear to be the best explanation. Garber and Hanson (1971a,b) showed that 0AA disposition is controlled by mitochondrial redox state. When it is reduced, less PEP and more aspartate and malate are formed. As these metab— olites are important in the rat in the transfer across the mitochondria, glucose production increases. Fatty acid ox— idation in this species increases total 0AA and malate (Sol- ing,1974). Medium chain triglycerides increase the lactate/ pyruvate ratio and the B—hydroxybutyrate/acetoacetate ratio in vivo in the rat also (Bach et al,l976). Arinze et a1 (1973) also perfused B-hydroxybutyrate to eliminate the effect of acetyl CoA resulting from fatty acid oxidation. B-hydroxy- butyrate is metabolized to acetoacetate, producing NADH. Rat liver showed the same response as with fatty acids, 21 once again suggesting the importance of mitochondrial redox state. In the guinea pig, cat, and human, PEPCK is about equally divided between the mitochondria and cytosol, and alterations in redox state influence glucose production differently than in the rat. In these species, based on data for the guinea pig, PEP may be formed in the cytosol, as in the rat, or in the mitochondria. In the mitochondria, a more reduced redox state would favor increased aspartate and malate formation and decreased PEP production (Garber and Hanson,l97la,b). Oleate (2mM) stimulated glucose prod- uction from lactate in rat liver, but inhibited it in guinea pig liver. Differences in fatty acid oxidation rate did not account for the differences in glucose production. Another study showed that after in vivo fat feeding to guinea pigs, the mitochondrial NAD+/NADH ratio was more reduced (Willms et al,l970). In the perfused guinea pig liver, prolonged exposure to fatty acids also increased the B-hydroxybutyrate/acetoacetate ratio. These data suggested that a more reduced intramitochondrial NAD+/NADH ratio might control gluconeogenesis from some substrates. Avail- ability of 0AA in the mitochondria is important in species with intramitochondrial PEPCK. Measurements of guinea pig liver showed increased oxidation with fasting (increased fatty oxidation). This shift increased 0AA 8 fold in the mitochondria. It also increased PEP formation from malate, while it decreased malate formation 3 fold (Garber and 22 Hanson,l97la,b). PEP was the only metabolite to increase when the redox state became more oxidized. In rabbit liver, a greater proportion of PEPCK is intramitochondrial (Garber and Hanson,l97la,b). Fasted rabbits also had a more oxidized redox state than fed animals. When isolated mitochondria were subjected to varying B—hydroxybutyrate/acetoacetate ratios, PEP synthesis from d-ketoglutarate increased with oxidized ratios, while malate formation decreased. When asketoglutarate and aspartate were substrates, oxidized ratios also increased PEP synthesis and decreased that of malate and citrate. Soling et a1 (1970) compared the distribution of PEPCK and the effects on gluconeogenesis in the rat, guinea pig, and pigeon. The pigeon differs from the others as it has almost totally intramitochondrial PEPCK (Soling and Kleineke, 1976). Gluconeogenesis was not increased in perfused pigeon liver with lactate and 2mM oleate over that of lactate alone. Glucose production from pyruvate was low, and was not in— creased with the addition of hexanoate, oleate, or xylitol. However, ethanol addition increased glucose production slight— ly. I Blocking aspartate aminotransferase (EC 2.6.1.1) with amino-oxyacetic acid will inhibit the conversion of aspartate to 0AA. In the rat and guinea pig liver, the addition of this inhibitor decreased lactate conversion to glucose, while it had no effect in pigeon liver (Soling et al,l97l). These data suggested that gluconeogenesis from lactate in pigeon 23 liver does not depend on aspartate transfer, consistant with the location of PEPCK in this species. Gevers (1967) suggested PEP as the mode of carbon transport out of the mitochondria in the chicken. Several other studies have corroborated that the pigeon and chicken have similar PEPCK distribution (Felicioli et al,l967; Peng et al,l973; Jo et al,l974). Thus, in studying chicken liver, one would expect to find gluconeogenic responses similar to pigeon, rabbit, and perhaps guinea pig, based on PEPCK distribution. GLUCOSE TURNOVER IN THE YOUNG CHICK USING VARIOUSLY LABELLED (3H, U-HC) GLUCOSE TRACERS Introduction The regulation of glucose production in the chick appears to vary from that of most mammals in several important aspects. The compartmentation of phosphoenolpyruvate carboxykinase is reported to be almost entirely mitochondrial, in contrast to most mammalian species where it is evenly distributed or al— most entirely cytosolic (Hanson,l974). Gluconeogenesis in avian liver is not stimulated by long or medium chain fatty acids, whereas in the rat, glucose production iSstimulated by these metabolites (Soling and Kleineke,l976). The lactate/ pyruvate ratio of avian liver is reported to become more ox— idized in situations Of increased gluconeogenesis, in contrast to the rat, where the lactate/pyruvate ratio becomes more re- duced (Leveille et al,l975). This ratio is often taken to be a measure of the cytosolic NADH/NAD ratio (Gumaa et al,l97l). Glucose turnover, percent glucose carbon recycling, and body glucose mass have also been found to be higher in the mature chicken than in most mammals (Belo et al,l976b). The purpose of the present study was to begin to char- acterize the rate and control of glucose production in the young chick. Although several studies have been conducted comparing various double labelled glucose tracers for est— imation of glucose turnover in other species, baseline data for the young chick have not been established. Estimates of "futile cycling" at the glucose" glucose-6-phosphate and fructose-6—phosphate ‘ fructose—1,6-diphosphate stages can also be obtained with certain of the double labelled tracers. It was also of interest to compare the results obtained for 24 25 various labelled tracers in estimation of glucose metabolism when a relatively large number of animals under similar circumstances were used. Materials and Methods Tracers. Glucose tracers were purchased from New England Nuc- lear (Boston, Mass.) or Amersham-Searle (Arlington Heights, 111.). The U-lnC was diluted in one batdiand used for all experiments. Each tritiated glucose was separated in suff- icient quantity for all experiments, then mixed with the U— 14C preparation. Aliquots of each tritiated tracer mixed . with U-luC were divided and frozen until used. The purity ‘ of glucose was determined by converting glucose to glucose— 6-phosphate, retaining it on a Dowex acetate column, and eluting it with 4 N formic acid (Katz et al,l975). Animals. Young male chicks (Fairview Farms, Remington,Ind) weighing 700-900 g were housed in pens with raised wire floors and fed a commercial high-carbohydrate diet (Mas- ter Mix Starter and Grower, Central Soya, Ft. Wayne, Ind). Water was available at all times. Chicks were fasted 24 hr prior to the tracer comparisons, direct body glucose deter— minations, and 3H20 incorporation into body glucose. Experiment 1. Indwelling catheters (Becton-Dickinson, Ruth- erford, N.J.) were fixed in the jugular vein of the fasted chicks and secured with cloth tape. Approximately 1 hr later, chicks were injected through the catheter with 5 uCi Of U-lnC glucose simultaneously with 40 pCi of either 2—tritiated (2T), 3T, 4T, 5T, or 6T glucose in 2 m1 of 26 saline. Blood samples (1 ml) were obtained through the catheter at appropriate time intervals up to 5 hr post injection. Initial samples were taken at 2 minute int- ervals. The volume of blood withdrawn was replaced by saline and a total of approximately 15-20 ml was drawn from each chicken. Blood was collected in chilled tubes containing heparin and sodium fluoride. Plasma was separ— ated and deproteinized with Ba(OH)2 and ZnSOh. Glucose was separated from its metabolites by passing the deproteinized plasma through a l x 5 cm column contain- ing equal mixtures of Dowex 1X8, Cl" and Dowex 50X8, H+. The column eluate was lyophilized, dissolved in .5 m1 of H20, and scintillation cocktail (3a70, Research Products Int- ernational, Elk Grove Village, Ill.) added. Liquid scint- illation counting was done on a Packard Tri Carb scintillation spectrophotometer. Spillover of 14C into the 3H channel was calculated. Efficiency of counting was obtained with an external radium standard. Plasma glucose concentration was also determined (Glu- costat, Worthington Biochemical, Freehold,NJ), and specific activity of 14C and 3H glucose in the plasma was calculated. Experiment 2. Either 4O pCi or 400 uCl of tritiated water in 5 ml saline was injected into a wing vein of 10 fasted chickens to determine the amount of cycling from the body water pool to glucose. Five hours after injection, a 10 ml blood sample was obtained via heart puncture. The plasma was extracted and deproteinized with 75-80% (v/v) ethanol and 27 boiled for a few minutes. The sample was centrifuged and the lipid removed with 5 volumes of chloroform. The upper layer containing the glucose was passed over the columns as in experiment 1. The samples were dried and redissolved in water three times to assure complete evaporation of trit- iated water. The samples were then counted by the liquid scintillation procedure for tritium, using the channels rat- io method to determine efficiency. Experiment 3. The total glucose mass of 10 chicks was est- imated. Chickens were fasted as in experiment 1, then killed by cervical dislocation and immediately submerged in liquid nitrogen ( 10 seconds elapsed between dislocation and im- mersion). The frozen carcasses were shattered and passed through a meat grinder until homogenous. Samples of 100 g were used for glucose extraction. Three volumes of hot wat- er were added and the mixture heated to 90-9500, then centrif- uged at 27000 x g for 30 minutes. The extraction of the pel- let was repeated three times. the total volume Of all ex— tracts combined, and glucose determined as in experiment 1, with dilution modifications. Calculations. The results were calculated by the graphical method (Katz et al, l974a,b). The rate Of glucose utilization, percent recycling, transit time and glucose mass were calcul— ated. Statistical Analysis. The results were analyzed by analysis of variance for a completely randomized design. Tukey's omega procedure was performed where significant treatment 28 differences were found (Steel and Torrie,l960). Results Plasma glucose values (not shown) were constant during sampling for any one chick, but ranged from 185-300 mg/100 ml among chicks fasted one day, which is much higher than values reported for most mammalian species (Be11,l971a). Table 1 contains the values for all parameters of gluc- ose metabolism calculated. The glucose replacement rate (rate of utilization) based on 2T-glucose was significantly higher than that from either 5T- or 6T—glucose. A 25% differ- ence existed between the 2T-glucose replacement rate and the 6T-g1ucose replacement rate, suggesting the presence of a "futile cycle” between glucose and g1ucose-6-phosphate. The 5T—glucose replacement rate was not significantly higher than the 6T-glucose replacement rate, suggesting no apparent "futile cycle" in the chicken between fructose-6-phosphate and fructose-1,6-diphosphate. No significant differences were found between 3T- and 6T—g1ucose, or between 3T- and 4T—glucose replacement rates. No significant difference was found among U-lQC-glucose replacement rates. Figure 1 presents the 3H/14C ratios of the tracers for the first 2 hr of the exper— iments and indicates the Similarity of 3T-, 4T-, 5T-, and 6T- glucose tracers in these experiments. The percent of glucose carbon recycling (Table 2) was Significantly higher for 2T-glucose than for the other tracers. No significant differences were found in estimates of tri— carbon recycling obtained with 3T-, 4T-, 5T-, or 6T-glucose. 29 Table 1. Glucose replacement rates in chicks, mg/min/kg*. Glucose tracer 3H (T) U-14C Significant; differences: l4 2T, U- C (9) 21.4 i 9.1 i .4 2T vs 5T, 6T (P<0.05) 3T, U-14C (5) 15.8 i 8.4 i 4T, U-14C (10) 15.6 i 1.0 9.2 i . 5T, U-14C (5) 17.0 i 1.6 10.4 i . 6T, U- C (10) 16.0 H- 0.7 5.6 H- *Values are mean i SEM for 5—10 chicks (700-900g) per group as indicated ( ). No significant differences were found for U-14C values. Comp— arisons made for 3H values were: 2T vs 5T, 2T vs 6T, 3T vs 6T and 3T vs 4T. .1. 30 Table 2. Glucose recycling in chicks*. Glucose tracer % :i%$:::::2:+ 2T, U-14C (9) 57 i 2 2T vs 5T, 6T 3T, U-14C (5) 47 1 2 (P