Estimates of Krebs Cycle Activity and Contributions of Gluconeogenesis to Hepatic Glucose Production in Fasting Healthy Subjects and IDDM Patients
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Diabetologia (1995) 38:831-838 Diabetologia 9Springer-Verlag 1995 Estimates of Krebs cycle activity and contributions of gluconeogenesis to hepatic glucose production in fasting healthy subjects and IDDM patients B.R. Landau 1, V. Chandramouli 1, W.C. Schumann 1, K. Ekberg 2, K. Kumaran 1, S.C. Kalhan 3, J. Wahren 2 1Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA 2Department of Clinical Physiology,Karolinska Hospital, Stockholm, Sweden 3Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio, USA Summary Normal subjects, fasted 60 h, and patients Krebs cycle flux and decarboxylation of pyruvate to with insulin-dependent diabetes mellitus (IDDM), acetyl-CoA, oxidized in the cycle, was less than one- withdrawn from insulin and fasted overnight, were 30th the fixation by pyruvate of CO2. Thus, in esti- given phenylacetate orally and intravenously infused mating the contribution of a gluconeogenic substrate with [3-14C]lactate and 13C-bicarbonate. Rates of he- to glucose production by measuring the incorpora- patic gluconeogenesis relative to Krebs cycle rates tion of label from the labelled substrate into glucose, were estimated from the 14C distribution in gluta- dilution of label at the level of oxaloacetate is rela- mate from urinary phenylacetylglutamine. Assuming tively small. Pyruvate cycling was as much as one- the 13C enrichment of breath CO 2 was that of the half the rate of conversion of pyruvate to oxaloace- CO 2 fixed by pyruvate, the enrichment to be expect- tate. Glucose and glutamate carbons were derived ed in blood glucose, if all hepatic glucose production from oxaloacetate formed by similar pathways if not had been by gluconeogenesis, was then estimated. from a common pool. In the 60-h fasted subjects, That estimate was compared with the actual enrich- over 80 % of glucose production was via gluconeo- ment in blood glucose, yielding the fraction of glu- genesis. In the diabetic subjects the percentages aver- cose production due to gluconeogenesis. Relative aged about 45 %. [Diabetologia (1995) 38: 831-838] rates were similar in the 60-h fasted healthy subjects and the diabetic patients. Conversion of oxaloace- Key words Gluconeogenesis, Krebs cycle, fasting, in- tate to phosphoenolpyruvate was two to eight times sulin-dependent diabetes mellitus, liver. It is a continuing challenge to develop a method for know the amount of label in the substrate entering reliably estimating the contribution of gluconeoge- the gluconeogenic process and the extent of dilution nesis to glucose production in humans, so that it can of label during that process, notably at the level of ox- be applied under both physiological and pathologi- aloacetate, an intermediate common to the Krebs cy- cal conditions [1, 2]. Measurements of incorporation cle and gluconeogenesis. of label into glucose on administration of a labelled Phosphoenolpyruvate (PEP) is the necessary in- gluconeogenic substrate are limited by the need to termediate formed from oxaloacetate via pyruvate in the production of glucose (Fig. 1). We therefore gave l~C-labelled bicarbonate to fasted normal sub- Received: 12 October 1994 and in revised form: 13 January jects, assuming the specific activity of expired 14CO2 1995 to be the specific activity of 14C fixed in the conver- sion of oxaloacetate to pyruvate [3]. We did that be- Corresponding author: Dr. B.R. Landau, Division of Molecu- cause of evidence that expired 14CO2 is a good mea- lar and Clinical Endocrinology,Case Western Reserve Univer- sity School of Medicine, 10900 Euclid Avenue, Cleveland, sure of mitochondrial t4CO2. We estimated the ex- Ohio 44106-4951, USA tent of dilution at the level of oxaloacetate from the Abbreviations: IDDM, Insulin-dependent diabetes mellitus; distribution of 14C from [3-14C]lactate in hepatic et- PEE phosphoenolpyruvate. ketoglutarate, estimated from the distribution of 14C 832 B. R. Landau et al.: Krebs cycle and gluconeogenesis in fasting and IDDM Glucose Lactate l Vl / stant rate for the next 6 h. Labelled bicarbonate (5 g) was in- v. V7 i, Fatty fused at a constant rate over the 6-h period. In the other two PEP Pyruvate ~, V=~ acids \ .V. cco ' v, CO,/ diabetic subjects infusion was for 8 h. Eighty ~Ci of the [3- w= f _ A~tyF-COA 14C]lactate was given, 14 ixCi as a bolus and the rest at a con- stant rate over the 8 h. Phenylacetate (4.8 g), was ingested in divided portions between the second and third hour. Five , ~[ , c,,,=~ grams of the labelled bicarbonate was infused, but the rate of Fumarate V~ infusion for the first hour was 55 % and the second hour 90 % =.~ V~ of the rate over the next 6 h. CO n - Katoglutarnte COs Blood (5 to 10 ml) was drawn at hourly intervals for deter- minations of plasma glucose and D-fl-hydroxybutyrate concen- Fig. 1 Scheme of pathways of gluconeogenesis and Krebs cycle trations. Blood for the isolation of glucose (70 to 110 ml), was metabolism in the liver. Rates are designated V 1, V 2, etc. in drawn 90 min before and at the end of the infusion. Urine, for keeping with previous usage [4] the isolation of urea and glutamate from excreted phenylace- tyl-glutamine, was collected at 90-min intervals over the last 3 h of infusion. Respiratory CO 2 was collected at hourly inter- in glutamine conjugated to phenylacetate. Our meth- vals by having the subjects breathe into balloons. Subjects od, while time consuming, provides details on the me- were encouraged to drink 180--240 ml of water hourly during the infusion. tabolic rates occurring in liver while offering a direct measure of the contribution of gluconeogenesis. We Measurements. Blood glucose, urinary urea, and glutamate have now used that approach to examine the rate of from the excreted phenylacetylglutamine were isolated and gluconeogenesis relative to Krebs cycle flux and the degraded as previously described [4]. The urea carbon was col- contribution of gluconeogenesis to glucose produc- lected as BaCO3 by using urease. Portions of the glucose and tion in insulin-dependent diabetic (IDDM) patients, glutamate were diluted with known quantities of unlabelled glucose and glutamate. Portions of these were then combusted withdrawn from insulin so as to develop mild ketosis. to CO 2 and the remainder degraded to yield each of the car- These findings have been compared with those ob- bons of the glucose and glutamate as CO 2 that was also collect- served in fasted normal subjects also in mild ketosis. ed as BaCO 3. Carbon 1 of the undiluted glutamate was isolated as BaCO 3 by treatment of a portion of the glutamate with chloramine T. Carbons 3 and 4 of the undiluted glucose were Subjects, materials and methods isolated as BaCO 3 by incubating the glucose with Lactobacil- lus delbrueckii to form lactate and decarboxylating the lac- Subjects. A healthy man and woman, ages 35 and 28 years, with tate. Respiratory CO2 was collected as barium carbonate by body mass indices 26.0 and 22.0 kg/m 2, respectively, and fasted bubbling the expired breath through carbonate-free sodium for 60 h, were studied. Five males with IDDM, ages 19 to hydroxide and then adding barium chloride. 23 years, with a mean body mass index of 22.9 kg/m 2 (range ~4C-specific activities of the barium carbonates from urea, 18.0 to 26.8) were also studied. The haemoglobin Ale concen- from the combustion of the diluted glucose and the diluted tration in four of the diabetic subjects ranged from 6.9 to and undiluted glutamate, from the individual carbons of the di- 8.0 % and in one subject, MP, it was 13.9 %. Insulin was with- luted glucose and glutamate, and from breath CO 2 were deter- drawn 24 h before the study and food was withheld for 12 h. mined by liquid scintillation spectrometry. The excess enrich- The diabetic subjects were encouraged to drink water through- ment of 1~C in the BaCOa from urea, carbon 1 of the undiluted out the period of insulin withdrawal. Approval for the study glutamate, the carboxyl group of lactate formed from the undi- was obtained from the Human Investigation Committees at luted glucose and breath CO 2 was determined by mass spectro- the Karolinska Hospital, Stockholm and University Hospitals metry [5]. Barium carbonates from glucose, urea glutamate of Cleveland. Informed consent was obtained from each and respiratory CO 2 collected at zero time were assayed for subject. the measure of ~aC natural abundance. Materials. [3-x4C]Lactate was prepared as previously described Calculations. Correction was made for the portion of the 14C [4] from D-[6J4C]glucose (purchased from Dupont-New Eng- incorporated into glutamate that was due to the fixation of land Nuclear, Boston, Mass., USA). 13C-labelled sodium bicar- 14CO2 formed from the [3JaC]lactate. The method for estimat- bonate, 99 % enriched, was purchased from Merck Isotopes ing the portion is based upon evidence that the bicarbonate Inc., St. Louis, Mo., USA. The [3-14C]lactate infused was dis- utilized in urea formation and formation of oxaloacetate from solved in 50 or 100 ml of a sterile 0.154 mold sodium chloride pyruvate derive from the same pool of CO2 in hepatic mito- solution. The 5 g of bicarbonate infused was dissolved in chondria [3]. Carbon in CO 2 fixed by pyruvate becomes car- 500 ml of a sterile 0.077 mold sodium chloride solution. The lac- bon 1 of ct-ketoglutarate and hence carbon 1 of glutamate. tate and bicarbonate solutions were shown to be pyrogen-free. 14CO2from [3J4C]lactate should then be incorporated into car- bon 1 of glutamate relative to its incorporation into urea in the Experimental protocol. The protocol was similar to that pre- same ratio as for 13C-bicarbonate.