Hormonal and metabolic effects of neuroglucopenia

Brain Research, 614 (1993) 99-108 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

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BRES 18904

Hormonal and metabolic effects of neuroglucopenia P a t r i c i a E. M o l i n a a K a m a l E l t a y e b a,1 H i s h a m H o u r a n i a,2 Koji O k a m u r a a,3, Lillian B. N a n n e y b,4, Phillip W i l l i a m s b,5 a n d N a j i N. A b u m r a d a b Department of Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (USA) and a Department of Surgery, Health Science Center, T-19, SUNE, Stony Brook, NYl1794-8191 (USA) (Accepted 12 January 1993)

We examined the role of central neuroglucopenia, induced by intracerebroventricular (i.c.v.) administration of 2-deoxyglucose (2-DG), on glucose and amino acid kinetics in conscious dogs. Group 1 received i.c.v. 2-DG at 2.5 m g ' k g - l - m i n -~ for 15 min. Group 2 received an equal intravenous (i.v.) amount of 2-DG. In the i.c.v, group, plasma glucose levels rose from 1065:4 m g / d l to a peak of 204_+ 12 mg/dl by 90 min. Blood lactate increased from 689_+ 1 to 2,812_+ 5 #mol/1 and blood alanine did not change from basal (256_+41 /xmol/l). The rate of hepatic glucose production, determined isotopically, was increased 2-fold over basal ( P < 0.01). Significant increases ( P < 0.001) over basal were also noted in plasma epinephrine, norepinephrine, insulin, glucagon and cortisol. Leucine rate of appearance (Ra) showed a 30% decrease from basal to 2.4 _+0.05/~mol-kg-1. m i n - I ( p < 0.01). In group 2 plasma glucose levels were not altered but plasma cortisol and glucagon showed a modest transient increase above basal ( P < 0.05). No significant changes were noted in amino acid kinetics. These findings suggest that periventricular neuroglucopenia, in the absence of peripheral glucose deprivation, is accompanied by hyperglycemia secondary to enhanced hepatic glucose production with decreased glucose utilization and by increased hepatic uptake of gluconeogenic precursors. These, however, were not accompanied by increased whole body proteolysis as was previously seen with generalized glucopenia resulting from insulin-induced hypoglycemia.

Key words: 2-Deoxyglucose; Intracerebroventricular; Neuroglucopenia; Glucose metabolism; Protein metabolism; Insulin; Glucagon; Catecholamine; Dog INTRODUCTION

It is generally accepted that insulin is a most important regulator of protein, carbohydrate and lipid metabolism 1'5. Several recent studies have demonstrated that physiologic elevations of plasma insulin levels, in the presence of euglycemia, suppress the rate of total body proteolysis and of amino acid oxidation 5'~°, decrease hepatic glycogen breakdown and gluconeogenesis 7 and decrease lipolysis and hepatic ketogenesis. Its effect on total body protein synthesis has been controversial, with some studies showing no change 26 or increase 2°, while others showing decreased rates~°; the latter response has been generally attributed to the

hypoaminoacidemia that usually accompanies insulin infusions. Despite elevated insulin levels, we and others have shown that hypoglycemia results in enhanced glycogenolysis, gluconeogenesis, lipolysis and ketogenesis it'12. We also showed that insulin-induced hypoglycemia is associated with enhanced proteolysis and amino acid oxidation and that most of this response was secondary to increased proteolysis of the nonhepatic splanchnic tissues, which comprise mostly the stomach, small and large intestines, mesenteric fat and the spleen tS. The exact mechanism for most of these changes has not been adequately assessed. There have been several attempts at examining the mechanism of

Correspondence: P. Molina, Department of Surgery, Health Science Center, T-19, Rm. 020, SUNY, Stony Brook, NY 11794-8191, USA. Fax: (1) (516) 444-7635. i Present address: c / o Office of the Academic Secretary, University of Gezira, P.O. Box 20, Medani, Sudan. 2 Present address: Baptist Medical Center, Medical Center Bid., Winstom, Salem, NC 27157, USA. 3 Present address: Otsuka Pharmaceutical Factory, Muya-CHO Naruto Tokushima, Japan. 4 Present address: Plastic Surgery Research Lab., Department of Plastic and Reconstructive Surgery, Vanderbilt University, MCN S-221, Nashville, TN 37232, USA. 5 Present address: Department of Surgical Research, Vanderbilt University, MCN T-2104, Nashville, TN 37232, USA.

100 the changes in glucose homeostasis. Several recent studies have shown that this effect is triggered by (a) CNS glucopenia z1'27'29 and (b) by changes in plasma catecholamines, glucagon and cortisol TM. The localization of neuroglucopenia necessary for triggering the counterregulatory response to insulin-induced hypoglycemia has been the subject of much controversy 28,8. It is generally believed that the localization of glucoreceptors in the CNS predominates in the ventromedial hypothalamus, i.e., periventricular nuclei ~3. This has been supported by studies in which the i.c.v, administration of 2-DG mimics many of the metabolic events in glucose homeostasis normally seen with insulin-induced hypoglycemia 19'22. Nevertheless, it remains unclear whether the same events will trigger the enhanced proteolysis seen with insulin-induced hypoglycemia and whether the effect on increased hepatic glucose production is mediated by increased glycogenolysis alone or in combination with enhanced gluconeogenesis. MATERIALS AND METHODS

Animal preparation Experiments were carried out on 14 18-h fasted mongrel dogs (18-25 kg) of either sex which had been fed dog chow for 4 weeks before their use. At least 3 weeks before the day of the experiment, silastic catheters were implanted under general anesthesia in the femoral artery, portal vein and hepatic vein as previously described ~8. After 1 week and under general anesthesia, the dogs were fitted with a cannula in the third ventricle. The head was attached to a stainless steel stereotaxic dog frame (David A. Kopf Instrument, Tujunga, CA). After prepping and draping, a midline incision was made in the scalp from the nasion to a point halfway between the bregma and the inion of the occiput. Muscles underlying the scalp were separated from the calvarias and retracted laterally. By use of a hand-operated drill press connected to the stereotaxic frame a hole was drilled through the skull at the coordinates that were chosen on the basis of the location of the third ventricle in the stereotaxic atlas of the dog brain 9. The third ventricle was cannulated via this hole in the skull. A stainless steel cannula of length 25-31 m m containing a stainless steel stylet was then inserted through the drill hole into the third ventricle. The cannula itself was constructed by soldering a 40m m piece of 22-gauge stainless steel tubing to one end of a hollowed-out stainless steel hub. The end of the hub connected to the tubing was threaded so that it could be secured to the skull by screwing it into the drill hole. The other end was threaded on the inside to

allow it to be attached to the drill press on the stereotaxic frame. Intra-operative access to the third ventricle was assumed when brisk outflow of CSF from the cannula was noted after the stylet had been removed. Proper positioning of the cannula in the third ventricle was subsequently confirmed at autopsy after injection of dye into the cannula and ventricular system postmortem. After insertion into the ventricle the cannula was further secured to the skull by a platform constructed of dental acrylic in which stainless steel screws attached to the skull were embedded. The stylet was re-inserted into the cannula, a sterile rubber septum was placed over the stylet and the open end of the hub of the cannula was closed with a stainless steel obturator threaded to fit the inner threads of the hub. Muscle layers and skin were then closed with interrupted vertical mattress sutures. By design the hub of the cannula was left exposed extending ~ 0.5 cm past the skin to allow ready access. Dogs were given an intramuscular injection of 1 × 106 units of penicillin pre-operatively and were given 1 g of Ampicillin orally for 3 days post-operatively. Animals were used in a study only if they had a white blood cell count less than 17,000 c e l l s / m m 3, hematocrit greater than 35%, consumed greater than two thirds of their daily rations and had normal stools 3 - 5 days prior to the study. On the day of the study the catheters were removed from a subcutaneous pocket through skin incisions made under local anesthesia. Each catheter was aspirated to remove its contents and then filled with heparinized saline until the experiment began. Dogs were then placed in a Pavlov harness and allowed to rest about one hour prior to the beginning of the experiment. Angiocatheters (No. 18 gauge, Abbot Laboratories, North Chicago, IL) were inserted percutaneously into the right and left cephalic and saphenous veins for the infusion of radioactive tracers and indocyanine green. The obturator, rubber septum and stylet were removed from the third ventricle cannula and a fresh rubber septum and obturator were then inserted in the hub. Two ml of CSF to be used as a vehicle for i.c.v. infusions were drawn over a 30-min period. The CSF was centrifuged (5 min at 2,000 r.p.m, in a refrigerated bench-top centrifuge) and kept on ice until the infusion began. 2-DG (37.5 m g / k g ) was then dissolved in CSF and this was infused into the third ventricle at a rate of 0.1638 m l / m i n using a Harvard infusion pump.

Experimental design All dogs received a priming dose of NaH14CO3 (4950 n C i / k g ) after which a primed constant infusion of [3-3H]glucose (3398 n C i - k g - l . m i n -1) and L-[114C]leucine (4250 n C i - k g - 1 , m i n - l ) w e r e given intra-

101 venously. A constant infusion of indocyanine green (0.10 m g / m 2. min) for the estimation of hepatic blood flow was also initiated at this time. All infusions were continued throughout the whole study period. Each experiment consisted of a 120-min tracer equilibration period followed by a 40-min basal sampling period. Infusion of 2-DG was then initiated at t = 0 min and continued for 15 min. The experimental period was extended to 180 min after initiating the 2-DG infusion. Two groups of dogs were studied: Group 1 (n = 7) received 2-DG (2.5 mg. kg -1- rain -l) through the third ventricle cannula. Group 2 (n = 7) received 2-DG (2.5 mg. kg- l. min- l) through a peripheral vein. An additional group of dogs (n = 3) received an infusion of CSF into the third ventricle at an identical rate used for the i.c.v, administration of 2-DG. This group served as a control to determine if the metabolic response observed after i.c.v. 2-DG could be related to the infusion (rate and volume) of CSF into the III ventricle. There were no changes detected in any of the parameters measured, including glucose and amino acid kinetics or circulating hormone levels. For the sake of brevity the data are not shown.

MA). Immunoreactive insulin was measured by Sephadex-bound antibody (Pharmacia, Piscataway, N J) procedure 33. Plasma used for the measurement of leucine and its ketoanalogue, a-ketoisocaproic acid (KIC), was deproteinated (1 : 1, vol/vol) with 6% sulfosalicylic acid. The specific activities of leucine and KIC were determined using a modified version 4 of the method of Nissen et al. 26.

Methods for sampling and processing blood and plasma

14CO2.

Blood was collected into EDTA-treated tubes. A 3 ml aliquot was directly transferred to a vacutainer tube containing ethyleneglycol-bis-(/3-aminoethyl ether)N , N , N ',N '-tetra acetic acid and reduced glutathione, after centrifugation plasma was analyzed for epinephrine and norepinephrine with the Upjohn Cat-a-Kit (Kalamazoo, MI). 1 ml of blood was deproteinated with 4% perchloric acid (PCA; 1:3 vol/vol). The supernatant was collected and stored at -20°C for later enzymatic assay of blood lactate, alanine and glycerol with a method developed by Lloyd et al. for the Technicon Autoanalyzer 23. The remaining blood was centrifuged (15 min at 3,500 r.p.m.) and plasma samples were analyzed for glucose by the glucose oxidase method on a Beckmann Glucose Analyzer II. A 1-ml aliquot for determination of plasma glucose-specific activity was deproteinated (1:10, vol/vol) with 4.5% barium hydroxide and 4.5% zinc sulfate• Recovery of glucose radioactivity was monitored as previously described 6. Remaining plasma was stored at -70°C and used later for determinations of insulin and cortisol. Aprotinin (Trasylol; 500 KIU/ml) was added to the samples used for assay of glucagon. Plasma glucagon was measured by established radioimmunoassay using Unger's 30K antibody obtained from the University of Texas, Southwestern Medical School 2. Plasma cortisol was measured using Clinical Assays Gammacoat radioimmunoassay kit (Travenol-Genentech, Cambridge,

Collection of expired air was carried out over a 2-min period every 15-30 min. The volume of expired gas was quantified by Wright Respirometer (Wright Instruments, UK) and percentage CO 2 in the expired gas was measured with a CO 2 analyzer (Cavitron Anarad Gas Analyzer, Santa Barbara, CA). The expired gas was then bubbled through a CO 2 trapping solution in scintillation vials containing 2 ml absolute ethanol, phenolphthalein indicator (0.25 mg) and hyamine hydroxide (25/~1) until the indicator changed color from purple to clear. The trapping vials were prepared in batches and titrated with 1 N HC1 to measure micro-equivalents of CO 2 needed to neutralize base in this solution. The trapped solution was counted to determine the specific activity of breath

Analytical procedures Net hepatic balances were calculated as: [0.28C a + 0.72Cpv - Chv] × Q. Hepatic fractional extraction: [0.28C a + 0.72Cp - Chv]/[0.28C a + 0.72Cw)]. Hepatic load of substrates: (0.28Q + 0.72 C w) x Q. For all three: C a, C w and Chv, are the substrate concentrations in the femoral artery, portal vein and hepatic vein, and Q is estimated hepatic blood flow (in ml. kg- 1. min- 1) as determined by the use of indocyanine green. The values of 0.28 and 0.72 represent the contribution of the hepatic artery and portal vein to the total hepatic blood flow. Increases in hepatic plasma flow during physiological perturbations were attributed to proportional changes in the flow of the portal vein and hepatic artery. Glucose rate of appearance (Glucose Ra; rag" kgmin-l), glucose utilization (Glucose Rd; mg-kg -x" min -1) and glucose clearance (C1; m l . k g -1 .min -1) were calculated according to the method of Wall et al. as simplified by DeBodo et al 7'32. The rate of leucine appearance (Leucine Ra) into plasma and its rate of disappearance (Leucine Rd) were estimated using both non-steady state and steady-state equations modeled after those used for glucose kinetics and as previously adapted and applied for the measurement of leucine kinetics ~'3°. Leucine-specific activity (SALeu) was utilized for the calculation of leucine entry into the plasma •

102 compartment. These calculations were compared to those obtained with the use of SAv,lC in 3 - 4 animals in each group. For reasons of economy and because no significant differences were noted between values obtained with the two different methods, we limited our analysis to the use of SAL~u. The rate of leucine oxidation was estimated using the plasma SALe u. It is unlikely that any significant differences in percent CO 2 fixation between the i.v. and i.c.v, groups occurred, given that there were no changes in leucine oxidation and CO 2 production. Although this is a possibility, no difference was allowed between the i.c.v, and the i.v. group and a fixed value of 80% for CO 2 recovery was used in the calculation of leucine oxidation. The rate of non-oxidative leucine Rd, an index of protein synthesis, was measured by subtracting the rate of leucine oxidation from that of the tracer determined Rd. Histological analysis. At the end of the experimental period, dogs were administered 5 /~Ci of [14C]2DG diluted in 400 /zl of CSF directly through the third ventricle cannula over a 5-rain period. Brains were removed 15 min later, immersed in formalin fixative, sectioned at 7/~m and dipped in Kodak NTB2 autoradiographic emulsion. After an exposure period of 28 days, sections were developed in D-19 stained with hematoxylin and eosin and photographed on an Olympus Vanox Alt light microscope. Statistical methods. Differences between the basal and experimental periods within the same group were determined by the paired t-test and repeated measures A N O V A . Differences between groups were determined using repeated measure A N O V A and unpaired t-test. All values in the text, figures and tables are expressed as mean_+ S.E.M. (standard error of the mean). Statistical significance was set at P < 0.05 ( * P < 0.05 compared to basal values; +P < 0.05 compared to time matched i.v. 2DG). RESULTS

Glucose kinetics (Fig. 1) Intravenous infusion of 2-DG did not alter glucose Ra or glucose Rd and as a result plasma glucose levels did not change from basal levels of 100 -I- 4 m g / d l . In contrast, i.c.v, administration of 2 - D G resulted in an immediate but transient rise ( + 84%) in glucose Ra (to 5.6 _+ 0.7 m g . kg-~ • min-~), with the rates returning to basal by 120 min. Glucose Rd showed a delayed (60 min) modest increase ( + 25% above basal), which was sustained throughout the duration of the experiment. This differential in the early changes in the rates of glucose appearance and the more delayed increase in the rates of glucose disappearance resulted in a signifi-

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Time (rain) Fig. 1. Plasma glucose (mg/dl), rate of appearance (Ra; m g . k g i. m i n - l ) and rate of disappearance (Rd; m g . k g I . m i n - i ) as a function of time after initiating 2-DG administration. Filled circles represent values of animals administered 2-DG into the third ventricle. Empty triangles represent values for animals administered 2-DG intravenously. Values are m e a n s + S.E.M.. * P < 0.05, denotes significant difference from basal (time = 0). + P < 0.05 denotes significant difference from time matched control.

cant hyperglycemia which peaked at 204 _+ 12 m g / d l by 90 min; this was followed by a later decline in plasma glucose levels, but the levels remained significantly higher than baseline by the end of the experimental (average 161 + 12 mg/dl). Glucose clearance rates were not affected by either i.v. or i.c.v. 2-DG, remaining constant at approximately 2.5-3.0 m l - k g - 1 , m i n - l in both groups (data not shown).

Gluconeogenic precursor balance (Fig. 2 and Table I): lactate Blood lactate levels as well as the lactate load delivered to the liver remained constant in the i.v. group at 600 _+ 70 /~mol/1 and 24 _+ 3 /~mol • k g - 1. min ~, respectively. Net hepatic lactate balance was neutral during the basal period (2 + 5/.~mol • kg-~ • min-~) and showed net uptake of 5 _+ 4 /~mol. kg -~. min -1 at 45

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6-fold increase in the lactate load reaching the liver (from 22 + 4 to 122 + 43 /zmol. kg -1 • min - I ) yet the hepatic fractional extraction was decreased from a basal average of 27 + 0.9% to 10 + 0.5% 2 h after i.c.v. 2-DG administration. The 'gut' balance of lactate switched from a net release of 1.6 + 0.9 /zmol. kg - l . min-1 during the basal period to a net uptake averaging 6 + 3.5 /xmol. kg -1" min -1 (data not shown); the fractional extraction by the 'gut' increased from 24 + 0.8% to 46 + 0.5% 45 min after i.v. 2-DG ( P < 0.05).

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Time ( r a i n ) Fig. 2. Blood alanine, lactate and glycerol ( m m o l / l ) , as a function of time after initiating 2-DG administration. Filled circles represent values of animals administered 2-DG into the third ventricle. Empty triangles represent values for animals administered 2-DG intravenously. Values are means_+ S.E.M. * P < 0.05, denotes significant difference from basal (time = 0). ÷ P < 0.05 denotes significant difference from time matched control.

min and later (75 min) decreasing towards basal, i.c.v. 2-DG resulted in a marked and sustained hyperlactacidemia which by 90 min averaged 2,680 + 600 /zmol/1 (4-fold over basal). This was accompanied by a

Within 45 min of i.v. 2-DG arterial blood alanine levels decreased by 25-30% with the levels remaining lower than basal until the end of the experimental period. Net alanine production by the gut remained the same (1.5 + 1 /zmol. kg -1. min -1) and the load of alanine reaching the liver as well as its net hepatic uptake remained stable• i.c.v, administration of 2-DG did not alter basal arterial alanine levels (312 + 60 /zmol/l). The gut alanine balance did not change from the basal period. Similarly, the load of alanine delivered to the liver did not change from basal. However, hepatic alanine uptake increased by an average of 2.4-fold over basal levels at 90 min and remained elevated throughout the remainder of the experimental period. Hepatic fractional extraction of alanine did not change from basal.

Glycerol i.v. administration of 2-DG resulted in a transient ( + 40%) increase in blood glycerol levels which peaked at 15 min (average 178 + 29 /zmol/1, P < 0.05) and returned to basal levels by 60 min. These changes were not accompanied by any significant increases in the load of glycerol reaching the liver (5 + 1 to 6 + 1/zmol kg - l • min-1), in its net hepatic uptake (3 + 1 to 4 + 1 /zmol • k g - 1 . m i n - l ) or in the hepatic fractional extraction (57-62%). i.c.v. 2-DG resulted in a more promi•

TABLE I

Net hepatic load and balance of the three main gluconeogenic precursors alanine, glycerol and lactate during basal and experimental periods Basal values are the average of at least three determinations prior to the administration of 2DG. Experimental values represent an average over the last 90 min of the experimental period. Positive values for balance denote net hepatic uptake. * P < 0.05 compared to basal, + P < 0.05 compared to i.v. 2DG.

Hepatic load Alanine (/xmol.kg 1 . m i n - l ) Lactate ( / z m o l . k g - l . m i n -1) Glycerol (p, m o l . k g - l . m i n -1)

Hepatic uptake

Basal

Experimental

Basal

Experimental

i.c.v. 11_+2 i.v. 1 4 + 4 i.c.v. 22_+4 i.v. 24+-3 i.c.v. 5 + 2 i.v. 6_+1

i.c.v. 18_+5 i.v. 14+_3 i.c.v. 122_+43 *-i.v. 2 7 ± 5 i.c.v. 11 + 2 * + i.v. 6 + 1

i.c.v. 3_+ 1 i.v. 3_+l i.c.v. 5+-3 i.v. 2+-5 i.c.v. 3 +- 1 i.v. 3_+l

i.c.v. 6_+2 * i.v. 4+-1 i.c.v. 12+-7 i.v. 5 ± 4 i.c.v. 7 _+ 1 * + i.v. 4_+1

104 nent rise in blood glycerol averaging 277 _+ 80 ~ m o l / 1 by 30 rain. Thereafter, the levels started to decline reaching baseline at 180 min. Simultaneously, the glycerol load delivered to the liver in the i.c.v. 2-DG was markedly increased from 5 _+ 1 /xmol • k g - 1 m i n - 1 at basal to a maximum of 15.1 + 1.4 /zmol • kg - ~ • m i n - 1 at 45 rain; thereafter, the values decreased approaching baseline at the end of the experimental period. This led to an increase in the net hepatic uptake of glycerol which was 5-fold at 30 min and remained higher than basal at the end of the experimental period. Hepatic fractional extraction did not change with 2-DG and averaged 0.56 to 0.63%. .

Amino acid metabolism (Fig. 3) i.v. 2-DG did not cause any significant alteration in either plasma leucine (126 _+ 12 /~mol/1), leucine Ra (3.1 + 0 . 3 / ~ m o l - k g -1 . r a i n - l ) , leucine oxidation (0.4 4-0.1 /~mol. kg - 1 . min l) or non-oxidative Rd (2.7 _+ 0.4 /~mol- k g - ~. min-~), i.c.v. 2-DG resulted in a sustained 20% decrease in plasma leucine levels from basal values of 111 _+ 7 p.mol/1 but reached statistical significance only at 90 min when the values averaged 81 _+ 6 ~ m o l / 1 ( P < 0.05). This appears to be the result of a significant drop in leucine Ra from a basal rate of 3.1 _+ 0.2/~mol • kg -~ • min -~ to 2.4 _+ 0.05/xmol.k- lg • m i n - ~ at 90 min ( P < 0.05). The changes in leucine Ra were significantly different from those in the i.v. group at 45 and 90 min ( P < 0 . 0 5 ) . The rate of leucine oxidation did not change. Non-oxidative Rd was signifi-

T A B L E II

Hormonal alterations of neuroglucopenia Plasma hormone concentrations and hormonal alterations of neuroglucopenia during basal and experimental periods in i.c.v, and i.v. 2DG groups, * P < 0.05 compared to basal, ÷ P < 0.05 compared to time matched i.v. 2DG. Basal values represent at least 3 samples prior to the administration of 2DG. Experimental values are an average over the last 90 min of the experimental protocol.

Insulin (/~U/ml) Glucagon (pg/ml) Cortisol

(,~g/d{) Epinephrine (pg/ml) Norepinephrine (pg/ml)

Basal

Experimental

1.c.v. 14_+3 l.v. 13.+2 l.c.v. 80.+8 i.v. 65_+7 l.c.v. 2.7-+0.4 i.v. 2.5.+0.4 i.c.v. 253 .+ 59 LV. 163.+41 LC.V. 340 5:83 i.v. 204 _+38

i.c.v. 30_+8 *+ i.v. 17.+3 i.c.v. 135_+ 19 *+ i.v. 66-+9 i.c.v. 9.4.+1 *+ i.v. 4.+1 i.c.v. 2246 _+558 * + i.v. 233.+54 i.C.V. 644 _+ 178 * + i.v. 258 _+55

cantly decreased from an average of 2.6 + 0.4 to = 1.7 + 0 . 3 t z m o l ' k g - ~ ' m i n -~ at 60 min and remained lower than basal throughout the rest of the experimental period, i.c.v, administration of 2-DG did not result in enhanced gut proteolysis, with gut leucine balance remaining neutral (data not shown).

Hormone levels (Table I1) i.v. 2-DG did not alter the circulating levels of insulin (13 + 2 ~ U / m l ) , epinephrine or norepinephrine (163 + 41, 204 + 38 p g / m l , respectively). However, i.v. 2-DG administration resulted in a modest and transient increase in plasma cortisol by 30 min

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Fig. 3. Plasma leucine (p, mol/l), leucine rate of appearance ( R a ; / ~ m o l . k g - X . m i n 1), leucine oxidation (p, mol.kg i.min 1) and non-oxidative rate of disappearance (p, mol. k g - i . m i n - t ) as a function of time after initiating 2-DG administration. Filled circles represent values of animals administered 2-DG into the third ventricle. Empty triangles represent values for animals administered 2-DG intravenously. Values are means_+S.E.M.. * P < 0.05, denotes significant difference from basal (time = 0). + P < 0.05 denotes significance from time matched i.v. 2-DG injected.

105 that was not different statistically from levels in the i.c.v, group at 30 and 60 min. Nevertheless, this increase was short-lived with levels returning to basal 90 rain after i.v. 2-DG administration. Plasma glucagon levels in the i.v. 2-DG group were slightly, yet significantly higher (P < 0.05) than basal, 30 min after administration, i.c.v, administration of 2-DG resulted in significant hormonal alterations; plasma insulin increased from 14 + 3 /xU/ml during

the basal period and peaked at 44 + 16/xU/ml by 45 min and declined to levels which averaged 30 _+8 /xU/ml during the last 90 min of the experimental period. Plasma glucagon levels increased and attained levels (peaked at 45 min at 213 + 53 pg/ml) that were higher than the basal levels and those attained in the i.v. group (P < 0.05 for both); the levels drifted towards basal but remained higher (P < 0.05) than basal or those achieved in the i.v. 2-DG group.

Fig. 4. a: low magnification view of the brain showing the third ventricle (v), cerebellum (c), thalamus (t) and hypothalamus (h). b: dark field view of the choroid plexus from the region of the fourth ventricle showing strong uptake of 2-deoxyglucose. c,d: bright field and dark field views of the thalamus and hypothalamic regions. Strongest uptake of 2-DG is present in thalamus and weak label is present in a few cells of the hypothalamus, e,f: bright field and dark field views of the cerebral cortex in the hippocampal region. Modest uptake of 2-DG is present in pyramidal neurons.

106 Plasma cortisol levels which averaged 2.7_+ 0.4 /.~g/dl in the basal period, showed an immediate and sustained increase reaching levels of 11 + 1 /~g/dl at 90 min; the levels remained elevated until the end of the experimental period. The changes in plasma epinephrine and norepinephrine were the most dramatic. With the onset of neuroglucopenia plasma epinephrine increased rapidly and peaked at 30 min at levels of 6187 + 2728 pg/ml which were --- 33-fold higher than basal; the levels then decreased but remained at an average 8-fold basal (2246_+ 558 pg/ml) by the end of the experimental period. Plasma norepinephrine which averaged 340 _+ 83 pg/ml in the basal period showed an early (30 rain) 3-fold increase (to 1195_+ 388 pg/ml) and then decreased but was still higher than basal at 180 min following i.c.v. 2-DG.

[14C]Deoxyglucose localization (Fig. 4) In an attempt to establish the areas of localization of the i.c.v.-administered 2-deoxyglucose, a preliminary survey was conducted of neuronal regions showing marked uptake. As expected, substantial labelling was observed in the ependymal cells lining the third and fourth ventricles (Fig. 4b). No uptake was detected in the ependymal cells lining the lateral ventricles or in those associated with the external surface of the cerebral cortex (data not shown). Prominent labeling was observed in certain thalamic and hypothalamic neurons (Fig. 4c,d). Slight uptake was detected in the pyramidal cells of the hippocampus (Fig. 4e,f) but no labeling was present in the somatosensory or frontal cortex. In the brainstem region, no uptake was present in the cerebellum, an occasional cell was labeled in the brainstem and some non-specifically associated 2-DG was present in tissues closest to the fourth ventricle (data not shown). DISCUSSION The aim of the present study was to determine if the whole body metabolic response to insulin-induced hypoglycemia can be reproduced by localized neuroglucopenia. The administration of 2-DG directly into the third ventricle caused a decrease in glucose oxidation by diminishing the rate of transfer of glucose from the plasma into the periventricular cells of the brain ~7. Furthermore it depresses the cerebral oxidation of glucose from within the cell and acts as a potent competitive enzymic inhibitor of glycolysis leading to glucopenia2~, mainly in the region of the periventricular nuclei. These properties of 2-DG make it suitable for use in recreating the localized changes in glucose metabolism seen during insulin-induced hypoglycemia.

Administration of 2-DG into the third ventricle resulted in a marked stress response similar in many respects to that seen with insulin-induced hypoglycemia14. These results are confirmatory of findings in previous studies and include profound increases in cortisol, epinephrine, norepinephrine and glucagon 3'16'24. The increases in plasma catecholamines and glucagon 16'19 appear to be CNS-mediated. Havel et al. 15 provided evidence that neuroglucopenia resulted in large increases in pancreatic norepinephrine output as well as in arterial epinephrine confirming the adreno-meduUary activation by neuroglucopenia. These rises in plasma epinephrine and norepinephrine are known to enhance glycogen breakdown in the extrasplanchnic tissues, particularly skeletal muscle and decrease glucose utilization and increase lipolysis. Our results are supportive as evidenced by three factors. (a) Increased blood lactate in the absence of increased splanchnic release of lactate suggesting enhanced glycogen breakdown by peripheral tissues, particularly skeletal muscle. (b) The lack of a drop in arterial alanine (as was seen with i.v. 2-DG), in the face of enhanced net hepatic uptake of alanine, suggesting increased alanine release by peripheral tissues, particularly skeletal muscle. (c) The increased blood glycerol, in the face of increased hepatic uptake of glycerol, suggesting that lipolysis must have been markedly increased. This increase in lipolysis, in conjunction with the elevated levels of plasma epinephrine, would in turn result in blunting of the expected rise in the rate of glucose disappearance. Ipp et al. m have also described a significant increase in glucagon released from the pancreas in response to the i.c.v, administration of 2-DG and further indicated that this response is inhibited by administration of somatostatin. In a similar fashion, it can be speculated that the rise in plasma glucagon together with that of epinephrine resulted in increased hepatic glucose production. Most likely, this could have resulted from an immediate enhancement of glycogenolysis preceding the increase in net hepatic uptake of lactate, glycerol and alanine, which would presumably serve to sustain increased rates of gluconeogenesis. Thus, our findings suggest that the majority of metabolic compensatory responses in carbohydrate metabolism seen during insulin-induced hypoglycemia are mimicked by i.c.v, administration of 2-DG. Interestingly, however, the same does not hold true for the changes observed in protein and amino acid metabolism during insulin-induced hypoglycemia. Previous studies from our laboratory have established that insulin-induced hypoglycemia is associated with enhanced rates of whole body rates of proteolysis and oxidation and a significant decrease in whole body

107 protein synthesis. We also noted that the primary source of proteolysis was the gastrointestinal tract t8. We had speculated that the enhanced gut proteolysis was the result of CNS glucopenia. The data from the present study indicate that glucopenia affecting primarily the periventricular area, while accounting for much of the change in glucose metabolism, does not account for the changes in protein metabolism seen with insulin induced hypoglycemia. On the contrary, i.c.v, administration of 2-DG resulted instead in decreased rates of whole body proteolysis, no change in the rate of leucine oxidation and decreased rates of amino acid disposal; there was no change in amino acid release by the GI tract. These changes are, in many respects, qualitatively similar to those observed with hyperinsulinemia, while euglycemia prevails. Because it is possible that some of the 2-DG administered into the third ventricle could have escaped into the systemic circulation we examined the effects following the intravenous administration of an identical dose of 2-DG. As expected, the dose administered was not sufficient to produce hyperglycemia; however, there were some other subtle systemic effects of this low i.v. dose. The pancreatic alpha cells responded by increasing, though transiently, plasma glucagon levels and similarly, there was a transient increase in circulating plasma levels of cortisol. These findings, though subtle, suggest the possibility that the pancreas and adrenal glands are quite sensitive to the inhibition of glycolysis by 2-DG, resulting in localized transient decreases in intracellular levels of ATP 21'31. Alternatively, these changes could be centrally (CNS) mediated secondary to a small amount of 2-DG entering the brain from the systemic circulation. Although unlikely, for this to occur the CNS hypothalamic-pituitary axis must be extremely sensitive to glucopenia, as those changes occurred without noticeable effects on glucose homeostasis. This would also indicate that there is a differential response between the hypothalamic-pituitary-adrenocortical axis and the sympatho-medullary axis, in response to central glucopenia, because of no change in the plasma levels of epinephrine or norepinephrine in the group that received i.v. 2-DG. Further studies are required to address the above discrepancies. Our results indicate that the localized neuroglucopenia that was elicited by i.c.v. 2-DG was sufficient to produce a marked stimulation of the hypothalamicpituitary-adrenal axis. This, however, was not responsible for the enhanced proteolysis seen with insulin-induced hypoglycemia as evidenced by a drop in leucine Ra, a drop in leucine oxidation and a failure to increase net amino acid production by the gut. While it is not possible from the present study to discern the exact

anatomic location of the centers responsible for regulating amino acid metabolism, it is clear that such center(s) is/are not localized in the periventricular area. Based on the autoradiographic analysis the radio-label distribution occurred into the hypothalamic region and into more distant areas including the thalamus and hippocampus. No radio-label was distributed in the brainstem area. Thus, it is possible that glucoreceptors located in the brainstem 25, could be responsible for triggering the increase in gut proteolysis previously described. Studies by DiRocco and Grill s would support this hypothesis, since their findings show that chronically decerebrate rats are capable of exhibiting sympathoadrenal hyperglycemia in response to glucopenia, thus providing evidence for efferent signals arising from the brainstem. Another possibility, albeit small, for the different responses in amino acid and glucose metabolism to i.c.v. 2-DG and insulin-induced hypoglycemia is the lack of peripheral glucopenia which could sensitize the enterocytes a n d / o r smooth muscle cells of the gut to the centrally activating responses resulting in a net increase in gut proteolysis. In summary, we report a whole body counter-regulatory response to periventricular neuroglucopenia, which is similar to that observed during whole body insulin-induced hypoglycemia as far as glucoregulation is concerned. Most importantly, our findings suggest that control of amino acid metabolism and the effect of insulin-induced hypoglycemia on the same, may not be regulated through the same pathways as that of glucose metabolism and that glycemia is a crucial regulator of amino acid kinetics even under marked stress-hormone activation. Acknowledgements. The authors wish to acknowledge the excellent technical assistance of Mrs. Mabel Collier, Tina Uselton, Becky Naukaum and Mr. Patrick Donahue. This work was supported by NIH Grant No. DK 42562.

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