The effects of acute elevations in plasma cortisol levels on alanine metabolism in the conscious dog

The Effects

of Acute

Elevations

Richard E. Goldstein,

in Plasma Cortisol Levels in the Conscious Dog

George W. Reed, David H. Wasserman,

Randall Buckspan,

Naji N. Abumrad,

on Alanine

Phillip E. Williams,

Metabolism

D. Brooks Lacy,

and Alan D. Cherrington

The present study was undertaken to determine whether an acute physiological increase in plasma cortisol level had significant effects on alanine metabolism and gluconeogenesis within 3 hours in conscious, overnight-fasted dogs. Each experiment consisted of an go-minute tracer and dye equilibration period, a 40-minute basal period, and a 3-hour experimental period. A primed, continuous infusion of [3-3H]glucose and continuous infusions of [U-14C]alanine and indocyanine green dye were initiated at the start of the equilibration period and continued throughout the experiment. Dogs were studied with (1) a hydrocortisone infusion ([CORT] 3.0 pg . kg-’ . min-‘, n = 5). (2) hydrocortisone infused as in CORT, but with pancreatic hormones clamped using somatostatin and basal intraportal replacement of insulin and glucagon (CLAMP + CORT, n = 5), or (3) saline infusion during a pancreatic clamp (CLAMP, n = 5). Glucose production and gluconeogenesis were determined using tracer and arteriovenous difference techniques. During CLAMP, all parameters were stable except for a modest 67% f 6% increase in gluconeogenic conversion of alanine to glucose and a 53% *‘26% increase in gluconeogenic efficiency. When plasma cortisol levels were increased fourfold during CLAMP + CORT, there was no change in the concentration, production, or clearance of glucose. Gluconeogenic conversion of alanine to glucose increased 10% f 34% and gluconeogenic efficiency increased 65% f 43%, while net hepatic alanine uptake (NHAU) increased 60% & 19% and hepatic fractional extraction of alanine increased 38% f 12%. Cortisol did not cause an increase in the arterial glycerol level or net hepatic glycerol uptake. When plasma cortisol levels were increased and the pancreatic hormones were allowed to change (CORT), there was a transient but significant decrease in plasma insulin levels, while plasma glucagon levels remained unchanged. There was no change in the concentration, production (R,), or clearance of glucose. However, gluconeogenic conversion of alanine increased 190% -C 59%, gluconeogenic efficiency increased 57% & 43%. NHAO increased 75% + 25%. and hepatic alanine fractional extraction increased 48% + 6%. These changes were all statistically significant by the first 90 minutes of hydrocortisone infusion. In addition, there was a tendency for both net hepatic production of lactate and net hepatic uptake of glycerol to be elevated compared with the other two groups. These results suggest that an acute physiologic increase in plasma cortisol level can increase the gluaoneogenic conversion of alanine to glucose by increasing both NHAU and hepatic fractional extraction of alanine. This increase in plasma cortisol level may also be associated with a transient decrease in plasma insulin level that may further promote gluconeogenesis. Copyright 0 1992 by W.B. Saunders Company

T

HE PLASMA CORTISOL level increases in response to various forms of stress; threefold to fivefold elevations have been shown to occur within the first few hours of trauma,’ sepsis,? exercise,’ and hypoglycemia.4 It has been clearly demonstrated that glucocorticoids can have significant effects on carbohydrate metabolism. In-vitro studies by Exton et a15.‘jshowed that 1 hour of glucocorticoid treatment restored glucose production to normal in adrenalectomized rats, and also restored the incorporation of [i4C]lactate into [r4C]glucose. Peterson et al7 showed that in H4IIE rat hepatoma cells, dexamethasone produced an increase in phosphoenolpyruvate carboxykinase gene transcription after only 30 minutes of exposure. Only a few studies have examined the effects of acute physiologic elevations in plasma cortisol level on carbohydrate metabolism in vivo.*.y Shamoon et al8 increased plasma cortisol levels twofold to threefold and were able to demonstrate a very pronounced decrease in glucose clearance accompanied by an increase in plasma glucose levels during 6 hours of hypercortisolemia. However, Simmons et al9 noted much less of a change in both plasma glucose level and clearance during 8 hours of a fourfold increase in plasma cortisol level. In neither of these studies was a change in plasma insulin level detected. Although in-vitro studies have focused on the ability of glucocorticoids to modulate gluconeogenesis, this issue has been addressed in only a few in-vivo studies.“JO Simmons et al’ demonstrated that both proteolysis and de-novo synthe-

Meta/~o/~sm, Vol41, No 12 (December), 1992: pp 1295-1303

sis of alanine increase after 4 hours of hypercortisolemia. thereby increasing the availability of a potential gluconeogenie substrate. They did not directly investigate the gluconeogenic conversion of alanine to glucose. Lecavalier et allo showed that lactate gluconeogenesis could be enhanced by 4 hours of hypercortisolemia and that cortisol augments glucagon-stimulated gluconeogenesis primarily by changes in gluconeogenic enzymes, rather than by alterations in substrate availability. However, the ability of cortisol to increase gluconeogenesis was studied only in the presence of hyperglycemia. The present study examines the direct effects of an acute fourfold physiologic increase in plasma cortisol level on carbohydrate metabolism in vivo. Particular emphasis is

From the Departments of Molecular Physiology and Biophy.sics, Surgery~ and Preventive Medicine, and the Diabetes Research and Training Center, Vanderbilt University School of Medicine, Nashville, TN. Address reprint requests to Richard E. Goldstein, MD, A-2219 Medical Center North, Vanderbilt University, 2lst and Garland Avenue, Nashville, TN 37232. Supported in part by Diabetes Research and Training Grant No. P60 DK20593, and Grants No. HHS DK30515 and No. SROI DKL8243 from the National Institutes of Health. Copyright 0 I992 by W.B. Saunders Company 00260495/9214112-0005$03.00i0

1295

1296

GOLDSTEIN ET AL

placed on the role that cortisol may play in stimulating gluconeogenesis and on the contribution that this may make to total hepatic glucose production (R,). MATERIALS

AND METHODS

Animals and Surgical Procedures Experiments were performed on a total of 15 mongrel dogs (16 to 25 kg) of either sex that had been fed a standard diet of meat and chow (31% protein, 52% carbohydrate, 11% fat, and 6% fiber based on dry weight; Kal Kan meat, Kal Kan Foods, Vernon, CA, and Wayne Dog Chow, Allied Mills, Chicago, IL). Two weeks before each experiment, a laparotomy was performed under general endotrachial anesthesia (sodium pentobarbital, 25 mgikg intravenously [IV]) with ventilation maintained using a Harvard Apparatus Respiratory Pump (Boston, MA). Using sterile surgical technique, silastic infusion catheters (0.76 mm internal diameter) were placed in jejunal and splenic veins. Silastic sampling catheters (1.02 mm internal diameter) were placed in the portal vein so that the tip was 1 inch below the liver, in the left common hepatic vein to sample blood draining 60% of the liver,” and in the left femoral artery. After insertion, the catheters were filled with saline containing 200 U/mL heparin, their free ends were knotted, and they were placed in a subcutaneous pocket so that complete closure of skin incisions was possible. All studies were performed on 18-hour overnight-fasted, conscious dogs. One day before the experiment, blood was drawn to determine the leukocyte count and hematocrit of each animal. Only animals that had (1) a leukocyte count below 18,000, (2) a hematocrit greater than 36%, (3) a good appetite (consuming all of the daily ration), (4) normal stools, and (5) no physical evidence of wound infection were used. On the day of the experiment, the subcutaneous ends of the catheters were exteriorized through small skin incisions made using local anesthesia (2% lidocaine; Astro, Worcester, MA). The contents of the catheters were aspirated and the catheters were flushed with saline. The conscious dog was then allowed to stand calmly in a Pavlov harness for 20 minutes before the start of the experiment. Exuerimental Procedures 1

Each experiment consisted of an 80-minute tracer and dye equilibration period (-120 to -40 minutes), a 40-minute basal period (-40 to 0 minutes), and a S-hour experimental period (0 to 180 minutes). At time -120 minutes, a primed, (50 uCi) constant infusion of [3-3H]glucose (0.35 uCi/min) and a constant infusion of [U-14C]alanine (0.026 uCi. kg-i min-‘) were started via an 18gauge angiocath in the cephalicvein, and continued throughout the entire experiment. The priming dose of [3-3H]glucose equaled the amount of tracer infused in 140 minutes. At time -120 minutes, a constant infusion of indocyanine green (0.1 mg rnm2. mini) was started in the cephalic vein and continued throughout the experiment. Three protocols were used. In the first protocol, CORT (n = 5) a constant infusion of hydrocortisone (3.0 ugikg min-‘) was begun at 0 minutes and continued throughout the experimental period. In the second protocol, CLAMP (n = 5). a peripheral infusion of somatostatin (0.8 pg. kg-’ . mini) was started at time -100 minutes to inhibit endogenous secretion of insulin and glucagon, while intraportal replacement infusions of insulin (250 p,U/kg . mini) and glucagon (0.65 ng kg-’ min-*) were started using the splenic infusion catheter. The plasma glucose level was monitored every 5 minutes, and the rate of insulin infused was adjusted to maintain euglycemia. The maximal change required in the insulin infusion rate was 140 pIJ_ kg-’ mini and the final

mean rate of infusion was 273 )LU . kg-’ mini. The last alteration in the insulin infusion rate was made at least 30 minutes before the start of the basal period. The glucagon infusion rate remained unchanged throughout the experiment. Saline was infused from 0 to 180 minutes. In the third protocol, CLAMP + CORT (n = 5), insulin and glucagon were clamped as described above, and a constant infusion of hydrocortisone (3.0 ug t kg-’ min-‘) was started at 0 minutes and continued throughout the experimental period. The maximal change required in the insulin infusion rate was 130 uI_l. kg-’ mini and the final mean rate of infusion was 298 uU. kg-i . min-I. Autopsies were performed at the conclusion of each experiment and proper catheter position was confirmed. Animals in this study were maintained and experiments were performed in accordance with the guidelines of the Animal Care Committee of Vanderbilt University in an AAALAC (American Association for Accreditation of Laboratory Animal Care)accredited facility. Processing of Blood Samples The collection and immediate processing of blood samples have been previously described. I2 Plasma glucose concentrations were determined by the glucose oxidase method in a Beckman glucose analyzer (Beckman Instruments, Fullerton, CA). Plasma glucose radioactivity (3H and i4C) was determined by liquid scintillation counting after deproteinization with barium hydroxide and zinc sulfate,i3 glucose isolation with cationic- and anionic-exchange resinsI and reconstitution in 1 mL distilled water and 10 mL aqueous counting scintillant (ACS; Amersham, Arlington Heights, IL). Whole-blood glucose values were assumed to equal 73% of plasma values, based on extensive comparisons between whole blood and plasma glucose performed in our laboratory.” Concentrations of indocyanine green were determined spectrophotometritally (805 nm) in arterial and hepatic vein plasma samples. Labeled and unlabeled plasma alanine and lactate concentrations were determined with a short-column, ion-exchange, chromatographic system that has been described previously.i2 Whole-blood lactate. alanine, glycerol, and 8-hydroxybutyrate concentrations were determined in samples deproteinized with 4% perchloric acid according to the method developed bv Lloyd et all6 for the Technicon AutoAnalyzer (Technicon Instruments, Tarrytown, NY). Immunoreactive glucagon concentrations were determined in plasma samples containing 50 uL Trasylol (FBA Pharmaceuticals, New York, NY) by radioimmunoassay (RIA) with 30K antiserum”; the interassay coefficient of variation (CV) was 22%. Plasma immunoreactive insulin was measured with the Sephadex-bound-antibody procedure is; the interassay CV was 11%. Plasma cortisol was measured with the Clinical Assays Gamma Coat RIA kit (Clinical Assays, Travenol-Genetech Diagnostics, Cambridge, MA); the interassay CV was 6%. Plasma epinephrine and norepinephrine levels were determined with the CAT-A-KIT radioenzymatic kit (Upjohn, Kalamazoo, MI) using the method described by Passon and Peuler.i9 The interassay CVs were 13% and 11% for epinephrine and norepinephrine, respectively. Materials [3-3H]glucose (New England Nuclear, Boston, MA) was used as the glucose tracer (500 uCi/O.OOSmg), and [U-14C]alanine (Amersham) was used as the labeled gluconeogenic precursor (171 mCi/mmol). Indocyanine green was purchased from Hynson, Westcott, and Dunning (Baltimore, MD). Phadebas insulin RIA kits were purchased from Pharmacia (Piscataway, NJ). Glucagon 30K antiserum was obtained from the University of Texas Southwestern Medical School (Dallas, TX), and the standard glucagon

1297

CORTISOL’S ACTION ON GLUCONEOGENESIS

and “SI-labeled glucagon were obtained from Novo (Copenhagen, Denmark). Catecholamine assay kits (CAT-A-KIT) were obtained from Upjohn. Insulin was obtained from Squibb-Novo (Princeton, NJ), and glucagon was obtained from Eli Lilly and Co. (Indianapolis, IN). Cortisol RIA kits were obtained from Micromedic Systems (Horsham, PA), and cyclic somatostatin was obtained from Bachem (Torrance, CA). Insulin, glucagon, and somatostatin solutions were prepared with 6% plasma in normal saline. The [3-3H]glucose infusate contained cold glucose so that its final concentration was 1 mg/mL. The indocyanine green infusate was prepared with sterile water, and the [U-‘“C]alanine and [3-sH]glucose infusates were prepared with normal saline.

Cakulatiom In all studies, measurement of hepatic extraction of indocyanine green dye?” was used to assess total hepatic plasma flow (HPF). The proportion of hepatic blood flow (HBF) provided by the hepatic artery was assumed to be 28%. based on a compilation of data by Greenway and Stark.” Recent studies in this laboratory usin; pulsed Doppler flow probes indicate that the proportion of blood flow provided by the hepatic artery may be closer to 20%. and results were also calculated using this percentage. This change had little effect on the data. and the conclusions reached were not altered by it. To be consistent with our previous reports in which indocyanine green was used to measure HBF, we chose to depict the data obtained using an assumption of 28% arterial flow. Net hepatic balances (whole blood) of alanine, lactate. glycerol, and P-hvdroxybutyrate were determined by the formula (H -- ]0.28A + 0.72P]) x HBF, where H, A, and P represent the concentrations of the given substrate in the hepatic vein, femoral artery. and portal vein, respectively. HBF was derived from the HPF‘, ie, HBF = HPFI(1 - hematocrit). All hepatic balances are depicted as positive values, but are labeled appropriately as either output or uptake. Net hepatic glucose balance was determined as described above after converting plasma glucose levels to blood glucose levels by multiplying by 0.73. Hepatic fractional extraction of alanine was determined by the formula ([0.28A + 0.72P] - H)/ (0.2NA + 0.72P). Glucose R, and hepatic glucose utilization (Rd) were determined by the equations for isotope dilution during a constant infusion of radioactive glucose ([3-3H]glucose), as described by Steele.?” The hepatic [‘“Clglucose production rate was determined using the tracer technique described by Chiasson et al.?? Once the [i4C]glucose production rate was determined, the gluconeogenic conversion rate was calculated by dividing the [“Cjglucose production rate (dpm kg-’ mini) by the specific activity (dpmikmol) of alanine entering the liver (weighting the arterial and portal specific activities). The efficiency of hepatic gluconeogenesis was calculated by dividing the [‘4C]glucose production rate by the rate of hepatic [14C]alanine uptake. The livers of these dogs were all net producers of labeled lactate during the basal and experimental periods, and therefore the net contribution at [‘.‘C]lactate to [i4C]glucose production was zero and [14C]lactate was not considered in the gluconeogenic calculations. The methods used for calculating gluconeogenic conversion rate and efficiency yield minimal estimates due to isotopic dilution in the oxaloacetate pool. Calculation of the absolute rate of gluconeogenesis from alanine would require the determination of a correction factor as described by HetenyiZ4 or the use of several tracers and specific activity determinations of the glucose skeleton as discussed by Katz.‘5 For this reason, it is most convenient to express data as a percent change from basal values. The validity of this method of expression depends on the assumption that dilution of the labeled gluconeogenic precursor changes little during the

course of the experiment. Experiments performed by Hetenyizj demonstrated that the correction factor varied little among normal. insulin-deprived, and steroid-treated dogs. Data are expressed as means * SE. Rates are expressed per kilogram body weight. Statistical analyses were performed using two-way and one-way ANOVA with repeated measures to compare values between groups and within groups, respectively. Results of one-way ANOVA within a particular group are reported as “across time.” When group differences were identified using two-way ANOVA, the Neuman-Keuls Test was applied to determine significance at specific time points. A P value of .05 was considered necessary to reach statistical significance. Statistical calculations were performed using SAS-PC (SAS Institute, Caty, NC). RESULTS

Arten’al Plasma Cortisol and Insulin Concentrations

In CLAMP, plasma cortisol levels remained unchanged, but in CLAMP + CORT and CORT, plasma cortisol levels increased approximately fourfold (Fig 1). In CLAMP and CLAMP + CORT, plasma insulin levels did not change significantly, remaining at 8 t 2 and 10 2 2 pU/mL, respectively. However, in CORT. insulin levels decreased significantly from 12 t 3 to 7 k 1 pU/mL (P < .0.5) during the first hour of hypercortisolemia, and then gradually increased to 10 +- 2 kU/mL by the conclusion of the experimental period.

I

+/-

Pancreatic

Clamp

ortirol

pg/kg-min)

(3.0

or Saline

Infusion

10 r

a-

o-o A-A 0-O

0 i

CLAMP CiAMP+CORT CORT

, -40

0

60 Time

120

180

(min)

Fig 1. Arterial plasma cortisol and insulin levels in l&hour overnight-fasted dogs studied with (1) CLAMP, (2) CLAMP + CORT, and (3) CORT + CLAMP without CORT. Data are means f SE for five dogs.

GOLDSTEIN

Arterial Plasma Glucagon, Epinephrine, and Norepinephrine Concentrations

In CLAMP, CLAMP + CORT, and CORT, glucagon levels (Fig 2) did not change significantly during the experimental period. The higher glucagon levels in CLAMP probably reflect the presence of a greater amount of cross-reacting, non-3,.500-molecular weight material. Plasma arterial epinephrine and norepinephrine levels remained unchanged in all three groups at approximately 85 and 120 pg/mL respectively.

I

+/-

Pancreatic Cortirol

(3.0

ET AL

Clomp @g/kg-min)

or Saline

lnfusio

150

.5

o-o

50

A-A O-0

ii z

CLAMP CUMP+CORT CORT

Glucose Concentration and Kinetics

The arterial plasma glucose concentration (Fig 3) did not change significantly across time in CLAMP, but decreased slightly across time in CLAMP + CORT (111 + 7 to 94 +- 8 mg/dL, P < .Ol). The arterial plasma glucose concentration did not change across time in CORT, and there was no +/-

t

Pancreatic -~

Clomp

Cortisol (3.0 ______

pg/kg-min)

or Salins

tnfusbn

120

o-o a--n O-0

CLAMP CLAMP+CORT CORT

0

I

-40

0

60

120

160

Time (min)

Fig 3. Effects in 18-hour overnight-fasted dogs of (I) CLAMP, (2) CLAMP + CORT, and (3) CORT on arterial plasma glucose levels, glucose production, and glucose clearance. Data are means + SE for five dogs.

-40

0

60

120

160

Time (min)

Fig 2. Arterial plasma glucagon. epinephrine, and norepinephrine levels in IS-hour overnight-fasted dogs studied with (1) CLAMP, (2) CLAMP + CORT, end (3) CORT. Data are means 2 SE for five dogs.

significant difference in the arterial plasma glucose concentration between the groups. Glucose R, decreased 10% to 15% across time in CLAMP (3.0 -t 0.3 to 2.5 2 0.4 mg kg-l min-*, P < *OS). Glucose R, did not change across time in CLAMP + CORT (2.6 ? 0.2 to 2.5 * 0.3 mg . kg-’ . min-I), but decreased slightly across time in CORT (3.0 2 0.3 to 2.7 2 0.3 mg kg-’ . min-l, P < .05). However, there was no significant difference in glucose R, between the groups. Net hepatic glucose output (Table 1) did not change across time within any group, and there was no significant difference between groups. Glucose clearance did not change across time in CLAMP or CLAMP + CORT, but decreased 15% across time CORT (2.9 ? 0.4 to 2.4 2 0.2 mL/kg . min-*, P < .OOl). Again, there was no significant difference between groups. HBF (data not shown), determined using indocyanine green, did not change significantly across time in either CLAMP, CLAMP +

1299

CORTISOL’S ACTION ON GLUCONEOGENESIS

Table 1. Net Hepatic Glucose Output Net Hepatic Glucose Output (mg

kg~

1 minm1)

Time (min) 60

0

-40

120

so

180

150

CLAMP

3.5 + 0.7

2.8 r 0.7

2.8 2 0.4

2.5 f 0.5

2.9 + 0.5

2.6 k 0.4

2.4 + 0.4

CLAMP + CORT

2.1 2 0.3

2.2 +- 0.2

2.1 f 0.2

2.6 k 0.6

2.9 It 0.6

2.6 r 0.3

2.7 + 0.2

CORT

2.5 I 0.8

2.4 2 0.5

2.8 t- 0.8

2.9 z 0.4

2.4 -+ 0.5

2.9 k 0.4

2.3 + 0.4

NOTE. Net hepatic glucose output during basal f-40

to 0 minutes) and experimental periods (0 to 180 minutes) in 18.hour overnight-fasted dogs

was studied with (1) CLAMP (n = 5). (2) CLAMP + CORT (n = 5). and (3) CORT (n = 5).

CORT, or CORT, between groups.

and

was no significant

there

difference

Gluconeogenic Indices In CLAMP, the rate at which alanine was converted to glucose (Fig 4) increased 67% ? 6% across time (P < .05). In CLAMP + CORT and CORT, gluconeogenic conversion of alanine to glucose increased 101% 2 34% and 190% t 59% across time, respectively. The increase in gluconeogenic conversion of alanine to glucose was significantly greater in CORT than in CLAMP after the first 90 minutes of hydrocortisone infusion (P < .OOl). There was also a greater increase in gluconeogenic conversion in CORT + CLAMP than in CLAMP, although it did not reach a Pvalue of .05 (.05 < P < .lO). Statistical differenti-

ancreofic orfitol

(3.0

Clamp pg/kg-min)

-or Saline

infusion

I B

ation could not be achieved with the given sample size to determine if CORT was significantly different from CLAMP + CORT. Gluconeogenic efficiency increased 53% * 26%, 65% * 43%, and 57% + 43% across time in CLAMP, CLAMP + CORT, and CORT (P < .05), respectively. There was no statistically significant difference in efficiency between groups. Alanine Metabolism In CLAMP, CLAMP + CORT, and CORT, arterial blood alanine levels (Fig 5) did not change significantly during the 3-hour experimental period. Net hepatic alanine uptake (NHAU) did not change across time in CLAMP. However, in CLAMP + CORT and CORT, NHAU increased across time from an average during the basal period of 3.7 2 0.4 to 5.6 + 1.0 krnol . kg-* . min-r and from 2.7 +0.5 to 5.0 t 1.0 p,mol . kg-r . min-r, respectively (P < .Ol). The increase in NHAU was significantly greater in both groups than in CLAMP after the first 90 minutes of hydrocortisone infusion (P < .05). There was no statistical difference in NHAU between CLAMP + CORT and CORT. Hepatic fractional alanine extraction did not change in CLAMP, but increased by 38% + 12% and 48% rt 6% in CLAMP + CORT and CORT, respectively (0.32 * 0.06 to 0.43 + 0.08 in CLAMP + CORT and 0.20 * 0.03 to 0.31 2 0.05 umol . kg-’ mm’ in CORT; P < .05 between groups). The increase in hepatic fractional alanine extraction was significantly greater in both groups than in CLAMP after the first 90 minutes of hydrocortisone infusion. Lactate Concentrations and Net Hepatic Lactate Balance

o-o

CLAMP

A-A a-0

CUYP+COAT CORT

Arterial blood lactate levels demonstrated no significant change across time within any group and were not different between groups (Table 2). Net hepatic lactate production also did not change significantly across time in any group and was not different between groups; however, there was a tendency for net hepatic lactate output to decrease with time in CLAMP.

; -40

0

60

120

180

Time (min)

Fig 4. Effects in 18-hour overnight-fasted dogs of (1) CLAMP, (2) CLAMP + CORT, and (3) CORT on gluconeogenic conversion of alanine to glucose and gluconeogenic efficiency. Data are means f SE for five dogs.

p-Hydroxybuqrate Concentrations and Net Hepatic fi-Hydroqbutyrate Production Arterial blood P-hydroxybutyrate levels averaged approximately 20 pmol/L, demonstrated no significant change across time within any group, and were not different between groups (Table 3). Similarly, net hepatic P-hydroxybutyrate production did not change significantly within any group and was not different between groups.

1300

GOLDSTEIN ET AL

+/-

Glycerol Concentration and Net Hepatic Glycerol Production

Pancreatic Clamp ortirol 3.0 pg/kg-min)

or Saline lnfusio

o-o

CLAMP

A-A O-0

CLAMP+CORT CORT

Arterial blood glycerol levels for CLAMP, CLAMP + CORT, and CORT remained unchanged between 50 and 90 kmol/L, did not show a significant change across time within any group, and were not different between groups (Table 4). Net hepatic glycerol uptake did not change across time in CLAMP or CLAMP + CORT, but increased across time in CORT from 1.7 + 0.6 to 2.8 ? 0.8 pmol . kg-l . min-’ (P < .0.5). However, there was no significant difference in net hepatic glycerol uptake between groups. DISCUSSION

-40

60 Time (min)

0

When plasma cortisol levels were increased fourfold and the pancreatic hormones were free to vary, the major effects in the present study were an 85% increase in NHAU, a 50% increase in hepatic fractional extraction of alanine, and a tripling in gluconeogenic conversion of alanine to glucose. All of these effects became significant after the first 90 minutes of the hydrocortisone infusion. When plasma cortisol levels were similarly increased while insulin and glucagon levels were clamped, similar increases occurred in NHAU and hepatic fractional extraction of alanine. However, there was the suggestion that gluconeogenic conversion of alanine to glucose may not have increased to the same extent. Several in-vitro studies have demonstrated that cortisol has a stimulatory effect on both gluconeogenic enzymes5,26 and alanine transporters2’.** within the first hour of exposure. Gluconeogenic enzymes stimulated include phosphoenolpyruvate carboxykinase’ and giucosedphosphatase.2g Although these changes would be expected to result in an increase in gluconeogenic efficiency in vivo, this was not detectable in the present study. It remains possible that an increase in gluconeogenic efficiency occurred, but that it was too small to detect with the methods used. If there was significant loss of 14C label up into glycogen, gluconeogenic efficiency would have been underestimated. However, Steiner et a130 demonstrated that liver glycogen of 18-hour overnight-fasted conscious dogs accumulated very little 14Clabel. Even when subjected to the intense glycogenogenic stimulation of a fourfold increase in arterial plasma insulin level, hepatic 14Cglyco-

160

120

Fig 5. Effects in 18-hour overnight-fasted dogs of (1) CLAMP, (2) CLAMP + CORT, and (3) CORT on arterial blood alanine levels, NHAU, and hepatic alanine fractional extraction. Data are means f SE for five dogs.

Table 2. Arterial Blood Lactate Levels and Net Hepatic Lactate Output Time (min) -40

0

60

90

120

150

160

Arterial blood lactate levels (~mol/L) CLAMP

672 k 83

688 k 87

779 + 128

747 + 138

757 e 143

778 -t 161

703 k 146

CLAMP + CORT

660 f 154

655 ‘- 149

718 r 213

661 + 160

662 2 160

674 -c 159

661 k 141

CORT

581 k 125

568 k 148

607 ? 134

609 + 141

692 k 227

614 -r 198

706 2 163

Net hepatic lactate output (pmol . kg-’ CLAMP

min-I) 6.5 2 3.6

3.9 A 3.0

3.2 i 2.7

4.4 r 3.9

3.8 + 4.6

0.3 z!z4.3

CLAMP t CORT

7.2 + 3.8

7.9 2 4.0

4.1 -+ 2.7

4.4 2 3.8

5.2 ‘- 2.5

3.1 2 3.3

-0.3

4.7 t 2.5

lr 3.2

CORT

8.8 ? 2.3

10.3 + 2.6

8.4 + 3.5

9.7 + 5.5

10.5 2 6.5

10.0 t- 6.8

10.6 k 6.7

NOTE. Arterial blood lactate levels and net hepatic lactate output during basal (-40 to 0 minutes) and experimental periods (0 to 180 minutes) in 18-hour overnight-fasted dogs were studied with (I) CLAMP (n = 5). (2) CLAMP + CORT (n = 5). and (3) CORT (n = 5).

1301

CORTISOL’S ACTION ON GLUCONEOGENESIS

Table 3. Arterial Blood B_Hydroxybutyrate

Levels and Net Hepatic B_Hydroxybutyrate Time

0

-40

60

Output

(min) 150

120

90

180

Arterial 6.hydroxybutyrate levels (~mol/L) 17?

6

20 ? 4

16 + 3

23 f 9

30-t

CLAMP + CORT

17 2 6

22 + 5

21 2 3

22 + 1

22 f 2

23 t 2

24 -t 2

CORT

18 + 3

17 + 5

21 i 5

18 + 5

24 _t 8

24 -t 8

23 t 7

7

18)

11

33 2 16

CLAMP

Net hepatic p-hydroxybutyrate output (Fmol . kg-’

. min-‘j

CLAMP

0.9 r 0.3

0.7 -t 0.2

0.8 ?I 0.3

0.6 + 0.2

0.7 2 0.4

1.5 t 0.8

1.4 + 0.6

CLAMP + CORT

0.7 + 0.1

0.9 * 0.2

0.9 t 0.2

1.0 + 0.2

1.0 2 0.2

0.8 + 0.2

0.9 * 0.2

CORT

0.7 lr 0.1

0.7 + 0.2

0.6 it 0.2

0.9 -t 0.2

1.o t 0.3

1.2 + 0.5

1.2 f 0.3

NOTE. Arterial blood p-hydroxybutyrate

levels and net hepatic f3-hydroxybutyrate output during basal (-40

to 0 minutes) and experimental

periods (0 to 180 minutes) in 18-hour overnight-fasted dogs were studied with (1) CLAMP (n = 5). (2) CLAMP + CORT (n = 5). and (3) CORT (n = 5).

without a decrease in the plasma alanine concentration. Their study indicated that at least 3 hours of hypercortisolemia were necessary for significant changes in alanine R, and Rd to be detectable. In the present study, an increase in net nonhepatic alanine release was significant 90 minutes into acute hypercortisolemia. The increase in alanine supply from the periphery could be due to increased proteolysis or increased de-novo alanine synthesis.” Either of these mechanisms should have caused a decrease in the arterial alanine specific activity; however, a change in the arterial alanine specific activity could not be detected in the present study. The arteriovenous mode of tracer delivery and blood sampling,” as used in this study, may not have allowed detection of the small decreases in alanine specific activity that would reflect increased entry of cold alanine into the plasma pool. Furthermore, any 14Crecycling”“J4 involving the transamination of i4C-pyruvate to alaninej5 might also lessen the decrease in alanine specific activity. However, another explanation for the failure to detect a decrease in alanine specific activity is decreased net peripheral muscle uptake of alanine. Decreased net peripheral muscle uptake of alanine occurring with an increase in NHAU would effectively increase the alanine load presented to the liver

gen deposition did not demonstrate a significant increase. It is doubtful that a 3-hour period of acute hypercortisolemia would cause any greater degree of 14Clabel deposition. The mechanism responsible for the increase in gluconeogenie conversion was for the most part related to an increase in NHAU. This could have resulted from an increased “push” of alanine toward the liver from nonhepatic tissues or from an increased rate of alanine transport into the hepatocyte. 28Since the concentration of alanine in plasma did not change, both processes must have increased in parallel. In an earlier study, when alanine was infused at 6 tlmol kg-* min-I,” a rate sufficient to double the arterial alanine concentration, NHAU doubled and conversion of alanine into glucose increased 87%. This occurred without a significant change in the fractional extraction of alanine by the liver, and did not result in changes in gluconeogenic efficiency. These data showed that when an increased supply of amino acid substrate is made available to the liver, the A transport system*” responds by delivering the amino acid into the hepatocyte. The experiments of Simmons et al” suggested that acute hypercortisolemia increased the net release of alanine from nonhepatic tissues. An 8-hour period of physiologic hypercortisolemia resulted in a significant increase in alanine R, and Rd

Table 4. Arterial Blood Glycerol Levels and Net Hepatic Glycerol Uptake Time 0

-40

Arterial

60

(min) 150

120

90

180

blood glycerol levels

(rJ.mol/L) CLAMP

69 + 21

61 k-8

742

54 + 7

73 + 13

75 z? 22

67 f 16

CLAMP + CORT

91 + 25

88 ‘- 20

99 2 23

79 r 22

932

83 + 25

81 c 22

56+

57 f 7

562

58+

59 f 9

62 + 10

81 2 15

CORT Yet hepatic glycerol uptake

(ymol

-

kg-’

12

15 IO

11

14

min.‘)

CLAMP

2.2 z 1.0

1.4 t 0.3

1.7 It 0.5

1.4 2 0.3

CLAMP + CORT

1.7 * 0.4

1.9 -t 0.4

2.6 2 0.5

2.1 2 0.6

CORT

1.6 f 0.5

1.8 2 0.4

1.7 * 0.3

2.2 + 0.7

2.1 & 0.6 2.4 2 0.5 2.6 + 0.7*

2.0 I? 0.9

1.9 ? 0.7

1.8 + 0.5

2.1 ‘- 0.6

2.3 ? 0.6”

2.8 2 0.8*

NOTE. Arterial blood glycerol levels and net hepatic glycerol uptake during basal (-40 to 0 minutes) and experimental periods (0 to 180 minutes) in 18-hour overnight-fasted dogs were studied with (1) CLAMP (n = 5), (2) CLAMP + CORT (n = 5), and (3) CORT (n = 5). *Significant differences (P < .05) between these parameters and the basal period.

1302

GOLDSTEIN

without decreasing alanine specific activity. This would also be compatible with the studies of Simmons et al.9 Although they did not detect increases in alanine R, and Rd until the fifth hour of acute hypercortisolemia, plasma alanine levels were supported relative to the saline control study much earlier in the study. The increase in gluconeogenic conversion occurred without any significant increase in glucose R,. The inability of cortisol to increase glucose R, despite the increase in gluconeogenic conversion of alanine to glucose is in agreement with other published studies in which glucose R, tended to decrease over several hours of hydrocortisone treatment.8.9.37 Net hepatic glucose output, determined in the present study using the arteriovenous-difference technique, also failed to increase significantly. However, Frizzell et aP8 demonstrated that the net contribution of gluconeogenesis to overall glucose production in the overnight-fasted dog is between 0% and 25%, with the remainder contributed through glycogenolysis. In the present study, the 2.5umol . kg-’ . min-l increase in NHAU could increase glucose production by a maximum of 0.23 mg . kg-i . min-l. Assuming that NHAU is 50%38.39of the total gluconeogenic amino acid uptake and that changes in the hepatic uptake of these amino acids mirror those of alanine, then the maximum possible change in glucose production as a result of changes in gluconeogenic amino acids would be only 0.46 mg . kg-r . min-l. This magnitude of change in the rate of glucose production is very close to the limits of detection in the present study, and this may account for our failure to detect an increase in glucose R,. There was a tendency for glucose R, to be slightly higher in the presence of cortisol than during a pancreatic clamp alone; thus, it is possible that by using a much larger sample size a small increase in glucose R, from enhanced gluconeogenesis could have been detected. However, it is also possible that there was no change in glucose R, because glycogenolysis decreased as gluconeogenesis increased, as has been suggested by the work of Connolly et a1.4n Other investigators8,9 have reported both a 10% to 20% decrease in glucose clearance occurring within 6 hours of acute hypercortisolemia and a slight increase in plasma glucose levels. This increase in plasma glucose levels occurred after the third hour of hypercortisolemia and appeared to be temporally related to the decrease in glucose clearance. In the present study, glucose clearance in the animals infused with cortisol alone did demonstrate a small decrease across time. Although this decrease in clearance was not significant when compared with that of the control group, it was consistent with the demonstrated transient decrease in plasma insulin levels. It is possible that species differences and/or the longer experimental period in the other studies8s9 accounted for the difference in results.

ET AL

One of the curious findings in the present study was the transient decrease in the plasma insulin level that occurred when cortisol was infused and pancreatic hormones were allowed to vary. Although Kalhan and Adam4t reported decreased insulin secretion in response to a glucose infusion after 24 hours of pretreatment with prednisone, Shamoon et al8 detected no decrease in insulin levels during 5 hours of physiologic hypercortisolemia, and Simmons et al” detected no decrease in plasma insulin levels or C-peptide values during 8 hours of physiologic hypercortisolemia. Recent in-vitro studies have demonstrated increases in phosphoenolpyruvate carboxykinase mRNA in response to a decrease in insulin Ievels.42 In the present study, whether plasma insulin levels were clamped or allowed to vary when plasma cortisol levels were increased, similar changes occurred in NHAU and hepatic fractional extraction of alanine. Although there was a tendency for the conversion of alanine to glucose to be further increased when insulin was allowed to transiently decrease, this effect was small. In the present study, cortisol did not exert statistically significant effects on lactate, ketone, or glycerol metabolism. Although Simmons et al9 observed increases in plasma levels of P-hydroxybutyrate, free fatty acids, and acetoacetate after 4 to 8 hours of hypercortisolemia, similar increases were also present in their saline controls. Engel and Engel, on the other hand, demonstrated a slight inhibitory effect of cortisol on ketosis in the rat. Lipolytic effects of glucocorticoids have been estabIished,44 but in-vitro studies have demonstrated that at least a 2-hour lag period is required45 for these effects to be manifested. In the present study, net hepatic glycerol uptake tended to increase across time within the group that received cortisol alone, while blood glycerol levels remained unchanged. This suggests that lipolysis may have been enhanced, and is also consistent with the transient decrease in plasma insulin levels. In conclusion, an acute physiologic increase in plasma cortisol level had significant effects on alanine metabolism in vivo. This was manifested by increased NHAU and increased hepatic fractional extraction of alanine, and was consistent with the increased delivery of alanine from the periphery to the liver. The changes in NHAU contributed to an increase in the gluconeogenic conversion of alanine to glucose without a detectable change in gluconeogenic efficiency. All of these increases were significant following just 90 minutes of acute hypercortisolemia. However, the cortisol-induced increase in gluconeogenesis was not of sufficient magnitude in the overnight-fasted dog to lead to a detectable increase in total glucose production. Although an acute physiologic increase in plasma cortisol level did appear to transiently decrease plasma insulin levels in the dog, the changes in alanine metabolism and gluconeogenesis were primarily the result of the elevation in plasma cortisol level.

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