0021-972X/91/7202-0308$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright© 1991 by The Endocrine Society

Vol. 72, No. 2 Printed in U.S.A.

Effects of Glucagon on Free Fatty Acid Metabolism in Humans* MICHAEL D. JENSEN, VALARIE J. HEILING, AND JOHN M. MILES Endocrine Research Unit, Department of Medicine, Mayo Medical School, Rochester, Minnesota 55905

was similar to baseline values (1.73 ± 0.12 vs. 1.75 ± 0.23 and 1.35 ± 0.18 vs. 1.35 ± 0.16 ^mol/kg-min, respectively, in IDDM and nondiabetic subjects). No significant changes in palmitate flux occurred in response to glucagon withdrawal or mild (nondiabetic volunteers) or high physiological (IDDM volunteers) hyperglucagonemia. Thus, undei conditions of normal FFA availability, changes in plasma glacagon concentrations within the physiological range have little or no effect on adipose tissue lipolysis. (J Clin Endocrinol Metab 72: 308-315, 1991)

ABSTRACT. To determine whether physiological changes in plasma glucagon concentrations are important in regulating basal adipose tissue lipolysis, FFA flux ([l-14C]palmitate) was measured in response to increases and decreases in plasma glucagon. Eight volunteers with insulin-dependent diabetes mellitus (IDDM) and nine healthy nondiabetic volunteers were studied using the pancreatic clamp technique to control plasma insulin, GH, and glucagon concentrations at desired levels. Palmitate flux at the chosen euglucagonemic hormone infusion rates

G

the export of FFA from adipose tissue under conditions of normal FFA availability. Plasma FFA concentrations alone, however, may not accurately reflect FFA turnover, because of differences in clearance between groups (18) and because changes in turnover may occur without the predicted changes in concentration (19). Measurement of FFA flux with isotope dilution techniques is required to quantitate effective adipose tissue lipolysis in vivo. We have examined the effects of both glucagon deficiency and physiological hyperglucagonemia on FFA turnover using the pancreatic clamp technique to control plasma insulin, glucagon, and GH concentrations at desired levels. Our results indicate that under conditions of normal FFA availability, changes in plasma glucagon concentrations within the physiological range have little or no effect on lipolysis.

LUCAGON is an important hormone in the regulation of glucose homeostasis in humans (1), and failure to increase its secretion in response to falling plasma glucose concentrations may represent the initial step in defective glucose counterregulation in patients with insulin-dependent diabetes mellitus (IDDM) (2). Glucagon may also affect amino acid metabolism, especially in the setting of insulin deficiency (3). If glucagon is an important lipolytic hormone in humans the hyperglucagonemia associated with diabetes (1) might contribute to the insulin resistance (4) via the glucose-fatty acid cycle (5, 6). Treatment of diabetes with somatostatin analogs that suppress glucagon secretion (7) might, therefore, also normalize FFA availability. Whether glucagon plays a physiological role in the regulation of effective adipose tissue lipolysis is controversial, however. Glucagon stimulates lipolysis in rat (8), but not human (9), adipocytes and plasma FFA or glycerol concentrations have increased in response to glucagon in some (10-13), but not all (14, 15), human studies. The ability of large doses of glucagon to stimulate catecholamine (16) and GH (17) release confounds the interpretation of some of these studies (10-12). The present studies were undertaken to determine whether glucagon plays an important role in regulating

Materials and Methods Subjects Nine normal healthy volunteers (three males and six females; age, 30 ± 1 yr; body mass index, 22.7 ± 0.4 kg/m2) and eight subjects with poorly controlled IDDM (two males and six females; age, 26 ± 2 yr; body mass index, 23.3 ± 0.5 kg/m2) participated in these studies. The glycosylated hemoglobin values of the IDDM subjects, were 15 ± 1% (normal, 4-7%), their duration of diabetes was 12 ± 2 yrs, and their insulin dose was 37 ± 3 U/day. All subjects had maintained a stable weight for at least 2 months before the study and consumed a diet containing greater than 200 g carbohydrate daily for more than 2 weeks before the studies. Informed written consent was obtained from all volunteers.

Received July 16,1990. Address all correspondence and requests for reprints to: Michael D. Jensen, M.D., Endocrine Research Unit, 5-164 West Joseph, Mayo Clinic, Rochester, Minnesota 55905. * This work was supported by USPHS Grants DK-38092, DK-40484, and RR-0585 and the Mayo Foundation.

308

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 August 2014. at 10:44 For personal use only. No other uses without permission. . All rights reserved.

EFFECTS OF GLUCAGON ON LIPOLYSIS Materials 14

[l- C]Palmitate (Research Products International Corp., Mount Prospect, IL) was prepared for iv infusion as previously described (20). Glucagon and human regular insulin (Eli Lilly Co., Indianapolis, IN), recombinant DNA human GH (Genentech, South San Francisco, CA), and somatostatin (Bachem, Inc., Torrence, CA) were used in these studies. Assays Palmitate concentration and specific activity (SA) were determined using a modification (21) of our high performance liquid chromatography technique (22). Plasma total FFA concentrations were determined by a microfluorometric enzymatic method (23). Plasma insulin (24), free insulin (25), C-peptide (26), glucagon (27), GH (28), and cortisol (Magic Core kit, Corning Medical, Medfield, MA) concentrations were measured by RIA. Plasma glucose concentrations were measured with a glucose analyzer (Yellow Springs Instruments, Yellow Springs, OH). Experimental design The IDDM volunteers received their last injection of intermediate-acting insulin 3 days before the study, only regular insulin was given, three to four times daily as needed, for those 3 days. The insulin doses were adjusted as needed on the basis of self-performed capillary blood glucose measurements. The last sc injection of regular insulin was given about 1700 h the day before the studies. All subjects were admitted to the Mayo Clinic General Clinical Research Center the evening before each study and given a standard evening meal. That evening, a blood sample was

obtained to provide a blank for the palmitate SA measurement. An 18-gauge infusion catheter was then placed in a forearm vein and kept patent by a controlled infusion of 0.9% NaCl (30 mL/h). An overnight iv insulin infusion was used to maintain euglycemia in the IDDM subjects (13) and was discontinued at the beginning of the pancreatic clamp study. A constant infusion of [l-14C]palmitate (~3 nCi/kg-min) was begun more than 30 min before the first morning blood sample in order to ensure isotopic equilibrium. On the morning of each study, an 18gauge catheter (Cathelon IV, Criticon, Tampa, FL) was inserted into a dorsal hand vein in a retrograde fashion, and the hand was placed in a heated (50-55 C) box for sampling of arterialized venous blood (29). Four baseline blood samples were obtained at 10-min intervals between 0730-0800 h on each study day. The pancreatic clamp studies were begun at 0800 h with infusions of somatostatin (120 ng/kg-min), GH (3 ng/kg-min), insulin (0.05 mU/kg-min in nondiabetic, 0.1 mU/kg-min in IDDM volunteers), glucagon (0.6 ng/kg-min), and 50% dextrose if needed to prevent plasma glucose concentrations from decreasing too rapidly during the hypoglucagonemic study interval. The hormones were suspended in 0.9% NaCl containing 1% human serum albumin infused via a Harvard pump (Harvard Apparatus, Reno, NV). Plasma glucose concentrations were determined at 10-min intervals throughout each study to assist in determining the amount of glucose to infuse. Each glucagon dose interval (euglucagonemia, hypo- and

309

hyperglucagonemia) was of 2-h duration. Plasma palmitate concentrations and SA were determined on plasma samples obtained at 30-min intervals over the first 90 min of each glucagon dose interval and at 10-min intervals for the last 30 min. During the high dose glucagon infusion, plasma palmitate concentration and SA were determined at 10-min intervals throughout the entire 120 min. Plasma hormone concentrations were measured on the last three blood samples obtained over the final 30 min of each glucagon dose interval. A diagram of the study protocol(s) is provided in Fig. 1. To determine whether glucagon is important in the maintenance of basal lipolysis the pancreatic clamp studies were begun, as described above, in nine nondiabetic subjects with the initial glucagon infusion rate (0.6 ng/kg-min) from 0800-1000 h, which was then discontinued from 1000-1200 h. The study was extended for an additional 4 h in seven of these subjects with the glucagon infusion resumed at 0.6 ng/kg-min from 12001400, and 1.2 ng/kg-min for the final 2 h. Each of these seven subjects and one additional nondiabetic subject underwent a pancreatic clamp study on a separate day during which they received 0.6 ng/kg-min glucagon for the first 2 h and 1.2 ng/ kg-min for the second 2 h. These same seven subjects also returned on a third occasion for another pancreatic clamp study, in which the first 2 h of euglucagonemia were followed by a second 2 h of euglucagonemia. This permitted us to determine time-dependent changes in FFA turnover that occur under pancreatic clamp conditions. Because there was no difference between the response of FFA turnover to hyperglucagonemia during the 4-h study day us. the 8-h study day in nondiabetic subjects (see Results), and because of the difficulties involved in withdrawing intermediate-acting insulin for 3 days, all IDDM subjects were studied on one occasion with four consecutive 2-h glucagon dose intervals. Using the pancreatic clamp study design described above, [1-14C]palmitate somatostatin + insulin + growth hormone IDDM (n-8)

8 hr study

3.0

4 hr study 4 hr study

nondiabetic (n-8)

nondiabetic (n»7

hyper-

O

eu-

• nondiabetic (n=7)

S 0.6

o

hypo-

r -1

nondiabetic (n-9)

r 4 Hours

FlG. 1. An outline of the study protocol(s) is provided. The infusion rates of somatostatin, insulin, and GH for nondiabetic volunteers and subjects with IDDM are provided in the text.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 August 2014. at 10:44 For personal use only. No other uses without permission. . All rights reserved.

JENSEN, HEILING, AND MILES

310

the sequential glucagon infusion rates in the IDDM subjects were 0.6, 0, 0.6, and 3.0 ng/kg-min. Analysis and calculations To maximize the possibility of detecting potential lipolytic properties of glucagon, careful consideration was given to the experimental design. The considerable intraindividual variability of overnight postabsorptive (30, 31) and pancreatic clamp (31) FFA flux made a separate study day (hypo-, eu-, and hyperglucagonemia) design untenable. The experimental protocol with the greatest potential to detect an effect of glucagon compared changes in FFA flux from an euglucagonemic study interval to that from a subsequent hypo- or hyperglucagonemic interval. Statistical power calculations were performed to determine how many experiments would be required to detect a change in FFA flux in response to changes in plasma glucagon concentrations. We analyzed data from previous pancreatic

clamp experiments in which euinsulinemia and euglucagonemia were maintained for two consecutive 2-h intervals to determine the inherent variability of palmitate flux under these circumstances; the variability was similar to that observed during the comparable study day in these experiments (see Results). From these data we found it would be necessary to study eight subjects to have an 84% chance of detecting a 20% difference in FFA turnover in response to increases or decreases in plasma glucagon using a two-tailed paired t test at a significance level of 0.05. All values are presented as the mean ± SE, except where indicated. Plasma hormone concentrations were stable over the final 20 min of the baseline and each glucagon dose interval; therefore, mean values for each subject were used to calculate group mean and SE. Similarly, plasma palmitate concentrations and SA were stable over the final 30 min of the baseline, euglucagonemic, and hypoglucagonemic study intervals, and mean SA values were used to calculate steady state palmitate and FFA flux (20). Because a transient lipolytic response to hyperglucagonemia is possible, the nonsteady state palmitate rate of appearance (Ra) was calculated (32) at 10-min intervals over the entire 120 min of the high dose glucagon interval. Comparisons of plasma hormone concentrations as well as palmitate and FFA concentrations and flux from one glucagon dose interval to another were made using a paired Student's t test. Comparisons of values obtained in nondiabetic and IDDM subjects were made using a nonpaired t test.

Results Plasma hormone concentrations (Table 1 and Figs. 2 and 3) On each occasion the 0.6 ng/kg-min glucagon infusion rate (euglucagonemia) resulted in plasma glucagon concentrations similar to those observed during the baseline interval in both groups of subjects. Glucagon withdrawal resulted in comparable significant reductions in plasma glucagon concentrations in IDDM and nondiabetic subjects. Plasma glucagon concentrations were stable during the two consecutive euglucagonemic study intervals in

JCE&M»1991 Vol 72 • No 2

the nondiabetic subjects. The 1.2 ng/kg-min glucagon infusion rate in nondiabetic subjects during the 4-h study day resulted in plasma glucagon concentrations approximately 40 pg/mL greater (P < 0.05) than those during euglucagonemia and similar to those obtained using the same infusion rate on the 8-h study day. The 3 ng/kgmin glucagon infusion rate in IDDM volunteers increased (P < 0.001) plasma glucagon concentrations to approximately 400 pg/mL, which was also significantly greater than those observsd during hyperglucagonemia in nondiabetic volunteers. Plasma total insulin cor.centrations (Figs. 2 and 3) in nondiabetic subjects were similar during the three baseline studies and slightly, but significantly, lower than baseline during some of ths study intervals. As expected, plasma C-peptide concentrations were lower (P < 0.001) during the somatostatin infusion. Plasma concentrations of insulin and C-peptide in nondiabetic subjects increased slightly (P = 0.08 and P = 0.03, respectively) from the first to the second euglucagonemic study interval. When euglucagonemia was followed by hyperglucagonemia, somewhat greater increases in plasma insulin and C-peptide concentrations occurred (both P < 0.01). As expected, plasma free insulin (Fig. 2) and free Cpeptide concentrations in the IDDM subjects were stable throughout the experiment. Plasma GH and cortisol concentrations during eaci pancreatic clamp study were not statistically different from baseline and stable throughout the studies in each group of subjects. Plasma glucose concentrations Baseline plasma glucose concentrations were 6.5 ± 0.4 and 5.2 ± 0.1 mM in IDDM and nondiabetic subjects, respectively (Figs. 2 and 3). Plasma glucose concentrations increased (P < 0.01) during the euglucagonemic study interval in both IDDM (to 8.4 ± 0.9 mM) and nondiabetic (to 9.2 ± 0.6 mM) subjects. During the hypoglucagonemic study interval IDDM and nondiabetic subjects' plasma glucose concentrations were allowed to decrease to similar levels (8.1 ± 0.6 and 8.2 ± 0.2 mM, respectively). Reinstitution of euglucagonemia increased plasma glucose concentrations to 8.6 ± 0.8 and 9.4 ± 0.5 mM, respectively, in IDDM and nondiabetic subjects, with a further increase (P < 0.001) during the hyperglucagonemic study interval in IDDM subjects (to 12.7 ± 0.9 mM), but not nondiabetic subjects (to 9.3 ± 0.7 mM). Plasma glucose concentrations remained relatively constant between the first and second glucagon dose intervals in the nondiabetic subjects who underwent two consecutive euglucagonemic study intervals. In contrast, when euglucagonemia was followed by hyperglucagonemia in nondiabetic subjects on the 4-h study day, plasma

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 August 2014. at 10:44 For personal use only. No other uses without permission. . All rights reserved.

EFFECTS OF GLUCAGON ON LIPOLYSIS

311

TABLE 1. Plasma hormone concentrations

IDDM (n = 8) Free insulin (pM) Free C-peptide (pM) Glucagon (ng/L) GH (Mg/L) Cortisol (nM)

Non-IDDM Insulin (pM) C-Peptide (pM) Glucagon (ng/L) GH (Mg/L) Cortisol (nM)

n=7 Insulin (pM) C-Peptide (pM) Glucagon (ng/L) GH (Mg/L) Cortisol (nM)

n=8 Insulin (pM) C-Peptide (pM) Glucagon (ng/L) GH (Mg/L) Cortisol (nM) a 6

Basal

Euglucagonemia

Hypoglucagonemia

Euglucagonemia

Hyperglucagonemia

44 ± 9 0.02 ± 0.01 161 ± 14 4.4 ± 1.5 366 ± 70

31 ± 3 0.02 ± 0.01 172 ± 13 1.5 ± 0.2 354 ± 70

32 ± 3 0.02 ± 0.01 123 ± 8° 1.5 ± 0.3 300 ± 65

35 ± 5 0.02 ± 0.01 175 ± 8 1.4 ± 0.2 299 ± 41

35 ± 6 0.02 ± 0.01 407 ± 13°'6 1.5 ± 0.3 304 ± 61

n=9

n=9

n=9

n=7

n=7

41 ± 3 0.39 ± 0.05 161 ± 18 6.2 ± 1.6 339 ± 73

32 ± 2 0.07 ± 0.01° 177 ± 20 2.0 ± 0.2 333 ± 65

33 ± 2 0.07 ± 0.01 124 ± 15° 2.0 ± 0.2 270 ± 39

36 ± 3 0.08 ± 0.01 159 ± 20 2.0 ± 0.2 250 ± 54

39 ± 3 0.10 ± 0.02 212 ± 19° 1.9 ± 0.2 307 ± 60

Basal

Euglucagonemia

Euglucagonemia

39 ± 3 0.33 ± 0.03 159 ± 21 4.1 ± 1.3 252 ± 52

33 ±3° 0.07 ± 0.01° 173 ± 23 1.8 ± 0.2 261 ± 43

38 ± 3 0.10 ± 0.01 177 ± 25 1.8 ± 0.2 269 ± 55

Basal

Euglucagonemia

Hyperglucagonemia

39 ± 2 0.37 ± 0.03 153 ± 16

30 ± 2° 0.08 ± 0.01° 167 ± 20

38 ±3° 0.12 ± 0.02° 204 ± 20°

5.6 ± 2.4

1.8 ± 0.3

1.7 ± 0.2

283 ± 62

197 ± 48

268 ± 66

P < 0.05 vs. basal and/or euglucagonemia. P < 0.05 us. non-IDDM same study interval.

glucose concentrations increased from 8.9 ± 0.4 to 10.5 ± 0.5 mM (P < 0.05). FFA kinetics (Table 2 and Figs. 3-6) In nondiabetic subjects, baseline plasma concentrations and flux of palmitate and FFA were similar on the 3 study days and not statistically different from those in the IDDM subjects who had received an overnight insulin infusion. The chosen infusion rates of insulin, glucagon, and GH in the IDDM and nondiabetic subjects were successful in maintaining plasma palmitate and FFA kinetics at basal levels for 2 h of euglucagonemia. When euglucagonemia was followed by hypoglucagonemia, no significant changes in palmitate or FFA concentrations, SA (Fig. 3), or flux (Table 2) were observed in either group of subjects. Slight decreases in plasma palmitate (Fig. 4) and FFA concentrations and flux occurred during the hyperglucagonemic study intervals in nondiabetic subjects. These decreases were not statistically significant, were similar

to those that occurred during 4 h of euglucagonemia (Fig. 6), and were coincident with the increases in plasma insulin concentrations (Table 1 and Fig. 3). Plasma insulin concentrations were more stable during hyperglucagonemia in IDDM subjects, and plasma palmitate Ra did not increase significantly; at no time during hyperglucagonemia did the mean palmitate Ra exceed 2 SD of the preceding euglucagonemic levels, and the mean palmitate flux over the final 30 min of hyperglucagonemia was not significantly increased.

Discussion The studies reported herein were designed to measure the lipolytic properties of glucagon in the context of normal basal (overnight postabsorptive) FFA turnover. This level of FFA availability was achieved in the presence of normal plasma concentrations of insulin, glucagon, and GH using the pancreatic clamp technique with maximum tolerated doses of somatostatin. After 2 h to allow FFA flux to stabilize with euglucagonemia, the

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 August 2014. at 10:44 For personal use only. No other uses without permission. . All rights reserved.

JENSEN, HEILING, AND MILES

312

eu-

|

JCE&M-1991 Vol 72 • No 2

hypo-

{

eu-

|

60 plasma insulin O

hyper-

|

-o-

nondiabetic

•«-

IDDM

40

E FIG. 2. Plasma free insulin (IDDM subjects) and total insulin (nondiabetic subjects) during the baseline (-30-0 min) and eu-, hypo-, eu-, and hyperglucagonemic study intervals of the 8-h study are provided in the top panel. Plasma glucose concentrations observed during these experiments are represented in the

20 15 -••'I I 11

plasma glucose

bottom panel.

c

10

E

-•— 1 I T I

L

ooo' I

I

-30

0

120

240

360

480

Minutes auglucagonamia

eu- or hyperglucagonemia

45

O

35

Q.

25

eu-

15

hyper-

plasma glucose

0

L

-30

60

120

180

240

Minutes FlG. 3. Plasma total insulin concentrations (top panel) and plasma glucose concentrations (bottom panel) are provided for the nondiabetic subjects for both 4-h study days. Euglucagonemia was maintained for the first 2 h, and the second 2 h consisted of persistent euglucagonemia (O) or hyperglucagonemia (•). For hormone doses, see the text.

glucagon infusion rate was either discontinued or increased 2-fold (nondiabetic subjects) or 5-fold (IDDM subjects). Neither hypoglucagonemia nor hyperglucagonemia resulted in significant changes in FFA availability. We conclude that glucagon normally plays little or no

role in regulating adipose tissue lipolysis in humans. Our findings are in contrast to several reports showing an increase in plasma glycerol or FFA concentrations during hyperglucagonemia (10-13). Significant increases in plasma FFA concentrations have been observed in response to infusions of pharmacological amounts of glucagon (10-12); however, large doses of glucagon can stimulate catecholamine (16) or GH (17) release. Endogenous GH secretion was prevented in the present study, and adrenergic stimulation should have increased lipolysis (30), which clearly did not occur. It is unlikely that GH or catecholamine availability is a confounding variable in the present study. In other studies, Gerich et al. (13) infused glucagon at 3 ng/kg-min during abrupt cessation of iv insulin in IDDM subjects withdrawn from sc insulin and found a significant increase in plasma FFA concentrations. Thus, a lipolytic effect of physiological hyperglucagonemia is demonstrable in the presence of near-absolute insulin deficiency, a condition that rarely occurs, however, even in diabetic ketoacidosis (33). In a previous study (31) we created insulin deficiency in two groups of nondiabetic volunteers with or without replacement amounts (0.6 ng/ kg-min) of glucagon. During euglucagonemia palmitate flux increased to 2.4 ± 0.3 jumol/kg-min, and in the absence of glucagon it was 2.5 ± 0.2 /umol/kg-min {P = NS). It appears that it nay be necessary to study Cpeptide-negative insulin-deficient IDDM subjects in or-

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 August 2014. at 10:44 For personal use only. No other uses without permission. . All rights reserved.

EFFECTS OF GLUCAGON ON LIPOLYSIS

313

TABLE 2. FFA kinetics

Basal IDDM (n = 8) Palmitate (/ZM) Palmitate flux (^mol/kg-min) FFA (MM) FFA flux (^mol/kg-min)

135 ± 9 1.73 ± 0.12 782 ± 88 9.97 ± 1.15 n=9

Non-IDDM Palmitate (^M) Palmitate flux (/imol/kgmin) FFA (MM) FFA flux (/xmol/kg-min)

111 ± 12 1.35 ± 0.18 753 ± 113 9.17 ± 1.68 Basal

n=7 Palmitate (HM) Palmitate flux (^mol/kg-min) FFA (HM) FFA flux (jtmol/kg-min)

97 ± 1 1 1.30 ± 0.12 642 ± 65 8.08 ± 0.98 Basal

n=8 Palmitate (^M) Palmitate flux (^mol/kg-min) FFA (fiM)

FFA flux (jimol/kg-min)

89 ± 1 1 1.1 ±0.11 615 ± 63 7.64 ± 0.64

Euglucagonemia 147 ± 1.75 ± 886 ± 10.55 ±

25 0.23 145 1.34

n=9

116 ± 1.35 ± 786 ± 9.09 ±

14 0.16 117 1.32

Euglucagonemia

106 ± 1.30 ± 718 ± 7.74 ±

14 0.11 66 0.38

Euglucagonemia

116 ± 1.35 ± 856 ± 10.10 ±

17 0.20 107 1.12

der to detect a lipolytic effect of high physiological amounts of glucagon. No effect of hyperglucagonemia on lipolysis was observed in diabetic subjects in our study despite achieving plasma glucagon concentrations similar to those observed during a prolonged fast (20) or during diabetic ketoacidosis (1). Although it is likely that plasma glucagon concentrations even higher than those obtained in the present study, especially when combined with complete insulin deficiency, are capable of stimulating lipolysis, the relevance of that situation to most human physiology or pathophysiology is questionable. Our data exclude a role for glucagon in the maintenance of basal lipolysis in nondiabetic or IDDM humans. It was especially important to include volunteers with poorly controlled IDDM in the present study. First, diabetes may increase the lipolytic response to glucagon (34), and if a lipolytic response to physiological hyperglucagonemia occurs, we would expect to detect it in these subjects. Second, IDDM subjects cannot increase insulin secretion in response to increases in plasma glucose concentrations. The nondiabetic subjects in the present study clearly had breakthrough insulin secretion

Hypoglucagonemia 132 ± 1.55 ± 796 ± 9.31 ±

18 0.15 111 0.95

n=9

119 ± 1.35 ± 736 ± 8.38 ±

15 0.14 110 1.03

Euglucagonemia

Hyperglucagonemia

123 ±18 1.39 ±0.19 682 ± 132 7.71 ±1.37

123 ± 17 1.53 ±0.20 675 ±92 8.36 ±1.06

n=7

n=7

86 ±11 1.10 ±0.15 589 ± 105 7.58 ± 1.44

92 ± 14 1.10 ±0.17 595 ± 104 7.60 ± 1.49

Euglucagonemia

85 ± 9 1.16 ± 0.13 655 ± 55 8.32 ± 1.00 Hyperglucagonemia

91 ± 1 1 1.10 ± 0.11 590 ± 69 7.89 ± 0.82

during glucagon-induced hyperglycemia, consistent with the results of our previous studies (31). This increase in plasma insulin concentration could have potentially offset a lipolytic effect of glucagon and may explain other reports in which glucagon has induced antilipolysis (35). The finding that hyperglucagonemia in IDDM volunteers did not increase palmitate Ra permits us to confidently discount the possibility that increased insulin secretion obscured a lipolytic effect. In the present study plasma glucose concentrations increased in both groups of subjects during the euglucagonemic study period, probably due to portal venous hypoinsulinemia relative to glucagon availability. If hyperglycemia per se resulted in suppression of adipose tissue lipolysis, this effect could have offset a lipolytic effect of hyperglucagonemia. Recent studies from our laboratory, however, have failed to demonstrate an independent antilipolytic effect of hyperglycemia (36) in the range observed in the present study. Moreover, plasma glucose concentrations changed relatively little during the hyperglucagonemic study compared with the euglucagonemic study in either group. No measure of plasma glycerol concentrations or glyc-

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 August 2014. at 10:44 For personal use only. No other uses without permission. . All rights reserved.

JCE&M»1991 Vol 72 • No 2

JENSEN, HEILING, AND MILES

314

|euglucagonemia|

200

hypjqlucaqonemia non-IDDM IDDM

plasma palmitate

O 140 3L

80

FlG. 4. Effects of glucagon withdrawal on plasma palmitate concentration and SA in IDDM (n = 8) and non-IDDM (n = 9) subjects.

10 Palmitate SA

O

E c

1= 2

L

90

150

120

210

180

240

Minutes [Euglucagonemia |

|euglucagonemia|

Hyperglucagonemia - O - non-IDDM - • - IDDM

180 i- Plasma palmitate

euglucagonemia

180 Plasma palmitate

120

0120 60 10

5>—6—6 r

Palmitate SA 60

L

10 Palmitate SA

Palmitate R,

-O—6

ex

•o

2 90

120

150

180

210

240

Minutes FlG. 5. Effects of sustained hyperglucagonemia on plasma palmitate concentration, SA, and Ra in eight IDDM subjects and eight nonIDDM subjects. The data from non-IDDM subjects are from the 4-h study day.

erol kinetics was made in these studies. While glycerol kinetics may, in theory, be an equal or superior measure of lipolysis, they may not be specific for adipose tissue lipolysis. Glycerol release into the circulation could result from lipolysis occurring not only in adipose tissue, but also in muscle (37). In summary, the present studies found no change in

L

90

120

150

180

210

240

Minutes FIG. 6. Changes in plasma pahritate concentration and SA in response to a second 2 h of euglucagonemia in seven non-IDDM subjects.

FFA kinetics in response to changes in plasma glucagon concentration within the physiological range. We conclude that glucagon playj, virtually no role in the maintenance of basal adipose tissue lipolysis and probably increases FFA availability only when it is administered in pharmacological amounts or in the setting of complete insulin deficiency in IEDM subjects. It follows that reduced FFA release via decreased glucagon secretion would not be an expected benefit of diabetes treatment

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 August 2014. at 10:44 For personal use only. No other uses without permission. . All rights reserved.

EFFECTS OF GLUCAGON ON LIPOLYSIS

modalities that act through this mechanism. The disadvantage of impaired glucose counterregulation from this therapy might not be offset by improvements in FFA metabolism. Finally, the use of a glucagon-deficient pancreatic clamp model (necessary to prevent unwanted endogenous insulin secretion) in our previous FFA insulin dose-response study (31) probably did not result in qualitatively or quantitatively inaccurate data. The glucagon-deficient pancreatic clamp model will be useful in future studies of the hormonal and substrate regulation of adipose tissue lipolysis in vivo.

Acknowledgments We gratefully acknowledge the expert technical assistance of M. Persson, J. King, J. Aikens, and the Mayo Clinical Research Center staff, as well as the editorial skills of J. Ashenmacher.

References 1. Unger RH, Aquilar-Parada E, Miiller WA, Eisentraut AM. Studies of pancreatic alpha cell function in normal and diabetic subjects. J Clin Invest. 1970;49:837-48. 2. Gerich JE, Langlois M, Noacco C, Karam JH, Forsham PH. Lack of glucagon response to hypoglycemia in diabetes: evidence for an intrinsic pancreatic alpha cell defect. Science. 1973;182:171-3. 3. Nair KS, Halliday D, Matthews DE, Welle SL. Hyperglucagonemia during insulin deficiency accelerates protein catabolism. Am J Physiol. 1987;253:E208-13. 4. Yki-Jarvinen H, Taskinen M-R, Kiviluoto T, et al. Site of insulin resistance in type I diabetes: insulin-mediated glucose disposal in vivo in relation to insulin binding and action in adipocytes in vitro. J Clin Endocrinol Metab. 1984;59:1183-92. 5. Randle PJ, Garland PB, Hales CN, Newsholme EA. The glucosefatty acid cycle; its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet. 1963;l:785-9. 6. Ferrannini E, Barrett EJ, Bevilacqua S, DeFronzo RA. Effect of fatty acids on glucose production and utilization in man. J Clin Invest. 1983;72:1737-47. 7. Gerich J, Gottesman I, Bolli G, Campbell P, Kennedy F. Treatment of diabetes with L363.586. In: Reichlin S, ed. Somatostatin: basic and clinical status. Int Conf on Somatostatin 1986. New York: Plenum Press; 1987;303-ll. 8. Hagen JH. Effect of glucagon on the metabolism of adipose tissue. J Biol Chem 1961;236:1023-7. 9. Burns TW, Langley P. Observations on lipolysis with isolated adipose tissue cells. J Lab Clin Med. 1968;72:813-23. 10. Pozza G, Pappalettera A, Meloglin 0, Viberti G, Ghidoni A. Lipolytic effect of intraarterial injection of glucagon in man. Horm Metab Res. 1971;3:291-2. 11. Liljenquist JE, Bomboy JD, Lewis SB, et al. Effects of glucagon on lipolysis and ketogenesis in normal and diabetic men. J Clin Invest. 1974;53:190-7. 12. Schade DS, Eaton RP. Modulation of fatty acid metabolism by glucagon in man. I. Effects in normal subjects. Diabetes. 1975;24:502-9. 13. Gerich JE, Lorenzi M, Bier DM, et al. Effects of physiologic levels of glucagon and growth hormone on human carbohydrate and lipid metabolism. J Clin Invest. 1976;57:875-84. 14. Miyoshi H, Shulman GI, Peters EJ, Wolfe MH, Elahi D, Wolfe RR. Hormonal control of substrate cycling in humans. J Clin Invest. 1988;81:1545-5.

315

15. Keller U, Schnell H, Sonnenberg GE, Gerber PPG, Stauffacher W. Role of glucagon in enhancing ketone body production in ketotic diabetic man. Diabetes. 1983;32:387-91. 16. Sarcione EJ, Black N, Sokal JE, Mehlman B, Knoblock E. Elevation of plasma epinephrine levels produced by glucagon in vivo. Endocrinology. 1963;72:523-6. 17. Wieland RG, Hallberg MC, Zorn EM. Growth hormone response to intramuscular glucagon. J Clin Endocrinol Metab. 1973;37:32930. 18. Nestel PJ, Ishikawa T, Goldrick RB. Diminished plasma free fatty acid clearance in obese subjects. Metabolism. 1978;27:589-97. 19. Havel RJ, Naimark A, Borchgrevink CF. Turnover rate and oxidation of free fatty acids of blood plasma in man during exercise: studies during continuous infusion of palmitate-1-C14. J Clin Invest. 1963;42:1054-63. 20. Jensen MD, Haymond MW, Gerich JE, Cryer PE, Miles JM. Lipolysis during fasting: decreased suppression by insulin and increased stimulation by epinephrine. J Clin Invest. 1987;87:20713. 21. Jensen MD, Rogers PJ, Ellman MG, Miles JM. Choice of infusionsampling mode for tracer studies of free fatty acid metabolism. Am J Physiol. 1988;254:E562-5. 22. Miles JM, Ellman MG, McClean KL, Jensen MD. Validation of a new method for determination of free fatty acid turnover. Am J Physiol. 1987;252:E431-8. 23. Miles J, Glasscock R, Aikens J, Gerich J, Haymond M. A microfluorometric method for the determination of free fatty acids in plasma. J Lipid Res. 1983;24:96-9. 24. Herbert V, Lav KS, Gottlieb GW, Bleicher SJ. Coated-charcoal immunoassay of insulin. J Clin Endocrinol Metab. 1965;25:137584. 25. Nakagawa S, Nakayam H, Sasaki T, et al. A simple method for the determination of serum free insulin levels in insulin treated patients. Diabetes. 1973;22:590-600. 26. Faber 0, Binder C, Markussen J, et al. Characterization of seven C-peptide antisera. Diabetes. 1978;27(Suppl l):170-7. 27. Faloona G, Unger RH. Glucagon. In: Jaffe B, Behrman H, eds. Methods of hormone radioimmunoassay. New York: Academic Press; 1974;317-30. 28. Peake GT. Growth hormone. In: Jaffe B, Behrman HR, eds. Methods of hormone radioimmunoassay. New York: Academic Press; 1974;103-21. 29. Abumrad NN, Rabin D, Diamond DP, Lacy WW. Use of a heated superficial hand vein as an alternative site for measurements of amino acid concentrations and for the study of glucose and alanine kinetics in man. Metabolism. 1981;30:936-40. 30. Galster AD, Clutter WE, Cryer PE, Collins JA, Bier DM. Epinephrine plasma threshold for lipolytic effects in man. J Clin Invest. 1981;67:1729-38. 31. Jensen MD, Caruso M, Heiling V, Miles JM. Insulin regulation of lipolysis in nondiabetic and IDDM subjects. Diabetes. 1989;38:1595-601. 32. Jensen MD, Heiling V, Miles JM. Measurement of nonsteady-state free fatty acid turnover. Am J Physiol. 1990;258:E103-8. 33. Schade DS, Eaton RP, Alberti KGMM, Johnston DG. Diabetic coma. Albuquerque: University of New Mexico Press; 1981;64-71. 34. Chatzipanteli K, Saggerson D. Streptozotocin diabetes results in increased responsiveness of adipocyte lipolysis to glucagon. FEBS Lett. 1983;155:135-8. 35. Wu M-S, Jeng CY, Hollenbeck CB, Chen Y-DI, Jaspan J, Reaven GM. Does glucagon increase plasma free fatty acid concentration in humans with normal glucose tolerance. J Clin Endocrinol Metab. 1990;70:410-6. 36. Caruso M, Divertie GD, Jensen MD, Miles JM. Lack of effect of hyperglycemia on lipolysis in humans. Am J Physiol. 1990;259:E452-7. 37. Wicklmayr M, Dietze G, Rett K, Mehnert H. Evidence for a substrate regulation of triglyceride lipolysis in human skeletal muscle. Horm Metab Res. 1985;17:471-5.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 August 2014. at 10:44 For personal use only. No other uses without permission. . All rights reserved.

Effects of glucagon on free fatty acid metabolism in humans.

To determine whether physiological changes in plasma glucagon concentrations are important in regulating basal adipose tissue lipolysis, FFA flux ([1-...
935KB Sizes 0 Downloads 0 Views