Control of insulin secretion during fasting in man

Control of Insulin Secretion During Fasting in Man Usah Lilavivathana,

R. G. Campbell,

During early starvation, declines in blood glucose and insulin levels occur concomitantly. To determine whether extracellular glucopenia is the primary signal for decreased insulin secretion, i.v. glucose was given as a continuous infusion to 7 lean male subjects throughout a 72-hr fast at the rate needed to maintain plasma glucose at the normal postabsorptive levels. Five lean male control subjects were fasted without glucose infusion. The acute insulin response to 5 g of i.v. glucose was determined after the fast in both groups. Significant glucopenia and concomitant insulinopenia were observed by 24 hr of fasting alone. In contrast, despite constant plasma glucose concentration of 86 f 3 mg/dl (&EM) in the subjects receiving glucose infusion, significant insulinopenia was observed by 48 hr (p < 0.01) and

and R. G. Brodows

there was essentially no difference in the mean insulin levels of the two groups. Significant free fatty acid increments were observed and comparable in both groups; however, the ketogenic response was significantly blunted by continuous glucose infusion (p < 0.001 at 72 hr). Plasma glucagon increased significantly (p < 0.001) by 48 hr in fasting alone and remained unchanged in the group receiving glucose. There was no significant change in urinary catecholamine excretion in the subjects receiving glucose. The acute insulin response to i.v. glucose was similar in both the starved and infused groups when compared to postabsorptive subjects. These data suggest extracellular glucopenia is not a prerequisite for insulinopenia in starvation.

D

IMINISHED INSULIN SECRETION during periods of total caloric deprivation in man is well recognized. Plasma insulin levels at 72 hr of fasting are approximately 40% less than postabsorptive levels.‘~’ Although the importance of this decline in modulating the metabolic response to starvation is firmly established,‘q3 the stimulus controlling insulin release is less-well understood. The simplest explanation is to attribute this fall in circulating insulin to the small decline in blood glucose concentration that occurs in the initial 48-72 hr of fasting.’ However, basal insulin levels do not always correlate with blood glucose after overnight or prolonged fasting.4 In the present study we attempted to clarify the importance of glucose as a signal for basal insulin secretion during starvation by preventing the usual decline in plasma glucose during the transition from the fed to the fasted state and evaluating the insulin secretory response. MATERIALS

AND

METHODS

Twelve healthy males within 10% of ideal body weight (according to Metropolitan Life Insurance Tables) between the ages of 20 and 34 yr were admitted to the Clinical Research Center

From the Endocrine-Metabolism Division, Genesee Hospital, the Endocrine-Metabolism l~nit. Monroe Community Hospital, and the Department of Medicine, Universit_v of Rochester School of Medicine and Dentistry, Rochester, N. Y. Receivedforpublication September 27, 1977. Supported in part by U.S. Public Health Service Training Grant Ah407092. Clinical Research Center Grant ECRC-RROO044. and the Carroll C. Colgan Fund. Dr. Campbell is the recipient oj-an Academic Career Development Awardfrom the National Institute of Health. Address reprint requests to Robert G. Brodows. M.D., Department of Medicine, The Genessee Hospital, 224 Alexander Street, Rochester, N. Y. 14607. o 1978 b.v Grune & Stratton, Inc. 0026-0495/78/2707-OOOS$Ol.CO/O Mefobolism, Vol. 27, No. 7 (July), 1978

815

LILAVIVATHANA,

816

CAMPBELL,

AND

BRODOWS

for 72 hr total fasting (5 males served as the control starvation group and 7 as the experimental group). All subjects were instructed to receive 300 g of carbohydrate daily for 3 days prior to the studies. The experimental group received a continuous 5% dextrose i.v. infusion by peristaltic infusion pump at the rate of 0.03 f .002 g/kg/hr (mean f SD) to maintain the plasma glucose concentration constant at the postabsorptive level throughout the 72-hr fast. Oral water was allowed up to the total daily fluid intake of 2500 ml. To prevent hypokalemia, all subjects in both groups received a potassium chloride supplement (0.5 meq/kg/24 hr). Blood samples were obtained each morning at 0, 24, 48, and 72 hr of starvation (0 hr represents a 12-hr overnight fast). Continuous 24-hr urine collections were made for catecholamines.5 Plasma was stored at -20°C until analyzed for free fatty acids (FFA), fl-hydroxybutyrate (BOH), glucose,’ and alanine.’ Insulin was assayed in triplicate by a double antibody method (Amersham-Searle) specially modified to obtain accurate measurements of insulin below 10 rU/ml. Within-assay and between-assay variability are 2.6 & 0.4 and 5.1 f 0.7 rU/ml, respectively. Glucagon was determined by radioimmunoassay in heparinized tubes containing 0.05 M benzamidine and utilizing reagents from Radioassay Systems Laboratories, Inc. (Carson, Calif.) Glucagon antibody cross-reactivity with gut glucagon is 0.018%. The assay is sensitive to 2.5 pg with intrassay variance of 6% at 70 pg/ml. Five controls and 5 glucose-infused subjects were given 5 g of 50% glucose by i.v. injection over 30 set at the end of the 72-hr fast, and blood samples for glucose and insulin were collected at 3, 5, and 10 min after the start of the injection. The incremental change in plasma glucose and insulin was calculated by subtracting the mean of the 3- and 5-min values from the mean of the control values (3-5 min AIR1 or glucose). In addition, the ratio of the 3-5 min AIR1 to 3-5 min A glucose (AIRI/AG) was calculated for each subject. These results were compared to those obtained in 11 lean postabsorptive subjects, ages 19-29, all with normal rates of glucose disappearance after i.v. glucose administration. Paired two-tailed t tests, Wilcoxan, or Mann and Whitney two-sample tests were used when applicable.8 Data are presented as mean + SE. RESULTS

Over 72 hr of fasting, the weight loss was 3.27 + 0.6 kg in the control group and 2.63 f 0.3 kg in the infused group. There were no significant changes in serum potassium concentrations in either group. Metabolic and hormonal responses in the control and glucose-infused groups are shown in Table 1. Both groups had similar postabsorptive plasma glucose Table

1. Plasma Metabolite and Hormone Concentrations for Glucose Infused

(I) and

Control (C) Group During 72-hr Fast (Mean f SEM) Hours of Fasting 24

0

48

72

Glucose (mg/lOO dl)

C I

87 f 2

89 f

FFA (PM)

C

664 f 61

1126 &70

I

751 z&443

1016rk116

&hydroxybutyrate

C

23 h-6

(PM) Alanine (@I)

I

82 +31

185 f 407

474 f

120*

682 f

C

457 f 42

377 f 33

286 f

16

266 f 28

409 f 25

429 f 22

403 f 22$

Insulin &J/ml

C

10.9 f 2.4

Glucagon (pg/ml)

C

90 f

tp < 0.05. sp < 0.01.

76 f 2 l*

996 zk417

67&2

61 zt4

88 f 2* 1481 ~1~666 1125 f

1217

2216 f 306

86 f 3* 1568 f 77 1360 f 89 3343 & 558 124*

409 f 301

4.9 f 0.7

3.7 f 0.7

3.0 f 0.5

5.7 f 0.7

4.7 f 0.6

127 f

127 k8

8.8 f

1.1

7.7 f

112 f

12

119+8

104 k4 *p < 0.001, control versus infused.

1

1.4

84 f 87

95 29

12

90 f 7t

INSULIN

SECRETION

IN FASTING

817

MAN

concentrations. In the control starvation group, glucose significantly declined to 76 + 2 mg/lOO dl (p < 0.001) by 24 hr and continued to fall to a nadir of 61 + 4 mg/lOO dl at 72 hr. During glucose infusion, the plasma glucose was maintained virtually constant throughout the 72-hr period. In contrast, a similar degree of insulinopenia was observed whether or not the plasma glucose was maintained at the postabsorptive level. In the control group, insulin fell significantly by 24 hr (p < 0.001) and reached the nadir by 72 hr. Despite constant glucose levels in the infused group, plasma insulin fell significantly by 48 hr (p < 0.01) and continued to decline at 72 hr. No statistically significant differences in insulin levels between the two groups were noted at any time during the fast. Figure 1 depicts the mean insulin/glucose ratios for the control and infused groups throughout 72 hr of fasting; the ratio declined in a similar fashion in both groups. FFA increased significantly by 24 hr in both groups ( p < 0.01) and continued to rise throughout the fast. In fasting alone, a marked rise of P-hydroxybutyrate was noted by 24 hr with maximal levels of 3343 =t 558 PM at 72 hr (p < 0.001). However, although a modest but significant increase of ,&hydroxybutyrate was noted by 24 hr (p < 0.05) in the infused group, the ketogenic response was markedly blunted compared to the noninfused group. Plasma glucagon levels rose modestly (but significantly) in the control group at 48 and 72 hr (p < 0.001, paired t test), but remained unchanged in the subjects infused with glucose. Alanine levels fell in the control group (p < 0.01) but remained constant in the infused group. The control group levels were lower than the infused group at 48 and 72 hr (p < 0.01). Urinary epinephrine excretion in the infused group was 37 f 9, 33 f 8, and 40 f 8 pg/24 hr and norepinephrine excretion was 146 f 29, 113 & 21 and 137 f 25 pg/24 hr at 24,48, and 72 hr, respectively. The acute insulin response to glucose (Table 2) was significantly less in both the infuseo and noninfused groups when compared with the postabsorptive group (p I 0.05). No difference was noted between starved and infused subjects. However, the 3-5 min AG was also diminished (p < 0.001) in both fasted

o .

Fig. 1. I/G ratio at 0, 24, 48, and 72 hr of fasting (mean f SEM). The I/G ratios between groups were not significantly different.

I

I

0

24

CONTROL

(n=5)

GLUCOSE

INFUSED

1

48

HOURS OF FASTING

72

(n=?)

818

LILAVIVATHANA, CAMPBELL, AND BRODOWS

Table 2. Acute Glucose and Insulin Response to 5 R lnlravenous in Postabsorptive,

Fasted (72-hr)

Infusion (72-hr)

and Fasted With Continuous Subjects (Mean

Glucose

=k SE) AIRI

3-5 min A IRI

3-5 min A GlUCO* Postabsorptive

Glucose Pulse

AG

45.2

& 1.26

47.7

f 0.99

1.13 f 0.2

36.7

f

1.07*

32.5

f

0.89

32.6

f 0.67*

27.6

f 3.2t

(n = 11) Fasted

1.567

f 0.10

(n = 5) Fasted with glucose

1.13 l 0.3

infusion

(n = 5)

*p < 0.001

from postabsorptive.

tp < 0.025.

groups compared to the postabsorptive rected for the degree of hyperglycemia all three groups.

group, so that the insulin response cor(AIRI/AG) was essentially the same in

DISCUSSION

Since pancreatic beta cell intracellular glucose concentration is essentially the same as extracellular glucose levels,9 and insulin secretion from the beta cell is proportional to peripheral blood levels in the basal state,‘O prevention of the usual decrement in plasma glucose during starvation should abolish the decline in plasma insulin if insulin is directly controlled by glucose. In the present study, continuous infusion of glucose at rates of 2.3 f 0.2 g/hr produced stable circulatory glucose levels during 72 hr of fasting without altering the typical pattern of insulin secretion associated with starvation. Decrements in plasma insulin in both the control group and the glucose-infused group were similar to those reported by others. ‘JI The insulin:glucose (I/G) ratios in both the noninfused and infused groups were similar to those recently reported by Merimee” in a large series of normal men and women using similar glucose and insulin methodology. If hypoinsulinemia is to be attributed to hypoglycemia, then one would predict the I/G ratio in the glucose-infused group to remain constant throughout the fast. On the contrary, the I/G ratio decreased in a similar fashion regardless of whether or not glucose was maintained at postabsorptive levels. These findings suggest that insulin secretion in early starvation is not controlled by glucose and concurs with the reported dissociation of glucose and insulin during fasting.4 A quantitative reduction in insulin response to glucose occurs during starvationlZ even though pancreatic insulin content is unchanged.13 The most consistent in vitro finding appears to be a block of glucose metabolism in one of the early glycolytic steps,14 although other abnormalities have been described.15 A decrease in the acute insulin response to glucose has been described by Fink I2 in 4%hr fasted subjects; however, the magnitude of the glucose stimulus was considerably greater than that used in the present study. Our results have failed to confirm a diminished glucose-stimulated insulin response during starvation utilizing a more physiologic glucose stimulus. The magnitude of the acute insulin response in this study is similar to that reported in normal subjects by

INSULIN

SECRETION

IN FASTING

MAN

819

Robertson et al.‘(‘*”in their extensive studies utilizing a 5-g intravenous glucose injection to evaluate the pancreatic glucose receptor in health and disease. The present protocol did not evaluate each subject’s insulin response before and after the fast. It is possible, but unlikely, that such a protocol would have documented a blunted acute insulin response during starvation. Alternatively, a greater hyperglycemic stimulus might be necessary to document insulin secretory deficiencies in starvation as has been demonstrated by Cerasi, Luft, and Efendic” in prediabetics. If glucose does not control fasting insulin secretion, what other potential control mechanisms exist? Enhanced adrenergic activity inhibits insulin release.19 Some investigators have proposed that the insulinopenia of starvation is the result of enhanced sympathetic activity since urinary and plasma catecholamines may be elevated during starvation; however, the present study has failed to confirm these findings. Furthermore, neither Misbin et a1.20nor Walter et al.2’ found adrenergic blockade effective in altering basal insulin levels during starvation. Previous studies from our laboratory have failed to document abnormal insulin secretion during starvation in catecholamine-deficient subjects,6 and recently, Young and Landsberg 22have suggested that sympathetic activity may even be decreased in starvation. Such observations make it unlikely that the sympathetic system is a major controlling influence on insulin secretion during early starvation. Enhanced lipolysis and ketogenesis were noted in all infused subjects, as expected, in the insulin deficient state. The finding that BOH levels were significantly lower in the infused group than in the noninfused group despite similar FFA concentrations is possibly due to the lack of rise in plasma glucagon in these subjects. Previous studies have demonstrated an increase in ketogenesis in insulin-deficient diabetics during physiologic glucagon infusion,23 and McGarry and Foster24 have recently stressed the importance of glucagon excess for activation of the hepatic ketogenic process. Glucagon has been reported to increase liver carnitine levels25 as well as decrease hepatic malonyl-CoA concentrations. Both of these effects would result in enhanced ketogenesis.27 It is conceivable, although unproven, that intravenous glucose infusion has resulted in increased malonyl-CoA production and thus diminished ketogenesis in the infused subjects. It is also possible that the small rise in plasma BOH in the infused group was, in part, related to the effect of glucose on renal ketone metabolism. Sapir et al. 28demonstrated decreased ketoacid excretion after ingestion of as little as 7.5 g of glucose daily in prolonged starvation. Thus, although BOH excretion was not measured in the current study, a modest increment in the plasma BOH levels could be the result of decreased BOH clearance from glucose administration. The hypoalaninemia noted following 72 hr of starvation has recently been attributed, in part, to the concomitant fasting hyperketonemia.29 Thus, the observation that plasma alanine levels failed to decline in our glucose-infused subjects could be related to their decreased circulating BOH concentrations. In addition, suppressed glucagon levels in these subjects could have resulted in diminished hepatic alanine extraction. Could the changes in FFA and BOH observed in the infused subjects account for their insulin response? It is unlikely, since neither endogenous FFA nor exogenous ketone infusions result in alterations in plasma insulin in man.29.30

LILAVIVATHANA,

820

CAMPBELL,

AND

BRODOWS

Insulin secretion may also be enhanced by amino acids-arginine, lysine, leucine, and threonine being the most potent. 31During early starvation, decrements in arterial concentrations of arginine and threonine have been noted; however, increased levels of leucine have also been observed.32 Is it possible that the continuous glucose infusion was responsible for diminished insulin secretion by decreasing insulinogenic amino acid levels? Although no amino acids other than alanine were measured in the present study, that explanation seems unlikely since infusion of glucose at rates three times greater than in the present study fails to significantly alter arterial concentrations of amino acids.33 Current evidence suggests that changes in amino acid metabolism during early starvation are a consequence of, rather than the cause for, the decline in insulin.32 The observation that insulin secretion is greater in response to ingested rather than intravenously administered glucose has resulted in an intense search for intestinal factors that could initiate this response. Secretin, gastrin, and cholecystokinin-pancreozymin have all been suggested as possible potentiators of pancreatic beta cell secretion. 34-36Current findings indicate that gastric inhibitory polypeptide (GIP) is a very important intestinal factor influencing insulin and glucagon secretion. GIP is secreted during glucose ingestion; endogenous as well as exogenous GIP enhanced insulin secretion stimulated by intravenous glucose.37*38GIP, however, fails to alter basal insulin levels, and would thus be an unlikely controlling factor of insulinopenia during starvation. In conclusion, insulinopenia during short-term starvation appears not to be due to the concomitant fall in plasma glucose. It is unlikely that the decline in plasma insulin is related to changes in free fatty acids, ketones, amino acids, or increased sympathetic tone. The exact mechanism remains to be elucidated. ACKNOWLEDGMENT The authors wish to thank Elaine King, Diane Nichols, and Elaine Rago for secretarial help.

and Karen

Baker for technical

assistance,

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PH: Effect of fasting upon insulin secretion in the rat. Am J Physiol213:843-848, 1967 14. Levy J, Herchuelz A, Sener A, et al: The stimulus-secretion coupling of glucose-induced insulin release. XX. Fasting: A model for aitered glucose recognition by the &cell. Metabolism 25:5833591, 1976 15. Howell SL, Green IC, Montague W: A possible role of adenylate cyclase in the longterm dietary regulation of insulin secretion from rat islets of Langerhans. Biochem J 136:343349, 1973 16. Robertson RP, Porte D Jr: The glucose receptor: A defective mechanism in diabetes mellitus distinct from the beta adrenergic receptor. J Clin Invest 52:870-876,1973 17. Robertson RP, Chen M: A role for prostaglandin E in defective insulin secretion and carbohydrate intolerance in diabetes mellitus. J Clin Invest 60:747-753, 1977 18. Cerasi E, Luft R, Efendic S: Decreased sensitivity of the pancreatic beta cells to glucose in prediabetic and diabetic subjects: A glucose dose-response study. Diabetes 21:224-234, 1972 19. Porte E Jr, Robertson RP: Control of insulin secretion by catecholamines, stress and the sympathetic nervous system. Fed Proc 32: 1792-1796, 1973 20. Misbin RI. Edgar PJ. Lockwood DH: Influence of adrenergic receptor stimulation on glucose metabolism during starvation in man: Effects on circulatory levels of insulin growth hormone and free fatty acids. Metabolism 20: 544-554, 1971 21. Walter RM, Dud1 RJ, Palmer JP, et al: The effect of adrenergic blockade on the glucagon responses to starvation in hypoglycemia in man. J Clin Invest 54:1214~1220, 1974 22. Young JD, Landsberg L: Decreased sympathetic activity during fasting. Clin Res 24: 640A, 1976 23. Schade DS, Eaton RP: Glucagon regulation of plasma ketone body concentration in human diabetics. J Clin Invest 56:1340-1344, 1975 24. McGarry JD, Foster DW: Ketogenesis and its regulation. Am J Med 61:9-13, 1976 25. McGarry JD, Robles-Valpes C, Foster DW: Role of carnitine in hepatic ketogenesis. Proc Nat1 Acad Sci USA 72:4385-4388, 1975 26. Cook GA. Lakshmanan MR. Veech RL:

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