Human islets chronically exposed in vitro to different stimuli become unresponsive to the same stimuli given acutely: evidence supporting specific desensitization rather than beta-cell exhaustion.

0021-972x/92/7404-0790$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society

Vol. 74, No. 4 Printed

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Human Islets Chronically Exposed in Vitro to Different Stimuli Become Unresponsive to the Same Stimuli Given Acutely: Evidence Supporting Specific Desensitization Rather Than ,&Cell Exhaustion ALBERT0 FEDERICO VALERIO

M. DAVALLI, ANTONIO E. PONTIROLI, BERTUZZI, BRUNO FATTOR, SIMONA DI CARLO, AND GUIDO POZZA

CARLO BRAGHI,

Istituto Scientifico San Raffaele, Cattedra di Clinica Medica (A.M.D., A.E.P., Cattedra di Patologia Chirurgica (C.S., V.D.C.), Uniuersitd. di Milano, Milan,

ABSTRACT. The aim of this study was to evaluate the effects of long term in vitro exposure of human pancreatic islets to different secretagogues on their subsequent secretory activity. Therefore, groups of 100 islets were cultured for 48 h in standard tissue culture medium (CMRL 1066) in the presence of 1 of the following: 5.5 mmol/L glucose, 16.7 mmol/L glucose, 5.5 mmol/ L glucose plus 10 mmol/L L-arginine, or 5.5 mmol/L glucose plus 100 rmol/L tolbutamide. Insulin levels in the culture medium declined with time under all culture conditions. Islets were then perifused and acutely stimulated with glucose (16.7 mmol/ L), L-arginine (10 mmol/L), and tolbutamide (100 rmol/L). Islets cultured in 16.7 mmol/L glucose showed no response to 16.7 mmol/L glucose [net area under the curve (A AUC), 11% of control], and a reduced response to acute tolbutamide (A AUC, 35% of control), but responded to L-arginine (A AUC, 75% of control). Islets cultured in the presence of 10 mmol/L Larginine had reduced responses to glucose (A AUC, 11% of control) and tolbutamide (A AUC, 27% of control), but responded to L-arginine (A AUC, 75% of control). Islets cultured

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F.B., B.F., S.B., G.P.) and Italy

in tolbutamide did not respond to tolbutamide (A AUC, 14% of control) and showed a reduced responses to acute glucose (A AUC, 36% of control) and L-arginine (A AUC, 24% of control). In a second set of experiments, islets cultured in 5.5 or 16.7 mmol/L glucose showed an insulin response to a supramaximal glucose stimulation (30 mmol/L glucose plus 0.5 mmol/L isobutylmethylxanthine) that was not statistically different. Similarly, islets that were cultured in the presence of 100 pmol/L tolbutamide still responded to 1 mmol/L tolbutamide. In conclusion, all stimuli evaluated in this study, chronically applied, reduced the insulin response to further acute stimulations. The different patterns of unresponsiveness observed together with the finding of a preserved insulin content in the islets after perifusions and a maintained capability to release insulin in response to supramaximal stimulations suggest that after chronic exposure to different stimuli, human islets become selectively desensitized to the same stimuli given acutely and do not become exhausted. (J Clin Endocrinol Metab 74: 790-794, 1992)

I

N ISLETS of Langerhans from rodents, insulin sensitivity to glucose and other stimuli is decreased after prolonged exposure to glucose, and this suggests that, at least in uitro, a third phase or a desensitization phase of insulin release may occur (l-4). The possibility exists that exhaustion or desensitization is a general phenomenon induced not only by glucose, but also by other secretagogues acting by similar or different mechanisms. In this study we evaluated the effect of prolonged in vitro exposure of human islets to different stimuli (glucose, L-arginine, and tolbutamide) on their subsequent sensitivity to the same stimuli given acutely.

Materials

and Methods

Islets isolation Isolated and purified human islets were obtained using the

automated method describedby Ricordi et al. (5), aspreviously reported (6). In uitro culture

After a 12-h overnight culture in CMRL 1066 (100% fetal calf serum, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 pg/mL streptomycin sulfate), groupsof 100islets (130-170 pm in diameter) were hand-picked and randomly transferred in four different media: standard CMRL 1066, as described above (at a glucose concentration of 5.5 mmol/L glucose); CMRL 1066 at 16.7 mmol/L glucose; CMRL 1066 plus 10 mmol/L L-arginine; and CMRL 1066plus 100 pmol/L tolbu-

Received January 15, 1991. Address all correspondence and requests for reprints to: Albert0 M. Davalli, Istituto San Raffaele, Via Olgettina 60, 20132 Milan, Italy. 790

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CHRONIC

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791

TABLE 1. Insulin releaseat different time periods under different culture conditions (pmol/islet/hour). Means + SE. n = 6 in all experiments

Hours of culture

Culture condition

6h

24 h

34.3 + 4.0” Glucose(5.5 mM) 211.0 f. 28.Sd Glucose(16.7 mM) Glucose(5.5 mM) + L-arginine (10 mM) 28.8 3~ 9.S8 Glucose(5.5 mM) + tolbutamide (100 pM) 150.6 f 10.6’ Valuesare the mean f SE, expressedas picomolesper islet/h (n = 6 in all experiments).

20.5 68.7 21.5 65.5

48 h

f 3.1* f 8.5’ + 5.gh _+ 9.2k

11.4 45.3 10.9 31.2

f. 2.6’ + 6.8’ + 3.8’ _+ 6.5’

‘vs. *, PC 0.025;” vs. ‘, PC 0.001; *IX ‘, P = NS; d us. e, P < 0.005; 'us.', P c 0.002; e us. f, P = NS;“us. h, P = NS;gus. ', P = NS; h us. ', P = NS; ' us. k, P < 0.001; j us. I, P < 0.001; k us. ', PC 0.02; a vs. d, PC 0.001; a vs. #, P = NS, a vs. j, P < 0.001; b US.e, P < 0.001; !JUS.h, P = NS; *vs. m, I’< 0.005; ' us. f, P < 0.005; ' us. i, P = NS; ' us. I, P c 0.05, d vs. t, P < 0.001; d US.j, P = NS; 8 vs. j, P < 0.001; e US.h, P < 0.001; e US.k, p = NS; h vs. k, P < 0.005;'~~. ',P < 0.005; fus. I, P = NS; 'us. ',P < 0.05. TABLE 2. Basal islet insulin release during the equilibration

Culture conditions

period of the perifusions

5.5 mM glusoce

Insulin release (pmol/islet . min)

0.16 f 0.01”

5.5 mM glucose +

0.22 + 0.01*

tolbutamide 0.18 + 0.01’

tamide. Islets were then cultured for a further 48 h in 95% air5% COZ at 37 C. Islets were plated in triplicate, and in the following perifusions each preparation was submitted to one acute stimulus only, with glucose, L-arginine, or tolbutamide. levels during

the culture

period

Insulin release during the culture period was evaluated by taking samples of culture media at different time periods (6). Perifusions In vitro perifusions were performed as previously described (6). The insulin responses to acute glucose (16.7 mmol/L), LGLUC.

3.3 mM 1

GLUC.

16.7 mM M @. -0 C - 4 A.*..A

06

lli’ I\

1 5.5 mM GLU 16.7 mM GLU 5.5 mM GLUtL-AAG 5.5 mM GLUtTOLB

Minutes

FIG. 1. Insulin responseto an acute glucose(GLU) stimulation after

a 48-h culture in different media. 0, Islets cultured in 5.5 mmol/L glucose; 0, islets cultured in 16.7 mmol/L glucose; A, islets cultured in 5.5 mmol/L glucose plus 100 rmol/L tolbutamide (TOLB); A, islets

cultured in 5.5 mmol/L glucoseplus 10 mmol/L L-arginine (ARG).

5.5 mM glucose + L-arginine 0.18 + 0.02d

periods of all perifusions performed. Values are the mean f

arginine (10 mmol/L), and tolbutamide (100 pmol/L) were evaluated. Perifusions lasted 60 min and consisted of a 40-min equilibration period (during which the glucose concentration of the perifusate was 3.3 mmol/L), followed by a ZO-min stimulation period, in which the perifusate was supplemented with glucose (to reach a final concentration of 3.3 or 16.7 mmol/L), 10 mmol/L L-arginine, or 100 pmol/L tolbutamide; the flow rate was 1 mL/min. In additional perifusions, islets were submitted to supramaximal stimulations in which the stimulus was alternatively 30 mmol/L glucose plus 0.5 mmol/L isobutylmethylxanthine (IBMX; Sigma, St. Louis, MO) or 3.3 mmol/ L glucose plus 1 mmol/L tolbutamide. Islet viability

and insulin

content

Islet viability after the 48-h culture period was assessedwith trypan blue exclusion and was greater than 95% after each culture condition. After culture and perifusions, the insulin content of islets was assessed (6). RIAs

Insulin content was assayed by RIA, using a commercial kit (Immunonuclear, Stillwater, MN). Calculations

Values are the mean + SE; n is the number of experiments.

glucose)

16.7 mM glucose

Data are calculated considering all insulin levels recorded during the equilibration SE (n = 66). a us. b, PC 0.001; a vs. ‘, P = NS;"vs. d,P = NS; *vs. ', P < 0.05.

Insulin

(3.3 mmol/L

and statistical

analysis

In all figures, n indicates the number of experiments performed, coincident with the number of pancreases studied if not otherwise specified. Data were expressed as the mean + SE. The integrated insulin release during perifusions was calculated by the linear trapezoidal method [net areas under the curve (A AUC)] and was expressed as picomoles per 100 islets/20 min). Comparisons were performed by factorial two-way analysis of variance; when analysis of variance yielded significant values (confidence interval, 95%)) variables were compared by Stu-


DAVALLI

792

ET AL.

JCE & M. 1992 Vol74.No4

TABLE 3. Integrated insulin response (AAUC; picomoles per 100 islets/20 min) to acute secretagogues after a period of 48 h in different culture conditions

Response to acute stimuli

Culture condition

Glucose (16.7

mM)

L-Arginine

(10

Tolbutamide

mM)

(100

PM)

Glucose (5.5 mM) 791 f. 168 (6)” 249 + 85 (9)* 1802 + 242 (7)' Glucose (16.7 mM) 89 f 80 (6)d 186 + 143 (9)' 639 + 200 (7)' Glucose (5.5 mM) + L-arginine (10 mM) 85 -+ 53 (6)g 186 f 91 (9)h 481 f 195 (7)’ Glucose (5.5 mM) + tolbutamide (100 pM) 284 f 65 (6)j 60 + 83 (9)' 254*147(7)' Values are the mean + SE; the number of experiments is in parentheses. Comparisons are made on the vertical lines; all other comparisons on the vertical lines are not significant. a us. d, P < 0.01; a vs.#, P < 0.01; a vs.j, P < 0.05;pvs.J,P < 0.05;'~~.', P < 0.05;'vs. ',P < 0.025;'vs. ', P < 0.01. 1

Results

GLUCOSE 3.3 mM [ L-ARGININE 10 mM

Insulin levels during culture periods 0.45 -

0.00

1 -10

1 -5

1 0

I 5

A--A

5.5

mt.4

GL”,L-ARG

L--CA

5.5

mt.i

GLU

I 10

I 15

‘TOLB

I 20

Minutes

2. Insulin response to an acute stimulation with L-arginine (ARG; 10 mmol/L) after a 48-hculture in different media. 0, Islets cultured in 5.5 mmol/L glucose (GLU); 0, islets cultured in 16.7 mmol/L glucose; A, islets cultured in 5.5 mmol/L glucose plus 100 pmol/L tolbutamide (TOLB); A, islets cultured in 5.5 mmol/L glucose plus 10 mmol/L Larginine. Values are the mean + SE; n is the number of experiments. FIG.

I

GLUCOSE 3.3 mM 1 TOLBUTAMIDE

2.1 1.8

O-.* &--.

,h

0.01 -10

-5

T 0

I 5

I 10

100

I.IM

Insulin release, calculated as insulin levels in the medium at 6,24, and 48 h, is summarized in Table 1. During the 48-h culture, insulin release progressively declined under all culture conditions: in islets cultured in the presence of L-arginine, insulin release was already low after 6 h, and the further decrease was not statistically significant. Perifusions Insulin releaseunder basalconditions

Insulin release during the equilibration period of the perifusions (at a glucose concentration of 3.3 mmol/L) is summarized in Table 2. Islets previously cultured in high glucose showed a higher basal insulin release than either control islets or islets cultured in the presence of tolbutamide.

55 mM GLU 16.7 mM GLU 5.5 mM GLU+L-ARG

I 15

I 20

Minutes

3. Insulin response to an acute stimulation with tolbutamide (TOLB; 100 rmol) after a 48-h culture in different media. 0, Islets cultured in 5.5 mmol/L glucose (GLU); 0, islets cultured in 16.7 mmol/ L glucose; A, islets cultured in 5.5 mmol/L glucose plus 100 pmol/L tolbutamide; A, islets cultured in 5.5 mmol/L glucose plus 10 mmol/L L-arginine (ARG). Values are the mean k SE; n is the number of experiments. FIG.

dent’s t test for paired or unpaired data as appropriate. P < 0.05 was considered statistically significant.

5.5 mM

16.7 mM

5.5 mM

5.5 mM + Tolb 100 )IM

4. Insulin response of the islets (A AUC insulin; picomoles per 100 islets/20 min) to supramaximal stimulations. A, The insulin response to an acute stimulation with 30 mmol/L glucose (GLU) plus 0.5 mmol/L IBMX; B, the insulin response to an acute stimulation with 1 mmol/L tolbutamide (TOLB). ARG, L-Arginine. Different culture conditions are given on the bottom. Values are the mean + SE; n indicates the number of perifusions performed. Both experiments shown in A and B were performed on islets obtained from three human pancreases. FIG.


CHRONIC

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ISLETS

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793

TABLE 4. Islet insulin content after culture

5.5mM glucose

Culture conditions Insulin content (pmol/islet)

16.7 mM glucose

381 + 70”

171 f 39*

5.5mM glucose + tolbutamide 160 + 27’

5.5mM glucose + L-arginine 217 f 42d

Values are the mean rt SE (n = 9). ’ us. b,p C 0.025; ’ us.‘, p < 0.001; ’ us. d, P = NS.

Acute stimulation with glucose

The insulin response to an acute glucose stimulus is shown in Fig. 1. The response to glucose was reduced after every kind of chronic stimulations; nevertheless, calculations of the integrated insulin responses (Table 3) showed that insulin release was maximally reduced in the islets cultured in the presence of high glucose or Larginine. In additional experiments, islets cultured for 48 h in the presence of 5.5 or 16.7 mmol/L glucose were challenged with a supramaximal glucose stimulus (30 mmol/L glucose plus 0.5 mmol/L IBMX); as shown in Fig. 4A, the insulin response to this stimulus was not significantly different in the two groups. Acute stimulation with L-arginine

Figure 2 shows the insulin response to an acute Larginine stimulus. The islet’s response to L-arginine was not affected by chronic exposure to glucose, arginine, or tolbutamide. In fact, the integrated insulin responses were similar in the different groups, with the exclusion of islets cultured in the presence of tolbutamide, which showed a decreased (although not significantly, P = 0.06) response to L-arginine (Table 3). Acute stimulation with tolbutamide

Figure 3 shows that insulin release in response to 100 hmol/L tolbutamide was decreased by all chronic stimulations. The response was maximally reduced in islets previously exposed to tolbutamide (Table 3). In additional experiments, a supramaximal tolbutamide stimulation (1 mmol/L) was able to elicit insulin response in islets preexposed to 100 bmol/L tolbutamide (Fig. 4B). Insulin content after experimental culture and perifusions

Table 4 summarizes the insulin content after culture and perifusions. The insulin content was reduced after all chronic stimulations, but the difference was statistically significant only for islets cultured in high glucose (45% of control) or in the presence of tolbutamide (42% of control). Discussion In the present study we evaluated the secretory activity of human islets chronically exposed to different stimuli.

Insulin release during the 48-h culture period decreased with time under all experimental conditions, suggesting that culture by itself is partially responsible for the time-related decrease in insulin release. During perifusions, islets cultured in the high glucose medium showed increased insulin release in the presence of low glucose, i.e. during the equilibration period. This finding agrees with the paradoxical insulin response to a low glucose concentration previously observed in islets cultured in high glucose (6) and contrasts with the hypothesis that such islets are exhausted. When challenged with acute stimuli, islets cultured in 16.7 mmol/L glucose showed a reduced insulin response to glucose and, to a lesser extent, to tolbutamide than islets cultured in 5.5 mmol/L glucose; in contrast, the response to L-arginine was preserved. The fact that the insulin response to a supramaximal glucose stimulus (30 mmol/L glucose plus 0.5 mmol/L IBMX) was not significantly different in islets cultured in 5.5 or in 16.7 mmol/ L glucose indicates that islets cultured in 16.7 mmol/L were not exhausted. A similar impaired response to acute stimulations was observed in islets chronically exposed to tolbutamide. These islets were mainly unresponsive to an acute equimolar tolbutamide stimulation and, to a lesser extent, to glucose. As happened with glucose, islets cultured in 100 pmol/L tolbutamide were responsive to a stronger tolbutamide stimulus (1 mmol/L). Perifusions performed on islets chronically exposed to L-arginine showed a decreased insulin response to acute glucose and acute tolbutamide, but not to L-arginine. However, in these islets, insulin levels were already low after 6 h of culture, and this suggests that, as reported for rodent islets (7), islets had been damaged by the long term exposure to L-arginine. In contrast, it is possible that the defective insulin release observed in islets exposed to high glucose and tolbutamide is due to desensitization to the same stimuli and not to a loss of islet viability or to P-cell exhaustion. In conclusion, this study shows that desensitization is a general phenomenon in human islets chronically exposed to different stimuli and is not limited to glucose. Since glucose and tolbutamide share some mechanisms involved in the stimulus-secretion coupling (8, 9), our study also supports the hypothesis that an impairment


DAVALLI

794 in one or two of these common pathways might at least in part for desensitization (10, 11).

account

References 1. Hoenig M, MacGregor LC, Matschinsky FM. In vitro exhaustion of pancreatic &cells. Am J Physiol. 1986;250:E502-11. 2. Bolaffi JL, Bruno L, Heldt A, Grodsky GM. Characteristics of desensitization of insulin secretion in fully in vitro systems. Endocrinology. 1988;122:1801-9. 3. Bolaffi JL, Heldt A, Lewis LD, Grodsky GM. The third phase of in vitro insulin secretion. Evidence for glucose insensitivity. Diabetes. 1986;35:370-3. 4. Grodsky GM. A new phase of insulin secretion. How will it contribute to our understanding of B-cell function? Diabetes. 198938673-8. 5. Ricordi C, Lacy PE, Finke EH, Olack BJ, Sharp DW. Automated method for isolation of human pancreatic islets. Diabetes. 1988;37:413-20.

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6. Davalli AM, Rico&i C, Socci C, et al. Abnormal sensitivity to glucose of human islets cultured in a high glucose medium: partial reversibility after an additional culture in a normal glucose medium. J Clin Endocrinol Metab. 1991;72:202-8. 7. Schafer G, Schatz H. Long-term effects of leucine and arginine on B-cell function of cultivated pancreatic rat islets. J Endocrinol. 1981;91:255-62. 8. Cook DL, Masatoshi I, Tolbutamide as mimic of glucose on P-cell electrical activity. ATP-sensitive K-channels as common pathway for both stimuli. Diabetes. 1989;38:416-21. 9. Zawalich WS, Zawalich KC. Induction of memory in rat pancreatic islets by tolbutamide. Dependence on ambient glucose level, calcium, and phosphoinositide hydrolysis. Diabetes. 1988;37:816-23. 10. Purrello F, Vetri M, Vinci C, Gatta C, Buscema M, Vigneri R. Chronic exposure to high glucose and impairment of K+-channel function in perifused rat pancreatic islets. Diabetes. 1990;39:3979. 11. Zawalich WS. Phosphoinositide hydrolysis and insulin secretion in response to glucose stimulation are impaired in isolated rat islets by prolonged exposure to the sulfonylurea tolbutamide. Endocrinology. 1989,125:281-6.


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