Regulation of insulin release in persistent hyperinsulinaemic hypoglycaemia of infancy studied in long-term culture of pancreatic tissue

Diabetologia, Aug 1990

Pancreatic tissue was obtained during therapeutic subtotal pancreatectomy from five infants with persistent hyperinsulinaemic hypoglycaemia of infancy (so-called nesidioblastosis). Collagenase digests of the specimens were cultured in RPMI 1640 medium on extracellular matrix-coated plates. Acute insulin secretion showed minimal sensitivity to changes in glucose concentration. Sensitivity to other nutrient secretagogues such as glyceraldehyde, leucine, α-ketoisocaproic acid and arginine was variable, showing either diminished or absent response. On the other hand, stimulators of Beta cell cAMP and modulators of the phosphoinositide-protein kinase C pathway were effective inducers of insulin release. The response to cAMP stimulators was independent of the glucose concentration. Although insulin output was high in the absence of glucose, this was not due to passive leak of hormone, since both removal of calcium and addition of somatostatin and epinephrine inhibited the secretion. Beta cells were more sensitive to somatostatin than epinephrine; however, both agents failed to completely suppress the release even at suprapharmacological concentrations. Although it cannot be excluded that the culture conditions affected Beta cell function, the present findings may suggest that cultured Beta cells in persistent hyperinsulinaemic hypoglycaemia of infancy behave like fetal Beta cells at early developmental stages.

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Regulation of insulin release in persistent hyperinsulinaemic hypoglycaemia of infancy studied in long-term culture of pancreatic tissue

Diabetologia Regulation of insulin release in persistent hyperinsulinaemic hypoglycaemia of infancy studied in long-term culture o f pancreatic tissue N. Kaiser s 0 P. Corcos 0 A. Tur-Sinai 0 Y. A r i a v 0 B. G l a s e r 0 H. L a n d a u 0 E. Cerasi 0 0 Departments of ~ Endocrinology and Metabolism and 2 Pediatrics, Hebrew University Hadassah Medical Center , Jerusalem , Israel Summary. Pancreatic tissue was obtained during therapeutic subtotal pancreatectomy from five infants with persistent hyperinsulinaemic hypoglycaemia of infancy (socalled nesidioblastosis). Collagenase digests of the specimens were cultured in R P M I 1640 medium on extracellular matrix-coated plates. Acute insulin secretion showed minimal sensitivity to changes in glucose concentration. Sensitivity to other nutrient secretagogues such as glyceraldehyde, leucine, c~-ketoisocaproic acid and arginine was variable, showing either diminished or absent response. On the other hand, stimulators of Beta cell c A M P and modulators of the phosphoinositide-protein kinase C pathway were effective inducers of insulin release. The response to c A M P stimulators was independent of the glucose concentration. Although insulin Nesidioblastosis; hyperinsulinaemic hypoglycaemia; insulin release; islet culture; Beta cell maturation; hypoglycaemia 9 Springer-Verlag1990 Persistent hyperinsulinaemic hypoglycaemia of infancy ( P H H I ) is a rare condition of which the aetiology is not clear. A wide spectrum of pancreatic morphological features has b e e n described; nesidioblastosis, initially thought to be the underlying cause, is c o m m o n to all neonates and infants [ 1-5 ]. The nuclear volume of B e t a cells is augmented [ 1, 5 ], but its significance in P H H I is unclear, since in accordance with observations in islets and in other endocrine cells [ 5-7 ] it may rather be the consequence of increased secretory activity. The biochemical defect of the endocrine pancreas in P H H I is also unknown, and only few investigators have succeeded in studying islet function u n d e r controlled in vitro conditions [ 8, 9 ]. Aynsley-Green et al. were successful in isolating islets from the pancreatic tissue of one out of three infants with P H H I ; during short-term incubations they observed an abnormal Beta cell sensitivity to glucose, with maximal stimulation at 4 mmol/1 glucose [9]. In the present study we utilized an improved m e t h o d for islet cell culture in order to characterize B e t a cell function in infants with P H H I . T h e m e t h o d enables us to obtain monolayer cultures of pancreatic islets from adult rats in which B e t a cells retain their differentiated function for several weeks output was high in the absence of glucose, this was not due to passive leak of hormone, since both removal of calcium and addition of somatostatin and epinephrine inhibited the secretion. B e t a cells were more sensitive to somatostatin than epinephrine; however, both agents failed to completely suppress the release even at suprapharmacological concentrations. Although it cannot be excluded that the culture conditions affected Beta cell function, the present findings may suggest that cultured Beta cells in persistent hyperinsulinaemic hypoglycaemia of infancy behave like fetal Beta cells at early developmental stages. in vitro [ 10 ]. In the present investigation we studied in some detail the regulation of insulin secretion by various physiological and pharmacological modulators using chronic cultures of pancreatic cells from infants with P H H I . Materials and methods Origin of tissue Pancreatic tissue was obtained during therapeutic subtotal pancreatectomy from five infants suffering from intractable PHHI. The diagnosis of PHHI was based on the following criteria: spontaneous hypoglycaemia with inappropriately high plasma insulin levels; glucose requirement in excess of 15 mg. kg- 1.min- 1in order to prevent hypoglycaemia;elevation ofblood glucoseby more than 1.67mmol/1 after glucagon administration; documentation of appropriate counterregulatory hormone responses; and low levels of non-esterifled fatty acids and ketone bodies. All infants were delivered at term after a normal pregnancy.Their relevant clinicaldata is presented in Table 1. Pancreatic cultures A, B, C, D and E were from infants aged 26, 10, 6, 9 and 3.5 weeks, respectively, at the time of surgery.Tissue resected from the head and the tail of the pancreas was immediately placed into ice-cold sterile RPMI 1640 medium and transferred to the laboratory for further processing. N. Kaiser et at.: Beta cell function in hyperinsulinaemichypoglycaemia of infancy Fig. 1 A-F. Cultures of pancreatic tissue from patients with persistent hyperinsulinaemic hypoglycaemia of infancy on extracellular matrix-coated plates. A Islet-like cell clusters after 1 day in culture. B 3-day-old culture. C 5-day-old culture. D 9-day-old culture. E 2-week-old primary culture showing a cluster of epithelial-likecells on top of a fibroblastoid cell layer. F An isolated group of epithelial cells forming a monolayer patch in a secondary culture (A-E, phase contrast x 100;F, phase contrast x 200) Cultureprocedure Pancreatic tissue (-1 g) was minced with scissors into 1-2 mm pieces which were digested by vigorous shaking at 37~ in 5 ml Hepes-buffered (10 mmol/1) Hanks' balanced salt solution (HBSS), pH 7.4, containing6 mg/ml collagenase (0.94 U/mg, Serva Feinbiochemica, Heidelberg, FRG). After 20-30 rain the digest was diluted with an equal volume of cold HBSS, centrifuged, and washed four times with HBSS at 4~ Since only few opaque islet-like structures could be identified under the stereomicroscope, no attempt was made to isolate pure islets, and the whole digest was resuspended in RPMI 1640 medium (GIBCO, Grand Island, NY, USA) containing antibiotics (105 U/1 penicillin and 100 mg/1 streptomycin). After three additional washes in this medium at room temperature, the pellet was resuspended in RPMI 1640 medium containing antibiotics and 10% fetal calf serum (FCS, Biological Industries,Beth Haemek, Israel) at 37~ The digest of i g pancreatic tissue was suspended in 60 ml culture medium and 2 ml of this cell suspension plated into 35 mm Falcon culture dishes coated with extracellular matrix (ECM) derived from bovine corneal endothelial cells as described previously [ 10, 11 ]. The cultures were maintained at 37~ under 6% CO2 in air with twice weekly changes of the medium. Most studies were performed a Glucose and insulin were assayed in the same plasma sample. Cortisol and growth hormone were from other episodes of hypoglycaemia (glucose less than 2.3 retool/l); b Age of admission at this hospital. PHHI = persistent hyperinsulinaemichypoglycaemiaof infancy with primary cultures. In some experiments secondary cultures were obtained from pancreatic cultures B and D by mild trypsin digestion and replating on new ECM-coated dishes; results were essentially similar in primary and secondary cultures. Study o f hormone content and release The culture medium was changed 16-24 h before each experiment and collected to determine chronic insulin secretion. At the start of experiments RPMI 1640 medium was replaced by 1 ml KrebsRinger bicarbonate buffer, pH 7.4, containing 10 retool/1 Hepes, 0.25% BSA (KRBH buffer) and 0 or 3.3 mmol/1 glucose. The cultures were preincubated in this buffer for i h at 37~ then the buffer was discarded, and the cultures were incubated in i ml of the same buffer for another h to give the 1st h insulin secretion rate. Thereafter, buffers containing various concentrations of glucose with or without other agents were added and the incubationcontinued for a further h (2nd h of incubation).The incubationbuffers were stored at - 20~ for insulinassay. In some experiments after glucose stimulation, the cultures were extracted for 24 h at 4~ with acid-ethanol (15 m112 tool/1HC1of 70% ethanol) for determinationof cellinsulin content. Insulinwas measured by radioimmunoassay,using guineapig anti-porcine insulin antibodies (Linco Research Inc., Eureka, Mo, USA) and a second antibody to separate bound and free hormone. Human insulin (Novo Research Institute, Bagsvaerd, Denmark) was used as a standard. The minimaldetectable concentration was 0.7 + 0.06 mU/1. Intraassay coefficient of variance (CV) was less than 6% across the entire range of the assay,while interassay CV was 13%, 9.7% and 10% at insulinconcentrations of 7, 23, and 83 mU/1, respectively. Data presentation Since results could not be related to Beta cell number or protein content in these mixed cell cultures, and insulin secretion rates were variable, each plate was subjected to consecutive treatments with glucose and other test compounds, the insulinsecretion rate during the 2nd h calculated as %, or fold increase, of the 1st h of incubation, and results were grouped and normalized to untreated control. Statistical analysis Data were computed as mean _+SEM and compared using the nonparametric Mann-WhitneyU test. When data obtained at different glucose concentrationswere pooled, e. g. when studyingthe effect of cAMP modulators on insulinsecretion, we used the Kruskal-Wallis one-way analysis of variance to test the effect of ambient glucose concentration. R e s u l t s M o n o l a y e r c u l t u r e s c o m p o s e d of a m i x e d cell p o p u l a t i o n w e r e o b t a i n e d f r o m all p a n c r e a t i c s p e c i m e n s . D u r i n g t h e 1st d a y of c u l t u r e , islet-like cell clusters of i r r e g u l a r s h a p e w e r e s e e n , w h i c h w i t h i n s e v e r a l h o u r s a t t a c h e d to t h e E C M - c o a t e d p l a t e s (Fig. 1 A ) . A f t e r 3 days, m o s t of t h e cell clusters w e r e f l a t t e n e d ; t h e c u l t u r e s c o n t a i n e d e p i t h e l i a l like cells, t o g e t h e r w i t h f i b r o b l a s t o i d cells a n d b i n u c l e a t e d cells, t h e l a t t e r p r o b a b l y of e x o c r i n e o r i g i n (Fig. 1 B). O n the 5 t h day of c u l t u r e discrete p a t c h e s of e p i t h e l i a l - l i k e cells w e r e a p p a r e n t ( c e n t r e of Fig. 1 C). T h e a p p e a r a n c e of the c u l t u r e did n o t c h a n g e m u c h o v e r t h e n e x t few days N. Kaiser et al.: Beta cell function in hyperinsulinaemichypoglycaemia of infancy i!ii!ii~ii!iiilii~iiiiiii!~iii:iili~~iil~i~iilli i~i!i~i!i i.i.i.i.:.il iiii!iiiiiiiiiii iii i ii iiiiiiiiiiiiiiiiiiiiiiiii iiiii!ii!iiiiiiiiiiiiiiiiiiii iii!iiiiiill :iiiii iiiiiii!iiiiiiiii!iiii iiiiiiiiiii!!iiiiliiiii~iiiiil iiiiii!iiiiiiiiiiiiiiilii!iiiiii !iiiii!iiii!iiiiiiiiiiii i~i:i~il i;il;'[i iiiiiiiiiiiiiiiiiiiiiiiiiii ~iiiiiiiiiiiiiiiiiiiiiiiliiiiiiiiit iiiiiiiil iiiiii?iii 0 3.3 0 6,9 0 16.7 0 ii~ilii~i iiiiiii ~:i~'i ~i!i i~:::!il 3.3 16.7 1.7 16.7 3.3 16.7 3,3 Glucose concentration (rnmol/I) F i g . 3 A - E . Effect o f m o d u l a t o r s o f c A M P on insulin secretion. Cultures were incubated for i h in glucose-free Krebs-Ringer bicarbonate-Hepes buffer or in buffer containingup to 16.7 mmol/1glucose. During the 2nd h of incubation,cultures were exposed to buffer containing varying concentrations of glucose (0-16.7 mmol/1) with or without 3-isobutyl-l-methyLxanthine (IBMX) (0.1mmol/1, black column), forskolin (25 gmol/1, hatched column) and glucagon (30 nmol/1,grey column). Insulinsecretion during the second h of incubationwith and without the cAMP modulators was calculated as % of 1st h insulinoutput. Results obtained at different concentrationsof glucose were pooled for each modulator, since by Kruskal-Wallis analysis of variance glucose was not found to affect islet response to the modulators. Pooled results were normalized to modulator-free controls (taken as 100%, dashed line) and presented for each culture as mean _+SEM of 3-24 plates, p values, calculated relative to control cultures by the Mann-WhitneyU test, were < 0.002 for IBMX and < 0.005for forskolin in all cultures tested. Glucagon caused a significant increase in insulinsecretion (p < 0.05) onlyincultures B, C and E (Fig. 1 D ) ; h o w e v e r , a f t e r 2 w e e k s i n c u l t u r e (Fig. 1 E), t h e f i b r o b l a s t o i d cells grew o v e r t h e e n d o c r i n e cells, t h e l a t t e r a p p e a r i n g as i s o l a t e d clusters o n t o p of a f i b r o b l a s t layer. T h e b i n u c l e a t e d cells w e r e n o l o n g e r p r e s e n t . S u b c u l t u r e of t h e cells b y m i l d t r y p s i n d i g e s t i o n a n d p l a t i n g o n n e w E C M c o a t e d plates, f o l l o w e d b y t h i m e r o s a l t r e a t m e n t to elimie,2 ._= e100' ~ ~ , ~ 80' Fig.4. Dose-response curve for the effect of epinephrine and somatostatin on insulin secretion. One to 4-week-old cultures of pancreatic tissue from patients B, C, D and E were incubated for two consecutive hours: first in presence of 3.3 mmol/1 glucose in KrebsRinger bicarbonate-Hepes buffer, followed by a 2nd h in the same buffer with and without epinephrine (open squares) or somatostatin (black squares). Insulin secretion during the 2nd h of incubation relative to the 1st h in control cultures containing glucose alone, is taken as reference (100%) for the effect of the inhibitors. Symbols and vertical lines denote mean  SEM of 5-15 plates n a t e f i b r o b l a s t s [ 10 ], r e s u l t e d in s e c o n d a r y c u l t u r e s w h i c h c o n t a i n e d r e d u c e d n u m b e r s of e p i t h e l i a l - l i k e e n d o c r i n e cells with little f i b r o b l a s t c o n t a m i n a t i o n (Fig. 1 F). H o w ever, d u e to excessive loss of v i a b l e cells, this p r o c e d u r e was a b a n d o n e d , m o s t s t u d i e s b e i n g p e r f o r m e d in p r i m a r y cultures. T h e c u l t u r e s of p a t i e n t s A a n d B s u r v i v e d for 8 w e e k s a f t e r w h i c h insulin s e c r e t i o n d i s a p p e a r e d a n d all cells b e c a m e f i b r o b l a s t o i d . T h e c u l t u r e s of p a t i e n t s C, D a n d E w e r e lost a f t e r 2, 4 a n d 3 w e e k s , respectively, b y bact e r i a l c o n t a m i n a t i o n d u e to the r e p e t i t i v e use of t h e plates. C h r o n i c insulin s e c r e t i o n was e v a l u a t e d b y m e a s u r i n g t h e a c c u m u l a t i o n of insulin in t h e R P M I 1640 m e d i u m (11.1 mmol/1 g l u c o s e ) 16-24 h a f t e r m e d i u m change. V e r y high levels w e r e o b s e r v e d o n d a y 4, w h i c h g r a d u a l l y dec r e a s e d d u r i n g t h e first w e e k of culture. M o s t of o u r studies w e r e p e r f o r m e d in 1-4 w e e k - o l d cultures. D u r i n g this p e r i o d islet f u n c t i o n was stable, a n d no effect o f cult u r e t i m e o n c h r o n i c o r a c u t e l y s t i m u l a t e d insulin secretion was o b s e r v e d . T h e 2 4 h insulin s e c r e t i o n was 9 5 + 1 2 % a n d 102 + 29% (n = 3) of tissue insulin c o n t e n t in c u l t u r e s D a n d E, respectively, w h i l e a s e c r e t i o n r a t e of 16 + 2% (n = 6) of c o n t e n t was f o u n d in c u l t u r e s e x p o s e d to gluc o s e - f r e e K R B H b u f f e r for I h. T h e v i a b i l i t y of B e t a cells was t e s t e d at v a r i o u s t i m e s d u r i n g c u l t u r e using 0.1 mmol/1 3 - i s o b u t y l - l - m e t h y l x a n t h i n e ( I B M X ) , o r 25 gmol/1 forskolin; cells w e r e cons i d e r e d v i a b l e if t h e y i n c r e a s e d t h e i r insulin o u t p u t b y at l e a s t 1.5 fold. B y this c r i t e r i o n , v i a b l e B e t a cells w e r e still p r e s e n t in s o m e of t h e m i x e d cell c u l t u r e s e v e n a f t e r 8 w e e k s (6 o u t o f 9 c u l t u r e s r e s p o n d e d to f o r s k o l i n w i t h 2 - 3 fold i n c r e a s e in insulin s e c r e t i o n ) . Cultures were incubated in buffer containing either no glucose or 3.3 mmol/1 glucose for I h followed by a 2nd h of incubation in the same buffer with no additives (control) or with the indicated nutrients each at 10 mmol/1. Results are expressed as mean + SEM. a Stimulation factor is computed for individualplates by calculating the ratio between 2nd and 1st h insulin secretion; the values were then nor_ malized in relation to control incubations (assigned a factor of 1.00). b p values are calculated relative to their respective controls using the Mann-Whitney U test. ~ KIC: c~-ketoisocaproicacid, PHHI = persistent hyperinsulinaemic hypoglycaemia of infancy Treatment Culture D T h e ability of B e t a cells to r e s p o n d a c u t e l y to glucose was a s s e s s e d in all cultures. A s s e e n in F i g u r e 2, i n c r e a s i n g glucose c o n c e n t r a t i o n f r o m 0 mmol/1 d u r i n g the 1st h o f i n c u b a t i o n to 1.7-16.7 mmol/1 d u r i n g the f o l l o w i n g h h a d little effect o n insulin r e l e a s e ; o n l y c u l t u r e s D a n d E res p o n d e d with a - 3 0 % i n c r e a s e . I n light of this o b s e r v a t i o n w e t e s t e d c u l t u r e s D a n d E for t h e i r a b i l i t y to s t o p insulin o u t p u t u p o n r e d u c t i o n of t h e glucose c o n c e n t r a t i o n f r o m 16.7 to 0 mmol/1. I n s p i t e o f t h e s m a l l B e t a - c e l l r e s p o n s e to an i n c r e a s e in m e d i u m glucose, t h e c u l t u r e s f a i l e d to res p o n d to its o m i s s i o n (1 h i n c u b a t i o n s ; d a t a n o t shown). T h e failure o f the c u l t u r e s to r e s p o n d to an a c u t e glucose stimulus p r o m p t e d us to s t u d y t h e i r a b i l i t y to r e a c t to o t h e r n u t r i e n t s ( T a b l e 2). E x p o s u r e to s u b s t r a t e s e c r e t a g o g u e s at e i t h e r 0 o r 3.3 mmol/1 glucose e v o k e d v a r i a b l e r e s p o n s e s . W h i l e c u l t u r e s B a n d C s h o w e d n o s e c r e t o r y r e s p o n s e to t h e t e s t e d n u t r i e n t s , c u l t u r e s D a n d E inc r e a s e d insulin s e c r e t i o n 1.3-2 fold in r e s p o n s e to glycera l d e h y d e , leucine, ~ - k e t o i s o c a p r o i c acid a n d arginine. T h e r e s p o n s e o f t h e c u l t u r e s to p h a r m a c o l o g i c a l a n d p h y s i o l o g i c a l m o d u l a t o r s of c A M P is s h o w n in F i g u r e 3. B o t h I B M X a n d f o r s k o l i n w e r e effective in a u g m e n t i n g insulin s e c r e t i o n , i r r e s p e c t i v e of t h e m e d i u m glucose conc e n t r a t i o n . G l u c a g o n c a u s e d o n l y a m o d e s t s t i m u l a t o r y effect in t h r e e out of f o u r t e s t e d c u l t u r e s (27 to 88% a b o v e control). T a b l e 3 shows t h e effects of c a r b a c h o l , a m u s c a r i n i c c h o l i n e r g i c a g o n i s t w h i c h s t i m u l a t e s p h o s p h o l i p i d b r e a k d o w n in n o r m a l islets [ 12 ], 1 - m o n o o l e o y l - r a c - g l y c e r o l ( M O G ) , an i n h i b i t o r of d i a c y l g l y c e r o l k i n a s e [ 13 ], a n d 12O - t e t r a d e c a n o y l p h o r b o l 1 3 - a c e t a t e ( T P A ) , an a c t i v a t o r of p r o t e i n k i n a s e C. T P A a n d c a r b a c h o l , b u t n o t M O G , s t i m u l a t e d insulin r e l e a s e w h e n t e s t e d at 3.3 mmol/1 glucose, c u l t u r e E b e i n g m o r e r e s p o n s i v e t h a n c u l t u r e D to t h e s e agents. C a l c i u m , a m a j o r r e g u l a t o r of insulin s e c r e t i o n in n o r m a l islets, was i m p o r t a n t also for insulin r e l e a s e f r o m Cultures were incubated in the presence of 3.3 mmol/1 glucose without additions for I h, followed by a 2nd h of incubation with and without (control) various agents as indicated. " Stimulation factor is computed for individual plates by calculating the ratio between 2nd and 1st h insulin secretion; the values were then normalized in relation to control incubations (assigned a factor of 1.00). b TPA = 12-0-tetradecanoylphorbo113-acetate, PHHI = persistent hyperinsulinaemic hypoglycaemiaof infancy, MOG = 1-monooleoyl-rac-glycerol Cultures were incubated in Krebs-Ringer bicarbonate-Hepes (KRBH) buffer modified as indicated in the Table. Ca-free buffer also contained 5 mmol/1 EGTA. Insulin secretion during i h of incubation is expressed as mean + SEM. The number of plates is in parenthesis. % change relative to control cells in regular KRBH buffer containing the same concentration of glucose, b p values are calculated relative to their respective controls using the Mann-Whitney U test. PHHI = persistent hyperinsulinaemic hypoglycaemia of infancy P H H I p a n c r e a t i c c u l t u r e s ( T a b l e 4). O m i s s i o n of c a l c i u m r e d u c e d insulin s e c r e t i o n b y - 8 5 % . This effect o f c a l c i u m free m e d i u m was o b s e r v e d b o t h in the p r e s e n c e a n d a b sence o f glucose. E p i n e p h r i n e a n d s o m a t o s t a t i n (Fig. 4) e l i c i t e d a d o s e d e p e n d e n t s u p p r e s s i o n of insulin s e c r e t i o n in all t e s t e d cultures. Maximal inhibitory response (40% of untreated control culture) was obtained with 6 nmol/1 somatostatin, whereas epinephrine caused a similar effect only at 5500 nmol/1. While full dose response curves were conducted only in the presence of 3.3 retool/1 glucose, the pancreatic tissue of patient B was also exposed to the inhibitors under glucose-free conditions. In the absence of glucose, epinephrine (5500nmol/1) and somatostatin (600 nmol/1) reduced the output of insulin by 58 + 10% and 32 +_1% (n = 3), respectively. Discussion Over the past decade 28 patients with PHHI have been studied in our medical centre [ 14 ]. It became possible to study the in vitro islet function in this disorder only recently by the development of improved islet cell culture techniques. Indeed, by using ECM-coated plates as a support surface, we have been able to culture rat islets with maintenance of normal B eta cell function for periods longer than 2 months [ 10 ]. This report represents our initial efforts to characterize Beta cell function, using the above technique, in the last five patients with PHHI to undergo subtotal pancreatectomy. In contrast to rat or adult human pancreas, and in accordance with the experience ofAynsley-Greenet al. [ 9 ], only a small number of islets could be isolated from the pancreas of PHHI patients by collagenase digestion; we therefore cultured the entire pancreatic digest. While this technique permitted us to use repeatedly the same plates for functional studies and thus compensated for the scarcity of tissue, we could not prevent the overgrowth of fibroblasts which precluded studying the biochemical characteristics of these islet cells. In several studies with adult and fetal human islets, elevation of Beta cell cAMP was shown to be the most effective stimulus of insulin secretion [ 15-18 ]. Using IBMX or forskolin we monitored the presence of Beta cells in our cultures. By this criterion, viable Beta cells were present for as long as 8 weeks in culture; this is similar to our experience with rat pancreatic Beta cells cultured on ECM-coated plates [10]. The salient feature of insulin release in our study is the deficient response to changes in ambient glucose concentration. Similar glucose unresponsiveness is typical of the human fetal pancreas at its early stages of development [ 16, 18, 19 ]. In freshly incubated islets of a PHHI patient, Aynsley-Green et al. [ 9 ] found an abnormal sensitivity to glucose, with maximal stimulation of insulin release at 4 mmol/l. It is not clear to us if this difference from our observations is due to the techniques used, or whether it reflects a characteristic of the one patient studied by Aynsley-Green et al. In the present studies, in addition to absent or limited responses to raised glucose levels, the omission of glucose failed to reduce the secretion. This too is similar to findingsin perifused islet-like cell clusters from pancreases of human fetuses at 17-20 weeks of gestation which, although showing a -1.6 fold increase in insulin secretion, did not shut off the release when the glucose concentration was reduced from 20 to 2 mmol/1 [ 18 ]. The response to other nutrient secretagogues was variable with some of the cultures showing a modest response. Also in vivo some PHHI patients showed sensitivityto leucine and arginine [ 9, 20 ]. Thus, some heterogeneity exists regarding the extent of nutrient non-recognition by these Beta cells. Insulin secretion in pancreatic cultures of PHHI is a regulated function despite the lack of sensitivity to nutrients. Indeed, agents that augment Beta cell cAMP stimulated insulin release in a manner quantitatively similar to the response of normal islets [ 10,21,22 ]. The insulinotropic effect ofIBMX, forskolin and glucagon was independent of ambient glucose. Also, human fetal islets respond to agents elevating intracellular cAMP in the absence of glucose [ 17 ], thus differingfrom the response of adult rat islets [ 23 ]. Insulin secretion could also be stimulated in PHHI cultures via the phosphoinositide-protein kinase C pathway, since both carbachol and TPA effectivelyincreased hormone release. The diacylglycerolkinase inhibitor MOG was inactive by itself and did not potentiate the effect of carbachol. The significance of this observation is not clear to us. The insulin secretory characteristics of PHHI discussed so far could indicate that in this disorder the normal maturation process that allows the full expression of substrate sensitivity in the Beta cell is deficient. However, these patients suffer from hypoglycaemia,i. e. inability to suppress insulin secretion when blood glucose falls below baseline. In agreement with the clinical picture, the insulin release rate in our cultures was not reduced in the total absence of glucose. However, insulin release is not caused by leakage from the Beta cell, since removal of calcium from the incubation medium reduced markedly the release. Thus, exocytosis appears to characterize secretion also in these Beta cells, which respond normally to the two physiologicregulators (calcium and cAMP) of insulin release. Another finding which may be of clinical significance was that even at high pharmacologic concentrations both somatostatin and epinephrine failed to abolish the insulin secretion. The sensitivity of the cultured Beta cells to somatostatin was several orders of magnitude greater than to epinephrine. This in vitro finding is of interest since some PHHI patients respond well to the administration of somatostatin or its analogues [ 24-27 ], while the endogenous catecholamine response to hypoglycaemiafailsto suppress insulinrelease. An additional finding that may be related to the intractable hypoglycaemia of PHHI is the high turnover rate of insulin observed in our cultures. Indeed, ~16% of cell content of insulin was secreted during i h in glucosefree medium, compared to 1.2 + 0.1% (n = 16) in adult rat islets cultured similarly on ECM-coated plates (unpublished observation). Others have shown a similar low turnover rate in cultured fetal and adult human islets [ 28, 29 ] and in suspended adult human islets [30]. Thus, P H H I Beta cells seem to show an insulinoma-like high rate of release of their insulin content. In summary, our data suggest that in PHHI the Beta cells resemble cells in the early fetal pancreas by their defective recognition of nutrient secretagogues and lack of glucose dependency of their cAMP mediated insulin release [ 16,19 ]. On the other hand, the substantial reduction of insulin release obtained with hormonal inhibitors (somatostatin and epinephrine) or with removal of calcium ions in the absence of glucose may suggest that even without substrate these Beta cells are in a "stimulated" state. S o m e c a u t i o n is h o w e v e r n e c e s s a r y f o r t h e i n t e r p r e t a t i o n o f t h e s e results. I n d e e d , a l t h o u g h l o n g - t e r m c u l t u r e s o n E C M h a v e b e e n s h o w n n o t t o m o d i f y t h e r e s p o n s i v e ness o f r a t islets [ 10 ], p a n c r e a s e s f r o m h e a l t h y i n f a n t s h a v e n o t b e e n t e s t e d in this s y s t e m . T h u s , a n a r t e f a c t t h a t m a y i n d u c e n u t r i e n t u n r e s p o n s i v e n e s s s e l e c t i v e l y in i n f a n t b u t n o t r a t B e t a cells c a n n o t b e e x c l u d e d . F u r t h e r s t u d i e s , inc l u d i n g c u l t u r e o f islet cells f r o m n o r m o g l y c a e m i c i n f a n t s , will b e n e c e s s a r y to v a l i d a t e t h e c o n c l u s i o n s o f t h e p r e s e n t i n v e s t i g a t i o n in r e l a t i o n to t h e a e t i o l o g y o f P H H I . Acknowledgments. The authors are grateful to Ms. D.Zemach for insulin determinations and to Ms. L. Granot for typing the manuscript. This work was supported by grants from the Wolfson Foundation, the George Grandis Endowment for Medical Research and the Fund for Basic Research Administered by the Israel Academy of Sciences and Humanities. A. R Corcos was a recipient of a Juvenile Diabetes Foundation International Scholarship. Dr. N. Kaiser Department of Endocrinology and Metabolism Hadassah University Hospital R O. Box 12000 91120 Jerusalem Israel 1. Jaffe R , Hashida Y , Yunis EJ ( 1980 ) Pancreatic pathology in hyperinsulinemic hypoglycemia of infancy . 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N. Kaiser, A. P. Corcos, A. Tur-Sinai, Y. Ariav, B. Glaser, H. Landau, E. Cerasi. Regulation of insulin release in persistent hyperinsulinaemic hypoglycaemia of infancy studied in long-term culture of pancreatic tissue, Diabetologia, 1990, 482-488, DOI: 10.1007/BF00405110