- Email: [email protected]
Dispersed adult rat pancreatic islet cells in culture: A, B, and D cell function
Dispersed
Adult Rat Pancreatic Islet Cells in Culture: A, B, and D Cell Function
G. C. Weir, The
availability
freshly
of suitably
dispersed
somatostatin culture.
adult
release
We
then
to Petri
mitochondria
clustered
of the
cell facing
function with
of the
those
for islets 17300
islets
responsiveness plus IBMX was
of islet
the
medium. islets
seen
with
freshly
cells
than
for glucagon.
structure-function of intact
it was
in culture
possible in parallel. either with
Somatostatin
Dispersed
determined
in primary
by islet
structure.
granules
concentrated
hormone
content
rates
cellular
of release
Whereas
the
appeared
and hormone
ratio
in glucagon insulin
Proinsulin
was
lower
reveal
from
several
ratios
dispersed
ways
In contrast
cells
in which
was similar ratios to the
to glucose
(incorporation than
to be a valuable the
being lack of
and glucose
of [“HI leucine)
from
model
as a
compared
content
in response
with
release
were
(glucagon:insulin
biosynthesis
thus appear
level,
the portion
or hormone
loss of A cells.
cells released
6 cells which
towards
content
or
monolayer
ultrastructural
of somatostatin:insulin
depleted or relative
islets.
at the
Since
glucagon,
islets
system
function
while
the
for the study
of dispersed
cells
islets.
AND
METHODS
(DMEM. Grand Island Biological Co.) containing 10% heat inactivated newborn calf serum and 5.5 mmol/L glucose was added. The cells were then centrifuged for ten minutes at 150 x g, and the supernatant discarded. Nine milliliters of DMEM was added to the pellet and the cells were again centrifuged. The pellet was resuspended in I mL of DMEM and cell counts were performed using a hemocytometer. Hormone
Loss With
Dispersion
Batches of ten intact islets were placed into IO x 75 mm plastic tubes and extracted in 500 ~1 of acid ethanol at 4 “C for 16 hours. The extracts were then stored at --20 “C until they were assayed. A portion of the islets (600) were dispersed using the method described above and suspended in 3 mL of culture medium 199 (Grand Island Biological Co.). containing IO’%heat inactivated newborn calf serum and 8.3 mmol/L glucose. The number of cells obtained from these 600 islets averaged I .3 x IO’. Fifty PL aliquots were pipetted into IO x 75 mm tubes and extracted as above. The remaining cells and islets were then cultured in medium 199 with glucose 8.3 mmol/L for two hours. These cells and islets were then washed twice with KRB-Hepes containing 2.8 mmol/L glucose and extracted for hormone content determination as described below.
of Islet Cells
Islets were obtained by collagenase digestion and hand-picking”’ from pancreases of male Wistar and Sprague-Dawley rats weighing between 160 and 240 g. The islets were washed with a modified Krebs-Ringer bicarbonate (KRB) buffer containing 10 mmol/L Hepcs (2-[IV-2-hydroxyethylpiperazin-N’yl] ethanesulphonic acid) [Grand Island Biological Co., Grand Island, NY), 5 mmol/L NaHco,. 5 mg/ml. bovine serum albumin (BSA, fraction V, from Behringwerke AC, Marbug/Lahn, FRG), and 2.8 mmol/L glucose. They were then incubated in calcium-free phosphate buffered saline (PBS), pH 7.4, containing 0.54 mmol/L EDTA and 0.1% Trypsin (1:250), (Dlfco Laboratories, Detroit, MI). for 12 to I5 minutes at 37 “C in a total volume of 5 mL contained in a 10 mL siliconized glass tube. The islets were then disaggregated by being aspirated 10 to 20 times through a siliconized Pasteur pipette (internal diameter of tip was about 0.9 mm). The tube was immediately placed on ice and then 4 ml, of Dulbecco’s modified Eagle’s tissue culture medium
Metabolism,
established
dish and secretory
release
HE STRUCTURE of the islets of Langerhans is highly organized and in man and rat the B cells are located in a central core with A, D, and PP cells forming a surrounding mantle.’ This organization, combined with the knowledge that the hormone released from a given cell type has potent effects upon the other islet cell types,’ ’ and the fact that specialized junctions are found between islet cells,‘.“,‘suggests that islet function is influenced by its structure. It has been difficult to investigate this likely contribution of structure to function with currently available in vitro techniques. However, methods have been described for dispersing adult islet cellsxm’3 and an understanding of the behavior of such cells could provide insights into the coordination of islet function. In this study tissue culture techniques were used to compare the function of dispersed adult islet cells with that of intact islets.
Dispersion
partially
function.
in insulin,
preserved
cultured
results
of islet
well
islet cells in monolayer and these
in studies (no alteration
cells were
were
degranulation
T
MATERIALS
days
islet cells, the cultured seen
assist
these
to estimate
fractional
may
to secretagogues
is at least
of four
L. Orci, and A. E. Renold
preparations to IBMX),
cell population
reflecting
to that
relationships,
response
to the Petri
not
dispersed
islets.
cell
appropriately
period
of cells,
dispersed
C. B. Wollheim,
islet function
attached
types
for cells)
comparable
in cultured
differs from that
the
modest
that
maintained
(-0.7:100),
islet
to respond
a culture
Since
of specific
and 4:lOO
was true
hypothesis during
P. Meda,
dispersed
change;
at the cell face
in a fashion
higher
opposite
glucose
dishes
the
for intact
for
after
number
and cells
characterized
rat islet cells failed
tested
had attached
P. A. Halban,
Vol
33, No 5
(May),
1984
From
the lnstitut
de Biochimie
logic et d’ Emhryologir.
Received for publication
(Frankfurt,
Switzerland).
the Kroc
from
is a recipient
National Address
Medical
c‘ol/ege
of
Virginia.
Endocrinolog_v.
Foundation,
the National
Institutes
of’ a Research requests
Medical
National
W, Germonyl.
Science Foundation reprint
25, 1983.
the Swiss
AG
and AM-00255 Meda
Juiv
by grants from
Hoechst
(Nyon.
d’ Hrsto-
Cencva. Switzerland
Virginia.
Supported tion,
and lnstitut
of Geneva.
of Endocrinology.
and the Division Richmond.
Cliniqur.
lJniver.ritj,
Scirnce
Founda-
Fondation
and grants.
oJ‘Health
Fellowship
from
Zyma
AM-20349 (USAI.
Dr
the Swiss
(83.896.O.Hl). to G. (I College
of
Weir,
Box
Virginia.
693.
Division
Richmond.
of VA
23298. (~11984 bv Grune
& Stratton,
Inc.
002~-04~5/~4/3305--nO13$0l.00/0
447
448
The hormone content of the cells is expressed in terms of islet equivalents (IE). Islet equivalents refer to the initial islet number. For instance 600 starting islets are considered 600 IEs regardless of cell loss, hormone loss, or the volume in which the cells are suspended. Culture of Cells and Islets Cells or islets were washed twice under sterile conditions with tissue culture medium 199. The islets were transferred in known numbers with the aid of a dissecting microscope to culture dishes (Falcon 1007 (60 x I5 mm) or 1008 (35 x 10 mm diameter), Falcon, Oxnard, CA), these dishes being chosen to prevent islet attachment. The resuspended cells were aliquoted with an Eppendorf pipette into type 3001 Falcon dishes (35 x 10 mm) which were noncoated, or coated with disrupted fibroblasts or poly-L-lysine. The fibroblast coating of Falcon 3001 dishes was carried out by growing pancreas-derived fibroblasts to contluence and then lysing them with sterile water as has been previously described.” Poly-L-lysine HBr (Sigma, St. Louis, MO) of 350,000 daltons was dissolved in sterile water to give a concentration of 300 pg/mL and I mL was added to groups of 20 to 40 Petri dishes (Falcon 3001). The solution remained in the dishes for one hour at room temperature and was then removed by suction. The dishes were then washed three times with sterile water and stored for up to one week at 4 “C wrapped in aluminum foil. All of the secretion and biosynthesis studies carried out on cultured cells employed cells which were plated on polyL-lysine-coated plates. Our preliminary studies led us to the conclusion that this coating enhanced the attachment of dispersed islet cells, but subsequent studies have revealed minor if any effect. Visual inspection during the culture period gave us the strong impression that poly-L-lysine promoted attachment, but we now suspect that this is only a weak electrostatic effect which is lost when the plates are washed. The fibroblast-coated dishes were used for the electron microscopy studies. In studies not included in this manuscript it was found that the secretory behavior of cells plated on these dishes was similar to that found with poly-L-lysine-coated or uncoated dishes. The insulin content of the cells remaining on the fibroblast-coated dishes after washing was sometimes lower than the content left on other dishes (range 42% to 100% of noncoated dishes). It must be emphasized that this is only a difference in insulin content and does not necessarily correlate with the efficiency of attachment. The culture medium for both the islets and the cells was 199 containing 8.3 mmol/L glucose, 10% fetal calf serum, penicillin (400 U/mL), streptomycin (200 pg/mL), and gentamycin (50 pg/mL). Cells were incubated in 2 mL of media at 37 “C in a humidified, 5% CO, atmosphere. The islets were similarly incubated in either 2 or 4 mL of medium. Hormone Release Studies in Cells and Islets In experiments examining hormone release by freshly dispersed cells and fresh islets, the cells and islets were preincubated for two hours in culture medium 199 with 8.3 mmol/L glucose. The cells and islets were washed twice with KRB-Hepes buffer with glucose 2.8 mmol/L, and then either 47,000 cells or ten islets were dispensed into siliconized glass tubes containing 1 mL of KRB-Hepes containing either 2.8 or 16.7 mmol/L glucose with or without 3-isobutyl1-methylxanthine (IBMX, Sigma Co, St. Louis, MO) and 600 U/mL of Aprotinin (Trasylol, Bayer A.G., Wuppertal FRG). The incubations lasted 90 minutes and were carried out at 37 OC.Tubes were then put on ice and centrifuged at 4 OCfor ten minutes at 150 x g. The supernatants were removed and frozen. Two millimeters of KRB-Hepes containing aprotinin (600 U/mL) was added to the pellets and the tissue was sonicated and frozen prior to measurement of hormone content.
WEIR ET AL
Hormone release was also studied from cells and islets which were cultured in parallel for four days. Floating cells and culture medium were removed by suction and the dishes containing attached dispersed cells were then washed three times with KRB-Hepes buffer with 2.8 mmol/L glucose at room temperature, with the addition and removal of 2 mL of KRB-Hepes at each stage. Islets were washed three times with the same buffer using siliconized tubes and centrifugation. The attached cells were then incubated in dishes containing 1 mL of KRB-Hepes buffer with glucose, IBMX, and aprotinin as above. The islets were distributed under microscopic observation into similar dishes (Falcon 3001) with 10 islets and I mL of the above buffers being contained in each dish. Dishes were incubated at 37 “C for 90 minutes and the incubation buffer was removed with particular care taken to leave the islets behind. The incubation buffer from both the cell and islet experiments was centrifuged for ten minutes at 150 x g to insure removal of the small number of contaminating cells (the hormone content of these cells was found to be less than 5% of the total content of the incubated cells). To extract the cells and islets, 1 mL of KRB-Hepes-aprotinin buffer was added to the dishes, and the bottoms were scraped with a rubber policeman. The buffer was then removed with a Pasteur pipette and placed into 10 x 75 mm glass tubes. The dishes were then washed with an additional one ml of buffer and this was added to the same glass tubes. The tissue was then sonicated and frozen pending hormone assay. Hormone release in all experiments was expressed as percent of content. The content value used for these calculations was the mean of determinations made on tissue studied at all three experimental conditions. In some of the experiments the insulin content after incubation with IBMX was slightly reduced, but this caused only a minimal reduction in the value used for the calculations. Insulin Biosynthesis The cultured ceils and islets used for the biosynthesis experiments were handled as described for the above secretion studies. The KRB-Hepes incubation buffer contained [4, 5-‘H] leucine (Radiochemical Centre, Amersham, Buckinghamshire, UK) with a specific radioactivity of 65 Ci/mmol, at a concentration of 50 &i/mL. Following incubation, a thorough washing was required to remove the extra cellular (‘H) leucine. Therefore the plates with attached cells were washed three times with KRB-Hepes buffer as described above. The islets were transferred to siliconized glass tubes and washed three times with the same buffer as has been described elsewhere.” One milliliter of glycine buffer 0.2 M with 0.25% bovine serum albumin, pH 8.8, was added to the dishes. After cells were dislodged with a rubber policeman, they were transferred to glass tubes, sonicated, and ultracentrifuged.” Immunoprecipitation of labelled proinsulin and insulin” was carried out with a large excess of guinea pig anti-pork insulin (Miles Research Products, Rehovot, Israel), followed by precipitation of the antibody-antigen complexes with protein A-Sepharose (Pharmacia, Zurich, Switzerland). Nonspecific binding was determined by performing parallel precipitations for each sample using nonimmune guinea pig serum in place of antiinsulin serum. The exact method and the relative specific and nonspecific binding properties of this method have been described in detail previously.” All results are expressed as the specifically immunoprecipitable radioactivity (total less nonspecific). The relative proportions of labeled proinsulin and insulin were not determined. Radioimmunoassays Immunoreactive insulin, glucagon, and somatostatin were determined by radioimmunoassay using the charcoal separation method of Herbert et a1.16Rat insulin, pork glucagon, and synthetic cyclic somatostatin were used as standards.
DISPERSED
ISLET
CELLS
IN
449
CULTURE
Data Presentation Data are expressed as mean and standard error of the mean. For statistical comparisons the two-tailed unpaired Student’s t-test was used.
Electron Microscopy At the end of an experiment, the medium was removed from the dishes and the cultures were fixed for one hour at room temperature in a 2.5% glutaraldehyde solution in 0.1 M phosphate buffer, pH 7.4, postfixed for one hour in 1% osmium tetroxide and dehydrated in graded ethanols. The cultures were then embedded in Epon and sectioned either parallel or perpendicular to the Petri dish bottom. Thin sections mounted on carbon-coated grids were stained with uranyl acetate and lead citrate and examined in a Philips EM 301 electron microscope. RESULTS
Cell Yield and Hormone Content Following Dispersion
After cells were dispersed they were washed in KRB-Hepes buffer once and counted. The cell yield was calculated from 20 dispersions. The starting number of islets was 1030 + 101 (range 550 to 2400) and the yield of cells per islet was 1425 * 80 (range 873 to 2 188). When the cell suspensions were examined on 14 separate occasions the percentage of cells which were single was 69 + 4 (range 48 to 86). An experiment was designed to determine the hormone loss which occurs during the acute dispersion process to gain some insight into degree of cell lysis and destruction which occurs during the procedure. As can be seen in Table 1 there was a fall of between 28% and 47% of hormone content occurring during the acute dispersion process. During the subsequent two-hour period in culture medium there was a modest additional drop in content to about 50% of the initial intact islet content, with all three hormones having been reduced to a similar degree by the end of the two hour period. The cell number during the two hour period in culture medium fell by a mean of 11%. The fall in hormone content of the intact islets during this twohour period was 33% for insulin, 16% for glucagon, and 17% for somatostatin (data not shown). Thus, the major ioss of hormone content of the dispersed cell preparation occurred during the acute dispersion pro-
cess with a smaller loss being observed during the subsequent two-hour period in culture medium. It was not practical to measure the number of cells in the initial islets, but these data suggest that the initial fall in hormone content can be ascribed to the cell loss which takes place during the traumatic dispersion process. Culture of Dispersed Islet Cells: Morphology
Regardless of the substratum employed there were certain general characteristics in the appearance of dispersed cells in culture. The attachment of cells seemed to be maximal after two to three days in culture and usually remained at that level until the fifth day of culture. The number of cells which attached, as judged by their stationary behavior when the dishes were moved, was usually about 30% to 60%. For the experiments in this study at least 47,000 cells were plated per dish and when examined after four to five days in culture about 30% to 50% of the cells were single with the rest consisting of groups of between two to five cells (Fig. 1). At times there were larger zones of aggregated cells in monolayer containing 20 or more cells. No more than 5% to 10% of the singlets or small groups (two to four cells) appeared spread out on the culture support and otherwise the rounded appearance seen in Fig. 1 was the norm. When the cultured cells were studied by electron microscopy it was usually observed that the single cells had a characteristic hemispheric shape (Fig. 2). Thus the overall shape was globular except where the cell had attached to the culture support (Fig. 2). At that site, the membrane appeared flattened against the culture support. In single attached B cells. mitochondria were consistently clustered at the portion of the cell facing the culture support, while most of the secretory granules were found at the portion of the cell facing the medium. Hormone Release From Freshly Prepared Dispersed Cells and Islets
The insulin release from freshly dispersed cells and from islets incubated in parallel (both groups having been preincubated for two hours in culture medium)
Table 1. Hormone Loss With islet Disruption isletsPnor
Cells lmmedlately
to Disruption
Following
Cells Followng
Disperswn
Dispersion and 2.hr Culture % Of
ngI1slet
Nt
ng/lE’
% of
Initial Islet Content
ng/lE’
lnltlal islet Content
Insulin
10
51.3
+ 7.0
28.2
-t 2.8
57.7
+ 4.4
25.1
i; 2.8
50.3
t
Glucagon
10
4.87
k 0.80
4.09
k 0.82
72.0
+ 4.9
3.14
i
0.52
58.3
+ 3 2
Somatostatin
10
0.660
+ 0.023
53.2
i: 3.3
i
0.025
47.4
+ 3.8
llE AND tN
is islet equivalent,
METHODS refers
which
*
IS an expression
0.344
0.057 of the
quantity
of cells obtained
section.
to the number
of replicates
contained
in two
separate
experiments.
from
each disrupted
0.313 islet.
It is described
in more
detail
4.0
in the MATERIALS
450
WEIR ET AL
Fig. 2. Electron micrograph of an isolated B cell which had been cultured for four days on osmotically disrupted fibroblasts. Culture medium was 199 containing 18.7 mmol/L glucose and 10% newborn calf serum. This thin section, which was cut perpendicular to the bottom of the culture dish, shows that, as it was consistently observed in our cultures, this single cell has a characteristic hemispheric shape. The arrow head points to an area of the flat portion where the plasma membrane of the 8 cell is closely opposed to the bottom of the culture dish. Note the good preservation of the cell ultrastructure and the peculiar distribution of intracellular organelles. Most of the mitochondria are clustered at the portion of the cell facing the culture support while most of the secretory granules are found at the portion of the cell facing the medium. The bar represents 2 pm; x 7,300.
Fig. 1. Dispersed attached adult islet cells cultured for three days on Poly-L-lysine-coated culture dishes as seen with phase contrast. Medium was 199 containing 8.3 mmol/L glucose and 10% fetal calf serum. Single cells, either round or spread. and clumps of two or more cells can be seen. The bar represents 50 pm: x160.
was quite different (Table 2). The release rates at low glucose were similar, but the response to high glucose (16.7 mmol/L) by the cells was negligible, whereas the release from islets increased 13-fold. In addition the insulin release from the cells in response to IBMX was considerably lower than was seen from islets. The glucagon release from cells was much higher than it was from islets, and with each there was modest stimulation by IBMX (2P < 0.01). Glucagon release from islets was slightly (2P -C0.05) decreased by high glucose. The somatostatin release from cells in the presence of both low and high glucose was higher than that from islets. High glucose failed to stimulate release from either cells or islets. The combination of glucose 16.7 mmol/L and IBMX did stimulate the release of somatostatin from islets (2P -C O.Ol), but not from cells. The residual hormone content tended to be similar for all three hormones when the three different incubation conditions were compared, with two exceptions.
The insulin content of islets after incubation in the combined presence of IBMX and high glucose was reduced relative to the other two conditions (2P -C 0.01). In contrast the islet somatostatin content after incubation at glucose 2.8 mmol/L was marginally lower than that seen after incubation at the higher glucose concentration, both with and without IBMX (2P < 0.05). Hormone Release From Cultured Dispersed Cells and Islets
Insulin release from higher than that from incubation conditions percent of stored insulin
cultured islets was slightly dispersed cells for all three studied when expressed as (Table 3). The stimulation of
Table 2. Hormone Release and Content of Freshly Dispersed Cells Versus Islets During a 90-Minute Incubation* Insulin
Glucagon
Cells
Islets
Celk
Somatostatin islets
Cells
Islets
Release (% of Content) (mmol/L) Glucose2.8
2.98 k 0.20
Glucose 16.7
3.88
+ 0.37
31.90
Glucose 16.7 + IBMX 1
16.5 k 0.96
73.1
content (ng) Glucose 2.8
334
187 k 13
14.3 + 1.26
14.4 Yk0.22
1.770
Glucose 16.7
326 k 31
171 + 6
12.3 + 0.85
16.8 + 0.48
1.990
Glucose 16.7 + IBMX 1
301
135 + 7
13.9 k 0.94
15.8 + 0.38
2.190
f 0.189
2.37 + 0.26
f 33 k 30
3.96 + 0.26
0.80 k 0.05t
11.40 + 1.27
+ 2.12t
3.34
f 0.32
0.65
r 0.07t
11.30
r 1.05
7.36
r 1.19t
+ 3.5t
4.84
k 0.38
1.32 + 0.19t
9.80
+ 0.65
19.60
k 3.36t
k 0.166
1.790
f .173
f 0.243
2.590
+ .316
2.720
+ ,389
lSecretion was carried with batches of either ten islets OT47,000 tDiffers from comparable result in cells, 2P < 0.05
6.84 + 1.31t
cells. There was a total of 22 to 36 replicates from four separate experiments.
451
DISPERSED ISLET CELLS IN CULTURE
Table 3. Hormone Release and Content of Four-Day-Cultured
Dispersed Cells Versus Islets During a g&Minute Incubation* Somatostatl”
GhKag0n
Insulin Islets
Cells
ISletS
Cells
ISletS
Cells
Release (% of Content) bmllol/L) Glucose 2.8
2.87
+ 0.28
4.09
t 0.3Jt
10.3 + 0.55
1.01
+ 0.08t
4.94
t 0.22
9.92
Glucose 16.7
10.0
+ 0.54
12.6 + 0.9Ot
10.2 + 1.17
1.02 k 0.16t
7.39
f 0.87
10.90
+ 1.21t
27.7
-t 3.05
37.7
16.6
+ 0.98
2.81
15.3
+ 2.15
20.90
*
Glucose 16.7 + IBMX
1
i
2.40t
+ 0.20t
k 0.91t 1.66
Content
(ng)
Glucose
2.8
184
+
19
127
i
12
7.47
+- 0.72
21.7
f
1.31
1.29
k 0.61
0.885
t
0.198
Glucose
16.7
198
* 24
141
+
18
7.62
+ 0.66
20.0
+
1.29
1.45
t
0.91
0.995
i
0.079
Glucose
16.7
7.86
+ 0.80
22.2
+ 1.1
1.28
+ 0.79
0.845
+ 0.113
+
IBMX 1
114 + 11
119 + 10
*Secretion was carried out in dishes which were originally plated with 80,000
cells and then washed, or in dishes containing ten islets each. There
were a total of 18 to 20 replicates from three separate experiments. tDiffers from comparable result in cells, 2P < 0.025
insulin release (as an increase relative to basal rates) was similar for cells and islets. IBMX led to a decreased insulin content in the dispersed cells. These results in cultured tissue clearly differ from those of freshly prepared cells and islets (Table 2). The ability of cultured cells to respond to high glucose and high giucose plus IBMX improved considerably. In contrast the responsiveness of the cultured islets has declined. The glucagon release from cultured cells was strikingly higher than that seen from cultured islets, and as was the case with freshly prepared tissues there was stimulation of glucagon release by IBMX. There was, however, no difference in the release of glucagon between the high and low glucose concentrations. Somatostatin release from cultured cells and islets was similar in that there was minimal or no enhancement by high glucose (2P < 0.05 for cells, and without significance for islets), and a comparable degree of stimulation by IBMX. The islet hormone content of insulin and somatostatin fell during the four day culture period, but the glucagon content showed less change (comparison between Tables 1, 2 and 3). It was not possible to
accurately count the number of cells which remained attached to the plates after the culture period, and therefore hormone content per cell could not be determined. Nonetheless, one can compare the ratios of hormone content between cultured cells and islets. The ratios of somatostatin to insulin were similar, being 0.65 + 0.15: 100 for islets and 0.69 +- 0.03: 100 for cells. The ratios of glucagon to insulin were, however, very different, being 17.1 2 1.O: 100 for islets and only 4.2 + 0.4 for cells (2P < 0.01). Insulin Biosynthesis
and Release: Comparison
of
Cultured Dispersed Cells and Cultured Islets
The rate of proinsulin and insulin biosynthesis was much higher in cultured dispersed cells than in similarly cultured islets, and in both cases this was stimulated by high glucose (Table 4). The amount of total protein biosynthesis was estimated by trichloroacetic acid (TCA) precipitation and this was also higher in cells than in islets. However, the percentage of protein biosynthesis accounted for by proinsulin and insulin biosynthesis was lower in cells than islets. In spite of the differences in the rates of (3H-) leucine incorpora-
Table 4. Biosynthesis of Proinsulin Plus Insulin and of Total Protein in Five-Day-Cultured Cells and Islets Promsulln/lnsulin
Proinsulmllnsul~n
Bwynthesls:
ProlnsulI”/lnsulln
Speclflc Radioactivity
Biosynrhesls:
dpmlng
Insulin
Total Proteln Biosynthesis (TCA-Precipltable
dpm/dish
or ten islets
+ 46(g)*
5.03
*
1.00
x
lo4
5.78
k
20.7
+ 4.43
x
lo4
14.30
dpmldlsh
Radioactwty)
or ten IS&S
RIosynthesIs
lmmunoreactwe
as % of
hl?.Ull” Release
Total Protein Biosynthesis
% of Content
Cells Glucose
Immol/L)
2.8
376
16.7
1470
173(9)
+ 0.54
x
lo5
8.1
+
1.1
4.5
+
1.54
x
105
13.7
k
1.8
23.1
+ 0.9(13) f
1.8(13)
Islets Glucose
(mmol/L)
2.8
125
+ 26(13)
2.36
k 0.42
x
lo4
1.51
t
0.18
x
lo5
14.5
+ 2.0
16.7
388
r
5.35
+ 0.47
x
10”
2.71
+ 0.16
x
lo5
19.6
+
*Numbers
in parentheses
originally plated with 56,000
refer
65(12)
1.55
2.9 25.0
+ 0.9(13) +
1.4(11)
to number of replicates from two experiments. Biosynthesis and secretion were carried out in dishes which were
cells, or in dishes containing ten islets each. The specific radioactivity, given as dpm/ng insulin, was calculated as dpm of
immunopreclpitable insulin and proinsulin divided by the content of immunoreactive insulin. All of the insulin and protein biosynthesis values for cells were statistically different from those of islets (2P < 0.05).
Release results between cells and islets were not statistically different.
452
WEIR ET AL
tion the insulin release was very similar in cells and islets. DISCUSSION
Even though techniques for dispersing cells of the islets of Langerhans are well described8-‘3 there is surprisingly little information available about the function of dispersed adult cells from islets9.10.‘7~20 or from the whole pancreas.*‘,** Depending on the study, the cells may or may not release insulin in response to glucose, and sometimes this response has only been observed in the presence of a phosphodiesterase inhibitor.” The factors responsible for the poor responsiveness of these freshly dispersed cells are unknown, but there are data available for insulin release which suggest that lack of cell-cell interaction could be responsible.*’ Acute injury to plasma membranes by trypsin or mechanical disruption could also be playing a role, and a phenomenon of passive hormone leakage has not been excluded. Certainly the dispersion process is very traumatic, as is indicated by the marked and irreversible hormone loss which has been demonstrated in the present study, and which we attribute primarily to cell death. When the dispersed cells were maintained in tissue culture it was found that some of the above peculiarities of release could be reversed. In the case of insulin release the functional characteristics of cultured cells and of islets cultured in parallel were very similar. In addition both glucagon and somatostatin release from cells became more responsive to IBMX. There were, however, interesting differences in the release of the two hormones between cultured cells and islets. Glucagon release from cells was far greater than that found from islets, whereas the somatostatin release from cells in the presence of the two glucose concentrations was less than from islets. The explanation for these differences is unknown, but it is possible that islet glucagon release is restrained by the local release of somatostatin, and that islet somatostatin release is enhanced by local glucagon secretion.’ Thus cells which have been dispersed may be exposed to lower concentrations of hormones released by other cell types. An important caveat about quantitating release in terms of hormone content is that we do not know that the hormone content for each cell type is similar in dispersed cells
and islets. We have not yet found a reliable method for quantifying the numbers of cultured dispersed A, B, and D cells. In addition one must be cautious about extrapolating results from in vitro islet studies to events occurring in vivo because islet blood flow may be an important determinant of function.23 The factors responsible for the recovery of secretory function are not known. Recovery from the acute insult by trypsin and mechanical disruption could play a role. Attachment of the cells to each other and the reestablishment of junctional complexes in culture could be responsible,*’ but this is not fully consistent with the observation that usually about half of the cells appeared to be single. On the other hand it is difficult to be confident that an apparently single cell is truly single or whether the release pattern seen reflects the whole or only a fraction of the cell population. We are, therefore, reluctant to draw any firm conclusions about the function of single cells. We might speculate, however, that attachment of cells to each other or to a substratum may influence their secretory behavior. In particular, the restored regulation of insulin release by dispersed B cells, upon attachment to the culture support, may be related to the concentration of their secretory granules in the portion of the cell facing the medium. Indeed, it is of interest that B cells of intact islets also have most secretory granules concentrated in one portion of their cytoplasm, facing capillaries.24325 The use of cultured dispersed islet cells which have attached to a substratum is an attractive investigational approach. Methods for separating specific islet cell types are already meeting with some success’3326~28 and the culture methods described in this study should provide an excellent way to study the function of these cells. This method could also have some advantages over intact islets in that the dispersed cells may be more accessible to various experimental materials such as secretagogues, metabolites, liposomes, or the addition of other cell types. This approach thus provides a promising method to study the individual components of the islet, such that the overall contribution of islet structure to its function can be better understood. ACKNOWLEDGMENTS We thank E. Barras, D. Boghikian. M. Thorimbert, and D. Wey for excellent technical assistance; J. Reynolds for preparation of the manuscript; and Dr S. Bonner-Weir for valued assistance.
REFERENCES 1. Orci L: Macro- and micro-domains in the endocrine pancreas. Diabetes 315388565, 1982 2. Samols E, Tyler JM, Marks V: Glucagon-insulin interrelationships, in: Lefebvre PJ, Unger RH (eds): Glucagon, Molecular Physiology, Clinical and Therapeutic Implications. Pergamon Press, Oxford p 151-173, 1972
3. Orci L, Unger RH: Functional subdivision of islets of Langerhans and possible role of D-cells. Lancet 2: 1243-1244, 1975 4. Weir CC, Knowlton SD, Atkins RF, et al: Glucagon secretion from the perfused pancreas of streptozotocin-treated rats. Diabetes 25:275-282,1976 5. Unger RH, Orci L: Glucagon and the A cell: Physiology and
453
DISPERSED ISLET CELLS IN CULTURE
pathophysiology. (Medical Progress) New Engl J Med 304:15181524, 1975 6. Meda P. Perrelet A, Orci L: Increase of gap junction between pancreatic B-cells during stimulation of insulin secretion. J Cell Biol 82:441-448. 1979 7. Orci L, Unger RH. Renold AE: Structural coupling between pancreatic islet cells. Experientia 29:1015-1018, 1973 8. Hellman B, Lernmark A: Methods for the isolation of cells with special reference to the pancreatic islets, in: Dubach UC, Schmidt U (eds): Recent advances in quantitative histo- and cytochemistry. Hans Huber Publishers. Vienna, 197 I, p 9 I 9. Krause U, Puchinger H, Wacher A: Inhibition of glucoseinduced insulin secretion in trypsin-treated islets of Langerhans. Horm Met Res 5~325-329, 1973 IO. Lernmark A: The preparation of, and studies on, free cell suspensions from mouse pancreatic islets. Diabetologia 10:431-438, 1974 11. Ono J, Takaki R, Fukuma M: Preparation of single cells from pancreatic islets of adult rat by the use of dispase. Endocrinol Japon 24:265-2X), 1977 12. Meda P, Hooghe-Peters EL. Orci L: Monolayer cultures of adult pancreatic islet cells on osmotically disrupted tibroblasts. Diabetes 29:497-500, 1980 13. Pipeleers DC. Pipeleers-Marichal MA: A method for the purification of single A, B and D cells and for the isolation of coupled cells from isolated rat islets. Diabetologia 20:654-663, 1981 14. Lacy PE, Kostianovsky M: Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes l6:35-39, 1967 15. Halban PA, Wollheim CB, Blonde1 B, et al: Long-term exposure of isolated pancreatic islets to mannoheptulose: evidence for insulin degradation in the B cell. Biochemical Pharmacol 291262552633, 1980 16. Herbert V, Lau KS, Gottlieb CV, et al: J Endocrinol Metab 25:137551384, 1965 17. Idahl LA, Lernmark A, Sehlin J, et al: The dynamics of
insulin release from mouse pancreatic islet cells in suspension. Pfleugers Arch 366:185-188.1976 18. Ono J, Takaki R, Okano H, et al: Long-term culture of pancreatic islet cells with special reference to the B-cell function. In Vitro 15:955102, 1979 19. Kostianovsky M, McDaniel ML, Still MF, et al: Monolayer cell culture of adult rat islets of Langerhans. Diabetologia 10:337344.1974 20. Halban PA, Wollheim CB, Blonde1 B, et al: The possible importance of contact between pancreatic islet cells for the control of insulin release. Endocrinology 111:86-94, 1982 21. Kawazu S, Kanazawa Y, Hayashi M, et al: Monolayer culture of human fetal and adult pancreas. Static and dynamic studies of insulin release in vitro. Horm Metab Res 12:354-360, 1980 22. Britt LD, Stojeba PC, Scharp CR, et al: Neonatal pig pseudo-islets: a product of selective aggregation. Diabetes 30:580583. 1981 23. Bonner-Weir S, Orci L: New perspectives on the microvasculature of the islets of Langerhans in the rat. Diabetes 31:883-889, 1982 24. Ferreira D: L’ultrastructure crine chez I’embryon et le rat 1:14-25. 1957
des cellules du pancreas nouveau-n&. J Ultrastruct
25. Bencosme SA, Pease DC: Electron microscopy creatic islets. Endocrinology 63:1-13, 1958
endoRes
of the pan-
26. Nielsen DA, Lernmark A, Berelowitz M, et al: Sorting of pancreatic islet cell subpopulations by light scattering using a fluorescence-activated cell sorter. Diabetes 3 1:299-306, 1982 27. Fletcher DJ, Weir GC, Grogan WM, et al: Preparation of islet B cell-enriched fractions by low-angle light scatter flowcytometry. J Cell Biol 9 I :393a. 198 1 28. Van de Winkel M. Maes E, Pipcleers D: Islet cell analysis and purification by light scatter and autofluorescence. Biochem Biophys Res Comm 107525-532, 1982
Adult Rat Pancreatic Islet Cells in Culture: A, B, and D Cell Function
G. C. Weir, The
availability
freshly
of suitably
dispersed
somatostatin culture.
adult
release
We
then
to Petri
mitochondria
clustered
of the
cell facing
function with
of the
those
for islets 17300
islets
responsiveness plus IBMX was
of islet
the
medium. islets
seen
with
freshly
cells
than
for glucagon.
structure-function of intact
it was
in culture
possible in parallel. either with
Somatostatin
Dispersed
determined
in primary
by islet
structure.
granules
concentrated
hormone
content
rates
cellular
of release
Whereas
the
appeared
and hormone
ratio
in glucagon insulin
Proinsulin
was
lower
reveal
from
several
ratios
dispersed
ways
In contrast
cells
in which
was similar ratios to the
to glucose
(incorporation than
to be a valuable the
being lack of
and glucose
of [“HI leucine)
from
model
as a
compared
content
in response
with
release
were
(glucagon:insulin
biosynthesis
thus appear
level,
the portion
or hormone
loss of A cells.
cells released
6 cells which
towards
content
or
monolayer
ultrastructural
of somatostatin:insulin
depleted or relative
islets.
at the
Since
glucagon,
islets
system
function
while
the
for the study
of dispersed
cells
islets.
AND
METHODS
(DMEM. Grand Island Biological Co.) containing 10% heat inactivated newborn calf serum and 5.5 mmol/L glucose was added. The cells were then centrifuged for ten minutes at 150 x g, and the supernatant discarded. Nine milliliters of DMEM was added to the pellet and the cells were again centrifuged. The pellet was resuspended in I mL of DMEM and cell counts were performed using a hemocytometer. Hormone
Loss With
Dispersion
Batches of ten intact islets were placed into IO x 75 mm plastic tubes and extracted in 500 ~1 of acid ethanol at 4 “C for 16 hours. The extracts were then stored at --20 “C until they were assayed. A portion of the islets (600) were dispersed using the method described above and suspended in 3 mL of culture medium 199 (Grand Island Biological Co.). containing IO’%heat inactivated newborn calf serum and 8.3 mmol/L glucose. The number of cells obtained from these 600 islets averaged I .3 x IO’. Fifty PL aliquots were pipetted into IO x 75 mm tubes and extracted as above. The remaining cells and islets were then cultured in medium 199 with glucose 8.3 mmol/L for two hours. These cells and islets were then washed twice with KRB-Hepes containing 2.8 mmol/L glucose and extracted for hormone content determination as described below.
of Islet Cells
Islets were obtained by collagenase digestion and hand-picking”’ from pancreases of male Wistar and Sprague-Dawley rats weighing between 160 and 240 g. The islets were washed with a modified Krebs-Ringer bicarbonate (KRB) buffer containing 10 mmol/L Hepcs (2-[IV-2-hydroxyethylpiperazin-N’yl] ethanesulphonic acid) [Grand Island Biological Co., Grand Island, NY), 5 mmol/L NaHco,. 5 mg/ml. bovine serum albumin (BSA, fraction V, from Behringwerke AC, Marbug/Lahn, FRG), and 2.8 mmol/L glucose. They were then incubated in calcium-free phosphate buffered saline (PBS), pH 7.4, containing 0.54 mmol/L EDTA and 0.1% Trypsin (1:250), (Dlfco Laboratories, Detroit, MI). for 12 to I5 minutes at 37 “C in a total volume of 5 mL contained in a 10 mL siliconized glass tube. The islets were then disaggregated by being aspirated 10 to 20 times through a siliconized Pasteur pipette (internal diameter of tip was about 0.9 mm). The tube was immediately placed on ice and then 4 ml, of Dulbecco’s modified Eagle’s tissue culture medium
Metabolism,
established
dish and secretory
release
HE STRUCTURE of the islets of Langerhans is highly organized and in man and rat the B cells are located in a central core with A, D, and PP cells forming a surrounding mantle.’ This organization, combined with the knowledge that the hormone released from a given cell type has potent effects upon the other islet cell types,’ ’ and the fact that specialized junctions are found between islet cells,‘.“,‘suggests that islet function is influenced by its structure. It has been difficult to investigate this likely contribution of structure to function with currently available in vitro techniques. However, methods have been described for dispersing adult islet cellsxm’3 and an understanding of the behavior of such cells could provide insights into the coordination of islet function. In this study tissue culture techniques were used to compare the function of dispersed adult islet cells with that of intact islets.
Dispersion
partially
function.
in insulin,
preserved
cultured
results
of islet
well
islet cells in monolayer and these
in studies (no alteration
cells were
were
degranulation
T
MATERIALS
days
islet cells, the cultured seen
assist
these
to estimate
fractional
may
to secretagogues
is at least
of four
L. Orci, and A. E. Renold
preparations to IBMX),
cell population
reflecting
to that
relationships,
response
to the Petri
not
dispersed
islets.
cell
appropriately
period
of cells,
dispersed
C. B. Wollheim,
islet function
attached
types
for cells)
comparable
in cultured
differs from that
the
modest
that
maintained
(-0.7:100),
islet
to respond
a culture
Since
of specific
and 4:lOO
was true
hypothesis during
P. Meda,
dispersed
change;
at the cell face
in a fashion
higher
opposite
glucose
dishes
the
for intact
for
after
number
and cells
characterized
rat islet cells failed
tested
had attached
P. A. Halban,
Vol
33, No 5
(May),
1984
From
the lnstitut
de Biochimie
logic et d’ Emhryologir.
Received for publication
(Frankfurt,
Switzerland).
the Kroc
from
is a recipient
National Address
Medical
c‘ol/ege
of
Virginia.
Endocrinolog_v.
Foundation,
the National
Institutes
of’ a Research requests
Medical
National
W, Germonyl.
Science Foundation reprint
25, 1983.
the Swiss
AG
and AM-00255 Meda
Juiv
by grants from
Hoechst
(Nyon.
d’ Hrsto-
Cencva. Switzerland
Virginia.
Supported tion,
and lnstitut
of Geneva.
of Endocrinology.
and the Division Richmond.
Cliniqur.
lJniver.ritj,
Scirnce
Founda-
Fondation
and grants.
oJ‘Health
Fellowship
from
Zyma
AM-20349 (USAI.
Dr
the Swiss
(83.896.O.Hl). to G. (I College
of
Weir,
Box
Virginia.
693.
Division
Richmond.
of VA
23298. (~11984 bv Grune
& Stratton,
Inc.
002~-04~5/~4/3305--nO13$0l.00/0
447
448
The hormone content of the cells is expressed in terms of islet equivalents (IE). Islet equivalents refer to the initial islet number. For instance 600 starting islets are considered 600 IEs regardless of cell loss, hormone loss, or the volume in which the cells are suspended. Culture of Cells and Islets Cells or islets were washed twice under sterile conditions with tissue culture medium 199. The islets were transferred in known numbers with the aid of a dissecting microscope to culture dishes (Falcon 1007 (60 x I5 mm) or 1008 (35 x 10 mm diameter), Falcon, Oxnard, CA), these dishes being chosen to prevent islet attachment. The resuspended cells were aliquoted with an Eppendorf pipette into type 3001 Falcon dishes (35 x 10 mm) which were noncoated, or coated with disrupted fibroblasts or poly-L-lysine. The fibroblast coating of Falcon 3001 dishes was carried out by growing pancreas-derived fibroblasts to contluence and then lysing them with sterile water as has been previously described.” Poly-L-lysine HBr (Sigma, St. Louis, MO) of 350,000 daltons was dissolved in sterile water to give a concentration of 300 pg/mL and I mL was added to groups of 20 to 40 Petri dishes (Falcon 3001). The solution remained in the dishes for one hour at room temperature and was then removed by suction. The dishes were then washed three times with sterile water and stored for up to one week at 4 “C wrapped in aluminum foil. All of the secretion and biosynthesis studies carried out on cultured cells employed cells which were plated on polyL-lysine-coated plates. Our preliminary studies led us to the conclusion that this coating enhanced the attachment of dispersed islet cells, but subsequent studies have revealed minor if any effect. Visual inspection during the culture period gave us the strong impression that poly-L-lysine promoted attachment, but we now suspect that this is only a weak electrostatic effect which is lost when the plates are washed. The fibroblast-coated dishes were used for the electron microscopy studies. In studies not included in this manuscript it was found that the secretory behavior of cells plated on these dishes was similar to that found with poly-L-lysine-coated or uncoated dishes. The insulin content of the cells remaining on the fibroblast-coated dishes after washing was sometimes lower than the content left on other dishes (range 42% to 100% of noncoated dishes). It must be emphasized that this is only a difference in insulin content and does not necessarily correlate with the efficiency of attachment. The culture medium for both the islets and the cells was 199 containing 8.3 mmol/L glucose, 10% fetal calf serum, penicillin (400 U/mL), streptomycin (200 pg/mL), and gentamycin (50 pg/mL). Cells were incubated in 2 mL of media at 37 “C in a humidified, 5% CO, atmosphere. The islets were similarly incubated in either 2 or 4 mL of medium. Hormone Release Studies in Cells and Islets In experiments examining hormone release by freshly dispersed cells and fresh islets, the cells and islets were preincubated for two hours in culture medium 199 with 8.3 mmol/L glucose. The cells and islets were washed twice with KRB-Hepes buffer with glucose 2.8 mmol/L, and then either 47,000 cells or ten islets were dispensed into siliconized glass tubes containing 1 mL of KRB-Hepes containing either 2.8 or 16.7 mmol/L glucose with or without 3-isobutyl1-methylxanthine (IBMX, Sigma Co, St. Louis, MO) and 600 U/mL of Aprotinin (Trasylol, Bayer A.G., Wuppertal FRG). The incubations lasted 90 minutes and were carried out at 37 OC.Tubes were then put on ice and centrifuged at 4 OCfor ten minutes at 150 x g. The supernatants were removed and frozen. Two millimeters of KRB-Hepes containing aprotinin (600 U/mL) was added to the pellets and the tissue was sonicated and frozen prior to measurement of hormone content.
WEIR ET AL
Hormone release was also studied from cells and islets which were cultured in parallel for four days. Floating cells and culture medium were removed by suction and the dishes containing attached dispersed cells were then washed three times with KRB-Hepes buffer with 2.8 mmol/L glucose at room temperature, with the addition and removal of 2 mL of KRB-Hepes at each stage. Islets were washed three times with the same buffer using siliconized tubes and centrifugation. The attached cells were then incubated in dishes containing 1 mL of KRB-Hepes buffer with glucose, IBMX, and aprotinin as above. The islets were distributed under microscopic observation into similar dishes (Falcon 3001) with 10 islets and I mL of the above buffers being contained in each dish. Dishes were incubated at 37 “C for 90 minutes and the incubation buffer was removed with particular care taken to leave the islets behind. The incubation buffer from both the cell and islet experiments was centrifuged for ten minutes at 150 x g to insure removal of the small number of contaminating cells (the hormone content of these cells was found to be less than 5% of the total content of the incubated cells). To extract the cells and islets, 1 mL of KRB-Hepes-aprotinin buffer was added to the dishes, and the bottoms were scraped with a rubber policeman. The buffer was then removed with a Pasteur pipette and placed into 10 x 75 mm glass tubes. The dishes were then washed with an additional one ml of buffer and this was added to the same glass tubes. The tissue was then sonicated and frozen pending hormone assay. Hormone release in all experiments was expressed as percent of content. The content value used for these calculations was the mean of determinations made on tissue studied at all three experimental conditions. In some of the experiments the insulin content after incubation with IBMX was slightly reduced, but this caused only a minimal reduction in the value used for the calculations. Insulin Biosynthesis The cultured ceils and islets used for the biosynthesis experiments were handled as described for the above secretion studies. The KRB-Hepes incubation buffer contained [4, 5-‘H] leucine (Radiochemical Centre, Amersham, Buckinghamshire, UK) with a specific radioactivity of 65 Ci/mmol, at a concentration of 50 &i/mL. Following incubation, a thorough washing was required to remove the extra cellular (‘H) leucine. Therefore the plates with attached cells were washed three times with KRB-Hepes buffer as described above. The islets were transferred to siliconized glass tubes and washed three times with the same buffer as has been described elsewhere.” One milliliter of glycine buffer 0.2 M with 0.25% bovine serum albumin, pH 8.8, was added to the dishes. After cells were dislodged with a rubber policeman, they were transferred to glass tubes, sonicated, and ultracentrifuged.” Immunoprecipitation of labelled proinsulin and insulin” was carried out with a large excess of guinea pig anti-pork insulin (Miles Research Products, Rehovot, Israel), followed by precipitation of the antibody-antigen complexes with protein A-Sepharose (Pharmacia, Zurich, Switzerland). Nonspecific binding was determined by performing parallel precipitations for each sample using nonimmune guinea pig serum in place of antiinsulin serum. The exact method and the relative specific and nonspecific binding properties of this method have been described in detail previously.” All results are expressed as the specifically immunoprecipitable radioactivity (total less nonspecific). The relative proportions of labeled proinsulin and insulin were not determined. Radioimmunoassays Immunoreactive insulin, glucagon, and somatostatin were determined by radioimmunoassay using the charcoal separation method of Herbert et a1.16Rat insulin, pork glucagon, and synthetic cyclic somatostatin were used as standards.
DISPERSED
ISLET
CELLS
IN
449
CULTURE
Data Presentation Data are expressed as mean and standard error of the mean. For statistical comparisons the two-tailed unpaired Student’s t-test was used.
Electron Microscopy At the end of an experiment, the medium was removed from the dishes and the cultures were fixed for one hour at room temperature in a 2.5% glutaraldehyde solution in 0.1 M phosphate buffer, pH 7.4, postfixed for one hour in 1% osmium tetroxide and dehydrated in graded ethanols. The cultures were then embedded in Epon and sectioned either parallel or perpendicular to the Petri dish bottom. Thin sections mounted on carbon-coated grids were stained with uranyl acetate and lead citrate and examined in a Philips EM 301 electron microscope. RESULTS
Cell Yield and Hormone Content Following Dispersion
After cells were dispersed they were washed in KRB-Hepes buffer once and counted. The cell yield was calculated from 20 dispersions. The starting number of islets was 1030 + 101 (range 550 to 2400) and the yield of cells per islet was 1425 * 80 (range 873 to 2 188). When the cell suspensions were examined on 14 separate occasions the percentage of cells which were single was 69 + 4 (range 48 to 86). An experiment was designed to determine the hormone loss which occurs during the acute dispersion process to gain some insight into degree of cell lysis and destruction which occurs during the procedure. As can be seen in Table 1 there was a fall of between 28% and 47% of hormone content occurring during the acute dispersion process. During the subsequent two-hour period in culture medium there was a modest additional drop in content to about 50% of the initial intact islet content, with all three hormones having been reduced to a similar degree by the end of the two hour period. The cell number during the two hour period in culture medium fell by a mean of 11%. The fall in hormone content of the intact islets during this twohour period was 33% for insulin, 16% for glucagon, and 17% for somatostatin (data not shown). Thus, the major ioss of hormone content of the dispersed cell preparation occurred during the acute dispersion pro-
cess with a smaller loss being observed during the subsequent two-hour period in culture medium. It was not practical to measure the number of cells in the initial islets, but these data suggest that the initial fall in hormone content can be ascribed to the cell loss which takes place during the traumatic dispersion process. Culture of Dispersed Islet Cells: Morphology
Regardless of the substratum employed there were certain general characteristics in the appearance of dispersed cells in culture. The attachment of cells seemed to be maximal after two to three days in culture and usually remained at that level until the fifth day of culture. The number of cells which attached, as judged by their stationary behavior when the dishes were moved, was usually about 30% to 60%. For the experiments in this study at least 47,000 cells were plated per dish and when examined after four to five days in culture about 30% to 50% of the cells were single with the rest consisting of groups of between two to five cells (Fig. 1). At times there were larger zones of aggregated cells in monolayer containing 20 or more cells. No more than 5% to 10% of the singlets or small groups (two to four cells) appeared spread out on the culture support and otherwise the rounded appearance seen in Fig. 1 was the norm. When the cultured cells were studied by electron microscopy it was usually observed that the single cells had a characteristic hemispheric shape (Fig. 2). Thus the overall shape was globular except where the cell had attached to the culture support (Fig. 2). At that site, the membrane appeared flattened against the culture support. In single attached B cells. mitochondria were consistently clustered at the portion of the cell facing the culture support, while most of the secretory granules were found at the portion of the cell facing the medium. Hormone Release From Freshly Prepared Dispersed Cells and Islets
The insulin release from freshly dispersed cells and from islets incubated in parallel (both groups having been preincubated for two hours in culture medium)
Table 1. Hormone Loss With islet Disruption isletsPnor
Cells lmmedlately
to Disruption
Following
Cells Followng
Disperswn
Dispersion and 2.hr Culture % Of
ngI1slet
Nt
ng/lE’
% of
Initial Islet Content
ng/lE’
lnltlal islet Content
Insulin
10
51.3
+ 7.0
28.2
-t 2.8
57.7
+ 4.4
25.1
i; 2.8
50.3
t
Glucagon
10
4.87
k 0.80
4.09
k 0.82
72.0
+ 4.9
3.14
i
0.52
58.3
+ 3 2
Somatostatin
10
0.660
+ 0.023
53.2
i: 3.3
i
0.025
47.4
+ 3.8
llE AND tN
is islet equivalent,
METHODS refers
which
*
IS an expression
0.344
0.057 of the
quantity
of cells obtained
section.
to the number
of replicates
contained
in two
separate
experiments.
from
each disrupted
0.313 islet.
It is described
in more
detail
4.0
in the MATERIALS
450
WEIR ET AL
Fig. 2. Electron micrograph of an isolated B cell which had been cultured for four days on osmotically disrupted fibroblasts. Culture medium was 199 containing 18.7 mmol/L glucose and 10% newborn calf serum. This thin section, which was cut perpendicular to the bottom of the culture dish, shows that, as it was consistently observed in our cultures, this single cell has a characteristic hemispheric shape. The arrow head points to an area of the flat portion where the plasma membrane of the 8 cell is closely opposed to the bottom of the culture dish. Note the good preservation of the cell ultrastructure and the peculiar distribution of intracellular organelles. Most of the mitochondria are clustered at the portion of the cell facing the culture support while most of the secretory granules are found at the portion of the cell facing the medium. The bar represents 2 pm; x 7,300.
Fig. 1. Dispersed attached adult islet cells cultured for three days on Poly-L-lysine-coated culture dishes as seen with phase contrast. Medium was 199 containing 8.3 mmol/L glucose and 10% fetal calf serum. Single cells, either round or spread. and clumps of two or more cells can be seen. The bar represents 50 pm: x160.
was quite different (Table 2). The release rates at low glucose were similar, but the response to high glucose (16.7 mmol/L) by the cells was negligible, whereas the release from islets increased 13-fold. In addition the insulin release from the cells in response to IBMX was considerably lower than was seen from islets. The glucagon release from cells was much higher than it was from islets, and with each there was modest stimulation by IBMX (2P < 0.01). Glucagon release from islets was slightly (2P -C0.05) decreased by high glucose. The somatostatin release from cells in the presence of both low and high glucose was higher than that from islets. High glucose failed to stimulate release from either cells or islets. The combination of glucose 16.7 mmol/L and IBMX did stimulate the release of somatostatin from islets (2P -C O.Ol), but not from cells. The residual hormone content tended to be similar for all three hormones when the three different incubation conditions were compared, with two exceptions.
The insulin content of islets after incubation in the combined presence of IBMX and high glucose was reduced relative to the other two conditions (2P -C 0.01). In contrast the islet somatostatin content after incubation at glucose 2.8 mmol/L was marginally lower than that seen after incubation at the higher glucose concentration, both with and without IBMX (2P < 0.05). Hormone Release From Cultured Dispersed Cells and Islets
Insulin release from higher than that from incubation conditions percent of stored insulin
cultured islets was slightly dispersed cells for all three studied when expressed as (Table 3). The stimulation of
Table 2. Hormone Release and Content of Freshly Dispersed Cells Versus Islets During a 90-Minute Incubation* Insulin
Glucagon
Cells
Islets
Celk
Somatostatin islets
Cells
Islets
Release (% of Content) (mmol/L) Glucose2.8
2.98 k 0.20
Glucose 16.7
3.88
+ 0.37
31.90
Glucose 16.7 + IBMX 1
16.5 k 0.96
73.1
content (ng) Glucose 2.8
334
187 k 13
14.3 + 1.26
14.4 Yk0.22
1.770
Glucose 16.7
326 k 31
171 + 6
12.3 + 0.85
16.8 + 0.48
1.990
Glucose 16.7 + IBMX 1
301
135 + 7
13.9 k 0.94
15.8 + 0.38
2.190
f 0.189
2.37 + 0.26
f 33 k 30
3.96 + 0.26
0.80 k 0.05t
11.40 + 1.27
+ 2.12t
3.34
f 0.32
0.65
r 0.07t
11.30
r 1.05
7.36
r 1.19t
+ 3.5t
4.84
k 0.38
1.32 + 0.19t
9.80
+ 0.65
19.60
k 3.36t
k 0.166
1.790
f .173
f 0.243
2.590
+ .316
2.720
+ ,389
lSecretion was carried with batches of either ten islets OT47,000 tDiffers from comparable result in cells, 2P < 0.05
6.84 + 1.31t
cells. There was a total of 22 to 36 replicates from four separate experiments.
451
DISPERSED ISLET CELLS IN CULTURE
Table 3. Hormone Release and Content of Four-Day-Cultured
Dispersed Cells Versus Islets During a g&Minute Incubation* Somatostatl”
GhKag0n
Insulin Islets
Cells
ISletS
Cells
ISletS
Cells
Release (% of Content) bmllol/L) Glucose 2.8
2.87
+ 0.28
4.09
t 0.3Jt
10.3 + 0.55
1.01
+ 0.08t
4.94
t 0.22
9.92
Glucose 16.7
10.0
+ 0.54
12.6 + 0.9Ot
10.2 + 1.17
1.02 k 0.16t
7.39
f 0.87
10.90
+ 1.21t
27.7
-t 3.05
37.7
16.6
+ 0.98
2.81
15.3
+ 2.15
20.90
*
Glucose 16.7 + IBMX
1
i
2.40t
+ 0.20t
k 0.91t 1.66
Content
(ng)
Glucose
2.8
184
+
19
127
i
12
7.47
+- 0.72
21.7
f
1.31
1.29
k 0.61
0.885
t
0.198
Glucose
16.7
198
* 24
141
+
18
7.62
+ 0.66
20.0
+
1.29
1.45
t
0.91
0.995
i
0.079
Glucose
16.7
7.86
+ 0.80
22.2
+ 1.1
1.28
+ 0.79
0.845
+ 0.113
+
IBMX 1
114 + 11
119 + 10
*Secretion was carried out in dishes which were originally plated with 80,000
cells and then washed, or in dishes containing ten islets each. There
were a total of 18 to 20 replicates from three separate experiments. tDiffers from comparable result in cells, 2P < 0.025
insulin release (as an increase relative to basal rates) was similar for cells and islets. IBMX led to a decreased insulin content in the dispersed cells. These results in cultured tissue clearly differ from those of freshly prepared cells and islets (Table 2). The ability of cultured cells to respond to high glucose and high giucose plus IBMX improved considerably. In contrast the responsiveness of the cultured islets has declined. The glucagon release from cultured cells was strikingly higher than that seen from cultured islets, and as was the case with freshly prepared tissues there was stimulation of glucagon release by IBMX. There was, however, no difference in the release of glucagon between the high and low glucose concentrations. Somatostatin release from cultured cells and islets was similar in that there was minimal or no enhancement by high glucose (2P < 0.05 for cells, and without significance for islets), and a comparable degree of stimulation by IBMX. The islet hormone content of insulin and somatostatin fell during the four day culture period, but the glucagon content showed less change (comparison between Tables 1, 2 and 3). It was not possible to
accurately count the number of cells which remained attached to the plates after the culture period, and therefore hormone content per cell could not be determined. Nonetheless, one can compare the ratios of hormone content between cultured cells and islets. The ratios of somatostatin to insulin were similar, being 0.65 + 0.15: 100 for islets and 0.69 +- 0.03: 100 for cells. The ratios of glucagon to insulin were, however, very different, being 17.1 2 1.O: 100 for islets and only 4.2 + 0.4 for cells (2P < 0.01). Insulin Biosynthesis
and Release: Comparison
of
Cultured Dispersed Cells and Cultured Islets
The rate of proinsulin and insulin biosynthesis was much higher in cultured dispersed cells than in similarly cultured islets, and in both cases this was stimulated by high glucose (Table 4). The amount of total protein biosynthesis was estimated by trichloroacetic acid (TCA) precipitation and this was also higher in cells than in islets. However, the percentage of protein biosynthesis accounted for by proinsulin and insulin biosynthesis was lower in cells than islets. In spite of the differences in the rates of (3H-) leucine incorpora-
Table 4. Biosynthesis of Proinsulin Plus Insulin and of Total Protein in Five-Day-Cultured Cells and Islets Promsulln/lnsulin
Proinsulmllnsul~n
Bwynthesls:
ProlnsulI”/lnsulln
Speclflc Radioactivity
Biosynrhesls:
dpmlng
Insulin
Total Proteln Biosynthesis (TCA-Precipltable
dpm/dish
or ten islets
+ 46(g)*
5.03
*
1.00
x
lo4
5.78
k
20.7
+ 4.43
x
lo4
14.30
dpmldlsh
Radioactwty)
or ten IS&S
RIosynthesIs
lmmunoreactwe
as % of
hl?.Ull” Release
Total Protein Biosynthesis
% of Content
Cells Glucose
Immol/L)
2.8
376
16.7
1470
173(9)
+ 0.54
x
lo5
8.1
+
1.1
4.5
+
1.54
x
105
13.7
k
1.8
23.1
+ 0.9(13) f
1.8(13)
Islets Glucose
(mmol/L)
2.8
125
+ 26(13)
2.36
k 0.42
x
lo4
1.51
t
0.18
x
lo5
14.5
+ 2.0
16.7
388
r
5.35
+ 0.47
x
10”
2.71
+ 0.16
x
lo5
19.6
+
*Numbers
in parentheses
originally plated with 56,000
refer
65(12)
1.55
2.9 25.0
+ 0.9(13) +
1.4(11)
to number of replicates from two experiments. Biosynthesis and secretion were carried out in dishes which were
cells, or in dishes containing ten islets each. The specific radioactivity, given as dpm/ng insulin, was calculated as dpm of
immunopreclpitable insulin and proinsulin divided by the content of immunoreactive insulin. All of the insulin and protein biosynthesis values for cells were statistically different from those of islets (2P < 0.05).
Release results between cells and islets were not statistically different.
452
WEIR ET AL
tion the insulin release was very similar in cells and islets. DISCUSSION
Even though techniques for dispersing cells of the islets of Langerhans are well described8-‘3 there is surprisingly little information available about the function of dispersed adult cells from islets9.10.‘7~20 or from the whole pancreas.*‘,** Depending on the study, the cells may or may not release insulin in response to glucose, and sometimes this response has only been observed in the presence of a phosphodiesterase inhibitor.” The factors responsible for the poor responsiveness of these freshly dispersed cells are unknown, but there are data available for insulin release which suggest that lack of cell-cell interaction could be responsible.*’ Acute injury to plasma membranes by trypsin or mechanical disruption could also be playing a role, and a phenomenon of passive hormone leakage has not been excluded. Certainly the dispersion process is very traumatic, as is indicated by the marked and irreversible hormone loss which has been demonstrated in the present study, and which we attribute primarily to cell death. When the dispersed cells were maintained in tissue culture it was found that some of the above peculiarities of release could be reversed. In the case of insulin release the functional characteristics of cultured cells and of islets cultured in parallel were very similar. In addition both glucagon and somatostatin release from cells became more responsive to IBMX. There were, however, interesting differences in the release of the two hormones between cultured cells and islets. Glucagon release from cells was far greater than that found from islets, whereas the somatostatin release from cells in the presence of the two glucose concentrations was less than from islets. The explanation for these differences is unknown, but it is possible that islet glucagon release is restrained by the local release of somatostatin, and that islet somatostatin release is enhanced by local glucagon secretion.’ Thus cells which have been dispersed may be exposed to lower concentrations of hormones released by other cell types. An important caveat about quantitating release in terms of hormone content is that we do not know that the hormone content for each cell type is similar in dispersed cells
and islets. We have not yet found a reliable method for quantifying the numbers of cultured dispersed A, B, and D cells. In addition one must be cautious about extrapolating results from in vitro islet studies to events occurring in vivo because islet blood flow may be an important determinant of function.23 The factors responsible for the recovery of secretory function are not known. Recovery from the acute insult by trypsin and mechanical disruption could play a role. Attachment of the cells to each other and the reestablishment of junctional complexes in culture could be responsible,*’ but this is not fully consistent with the observation that usually about half of the cells appeared to be single. On the other hand it is difficult to be confident that an apparently single cell is truly single or whether the release pattern seen reflects the whole or only a fraction of the cell population. We are, therefore, reluctant to draw any firm conclusions about the function of single cells. We might speculate, however, that attachment of cells to each other or to a substratum may influence their secretory behavior. In particular, the restored regulation of insulin release by dispersed B cells, upon attachment to the culture support, may be related to the concentration of their secretory granules in the portion of the cell facing the medium. Indeed, it is of interest that B cells of intact islets also have most secretory granules concentrated in one portion of their cytoplasm, facing capillaries.24325 The use of cultured dispersed islet cells which have attached to a substratum is an attractive investigational approach. Methods for separating specific islet cell types are already meeting with some success’3326~28 and the culture methods described in this study should provide an excellent way to study the function of these cells. This method could also have some advantages over intact islets in that the dispersed cells may be more accessible to various experimental materials such as secretagogues, metabolites, liposomes, or the addition of other cell types. This approach thus provides a promising method to study the individual components of the islet, such that the overall contribution of islet structure to its function can be better understood. ACKNOWLEDGMENTS We thank E. Barras, D. Boghikian. M. Thorimbert, and D. Wey for excellent technical assistance; J. Reynolds for preparation of the manuscript; and Dr S. Bonner-Weir for valued assistance.
REFERENCES 1. Orci L: Macro- and micro-domains in the endocrine pancreas. Diabetes 315388565, 1982 2. Samols E, Tyler JM, Marks V: Glucagon-insulin interrelationships, in: Lefebvre PJ, Unger RH (eds): Glucagon, Molecular Physiology, Clinical and Therapeutic Implications. Pergamon Press, Oxford p 151-173, 1972
3. Orci L, Unger RH: Functional subdivision of islets of Langerhans and possible role of D-cells. Lancet 2: 1243-1244, 1975 4. Weir CC, Knowlton SD, Atkins RF, et al: Glucagon secretion from the perfused pancreas of streptozotocin-treated rats. Diabetes 25:275-282,1976 5. Unger RH, Orci L: Glucagon and the A cell: Physiology and
453
DISPERSED ISLET CELLS IN CULTURE
pathophysiology. (Medical Progress) New Engl J Med 304:15181524, 1975 6. Meda P. Perrelet A, Orci L: Increase of gap junction between pancreatic B-cells during stimulation of insulin secretion. J Cell Biol 82:441-448. 1979 7. Orci L, Unger RH. Renold AE: Structural coupling between pancreatic islet cells. Experientia 29:1015-1018, 1973 8. Hellman B, Lernmark A: Methods for the isolation of cells with special reference to the pancreatic islets, in: Dubach UC, Schmidt U (eds): Recent advances in quantitative histo- and cytochemistry. Hans Huber Publishers. Vienna, 197 I, p 9 I 9. Krause U, Puchinger H, Wacher A: Inhibition of glucoseinduced insulin secretion in trypsin-treated islets of Langerhans. Horm Met Res 5~325-329, 1973 IO. Lernmark A: The preparation of, and studies on, free cell suspensions from mouse pancreatic islets. Diabetologia 10:431-438, 1974 11. Ono J, Takaki R, Fukuma M: Preparation of single cells from pancreatic islets of adult rat by the use of dispase. Endocrinol Japon 24:265-2X), 1977 12. Meda P, Hooghe-Peters EL. Orci L: Monolayer cultures of adult pancreatic islet cells on osmotically disrupted tibroblasts. Diabetes 29:497-500, 1980 13. Pipeleers DC. Pipeleers-Marichal MA: A method for the purification of single A, B and D cells and for the isolation of coupled cells from isolated rat islets. Diabetologia 20:654-663, 1981 14. Lacy PE, Kostianovsky M: Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes l6:35-39, 1967 15. Halban PA, Wollheim CB, Blonde1 B, et al: Long-term exposure of isolated pancreatic islets to mannoheptulose: evidence for insulin degradation in the B cell. Biochemical Pharmacol 291262552633, 1980 16. Herbert V, Lau KS, Gottlieb CV, et al: J Endocrinol Metab 25:137551384, 1965 17. Idahl LA, Lernmark A, Sehlin J, et al: The dynamics of
insulin release from mouse pancreatic islet cells in suspension. Pfleugers Arch 366:185-188.1976 18. Ono J, Takaki R, Okano H, et al: Long-term culture of pancreatic islet cells with special reference to the B-cell function. In Vitro 15:955102, 1979 19. Kostianovsky M, McDaniel ML, Still MF, et al: Monolayer cell culture of adult rat islets of Langerhans. Diabetologia 10:337344.1974 20. Halban PA, Wollheim CB, Blonde1 B, et al: The possible importance of contact between pancreatic islet cells for the control of insulin release. Endocrinology 111:86-94, 1982 21. Kawazu S, Kanazawa Y, Hayashi M, et al: Monolayer culture of human fetal and adult pancreas. Static and dynamic studies of insulin release in vitro. Horm Metab Res 12:354-360, 1980 22. Britt LD, Stojeba PC, Scharp CR, et al: Neonatal pig pseudo-islets: a product of selective aggregation. Diabetes 30:580583. 1981 23. Bonner-Weir S, Orci L: New perspectives on the microvasculature of the islets of Langerhans in the rat. Diabetes 31:883-889, 1982 24. Ferreira D: L’ultrastructure crine chez I’embryon et le rat 1:14-25. 1957
des cellules du pancreas nouveau-n&. J Ultrastruct
25. Bencosme SA, Pease DC: Electron microscopy creatic islets. Endocrinology 63:1-13, 1958
endoRes
of the pan-
26. Nielsen DA, Lernmark A, Berelowitz M, et al: Sorting of pancreatic islet cell subpopulations by light scattering using a fluorescence-activated cell sorter. Diabetes 3 1:299-306, 1982 27. Fletcher DJ, Weir GC, Grogan WM, et al: Preparation of islet B cell-enriched fractions by low-angle light scatter flowcytometry. J Cell Biol 9 I :393a. 198 1 28. Van de Winkel M. Maes E, Pipcleers D: Islet cell analysis and purification by light scatter and autofluorescence. Biochem Biophys Res Comm 107525-532, 1982