1 Department of Molecular Medicine, Endocrine and Diabetes Unit, Rolf Luft Center for Diabetes Research, Karolinska Institutet, Karolinska Hospital, SE-171 76 Stockholm; 2 BioImage Novo Nordisk, DK-2860 Soborg, Denmark; and 3 Department of Medical Cell Biology, University of Uppsala, SE-751 23 Uppsala, Sweden
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ABSTRACT |
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To further evaluate the role of polyamines in insulin production and cell replication in diabetic pancreatic islets, we have studied hyperplastic islets of obese-hyperglycemic mice of different ages and normal islets of the same strain. The aims of the study were to investigate the impact of the diabetic state and aging on polyamine contents and requirements in these islets. Cultured islets from lean and obese animals contained significantly less polyamines than freshly isolated islets. Spermine-to-spermidine ratio was elevated in freshly isolated islets from young obese mice compared with those from lean mice. In islets from old obese animals, spermidine content was decreased, whereas the content of spermine was not different from that of young obese mice. The physiological significance of polyamines was investigated by exposing islets in tissue culture to inhibitors of polyamine synthesis. This treatment caused a partial polyamine depletion in whole islets but failed to affect polyamine content of cell nuclei. Insulin content was not affected in polyamine-deficient islets of obese mice, irrespective of age, in contrast to decreased islet insulin content in polyamine-depleted young lean animals. Polyamine depletion depressed DNA synthesis rate in obese mouse islets; in lean mice it actually stimulated DNA synthesis. We concluded that important qualitative and quantitative differences exist between islets from obese-hyperglycemic and normal mice with respect to polyamine content and requirements of polyamines for regulation of insulin content and cell proliferation. The results suggest that spermine may be involved in mediating the rapid islet cell proliferation noted early in obese-hyperglycemic syndrome, but changes in spermine concentration do not seem to account for the decline in islet cell DNA synthesis in aged normoglycemic animals.
diabetes mellitus; insulin secretion; aging
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INTRODUCTION |
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THE RECESSIVELY
INHERITED obese-hyperglycemic syndrome in mice (gene symbol
ob) is characterized by hyperphagia, insulin resistance, nonketotic hyperglycemia, marked -cell hyperplasia, and
hypersecretion of insulin (12, 42). It has frequently been
used as an animal model of diabetes mellitus, resembling human type 2 diabetes. Studies of the development of the syndrome suggest that an
early peripheral insulin resistance leads to hyperglycemia and an
increased demand for insulin, which induces excessive replication of
the pancreatic
-cells (42). Despite this expansion of
the
-cell mass and elevated insulin secretion, the mice develop
hyperglycemia. However, this is a transient state, and late in the
syndrome the animals become normoglycemic again and islet cell
replication is markedly decreased (3, 42).
The obese-hyperglycemic syndrome has recently engendered renewed interest because of the discovery of the leptin system. Leptin, the protein encoded by the ob gene, is secreted from white adipose tissue and regulates satiety and energy expenditure through hypothalamic receptors (15, 22, 25). Interestingly, ob/ob mice have a mutation in the leptin gene and are thus deficient in leptin (22, 25). Islets from obese-hyperglycemic mice show hyperplastic changes (3, 13, 40, 42) and abnormal insulin secretory behavior (4-6, 8-11, 14, 21, 42).
We previously implicated polyamines as stimulatory or permissive factors for DNA synthesis and insulin production in pancreatic islets isolated from normoglycemic mice and fetal rats (32-34, 41). In an attempt to further elucidate the role of polyamines in diabetic pancreatic islets, isolated islets from obese-hyperglycemic mice at different stages of the syndrome have now been studied with specific attention to the relation between the polyamine content, on one hand, and the proliferative activity and insulin content of the islet cells, on the other. Thus the two aims of this study were to investigate the impact of the diabetic state and aging on polyamine contents and requirements in these islets.
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MATERIALS AND METHODS |
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Materials. Difluoromethylornithine (DFMO) and methylacetylenic putrescine (MAP) were generously provided by Dr. Peter P. McCann (Merrell Dow Research Center, Cincinnati, OH), and ethylglyoxal bis(guanylhydrazone) (EGBG) was provided by Dr. Juhani Jänne (University of Helsinki). RPMI 1640 culture medium, L-glutamine, penicillin, streptomycin, and donor calf serum were obtained from Flow Laboratories (Irvine, CA); cyanogen bromide-activated Sepharose 4B was from Pharmacia Fine Chemicals (Uppsala, Sweden); anti-bovine insulin serum was from Miles-Yeda (Rehovot, Israel); crystalline mouse insulin and 125I-insulin were from Novo; and spermidine, spermine, L-proline, dansyl chloride, and HEPES were from Sigma Chemical (St. Louis, MO). Tissue culture dishes were supplied by Heger (Stockholm, Sweden). Collagenase (type CLS, EC 3.4.24.3) was obtained from Boehringer-Mannheim (Mannheim, Germany); Hanks' balanced salt solution was from Statens Bakteriologiska Laboratorium (Stockholm, Sweden); and [methyl-3H]thymidine (5 Ci/mmol) and L-[4,5-3H]leucine (40 Ci/mmol) were from Amersham (Amersham, UK). Unisolve was supplied by New England Nuclear (Boston, MA), and Soluene was purchased from Packard Instruments (Downers Grove, IL). All other chemicals of analytic grade were obtained from E. Merck (Darmstadt, Germany).
Preparation and culture of isolated islets. Genetically obese mice (genotype ob/ob) of both genders and lean mice of the same strain (+/?) were obtained from the inbred Uppsala colony, which originated from a breeding couple obtained from Jackson Laboratories (Bar Harbor, ME) (12). Obese mice were used at 2 and 8 mo of age and lean animals, at 2 mo of age only. Islets from animals starved overnight were isolated by a collagenase digestion technique (19) and subsequently picked free of exocrine tissue by means of a braking pipette. They were used immediately or cultured freely floating (1) for 2 days at 37°C in RPMI 1640 medium containing 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10% calf serum, and the polyamine synthesis inhibitors as indicated.
Use of polyamine synthesis inhibitors. We have in this study used inhibitors of key enzymes in polyamine biosynthesis, i.e., DFMO, MAP, and EGBG. DFMO and MAP are specific and irreversible inhibitors of ornithine decarboxylase, the enzyme regulating putrescine formation (24, 26). EGBG is a highly selective and potent, albeit not completely specific, inhibitor of S-adenosylmethionine decarboxylase, which controls synthesis of spermidine and spermine (31). The rationale for using both MAP and DFMO is, as we previously reported, that MAP alone is not very effective in inhibiting ornithine decarboxylase (41). Throughout our islet studies, we found that partial putrescine and spermidine depletion by DFMO/MAP has little impact on islet hormone production and does not at all affect islet cell replication (32-34, 41). In contrast, when EGBG is included, pronounced effects are seen on both of these parameters. It does not seem to matter, however, whether EGBG is used alone or in combination with the ornithine decarboxylase inhibitors, because both alternatives produce virtually identical results. For these reasons, we used EGBG in combination with the ornithine decarboxylase inhibitors.
Whole cell polyamine content.
The total islet polyamine content was determined by one-dimensional TLC
(30, 34, 41). For this purpose, the islets were washed in
PBS and homogenized by sonication in 20 µl of 0.3 M perchloric acid
at 4°C. After centrifugation (5 min, 12,000 g), the
supernatant was alkalinized by addition of 10 µl of 1 M
Na2CO3 followed by 75 µl of 10 mg/ml dansyl
chloride in acetone. After incubation at room temperature overnight in
the dark, excess reagent was reacted with 5 µl of
L-proline (250 mg/ml) and then sonicated for 2 min. Dansyl
polyamines were extracted in 100 µl of toluene. The toluene was
evaporated, and the residue was redissolved in 5 µl of toluene for
application on TLC plates (high-performance TLC Fertigplatten,
Kieselgel 60 F254, E. Merck). The dansylated polyamines
were separated by one run with ethyl acetate-cyclohexane (1:1, vol/vol)
followed by two runs with diethyl ether-cyclohexane (2:3, vol/vol). The
spots were scraped off the plates, and the fluorescence intensities of
the supernatants were measured in a luminescence spectrometer (model
LS5, Perkin-Elmer) connected to a plate reader at an excitation
wavelength of 360 nm and an emission wavelength of 510 nm. This method
has, in our hands, an intra-assay variability (SE/mean) of 6%, an
interassay variability of 3%, and a sensitivity of ~15 pmol (i.e.,
an amount of polyamine resulting in a fluorescence intensity 2 SD above
the blank reading). Standard curves were linear up to 1,000 pmol and
showed correlation coefficients of
0.99.
Polyamine content in cell nuclei. Cultured islets in groups of 300 were washed in ice-cold PBS and mechanically homogenized in 100 µl of a buffer containing 250 mM sucrose, 2% Triton X-100, 2 mM EDTA, and 20 mM Tris (pH 7.5). After sedimentation of nuclei (10 s, 12,000 g), the pellet was suspended once in the homogenization buffer and recentrifuged. Twenty microliters of 0.3 M perchloric acid were subsequently added, and the polyamine content of this nuclear fraction was analyzed as described above. All steps of the fractionation procedure were carried out at 0-4°C to prevent possible redistribution of polyamines between cellular compartments. Because previous studies have shown that polyamines are found mainly in the insulin secretory granules (18), it was important to ascertain the purity of the nuclear fraction. For this purpose, we determined the insulin content in acid-ethanol extracts of the pellets obtained after perchloric acid extraction. The results revealed that the nuclear fraction contained ~1% of total cellular insulin as determined by RIA (16). This means that the nuclear fraction was essentially devoid of secretory granule contamination.
Islet DNA synthesis and contents of DNA. Islets in groups of 50-150 were cultured for 2 days as described above. During the last 5 h of culture, 1 µCi/ml [methyl-3H]thymidine was present in the culture medium. At the end of the labeling period, islets were washed in PBS and homogenized in 0.3 M perchloric acid, and the acid-insoluble material was pelleted by centrifugation, solubilized in redistilled water, sonicated, and precipitated in ice-cold 10% TCA. The precipitate was washed twice in TCA and dissolved in 50 µl of Soluene. The radioactivity incorporated was determined by scintillation counting after addition of 1 ml of Unisolve. Duplicate samples of the aqueous homogenates were analyzed fluorometrically for DNA (17, 20).
For determination of labeling indexes, batches of 25-30 islets cultured as described above in the presence of 1 µCi/ml [3H]thymidine for the final 5 h were used. After careful rinsing, islets were fixed in Bouin's solution and subsequently processed for autoradiography as previously described (3, 36). A minimum of 2,000 cells (in some cases 4,000 cells) were counted, and a labeled cell was defined as carryingInsulin production and secretion. Cultured islets in duplicate groups of 20 were incubated at 37°C and pH 7.4 for 2 h in a bicarbonate buffer (41) containing 10 mM HEPES, 2 mg/ml BSA, 16.7 mM glucose, and 50 µCi/ml L-[4,5-3H] leucine. After incubation, the islets were washed in PBS and ultrasonically homogenized in redistilled water. Rates of (pro)insulin biosynthesis were measured using an immunoprecipitation technique (41).
The islet insulin content (extracted from sonicates overnight at 4°C in 70% ethanol plus 0.18 M HCl) was measured by RIA (16). For short-term insulin release experiments, duplicate batches of 10 islets were selected and preincubated at 37°C for 45 min in a bicarbonate buffer (41) supplemented with 2 mg/ml BSA, 3.3 mM glucose, and 10 mM HEPES (pH 7.4). The preincubation media were discarded, incubations were continued for another 60 min in fresh buffer, and media were frozen for subsequent analysis of their insulin concentration (16). Fresh media, now containing 16.7 mM glucose, were added to the same islets, and incubations were continued for another 60 min; media were then frozen for subsequent analysis of their insulin concentration (16). Polyamine synthesis inhibitors were not present during these short-term experiments.Statistical analysis. Means ± SE were calculated, and groups of data were compared using Student's t-test for unpaired data. In case of multiple comparisons, data were compared by multiway factorial ANOVA in combination with Bonferroni's modified t-statistics by use of a StatView 512+ (version 1.0) software package from Abacus Concepts and BrainPower (Calabasas, CA).
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RESULTS |
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Whole cell and nuclear polyamine content.
The spermine-to-spermidine ratio was markedly increased in islets from
2-mo-old obese animals compared with lean mice of the same age (Table
1). In fresh islets isolated from aged
(8-mo-old) obese mice, the spermidine content was significantly reduced
compared with that in 2-mo-old obese animals (Table 1). The content of spermine was, however, not altered in the islets of the older mice.
Also, the spermine-to-spermidine ratio was further elevated in the
islets from the 8-mo-old obese mice compared with the young animals. In
all groups, the islet content of spermidine decreased markedly during
the 2-day culture period compared with freshly isolated islets (Table
1). In contrast, the spermine content was decreased only in cultured
islets of 8-mo-old obese mice. We are not quite sure why polyamines
fall during culture, but we have repeatedly observed it in previous
islet experiments. It could of course be an artifact of culture,
meaning that the tone of control of polyamine formation normally
maintained in vivo may be lost in vitro. The islet contents of
spermidine and spermine were significantly reduced in all groups
cultured for 2 days in the presence of DFMO (5 mM) + MAP (200 µM) + EGBG (100 µM).
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Islet DNA synthesis.
After 2 days of culture, there was a significant decrease in
[3H]thymidine incorporation in islets from obese mice
compared with lean mice (Table
3). In islets from lean 2-mo-old
mice, treatment with DFMO + MAP + EGBG evoked a significant
increase in [3H]thymidine incorporation and labeling
index (Table 3). In contrast, [3H]thymidine incorporation
rates were markedly depressed by the polyamine synthesis inhibitors in
islets from the obese animals at 2 and 8 mo of age. Labeling indexes in
the obese islets were decreased as well, but the difference did not
attain statistical significance for the islets of the 8-mo-old mice. In
a separate set of experiments conducted with islets from 8-mo-old obese
mice only, DFMO (5 mM) alone failed to affect the
[3H]thymidine incorporation rate, whereas EGBG (100 µM)
alone was not as effective as DFMO + EGBG in this respect, evoking
a 65-70% decrease in DNA synthesis (Fig.
1).
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Insulin production and secretion.
The insulin content in islets isolated from 2-mo-old lean, but not
obese, mice was significantly reduced by treatment with DFMO + MAP + EGBG (Table 3). These drugs also failed to influence the
content of insulin in islets isolated from 8-mo-old obese animals. In a
separate set of experiments conducted with islets from 8-mo-old obese
mice only, DFMO (5 mM) alone failed to affect insulin production,
whereas EGBG (100 µM) alone was as effective as DFMO + EGBG in
this respect, significantly impairing (pro)insulin biosynthesis (Fig.
2). When islets from this latter series
were incubated for 60 min in low or high glucose concentrations,
glucose-sensitive, but not basal, insulin release was significantly
impaired in islets cultured in the presence of EGBG but not in
islets treated with DFMO alone (Fig. 3).
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DISCUSSION |
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We have 1) analyzed the polyamine content of fresh islets isolated from diabetic obese-hyperglycemic and lean mice of different ages, assumed to reflect the in vivo situation, and 2) compared differences in requirement of polyamines for proliferation and hormone content between young and senescent islets of obese-hyperglycemic mice, on one hand, and lean and obese mouse islets, on the other. This was done in an attempt to delineate any differences in polyamine synthesis and requirements between diabetic mice and healthy animals and whether these parameters are altered in aged islets. The obese-hyperglycemic syndrome in these mice in many respects resembles type 2 diabetes mellitus and has frequently been used as an animal model for human diabetes (12, 13).
Direct addition of spermidine and spermine to cell cultures is not
always feasible because of toxic effects due to polyamine-catabolizing enzymes present in serum and also interactions with cell membranes (29, 37). A way of obviating this problem is to use
selective inhibitors of rate-limiting enzymes in polyamine
biosynthesis. The results show that freshly isolated islets from
2-mo-old obese-hyperglycemic mice showed an increased
spermine-to-spermidine ratio compared with those of lean animals.
Considering that the islet cell replicatory activity in the obese mice
is maximally enhanced at this age (3), and because
spermine is the polyamine essential for DNA replication (31, 33,
41), the elevated ratio likely represents a preferential spermine synthesis related to the enhanced rate of proliferation. The
crucial issue of whether this event is the cause or the consequence of
the increased -cell replication was addressed by the use of polyamine synthesis inhibitors. This treatment elicited not only the
expected decrease in polyamine content but also resulted in a marked
decrease in DNA synthesis. These findings suggest that spermine indeed
may be involved in mediating the rapid
-cell replication associated
with the obese-hyperglycemic syndrome. Nevertheless, because the
specificity of EGBG is not absolute (31), it cannot be
excluded that part of the actions of EGBG observed here may not be
attributable solely to spermine depletion.
Our present findings indicate that the labeling index of islets from diabetic obese mice was not different from that of lean mice after culture. This means that the enhanced DNA synthesis known to occur in vivo in islets of obese mice (3) is normalized by 2 days of tissue culture. Unfortunately, it is not possible for technical reasons to perform reliable DNA synthesis measurements in vitro on freshly isolated islets, because the collagenase isolation procedure seems to severely interfere with thymidine uptake and metabolism (36). The use of labeling index measurements is a well-established method for studies of islet cell DNA synthesis (technique reviewed in Ref. 36). While it is clear that changes in this index do not necessarily imply that mitotic cell division is altered, a net change in cell number would be impossible to detect after 2 days in culture, because only ~2% of adult islet cells traverse the cell cycle. In all groups, there was a decrease in polyamines, particularly spermidine, during culture. This phenomenon was reported by us previously (41), but it is not known why it is occurring.
The -cell replicatory capacity decreases during senescence (3,
35), and so does the expression of polyamine biosynthetic enzymes in other cell systems (7), suggesting a possible
interrelationship between these two phenomena. In the presently studied
fresh islets isolated from aged obese mice, there was a clear decrease
in the content of spermidine, whereas the spermine content did not
differ from that of young obese controls, suggesting that the lowered proliferative activity of the aged cells was not due to a change in
spermine content. Further characterization of the subcellular polyamine
distribution revealed that the nuclear polyamine content was not
altered by the polyamine synthesis inhibitors in any group. Thus it
appears that the
-cell is equipped with a mechanism translocating spermidine and spermine into the nucleus, when there is a depletion of
cytoplasmic polyamines. This may serve as a means by which the
-cell
in islets of the lean mice can amplify its rate of DNA synthesis,
despite a decrease in the cytosolic polyamine content. Such reversible
fluxes of polyamines between nuclei and cytosol are also likely to take
place in normal conditions. The elevated DNA synthesis in lean
mouse islets, occurring despite a decreased cytosolic polyamine
content, may be conveyed also by other mechanisms that transduce the
mitogenic message into the nucleus. One such mechanism could be the
polyamine-dependent casein kinase II, which has been shown to localize
in the cytosol and the nucleus (23) and has also been
demonstrated in mouse
-cells (39). Apparently,
-cells from obese diabetic mice differ from those of normal mice in
their sensitivity to polyamine depletion, in that the DNA synthesis of
the former cells is decreased by DFMO, MAP, and EGBG. The
-cells from obese diabetic animals may have different polyamine turnover rates
or differences in the regulation of polyamine-dependent protein
kinases, which make them more susceptible to polyamine synthesis
inhibitors than
-cells from normal mice. In agreement with this,
previous reports have indicated a number of functional and secretory
abnormalities in islets from the diabetic ob/ob mice
(4-6, 8-11, 14, 21, 40, 43).
Another area in which polyamines in general, and spermine in particular, have been implicated is insulin production and secretion (18, 32-34, 41). These studies were conducted in normal mouse or rat islets and demonstrated a glucose-regulated spermine content, suggesting a stimulatory or permissive role of spermine at multiple sites of insulin production and glucose-sensitive insulin release, results reproduced in the present study. Such a role is also in conformity with the present observations of an increased spermine-to-spermidine ratio in conjunction with a previously reported enhanced (pro)insulin biosynthesis (2) and hypersensitive secretory response to glucose (5, 11, 21, 40) in islets of the obese-hyperglycemic mouse.
Because pancreatic islets contain a mixed population of
hormone-secreting cells, it cannot completely be excluded that the effects reported here to a minor extent may result from non--cells. However, because in the islets of obese-hyperglycemic mice there is a
marked enrichment in insulin-producing
-cells (40, 42), this possibility seems remote. Likewise, the proportion of
-cells within the islet changes with time and also between lean and obese mice. Although the differences are not great, they should be taken into
consideration when viewing the results.
It is concluded from the present data that islets from
obese-hyperglycemic mice exhibit important qualitative and quantitative differences in their polyamine content and requirement of polyamines for cell proliferation compared with lean mice of the same strain. The
results furthermore conform to the view that spermine may be involved
in mediating the rapid islet cell proliferation noted early in the
obese-hyperglycemic syndrome, but changes in spermine do not seem to
account for the decline in DNA synthesis in senescent -cells.
Whether these differences in the formation and requirements of
polyamines between islets from lean and obese-hyperglycemic mice also
exist in islets from diabetic patients remains to be elucidated.
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ACKNOWLEDGEMENTS |
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Critical review of the manuscript by Prof. Claes Hellerström is gratefully acknowledged. The skillful technical assistance by Margareta Engkvist, Eva Törnelius, Cristina Bittkowski, and Astrid Nordin is gratefully acknowledged.
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FOOTNOTES |
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Financial support was received from the Karolinska Institutet, Swedish Medical Research Council Grants K98-03X-12550-01A, 72X-00034, 72X-09890, 72XS-12708, 72X-09891, K99-72X-00109-35B, K99-72X-00109-35B, and 12P-10151, the Swedish Diabetes Association, the Swedish Society of Medicine, the Nordic Insulin Foundation, Barndiabetesfonden, Berth von Kantzow's Foundation, Magnus Bergvalls Foundation, Torsten and Ragnar Söderberg's Foundations, Novo-Nordisk Sweden Pharma, Harald Jeansson's and Harald and Greta Jeansson's Foundations, Tore Nilsson's Foundation for Medical Research, Åke Wiberg's Foundation, Syskonen Svensson's Fund, and Fredrik and Inger Thuring's Foundation.
Address for reprint requests and other correspondence: A. Sjöholm, Dept. of Molecular Medicine, Endocrine and Diabetes Unit, Rolf Luft Center for Diabetes Research, Karolinska Institutet, Karolinska Hospital (L6:B01), SE-171 76 Stockholm, Sweden (E-mail: ake{at}enk.ks.se).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 April 2000; accepted in final form 7 September 2000.
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