1 Diabetes Research Laboratory, Program in Nutrition and Biochemistry and 2 Program in Physiology, Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia 31207; 3 National Space Biomedical Research Institute, Houston 77030; 4 Nutritional Biochemistry Laboratory and 5 Laboratory for Human Immune Function and Signal Transduction, Life Sciences Research Laboratories, National Aeronautics and Space Administration Lyndon B. Johnson Space Center, Houston, Texas 77058
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ABSTRACT |
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The present
studies were designed to determine effects of a microgravity model
system upon lipopolysaccharide (LPS)-stimulated tumor necrosis
factor- (TNF-
) activity and indexes of insulin and fuel
homeostasis of pancreatic islets of Langerhans. Islets (1,726 ± 117, 150 islet equivalent units) from Wistar-Furth rats were treated as
1) high aspect ratio vessel (HARV) cell culture, 2) HARV plus LPS, 3) static culture, and
4) static culture plus LPS. TNF-
(L929 cytotoxicity
assay) was significantly increased in LPS-induced HARV and static
cultures; yet the increase was more pronounced in the static culture
group (P < 0.05). A decrease in insulin concentration
was demonstrated in the LPS-stimulated HARV culture (P < 0.05). We observed a greater glucose concentration and increased
disappearance of arginine in islets cultured in HARVs. Although
nitrogenous compound analysis indicated a ubiquitous reliance on
glutamine in all experimental groups, arginine was converted to
ornithine at a twofold greater rate in the islets cultured in the HARV
microgravity model system (P < 0.05). These studies
demonstrate alterations in LPS-induced TNF-
production of pancreatic
islets of Langerhans, favoring a lesser TNF activity in the HARV. These
alterations in fuel homeostasis may be promulgated by gravity-averaged
cell culture methods or by three-dimensional cell assembly.
tumor necrosis factor-; cytokines; diabetes; amino acids
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INTRODUCTION |
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FACTORS THAT
MODULATE glucose tolerance and hyperglycemia in diabetes and its
related syndromes include pancreatic islet insulin secretory function,
hepatic and peripheral insulin sensitivity, and glucose effectiveness
(7, 16, 42). An intriguing candidate peptide that has
recently been implicated in the glucose intolerance of diabetes and
obesity is the cytokine tumor necrosis factor- (TNF-
). TNF-
is
a potent modulator of insulin sensitivity in adipocytes (1,
14). Increased TNF-
induces peripheral insulin resistance, which can decrease glucose utilization. Although it was
once believed that macrophages and adipocytes were the primary sites of
TNF-
production, we have recently demonstrated that purified
pancreatic islets of Langerhans are a potent source of TNF-
(20). These observations suggest a putative islet-linked site of action for TNF-
in glucose intolerance.
Prolonged bed rest has long been utilized as a paradigm for insulin resistance and glucose intolerance; it also resembles metabolic alterations observed in microgravity (29). Human bed rest studies (21, 22) demonstrate increased peripheral tissue insulin resistance and reduced glucose tolerance. Similarly, postflight investigations of cosmonauts (12) after free-fall orbit demonstrate increased plasma glucose and decreased glucose tolerance. Other spaceflight investigations illustrate altered C-peptide excretion (33) and insulin resistance (8) during earth orbit. These observations and others (24) suggest that bed rest and in-flight experiments support changes similar to those observed in the glucose intolerance of prediabetes, aging, physical inactivity, or obesity.
A series of in vitro studies illustrate altered immune cell cytokine
activity in microgravity that may favor increased insulin resistance
via altered TNF- release. The Skylab 3 mission demonstrated that immune responses may be altered by spaceflight (32).
On Space Transportation System (STS)-56, MC3T3-E1 osteoblasts activated in microgravity utilized less glucose and had reduced prostaglandin E2, a proposed regulator of cytokine production
(15). As short a duration as 8 s of microgravity has
been demonstrated to alter macrophage responses (2).
Lipopolysaccharide (LPS)-activated macrophages secrete more
interleukin-1 and TNF-
when stimulated in microgravity than on earth
(6). These spaceflight studies may provide insight into a
mechanistic link between immune modulations and glucose tolerance.
However, serious questions remain as to which observations are due to
stressors of lift-off and reentry and which are true microgravity effects.
Although there is disagreement about whether ground-based paradigms
truly simulate microgravity (38), certain cell culture systems provide a model system for investigating altered gravity effects. One such microgravity model system is the "high aspect ratio
vessel" (HARV) developed at the Johnson Space Center. The HARV is a
self-contained, horizontally rotating cell culture system that allows
for diffusion of oxygen and carbon dioxide across a semipermeable
membrane. The HARV demonstrates a very low shear stress (0.5 dyn/cm2) for 1- or 2-mm cellular aggregates
(38). It has a time-averaged gravity vector of
102 g (31) compared with that of
near-earth free-fall orbit, which is between 10
4 and
10
6 g. Thus the HARV is a useful paradigm for
studying cellular physiology in a ground-based cell culture system that
demonstrates both low shear stress and a gravity-averaged free-fall paradigm.
The present studies were designed to determine the main and interactive
effects of microgravity and addition of LPS on TNF- activity,
insulin secretion, and glucose concentrations in cultured islets of
Langerhans. An additional aim was to determine alterations in amino
acid and nitrogenous compound utilization as an explanation for altered
glucose concentrations in the microgravity model system.
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SUBJECTS AND METHODS |
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Animal Care
Male Wistar-Furth rats (Harlan Sprague-Dawley, Houston, TX and Indianapolis, IN) were obtained at 9-10 wk of age and ~220 g body wt and were housed in shoebox cages (n = 3/cage) with cellulose bedding. Animals were maintained on a 12:12-h light-dark cycle (lights on at 0700) and had access to food and water ad libitum. All procedures were carried out in accordance with the guidelines of the National Institutes of Health and were approved by the NASA-Johnson Space Center and Mercer University Institutional Animal Care and Use Committees.Study Design
There were two studies carried out in these experiments (Fig. 1A): In study A, we contrasted the HARV cell culture technique vs. the static plate (PLATE) culture paradigm; in study B, those groups were further subdivided into LPS-stimulated and nonstimulated treatment groups. This experimental design allowed us to differentiate the independent effects of the cell culture method (HARV vs. PLATE) under basal conditions on TNF-
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Pancreatectomy and Islet Isolation: Days 1 and 2
All animals were acclimatized to the laboratory for 1-2 wk before beginning experiments. We removed food from the cages of donor male Wistar-Furth rats at ~1800; at 0800 the next morning (day 1, Fig. 1A), the rats were anesthetized with 55 mg/kg intraperitoneal pentobarbital sodium and were pancreatectomized. Animals were exsanguinated under surgical anesthesia after pancreatectomy. Islets were isolated using established methods (3) with modifications previously described (11). Purified islets were hand-picked to obtain ~1,700 islets per treatment group (Fig. 1B) by use of ×25 magnification with a green illuminated background and white fiber optic side illumination.Static Culture of Islets After Islet Isolation: Days 1 and 2
Freshly isolated islets of Langerhans were placed into Medium-199 (with additional 100 U/ml penicillin, 100 µg/ml streptomycin, 10% fetal bovine serum, 25 mmol/l HEPES buffer, and 0.68 mmol/l L-glutamine) as previously described (11). Islets were isolated over a 2-day period. The islets from two rats each (six rats/day of isolation), were placed into 6-well polystyrene culture plates with 10 ml Medium-199/well (~1,200 islets). Islets were introduced into static culture at ~5 PM. Islets remained on static culture for 24-48 h before being aliquoted into HARV cultures for experiments.HARV Cell Culture Techniques for Pancreatic Islets: Days 3 and 4
Islets were aspirated from culture plates, and cells were transferred into a 50-ml conical vial and brought to 40 ml with M-199. Islets from all pancreatic isolations were mixed and then aliquoted into four 15-ml conical vials and centrifuged at 1,000 rpm. The vials were vortexed, and aliquots of 100 µl (3×) were removed for determination of islet diameter, which was estimated according to standards established by Ricordi et al. (30). A 1-ml sample was obtained, designated 0 h (study A), and frozen atIslets [1,726 ± 117, 150 islet equivalent units (IEU)] were aliquoted into two 10-ml HARVs (Synthecon, Houston, TX) and into culture plate wells and were placed in a 5% CO2, 95% humidified, 37°C incubator. The initial HARV speed was set at ~12 rpm. All bubbles were carefully removed from the HARVs before initiation of revolutions.
Preparation for LPS Stimulation: Day 5
After 48 h of HARV or static culture, 1 ml of islet media was aliquoted for analysis and designated 48 h (study A). Both HARV and static cultures had 5 ml of freshly prepared Medium-199 added to bring the total volume to 10 ml. All subsequent samples were taken by isovolumetric techniques. In the HARV, this was accomplished by infusing 300 µl of cold (4°C) Medium-199 into the bottom port of a vertical, stationary HARV while removing 300 µl of sample from the upper port. A similar technique was used in static cultures to ensure equal treatment perturbations.LPS Stimulation, Samples for TNF, Insulin, Glucose, and Lactate: Days 5 and 6
Before LPS addition, a 300-µl sample was removed from all treatments and designated as 0 h post-LPS stimulation. At time 0, 100 µl of 100 µg/ml LPS (Escherichia coli 026:B6, Sigma, St. Louis, MO) dissolved in Medium-199 were added to HARVs and static cultures. Islet medium samples were obtained (300 µl) for TNF, glucose, insulin, and lactate analysis at 3, 6, 12, 24, and 48 h post-LPS stimulation. The samples were placed immediately into 1.8-ml cryovials and stored atIslet and Cell Image Analysis: Days 3 and 5 (Cohort Group)
Islets were obtained from HARV and static cultures for image analysis by phase-contrast and confocal microscopy. Representative images of islets of Langerhans from the initial static culture period (day 3) and islet cells cultured in HARVs (day 5) were obtained on a Hund Willovert S inverted microscope equipped with phase-contrast optics, a green filter, and white fiber optic side illumination. Islets were maintained in culture medium, and images were acquired using a Cannon EOS 1N 35 mm camera and a ×40 air objective.Sample Analysis
TNF assay.
The L929 mouse fibroblast assay, as previously described (18, 19,
20), was used to measure islet medium TNF activity. Briefly,
L929 cells were grown to confluence overnight in 96-well culture
plates. Actinomycin D (5 µg/ml; Merck Sharp and Dohme, Weston, PA)
was added to each well, and serial dilutions of experimental islet
medium were added to duplicate L929 wells. After incubation, adherent
L929 cells were stained with 0.5% crystal violet, optical density of
each well was spectrophotometrically measured, and percent cytotoxicity
of L929 cells was determined (27). TNF activity was then
converted to units per milliliter; 1 unit of TNF activity was defined
as = 50% L929 cytotoxicity in the appropriate dilution of islet
medium. Dose-response inhibition of TNF activity with the use of
rat-specific TNF- antibodies (Endogen, Boston, MA) was performed on
islet media samples to confirm that L929 cytotoxicity was due to
TNF-
activity.
Insulin analysis. Islet medium was diluted 1:500 in Medium-199, and insulin was determined by competitive binding radioimmunoassay (Linco, St. Charles, MO) with antibodies raised against rat insulin and with use of rat insulin standards (25). Previous determination of the within-assay coefficient of variation for eight assays of pooled rat plasma was estimated at 6.2%.
Glucose and lactate analysis. Samples were analyzed in duplicate as is or were diluted 1:10 with distilled water for glucose and lactate analysis by means of a YSI 2300 Stat Plus Analyzer (Yellow Springs Instruments, Yellow Springs, OH).
Amino acid/metabolite assay. Analysis of nitrogenous metabolites was performed post hoc to probe an explanation for altered glucose and lactate data in study A. There was insufficient sample for amino acid analysis of study B. The cell culture medium (400 µl) was prepared for analysis by precipitation of protein from the samples with an equal amount of sulfosalicylic acid solution (Pickering Seraprep) and centrifugation. The supernatant (300 µl) was aliquoted, and an internal standard (glucosaminic acid, Sigma) was added. The sample was filtered (0.2 µm) and placed on the amino acid analyzer (Beckman 6300).
The Beckman System 6300 High Performance Amino Acid Analyzer uses ion exchange chromatography with postcolumn ninhydrin reaction and visible colorimetric detection for the analysis of amino acids. Depending on their dissociation characteristics, the amino acids are differentially eluted from the column with a series of lithium buffers of increasing pH and ionic strength and are further positioned by increasing the column temperature during the run. Ninhydrin, mixed continuously with the column eluent, reacts in a highly specific manner with the separated amines to form colored products, the intensity of which is proportional to the concentration of the amino acid present.Lactate dehydrogenase. Lactate dehydrogenase (LDH) assays were performed in the supernatant of all samples from study B by means of an in vitro toxicology assay (TOX-7, Sigma).
Statistical Analysis
There were multiple replications of the experimental protocol in which two HARVs and two static plates were studied concurrently. In the initial studies, five replications (islets from 12 pancreases) resulted in n = 10 per experimental treatment group for TNF-Study A was analyzed by two-way (between/within) repeated-measures ANOVA with interaction (GLM, METHOD vs. TIME, SAS version 6.11, Cary NC). Post hoc differences between treatments within a time period were determined by Duncan's post hoc test. A value of P < 0.05 was considered statistically significant. Means with similar superscripts are not significantly different by post hoc analysis (P > 0.05).
Study B was analyzed by three-factor (between/between/within) repeated-measures ANOVA (METHOD vs. LPS vs. TIME); main effects and interactions were determined. Raw data were used for all analyses with the exception of the insulin data. Due to a twofold greater basal insulin concentration in experiment 4, all data were transformed and normalized to percent baseline before statistical analysis. At discreet time points, a one-way between-subjects ANOVA was performed, and a Duncan's post hoc test was used to determine statistical significance between groups (P < 0.05). Means with similar superscripts are not significantly different by post hoc analysis (P > 0.05).
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RESULTS |
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Pancreatectomy and Islet Isolation
Islets were isolated from living pancreas donors and were placed into cell culture within 5 h after pancreatectomy. Five complete experiments were performed on separate islet populations, with four treatment groups per experiment for study A; a subset of four islet populations per treatment was studied in study B. There were no significant differences for the 150-µm islet equivalent mass in the islet populations between experimental groups (P > 0.05, Fig. 1B). The average number of islets per treatment group was 1,726 ± 117 IEU. All values as shown in Figs. 1-5 are means ± SE.
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LPS-Stimulated TNF- Production
After stimulation with LPS, there were significant increases in TNF-
of both static and HARV-cultured islets (Fig. 2B). Three-way repeated-measures ANOVA indicated significant between-subject effects
for METHOD (P = 0.0049, PLATE > HARV),
LPS (P = 0.0001, LPS > non-LPS), and HARV-LPS
(P = 0.0046, PLATE-LPS > HARV-LPS). Within-subject effects were significant for TIME (P = 0.0001) and an interaction of TIME-LPS (P = 0.0001). Islets stimulated with LPS in static culture retained
a significantly greater TNF-
concentration than LPS-stimulated HARV
cultures throughout the entire 48 h of the study. There were no
significant changes in TNF-
among control cultures.
Insulin Concentration
At time 0, insulin concentrations were <100 nmol/l and were not significantly different between HARV and static culture treatment groups (Fig. 3A). Repeated-measures ANOVA indicated a significant effect of TIME (P = 0.0004, 48 HRS > 0 HRS). After 48 h of culture, there was a greater than threefold increase in immunoreactive rat insulin concentrations. However, there were no significant differences between HARV and PLATE at 48 h.There was a marked linear increase in the insulin concentration of both HARV and static culture islets that were not stimulated with LPS (Fig. 3B). Repeated-measures ANOVA indicated no significant between-subject differences; however, within-subject effects of TIME and TIME-LPS were significant (P = 0.0001, P = 0.0001, respectively). In both HARV and static cultures that were stimulated with LPS, insulin secretion was attenuated relative to nonstimulated treatment paradigms. At 48 h post-LPS stimulation, both control groups were different from the HARV-LPS group. However, the static (PLATE) culture islets with LPS, although intermediate in value at 48 h, did not differ from any other group in their insulin secretion.
Glucose Concentration
Before cell culture, there were no significant differences in the amount of glucose in islet medium between HARV and static culture groups (Fig. 4A). Two-way repeated-measures ANOVA demonstrated a significant interaction of TIME and METHOD (P = 0.0488). The media from islets in the PLATE culture had the lowest glucose concentration at 48 h (P < 0.05).After stimulation with LPS, there were relatively minor differences in the glucose concentration during 12 h of culture (Fig. 4B). Repeated-measures ANOVA indicated a significant effect of METHOD (P = 0.0203, HARV > PLATE). The within-subject effect of TIME was also significant (P = 0.0026). At 48 h post-LPS stimulation, the static culture group had metabolized the greatest amount of glucose from the medium. The highest glucose concentrations were observed in the non-LPS-stimulated HARV cultures, which were significantly different from the LPS-stimulated static culture islets.
Lactate Concentration
Media lactate concentrations increased during the initial 48-h culture period to a level that was significantly different from the 0 HRS value (Fig. 5A). Repeated-measures ANOVA illustrated a significant effect of TIME (P = 0.0001, 48 HRS > 0 HRS). However, there were no significant differences between the lactate concentrations of HARV and static culture groups.During the LPS stimulation studies, all treatment groups increased their lactate concentrations (Fig. 5B). Repeated-measures ANOVA indicated a significant main effect only for TIME (P = 0.0001). At 48 h of study, there were no significant differences between groups.
Amino Acid/Nitrogenous Metabolite Concentrations
Amino acids and nitrogenous analytes were arranged into functional groupings and are depicted in Table 1. Urea cycle metabolites of arginine and ornithine both demonstrated an equimolar bidirectional shift in concentration favoring a greater difference in HARV (TIME, METHOD, P = 0.0027, 0.0001, respectively). Urea concentrations increased over time (Within, TIME, P = 0.0001) as did NH3, which was equimolar in both HARV and PLATE (Within, TIME, P = 0.0001).
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Nitrogen transfer metabolites illustrated that glutamate production and glutamine utilization were equimolar in both HARV and PLATE (Within, Time, P = 0.0001 for both). Alanine concentrations increased in the islet media during the 48 h of study (Within, TIME, P = 0.0223).
Neurotransmitter synthesis metabolites of -aminobutyric acid and
phosphatidylserine increased with time (Within, TIME, P = 0.0001). Glycine, an amino acid associated with creatine, heme, and
purine biosynthesis, increased with time in both HARV and PLATE
(Within, TIME, P = 0.0341).
LDH
There were no significant differences in LDH values at any time point in study B (Table 2). Repeated-measures ANOVA with interaction determined significant between-subjects overall effects of METHOD (HARV < PLATE, P = 0.0505). Within-subjects analysis was significant for TIME only (P = 0.001).
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HARV-Generated Islet Cell Aggregates
Phase-contrast microscopy of isolated islets of Langerhans cultured on static plates for 24-48 h illustrates intact islets with apparently intact collagen capsules as well as some freely circulating cells from islets that were overdigested (Fig. 6A). After 48 h of HARV culture, cellular aggregation is apparent as a ubiquitous phenomenon in islet cell aggregates (ICAs) cultured in the microgravity model system (Fig. 6B). A prominent capsular appearance in the periphery of the matrix of ICAs is apparent.
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DISCUSSION |
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The present studies were designed to investigate the influence of
the HARV microgravity model system and LPS administration on TNF-
production of pancreatic islets of Langerhans and to elucidate related
changes in insulin and glucose homeostasis. Studies were also designed
to determine the independent influence of cell culture method and LPS
stimulation on TNF-
activity and hormonal production and to
elucidate potential interactions between these variables. Additionally,
investigation of nitrogenous analytes was performed to elucidate an
explanation for glucose and lactate data. The current studies
demonstrate five novel observations. 1) LPS-induced TNF-
production is less in the HARV islet medium than in static cultures;
2) the increase in TNF-
is associated with a lesser basal
insulin secretion, especially in HARV cultures; 3) islets
cultured with the HARV are associated with a lesser glucose consumption
than are islets from static culture; 4) HARV islets
demonstrate an equimolar increase in the utilization of arginine and
production of ornithine; and 5) islets cultured with HARVs
will aggregate into "tissue-like units", which we have designated ICAs.
A most interesting and novel result in our present study is that
isolated islet preparations in HARVs had increased TNF levels with
exposure to LPS, indicating that TNF-producing cells are present in
ICAs and that LPS stimulates TNF secretion in these islets. Our present
studies indicate the possible involvement of TNF at the site of insulin
secretion, the pancreatic islets of rats. Others have suggested that
TNF gene expression may be located in the pancreas and may play a role
in islet function. Norman et al. (28) noted increased TNF
messenger RNA expression and intrapancreatic TNF that were detectable
1-6 h after onset of acute pancreatitis induced in mice by
infusion of a cholecystokinin analog. They attributed this TNF
expression to acute infiltration of macrophages during the pancreatic
inflammation. Toyoda et al. (37) detected TNF- in mouse
islets during development of diabetes in the nonobese diabetic mouse
strain NOD-Sansum. They noted that <1% of islet immune cells in this
strain were macrophages and suggested that TNF-
may be produced by
islet T cells during an autoimmune reaction in the islets of this
diabetic mouse. Our data provide further evidence that islets can be
sites of TNF secretion in rats. We believe that this is an intriguing
observation with respect to islet secretory function. The lesser
LPS-induced TNF-
production in the HARV was not expected and is
contrary to what has been previously observed during in-flight studies of LPS-stimulated macrophages (6). The present
investigations suggest three possibilities: 1) that islets
behave differently from macrophages after LPS stimulation in a
microgravity model system, 2) that results obtained in the
HARV are not directly comparable with in-flight experiments, or
3) that the aggregation of cells into ICAs differentially
modulates the production of TNF-
after stimulation with LPS. The
direct applicability of these data to in-flight studies remains to be determined.
That TNF- is associated with a decreased basal insulin secretion is
intriguing, both as it relates to in-flight studies and ground-based bed rest models, and as it suggests insight into the
pathophysiology of type 1 and type 2 diabetes mellitus. In-flight studies during the Skylab mission (17) illustrated a
consistent decrease in the plasma insulin concentration from 38 to 82 days. A decrease in pancreatic insulin secretion is seen both after autoimmune insulitis of type 1 diabetes (10) and after
prolonged peripheral insulin resistance with compensatory
pancreatic-derived hyperinsulinemia in type 2 diabetes
(39). It is not possible to say whether these data are
comparable, because serum insulin values are affected by rates of
insulin secretion, insulin sensitivity, and insulin clearance, factors
that were not measured on Skylab. In addition, plasma glucose values
were also decreased in Skylab crewmembers, suggesting improved glucose
control. However, the Skylab data have been viewed as conflicting,
since a sharp drop in insulin was followed by a trend toward an
increase, which was subsequently followed weeks later by a spike in
insulin during the 3rd and 4th weeks of flight (26). This
could occur if there were a compensatory increase in insulin production
secondary to reduced insulin sensitivity, as suggested by Stein et al.
(36); however, this would have to occur in a
time-dependent process with pancreatic function reacting secondarily
and/or primarily to spaceflight stressors. The present studies raise
the possibility of an endogenously mediated decrease in insulin
secretion as a secondary consequence of altered cytokine production in
the HARV microgravity model system. That the lowest basal insulin
secretion was observed in LPS-stimulated HARV cultures is a novel
observation and suggests that islet-derived TNF has a potent capacity
to downregulate islet insulin secretion in microgravity model systems.
We hypothesize that this reduction in insulin secretion occurs at a
lower medium concentration of TNF-
in the HARV than is observed in
the static plate controls, possibly due to higher intracellular
concentrations of TNF in the ICAs. This hypothesis is currently under
investigation in ongoing studies.
Ground-based bed rest studies support the hypothesis of a progressive
increase in insulin resistance that is accompanied by a decrease in
muscle mass after prolonged physical inactivity (4). TNF
has not been related to this effect, but it has been suggested as a
mechanism of obesity-induced insulin resistance, which may accompany
decreased activity levels (1, 14). Others (37) have
suggested that TNF may play a direct role in islet cell function during
the autoimmune response in the development of insulin-dependent
diabetes mellitus. Dunger et al. (9) found that direct TNF
exposure inhibited insulin secretion and caused significant DNA strand
breakage in isolated rat islets. Others reported that TNF attenuated
islet cell function and proposed that direct stimulation by TNF may be
involved in modulation of insulin secretion from -cells during the
progressive autoimmune development of insulin-dependent diabetes
mellitus. The pathophysiology of type 2 diabetes is known to
involve a decrease in insulin sensitivity and an initial compensatory
hyperinsulinemia followed by a subsequent decline in pancreatic insulin
secretion (7). When sequentially combined, these events
promulgate hyperglycemia. The influence of TNF-
in the
peripheral insulin resistance of adipose tissue has been implicated in
the pathophysiology of type 2 diabetes (13, 14). The
present studies provide an additional mechanism by which islet-derived
TNF-
may contribute to the development of type 2 diabetes by
suppressing insulin secretion and promulgating hyperglycemia. The
clinical relevance of these observations is unknown. The consistent
increase in media insulin concentration and the similar LDH activity of
all groups demonstrate two measures of cell viability throughout the study.
It has been proposed that insulitis with inflammatory cell influx into
islets is responsible for any islet expression of TNF that may then
influence islet function. Although macrophages are present in islets
and may be producing TNF in response to LPS, we propose that other
possible sites of TNF production may also exist in the islet
infrastructure. The many endocrine cell types located in islets could
be possible sources of TNF, but vascular smooth muscle cells of the
complex intraislet vasculature may also be secreting TNF. In previous
studies (27), we have shown that human blood vessels can
be a significant source of TNF. We found that when stimulated with LPS,
the time-dependent release of TNF from human vascular tissue was
significantly increased compared with time-matched nonstimulated
control vascular tissue. In human smooth muscle cells cultured from
both internal mammary arteries and saphenous veins, the release of TNF
into the medium essentially mimicked that seen in the intact vascular
segments. Our experiments showed that TNF release occurred from intact
blood vessels and from smooth muscle cells. Collectively, these
findings suggested that at least one source of TNF may be the smooth
muscle cell within vascular tissue. Because all islets have afferent arterioles that branch into numerous capillaries to form
glomerular-like structures that then form an extensive network of
peri-insular collecting venules, it is an intriguing possibility that
cells other than macrophages located in or near such highly
vascularized islet beds may also synthesize and secrete TNF.
Preliminary immunohistochemical studies (data not shown) illustrate
that TNF- is present in the cytosol of endothelial cells of the
islet vasculature and in the cytosol of islet endocrine cells after LPS
stimulation; TNF-
is not present in exocrine tissue. Thus the exact
sites of TNF-
gene transcription and translation remain to be
delineated in future studies.
The present studies demonstrated alterations in disappearance of glucose and arginine and differences in appearance of ornithine as a result of time and the cell culture method used. The lack of a change in medium glucose concentration of HARV cultures during 48 h of study A and during 48 h of LPS culture in study B suggests that alternative fuel sources have been utilized or that basal energy expenditure was significantly reduced in the microgravity model system. The equivalent values in lactate concentration between groups over time indicate that this phenomenon is not explained by altered glycolytic activity. In the basal state, 30% of islet metabolic energy requirements are met by the oxidation of the amino acid glutamine (23). Glutamine is the most abundant amino acid in the body and plays a primary role as a carrier of nitrogen between organs, with its amide group used for nucleic acid biosynthesis (41). We added 0.68 mmol/l L-glutamine to the Medium-199 utilized in the present studies, an amount sufficient to support basal energy expenditure. The nitrogenous compounds analyzed in the current experiments illustrate that glutamine is used ubiquitously by islets of Langerhans regardless of the experimental paradigm. The equimolar increase in alanine in HARV islets is consistent with the concept of an increased utilization of glutamine for energy by the splanchnic organs in times of catabolic stress. However, arginine disappearance and ornithine appearance coexist with a twofold greater change in urea concentrations in HARVs. Taken together, these observations suggest increased arginase activity in HARV-cultured islets of Langerhans. Because insulin was not altered in study A, we hypothesize that the decrease in arginine concentration observed in the present studies was not sufficient to alter basal insulin secretion. However, if there were an increase in insulin resistance during spaceflight that was present during the postprandial state, an increased conversion of arginine to ornithine might modulate a decreased ability for compensatory insulin secretion. The lack of a significant increase in the amino acid citrulline rules out a more active nitric oxide synthase vs. arginase. Thus nitric oxide production via arginine-to-citrulline conversion is likely not greater in the HARV, and islet blood perfusion, vascular reactivity, or membrane integrity is in all likelihood not compromised. The equimolar increase in urea, however, would suggest that an increase in total urea cycle activity is not a plausible explanation for these data. Decreases in lean body mass and altered amino acid utilization illustrate that nitrogenous metabolites are influenced by spaceflight (33-35). In ground-based studies, the reduction in lean body mass occurs concomitantly with altered glucose and insulin metabolism (21, 22, 29). At present, there is no unified hypothesis to explain these observations, although it has been suggested that a loss of lean body mass and subsequent release of nitrogenous metabolites may be related to an increase in insulin resistance (36). The present studies are the first to document decreased glucose utilization concomitant with alterations in nitrogenous analyte utilization with respect to an in vitro microgravity model system; this both supports and extends the data and hypotheses of Stein and colleagues (33-36).
The aggregation of isolated islets of Langerhans into ICAs was a
serendipitous discovery. From the four experiments conducted herein, we
observed that islet aggregation occurs within 15 h of HARV
culture. That cells will aggregate within the HARV is a well known
phenomenon and has been documented elegantly in a recent review
(38). Islet transplantation research currently underway,
which utilizes a microgravity model system for cell assembly, is aimed
at developing an islet -cell/sertoli cell aggregate for
transplantation in type 1 diabetes (5). The present studies illustrate that ~1,700 whole islets will aggregate into ICAs
that measure ~3 mm in diameter. The importance of such a large tissue
mass may be salient to the success of pancreatic islet transplantation
as a cure for type 1 diabetes mellitus. At present, purified islets of
Langerhans have been transplanted intraportally into the livers of type
1 patients, resulting in normalization of plasma glucose concentrations
(40). The requirement for immunosuppression is
contraindicated for effective islet function, as most immunosuppresive
agents decrease insulin sensitivity. Sertoli-islet cell aggregates may
provide an immunoprivileged graft; however, these cells will still need
to be transplanted into a highly vascularized organ site such as the
liver. The potential of ICAs lies in the possibility of creating a
greater tissue mass, with potential vascularization, which can be
transplanted into a readily accessible vascularized organ such as the
kidney capsule. Such an approach, if combined with sertoli cell
technology, would provide a graft that is immunoprivileged and would
allow for removal of the graft in the presence of rejection. Such
options are not currently available for intraportal islet transplants,
and at present, recipients of purified islets require lifelong
immunosuppression. Such possibilities are encouraging for ultimate
success in the search for a cure for type 1 diabetes, a cure that
ideally will ameliorate the need for daily exogenous insulin
administration and prevent the devastating secondary complications of
this disease.
In conclusion, the present studies demonstrated alterations in
LPS-induced TNF- production of pancreatic islets of Langerhans, favoring a lesser TNF production in the microgravity model system HARV
paradigm. We conclude that rat pancreatic islets are possible sources
of TNF during exposure to bacterial LPS and that insulin secretion may
be altered by LPS-induced TNF secretion. Our results are most
interesting because TNF has been implicated as possibly playing a
significant role in the obesity-related development of insulin
resistance, glucose intolerance, and non-insulin-dependent diabetes
mellitus. If one proposes that an infectious agent, or even increased
levels of circulating lipoprotein or glucose, could stimulate TNF
secretion within the islet, which then may alter insulin secretion,
intriguing hypotheses could be developed as to the local production of
islet-derived TNF and its action on islet cell function in the whole
body. The present studies also illustrated that basal insulin secretion
was suppressed concomitantly with an increase in TNF-
and may be
implicated in the hormonal alterations of spaceflight and the
pathophysiology of type 2 diabetes. Fuel homeostasis appears to be
different in the HARV culture, as virtually no glucose was utilized in
this paradigm, suggesting an alternative fuel source for ICAs. Although
nitrogenous compound analysis indicated a ubiquitous reliance on
glutamine in all experimental groups, arginine was converted to
ornithine at a twofold greater rate in the islets cultured in the HARV
microgravity model system. Such observations suggest cell-specific
effects of this system upon nitrogenous compound utilization. Finally,
islets that are cultured in HARVs will aggregate into ICAs. A limited
number of biochemical and physiological characteristics of these
aggregates have been documented in the present studies. The clinical
significance of these observations to the pathophysiology of diabetes
or their relation to manned spaceflight, however, remains to be determined.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the important advice and assistance of Drs. Neal Pellis and Judith Campell-Washington of the National Aeronautics and Space Administration-Johnson Space Center (NASA-JSC) for discussing microgravity model systems with the authors and advocating the HARV system as appropriate for these studies. Dr. Diana Risen (NASA-JSC) gave timely advice, technical support, and guidance in the use of HARV techniques for cell culture work. Dr. Peter Farrell at The Pennsylvania State University was most helpful in sharing his personal expertise and unpublished data on islet secretory function in free fall and microgravity model systems. Without the gracious interdisciplinary assistance of these individuals at several institutions, these studies would not have been carried to successful completion. Kimberly Welch-Holland performed the analyses of insulin, glucose, and lactate in islet medium. Myra D. Smith (NASA-JSC) performed the analyses of nitrogenous compounds. Paula Ezell performed photomicroscopy.
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FOOTNOTES |
---|
These studies were supported by a Summer Faculty Fellowship from the American Society for Engineering Education and NASA, NASA-JSC Director's Grant NAG 9-1021, and the Robert W. Hansen Diabetes Fund of the Fraternal Order of Eagles.
Address for reprint requests and other correspondence: B. W. Tobin, Mercer University School of Medicine, 1550 College St., Macon, GA 31207 (E-mail: tobin.b{at}gain.mercer.edu).
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 30 December 1999; accepted in final form 25 August 2000.
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