Altered TNF-alpha , glucose, insulin, and amino acids in islets of Langerhans cultured in a microgravity model system

Brian W. Tobin1, Sandra K. Leeper-Woodford2, Brian B. Hashemi3, Scott M. Smith4, and Clarence F. Sams5

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present studies were designed to determine effects of a microgravity model system upon lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-alpha ) 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-alpha (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-alpha 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-alpha ; cytokines; diabetes; amino acids


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha (TNF-alpha ). TNF-alpha is a potent modulator of insulin sensitivity in adipocytes (1, 14). Increased TNF-alpha induces peripheral insulin resistance, which can decrease glucose utilization. Although it was once believed that macrophages and adipocytes were the primary sites of TNF-alpha production, we have recently demonstrated that purified pancreatic islets of Langerhans are a potent source of TNF-alpha (20). These observations suggest a putative islet-linked site of action for TNF-alpha 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-alpha 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-alpha 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 10-2 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-alpha 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.


    SUBJECTS AND METHODS
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ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha activity, glucose, insulin, lactate, and amino acids (study A), and to determine whether interactions of LPS administration and the cell culture method were operative in regulating glucose and insulin levels (study B).


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Fig. 1.   Experimental design (A) and the islet equivalent units (IEU) per treatment group (B). HRV-CN, high aspect ratio vessel (HARV) control; HRV-LPS, HARV with lipopolysaccharide (LPS) stimulation; PLT-CN, static culture (PLATE) control; PLT-LPS, static culture plate stimulated with LPS; TNF, tumor necrosis factor. Data are means ± SE.

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 at -70°C.

Islets [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 at -70°C for subsequent analyses.

Islet 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-alpha antibodies (Endogen, Boston, MA) was performed on islet media samples to confirm that L929 cytotoxicity was due to TNF-alpha 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-alpha , insulin, glucose, lactate, and amino acid analysis. Study B contained a subset of n = 4 different islet populations per group throughout. The experimental paradigm included dependent variable analysis consisting of 1) basal TNF, insulin, glucose, lactate, and nitrogenous analytes during 48 h of cell culture and 2) LPS stimulation of TNF-alpha secretion and subsequent insulin, glucose, and lactate measures during 48 h of cell culture. The independent variables used for statistical analysis included METHOD [HARV vs. PLATE culture], LPS (LPS stimulated vs. non-LPS-stimulated), and TIME (0 vs. 48 h for study A or 0, 3, 6, 12, 24, or 48 h for study B).

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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 2.   Tumor necrosis factor-alpha (TNF-alpha ) in islet medium during 48-h culture (A) and post-LPS stimulation (B). Data are means ± SE. There were no significant differences in L929 TNF-alpha cytotoxicity for any treatment group in study A. After stimulation with LPS, there were significant increases in TNF-alpha (study B). Significant between-subject effects were noted 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 interaction of TIME-LPS (P = 0.0001).



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Fig. 3.   Insulin in islet medium during 48-h culture (A) and post-LPS stimulation (B). Data are means ± SE. At time 0, insulin values were not different in study A; however, ANOVA indicated a significant effect of TIME (P = 0.0004). In study B, insulin concentration increased in both HARV and PLATE islets not stimulated with LPS. There were no significant differences between subjects; however, within-subject effects of TIME and TIME-LPS were significant (P = 0.0001, P = 0.0001). In both LPS-stimulated cultures, insulin secretion was less compared with controls. At 48 h, both control groups were different from the HARV-LPS group; the static plate culture islets with LPS were intermediate in insulin secretion.



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Fig. 4.   Glucose in islet medium during 48-h culture (A) and post-LPS stimulation (B). Data are means ± SE. At time 0, there were no significant differences in glucose concentration in islet medium in study A; however, after 48 h, there was 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). In study B, there were minor differences in medium glucose concentration <= 12 h of culture. ANOVA indicated a significant effect of METHOD (P = 0.0203, HARV > PLATE) and TIME (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 medium glucose concentrations were observed in the non-LPS-stimulated HARV cultures, which were significantly different from the LPS-stimulated PLATE islets.



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Fig. 5.   Lactate in islet medium during 48-h culture (A) and post-LPS stimulation (B). Data are means ± SE. The 48-h value was significantly different from the 0 HRS value, as the repeated-measures ANOVA illustrated a significant effect of TIME in study A (P = 0.0001, 48 HRS > 0 HRS). During LPS stimulation (study B), all treatment groups increased their lactate concentrations; ANOVA main effects were significant only for TIME (P = 0.0001).

LPS-Stimulated TNF-alpha Production

Before LPS stimulation, there were no significant differences in L929 TNF-alpha cytotoxicity for any treatment group in any statistical measure (Fig. 2A).

After stimulation with LPS, there were significant increases in TNF-alpha 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-alpha concentration than LPS-stimulated HARV cultures throughout the entire 48 h of the study. There were no significant changes in TNF-alpha 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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Table 1.   Nitrogenous metabolite concentrations measured during the initial basal period of cell culture in HARV and static plates

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 gamma -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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Table 2.   LDH measured during 48 h of cell culture in HARV and static plates with and without LPS stimulation

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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Fig. 6.   Phase-contrast photomicrographs of freshly isolated islets of Langerhans from male Wistar-Furth rats (A) and islets cultured for 48 h in the HARV (B). All images were obtained using a ×40 air objective with a green filter and white fiber-optic side illumination.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present studies were designed to investigate the influence of the HARV microgravity model system and LPS administration on TNF-alpha 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-alpha 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-alpha production is less in the HARV islet medium than in static cultures; 2) the increase in TNF-alpha 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-alpha 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-alpha 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-alpha 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-alpha after stimulation with LPS. The direct applicability of these data to in-flight studies remains to be determined.

That TNF-alpha 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-alpha 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 beta -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-alpha 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-alpha 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-alpha is present in the cytosol of endothelial cells of the islet vasculature and in the cytosol of islet endocrine cells after LPS stimulation; TNF-alpha is not present in exocrine tissue. Thus the exact sites of TNF-alpha 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 beta -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-alpha 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-alpha 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.


    ACKNOWLEDGEMENTS

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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
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