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INSULIN-GLUCAGON-GI PEPTIDES-DIABETES MELLITUS |
Department of Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama 35233-1711
Address all correspondence and requests for reprints to: Dr. Gail Mick, University of Alabama at Birmingham, ACC 608, 1600 7th Avenue South, Birmingham, Alabama 35233-1711. E-mail: . gmick{at}peds.uab.edu
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Abstract |
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Insulin-stimulated VEGF release was mediated in part via PI3K, but not PKC. Additional hormones/agents were tested, including steroids, leptin, an adenosine analog, and norepinephrine. Only the latter compound increased VEGF production, and this effect was mediated by adenylate cyclase. Adjusting the incubation glucose concentration between 0–20 mM did not alter adipocyte VEGF release. An experimental mimic of hypoxia, CoCl2, also increased adipocyte VEGF, and this effect was additive with 100 nM insulin. These studies demonstrate that physiological insulin concentrations stimulate VEGF formation and expression in cultured rodent white adipocytes. Although the biological significance of this observation remains to be determined, if white adipocyte-derived VEGF has paracrine or systemic endocrine actions, these might hypothetically impact on adipose expansion or the vascular comorbidities of obesity.
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Introduction |
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Angiogenesis, or the formation of new blood vessels from preexisting vessels, is crucial to wound healing, embryonic development, normal growth and development, and reproduction. Regarding adipose tissue, the role of VEGF as a stimulus for new vessel growth in brown fat has been described (2). White adipocytes, in contrast, are most widely recognized as a repository for triglyceride. In this depot capacity, white adipocytes remain in perpetual contact with both circulating and local metabolic and hormonal stimuli. Moreover, white adipose tissue has garnered new attention for its abilities as a major endocrine organ (3). Given the growth and hypertrophy of adipose tissue in obesity, the potential that the excess production of adipose-derived factors may contribute to obesity-related insulin resistance is a tenable concern (4, 5, 6).
We describe a new hormone-regulated function of white adipocytes, namely, insulin-stimulated VEGF formation. The stimulation by insulin occurs within a physiological insulin concentration range and is unique to adipocytes as opposed to neighboring stromal cells.
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Materials and Methods |
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Adipose tissue
After euthanasia by CO2 asphyxiation between 1400–1600 h, adipose tissue was obtained from epididymal fat pads, retroperitoneum, omental, and sc regions of 150- to 300-g male, fed Sprague Dawley rats and placed in sterile pH 7.4 DMEM containing 20 mM HEPES, 5 mM glucose, and 1% (wt/vol) BSA with 50 µg/ml gentamicin (hereafter called DMEM). The tissue was meticulously trimmed of excess vessel and then prepared as minced tissue, isolated fat cells, or stromal cells. Minced tissue (2- to 4-mm3 pieces) was prepared for culture in the above DMEM adjusted to 2–4% BSA (100 mg tissue/ml) (7). Isolated cells (adipocyte and stromal) were released by digestion at 37 C for 30–40 min with shaking (80 cycles/min) in Krebs-Ringer buffer containing 1.5–2.0 mg/ml collagenase (type 1, Worthington Biochemical Corp., Lakewood, NJ) (8). Dispersed cells were filtered through nylon mesh and allowed to settle passively for 1–3 min. The infranatant (below upper fat cell layer) was removed and centrifuged for 10 min at 200 x g. The resulting cell pellet was washed three times in DMEM (2–4% BSA) and used for stromal cell studies (1–2 x 106 cells/ml) (9). The remaining floating fat cell layer was also washed three times in fresh DMEM (2–4% BSA) buffer before culture (6–7 x 105 cells/ml). All tissue and cell samples were incubated at 37 C under 5% CO2 for 16–24 h (7). Stromal cells were examined in suspension culture because the cells did not uniformly adhere to the cell plates during the incubation. To address concerns that the fat cells be evenly exposed to medium throughout the incubation period despite their buoyancy, we manually rocked the cell plates four or five times during a 16-h incubation period. Of note, we do not mechanically rock the cells because in our hands this leads to excessive cell rupture. After incubation, an aliquot of the medium was removed to measure VEGF (EIA kit for murine VEGF165), and mRNA was isolated from adipocytes.
VEGF assay
VEGF was measured with an enzyme immunoassay kit for murine VEGF165 (R\|[amp ]\|D Systems, Inc., Minneapolis, MN). The sensitivity of this assay was 3.0 pg/ml. The intra- and interassay coefficients of variations were 5.7% and 6.8%, respectively.
RNA extraction and analysis
Total RNA was isolated from adipocytes and analyzed by Northern blot using a 980-bp murine VEGF164 cDNA (Kevin Claffey, University of Connecticut, Farmington, CT) and mouse actin cDNA probe (Ambion, Inc., Austin, TX) as a control for RNA integrity and loading. Band densitometry was performed using a Macintosh computer (Apple Computer, Inc., Cupertino, CA) and the NIH Image program.
Statistics
Cell samples, pooled from three or four rats (n = 1), were tested (control vs. hormone, etc.) in triplicate unless otherwise noted. Rates of VEGF released into culture medium are given as picograms of VEGF per 106 cells or 100 mg tissue/12 h unless stated otherwise. Results are presented as the mean ± SD, with the number of determinations given as "n." Paired t test and ANOVA were used for statistical analysis.
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Results |
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). The rates of VEGF release into the medium were comparable between adipocytes and stromal cells (294 ± 87 and 200 ± 96 pg VEGF/106 cells·12 h, respectively). Of significance, only the isolated fat cells (or whole fat) demonstrated insulin-responsive VEGF secretion. Throughout the incubation, adipocyte and stromal cell number and viability (by propidium iodide staining) remained at more than 95% of initial values. Adipocyte VEGF release was linear for 24 h (data not shown). The effect of insulin on VEGF secretion was first apparent after 4–6 h in culture (not shown).
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). Although omental cells had slightly higher rates of basal and insulin-stimulated VEGF release, these differences were not statistically significant. Moreover, adipocytes from each cell group were stimulated to a comparable (not statistically different) degree by 100 nM insulin.
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). The insulin concentration yielding 50% stimulation in VEGF release was physiological (
1 nM insulin). Other hormones and modifiers were tested for their ability to regulate VEGF secretion in adipocytes (Table 2). Both pharmacological steroids (100 nM dexamethasone) and physiological steroids (400 nM corticosterone) were without significant effect on VEGF formation. Norepinephrine at pharmacological concentrations (1–10 µM) stimulated VEGF. This adrenergic effect was mimicked by forskolin, N,O'-dibutyryl cAMP (Bu)2cAMP, as well as the selective ß3-adrenergic receptor agonist BRL37344 (Table 2). The addition of a nonhydrolysable adenosine agonist [N6-(2-phenylisopropyl)-adenosine] did not alter basal or insulin-stimulated VEGF release from adipocytes.
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).
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). None of these experimental manipulations, including glucose deprivation, affected VEGF release.
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). In separate control experiments (not included) there was no effect of 100 µM MgCl2 on VEGF release. Most intriguing, the ability of CoCl2 to increase VEGF release was entirely additive with that caused by maximal insulin stimulation (100 nM).
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, A and B). Similar to the protein data, insulin increased the amount of VEGF mRNA. When normalized to actin, the effect of insulin was dose dependent (Fig. 5B).
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Discussion |
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The potential role of adipose tissue microcirculation as a direct modifier of fat tissue expansion or intrinsic regulator of fat metabolism has historic precedent replete with forward-thinking experimental physiology (13, 14, 15, 16, 17). Indeed, it is plausible that a high degree of plasticity might be required of the adipose vascular bed given the unique propensity of this endocrine organ (6, 18) to grow and expand during both normal maturation and obesity (19). Furthermore, the unusual angiogenic activity of fat tissue has long been recognized (20), and in this regard fat tissue continues to have unique surgical applications as a topical promoter of revascularization.
Herein we appraise insulin-regulated VEGF formation in cultured rodent adipocytes. Freshly isolated fat cells (prepared by collagenase digestion and multiple washes) are a highly uniform cell suspension given the buoyancy of fat cells (21). Whole fat tissue, on the other hand, is replete with many cell types (including blood cells, endothelial cells, stromal cells, macrophages, and preadipocytes), some of which have the capacity to produce VEGF. Because simultaneously isolated, mixed stromal cell preparations from each fat tissue sample examined did not demonstrate insulin-responsive VEGF formation, we conclude that the insulin-stimulated VEGF release that was measured in adipocytes originated from the fat cells, rather than a potential trace contamination of these buoyant cells with nonadipose or stromal cells.
The effect of insulin on fat cell VEGF release was dose responsive and physiological. As this hormone response was first detected 4–6 h after insulin treatment and was accompanied by an increase in adipocyte VEGF165 mRNA levels, it is likely that the mechanism involves de novo protein synthesis rather than the release of stored protein. Although both white (22, 23) and brown adipocytes (24) have been shown to express or secrete VEGF, the regulation of adipocyte VEGF by insulin has not been reported.
There were no significant depot-specific differences in adipocyte VEGF release despite the slightly higher mean rate of omental fat cell VEGF release. This is in contrast to previous work (23) in which omental adipocytes were shown to have significantly greater in vitro rates of VEGF release compared with epididymal fat cells. Moreover, similar differences were apparent between omental and epididymal fat tissues regarding the picograms of VEGF per mg tissue. We examined VEGF production in minced fat tissue from the various depots. In a representative experiment the rates [basal (100 nM insulin)] were 303 (647), 159 (245), 936 (1872), and 126 (307) pg/100 mg tissue·12 h for epididymal, sc, omental, and retroperitoneal fat tissue, respectively. Hence, omental fat tissue has a relatively higher rate of VEGF secretion compared with other depots. This is consistent with our isolated omental adipocytes data. Of interest, cultured omental stromal cells (not shown) had similar (and non-insulin-responsive) rates of VEGF secretion to those of epididymal stromal cells. Although we did not demonstrate statistically higher rates of omental fat cell VEGF release as reported previously (23), it is possible that minor differences in fat cell preparation or incubation methods are accountable. Additionally, regarding the omental fat cell incubation, doubling the albumin concentration (to 4% BSA) or adding 200 nM (-)N6-(2-phenylisopropyl)-adenosine did not alter our findings.
Besides insulin, other potential hormone regulators of adipocyte VEGF secretion were examined. Both physiological (400 nM corticosterone) and pharmacological (100 nM dexamethasone) steroids caused a slight (<10%), but insignificant, inhibition of adipocyte VEGF release. Leptin had no effect on VEGF release. This is consistent with the recent observation that leptin stimulates angiopoiten-2 (a Tie-2 receptor antagonist), but not VEGF or angiopoietin-1 (Tie-2 receptor agonist), in differentiated 3T3-F442A cells (25). Other reports indicate that leptin has either a direct (26, 27) or a synergistic (28) effect on angiogenesis or vascular permeability. Together these data and ours point to complex hormonal interactions between insulin and leptin on adipose vasculature.
Norepinephrine (NE) stimulated VEGF secretion from cultured adipocytes. This effect was mimicked by 100 µM forskolin and 100 µM (Bu)2cAMP, suggesting that adenylate cyclase was activated. Brown adipocytes stimulate VEGF secretion via the ß3-adrenergic cascade (2). Likewise, the ß3-selective agonist, BRL 37344, also augmented white adipocyte VEGF release. It is not entirely surprising that white fat, analogous to brown fat, is capable of adrenergic-mediated VEGF secretion. It should be mentioned that the concentration of NE required to alter VEGF release is supraphysiological (1–10 µM) (29) in contrast to the physiological dose response for insulin.
Experiments in which maximal insulin (100 nM) and NE (10 µM) were added together were largely nonadditive for VEGF release (combined hormones were 10–20% above either hormone alone). This observation is intriguing because insulin and NE are usually considered to be counterregulatory hormones both in vivo and in vitro (30). Although unresolved in our current studies, various mechanisms by which NE’s intracellular actions might converge with those of insulin in the stimulation of VEGF release can be postulated. Potential mechanisms include the activation of 
1-adrenergic receptors by NE (31) or the phosphorylation by cAMP-dependent PKA of regulatory proteins other than hormone-sensitive lipase (32). Regarding 1-adrenergic receptors, it is known that NE increases glucose transport and glycolytic flux in isolated white adipocytes (33, 34). This may be relevant given observations that glucose metabolism per se regulates gene expression and cellular hormone/metabolic function (35, 36, 37). Future studies will hopefully provide greater insight regarding these possibilities.
Potential mechanisms for insulin-regulated VEGF were examined. Strategic modifiers or effectors of PKC [calphostin-C, phorbal myristate acetate, and calcium (A23187)] and PI3K (wortmannin) were tested. Although wortmannin entirely blocked insulin-stimulated VEGF secretion at 1 nM insulin, it only partially inhibited the response noted at 100 nM insulin. This suggests that more than one intracellular mechanism of action underlies insulin-stimulated VEGF secretion. PI3K mediates insulin-stimulated VEGF mRNA expression in insulin or IGF-I receptor-transfected NIH-3T3 fibroblasts (38) as well as in endothelial cells (39). In Ras-transformed cells (40) and human prostate cancer cells (41), PI3K was, furthermore, shown to couple hypoxia- and epidermal growth factor-related increases in VEGF. It is worth mentioning that multiple hormonal and postreceptor signal mechanisms have been shown to regulate VEGF formation even within the same cell (42, 43).
The potential regulatory action of glucose on insulin-stimulated VEGF secretion was examined. Both hypoglycemia (44) and hyperglycemia (45, 46, 47) have been shown to regulate VEGF secretion. Hypoglycemia, like hypoxic stress, triggers VEGF synthesis via the transcription complex entitled hypoxia-inducible factor-1/aryl hydrocarbon-receptor nuclear translocator (HIF-1/ARNT). We found no effect of absent, physiological (5 mM), or elevated (20 mM) glucose on basal or insulin-stimulated VEGF secretion in cultured adipocytes. This is in contrast to similar in vitro studies using human vascular smooth muscle cells (45) or renal mesangial cells (47) in which glucose (up to 30 mM) increased VEGF165 peptide production and mRNA expression over 3–14 h in culture. It is reasonable to propose that the potential (and to date unproven) damaging effects caused by glycemia-related VEGF secretion could be limited to vascular cell types.
To further characterize the potential role of the HIF-1/ARNT as a mediator of VEGF formation in white fat cells, we tested the effect of 100 µM CoCl2, a compound that mimics hypoxia by binding heme-sensitive enzymes (48). Adipocytes increased VEGF secretion impressively in response to CoCl2, and this effect was additive with the maximal concentrations of insulin. These data indicate that adipocyte VEGF induction can occur via the well described and ubiquitous transcription regulatory complex HIF-1/ARNT (49). The additive interaction between insulin and CoCl2 suggests that insulin-stimulated VEGF formation is not entirely mediated through the HIF-1/ARNT complex. Future studies will need to examine this more closely, however, given evidence in Hep-G2 hepatoma cells (50) that insulin induces both target genes (glycolytic enzymes and glucose transporters, Glut-1 and Glut-3) and VEGF via the HIF-1/ARNT complex.
The delayed time course (not shown) before insulin-stimulated VEGF release was detected (4–6 h) is consistent with de novo VEGF synthesis. VEGF165 mRNA expression was analyzed, and similar to the secreted protein data, insulin treatment was associated with an increase in measured VEGF mRNA in cultured fat cells.
As for a potential physiological or pathologic role of VEGF in white adipose tissue, the possibilities, although admittedly speculative, are alluring. Nonetheless, it is unarguable that blood flow to fat tissue is essential for substrate, hormone, and carrier protein delivery and, on the other hand, for mobilization and efflux of fatty acids, glycerol, and other metabolites during lipolytic conditions (18). The proliferation of fat tissue is dependent on the growth and new formation of capillaries (51). It is conceivable that paracrine factors, amid the primary constituent cells that comprise fat tissue, govern the overall volume and mass of this tissue (52). Beyond this hypothetical control of adipose tissue mass via local angiogenesis, others have shown that fat tissue blood flow, as expressed per unit weight, is attenuated in obesity (53). Whether this disturbance in adipose hemodynamics contributes to any of the metabolic derangements of obesity, including type II diabetes, remains unproven. Evidence is accumulating, however, that lipolysis may be compromised in obesity because of disturbed blood flow (54). Finally, it remains to be determined whether adipocyte VEGF has biologically relevant systemic actions on vessel growth and permeability, particularly regarding the profound vascular morbidity associated with hyperinsulinemia, obesity, and type 2 diabetes.
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Footnotes |
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Received July 30, 2001.
Accepted for publication October 29, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the pathogenesis of insulin resistance and type 2 diabetes. Trends Endocrinol Metab 11:212–217[CrossRef][Medline]
-1 Adrenergic stimulation of glucose uptake in rat white adipocytes. J Pharmacol Exp Ther 286:607–610-1 Adrenergic regulation of lactate production by white adipocytes. J Pharmacol Exp Ther 277:235–238/ARNT. EMBO J 17:5085–5094[CrossRef][Medline]
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