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White adipose tissue from rats was examined for insulin- responsive vascular endothelial growth factor 165 (VEGF) secretion and mRNA expression. When separated into it constituent fat vs. stromal-vascular cells using collagenase digestion methods, only the adipocytes (or whole fat tissue) responded to physiological insulin concentrations by doubling VEGF release over 4 and 24 h in culture. Adipocyte VEGF mRNA expression increased similarly. Several adipose depots were tested. Although omental fat cells had the highest rates of VEGF release, the differences were not significant. 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, CoCl(2), 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.

Leptin, the gene product of adipose tissue that signals caloric plentitude via central nervous system receptors, may also have diverse peripheral metabolic actions. Of paramount interest has been the potential interaction(s) between leptin and insulin. Insofar as insulin alters leptin secretion/action (or vice versa), dysregulation of this system could contribute to disease states such as diabetes. The purpose of this study was to examine the effect of exogenous insulin on serum leptin in children with newly-diagnosed Type 1 diabetes. Since these patients are hypoinsulinemic (insulin-depleted) at diagnosis, they present an ideal opportunity to examine the effect of insulin repletion on serum leptin. Seventeen patients were enrolled. At baseline (prior to insulin therapy), leptin levels were 4.3 +/- 1.1 ng/ml; they were not statistically related to the baseline serum insulin or illness severity. There was no significant change in serum leptin before, shortly (1-6 days) or several weeks (3-26 weeks) after insulin treatment even when the data was corrected for changes in BMI, hemoglobin A1C, and daily insulin dose. Since repletion of the insulin deficiency that is present in non-acidotic, ambulatory patients with new onset Type 1 diabetes did not alter serum leptin, these results argue against an effect of insulin on serum leptin in the absence of the acute diabetic ketoacidosis. Because as the recuperative months following the diagnosis of new onset Type 1 diabetes are marked by weight gain, the absence of a rise in serum leptin might also indicate either an adaptive (weight permissive) or pathologic (impaired secretory) deficit.

Leptin's role in the regulation of food intake, energy expenditure and weight control are widely recognized, especially in rodents. Likewise, the potential regulation of leptin secretion by insulin (and vice versa) has been of particular interest insofar as these nutrient signals may have meaningful, even adverse (inter)actions, in diabetes. We used a freshly isolated rat adipose tissue culture model to examine the effect of insulin, metformin and glibenclamide on basal and steroid-stimulated leptin secretion. This model was selected because of its physiologic rates of leptin formation and preservation of potentially significant cell-cell interactions compared to isolated cells. The basal rate of leptin secretion was 3. 4+/-1.2 ng/100 mg tissue/24 h. The addition of 100 nM dexamethasone or 400 nM hydrocortisone stimulated leptin secretion by 3-4 fold over basal (no steroid). Insulin inhibited both basal and steroid-activated leptin secretion by 35-50%. This inhibition was present with either 1 mM pyruvate or 5 mM glucose as a substrate suggesting that glycolysis was not required. Metformin inhibited basal and dexamethasone-stimulated leptin secretion in a dose dependent manner (50% inhibition occurred at 1 mM metformin) while glibenclamide was ineffective. The effect of insulin on isolated fat cells versus fat tissue was tested in parallel. After 24 h in culture, insulin inhibited leptin secretion similarly in both adipose preparations. The addition of 200 nM (-)N6-(2-phenylisopropyl)-adenosine did not alter the results.

It is widely accepted that hyperglycemia per se incites and perpetuates the diabetic state by adverse effects on beta cell insulin secretion and peripheral insulin action. Examination of the latter locus has revealed glucose-related abnormalities in facilitated glucose transport. Beyond the plasma membrane, however, there is scant data examining whether hyperglycemia influences important intracellular metabolic events. We recently described a sizable reduction in post-transport, in situ metabolism in permeabilized fat cells from streptozocin-induced diabetic rats. Of importance, the diabetes-related deficit was entirely ameliorated by insulin therapy. In this study we examined whether hyperglycemia per se contributes to this altered intracellular metabolic effect. By infusing phlorizin, near euglycemia was achieved for at least four days in streptozocin-induced diabetic rats. The phlorizin-treated diabetic rats had improved (intact cell) rates of insulin-stimulated 2-deoxyglucose uptake. Despite this, permeabilized fat cell studies revealed no improvement or deterioration in diabetic intracellular metabolism as measured by both the oxidation of [6-14C]glucose-6-phosphate via the citric acid cycle or its incorporation into triglyceride. We conclude that hypoinsulinemia, and not hyperglycemia, mediates the disturbance in porous diabetic adipocyte cellular metabolism.