Leptin in children with newly diagnosed type 1 diabetes: effect of insulin therapy.

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.

Inhibition of leptin secretion by insulin and metformin in cultured rat adipose tissue.

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.

Inhibition of acetyl CoA carboxylase by GTP gamma S.

The effect of nonhydrolyzable guanine nucleotides on mammalian acetyl CoA carboxylase (ACC) activity was examined. Using porous rat adipocytes and crude fat cell homogenates to study metabolic pathway flux, GMPPNP and/or GTP gamma S inhibited [14C]fatty acid formation by up to 95% when either [6-14C]glucose-6-phosphate or [1-14C]acetyl CoA was used as substrate. If [2-14C]malonyl CoA initiated flux, however, no inhibition was apparent. These pathway flux studies suggested that ACC was the locus of inhibition, and that the mechanism might involve a disruption of guanine nucleotide hydrolysis by the nonhydrolyzable analogues. Using partially and avidin-sepharose-purified ACC preparations from rat fat, liver and mammary tissue, citrate-stimulated ACC activity was inhibited by 25-75% with 50 microM GTP gamma S. Related compounds and nucleotides had absent-to-minimal effects on ACC. ATP gamma S was inhibitory (10-30% at 5-15 microM), but always to a lesser degree than equimolar GTP gamma S. Filter binding assays with [alpha-32P]GTP or [35S]GTP gamma S were negative, but low-level GTPase activity was detected. Using photoaffinity labelling techniques, [alpha-32P]GTP was found to bind ACC and not pyruvate carboxylase. The hypothesis that citrate-responsive ACC activity may be modulated by an intrinsic or associated GTP binding site is explored. Since ACC forms polymers, as does the cytoskeletal protein beta-tubulin, amino acid sequence comparisons between ACC and atypical GTP binding domain of beta tubulin are presented.

Neonatal cord blood leptin: its relationship to birth weight, body mass index, maternal diabetes, and steroids.

Leptin is a 16-kD protein encoded by the ob/ob (obesity) gene. In rodents it plays a role in obesity, diabetes, fertility, and neuroendocrine function. In humans serum concentrations of leptin correlate with total body fat in both adults and children. We measured cord blood leptin in 186 neonates that included 82 appropriate for gestational age (AGA), 47 large for gestational age (LGA), 20 infants of diabetic mothers, 52 preterm infants, and 15 intrauterine growth-retarded (IUGR) infants. There were 16 pairs of twins. The mothers of 17 preterm infants were treated with steroids before delivery. Leptin (mean +/- SD) concentration in term, AGA infants (39.4 +/- 1.1 wk) with birth weight (BW) of 3.2 +/- 0.3 kg, body mass index (BMI) of 12.6 +/- 1.1 was 4.01 +/- 3.5 ng/mL. BW correlated with cord leptin (p = 0.002) in a multivariate analysis controlling for potential confounders. Both LGA infants and infants of diabetic mothers had higher cord leptin concentration 7.3 +/- 3.8 and 6.1 +/- 4.8 ng/mL, respectively, compared with AGA infants (p < 0.05). Preterm infants had a mean leptin level of 1.8 +/- 0.97 ng/mL and a 3-fold elevation was seen if mothers received steroids antenatally (p = 0.006). IUGR infants had increased leptin (6.5 +/- 3.9 ng/mL, p = 0.03). Concerning the twin pairs, the smaller had a higher leptin level compared with larger twin (4.1 +/- 9.51 versus 2.8 +/- 5.14, p = NS). Neonatal cord leptin concentrations correlate well with BW and BMI. No gender differences were found in cord blood leptin. Maternal obesity had no effect on cord leptin, whereas exogenous maternal steroids increased neonatal leptin concentrations.

Persistence of disturbed adipocyte metabolism in streptozocin-induced diabetic rats despite near-euglycemia with phlorizin.

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.

Activation of in situ glycolytic flux by bisphosphorylated compounds: studies in porous rat adipocytes.

By carefully permeabilizing eukaryotic cells such that intracellular enzymes are largely retained, an opportunity is created to explore the regulation of in situ flux. This is particularly important since the latter may not be accurately represented by kinetic measurements of isolated, solubilized enzymes from disrupted cells. In this study the action of fructose 2,6-diphosphate (F2,6DP) and other bisphosphorylated sugars which purportedly activate phosphofructokinase-1 (PFK-1; EC 2.7.1.11) were studied. Using porous adipocytcs and initiating flux with radiolabeled glucose 6-phosphate, the regulation of lactate production under both 0.1 and 1.0 mM ATP conditions by F2,6DP, glucose 1,6-diphosphate (G1,6DP), ribulose 1,5-diphosphate (R1,5DP), 2,3 diphosphoglycerate (2,3DPG), and mannose 6-phosphate (M6P) was examined. Studied at 1, 5, and 25 microM concentrations, F2,6DP and 2,3DPG significantly (and to the same extent) augmented glycolysis compared to control (at 0.1 mM ATP, the respective glycolytic rates--as % above control--at these three above-mentioned concentrations for F2,6DP were 60, 84, and 77%, whereas for 2,3DPG they were 84, 105, and 179%; at 1 mM ATP, the F2,6DP effect was 88, 99, and 121%, and for 2,3DPG it was 52, 89, and 96%). Stimulation by these compounds was less obvious at higher glycolytic flux rates (saturating amounts of G6P). Amongst this group, and only at 1.0 mM ATP, the sole other positive effector was 25 microM R1,5DP. The measured fat cell content of G1,6DP was 24 +/- 4 microM (n = 3); at this concentration no significant effect on glycolysis was observed. Examining the effects of 2,3DPG (25 microM) on proximal glycolysis (to triose phosphates) revealed there was a modest, but significant, 41% increase over basal; in contrast, under the exact same conditions, F2,6DP caused a 123% increase. Separate experiments also examined the effect of F2,6DP, 2,3DPG, and G1,6DP on glycolysis at 5 and 25 microM in the presence of a physiologic cytosolic ATP/ADP ratio and free cation concentrations. Under these conditions, F2,6DP and 2,3DPG remained pre-eminent in their stimulatory prowess, inducing 27-71% increases over control, while G1,6DP remained ineffectual. These studies support a locus of action of 2,3DPG on overall glycolysis which is distal to the triose phosphates. M6P was ineffective at all concentrations. In conclusion, F2,6DP is the pre-eminent in situ regulator of in situ adipocyte glycolysis, especially at higher ATP levels, although other sugars containing two phosphoryl groups may under certain conditions cause activation.

Selective stimulation of in situ intermediary metabolism by free calcium in permeabilized rat adipocytes.

The hypothesis that ionized calcium [Ca2+]i may stimulate in situ rat adipocyte intermediary metabolism distal to glucose transport was tested. A metabolically active porous adipocyte model was employed in which pathway metabolism is exclusively pore-dependent using glucose 6-phosphate (G6P) as substrate. Cellular [Ca2+]i was, furthermore, directly adjusted to between 0-2.5 microM via the membrane pores. Three metabolic fluxes were examined, (1) glycolysis-Krebs ([6-14C]G6P oxidation), (2) glycolysis to lactate ([U-14C]G6P to [14C]lactate) and (3) pentose pathway ([1-14C]G6P oxidation). Glycolysis-Krebs oxidation was was found to be selectively (33% above basal P less than 0.001) stimulated by 0.625 microM free calcium. In contrast, there was no effect of [Ca2+]i on the other, exclusively cytoplasmic, pathways. The stimulation of glycolysis-Krebs by [Ca2+]i was inhibited by a mitochondrial calcium channel blocker (Ruthenium red) and persisted over a range of ATP/ADP ratios. Separate studies demonstrated that 2-[1-14C]ketoglutarate oxidation was also calcium-stimulated in the porous adipocytes (160% over baseline at 1 microM [Ca2+]i). These studies thus demonstrate that physiologically relevant increments in porous adipocyte [Ca2+]i enhance overall in situ glycolytic-Krebs pathway oxidation by a mechanism which entails mitochondrial calcium uptake. Methodologically, this metabolically active porous adipocyte model presents a novel experimental approach to investigations regarding the effects of ionized calcium on intermediary metabolism beyond glucose transport.

Diminished in situ glucose-6-phosphate flux in permeabilized adipocytes from streptozocin-induced diabetic rats.

With metabolically active, saponin-permeabilized adipocytes, in situ pathway metabolism, which was distal to glucose transport, was examined in acute streptozocin-induced diabetic (STZ-D) rats. Metabolic reactions were initiated with selectively radiolabeled glucose-6-phosphate (G6P), an otherwise inert substrate with intact cells. Thus, the membrane pores permitted a direct comparison of cellular flux between control and STZ-D adipocytes at identical initial substrate concentrations. Three metabolic pathways were studied: 1) proximal glycolysis through the triosephosphates ([3-3H]G6P to 3H2O), 2) glycolysis-Krebs ([6-14C]G6P) oxidation, and 3) lipogenesis ([6-14C]G6P incorporation into triglyceride). The extent of membrane porosity was assessed by both propidium iodide staining and lactate dehydrogenase leakage to assure that porosity was comparable between the cell groups. Porous adipocytes from STZ-D rats had markedly attenuated rates of G6P metabolism compared with controls. At enzyme-saturating concentrations of G6P (4 mM), this deficit ranged from 44% for glycolysis-Krebs oxidation to 88% for lipogenesis. The reduction in glycolysis-Krebs oxidation was also evident between 0.5 and 6 mM G6P. These porous-cell data were compared with parallel studies of glucose metabolism and clearance in intact adipocytes. Finally, several glycolytic enzymes and acetyl-CoA carboxylase were measured in cell-free (sonicated) extracts with traditional in vitro methods under Vmax conditions. Overall, the in situ porous-cell flux measurements uncovered larger deficits in posttransport cellular metabolism than were apparent in the cell-free, in vitro assays. We conclude that, in actively metabolizing porous rat adipocytes, there exists a striking and unequivocal transport-independent defect in intermediary metabolism after acute STZ-D.

Kinetic superiority of intra- vs. extracellular pentose pathway flux: studies in porous adipocytes.

Considering how the cytosol is typically prepared, to wit, by cell disruption and ultracentrifugation, historically this compartment has been deemed unstructured and kinetically analogous to a solubilized system. By prudently permeabilizing rat adipocytes so that there is scant enzyme egress, intermediary metabolism can be explored intracellularly. Herein, cytoplasmic flux vs. a soluble reference system was compared. The three-enzyme oxidative portion of the pentose pathway was examined using [1-14C]glucose 6-phosphate (G-6-P); both the steady-state velocities (nu o) and transient times (tau ss) were compared in each system. At low G-6-P levels (much less than km), nu o is dependent solely on the activity of the first enzyme, and tau ss is determined by the distal two enzymes. In our experiments, the need also arose to compare tau ss between preparations, wherein the enzyme concentrations were unequal. It is shown that tau ss.nu o/G-6-P serves as an index of kinetic efficiency. Over various dilutions, the kinetic value (nu o/G-6-P) for the porous cells ranged from 2.0 to 23.9 x 10(-6) l/min, with corresponding tau ss values of 18-1.0 min. The respective values for the solubilized enzyme system were from 4.9 to 42.2 x 10(-6) l/min and from 15.2 to 1.1 min. This abbreviated pathway occurring in porous cells was nearly twice as fast at reaching steady state than the corresponding solubilized system. We conclude that cytoplasmic flux is kinetically efficient and that metabolic studies conducted only under Vmax conditions and ignoring tau ss could overlook the cellular effects of hormones or pathological states.

Highly specific micromethod for the enzymatic determination of radioactive [14C]lactate.

By collecting released 14CO2 following the enzymatic decarboxylation of radiolabeled lactate, picomoles of the latter can be precisely, easily, and reproducibly measured in small biological fluids. This radioactive [14C]lactate microassay does not require a neutralization step, nor does it require chemical extractions and partioning procedures, ion exchange, or pyruvate derivatization. Under our specified conditions this simple reaction goes to completion in 90 min. Using this assay in porous adipose cells, with the cell number logarithmically less than that found in other literature methods, the measured glycolytic flux rates were consistent with those previously reported. In these studies, glycolysis was initiated with [U-14C]glucose 6-phosphate. This microradioactive lactate assay is useful when dealing with minute tissue samples and/or microliter aliquots of biological fluids.

Massive breast enlargement in an infant girl with central nervous system dysfunction.

A 6-month-old female is described who presented with severe idiopathic macromastia. The breast enlargement began at 2 months of age and progressed such that subtotal mastectomies were necessary at 23 months. Extensive hormonal evaluation prior to surgery revealed no evidence of estrogenization or precocious puberty. There was no galactorrhea. A breast biopsy showed immature mammary tissue. In vitro analysis of the patient's serum using a mouse mammary thymidine incorporation assay revealed similar mitogenic activity in the patient's serum compared to adult controls. Post surgical follow up of this patient, 3.5 years later, has revealed no breast enlargement, precocious sexual development, or growth acceleration. Of interest, however, she has manifested an idiopathic degenerative neurologic condition characterized by psychomotor delay, ataxia, and seizures. Remarkably, hormone studies at age 5.5 years showed an exaggerated gonadotrophin response to intravenous gonadotrophin releasing hormone and prepubertal estrogen levels. While this case may represent an extraordinary example of idiopathic premature thelarche, the severe nature of this infant's macromastia in association with neurologic dysfunction and increased gonadotrophins suggests that central nervous system factors were etiologic.

Adjunctive use of tolazamide in newly-diagnosed diabetic children.

Recent data suggests that one of the major actions of sulfonylureas is to potentiate the anabolic cellular effects of insulin. This is the first study to examine the use of sulfonylureas as adjunctive therapy in newly-diagnosed type I diabetic children. A random, prospective, double blind study over 15 months, stratified by age at diagnosis, was conducted. The treatment group (n = 13) received daily oral weight-adjusted tolazamide whereas the control group (n = 11) received placebo. Monthly comparison of the HbA1 values between groups revealed no statistical difference; likewise, the fasting serum C-peptide values were not dissimilar. The mean daily insulin dose per kilogram, however, was less in the tolazamide group (P less than 0.001). The data suggests that the addition of tolazamide may not be of therapeutic benefit in newly diagnosed juvenile diabetics, although insulin requirements may be reduced.

Carnitine palmitoyltransferase: effects of diabetes, fasting, and pH on the reaction that generates acyl CoA.

Although carnitine palmitoyltransferase (CPT) has received considerable attention, particularly its regulation by malonyl CoA, most studies have monitored the forward reaction, ie, the formation of acylcarnitine. We examined the opposite or reverse reaction, in which palmitoyl CoA is generated, in osmotically-disrupted rat hepatic mitochondria. Specifically, the effects of pH, fasting, and untreated recent-onset diabetes were investigated. As with the forward (f) reaction, the CPT reverse (r) velocity v pH curve was somewhat parabolic with a pH maximum at approximately 7.2 (except the CPT that was from the diabetic rats). However, as the pH rose, the CPT reverse and forward curves diverged due to a precipitous decline in the forward reaction. This discordance in rates in the alkaline range was apparent in all three groups of CPT but was most prominent in the diabetic preparation (for example, as the pH increased from 7.3 to 8.8, the respective declines in the f and r velocities were 74% and 2%). In addition, under our assay conditions the CPTr from diabetic rats not only had a higher velocity (55.4 +/- 1.4 nmol/min/mg protein) than that from the fed (32.1 +/- 3.1) or fasted (43.1 +/- 3.4) animals, but also the Vmax was found to be twofold greater, even though there was no difference in the Km for palmitoylcarnitine. In summary, diabetes affects the kinetics of the reverse reaction and, regardless of the animal's premortem condition, but more so in the diabetes, this reaction is less attenuated than the forward one as the pH rises.

Preservation of intermediary metabolism in saponin-permeabilized rat adipocytes.

A unique permeabilized adipocyte model is described in which vigorous intracellular intermediary metabolism is preserved through both the pentose and glycolytic-Krebs pathways following saponin-induced pore formation in plasma membranes. Upon addition of appropriate cofactors, the cells metabolize both glucose and glucose 6-phosphate at rates which are severalfold greater than control (membrane-intact) cells. Saponin is shown to mediate these metabolic effects solely by creating membrane pores through which substrate influx occurs. This cell model provides an unprecedented opportunity to examine intermediary metabolism in situ because it permits the entry into the cytosol of previously restricted substrates, modifiers, and radiolabeled compounds. By circumventing the glucose transporter while, for the most part, preserving plasma membrane integrity, these metabolically active, porous adipocytes may permit the direct exploration of postinsulin receptor glucose metabolism by various hormones or drugs.