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Gamma aminobutyric acid (GABA) is synthesized from glutamate by glutamic decarboxylase (GAD). The entero-pancreatic biology of GABA, which is produced by pancreatic islets, GAD-expressing microbiota, enteric immune cells, or ingested through diet, supports an essential physiologic role of GABA in the health and disease. Outside the central nervous system (CNS), GABA is uniquely concentrated in pancreatic β-cells. They express GAD65, which is a type 1 diabetes (T1D) autoantigen. Glutamate constitutes 10% of the amino acids in dietary protein and is preeminently concentrated in human milk. GABA is enriched in many foods, such as tomato and fermented cheese, and is an over-the-counter supplement. Selected microbiota in the midgut have the enzymatic capacity to produce GABA. Intestinal microbiota interact with gut-associated lymphoid tissue to maintain host defenses and immune tolerance, which are implicated in autoimmune disease. Although GABA is a widely known inhibitory neurotransmitter, oral GABA does not cross the blood brain barrier. Three diabetes-related therapeutic actions are ascribed to GABA, namely, increasing pancreatic β-cell content, attenuating excess glucagon and tamping down T-cell immune destruction. These salutary actions have been observed in numerous rodent diabetes models that usually employed high or near-continuous GABA doses. Clinical studies, to date, have identified positive effects of oral GABA on peripheral blood mononuclear cell cytokine release and plasma glucagon. Going forward, it is reassuring that oral GABA therapy has been well-tolerated and devoid of serious adverse effects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mick and McCormick.)

Type 1 diabetes (T1D) is an autoimmune disease culminating in the destruction of insulin-producing pancreatic cells. There is a need for the development of novel antigen-specific strategies to delay cell destruction, including combinatorial strategies that do not elicit systemic immunosuppression. Gamma-aminobutyric acid (GABA) is expressed by immune cells, β-cells, and gut bacteria and is immunomodulatory. Glutamic-acid decarboxylase 65 (GAD65), which catalyzes GABA from glutamate, is a T1D autoantigen. To test the efficacy of combinatorial GABA treatment with or without GAD65-immunization to dampen autoimmune responses, we enrolled recent-onset children with T1D in a one-year clinical trial (ClinicalTrials.gov NCT02002130) and examined T cell responses. We isolated peripheral blood mononuclear cells and evaluated cytokine responses following polyclonal activation and GAD65 rechallenge. Both GABA alone and GABA/GAD65-alum treatment inhibited Th1 cytokine responses over the 12-month study with both polyclonal and GAD65 restimulation. We also investigated whether patients with HLA-DR3-DQ2 and HLA-DR4-DQ8, the two highest-risk human leukocyte antigen (HLA) haplotypes in T1D, exhibited differences in response to GABA alone and GABA/GAD65-alum. HLA-DR4-DQ8 patients possessed a Th1-skewed response compared to HLA-DR3-DQ2 patients. We show that GABA and GABA/GAD65-alum present an attractive immunomodulatory treatment for children with T1D and that HLA haplotypes should be considered.

Gamma aminobutyric acid(GABA) is synthesized by glutamate decarboxylase(GAD) in β-cells. Regarding Type 1 diabetes(T1D), animal/islet-cell studies found that GABA promotes insulin secretion, inhibits α-cell glucagon and dampens immune inflammation, while GAD immunization may also preserve β-cells. We evaluated the safety and efficacy of oral GABA alone, or combination GABA with GAD, on the preservation of residual insulin secretion in recent-onset T1D. Herein we report a single-center, double-blind, one-year, randomized trial in 97 children conducted March 2015 to June 2019(NCT02002130). Using a 2:1 treatment:placebo ratio, interventions included oral GABA twice-daily(n = 41), or oral GABA plus two-doses GAD-alum(n = 25), versus placebo(n = 31). The primary outcome, preservation of fasting/meal-stimulated c-peptide, was not attained. Of the secondary outcomes, the combination GABA/GAD reduced fasting and meal-stimulated serum glucagon, while the safety/tolerability of GABA was confirmed. There were no clinically significant differences in glycemic control or diabetes antibody titers. Given the low GABA dose for this pediatric trial, future investigations using higher-dose or long-acting GABA formulations, either alone or with GAD-alum, could be considered, although GABA alone or in combination with GAD-alum did nor preserve beta-cell function in this trial., (© 2022. The Author(s).)

Background: Evidence suggests that GABA may reduce pancreatic inflammation, protect β-cells from autoimmune destruction, and potentiate the regeneration of new β-cells in the setting of type 1 diabetes mellitus (T1DM). The enzyme GAD, also expressed in human pancreatic β-cells, is an antigenic target of reactive T cells. We hypothesized that treatment of children with recent onset T1DM with GABA or combination GABA with GAD will preserve β-cell function and ameliorate autoimmune dysregulation., Methods: This is a one-year, prospective, randomized, double-blind, placebo-controlled trial. Ninety-nine patients aged 4-18 years with newly diagnosed T1DM are randomized into three treatment groups: 1) oral GABA twice daily in addition to two injections of recombinant GAD enzyme, 2) oral GABA plus placebo GAD injections, or 3) placebo GABA and placebo GAD. Patients are evaluated at baseline and months 1, 5, 8 and 12. Mixed meal tolerance testing is performed at all but the 8-month visit. Laboratory studies will assess indices of beta and alpha cell function, glycemic control, immunophenotyping, and diabetes-related autoantibodies., Results: The primary outcome is the effect on pancreatic β-cell function as measured by meal-stimulated c-peptide secretion compared between the treatment groups before and after one year of treatment. Secondary outcomes include: 1) fasting and meal stimulated glucagon and proinsulin levels, 2) response in insulin usage by participants, 3) indices of immune cell function, and 4) effect on autoantibodies GAD65, ICA512, and ZnT8., Conclusions: This trial will determine the safety and efficacy of GABA and combination GABA/GAD therapy to delay T1DM progression in children., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background/aims: Newborn screening protocols for congenital hypothyroidism (CH) vary as to whether a TSH or T4 algorithm or some combination is performed. We aimed to determine the 3-year clinical outcome of infants diagnosed with CH and screen-positive for CH using a 2-screen protocol that measures both T4 and TSH on all specimens., Methods: Retrospective analysis of patients with CH who were detected by first (NBS1) or second (NBS2) newborn screen in Alabama (2009-2016) and followed at our university-based practice. Clinical follow-up established the final diagnoses in 146 patients, including a subset of 72 patients with eutopic glands., Results: 168 patients were studied: 139 (83%) were detected by NBS1 and 29 (17%) by NBS2. Screening T4 concentrations were 45% reduced in NBS2 compared to NBS1 (p= 0.0002). Thyroid dysgenesis was present in 55% of NBS1 patients while all in NBS2 were eutopic. Follow-up of 146 patients confirmed permanent CH in 92 patients in NBS1 (75%) and 5 in NBS2 (20%). Hispanic infants were only detected by NBS1, and 93% had permanent CH. Transient CH was associated with congenital heart disease. In patients with eutopic, permanent CH, dyshormonogenesis was confirmed in 23% of NBS1 patients and 40% of NBS2. One case of central CH was detected by each screen., Conclusions: This 8-year, retrospective study buttresses the importance of a 2-screen approach for CH by identifying 5 infants with clinically significant permanent thyroid dysfunction including dyshormonogenesis and central hypothyroidism. It is the first 2-screen study to incorporate thyroid ultrasound. Disconcertingly, 4 of 5 second-screen infants with permanent CH had no risk factors for CH, and these infants would otherwise not have been detected., (© 2019 S. Karger AG, Basel.)

Both increased adrenal and peripheral cortisol production, the latter governed by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), contribute to the maintenance of fasting blood glucose. In the endoplasmic reticulum (ER), the pyridine nucleotide redox state (NADP/NADPH) is dictated by the concentration of glucose-6-phosphate (G6P) and the coordinated activities of two enzymes, hexose-6-phosphate dehydrogenase (H6PDH) and 11β-HSD1. However, luminal G6P may similarly serve as a substrate for hepatic glucose-6-phophatase (G6Pase). A tacit belief is that the G6P pool in the ER is equally accessible to both H6PDH and G6Pase. Based on our inhibition studies and kinetic analysis in isolated rat liver microsomes, these two aforesaid luminal enzymes do share the G6P pool in the ER, but not equally. Based on the kinetic modeling of G6P flux, the ER transporter for G6P (T1) preferentially delivers this substrate to G6Pase; hence, the luminal enzymes do not share G6P equally. Moreover, cortisol, acting through 11β-HSD1, begets a more reduced pyridine redox ratio. By altering this luminal redox ratio, G6P flux through H6PDH is restrained, allowing more G6P for the competing enzyme G6Pase. And, at low G6P concentrations in the ER lumen, which occur during fasting, this acute cortisol-induced redox adjustment promotes glucose production. This reproducible cortisol-driven mechanism has been heretofore unrecognized., (© 2016 Society for Endocrinology.)

Dysregulation of adrenal glucocorticoid production is increasingly recognized to play a supportive role in the metabolic syndrome although the mechanism is ill defined. The adrenal cytochrome P450 (CYP) enzymes, CYP17 and CYP21, are essential for glucocorticoid synthesis. The omega-3 and omega-6 polyunsaturated fatty acids (PUFA) may ameliorate metabolic syndrome, but it is unknown whether they have direct actions on adrenal CYP steroidogenic enzymes. The aim of this study was to determine whether PUFA modify adrenal glucocorticoid synthesis using isolated porcine microsomes. The enzyme activities of CYP17, CYP21, 11β-hydroxysteroid dehydrogenase type 1, hexose-6-phosphate dehydrogenase (H6PDH), and CYP2E1 were measured in intact microsomes treated with fatty acids of disparate saturated bonds. Cortisol production was measured in a cell-free in vitro model. Microsomal lipid composition after arachidonic acid (AA) exposure was determined by sequential window acquisition of all theoretical spectra-mass spectrometry. Results showed that adrenal microsomal CYP21 activity was decreased by docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), eicosapentaenoic acid, α-linolenic acid, AA, and linoleic acid, and CYP17 activity was inhibited by DPA, DHA, eicosapentaenoic acid, and AA. Inhibition was associated with the number of the PUFA double bonds. Similarly, cortisol production in vitro was decreased by DPA, DHA, and AA. Endoplasmic enzymes with intraluminal activity were unaffected by PUFA. In microsomes exposed to AA, the level of AA or oxidative metabolites of AA in the membrane was not altered. In conclusion, these observations suggest that omega-3 and omega-6 PUFA, especially those with 2 or more double bonds (DPA, DHA, and AA), impede adrenal glucocorticoid production.

The role of adrenal hexose-6-phosphate dehydrogenase in providing reducing equivalents to P450 cytochrome steroidogenic enzymes in the endoplasmic reticulum is uncertain. Hexose-6-phosphate dehydrogenase resides in the endoplasmic reticulum lumen and co-localizes with the bidirectional enzyme 11β-hydroxysteroid dehydrogenase 1. Hexose-6-phosphate dehydrogenase likely provides 11β-hydroxysteroid dehydrogenase 1 with NADPH electrons via channeling. Intracellularly, two compartmentalized reactions generate NADPH upon oxidation of glucose-6-phosphate: cytosolic glucose-6-phosphate dehydrogenase and microsomal hexose-6-phosphate dehydrogenase. Because some endoplasmic reticulum enzymes require an electron donor (NADPH), it is conceivable that hexose-6-phosphate dehydrogenase serves in this capacity for these pathways. Besides 11β-hydroxysteroid dehydrogenase 1, we examined whether hexose-6-phosphate dehydrogenase generates reduced pyridine nucleotide for pivotal adrenal microsomal P450 enzymes. 21-hydroxylase activity was increased with glucose-6-phosphate and, also, glucose and glucosamine-6-phosphate. The latter two substrates are only metabolized by hexose-6-phosphate dehydrogenase, indicating that requisite NADPH for 21-hydroxylase activity was not via glucose-6-phosphate dehydrogenase. Moreover, dihydroepiandrostenedione, a non-competitive inhibitor of glucose-6-phosphate dehydrogenase, but not hexose-6-phosphate dehydrogenase, did not curtail activation by glucose-6-phosphate. Finally, the most compelling observation was that the microsomal glucose-6-phosphate transport inhibitor, chlorogenic acid, blunted the activation by glucose-6-phosphate of both 21-hydroxylase and 17-hydroxylase indicating that luminal hexose-6-phosphate dehydrogenase can supply NADPH for these enzymes. Analogous kinetic observations were found with microsomal 17-hydroxylase. These findings indicate that hexose-6-phosphate dehydrogenase can be a source, but not exclusively so, of NADPH for several adrenal P450 enzymes in the steroid pathway. Although the reduced pyridine nucleotides are produced intra-luminally, these compounds may also slowly transverse the endoplasmic reticulum membrane by unknown mechanisms., (Copyright © 2013 Elsevier B.V. All rights reserved.)

With the exception of the oxidation of G6P (glucose 6-phosphate) by H6PDH (hexose-6-phosphate dehydrogenase), scant information is available about other endogenous substrates affecting the redox state or the regulation of key enzymes which govern the ratio of the pyridine nucleotide NADPH/NADP. In isolated rat liver microsomes, NADPH production was increased, as anticipated, by G6P; however, this was strikingly amplified by palmitoylcarnitine. Subsequent experiments revealed that the latter compound, well within its physiological concentration range, inhibited 11β-HSD1 (11β-hydroxysteroid dehydrogenase 1), the bidirectional enzyme which interconnects inactive 11-oxo steroids and their active 11-hydroxy derivatives. Notably, palmitoylcarnitine also stimulated the antithetical direction of 11β-HSD1 reductase, namely dehydrogenase. This stimulation of H6PDH may have likewise contributed to the NADPH accretion. All told, the result of these enzyme modifications is, in a conjoint fashion, a sharp amplification of microsomal NADPH production. Neither the purified 11β-HSD1 nor that obtained following microsomal sonification were sensitive to palmitoylcarnitine inhibition. This suggests that the long-chain amphipathic acylcarnitines, given their favourable partitioning into the membrane lipid bilayer, disrupt the proficient kinetic and physical interplay between 11β-HSD1 and H6PDH. Finally, although IDH (isocitrate dehydrogenase) and malic enzyme are present in microsomes and increase NADPH concentration akin to that of G6P, neither had an effect on 11β-HSD1 reductase, evidence that the NADPH pool in the endoplasmic reticulum shared by the H6PDH/11β-HSD1 alliance is uncoupled from that governed by IDH and malic enzyme.

The effect of intracerebroventricular (ICV) antileptin antibody on the onset of puberty in the female rat and the relationship between serum leptin, luteinizing hormone (LH), and body weight were investigated. Antileptin antibody (group A) was infused ICV from days 23-36 in prepubertal female rats whereas the control (group B) received ICV goat immunoglobulin G (IgG). In the antileptin group, mean day of vaginal opening (VO) was postponed (day 34 versus day 30, P < .01 ). Body weight trended higher after 30 days in the antileptin group but not significantly. However, there was no difference in serum leptin and LH between the two groups on the day of VO. Serum leptin was relatively constant from day 23 through day 31 and did not correlate with LH (r = 0.14, P = .10). These studies demonstrate that central leptin promotes the onset of female rat puberty as evidenced by VO. Finally, central leptin impacts female rat pubertal onset in distinction from serum leptin and body weight.

11beta-Hydroxysteroid dehydrogenase1(11beta-HSD1) can serve either as an oxo-reductase or dehydrogenase determined by the redox state in the endoplasmic reticulum (ER). This bidirectional enzyme governs paracrine glucocorticoid production. Recent in vitro studies have underscored the key role of cytoplasmic glucose-6-phosphate (G6P) in controlling the flux direction of 11betaHSD-1 by altering the intraluminal ER NADPH/NADP ratio. The hypothesis that other hexose phosphoesters or the plentiful cellular oxidative protector glutathione could also regulate microsomal 11betaHSD-1 activity was tested. Fructose-6-phosphate increased the activity of 11beta-HSD1 reductase in isolated rat and porcine liver microsomes but not porcine fat microsomes. Moreover, oxidized glutathione (GSSG) attenuated 11beta-HSD1 reductase activity by 40% while reduced glutathione (GSH) activated the reductase in liver. Fat microsomes were unaffected because they lack glutathione reductase. Nonetheless, another oxidizing agent, hydrogen peroxide (0.5mM), inhibited both fat and liver 11beta-HSD1 reductase. Consistent with the major role of the redox state, 2.5mM GSSG and hydrogen peroxide augmented the 11beta-HSD1 dehydrogenase, antithetical to the reductase, by 20-30% in liver microsomes. Given the key role of reactive oxygen species and hexose phosphate accumulation in the pathoetiology of obesity and diabetes, these compounds might also modify 11beta-HSD1 in these conditions.

11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) catalyzes the interconversion of biologically inactive 11 keto derivatives (cortisone, 11-dehydrocorticosterone) to active glucocorticoids (cortisol, corticosterone) in fat, liver, and other tissues. It is located in the intraluminal compartment of the endoplasmic reticulum. Inasmuch as an oxo-reductase requires NADPH, we reasoned that 11 beta-HSD1 would be metabolically interconnected with the cytosolic pentose pathway because this pathway is the primary producer of reduced cellular pyridine nucleotides. To test this theory, 11 beta-HSD1 activity and pentose pathway were simultaneously measured in isolated intact rodent adipocytes. Established inhibitors of NAPDH production via the pentose pathway (dehydroandrostenedione or norepinephrine) inhibited 11 beta-HSD1 oxo-reductase while decreasing cellular NADPH content. Conversely these compounds slightly augmented the reverse, or dehydrogenase, reaction of 11 beta-HSD1. Importantly, using isolated intact microsomes, the inhibitors did not directly alter the tandem microsomal 11 beta-HSD1 and hexose-6-phosphate dehydrogenase enzyme unit. Metabolites of 11 beta-HSD1 (corticosterone or 11-dehydrocorticosterone) inhibited or increased pentose flux, respectively, demonstrating metabolic interconnectivity. Using isolated intact liver or fat microsomes, glucose-6 phosphate stimulated 11 beta-HSD1 oxo-reductase, and this effect was blocked by selective inhibitors of glucose-6-phosphate transport. In summary, we have demonstrated a metabolic interconnection between pentose pathway and 11 beta-HSD1 oxo-reductase activities that is dependent on cytosolic NADPH production. These observations link cytosolic carbohydrate flux with paracrine glucocorticoid formation. The clinical relevance of these findings may be germane to the regulation of paracrine glucocorticoid formation in disturbed nutritional states such as obesity.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.