Evidence that adrenal hexose-6-phosphate dehydrogenase can effect microsomal P450 cytochrome steroidogenic enzymes.

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 11ß-hydroxysteroid dehydrogenase 1. Hexose-6-phosphate dehydrogenase likely provides 11ß-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 11ß-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.

GABA and Combined GABA with GAD65-Alum Treatment Alters Th1 Cytokine Responses of PBMCs from Children with Recent-Onset Type 1 Diabetes.

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, ß-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.

Manifold effects of palmitoylcarnitine on endoplasmic reticulum metabolism: 11ß-hydroxysteroid dehydrogenase 1, flux through hexose-6-phosphate dehydrogenase and NADPH concentration.

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 11ß-HSD1 (11ß-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 11ß-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 11ß-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 11ß-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 11ß-HSD1 reductase, evidence that the NADPH pool in the endoplasmic reticulum shared by the H6PDH/11ß-HSD1 alliance is uncoupled from that governed by IDH and malic enzyme.

A randomized trial of oral gamma aminobutyric acid (GABA) or the combination of GABA with glutamic acid decarboxylase (GAD) on pancreatic islet endocrine function in children with newly diagnosed type 1 diabetes.

Gamma aminobutyric acid(GABA) is synthesized by glutamate decarboxylase(GAD) in ß-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 ß-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.

Congenital Hypothyroidism: 8-Year Experience Using 2 Newborn Screens in Alabama.

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.

Effect of gamma aminobutyric acid (GABA) or GABA with glutamic acid decarboxylase (GAD) on the progression of type 1 diabetes mellitus in children: Trial design and methodology.

BACKGROUND: Evidence suggests that GABA may reduce pancreatic inflammation, protect ß-cells from autoimmune destruction, and potentiate the regeneration of new ß-cells in the setting of type 1 diabetes mellitus (T1DM). The enzyme GAD, also expressed in human pancreatic ß-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 ß-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 ß-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.

Modification of microsomal 11beta-HSD1 activity by cytosolic compounds: glutathione and hexose phosphoesters.

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.

Cortisol promotes endoplasmic glucose production via pyridine nucleotide redox.

Both increased adrenal and peripheral cortisol production, the latter governed by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-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 11ß-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 11ß-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.

Effect of n-3 and n-6 Polyunsaturated Fatty Acids on Microsomal P450 Steroidogenic Enzyme Activities and In Vitro Cortisol Production in Adrenal Tissue From Yorkshire Boars.

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, 11ß-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.

White adipocyte vascular endothelial growth factor: regulation by insulin.

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.

Effect of central antileptin antibody on the onset of female rat puberty.

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.

Evidence that the 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD1) is regulated by pentose pathway flux. Studies in rat adipocytes and microsomes.

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.