Srinivas B. Narayan

Mechanism of Hyperinsulinism in Short-chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency Involves Activation of Glutamate Dehydrogenase

The mechanism of insulin dysregulation in children with hyperinsulinism associated with inactivating mutations of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) was examined in mice with a knock-out of the hadh gene (hadh−/−). The hadh−/− mice had reduced levels of plasma glucose and elevated plasma insulin levels, similar to children with SCHAD deficiency. hadh−/− mice were hypersensitive to oral amino acid with decrease of glucose level and elevation of insulin. Hypersensitivity to oral amino acid in hadh−/− mice can be explained by abnormal insulin responses to a physiological mixture of amino acids and increased sensitivity to leucine stimulation in isolated perifused islets. Measurement of cytosolic calcium showed normal basal levels and abnormal responses to amino acids in hadh−/− islets. Leucine, glutamine, and alanine are responsible for amino acid hypersensitivity in islets. hadh−/− islets have lower intracellular glutamate and aspartate levels, and this decrease can be prevented by high glucose. hadh−/− islets also have increased [U-14C]glutamine oxidation. In contrast, hadh−/− mice have similar glucose tolerance and insulin sensitivity compared with controls. Perifused hadh−/− islets showed no differences from controls in response to glucose-stimulated insulin secretion, even with addition of either a medium-chain fatty acid (octanoate) or a long-chain fatty acid (palmitate). Pull-down experiments with SCHAD, anti-SCHAD, or anti-GDH antibodies showed protein-protein interactions between SCHAD and GDH. GDH enzyme kinetics of hadh−/− islets showed an increase in GDH affinity for its substrate, α-ketoglutarate. These studies indicate that SCHAD deficiency causes hyperinsulinism by activation of GDH via loss of inhibitory regulation of GDH by SCHAD.

GREEN TEA POLYPHENOLS CONTROL DYSREGULATED GLUTAMATE DEHYDROGENASE IN TRANSGENIC MICE BY HIJACKING THE ADP ACTIVATION SITE

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of l-glutamate and, in animals, is extensively regulated by a number of metabolites. Gain of function mutations in GDH that abrogate GTP inhibition cause the hyperinsulinism/hyperammonemia syndrome (HHS), resulting in increased pancreatic β-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. We have previously shown that two of the polyphenols from green tea (epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)) inhibit GDH in vitro and that EGCG blocks GDH-mediated insulin secretion in wild type rat islets. Using structural and site-directed mutagenesis studies, we demonstrate that ECG binds to the same site as the allosteric regulator, ADP. Perifusion assays using pancreatic islets from transgenic mice expressing a human HHS form of GDH demonstrate that the hyperresponse to glutamine caused by dysregulated GDH is blocked by the addition of EGCG. As observed in HHS patients, these transgenic mice are hypersensitive to amino acid feeding, and this is abrogated by oral administration of EGCG prior to challenge. Finally, the low basal blood glucose level in the HHS mouse model is improved upon chronic administration of EGCG. These results suggest that this common natural product or some derivative thereof may prove useful in controlling this genetic disorder. Of broader clinical implication is that other groups have shown that restriction of glutamine catabolism via these GDH inhibitors can be useful in treating various tumors. This HHS transgenic mouse model offers a highly useful means to test these agents in vivo.

Juvenile Neuronal Ceroid-Lipofuscinosis (Batten Disease): A Brief Review and Update

Juvenile neuronal ceroid-lipofuscinosis (JNCL, Batten disease, Spielmeyer-Vogt-Sjogren disease, CLN3) is the most common inherited, autosomal recessive, neurodegenerative disorder in man. Like the other neuronal ceroid-lipofuscinoses, it is characterized by progressive loss of vision, seizures, and loss of cognitive and motor functions, leading to premature demise. JNCL is caused by mutations of CLN3, a gene that encodes a hydrophobic transmembrane protein, which localizes to membrane lipid rafts in lysosomes, endosomes, synaptosomes, and cell membrane. While the primary function of the CLN3 protein (CLN3P) may be debated, its absence affects numerous cellular functions including pH regulation, arginine transport, membrane trafficking, and apoptosis. We have recently suggested that the unifying primary function of CLN3P may be in a novel palmitoyl-protein Delta-9 desaturase (PPD) activity that in our opinion could explain all of the various functional abnormalities seen in the JNCL cells. Another group of researchers has recently shown a correlation between the CLN3P expression and the synthesis of bis(monoacylglycerol)phosphate (BMP) and suggested that CLN3P may play a role in the biosynthesis of BMP. In this review, following an introduction to the neuronal ceroid-lipofuscinoses, we provide a brief overview and an update of the most recent research in JNCL, specifically that related to the function of CLN3P.

Measurement of plasma amino acids by Ultraperformance® Liquid Chromatography

Abstract Background: Since the early 1960s, quantitative amino acid analysis (AAA) has traditionally been performed using ion-exchange chromatography with post-column ninhydrin derivatization. This established technology has many advantages, including relatively low cost of operation and ease of use. However, analysis times of 120+ min and high maintenance requirements make this technology unsuitable for the modern clinical laboratories with a requirement for rapid turnaround times. The work described here is a summary of our experiences with a new approach to full profile analysis of physiological amino acids. Methods: Amino acids were derivatized in batches with a proprietary reagent, AccQTag®, which reacts with primary and secondary amines. The derivatized amino acids were separated using Ultraperformance® Liquid Chromatography (UPLC). In a prospective method comparison, quantitative plasma amino acid data obtained from approximately 170 patient samples using both the UPLC method and a traditional ion-exchange chromatography amino acid analyzer were evaluated. Results: The data obtained from the two methods were found to agree well. Correlation coefficients for the most important amino acids seen in inborn errors of metabolism, such as phenylalanine, tyrosine and branched chain amino acids varied from 0.8658 to 0.9932 with minor slope biases. This approach also reduced the run time from 120 to 45 min per sample using a sample volume of 0.1 mL, compared to the 0.5 mL volume required for ion-exchange chromatography. Conclusions: This new approach for the full profile analysis of physiological amino acids has been shown to be a viable substitute for current ion-exchange methodologies. It provides substantial benefits including significant reductions in runtime and necessary sample volume for the investigation and monitoring of patients with metabolic disorders and for nutritional management of a variety of patients.

Screening for calcium channel modulators in CLN3 siRNA knock down SH-SY5Y neuroblastoma cells reveals a significant decrease of intracellular calcium levels by selected L-type calcium channel blockers

BACKGROUND Defects of the CLN3 gene on chromosome 16p12.1 lead to the juvenile form of neuronal ceroid-lipofuscinosis (JNCL, Batten Disease), the most common recessive inherited neurodegenerative disorder in children. Dysregulation of intracellular calcium homeostasis in the absence of a functional CLN3 protein (CLN3P, Battenin) has been linked to synaptic dysfunction and accelerated apoptosis in vulnerable neuronal cells. Prolonged increase of intracellular calcium concentration is considered to be a significant trigger for neuronal apoptosis and cellular loss in JNCL. METHODS We examined the potential effect of 41 different calcium channel modulators on intracellular calcium concentration in CLN3 siRNA knock down SH-SY5Y neuroblastoma cells. RESULTS Six drugs belonging to the group of voltage dependent L-type channel blockers show significant lowering of the increased intracellular calcium levels in CLN3 siRNA knock down cells. CONCLUSIONS Our studies provide important new data suggesting possible beneficial effects of the tested drugs on calcium flux regulated pathways in neuronal cell death. Therapeutic intervention in this untreatable disease will likely require drugs that cross the blood-brain barrier as did all of the positively screened drugs in this study. GENERAL SIGNIFICANCE Better comprehension of the mechanism of neurodegeneration in rare recessive disorders, such as neuronal ceroid-lipofuscinoses, is likely to help to better understand mechanisms involved in more complex genetic neurodegenerative conditions, such as those associated with aging.

Amlodipine prevents apoptotic cell death by correction of elevated intracellular calcium in a primary neuronal model of Batten disease (CLN3 disease).

CLN3 disease (Spielmeyer-Vogt-Sjogren-Batten disease) is a severe pediatric neurodegenerative disorder for which there is currently no effective treatment. The disease is characterized by progressive neuronal death, which may be triggered by abnormal intracellular calcium levels leading to neuronal apoptosis. Previously, we demonstrated reversal of the calcium effect in a neuroblastoma cell line using amlodipine and other calcium channel antagonists. In the present studies, we developed a CLN3 siRNA-inhibited primary rat neuron model to further study etoposide-induced calcium changes and apoptosis in CLN3 disease followed by recovery experiments with amlodipine. Our results show that intracellular calcium is significantly elevated in siRNA-inhibited cortical neurons after potassium chloride-induced depolarization. We were also able to show that amlodipine, a predominantly L-type dihydropyrimidine calcium channel antagonist can reverse the aberrant calcium elevations in this model of the disease. We performed an in situ TUNEL assay following etoposide-exposure to siRNA inhibited primary neurons, and apoptotic nuclei were detected providing additional evidence that increased neuronal apoptosis is associated with increased calcium levels. Amlodipine also reduced the absolute number of apoptotic cells in this experimental model.

Short-chain 3-hydroxyacyl-coenzyme A dehydrogenase associates with a protein super-complex integrating multiple metabolic pathways.

Proteins involved in mitochondrial metabolic pathways engage in functionally relevant multi-enzyme complexes. We previously described an interaction between short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD) and glutamate dehydrogenase (GDH) explaining the clinical phenotype of hyperinsulinism in SCHAD-deficient patients and adding SCHAD to the list of mitochondrial proteins capable of forming functional, multi-pathway complexes. In this work, we provide evidence of SCHADs involvement in additional interactions forming tissue-specific metabolic super complexes involving both membrane-associated and matrix-dwelling enzymes and spanning multiple metabolic pathways. As an example, in murine liver, we find SCHAD interaction with aspartate transaminase (AST) and GDH from amino acid metabolic pathways, carbamoyl phosphate synthase I (CPS-1) from ureagenesis, other fatty acid oxidation and ketogenesis enzymes and fructose-bisphosphate aldolase, an extra-mitochondrial enzyme of the glycolytic pathway. Most of the interactions appear to be independent of SCHADs role in the penultimate step of fatty acid oxidation suggesting an organizational, structural or non-enzymatic role for the SCHAD protein.

Measurement of β1- and β2-Globulins Improves Detection of M-Spikes on High-Resolution Electrophoresis

High-resolution serum protein electrophoresis (SPE) provides a clearer separation of β1- and β2-globulins than does low-resolution SPE. Currently, immunofixation electrophoresis (IFE) is performed in most laboratories based on high clinical suspicion of a disease associated with a monoclonal gammopathy or an abnormality in the pattern displayed on the electrophoretogram. The aim of this study was to prospectively determine the utility of performing IFE on specimens with increased β1- or β2-globulins on high-resolution SPE but with a normal electrophoretic pattern on visual inspection. SPE was performed with a Sebia-Hydrasys automated electrophoresis system (Hydragel β-1 + β-2 gels; Sebia). The same system was used for IFE with polyclonal anti-human serum for identifying immunoglobulin heavy and light chains. The reference intervals for β1 and β2 concentrations, determined from 100 fresh samples from healthy volunteers in this laboratory, were 4–8 and 1–5 g/L (mean ± 2 SD), respectively. This study received approval from our Institutional Review Board. During the period from March to December 2001, 3179 samples were submitted for SPE. Of these, 963 had IFE performed because of clinical suspicion or abnormalities in the SPE. All serum specimens submitted for SPE with increased β1 or β2 (n = 51) were prospectively evaluated by IFE. Fifteen of the samples had an obvious M-spike that warranted an IFE and were excluded from the analyses. As a control group, 50 samples with protein concentrations within the reference intervals and a visually normal electrophoretogram were also subjected to IFE. Criteria for inclusion were ( a ) no distinct protein band evident outside of the usual six bands (albumin, α1, α2, β1, β2, and γ) and ( b ) an increased β1 or β …

Molecular assay for detection of the common carnitine palmitoyltransferase 1A 1436(C>T) mutation.

Abstract Background: Carnitine palmitoyltransferase 1A (CPT1A) deficiency is a metabolic disorder that occurs at a key checkpoint of fatty acid metabolism. A new form of CPT1A deficiency caused by a mutation at nucleotide 1436 (C>T), resulting in an amino acid substitution of leucine for proline at position 479 (P479L), has been isolated in Canadian First Nations and Inuit populations. The present study offers a molecular method for assessing CPT1A 1436 (C>T) mutation status. Methods: CPT1A-deficient fibroblasts from four patient fibroblast cell lines and ten patient peripheral blood spots were all analyzed by polymerase chain reaction (PCR) coupled to restriction endonuclease (RE) treatment. Genomic DNA was PCR-amplified and treated with an RE specific for normal DNA. CPT1A 1436 (C>T) mutations were identified by resistance to RE treatment. Results: The RE-PCR assay identified homozygosity for the 1436 (C>T) mutation in four fibroblast cell lines and nine blood spots with CPT1A enzyme deficiency. In addition, the assay identified one blood spot that corresponded to the heterozygous genotype. Conclusions: RE-PCR assay for the 1436 (C>T) mutation provides a rapid assay for the diagnosis of CPT1A deficiency resulting from this mutation. The assay will have utility in screening populations with a high prevalence of this genotype. Clin Chem Lab Med 2006;44:1090–1.

Flow injection tandem mass spectrometric measurement of ceramides of multiple chain lengths in biological samples

A method is presented for the measurement of ceramide species in biological fluids using flow injection tandem mass spectrometry. Ceramides are important signaling compounds in a number of cell:cell interactions including apoptosis and neurodegeneration. Because of the large number of potential fatty acid constituent moieties on ceramide molecules, a method which accurately distinguishes different chain-length species was required. The present method does not require HPLC separation and is designed to be applicable to high throughput analysis required for clinical studies. We provide a reference range for all measurable ceramide species in normal human plasma and an example of the utility of the assay in providing biomarkers in an in vitro apoptotic cell death study using murine hematopoietic cells treated with daunorubicin.