Alisa Gutman

Characterization of stages in development of obesity-diabetes syndrome in sand rat (Psammomys obesus).

Sand rats (Psammomys obesus) maintained on a diet providing a free choice between laboratory chow and salt bush (Atriplex halimus) were classified into four groups differing in extent of the diabetic syndrome: A, normoglycemic-normoinsulinemic; B, normoglycemichyperinsulinemic; C, hyperglycemic-hyperinsulinemic; or D, hyperglycemic with reduced insulin levels. The metabolic pattern of these groups was characterized by measuring 1) the uptake of fatty acid-labeled, very-lowdensity lipoprotein-borne triglycerides (VLDL-TG) and [3H]%-2-deoxyglucose (2-DOG) into muscle and adipose tissues; 2) incorporation of [14C]alanine into glycogen in vivo; 3) gluconeogenesis from lactate, pyruvate, and alanine in hepatocytes; 4) the effect of insulin on glycogen synthesis from glucose; 5) the oxidation of albumin-bound [1-14C]palmitate and [14C]glucose in strips of soleus muscle; 6) activities of muscle and adipose tissue lipoprotein lipase; and 7) activities of rate-limiting enzymes of glycolysis, gluconeogenesis, and fatty acid synthesis in liver. In group A, uptake of VLDL-TG and activity of lipoprotein lipase were higher in adipose tissue and lower in muscle than in albino rats. In the liver, gluconeogenesis and the activity of phosphoenolpyruvate carboxykinase, as well as lipid synthesis and the activity of NAOP-malate dehydrogenase,were higher than in albino rats, whereas activity of pyruvate kinase was lower. In group B, uptake of VLDL-TG by adipose tissue and muscle and lipoprotein lipase activity were similar or higher than in group A. Uptake of 2-DOG by muscle and adipose tissue and activity of liver phosphoenolpyruvate carboxykinase were lower than in group A. In groups C and D, uptake of VLDL-TG and lipoprotein lipase activity in muscle were further increased. In adipose tissue a progressive decrease in VLDL-TG uptake and lipoprotein lipase activity was found, and uptake of 2-DOG by muscle and adipose tissue was further reduced. In the liver, gluconeogenesis was increased, and activity of phosphoenolpyruvate carboxykinase reached a maximum in group D. These results suggest that in the hyperinsulinemic stage (group B), uptake of glucose by muscle and adipose tissue is reduced, but insulin suppresses gluconeogenesis and stimulates hepatic synthesis and adipose tissue uptake of TG. Hyperglycemia manifests itself when insulin resistance results in increased gluconeogenesis and a further reduction in peripheral glucose uptake. These characteristics can be regarded as a model for the development of type II diabetes in humans evoked by nutritional affluence.

Molecular basis of hepatic carnitine palmitoyltransferase I deficiency.

Mitochondrial fatty acid beta-oxidation is important for energy production, which is stressed by the different defects found in this pathway. Most of the enzyme deficiencies causing these defects are well characterized at both the protein and genomic levels. One exception is carnitine palmitoyltransferase I (CPT I) deficiency, of which until now no mutations have been reported although the defect is enzymatically well characterized. CPT I is the key enzyme in the carnitine-dependent transport across the mitochondrial inner membrane and its deficiency results in a decreased rate of fatty acid beta-oxidation. Here we report the first delineation of the molecular basis of hepatic CPT I deficiency in a new case. cDNA analysis revealed that this patient was homozygous for a missense mutation (D454G). The effect of the identified mutation was investigated by heterologous expression in yeast. The expressed mutant CPT IA displayed only 2% of the activity of the expressed wild-type CPT IA, indicating that the D454G mutation is the disease-causing mutation. Furthermore, in patients fibroblasts the CPT IA protein was markedly reduced on immunoblot, suggesting that the mutation renders the protein unstable.

Phenotype and genotype variation in primary carnitine deficiency

Purpose: Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation resulting from defective carnitine transport. This disease is caused by mutations in the carnitine transporter gene SLC22A5. The objective of this study was to extend mutational analysis to four additional families with this disorder and determine whether recurrent mutations could be found.Methods: The SLC22A5 gene encoding the OCTN2 carnitine transporter was sequenced, and the missense mutations identified were expressed in Chinese hamster ovary (CHO) cells.Results: DNA sequencing revealed four novel mutations (Y4X; dup 254–264, 133X; R19P; R399Q). Alleles introducing premature STOP codons reduced the levels of OCTN2 mRNA. Carnitine transport in CHO cells expressing the R19P and R399Q mutations was reduced to < 5% of normal. The 133X mutation was found in two unrelated European families. Two patients within the same family, both homozygous for the same mutation (R399Q) had completely different clinical presentation.Conclusions: Heterogeneous mutations in the SLC22A5 gene cause primary carnitine deficiency. Different presentations are observed even in children with identical mutations.

Neurologic Presentations of Mitochondrial Disorders

This article describes the neurologic presentations of children with mitochondrial disorders. The charts of 42 children with highly suspect mitochondrial disorders were reviewed. Thirty-seven children were diagnosed as having definite mitochondrial disorders based on a suggestive clinical presentation and at least one accepted criteria, while in five patients the diagnosis remained probable. All patients had nervous system involvement, but it was the presenting symptom in 28 of 42. Eighteen children had normal intelligence and 24 had mental retardation or developmental delay at the onset of their disease. Twenty-five patients had either an acute regression or a progressive encephalopathy. The most frequent neurologic manifestations were abnormal tone, seizures, extrapyramidal movements, and autonomic dysfunction. The eyes were involved in 11 children. Nerve deafness was found in seven patients. Myopathy was found in only six patients. In conclusion, a complex neurologic picture, especially with other organ involvement, warrants a full mitochondrial evaluation. (J Child Neurol 2000; 15:44-48).

Pitfalls in the diagnosis of glycine encephalopathy (non-ketotic hyperglycinemia).

Non-ketotic hyperglycinemia (NKH), also termed glycine encephalopathy (MIMa 605899), is an autosomal recessive inborn error of glycine degradation which leads to severe neurological symptoms and profound psychomotor disability. In NKH, glycine accumulates in all body fluids and tissues, including the CNS. The biochemical hallmark of NKH is increased glycine concentration in the plasma and to an even greater extent in the CSF, leading to an elevation of the CSF:plasma glycine ratio (C:PGR) to above 0.08 (normal <0.04). The fundamental defect is in the glycine cleavage system (GCS), a multienzyme complex located in the inner mitochondrial membrane of the liver, kidney, brain, and placenta. It consists of four individual protein components termed P (a pyridoxal phosphate-dependent glycine decarboxylase), H (a lipoic acid-containing hydrogen carrier protein), T (a tetrahydrofolate-dependent protein), and L (a lipoamide dehydrogenase). In more than 80% of patients the defect is in the P protein (MIM 238300), but defects in the T (MIM 238310) and H (MIM 238330) proteins have also been described. The pathogenesis of NKH is related to the properties of glycine as an excitatory neurotransmitter acting via the N-methyl-Daspartate receptor in the cortex and an inhibitory neurotransmitter in the brainstem and spinal cord.1–3 Classically, NKH presents in the early neonatal period with progressive lethargy, hypotonia, myoclonic jerks, hiccups, and apnea, usually leading to total unresponsiveness, coma, and death unless the patient is supported through this stage with mechanical ventilation. Survivors almost invariably display profound neurological disability and intractable seizures. In a minority of NKH cases the presentation is atypical with a later onset and features including seizures, developmental delay and/or regression, hyperactivity, spastic diplegia, spinocerebellar degeneration, optic atrophy, vertical gaze palsy, ataxia, chorea, and pulmonary hypertension.4–20 Atypical cases are more likely to have milder elevations of glycine concentrations and C:PGR with residual GCS activity. A handful of transient NKH cases have been reported21–26 with neonatal onset and characteristic EEG and biochemical abnormalities which, however, return to normal by 2 months of age, usually with complete clinical resolution. Such cases have been attributed to delayed maturation of the hepatic and cerebral GCS. NKH is generally considered to be a rare disease, but relatively higher incidences have been reported in Northern Finland,27 British Columbia,28,29 and Israel.30–32 In our own laboratory, one of four in Israel performing amino acid analyses, we have diagnosed 15 new unrelated cases of NKH in the past 3 years. Ideally, the diagnosis of NKH should be confirmed by demonstrating deficient GCS activity using the [1-14C] glycine decarboxylation assay, together with assay of the individual components of the complex.33 This necessitates a liver biopsy, as NKH activity is not expressed in fibroblasts nor untransformed lymphocytes. An open liver biopsy is necessary in order to obtain the required amount of tissue for the complete evaluation. In most cases, the treating physician and/or family are unwilling to perform this procedure, particularly in a critically ill infant. Furthermore, the assay is available in only a few centres worldwide necessitating transport of a limited and crucial sample under stringent conditions. Enzymatic diagnosis of NKH due to P-protein deficiency using Epstein-Barr virus transformed lymphoblasts has been reported.34 However, the normal GCS activity in lymphoblasts is low, and some other laboratories have been unable to reproduce these results consistently.35 Furthermore, lymphoblast GCS activity may be normal despite unequivocally deficient hepatic GCS activity.17 At present, molecular diagnosis of NKH by mutation analysis is not a practicable alternative for most sporadic cases. With the exception of a common mutation in the P-protein gene among Finnish patients36 representing a founder effect, there have been few reports of recurring mutations in the Por T-protein genes in unrelated patients.37–39 Thus, A nntation

Multiple presentation of mitochondrial disorders

The aim of this study was to assess the heterogeneous clinical presentations of children with mitochondrial disorders evaluated at a metabolic neurogenetic clinic. The charts of 36 children with highly suspected mitochondrial disorders were reviewed. Thirty one children were diagnosed as having a mitochondrial disorder, based on a suggestive clinical presentation and at least one of the accepted laboratory criteria; however, in five children with no laboratory criteria the diagnosis remained probable. All of the patients had nervous system involvement. Twenty seven patients also had dysfunction of other systems: sensory organs in 15 patients, cardiovascular system in five, gastrointestinal system in 20, urinary system in four, haematopoietic system in four, and endocrine system in nine. The clinical presentation was compatible with an established syndrome in only 15 children. Severe lactic acidosis or ragged red muscle fibres were encountered in very few patients. These results suggest that mitochondrial disorders should be evaluated in children presenting with a complex neurological picture or multisystem involvement. Key messages A full mitochondrial evaluation is warranted in children with a complex neurological picture or a single neurological symptom and other system involvement When the presentation is classic for a maternally inherited mitochondrial syndrome—for example, mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), myoclonic epilepsy and ragged red muscle fibres (MERRF), Leber’s hereditary optic neuropathy (LHON), appropriate mitochondrial DNA studies should be obtained first When the clinical picture is classic for a nuclear DNA inherited syndrome and the gene or the linkage is known—for example, MNGIE (mitochondrial neurogastrointestinal encephalopathy) or DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness), proceed with genetic studies When the clinical picture is non-specific but highly suggestive of a mitochondrial disorder, start with serum and/or cerebrospinal fluid lactic acid and urinary organic acids and proceed with muscle biopsy and assessment of the respiratory chain enzymes Normal serum or cerebrospinal fluid lactic acid does not exclude a mitochondrial disorder

Persistent NKH with transient or absent symptoms and a homozygous GLDC mutation

Three of four nonketotic hyperglycinemia patients homozygous for a novel GLDC mutation (A802V) were treated by assisted respiration and/or sodium benzoate with or without ketamine and had transient neonatal or absent symptoms and normal developmental outcome, despite persisting biochemical evidence of nonketotic hyperglycinemia. This exceptional outcome may be related to the high residual activity of the mutant protein (32% of wild type) and therapeutic intervention during a critical period of heightened brain exposure and sensitivity to glycine. Ann Neurol 2004;56:139–143

Treatment from birth of nonketotic hyperglycinemia due to a novel GLDC mutation

To determine whether the devastating outcome of neonatal‐onset glycine encephalopathy (NKH) could be improved by instituting treatment immediately at birth rather than after symptoms are already well established.

Mechanism of placental glycogen deposition in diabetes in the rat

SummaryThe metabolic basis for glycogen accumulation in the placenta of rats with diabetes induced by streptozotocin on day 12 of pregnancy was studied on days 15 and 20. On day 15 glycogen content of the placenta was 1.5-fold higher in the diabetic than in the control rats and this difference increased to > fivefold on day 20 of gestation whether calculated per g tissue or per total placenta. Accumulation of glycogen was associated with increased specific activities of both glycogen synthase and phosphorylase. The activities of these enzymes regulating synthase and phosphorylase activities and the activity of acid α-glucosidase were not significantly affected by diabetes. Glucose-6-phosphate concentration of the placenta was 67 and 23 nmol/g in diabetic and control rats, respectively. Incubation of placental homogenates with glucose increased the rate of inactivation of phosphorylase and activation of glycogen synthase. These results indicate that the enhanced glucogenesis in diabetes is not due to changes in the activities of these enzymes, as measured in vitro under standard conditions. The factors promoting glycogen accumulation in vivo are related to the abundance of glucose and glucose-6-phosphate as substrates for glycogen synthesis, which may also cause an increase in the activity ratio glycogen synthase a/ phosphorylase a. In addition, the high intracellular glucose-6-phosphate concentration is likely to enable glycogen synthase b to contribute to glycogen synthesis.

Patterns of Decrease of Free Fatty Acids During Glucose Tolerance Tests

In normal subjects the decrease of serum FFA levels after an oral glucose load was prompt and persistent with a drop to below 200 μEq./L. at one hour. Among patients with mild diabetes about 40 per cent showed FFA response similar to normal despite abnormal GT. Another 40 per cent diabetic patients had a delayed FFA decrease with a drop to minimum levels only after two hours and about 15 per cent of the patients had a deficient response with only a small decrease in FFA after glucose ingestion. In the two latter groups the slopes of FFA decrease (—log [FFA]/t) were less steep, indicating that the failure to reach the minimum level of normal subjects was not related to the higher initial serum FFA concentration. The occurrence of normal pattern of FFA decrease among a large proportion of diabetics was taken as an indication of normal function of adipose tissue. The tissue remained sensitive to ILA even when available in diminished amounts as assessed by comparisons of rises in the glucose uptake of rat diaphragm. A delayed FFA decrease appeared associated with late appearance of ILA, whereas the inability of adipose tissue to arrest the outflow of FFA was consistent with scarcity of ILA. The differential effectiveness of insulin as a result of selective antagonism toward its action on tissues is discussed.