Competitive inhibition by glucose of myo-inositol incorporation into cultured porcine aortic endothelial cells.

To explore the significance of hyperglycaemia as a causal factor for the appearance of diabetic angiopathies we investigated aspects of myo-inositol metabolism in porcine aortic endothelial cells. myo-Inositol was shown to be a long-living metabolite. Its uptake into the cells was mediated by a high-affinity, Na(+)-dependent uptake system inhibitable by ouabain with an apparent KM of 18.6 mumols/l, which was responsible for more than 80% of total uptake at physiological myo-inositol concentrations. Inhibition of inositol uptake by D-glucose was exclusively competitive with an apparent Ki of 24 mmol/l as shown by Lineweaver-Burk- and Dixon-plot analysis. The specificity of competitive inhibition was studied. L-Glucose which is stereochemically related to myo-inositol in the same way as the D-isomer proved to be an equally potent inhibitor. The hexoses D-galactose, D-mannose and D-fructose inhibited myo-inositol uptake to a minor extent. D-allose and 3-O-methyl-D-glucose had no inhibitory effect indicating that the OH-group of the carbon atom in 3 position is essential for the interaction with the carrier. The acyclic hexitol sorbitol also did not compete. As expected, the aldose reductase blocker sorbinil did not influence the carrier since there is no polyol pathway operating in porcine aortic endothelial cells. In accordance with the results of the uptake experiments, the incorporation of exogenous myo-inositol into membrane phosphatidylinositol was reduced at elevated extracellular glucose levels. The results raise the possibility that hyperglycaemia impairs endothelial inositol supply.

Mitochondrial diabetes mellitus: a review.

We review the relationship between various types of mitochondrial DNA mutations and the prevalence as well as the pathobiochemical and clinical features of mitochondrial diabetes mellitus. An A to G transversion mutation in the tRNA(Leu(UUR)) gene is associated with diabetes in about 1.5% of the diabetic population in different countries and races. Phenotypically this type of mitochondrial diabetes is combined with deafness in more than 60% and is clinically distinguishable with respect to several characteristics from the two idiopathic forms of diabetes. The underlying pathomechanism is probably a delayed insulin secretion due to an impaired mitochondrial ATP production in consequence of the mtDNA defect.

Mitochondria and diabetes. Genetic, biochemical, and clinical implications of the cellular energy circuit.

Physiologically, a postprandial glucose rise induces metabolic signal sequences that use several steps in common in both the pancreas and peripheral tissues but result in different events due to specialized tissue functions. Glucose transport performed by tissue-specific glucose transporters is, in general, not rate limiting. The next step is phosphorylation of glucose by cell-specific hexokinases. In the beta-cell, glucokinase (or hexokinase IV) is activated upon binding to a pore protein in the outer mitochondrial membrane at contact sites between outer and inner membranes. The same mechanism applies for hexokinase II in skeletal muscle and adipose tissue. The activation of hexokinases depends on a contact site-specific structure of the pore, which is voltage-dependent and influenced by the electric potential of the inner mitochondrial membrane. Mitochondria lacking a membrane potential because of defects in the respiratory chain would thus not be able to increase the glucose-phosphorylating enzyme activity over basal state. Binding and activation of hexokinases to mitochondrial contact sites lead to an acceleration of the formation of both ADP and glucose-6-phosphate (G-6-P). ADP directly enters the mitochondrion and stimulates mitochondrial oxidative phosphorylation. G-6-P is an important intermediate of energy metabolism at the switch position between glycolysis, glycogen synthesis, and the pentose-phosphate shunt. Initiated by blood glucose elevation, mitochondrial oxidative phosphorylation is accelerated in a concerted action coupling glycolysis to mitochondrial metabolism at three different points: first, through NADH transfer to the respiratory chain complex I via the malate/aspartate shuttle; second, by providing FADH2 to complex II through the glycerol-phosphate/dihydroxy-acetone-phosphate cycle; and third, by the action of hexo(gluco)kinases providing ADP for complex V, the ATP synthetase. As cytosolic and mitochondrial isozymes of creatine kinase (CK) are observed in insulinoma cells, the phosphocreatine (CrP) shuttle, working in brain and muscle, may also be involved in signaling glucose-induced insulin secretion in beta-cells. An interplay between the plasma membrane-bound CK and the mitochondrial CK could provide a mechanism to increase ATP locally at the KATP channels, coordinated to the activity of mitochondrial CrP production. Closure of the KATP channels by ATP would lead to an increase of cytosolic and, even more, mitochondrial calcium and finally to insulin secretion. Thus in beta-cells, glucose, via bound glucokinase, stimulates mitochondrial CrP synthesis. The same signaling sequence is used in the opposite direction in muscle during exercise when high ATP turnover increases the creatine level that stimulates mitochondrial ATP synthesis and glucose phosphorylation via hexokinase. Furthermore, this cytosolic/mitochondrial cross-talk is also involved in activation of muscle glycogen synthesis by glucose. The activity of mitochondrially bound hexokinase provides G-6-P and stimulates UTP production through mitochondrial nucleoside diphosphate kinase. Pathophysiologically, there are at least two genetically different forms of diabetes linked to energy metabolism: the first example is one form of maturity-onset diabetes of the young (MODY2), an autosomal dominant disorder caused by point mutations of the glucokinase gene; the second example is several forms of mitochondrial diabetes caused by point and length mutations of the mitochondrial DNA (mtDNA) that encodes several subunits of the respiratory chain complexes. Because the mtDNA is vulnerable and accumulates point and length mutations during aging, it is likely to contribute to the manifestation of some forms of NIDDM.(ABSTRACT TRUNCATED)

Upregulation of myo-inositol transport compensates for competitive inhibition by glucose. An explanation for the inositol paradox?

High glucose concentrations inhibit the uptake of myo-inositol into cells. However, whether this leads to a depletion of intracellular myo-inositol levels has been debated, because unchanged, decreased, and increased cellular myo-inositol levels all have been reported for diabetic tissues. To evaluate whether cells are capable of counterregulating impaired uptake, we have investigated myo-inositol uptake in porcine aortic endothelial cells under short- and long-term hyperglycemic conditions. Although increasing glucose concentrations inhibited acute myo-inositol uptake competitively, the uptake was increased markedly, when cells were already preincubated in a high glucose medium for > 6 h. The stimulation was maximal at 20 mM of glucose with no further increase at 40 mM glucose. The same stimulation of uptake could be induced by 5 mM of glucose plus 35 mM of raffinose, whereas 35 mM of sorbitol or mannitol, which do not compete for myo-inositol uptake, were ineffective. Lineweaver-Burk analysis revealed an increased Vmax for the induced myo-inositol transport activity, whereas the Km for myo-inositol remained constant (18 microM). The upregulated inositol transporter was still Na+ and ATP dependent, indicating that the same carrier system was operating. Uptake returned to control values when cells were again exposed to normoglycemic medium conditions for an additional 24 h. When endothelial cells were incubated with D-[U-14C]glucose and 10 microM myo-[2-3H]inositol of equal specific radioactivity for 24 h, no 14C radioactivity was found in intracellular myo-inositol, indicating that conversion of glucose to myo-inositol was rather low.

Genetic and structural characterization of the human mitochondrial inner membrane translocase.

Translocation of nuclear-encoded mitochondrial preproteins is mediated by translocases in the outer and inner membranes. In the yeast Saccharomyces cerevisiae, translocation of preproteins into the matrix requires the membrane proteins Tim23, Tim17 and Tim44, which drive translocation in cooperation with mtHsp70 and its co-chaperone Mge1p. We have cloned and functionally analyzed the human homologues of Tim17, Tim23 and Tim44. In contrast to yeast, two TIM17 genes were found to be expressed in humans. TIM44, TIM23 and TIM17a genes were mapped to chromosomes 19p13.2-p13.3, 10q11. 21-q11.23 and 1q32. The TIM17b gene mapped to Xp11.23, near the fusion point where an autosomal region was proposed to have been added to the "ancient" part of the X chromosome about 80-130 MY ago. The primary sequences of the two proteins, hTim17a and hTim17b, are essentially identical, significant differences being restricted to their C termini. They are ubiquitously expressed in fetal and adult tissues, and both show expression levels comparable to that of hTim23. Biochemical characterization of the human Tim components revealed that hTim44 is localized in the matrix and, in contrast to yeast, only loosely associated with the inner membrane. hTim23 is organized into two distinct complexes in the inner membrane, one containing hTim17a and one containing hTim17b. Both TIM complexes display a native molecular mass of 110 kDa. We suggest that the structural organization of TIM23.17 preprotein translocases is conserved from low to high eukaryotes.

Frequency of mitochondrial transfer RNA mutations and deletions in 225 patients presenting with respiratory chain deficiencies.

OBJECTIVE: To evaluate the frequency of pathogenic mtDNA transfer RNA mutations and deletions in biochemically demonstrable respiratory chain (RC) deficiencies in paediatric and adult patients. METHODS: We screened for deletions and sequenced mitochondrial transfer RNA genes in skeletal muscle DNA from 225 index patients with clinical symptoms suggestive of a mitochondrial disorder and with biochemically demonstrable RC deficiency in skeletal muscle. RESULTS: We found pathogenic mitochondrial DNA mutations in 29% of the patients. The detection rate was significantly higher in adults (48%) than in the paediatric group (18%). Only one pathogenic mutation was detected in the neonatal group. In addition, we describe seven novel transfer RNA sequence variations with unknown pathogenic relevance (six homoplasmic and one heteroplasmic) and 13 homoplasmic polymorphisms. One heteroplasmic transfer RNA(Leu(UUR)) A>G mutation at position 3274 is associated with a distinct neurological syndrome. CONCLUSIONS: We provide an estimation of the frequency of mitochondrial transfer RNA mutations and deletions in paediatric and adult patients with respiratory chain deficiencies.

Clinical utility of nonenzymatically glycosylated blood proteins as an index of glucose control.

This study compares the utility of nonenzymatically glycosylated serum proteins (lys-GSP) to glycosylated hemoglobins (HbA1a-c) as control indices of glucose homeostasis in patients with IDDM. The diagnostic value of lys-GSP was also examined in patients with non-insulin-dependent diabetes mellitus, in subjects with impaired glucose tolerance, and in two patients with insulinoma. The intraindividual fluctuation of lys-GSP in normoglycemic subjects is very small, resulting in an interindividual range of 3.0 +/- 0.3 lysine-bound glucose/mg protein (means +/- SD, N = 52). HbA1a-c with a normal range of 6.4 +/- 0.9% (N = 52) shows greater variability. In IDDM there is no overlap of lys-GSP levels between the normal and the diabetic range at the 95% confidence level. In patients treated with an open-loop insulin delivery system failure of normalization of the glucose balance was clearly discernible by an elevation of GSP. In contrast, in about 40% of the patients with incomplete glycemic control the HbA1a-c levels fell within the normal range. The utility of lys-GSP for diagnosis of diabetes is compared with the results of 60 oral glucose tolerance tests. Two patients suffering from insulinoma displayed decreased lys-GSP values. From these results it appears that determination of lys-GSP represents a more sensitive parameter for long-term control than HbA1a-c and is suitable for monitoring even small fluctuations of blood glucose.

Diabetes mellitus is one of the heterogeneous phenotypic features of a mitochondrial DNA point mutation within the tRNALeu(UUR) gene.

A heteroplasmic point mutation (transition A-to-G at nucleotide position 3,243 in the mitochondrial tRNALeu(UUR) gene) is found in a family suffering from a syndrome with diabetes, deafness and cardiomyopathy as the predominant clinical features.

High concentrations of low density lipoprotein decrease basement membrane-associated heparan sulfate proteoglycan in cultured endothelial cells.

The effect of increasing low density lipoprotein (LDL) concentrations on the synthesis of basement membrane components was investigated in proliferating porcine aortic endothelial cells (PAEC) in culture. Basement membrane-associated heparan sulfate proteoglycan (HSPG) and fibronectin were determined by enzyme immunoassay. Low extracellular LDL-levels increase, high extracellular LDL-levels decrease the HSPG content of PAEC. Fibronectin synthesis was only slightly affected while proliferation and metabolic activity as assessed by lactate production were constant. Insulin or high extracellular glucose did not influence the effect of LDL on basement membrane components.

No genetic differences between affected and unaffected members of a German family with Leber's hereditary optic neuropathy (LHON) with respect to ten mtDNA point mutations associated with LHON.

In order to investigate possible synergistic influences of different mtDNA mutations on penetrance and severity of Leber's hereditary optic neuropathy (LHON), a large German LHON pedigree is characterized with respect to 10 different mutations associated with LHON. All members of the family carry three different mtDNA mutations (at nucleotide 4,216, 11,778 and 13,708) in a homoplasmic form, regardless of whether or not they are clinically affected. Testing for another 7 mutations reveals negative results in all family members. Hence, the variable disease expression of the family members cannot be explained by varying combinations of LHON-associated mtDNA mutations.

Respiratory chain activity in tissues from patients (MELAS) with a point mutation of the mitochondrial genome [tRNA(Leu(UUR))].

A heteroplasmic point mutation (transition A to G at position 3243 in the mitochondrial tRNA(Leu(UUR)) gene is indicative for myo-encephalopathy with lactic acidosis and stroke-like episodes (MELAS). Decreased respiratory chain complex activities measured in different tissues from four patients with MELAS syndrome do not correlate with the proportion of mutated mitochondrial genome.

Homozygosity (E140K) in SCO2 causes delayed infantile onset of cardiomyopathy and neuropathy.

OBJECTIVE: To report three unrelated infants with a distinctive phenotype of Leigh-like syndrome, neurogenic muscular atrophy, and hypertrophic obstructive cardiomyopathy. The patients all had a homozygous missense mutation in SCO2. BACKGROUND: SCO2 encodes a mitochondrial inner membrane protein, thought to function as a copper transporter to cytochrome c oxidase (COX), the terminal enzyme of the respiratory chain. Mutations in SCO2 have been described in patients with severe COX deficiency and early onset fatal infantile hypertrophic cardioencephalomyopathy. All patients so far reported are compound heterozygotes for a missense mutation (E140K) near the predicted CxxxC metal binding motif; however, recent functional studies of the homologous mutation in yeast failed to demonstrate an effect on respiration. METHODS: Here we present clinical, biochemical, morphologic, functional, MRI, and MRS data in two infants, and a short report in an additional patient, all carrying a homozygous G1541A transition (E140K). RESULTS: The disease onset and symptoms differed significantly from those in compound heterozygotes. MRI and muscle morphology demonstrated an age-dependent progression of disease with predominant involvement of white matter, late appearance of basal ganglia lesions, and neurogenic muscular atrophy in addition to the relatively late onset of hypertrophic cardiomyopathy. The copper uptake of cultured fibroblasts was significantly increased. CONCLUSIONS: The clinical spectrum of SCO2 deficiency includes the delayed development of hypertrophic obstructive cardiomyopathy and severe neurogenic muscular atrophy. There is increased copper uptake in patients' fibroblasts indicating that the G1541A mutation effects cellular copper metabolism.

Atherogenic levels of low density lipoprotein alter the permeability and composition of the endothelial barrier.

In the present study we investigated the influence of elevated low density lipoprotein (LDL) concentration on endothelial permeability. Endothelial cells were cultured on microporous membranes until confluence and albumin, dextran and LDL transfer across endothelial monolayers was determined to assess macromolecular permeability. Exposure of proliferating aortic endothelial cells to LDL levels of more than 1 mg/ml LDL-cholesterol induced a concentration-dependent exponential increase in the permeability of confluent endothelial monolayers. Acute addition of high LDL concentration did not alter macromolecular permeability. Once elevated permeability was induced, it persisted. It was not readily reversible after addition of normal LDL levels. Change in permeability was accompanied by a selective decrease in basement membrane associated heparan sulfate proteoglycan (HSPG) content. The apparent parallel between the loss in endothelial barrier function and HSPG decrease implicates a connection between the two events. Prolonged, but not acute, incubation with antiserum directed against the core-protein of HSPG also led to increased permeability, suggesting a causal role of HSPG for the proper function of endothelium. The fact that non-atherogenic LDL-cholesterol levels had no effect indicates that a 'threshold' concentration for LDL-cholesterol may exist, leading to nondenuding injury in the endothelial barrier as an early event in development of atherosclerosis.

In situ hybridization of mitochondrial DNA in the heart of a patient with Kearns-Sayre syndrome and dilatative cardiomyopathy.

Previous studies have revealed cytochrome-c-oxidase-deficient cardiomyocytes and the 4,977 base pair deletion ("common deletion") of mitochondrial DNA (position 8,482-13,459) in the heart of a patient with dilatative cardiomyopathy and Kearns-Sayre syndrome. In the present investigation the co-localization of the enzymatic and genomic defects was studied. In situ hybridization of mitochondrial DNA (mtDNA) revealed different hybridization patterns in the cytochrome-c-oxidase-deficient cells: (1) a selective reduction of the hybridization signal with an mtDNA probe recognizing the common deletion, indicating predominance of the deleted over the nondeleted mtDNA molecules in the cytochrome-c-oxidase-deficient cells; (2) a reduced hybridization signal with different mtDNA probes, indicating depletion of mtDNA; and (3) normal hybridization signals with different probes in single cytochrome-c-oxidase-deficient cardiomyocytes. These results indicate that different mechanisms may co-exist in Kearns-Sayre syndrome and may lead to defective respiratory chain function. The question of the pathogenetic interrelationship is discussed.

Mitochondrial myopathies: divergences of genetic deletions, biochemical defects and the clinical syndromes.

Genomic Southern analysis of muscle mitochondrial (mt) DNA from 16 patients with mitochondrial myopathies was performed; 14 of 16 patients had chronic progressive external ophthalmoplegia (CPEO), while 2 patients had mitochondrial myopathies without CPEO. Eleven patients with CPEO, including 5 who exhibited the complete triad of symptoms characteristic of the Kearns-Sayre syndrome (i.e. CPEO, retinal degeneration and heart block) had heteroplasmic mtDNA with deletions ranging from 2.0 to 8.0 kb in length. There was no clear-cut correlation between the size and location of the deletions, on the one hand, and the histochemical and biochemical data or the severity of the disease, on the other.

Screening for mitochondrial DNA (mtDNA) point mutations using nonradioactive single strand conformation polymorphism (SSCP) analysis.

OBJECTIVES: Mitochondrial cytopathies such as Leber's hereditary optic neuropathy (LHON), mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), and myoclonus epilepsy with red ragged fibers (MERRF) are associated with distinct mtDNA point mutations (for review see 1). LHON, for example, is related to at least 14 mtDNA point mutations within different mitochondrially encoded respiratory subunit genes. In addition, the number of newly found LHON-related mutations is increasing. In the light of the large number and the dispersed distribution of these point mutations throughout the mitochondrial genome, screening for these by sequencing all of suspected loci is laborious and time-consuming. In order to facilitate a rapid screening for mitochondrial point mutations we have evaluated the use of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) for the analysis of the human mitochondrial genome. DESIGN AND METHODS: In a first evaluation step we created a variety of pUC18 clones derived from mitochondrial control region amplifications with defined sequence differences and length. These clones were used as standard material for an optimization of the PCR-SSCP analysis. The optimized PCR-SSCP was then applied to large cohorts of patients with known, i.e., sequenced mtDNA point mutations and to healthy controls in order to evaluate its sensitivity. RESULTS: The most common LHON-related mtDNA point mutations at nucleotide positions (nps) 11778, 14484, 4216, could be detected by SSCP analysis, as well as the heteroplasmic np 3243 MELAS associated point mutation. Several new polymorphisms and point mutations were found. A sensitivity, i.e., the ability to detect defined point mutations, of 93% (clones) and 98% (disease controls) was achieved when comparing SSCP- and direct sequencing results. CONCLUSION: The PCR-SSCP approach using a non-radioactive silver staining method is suited for the detection of human mitochondrial point mutations, as well as a helpful screening tool for novel mt DNA mutations.

Human alkaline phosphatases. II. Metalloenzyme properties of the enzyme from human liver.

Human liver alkaline phosphatase is a metalloenzyme requiring Zn2 and Mg2 for full activity. Zn2 cannot be replaced by manganese, cobalt or calcium, whereas Mg2 can be replaced by manganese or calcium. The binding constants of the enzyme for different divalent cations were determined by the use of complexing agents. The enzyme is inhibited by a number of reducing and complexing agents such as 2-mercaptoethanol, cyanide, nitrilotriacetic acid and EDTA. From studies using these inhibitors it is suggested that there are different mechanisms of inhibition. Reversible inhibition occurs if the free Zn2 concentration is not significantly lower than 10(-12)M. Inhibition is irreversible at lower Zn2 concentrations. Evidence is given, that the human liver alkaline phosphatase possesses different zinc binding sites, which are responsible for the catalytic function and for the integrity of the enzyme structure.