Ketohexokinase knockout mice, a model for essential fructosuria, exhibit altered fructose metabolism and are protected from diet-induced metabolic defects.

Fructose consumption in humans and animals has been linked to enhanced de novo lipogenesis, dyslipidemia, and insulin resistance. Hereditary deficiency of ketohexokinase (KHK), the first enzymatic step in fructose metabolism, leads to essential fructosuria in humans, characterized by elevated levels of blood and urinary fructose following fructose ingestion but is otherwise clinically benign. To address whether KHK deficiency is associated with altered glucose and lipid metabolism, a Khk knockout (KO) mouse line was generated and characterized. NMR spectroscopic analysis of plasma following ingestion of [6-13C] fructose revealed striking differences in biomarkers of fructose metabolism. Significantly elevated urine and plasma 13C-fructose levels were observed in Khk KO vs. wild-type (WT) control mice, as was reduced conversion of 13C-fructose into plasma 13C-glucose and 13C-lactate. In addition, the observation of significant levels of fructose-6-phosphate in skeletal muscle tissue of Khk KO, but not WT, mice suggests a potential mechanism, whereby fructose is metabolized via muscle hexokinase in the absence of KHK. Khk KO mice on a standard chow diet displayed no metabolic abnormalities with respect to ambient glucose, glucose tolerance, body weight, food intake, and circulating trigylcerides, ß-hydroxybutyrate, and lactate. When placed on a high-fat and high-fructose (HF/HFruc) diet, Khk KO mice had markedly reduced liver weight, triglyceride levels, and insulin levels. Together, these results suggest that Khk KO mice may serve as a good model for essential fructosuria in humans and that inhibition of KHK offers the potential to protect from diet-induced hepatic steatosis and insulin resistance.

Hereditary fructose intolerance and celiac disease: a novel genetic association.

BACKGROUND & AIMS: Celiac disease (CD) has been associated with several genetic disorders, but has not been associated with hereditary fructose intolerance (HFI). METHODS: We identified CD in 4 female patients affected by HFI from among 38 Italian HFI patients. RESULTS: Three of these patients were children in whom the CD-associated signs were hypertransaminasemia, failure to thrive, low weight, and short stature, whereas the adult patient had protracted diarrhea notwithstanding a fructose-free diet. The incidence of CD in our group of HFI patients was higher (>10%) than in the general population (1%-3%) (P<.02). CONCLUSIONS: The possibility of an association between these 2 gastrointestinal disorders is important, particularly in the management of HFI patients with persisting symptoms.

Properties of normal and mutant recombinant human ketohexokinases and implications for the pathogenesis of essential fructosuria.

Alternative splicing of the ketohexokinase (fructokinase) gene generates a "central" predominantly hepatic isoform (ketohexokinase-C) and a more widely distributed ketohexokinase-A. Only the abundant hepatic isoform is known to possess activity, and no function is defined for the lower levels of ketohexokinase-A in peripheral tissues. Hepatic ketohexokinase deficiency causes the benign disorder essential fructosuria. The molecular basis of this has been defined in one family (compound heterozygosity for mutations Gly40Arg and Ala43Thr). Here we show that both ketohexokinase isoforms are indeed active. Ketohexokinase-A has much poorer substrate affinity than ketohexokinase-C for fructose but is considerably more thermostable. The Gly40Arg mutation seems null, rendering both ketohexokinase-A and ketohexokinase-C inactive and largely insoluble. The Ala43Thr mutant retains activity, but this mutation decreases the thermal stability of both ketohexokinase-A and ketohexokinase-C. At physiologic temperature, this results in significant loss of ketohexokinase-C activity but not of ketohexokinase-A. Affected individuals who carry both mutations therefore probably have a selective deficiency of hepatic ketohexokinase, with peripheral ketohexokinase-A being preserved. These findings raise the possibility that ketohexokinase-A serves an unknown physiologic function that remains intact in essential fructosuria. Further mutation analysis in this rare disorder could illuminate the question of whether ketohexokinase-A activity is, unlike that of ketohexokinase-C, physiologically indispensable.

Hereditary fructose intolerance caused by a nonsense mutation of the aldolase B gene.

The nucleotide sequence of a patient's aldolase B gene was determined and showed a substitution of a single nucleotide (C----A) at position 720 in the coding region, which resulted in the 240th amino acid, a cysteine, being changed to a stop codon (TGC----TGA). By an allele-specific oligonucleotide probe and polymerase chain reaction, the patient was shown to be homozygous for the mutation. To examine whether this mutation causes functional defect of the enzyme, the activity of the aldolase B from the patient, expressed in Escherichia coli by using expression plasmid, was measured. No activity was observed, and the predicted product was recovered from E. coli expression plasmid, indicating that this nonsense mutation was the cause of aldolase B deficiency.

Partial aldolase B gene deletions in hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is an autosomal recessive condition caused by a deficiency of aldolase B. We have recently shown that three point mutations in this gene account for approximately 85% of HFI alleles in Europe and the United States and are thus of diagnostic importance. In this paper we define three new lesions in the aldolase B gene: two are large deletions, one of 1.65 kb and one of 1.4 kb; the third is a small deletion of 4 bp. We have determined the breakpoints of these deletions and have demonstrated that the presence of such lesions may complicate the genotyping of individuals for diagnosis of HFI.

Molecular evidence for compound heterozygosity in hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is an inborn error of metabolism, inherited as an autosomal recessive disorder and caused by a decrease in the activity of fructose-1-phosphate aldolase (aldolase B) in affected individuals. Investigation of the molecular basis of HFI is reported here by the identification of two molecular lesions in the aldolase B gene of the HFI individual. Using polymerase chain reaction to specifically amplify exons at this locus and T7 polymerase for the sequence determination of these double-stranded fragments, we show the mutational heterogeneity of the proband. One allele, previously indicated by restriction analysis, was confirmed as A149P (Ala 149 to Pro in exon 5). The other allele was identified as a 4-bp deletion found in exon 4, a deletion which causes a frameshift at codon 118, resulting in a truncated protein of 132 amino acids. Segregation of these mutant alleles in the proband's family was shown by using allele-specific oligodeoxynucleotides to probe blots of amplified DNA. The techniques employed here represent a rapid and efficient method for detection of other mutations in families with this disease. In addition, the ability to detect mutant alleles by allele-specific hybridization offers a new method for definitive diagnosis, a method which avoids a fructose loading or liver-biopsy examination.

Molecular analysis of aldolase B genes in hereditary fructose intolerance.

The molecular basis of hereditary fructose intolerance (HFI) was studied in 50 subjects (41 pedigrees, 82 apparently independent mutant alleles of aldolase B) by direct analysis of aldolase B genes amplified by means of the polymerase chain reaction. The mutation A149P (ala 149----pro) was found in 67% of alleles but was significantly more common in patients from northern than from southern Europe. Two other point mutations of aldolase B were identified. A174D (C----A; ala 174----asp) was found in subjects from Italy, Switzerland, and Yugoslavia (overall frequency 16%) but not in those from the United Kingdom, France, or the United States. L288 delta C carried a single base-pair deletion causing frameshift at codon 288 and was restricted to Sicilian subjects. By testing for these mutations in amplified DNA with a limited panel of allele-specific oligonucleotides, more than 95% of HFI patients will be susceptible to genetic diagnosis.

[Hereditary fructose intolerance]. / Hereditäre Fruktoseintoleranz.

The paper gives an overview of current diagnostic procedures in patients with clinical suspicion of hereditary fructose intolerance. On the basis of the literature and of a 9 years' experience at the Department of Paediatrics of the University of Graz a different approach according to the clinical condition of the patients is proposed (good clinical condition, severe liver disease, or bad clinical condition--i.e. liver biopsy is ethically not justified). The aim of this approach is to minimize invasive procedures for the children.

Inherited disorders of carbohydrate metabolism in children studied by 13C-labelled precursors, NMR and GC-MS.

Glucose carbon recycling, glucose production and glucose turnover in glycogen storage disease type I and type II patients and control subjects were determined by a novel approach--mass isotopomer analysis of plasma 13C glucose. Changes in the isotopomer distribution of plasma 13C glucose were found only in glycogen storage disease type III patients and control subjects. Glucose carbon recycling parameters were also derived from 13C NMR spectra of plasma glucose C-1 splitting pattern. Our results eliminate a mechanism for glucose production in glycogen storage disease type I children involving gluconeogenesis. However, glucose release by amylo-1,6-glucosidase activity is in agreement with our results. A quantitative determination of the metabolic pathways of fructose conversion to glucose in normal children, and in children with disorders of fructose metabolism was derived from 13C NMR measurement of plasma 13C glucose isotopomer populations following [U-13C]fructose administration. A direct pathway from fructose, bypassing fructose-1-phosphate aldolase, to fructose-1,6-diphosphate in controls and hereditary fructose intolerant children (47% and 27%, respectively) was identified. In children with fructose-1,6-diphosphatase deficiency, only the gluconeogenic substrates were 13C labelled but no synthesis of glucose from [U-13C]fructose occurred. The significantly lower (by 68%) conversion of fructose to glucose in hereditary fructose intolerance, as compared to control subjects, and non-conversion in fructose-1,6-diphosphatase deficient subjects after [U-13C]fructose (approximately 20 mg/kg) administration can serve as the basis of a safe diagnostic test for patients suspected of inborn errors of fructose metabolism and other defects involving gluconeogenesis.

Molecular analysis of aldolase B genes in the diagnosis of hereditary fructose intolerance in the United Kingdom.

To investigate the molecular basis of hereditary fructose intolerance, we have studied 12 British patients, all of whom were found to carry a single mutation in the gene coding for aldolase B. We have estimated the frequency of this lesion, termed A149P, amongst affected individuals in the population and predict that a diagnosis may be made non-invasively in more than 83 per cent of cases by demonstrating the presence of this allele. Genetic diagnosis and detection of asymptomatic carriers of the disease may be achieved by the specific amplification of DNA derived from mouthwash samples followed by hybridization to allele-specific oligonucleotides.

Subgingival microflora, dental and periodontal conditions in patients with hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is a rare autosomally recessive disease which leads to severe hypoglycemia. The fructose-free diet of these patients apparently influences dental health. Half of the patients are free of caries, but there are no reports on their periodontal condition nor on the composition of their subgingival microflora. Therefore 18 patient with HFI were examined for the following parameters: radiographic bone loss, caries rate, gingival bleeding, occurrence of calculus, pocket depths, salivary flow rate, buffer capacity and pH from paraffin wax-stimulated saliva. Salivary S. mutans and lactobacilli were also enumerated. Specific antibody titers from whole saliva and serum to Actinobacillus actinomycetemcomitans gamma 4, Bacteroides gingivalis, and Capnocytophaga ochracea were determined with ELISA. Finally the subgingival plaque was analyzed by culture. Altogether 196 anaerobic or microaerophilic isolates representing 29 different species were obtained from the HFI patients and 164 isolates of 26 species from the controls. The frequency of Actinomyces odontolyticus, Veillonella parvula, and Wolinella recta in the HFI patients was significantly high The DMF-induces were lower in the study group than in the controls and so was the occurrence of S. mutans and lactobacilli in the saliva. Of the periodontal parameters examined, the only difference found was an increased incidence of gingival bleeding and calculus formation in the HFI patients. The results suggest that in the HFI patients the common gingival bleeding is associated with the more frequent occurrence of Actinomyces odontolyticus, Veillonella parvula, and Wolinella recta.

Congenital hereditary fructose intolerance and pregnancy.

Congenital hereditary fructose intolerance is associated with the inability to tolerate fructose and carbohydrates, which are converted into fructose. We describe management of a pregnancy complicated by this disease in the mother and its implications for the neonate.