Activation of AMPD2 drives metabolic dysregulation and liver disease in mice with hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is a painful and potentially lethal genetic disease caused by a mutation in aldolase B resulting in accumulation of fructose-1-phosphate (F1P). No cure exists for HFI and treatment is limited to avoid exposure to fructose and sugar. Using aldolase B deficient mice, here we identify a yet unrecognized metabolic event activated in HFI and associated with the progression of the disease. Besides the accumulation of F1P, here we show that the activation of the purine degradation pathway is a common feature in aldolase B deficient mice exposed to fructose. The purine degradation pathway is a metabolic route initiated by adenosine monophosphate deaminase 2 (AMPD2) that regulates overall energy balance. We demonstrate that very low amounts of fructose are sufficient to activate AMPD2 in these mice via a phosphate trap. While blocking AMPD2 do not impact F1P accumulation and the risk of hypoglycemia, its deletion in hepatocytes markedly improves the metabolic dysregulation induced by fructose and corrects fat and glycogen storage while significantly increasing the voluntary tolerance of these mice to fructose. In summary, we provide evidence for a critical pathway activated in HFI that could be targeted to improve the metabolic consequences associated with fructose consumption.

Cerebral Fructose Metabolism as a Potential Mechanism Driving Alzheimer's Disease.

The loss of cognitive function in Alzheimer's disease is pathologically linked with neurofibrillary tangles, amyloid deposition, and loss of neuronal communication. Cerebral insulin resistance and mitochondrial dysfunction have emerged as important contributors to pathogenesis supporting our hypothesis that cerebral fructose metabolism is a key initiating pathway for Alzheimer's disease. Fructose is unique among nutrients because it activates a survival pathway to protect animals from starvation by lowering energy in cells in association with adenosine monophosphate degradation to uric acid. The fall in energy from fructose metabolism stimulates foraging and food intake while reducing energy and oxygen needs by decreasing mitochondrial function, stimulating glycolysis, and inducing insulin resistance. When fructose metabolism is overactivated systemically, such as from excessive fructose intake, this can lead to obesity and diabetes. Herein, we present evidence that Alzheimer's disease may be driven by overactivation of cerebral fructose metabolism, in which the source of fructose is largely from endogenous production in the brain. Thus, the reduction in mitochondrial energy production is hampered by neuronal glycolysis that is inadequate, resulting in progressive loss of cerebral energy levels required for neurons to remain functional and viable. In essence, we propose that Alzheimer's disease is a modern disease driven by changes in dietary lifestyle in which fructose can disrupt cerebral metabolism and neuronal function. Inhibition of intracerebral fructose metabolism could provide a novel way to prevent and treat this disease.

Fructose contributes to the Warburg effect for cancer growth.

Obesity and metabolic syndrome are strongly associated with cancer, and these disorders may share a common mechanism. Recently, fructose has emerged as a driving force to develop obesity and metabolic syndrome. Thus, we assume that fructose may be the mechanism to explain why obesity and metabolic syndrome are linked with cancer. Clinical and experimental evidence showed that fructose intake was associated with cancer growth and that fructose transporters are upregulated in various malignant tumors. Interestingly, fructose metabolism can be driven under low oxygen conditions, accelerates glucose utilization, and exhibits distinct effects as compared to glucose, including production of uric acid and lactate as major byproducts. Fructose promotes the Warburg effect to preferentially downregulate mitochondrial respiration and increases aerobic glycolysis that may aid metastases that initially have low oxygen supply. In the process, uric acid may facilitate carcinogenesis by inhibiting the TCA cycle, stimulating cell proliferation by mitochondrial ROS, and blocking fatty acid oxidation. Lactate may also contribute to cancer growth by suppressing fat oxidation and inducing oncogene expression. The ability of fructose metabolism to directly stimulate the glycolytic pathway may have been protective for animals living with limited access to oxygen, but may be deleterious toward stimulating cancer growth and metastasis for humans in modern society. Blocking fructose metabolism may be a novel approach for the prevention and treatment of cancer.

Osmotic Nephrosis and Acute Kidney Injury Associated With SGLT2 Inhibitor Use: A Case Report.

We report a case of a patient who developed dialysis-requiring acute kidney injury (AKI) after the use of canagliflozin. A 66-year-old man with type 2 diabetes who was recovering from left knee septic arthritis at a rehabilitation facility was admitted with oliguric AKI 5 days after starting treatment with canagliflozin, an inhibitor of sodium/glucose cotransporter 2 (SGLT2). The patient presented with hematuria, non-nephrotic-range proteinuria, and serum creatinine level of 6.8 (baseline, 1.1-1.3) mg/dL. There was no recent use of radiocontrast agents or exposure to other nephrotoxins. The patient subsequently required hemodialysis. Due to recent antibiotic use (ampicillin-sulbactam), acute interstitial nephritis was considered in the differential diagnosis. Kidney biopsy was performed, which showed the presence of osmotic nephropathy. The patient's kidney function returned to baseline after 2 weeks of hemodialysis. This case provides evidence of an association of osmotic nephropathy with the use of canagliflozin and discusses potential mechanisms. We recommend kidney biopsy for cases of severe AKI associated with SGLT2 inhibitors to better understand the relationship of this complication with the use of this class of medications.

Ketohexokinase C blockade ameliorates fructose-induced metabolic dysfunction in fructose-sensitive mice.

Increasing evidence suggests a role for excessive intake of fructose in the Western diet as a contributor to the current epidemics of metabolic syndrome and obesity. Hereditary fructose intolerance (HFI) is a difficult and potentially lethal orphan disease associated with impaired fructose metabolism. In HFI, the deficiency of aldolase B results in the accumulation of intracellular phosphorylated fructose, leading to phosphate sequestration and depletion, increased adenosine triphosphate (ATP) turnover, and a plethora of conditions that lead to clinical manifestations such as fatty liver, hyperuricemia, Fanconi syndrome, and severe hypoglycemia. Unfortunately, there is currently no treatment for HFI, and avoiding sugar and fructose has become challenging in our society. In this report, through use of genetically modified mice and pharmacological inhibitors, we demonstrate that the absence or inhibition of ketohexokinase (Khk), an enzyme upstream of aldolase B, is sufficient to prevent hypoglycemia and liver and intestinal injury associated with HFI. Herein we provide evidence for the first time to our knowledge of a potential therapeutic approach for HFI. Mechanistically, our studies suggest that it is the inhibition of the Khk C isoform, not the A isoform, that protects animals from HFI.

Phosphoinositide 3-Kinase Regulates Glycolysis through Mobilization of Aldolase from the Actin Cytoskeleton.

The phosphoinositide 3-kinase (PI3K) pathway regulates multiple steps in glucose metabolism and also cytoskeletal functions, such as cell movement and attachment. Here, we show that PI3K directly coordinates glycolysis with cytoskeletal dynamics in an AKT-independent manner. Growth factors or insulin stimulate the PI3K-dependent activation of Rac, leading to disruption of the actin cytoskeleton, release of filamentous actin-bound aldolase A, and an increase in aldolase activity. Consistently, PI3K inhibitors, but not AKT, SGK, or mTOR inhibitors, cause a significant decrease in glycolysis at the step catalyzed by aldolase, while activating PIK3CA mutations have the opposite effect. These results point toward a master regulatory function of PI3K that integrates an epithelial cell's metabolism and its form, shape, and function, coordinating glycolysis with the energy-intensive dynamics of actin remodeling.

Enzyme Substrate Specificity Conferred by Distinct Conformational Pathways.

Substrate recognition is one of the hallmarks of enzyme catalysis. Enzyme conformational changes have been linked to selectivity between substrates with little direct evidence. Aldolase, a glycolytic enzyme, must distinguish between two physiologically important substrates, fructose 1-phosphate and fructose 1,6-bisphosphate, and provides an excellent model system for the study of this question. Previous work has shown that isozyme specific residues (ISRs) distant from the active site are responsible for kinetic distinction between these substrates. Notably, most of the ISRs reside in a cluster of five surface α-helices, and the carboxyl-terminal region (CTR), and cooperative interactions among these helices have been demonstrated. To test the hypothesis that conformational changes are at the root of these changes, single surface-cysteine variants were created with the cysteine located on helices of the cluster and CTR. This allowed for site-specific labeling with an environmentally sensitive fluorophore, and subsequent monitoring of conformational changes by fluorescence emission spectrophotometry. These labeled variants revealed different spectra in the presence of saturating amounts of each substrate, which suggested the occurrence of different conformations. Emission spectra collected at various substrate concentrations showed a concentration dependence of the fluorescence spectra, consistent with binding events. Lastly, stopped-flow fluorescence spectrophotometry showed that the rate of these fluorescence changes was on the same time-scale as catalysis, thus suggesting a link between the different fluorescence changes and events during catalysis. On the basis of these results, we propose that different conformational changes may be a common mechanism for dictating substrate specificity in other enzymes with multiple substrates.

Targeting of several glycolytic enzymes using RNA interference reveals aldolase affects cancer cell proliferation through a non-glycolytic mechanism.

In cancer, glucose uptake and glycolysis are increased regardless of the oxygen concentration in the cell, a phenomenon known as the Warburg effect. Several (but not all) glycolytic enzymes have been investigated as potential therapeutic targets for cancer treatment using RNAi. Here, four previously untargeted glycolytic enzymes, aldolase A, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase, and enolase 1, are targeted using RNAi in Ras-transformed NIH-3T3 cells. Of these enzymes, knockdown of aldolase causes the greatest effect, inhibiting cell proliferation by 90%. This defect is rescued by expression of exogenous aldolase. However, aldolase knockdown does not affect glycolytic flux or intracellular ATP concentration, indicating a non-metabolic cause for the cell proliferation defect. Furthermore, this defect could be rescued with an enzymatically dead aldolase variant that retains the known F-actin binding ability of aldolase. One possible model for how aldolase knockdown may inhibit transformed cell proliferation is through its disruption of actin-cytoskeleton dynamics in cell division. Consistent with this hypothesis, aldolase knockdown cells show increased multinucleation. These results are compared with other studies targeting glycolytic enzymes with RNAi in the context of cancer cell proliferation and suggest that aldolase may be a useful target in the treatment of cancer.

Increased prevalence of mutant null alleles that cause hereditary fructose intolerance in the American population.

Mutations in the aldolase B gene (ALDOB) impairing enzyme activity toward fructose-1-phosphate cleavage cause hereditary fructose intolerance (HFI). Diagnosis of the disease is possible by identifying known mutant ALDOB alleles in suspected patients; however, the frequencies of mutant alleles can differ by population. Here, 153 American HFI patients with 268 independent alleles were analyzed to identify the prevalence of seven known HFI-causing alleles (A149P, A174D, N334K, Delta4E4, R59Op, A337V, and L256P) in this population. Allele-specific oligonucleotide hybridization analysis was performed on polymerase chain reaction (PCR)-amplified genomic DNA from these patients. In the American population, the missense mutations A149P and A174D are the two most common alleles, with frequencies of 44% and 9%, respectively. In addition, the nonsense mutations Delta4E4 and R59Op are the next most common alleles, with each having a frequency of 4%. Together, the frequencies of all seven alleles make up 65% of HFI-causing alleles in this population. Worldwide, these same alleles make up 82% of HFI-causing mutations. This difference indicates that screening for common HFI alleles is more difficult in the American population. Nevertheless, a genetic screen for diagnosing HFI in America can be improved by including all seven alleles studied here. Lastly, identification of HFI patients presenting with classic symptoms and who have homozygous null genotypes indicates that aldolase B is not required for proper development or metabolic maintenance.

Aminoethylation in model peptides reveals conditions for maximizing thiol specificity.

Control of pH in aminoethylation reactions is critical for maintaining high selectivity towards cysteine modification. Measurement of aminoethylation rate constants by liquid chromatography mass spectrometry demonstrates reaction selectivity of cysteine>>amino-terminus>>histidine. Lysine and methionine were not reactive at the conditions used. For thiol modification, the acid/base property of the gamma-thialysine residue measured by NMR results in a 1.15 decrease in pK(a) (relative to a lysine residue). NMR confirms ethylene imine is the reactive intermediate for alkylation of peptide nucleophiles with bromoethylamine. Conversion of bromoethylamine into ethylene imine prior to exposure to the target thiol, provides a reagent that promotes selectivity by allowing precise control of reaction pH. Reaction selectivity plots of relative aminoethylation rates for cysteine, histidine, and N-terminus imine demonstrate increasing alkaline conditions favors thiol modification. When applied to protein modification, the conversion of bromoethylamine into ethylene imine and buffering at alkaline pH will allow optimal cysteine residue aminoethylation.

Intein-mediated purification of a recombinantly expressed peptide.

A 26 amino acid peptide has successfully been purified via recombinant expression as an intein fusion protein accompanied by cleavage without the need for any exogenous proteases or cofactors, thus offering a practical, inexpensive approach to produce isotopically labelled peptides.

Intraneuronal Abeta42 accumulation in Down syndrome brain.

Alzheimer's disease (AD) brains display A beta (Abeta) plaques, inflammatory changes and neurofibrillary tangles (NFTs). Converging evidence suggests a neuronal origin of Abeta. We performed a temporal study of intraneuronal Abeta accumulation in Down syndrome (DS) brains. Sections from temporal cortex of 70 DS cases aged 3 to 73 years were examined immunohistochemicallyf or immunoreactivity (IR) for the Abeta N-terminal, the Abeta40 C-terminus and the Abeta42 C-terminus. N-terminal antibodies did not detect intracellular Abeta. Abeta40 antibodies did not detect significant intracellular Abeta, but older cases showed Abeta40 IR in mature plaques. In contrast, Abeta42 antibodies revealed clear-cut intraneuronal IR. All Abeta42 antibodies tested showed strong intraneuronal Abeta42 IR in very young DS patients, especially in theyoungest cases studied (e.g., 3 or 4yr. old), but this IR declined as extracellular Abeta plaques gradually accumulated and matured. No inflammatory changes were associated with intraneuronal Abeta. We also studied the temporal development of gliosis and NFT formation, revealing that in DS temporal cortex, inflammation and NFT follow Abeta deposition. We conclude that Abeta42 accumulates intracellularly prior to extracellular Abeta deposition in Down syndrome, and that subsequent maturation of extracellular Abeta deposits elicits inflammatory responses andprecedes NFTs.

Chemical-modification rescue assessed by mass spectrometry demonstrates that gamma-thia-lysine yields the same activity as lysine in aldolase.

The role of active site residues in fructose 1,6-bisphosphate aldolase is investigated by chemical-modification rescue. An active-site mutation, K107C, is constructed in a background where the four solvent-accessible cysteine residues are converted to alanine. The resulting mutant, tetK107C, when reacted with bromoethylamine (BrEA), shows a 40-fold increase in activity (to 80% that of wild type). Determination of the sites and their degree of modification using electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) is developed, allowing correlation of activity after chemical modification rescue to the degree of modification. The stoichiometry of the reaction is 2.5 aminoethylations per subunit, as measured by ESI-FTMS. Protein modification with a double-labeled mix (1:1) of natural abundance isotope (d(0)-BrEA) and 2-bromoethyl-1,1,2,2-d4-amine hydrobromide (d(4)-BrEA), followed by dialysis and trypsin digestion, shows aminoethylated peptides as "twin peptides" separated by four mass units in ESI-FTMS analysis. Using this detection procedure under nondenaturing (native) conditions, C107 is aminoethylated, whereas the four buried thiols remain unlabeled. Aminoethylation of other residues is observed, and correlates with those peptides containing histidine, methionine, and/or the amino terminus. Quantification of the aminoethylation reaction is achieved by labeling with nondeuterated d(0)-BrEA under denaturing conditions following double labeling under native conditions. In addition to complete labeling all five thiols, the intensity of the d(0)-BrEA peak for C107 containing peptides increases, and the change in the d(0)/d(4) ratio between native and denaturing conditions shows 82 +/- 4.5% aminoethylation at C107. This correlation of modification with the recovered activity, indicates that gamma-thia-lysine replaces lysine in the catalytic mechanism. Kinetic constants measured for the rescued K107C mutant enzyme with the substrates fructose 1-phosphate and fructose 1,6-bisphosphate are consistent with the role of the positively charged lysine binding to the C6-phosphate. ESI-FTMS, combined with this double-labeling procedure, allows precise identification of sites and measurement of degree of protein modification.