Cellular Intermediary Metabolism Research Articles

Mitigation of Cadmium Toxicity through Modulation of the Frontline Cellular Stress Response

Cadmium (Cd) is an environmental toxicant of public health significance worldwide. Diet is the main Cd exposure source in the non-occupationally exposed and non-smoking populations. Metal transporters for iron (Fe), zinc (Zn), calcium (Ca), and manganese (Mn) are involved in the assimilation and distribution of Cd to cells throughout the body. Due to an extremely slow elimination rate, most Cd is retained by cells, where it exerts toxicity through its interaction with sulfur-containing ligands, notably the thiol (-SH) functional group of cysteine, glutathione, and many Zn-dependent enzymes and transcription factors. The simultaneous induction of heme oxygenase-1 and the metal-binding protein metallothionein by Cd adversely affected the cellular redox state and caused the dysregulation of Fe, Zn, and copper. Experimental data indicate that Cd causes mitochondrial dysfunction via disrupting the metal homeostasis of this organelle. The present review focuses on the adverse metabolic outcomes of chronic exposure to low-dose Cd. Current epidemiologic data indicate that chronic exposure to Cd raises the risk of type 2 diabetes by several mechanisms, such as increased oxidative stress, inflammation, adipose tissue dysfunction, increased insulin resistance, and dysregulated cellular intermediary metabolism. The cellular stress response mechanisms involving the catabolism of heme, mediated by heme oxygenase-1 and -2 (HO-1 and HO-2), may mitigate the cytotoxicity of Cd. The products of their physiologic heme degradation, bilirubin and carbon monoxide, have antioxidative, anti-inflammatory, and anti-apoptotic properties.

Autophagy inhibition by biotin elicits endoplasmic reticulum stress to differentially regulate adipocyte lipid and protein synthesis.

Biotin is an indispensable adipogenic agent, and its ability to coordinate carbohydrate, lipid, and amino acid metabolism sensitizes insulin signaling in adipocytes. This enables the organism to adapt and survive under nutrient stress by synthesis and storage of lipids. Biotin deficiency mimics insulin resistance with alterations in cellular intermediary metabolism. Though the mechanism of lipogenesis is well established across cell types, considering its predisposition to accumulate only lipids, it is necessary to elucidate the mechanism that minimizes the effects of biotin on adipocyte protein synthesis. In order to determine the differential metabolic phenotype by biotin, the primary cultures of adipocytes were induced to differentiate in the presence and absence of excess biotin. Serum pre-incubated with avidin was used to limit biotin availability in cultured cells. Biotin restricts cellular signaling associated with protein synthesis without altering total protein content. The decline in autophagy elicits endoplasmic reticulum stress to inhibit protein synthesis by eIF2α phosphorylation possibly via accumulation of misfolded/long-lived proteins. Furthermore, the compensatory increase in Unc51 like autophagy activating kinase 1 possibly competes with eukaryotic initiation factor 4E-binding protein 1 and ribosomal p70 S6kinase phosphorylation by mechanistic targets of rapamycin complex 1 to uncouple its effect on protein synthesis. In conclusion, autophagy inhibition by biotin uncouples protein synthesis to promote lipogenesis by eliciting endoplasmic reticulum stress and differential phosphorylation of mechanistic targets of rapamycin complex 1 substrates.

Abstract 569: Distinct Metabolomic Features in Dahl salt-sensitive rats and consomic SS.13BN rats

Our previous proteomic study revealed substantial differences in several proteins between Dahl salt-sensitive (SS) rats and salt-insensitive consomic SS.13BN rats. Subsequent studies indicated a role of fumarase insufficiency in the development of hypertension in SS rats. A global metabolic profiling study using gas chromatography/mass spectrometry (GC/MS) was performed in plasma of SS rats (n=9) and SS.13BN rats (n=8) on a 0.4% NaCl diet to gain further insights into the relationship between alterations in cellular intermediary metabolism and predisposition to hypertension. Principal component analysis of the data revealed a clear clustering and separation of metabolic profiles between SS rats and SS.13BN rats. 23 differential metabolites were identified (P<0.05). Levels of five TCA cycle-related metabolites, fumarate, cis-aconitate, isocitrate, citrate and succinate, were higher in SS rats. Pyruvate, which connects TCA cycle and glycolysis, was also increased in SS rats. Moreover, enzymatic activities of fumarase, aconitase, α-ketoglutarate dehydrogenase and succinyl-CoA synthetase were lower in the heart, liver or skeletal muscles of SS rats. The distinct metabolic features in SS and SS.13BN rats supports a role of abnormal TCA cycle in predisposing SS rats to developing salt-sensitive hypertension.

Analysis of metabolites in plasma reveals distinct metabolic features between Dahl salt-sensitive rats and consomic SS.13BN rats

Salt-sensitive hypertension is a major risk factor for cardiovascular disorders. Our previous proteomic study revealed substantial differences in several proteins between Dahl salt-sensitive (SS) rats and salt-insensitive consomic SS.13BN rats. Subsequent experiments indicated a role of fumarase insufficiency in the development of hypertension in SS rats. In the present study, a global metabolic profiling study was performed using gas chromatography/mass spectrometry (GC/MS) in plasma of SS rats (n=9) and SS.13BN rats (n=8) on 0.4% NaCl diet, designed to gain further insights into the relationship between alterations in cellular intermediary metabolism and predisposition to hypertension. Principal component analysis of the data sets revealed a clear clustering and separation of metabolic profiles between SS rats and SS.13BN rats. 23 differential metabolites were identified (P<0.05). Higher levels of five TCA cycle metabolites, fumarate, cis-aconitate, isocitrate, citrate and succinate, were observed in SS rats. Pyruvate, which connects TCA cycle and glycolysis, was also increased in SS rats. Moreover, lower activity levels of fumarase, aconitase, α-ketoglutarate dehydrogenase and succinyl-CoA synthetase were detected in the heart, liver or skeletal muscles of SS rats. The distinct metabolic features in SS and SS.13BN rats indicate abnormalities of TCA cycle in SS rats, which may play a role in predisposing SS rats to developing salt-sensitive hypertension.

PPARα Activation Induces Nε‐Lys‐Acetylation of Rat Liver Peroxisomal Multifunctional Enzyme Type 1

Peroxisomes are ubiquitous subcellular organelles that participate in metabolic and disease processes, with few of its proteins undergoing posttranslational modifications. As the role of lysine-acetylation has expanded into the cellular intermediary metabolism, we used a combination of differential centrifugation, organelle isolation by linear density gradient centrifugation, western blot analysis, and peptide fingerprinting and amino acid sequencing by mass spectrometry to investigate protein acetylation in control and ciprofibrate-treated rat liver peroxisomes. Organelle protein samples isolated by density gradient centrifugation from PPARα-agonist treated rat liver screened with an anti-N(ε)-acetyl lysine antibody revealed a single protein band of 75 kDa. Immunoprecipitation with this antibody resulted in the precipitation of a protein from the protein pool of ciprofibrate-induced peroxisomes, but not from the protein pool of non-induced peroxisomes. Peptide mass fingerprinting analysis identified the protein as the peroxisomal multifunctional enzyme type 1. In addition, mass spectrometry-based amino acid sequencing resulted in the identification of unique peptides containing 4 acetylated-Lys residues (K¹⁵⁵, K¹⁷³, K¹⁹⁰, and K⁵⁸³). This is the first report that demonstrates posttranslational acetylation of a peroxisomal enzyme in PPARα-dependent proliferation of peroxisomes in rat liver.

A review of the important central role of altered citrate metabolism during the process of stem cell differentiation

Stem cells are highly proliferating cells that have the potential for differentiation leading to the development of specialized functional cell types. The process of stem cell differentiation requires an increase in the recruitment and population of the undifferentiated stem cells, which are then differentiated to specific functional cell types. Genetic/metabolic transformations in the cellular intermediary energy metabolism are required to provide the bioenergetic, synthetic, and catabolic requirements of the stem cells during this process. However, the identification of the intermediary energy metabolism pathways and their alterations during the proliferation and differentiation of stem cells remain largely unknown; mainly due to the lack of attention and/or required research that focuses on this relationship. In the absence of such information, a full understanding of the factors and conditions required to promote stem cell differentiation leading to development of normal functional metabolic specialized cells cannot be achieved. The purpose of this review is to provide the background and bring attention to the essential relationship of altered cellular intermediary metabolism in the context of the process of stem cell proliferation and differentiation. Citrate metabolism is central to the genetic and metabolic transformation leading to the development of the specialized functional cells. This review identifies the involvement of altered citrate metabolism and the associated genetic alterations of key pathways, enzymes, and transporters; as well as the bioenergetic implications. The importance is emphasized for identification and employment of required conditions to insure that the process of experimental stem cell differentiation results in the development of specialized cells that represent the functional metabolic characteristics and capabilities of their native specialized cells. This is an essential requirement for the successful application of stem cell therapy and regenerative medicine for many pathological conditions.

The genetic/metabolic transformation concept of carcinogenesis

The carcinogenesis process is poorly understood and subject to varying concepts and views. A rejuvenated interest has arisen regarding the role of altered cellular intermediary metabolism in the development and progression of cancer. As a result, differing views of the implications of altered metabolism in the development of cancer exist. None of the concepts recognize and incorporate the principles of cell metabolism to cell activity, which are applicable to all cells including the carcinogenesis process. This presentation incorporates a novel concept of carcinogenesis that includes a "genetic/metabolic" transformation that encompasses these principles of cell metabolism to cell activity. The intermediary metabolism transformation is essential to provide the bioenergetic/synthetic, growth/proliferation, and migration/invasive events of malignancy. The concept invokes an "oncogenetic transformation" for the development of neoplastic cells from their precursor normal cells; and a required "genetic/metabolic" transformation for facilitation of the development of the neoplastic cells to malignant cells with the manifestation of the malignant process. Such a concept reveals stages and events of carcinogenesis that provide approaches for the identification of biomarkers and for development of therapeutic agents. The presentation discusses the contemporary application of genetics and proteomics to altered cellular metabolism in cancer; and underscores the importance of proper integration of genetics and proteomics with biochemical and metabolic studies, and the consequences of inappropriate studies.

Tumor cell metabolism: the marriage of molecular genetics and proteomics with cellular intermediary metabolism; proceed with caution!

Metabolic transformations of malignant cells are essential to the development and progression of all cancers. The understanding of the pathogenesis and progression of cancer requires the establishment of the altered genetic/metabolic factors that are essential to the development, growth, and proliferation of the malignant cells. Recognition of this important relationship has resulted in a resurgence of interest in the intermediary metabolism of tumor cells. The role of molecular genetics and proteomics and the application of molecular technology in assessing altered cellular metabolism has become a major area of biomedical research. The contemporary generation of biomedical scientists is exceptionally well trained in all areas of molecular biology and molecular technology, which are now important tools to be applied to the regulation of cellular intermediary metabolism. Simultaneously, the didactic and methodological training associated with the principles and operation of metabolic pathways, enzymology, cellular enzyme activity, and associated biochemical implications has been diminished and often eliminated from the pre- and post-doctoral programs. Interpretations and conclusions of alterations in cellular enzyme activity and associated metabolic pathways based on genetic/proteomic changes can and will result in misrepresentation of important metabolic implications in malignancy and other diseases. It is essential that the genetic/proteomic studies be coupled to biochemical/metabolic cellular events to satisfy the axiom: "genetic transformations and proteomic alterations will have little relevancy to disease processes if the genetic/proteomic alterations are not manifested in altered and impaired cellular and metabolic function". The appropriate marriage of molecular genetics/proteomics with the regulation of cellular intermediary metabolism will provide new revelations and understanding of malignancy that could not be achieved in earlier generations.

Small molecule inhibition of hypoxia inducible factor-1α: A viable therapeutic approach?

Small molecule inhibition of hypoxia inducible factor-1α: A viable therapeutic approach?

A novel mechanism for coupling cellular intermediary metabolism to cytosolic Ca2+signaling via CD38/ADP‐ribosyl cyclase, a putative intracellular NAD+sensor

CD38 is an ectocyclase that converts NAD+ to the Ca2+-releasing second messenger cyclic ADP-ribose (cADPr). Here we report that in addition to CD38 ecto-catalysis, intracellularly expressed CD38 may catalyze NAD+-->cADPr conversion to cause cytosolic Ca2+ release. High levels of CD38 were found in the plasma membranes, endoplasmic reticulum, and nuclear membranes of osteoblastic MC3T3-E1 cells. More important, intracellular CD38 was colocalized with target ryanodine receptors. The cyclase also converted a NAD+ surrogate, NGD+, to its fluorescent product, cGDPr (Km approximately 5.13 microM). NAD+ also triggered a cytosolic Ca2+ signal. Similar results were obtained with NIH3T3 cells, which overexpressed a CD38-EGFP fusion protein. The Delta(-49)-CD38-EGFP mutant with a deleted amino-terminal tail and transmembrane domain appeared mainly in the mitochondria with an expected loss of its membrane localization, but the NAD+-induced cytosolic Ca2+ signal was preserved. Likewise, Ca2+ release persisted in cells transfected with the Myr-Delta(-49)-CD38-EGFP or Delta(-49)-CD38-EGFP-Fan mutants, both directed to the plasma membrane but in an opposite topology to the full-length CD38-EGFP. Finally, ryanodine inhibited Ca2+ signaling, indicating the downstream activation of ryanodine receptors by cADPr. We conclude that intracellularly expressed CD38 might link cellular NAD+ production to cytosolic Ca2+ signaling.

Differential Effect of Glycolytic Intermediaries upon Cyclic ADP-ribose-, Inositol 1′,4′,5′-Trisphosphate-, and Nicotinate Adenine Dinucleotide Phosphate-Induced Ca2+ Release Systems

We investigated the effect of glycolytic pathway intermediaries upon Ca2+ release induced by cyclic ADP-ribose (cADPR), inositol 1′,4′,5-trisphosphate (IP3), and nicotinate adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenate. Fructose 1,6,-diphosphate (FDP), at concentrations up to 8 mM, did not induce Ca2+ release by itself in sea urchin egg homogenate. However, FDP potentiates Ca2+ release mediated by agonists of the ryanodine channel, such as ryanodine, caffeine, and palmitoyl-CoA. Furthermore, glucose 6-phosphate had similar effects. FDP also potentiates activation of the ryanodine channel mediated by the endogenous nucleotide cADPR. The half-maximal concentration for cADPR-induced Ca2+ release was decreased approximately 3.5 times by addition of 4 mM FDP. The reverse was also true: addition of subthreshold concentrations of cADPR sensitized the homogenates to FDP. The Ca2+ release mediated by FDP in the presence of subthreshold concentrations of cADPR was inhibited by antagonists of the ryanodine channel, such as ruthenium red, and by the cADPR inhibitor 8-Br-cADPR. However, inhibition of Ca2+ release induced by IP3 or NAADP had no effect upon Ca2+ release induced by FDP in the presence of low concentrations of cADPR. Furthermore, FDP had inhibitory effects upon Ca2+ release induced by both IP3 and NAADP. We propose that the state of cellular intermediary metabolism may regulate cellular Ca2+ homeostases by switching preferential effects from one intracellular Ca2+ release channel to another.

Valproate inhibits the mitochondrial pyruvate-driven oxidative phosphorylation in vitro.

Valproic acid (2-n-propylpentanoic acid; VPA), a medium-chain branched fatty acid, has proved to be an effective antiepileptic drug. It has also been used widely in the symptomatic treatment of children for a variety of types of seizure. Hepatotoxicity is one of the adverse effects. Several groups have studied the in vitro deleterious effects of VPA on cellular intermediary metabolism. However, the exact mechanisms of VPA-associated hepatotoxicity are still unknown. Despite the structural simplicity of valproate, its metabolic profile is quite complex. Glucuronidation and β-oxidation are quantitatively the most important routes of biotransformation, generating a complex pattern of intermediates (Baillie 1992) which could potentially interfere with mitochondrial metabolism at different levels. In the past, several papers have described inhibitory effects of VPA and its metabolites on mitochondrial oxidative phosphorylation, both in vitro (Haas et al 1981; Becker and Harris 1983; Ponchaut et al 1992) and in vivo (Rumbach et al 1983). However, the results are not easily comparable and the reported data are quite contradictory. Although our primary interest was the effect of VPA on β-oxidation, we decided to study in parallel the interference of VPA with mitochondrial substrate oxidations. We therefore investigated the effect of this drug on mitochondrial oxidative phosphorylation, in concentrations similar to those found in man during therapy. The effects of two of its unsaturated metabolites, 2-propyl-2-pentenoic acid (1 mmol/L) (Δ 2(E) -VPA; mitochondrial β-oxidation intermediate) and 2-propyl-4-pentenoic acid (1 mmol/L) (Δ 4 -VPA; microsomal dehydrogenation product), were also studied. Two experimental approaches were used for this study: digitonin-permeabilized rat hepatocytes for the study of the rate of ATP synthesis, and rat liver mitochondria for the study of oxygen consumption by polarography, both in the presence of selected respiratory chain substrates.

Metabolic alterations in liver and testes of adult and newborn rats following cadmium administration.

Cadmium is a well known toxic heavy metal responsible for many toxic manifestations. A large number of studies have been conducted to understand the effect of cadmium on cellular intermediary metabolism. Although, most of the metal is stored in liver and kidney, the organ affected most in acute toxicity is testis. Cadmium has been shown to have high affinity to sulfhydryl groups. Reducing equivalents such as NADH or NADPH are important for keeping the sulfhydryl groups at a reduced state and maintaining the cellular integrity. The present experiment was designed to study the effect of acute cadmium administration on the activities of some of the tissue enzyme systems that provide the reducing equivalent NADPH. The levels of NADH and NADPH were measured. The effect of simultaneous administration of zinc on cadmium-induced changes was also determined. Because newborn animals have been found to be resistant to many effects of cadmium, the studies were also conducted to compare the effects of cadmium on adult and newborn rats.

Gastrointestinal and hepatic manifestations of chronic alcoholism

Chronic alcoholism is a multisystem disease that adversely affects all of the organs of the body. Such adverse effects within the gastrointestinal tract begin immediately after ingestion and continue after absorption within the liver as well as in other organs. The liver is particularly susceptible to alcoholassociated injury as it is the site within the body wherein most of the subsequent metabolism of ethanol occurs. Until the liver has completed this oxidative function however, alcohol is present in the bloodstream and can thus adversely affect other organs. These include the brain, heart, muscles, and gonads. Not only is ethanol toxic per se, but also its metabolism may produce toxic metabolites such as acetaldehyde and acetate. In addition, changes in cellular redox potential occurring as a result of ethanol metabolism can disturb cellular intermediary metabolism at multiple sites. Finally, other alcohols, aldehydes, and organic molecules (collectively termed congeners) in alcoholic beverages may contribute, at least in part, to the pathogenesis of tissue injury produced as a result of ingestion of such beverages. Although the liver is most susceptible, other organs suffer the effects of ethanol as well. We will address these problems in this presentation. It is estimated that there are 9.3-10 million problem drinkers in the adult population of the United States (1). This figure represents 7% of the total adult population age 18 yr and over. In addition to this massive adult problem-drinker population, there are an estimated 3.3 million problem drinkers among

Alterations in cellular intermediary metabolism by 4-dimethylaminophenol in the isolated perfused rat liver and the implications for 4-dimethylaminophenol toxicity

To evaluate the influence of 4-dimethylaminophenol (DMAP) on cellular intermediary metabolism, isolated rat livers were single pass perfused with subtoxic (0.3 mM) and toxic (1 mM) concentrations of DMAP. The rate of glycolysis and oxygen consumption both increased with biphasical kinetics immediately after the onset of DMAP infusion. After a transient reduction, the cytosolic NAD system was oxidized by DMAP; the mitochondrial NAD system, except for a brief initial oxidation, remained almost unaffected. DMAP caused an intracellular alkalinization. At 0.3 mM, this alkalinization was confined to the cytosol (+0.6pH units); at 1 mM the mitochondria were alkalized in addition (+0.8pH units). In freeze-clamped and lyophilized tissue, following 0.3mM DMAP, the ATP/ADP ratio was lowered by two-thirds and 2-oxoglutarate decreased by one-half; citrate, malate and the sum of adenine nucleotides were unchanged. After 1 mM DMAP, all metabolites were decreased, the ATP/ADP ratio was lowered by three-quarters. Subcellular fractionation revealed inhibition of the citric acid cycle by DMAP, resulting in lowered mitochondrial/cytosolic gradients for Krebs cycle intermediates. The cellular content of CoA was unchanged at 0.3 mM but diminished by 65 per cent at 1 mM, in accordance with the unchanged rate of ketogenesis at 0.3 mM and inhibition at 1 mM. We conclude that mitochondrial CoA depletion with subsequent inhibition of the citric acid cycle and of oxidative energy metabolism suffices to explain DMAP toxicity.