Molecular Basis of Disrupted Polyamine Metabolism in Diabetes and Effects of Triethylenetetramine

Abstract

Type 2 diabetes is a growing epidemic throughout the world. Although defects in secretion and action of insulin are both fundamental to disease causation, they increasingly appear not to explain the complications and organ pathology that causes most diabetic morbidity and mortality. An increase in understanding of diabetes pathobiology has led to the investigation of several other pathways including polyamine metabolism. The polyamines are small aliphatic molecules that play roles in many cellular processes. Their levels are very tightly regulated and disruptions are associated with many disease states, including diabetes. Triethylenetetramine (TETA), a tetra-amine, is a divalent-copper-selective chelator that is also a synthetic polyamine analogue. There is substantive evidence that in vivo acetylation of TETA can be catalyzed by either of two enzymes: spermidine/spermine-N1-acetyltransferase (SSAT1), which catalyzes the physiological acetylation of the polyamines spermidine and spermine, and its homologue, thialysine N-ε-acetyltransferase (SSAT2), to yield the two known metabolites, monoacetyl- TETA (MAT) and diacetyl-TETA (DAT). However, the role played by SSAT2 in physiological polyamine metabolism, if any, is yet to be ascertained. The endogenous polyamines and TETA can exert influences on diabetic pathobiology. Treatment with TETA has been shown to ameliorate the effects of diabetes in the heart and kidney at both the functional and molecular levels, whereas polyamine metabolism is linked to cellular and molecular aspects of diabetes pathobiology, including amongst others the following processes: nitric oxide metabolism; advanced-glycation endproduct (AGE) formation; inflammation; insulin production; and glucose transport. The main aim of this thesis was to investigate the links between TETA metabolism, polyamine metabolism and the pathobiology of diabetes through the measurement of levels of specific mRNAs, proteins, enzyme activities and metabolites. These investigations were performed using the widelyaccepted streptozotocin (STZ)-induced model of diabetes in the rat. The levels of mRNAs corresponding to key enzymes and regulatory proteins that control polyamine metabolism were measured in cardiac left ventricle, kidney cortex, and liver, whereas protein and activity levels were measured in liver only due to feasibility constraints. These organs were chosen since each of them can become severely damaged in diabetes, and they therefore serve as exemplars of diabetic organ damage relevant to the molecular pathogenesis of the complications. This study identified previously unreported changes in polyamine metabolism in diabetes and showed that TETA can improve the diabetes-associated disruption of some of the key enzymes in the pathways of polyamine biosynthesis and catabolism. The transcriptional analysis showed a diabetes-dependent increase in levels of mRNAs corresponding to several enzymes of polyamine biosynthesis in liver and kidney. Furthermore, levels of the key enzyme ODC (ornithine decarboxylase) were also increased at the protein level. In the kidney, TETA alleviated the diabetes-dependent-increase of Arg2, which encodes the enzyme arginase II. ODC is regulated by the action of antizyme (AZ) and antizyme inhibitor (Azin) proteins, whose biology with respect to diabetes was investigated for the first time in this study. Decreases in the levels of the mRNA, Oaz1, and protein corresponding to AZ2 protein were here demonstrated in diabetes, which is interpreted as consistent with the higher levels of active ODC also observed in diabetes. The reactions of polyamine catabolism involve the following enzyme-mediated catalytic processes: acetylation; back-conversion; and terminal degradation of polyamine metabolites. STZ-induced diabetes was associated with decreased mRNA (Ssat1) and protein levels of the acetylation enzyme SSAT1, and increased mRNA (Ssat2) levels of SSAT2. This result gives new insights into a potential mechanism for the increased acetylation of TETA that has been reported in diabetic patients. There was also a significant increases in Smox and Paox mRNA in diabetic kidney, which was significantly decreased with TETA-treatment. Also investigated were the effects of diabetes and of TETA treatment on a newly described enzyme similar to diamine oxidase, diamine oxidase-like protein 1 (Doxl-1), results showed lower mRNA (Doxl1) levels in diabetes. Hypusine is a unique amino acid derived from spermidine, which is found only in active eukaryotic translation initiation factor 5A-1 (eIF5A). Hypusine is incorporated into the protein biosynthetically through two enzyme-catalyzed reactions, namely: (i) transfer of the 4-aminobutyl moiety of the polyamine spermidine to the epsilon-amino group of a single specific lysine residue in the eIF5A precursor protein, catalyzed by the enzyme deoxyhypusine synthase (DHPS), to form an intermediate, deoxyhypusine; and (ii) subsequent hydroxylation of this 4-aminobutyl moiety catalyzed by the enzyme deoxyhypusine hydroxylase (DOHH). Increases in the mRNA levels corresponding to both of these enzymes (respectively Dhps and Dohh) were measured in the kidney in diabetes and deoxyhypusine synthase mRNA was found to be significantly lower with TETA treatment in this tissue. Active eIF5A is thought to be necessary for some processes that can lead to islet beta-cell death in diabetes and this is the first report to the candidate’s knowledge of the levels of these transcripts in liver, kidney and heart. Overall this study showed that several key steps in polyamine metabolism become disrupted in diabetes, and that TETA has a normalizing effect on several of these enzymes. This thesis also aimed to determine through metabolomic methods whether changes measured at the transcript and/or protein levels might also be reflected in the levels of metabolites in the liver, and in addition to determine whether TETA or its known acetyl-derivatives might be acted on by degradation enzymes within the polyamine pathway. A non-targeted metabolomic approach was applied as is usual at the beginning of such an investigation. It showed that KEGG (Kyoto Encyclopaedia of Genes and Genomes) pathways involved in polyamine metabolism were changed in diabetes, and that many of these changes were reversed by TETA treatment. To give further insight into TETA metabolism, a theoretical enzyme analysis was performed by examining the putative ability of different polyamine enzyme systems to metabolize TETA and its known acetyl metabolites. Several enzymes in the polyamine catabolic pathway have loose substrate specificity and have been shown to metabolize a number of synthetic polyamine analogues: through this analysis, several putative theoretical TETA metabolites were identified. However, subsequent targeted metabolomic analysis did not reproducibly identify any previouslyunknown TETA metabolite in liver extracts, and further experiments will be required to determine whether TETA is degraded or back converted as part of its in vivo metabolism in mammals.