S.J. and R.-Z.Y. contributed equally to this work.
Liver Biology and Pathobiology
Murine alanine aminotransferase: cDNA cloning, functional expression, and differential gene regulation in mouse fatty liver
Article first published online: 26 APR 2004
Copyright © 2004 American Association for the Study of Liver Diseases
Volume 39, Issue 5, pages 1297–1302, May 2004
How to Cite
Jadaho, S. B., Yang, R.-Z., Lin, Q., Hu, H., Anania, F. A., Shuldiner, A. R. and Gong, D.-W. (2004), Murine alanine aminotransferase: cDNA cloning, functional expression, and differential gene regulation in mouse fatty liver. Hepatology, 39: 1297–1302. doi: 10.1002/hep.20182
- Issue published online: 26 APR 2004
- Article first published online: 26 APR 2004
- Manuscript Accepted: 16 JAN 2004
- Manuscript Received: 19 DEC 2003
- NIH. Grant Number: R03DK60563
Alanine aminotransferase (ALT) is a widely used index of liver integrity or hepatocellular damage in clinics as well as a key enzyme in intermediatary metabolism. In this study, we have cloned the complementary DNAs of murine homologues of human alanine aminotransferase 1 and 2 (ALT1 and ALT2). The deduced peptides of murine ALT1 (mALT1) and ALT2 (mALT2) share 87% and 93% identity, respectively, with their human counterparts at the amino acid level. Murine ALT genes localize to separate chromosomes, with mALT1 gene (gpt1) on chromosome 15 and mALT2 gene (gpt2) on chromosome 8. The murine gpt1 and gpt2 differ in messenger RNA expression: gpt1 is mainly expressed in liver, bowel, and white adipose tissue and gpt2 is highly expressed in muscle, liver, and white adipose tissue. Expression of recombinant mALT1 and mALT2 proteins in Escherichia coli (E. coli) produced functional enzymes that catalyze alanine transamination. The potential diagnostic value of ALT isoenzymes in liver disease was evaluated in an obese animal model. In fatty livers of obese mice, ALT2 gene expression is induced 2-fold, but ALT1 remains the same. Furthermore, in fatty liver, total hepatic ALT activity is elevated significantly by 30% whereas aspartate aminotransferase (AST) activity remains unchanged. In conclusion, these results indicate that ALT2 may be responsible for the increased ALT activity in hepatic steatosis and provide evidence that an ALT isoenzyme-specific assay may have more diagnostic value than the total ALT activity assay currently in clinical use. (HEPATOLOGY 2004;39:1297–1302.)
Alanine aminotransferase (ALT; EC 126.96.36.199., formerly glutamate pyruvate transaminase [GPT]) is a pyridoxal enzyme catalyzing reversible transamination between alanine and 2-oxoglutarate to form pyruvate and glutamate. By mediating the conversion of these four major intermediate metabolites, ALT plays an important role in gluconeogenesis and amino acid metabolism. In muscle and certain other tissues, ALT degrades amino acids for fuel, and amino groups are collected from glutamate by transamination. ALT transfers the α-amino group from glutamate to pyruvate to form alanine, which is a major amino acid in blood during fasting. Alanine is taken up by the liver for generating glucose from pyruvate in a reverse ALT reaction, constituting the so-called alanine-glucose cycle.1 This cycle is also important during intensive exercise when skeletal muscles operate anaerobically, producing not only ammonia groups from protein breakdown but also large amounts of pyruvate from glycolysis.
ALT activities exist in many tissues, including liver, muscle, heart, kidney, and brain. Early biochemical and cytogenetic studies have suggested the existence of two ALTs in mice, rats, pigs, and humans. We and others have reported molecular cloning of the complementary DNAs (cDNAs) of two human ALT isoenzymes, hALT1 and hALT2,2, 3 and demonstrated that gpt1 and gpt2 localize to separate chromosomes in humans and have distinctive tissue distribution patterns, suggesting a tissue-dependent role for ALT isoenzymes.
Perhaps the most well-known aspect of ALT is that it is used clinically as an index of liver integrity or hepatocellular damage; serum ALT activity is significantly elevated in a variety of liver damage conditions including viral infection, alcoholic steatosis, nonalcoholic steatohepatitis (NASH), and drug toxicity, although the underlying mechanism is not well understood.4, 5 The identification of two ALT isoenzymes enables us to hypothesize that an ALT isoenzyme-specific assay may provide more diagnostic value than the currently used ALT assay, which presumably measures the catalytic activities of both ALTs. To test our hypothesis, we cloned the murine homologues of human ALT1 and ALT2 genes and studied their tissue distribution and regulation in obesity-associated liver steatosis. Our results suggest that ALT2 gene expression is specifically induced in fatty liver and potentially can be a diagnostic marker for NASH.
Materials and Methods
Cloning of Murine ALT1 and ALT2 cDNAs.
Peptide sequences of human ALT1 and ALT2 were used as probes to search the mouse murine expressed sequence tag (EST) database using tBLASTn. (http://www.ncbi.nlm.nih.gov/blast/). The EST clones that showed closest homology to the probes (IMAGE clone 4195300/BC022625 tohALT1, and IMAGE clone 5065322/BC34219 to hALT2) were purchased from Invitrogen (Carlsbad, CA).
Northern Blot Analysis.
Male obese mice (ob/ob), littermate control (+/?), and C57BL/6J, 6 to 8 weeks old, were purchased from the Jackson Laboratory (Bar Harbor, ME) and euthanized with CO2 according to protocol approved by Institutional Animal Care and Use Committee. Tissues were immediately frozen in liquid nitrogen until use for RNA extraction or enzyme activity assay. Total RNA was prepared with Trizol (Life Technologies Inc., Gaithersburg, MD) from the snap-frozen tissues. For the tissue distribution study, pooled 15 μg of total RNA from 3 to 4 mice were electrophoresed on a 1.2% agarose gel and blotted to a Nitro-plus membrane (Schleicher & Schuell, Dassel, Germany). The DNA probes of murine ALT1 (mALT1) (1.4 kb) and mALT2 (2.4 kb) were derived from restriction enzyme digestion of IMAGE clone 4195300 (Sal I/Not I) and clone 5065322 (Sal I/Not I), respectively. Probes were random-labeled (Stratagene, La Jolla, CA) with 32P-dCTP, hybridization was carried out at 65°C in Rapid-hyb buffer (Amersham Bioscience, Piscataway, NJ), and blots were washed twice with 0.5 × SSC/1% SDS at 65°C (stringent wash) and visualized by PhosphorImager (Amersham Biosciences) or x-ray film.
Chromosomal Localization of Murine gpt1 and gpt2.
Full-length cDNAs of mALT1 (BC022625) and mALT2 (BC34219) were searched against the mouse genome sequence database with BLASTn (http://www.ncbi.nlm.nih.gov/genome/seq/MmBlast.htmland), and their chromosomal localizations were determined by MapViewer (www.ncbi.nlm.nih.gov/mapview).
Expression of Recombinant Protein in Bacteria.
The coding region of mALT1 cDNA was amplified by polymerase chain reaction (PCR) at 28 cycles at 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 1.5 minutes, with a final extension of 7 minutes at 72°C using the Turbo Pfu PCR system (Stratagene) with an Nde I-linked primer, p1408 5′-GGAAG ATCTCATATGGCCTCACAAAGGAATGAC-3′ (nt 106-126, BC022625), and a Not I;-linked primer, p1409 5′-AATGCGGCCGCTCAGGAGTACTCATGAGTGAA-3′ (nt 1596-1576, BC022625), using IMAGE clone 4195300 as a template. The resulting PCR product was digested with Nde I/Not I and subcloned into PET28a (Novagen, Madison, WI), creating plasmid PET28-mALT1. The absence of mutations in the inserted murine ALT1 cDNA was verified by DNA sequence analysis. The same approach was used to clone the coding region of murine ALT2 cDNA from IMAGE clone 5065322 into PET28a by PCR using the IMAGE clone 5065322 as template with primer p1405 5′- GGAAGATC TCCATGGCCCATATGCAGCGGGCAGCGGTGCTGGT-3′ (nt 128-150, BC034219) and p1406 5′-AATGCGGCCGCTCATGAGTACTGCTCCAGGAA-3′ (nt 1696-1676, BC034219), creating plasmid PET28-mALT2.
To express mALT1 and mALT2 proteins, plasmid PET28-mALT1, PET28-mALT2, or empty vector PET28 were used to transform competent E. coli. (Tuner DE3, Novagen). A fresh colony of the transformants was grown in 50 mL Luria-Bertani (LB) media containing 30 μg/mL kanamycin to an OD600 of 0.7, at which time isopropyl-beta-D-thiogalactopyranoside (IPTG) was added (1 mM, final concentration) to induce expression of the recombinant proteins. Cell pellets were harvested from 20 mL cultures before and after 4 hours of induction and were resuspended in 5 mL of TE buffer [10 mM Tris-HCl (pH 7.4), 0.1 mM ethylenediaminetetraacetic acid], followed by a brief sonication (3 × 10 seconds, setting 3; Fisher 550 Sonic Dismembrator, Pittsburgh, PA). Cell lysates were centrifuged at 10,000 × g for 15 minutes at 4°C, and supernatants were analyzed immediately for enzyme activity and protein analysis.
For ALT activity assay of bacterially expressed recombinant proteins, the GPT Optimized Alanine Aminotransferase kit (Sigma Dignostics, catalog no. DG159-K, St. Louis, MO) was used to measure ALT activities as specified in the manual. Briefly, 0.5 mL of cell lysate was incubated with a 2.5 mL mixture of reagent A and B containing L-alanine, NADH, lactate dehydrogenase, and 2-oxoglutarate at 25°C. Absorbance at 340 nm was recorded at 1, 2, and 3 minutes after incubation. The slope of absorbance decrease is proportional to ALT activity. Protein concentration of cell lysates were determined by Coomassie Brilliant Blue G250 (BioRad, Hercules, CA) using bovine serum albumin as a standard. Final ALT activities were corrected by protein concentration of cell lysates. One unit of ALT activity was defined as the amount of enzyme that catalyzes the formation of 1 μmol/L of NAD per minute at 25°C.
For hepatic ALT and aspartate aminotransferase (AST) activity assay, a piece of snap-frozen liver (50-60 mg) was thawed on ice and then minced and homogenized in 19 volumes of TE buffer (wt/vol) with Dounce homogenizer (×40 times). The resulting homogenate was further sonicated (3 × 10 seconds, setting 4, Fisher 550 Sonic Dismembrator) followed by centrifugation at 10,000 × g for 15 minutes at 4°C. The supernatant was assayed for hepatic ALT using an L-type GPT J2 kit (Wako Chemicals, Osaka, Japan) because the Sigma GPT kit had been discontinued; however, both the Sigma and Wako GPT kits yielded comparable results (data not shown). AST activity was measured with an AST/GOT Liqui-UV kit (Stanbio Laboratories, Boerne, TX) according to the manufacturer's instructions.
Data Presentation and Statistical Methods.
Data are presented as mean ± SD. Statistical significance was determined by Student unpaired t test. A P value less than .05 was considered significant.
Cloning of Murine ALT1 and ALT2 cDNAs.
A search of the mouse EST database using hALT1 and hALT2 peptide sequences as probes yielded several highly homologous EST clones (data not shown). Of them, IMAGE clones 4195300 and 5065322 were fully sequenced and revealed the highest homology to human ALT1 and ALT2, respectively, in the entire protein-coding region. The DNA nucleotide sequences of these two clones were confirmed by sequence analysis and are predicted to encode proteins of 496 (4195300) and 522 (5065322) amino acids. Comparison of the mouse and human peptide sequences revealed that IMAGE clone 4195300 shares 87% identity and 89% similarity to hALT1, but 70% identity and 72% similarity to hALT2, whereas clone 5065322 shares 93% identity and 95% similarity with hALT2, but 69% identity and 78% similarity to hALT1 (Figs. 1A and B). Thus, we named the cDNA clone 4195300 “mouse ALT1” (mALT1) and the clone 5065322 “murine ALT2” (mALT2). Sixty-seven percent of amino acids are identical between mALT1 and mALT2; a similar degree of conservation is found between hALT1 and hALT2 (68%).3
Tissue Distribution and Chromosomal Localization.
Next, we examined the gene expression of ALT1 and ALT2 in mouse tissues by Northern analysis. The ≈3.3 kb ALT2 messenger RNA (mRNA) was expressed at high levels in muscle, liver, and white adipose tissue (WAT), at moderate levels in brain and kidney, and at a low level in heart. By contrast, the ≈1.8 kb mALT1 mRNA was highly expressed in liver and considerably in WAT, intestine, and colon (Fig. 2). Notably, some tissues appeared to selectively express one ALT isoenzyme over the other. For instance, mALT2 was significantly expressed—but mALT1 barely expressed—in muscle and brain. In contrast, bowel expressed only ALT1, not ALT2. To determine the chromosomal localization of murine gpt1 and gpt2, we searched the corresponding cDNAs against the mouse genome and localized gpt1 to murine chromosome 15D3 and gpt2 to chromosome 8C2. Both of these regions are syntenic to the chromosomal regions where human gpt1 (chromosome 8q24.3) and gpt2 (chromosome 16q12.2) reside.
Functional Expression of mALT1 and mALT2 in Bacteria.
High conservation between mouse and human ALTs at the peptide level suggests that the murine ALTs would function as ALT. To validate this assumption, the plasmids PET28-mALT1, PET28-mALT2, or empty vector PET28 were expressed in E. coli with or without IPTG induction. Soluble fractions of cell lysates were assayed for ALT activity and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. As shown in Fig. 3A, under induction of IPTG, significant ALT activity was observed in cell lysates from bacteria transformed with PET28-mALT1 (2.62 units/mg protein) and PET28-mALT2 (0.72 units/mg protein), compared to empty vector control (0.19 units/mg protein). Accordingly, protein bands at approximate molecular weights (MW) of 58 kd and 62 kd were clearly visible (Fig. 3B) in bacterial cell pellets after IPTG induction, corresponding to mALT1 (calculated MW 55 kd) and mALT2 (calculated MW 58 kd). It should be noted that ALT activities detected in the above cell lysates do not reflect the specific activity of the ALT isoenzymes, since most of the recombinant proteins were expressed in the insoluble fraction of inclusion bodies (data not shown), and, therefore, the actual amount of ALT in the cell lysates was not known. Nevertheless, these data confirm that murine ALT1 and ALT2 cDNAs encode functional ALT.
ALT2 Gene Expression is Specifically Induced in Fatty Liver.
The distinctive tissue distribution patterns of ALT1 and ALT2 mRNAs are likely due to a difference in their gene regulation. Gene expression differences were examined in fatty livers of obese mice (ob/ob). Compared to the normal liver of lean mice (+/?), the expression of mALT2 mRNA was elevated about twofold, but that of mALT1 remained unchanged (Fig. 4). Furthermore, a significant elevation (30%, P < .01) of ALT enzymatic activity was observed in fatty liver relative to normal liver. These data suggest that ALT2 induction is probably responsible for the increased ALT activity in fatty liver. Interestingly, AST activity was barely increased in fatty liver (5%, P = .5) compared with normal liver (Fig. 5).
In the current study, we report the molecular cloning of murine homologues of human ALT1 and ALT2. ALT is an important intermediary enzyme involved in the metabolism of amino acids, glucose, and possibly fatty acids and is well known for its use as a surrogate marker for liver damage in clinical diagnostics. The existence of two ALT isoenzymes has been suggested by early biochemical studies, but the isoenzymes' molecular identities were not known until the recent cloning of human ALT1 and ALT2 cDNAs.2, 3 In addition to the dissimilarity of the peptide sequences of ALT1 and ALT2 (68% identity), the ALT genes reside on separate chromosomes and have distinct tissue distributions and possibly cellular localizations. These characteristics suggest that ALT isoenzymes may behave discordantly in various clinical conditions. In other words, under certain clinical conditions, one isoenzyme may be elevated but not the other, or vice versa. By virtue of this feature, ALT isoenzymes may be better diagnostic markers than total ALT activity. To this end, we cloned murine ALT1 and ALT 2 cDNAs and studied their regulation in ob/ob mice and their lean littermates.
ALT activities are present in many tissues, including liver, heart, kidney, muscle, brain, and adipose tissue in rodents.6, 7 Our Northern blot data are in agreement with the early functional studies: one or both of the ALT genes are expressed in the tissues where ALT activity has been observed. Interestingly, mALT1 is mainly expressed in liver and bowels, whereas mALT2 is highly expressed in muscle, liver, fat, and kidney, a tissue pattern reminiscent of human ALT1 and ALT2 tissue distribution. The conservation between murine and human ALT isoenzymes in protein sequence, gene localization, and tissue distribution forms a basis for using the mouse as a model for exploring the diagnostic value of ALT isoenzymes.
ALT and AST activity levels have been used in clinic diagnostics since 1953.8 Elevation of these two enzyme activities in serum are regarded as evidence of liver damage, as in viral hepatitis, NASH, or drug hepatoxicity. However, the mechanism for the serum ALT increase is not well understood and is thought to be caused by “leakage” of the cellular enzyme into the systemic circulation. Moreover, which ALT isoenzyme is responsible for the serum elevation is not known because the current ALT assay measures total catalytic activity of ALT—presumably the combined activity of ALT1 and ALT2. Molecular cloning of ALT isoenzymes in mice and humans provides a means to study the underlying mechanism(s) as well as an interpretation of clinical ALT observations. For example, ALT elevation in muscle disease may be due to a “leak” of ALT—presumably ALT2—from muscle, where ALT2, but not ALT1, is abundantly expressed. Similarly, a specific ALT isoenzyme may be induced in a given clinical condition. Thus, we examined hepatic ALT1 and ALT2 gene expression in obese mice because hepatic steatosis is always associated with this genetically obese model.9 Indeed, mALT2—but not ALT1—gene expression is specifically induced. Furthermore, total ALT enzymatic activity is increased by 30% in fatty liver over nonfatty liver, suggesting that ALT2 may be primarily responsible for the increased serum ALT activity in liver steatosis. Interestingly, AST activity remains unchanged in the same condition. These animal findings are in agreement with clinical observations in which serum ALT is generally increased to a greater extent than AST in patients with liver steatosis.10–12 Further research is warranted to determine whether serum ALT2 could be a specific diagnostic marker for liver steatosis in humans.
Additionally, the ALT isoenzymes may be present in different cellular compartments, which can also be utilized for diagnostic purposes: the release of a given ALT isoenzyme into the circulation may reflect the nature of the liver damage. By immunodepletion of cytosolic AST, Darling et. al. found that serum mitochondrial AST content is specifically increased in patients treated with halothane and suggested that this AST isoenzyme is a sensitive marker for halothane-induced hepatic injury.13 Both cytosolic and mitochondrial ALT activities were found in liver, kidney, and skeletal and cardiac muscles.14–16 At present, which ALT isoenzyme, ALT1 or ALT2, is cytosolic or mitochondrial remains unclear. ALT isoenzyme-specific antibodies will help to elucidate the cellular localization of ALT isoenzymes and their changes in disease states.
In summary, differences in ALT isoenzymes in tissue distribution, gene regulation, and possible cellular localization suggest that the ability to measure ALT isoenzyme-specific activity levels would be a significant improvement over measurement of total ALT activity in clinical diagnostics. The cloning of murine homologues of human ALT isoenzymes in mice provides a novel tool to study potential clinical applications of ALT isoenzymes as molecular markers for nonalcoholic fatty liver diseases as well as other clinical conditions.
The authors thank Toni Pollin for critical reading of the manuscript and Bob Stephens and Toni Harbaugh-Blackford for bioinformatics support at Advanced Biomedical Computing Center, NCI-FCRDC.