Determination of relative protein degradation activity at different life stages in rainbow trout (Oncorhynchus mykiss)

Rainbow trout were reared from 5 g to ~ 400 g on a diet formulated to supply the required protein from either fishmeal or plant proteins. The fish were sampled at every weight doubling and liver and muscle samples were obtained. From these tissue samples RNA and protein were isolated and analyzed for the expression of a number of muscle regulatory and protein degradation genes and enzymatic activity for proteins involved in the caspase, calpain, and ubiquitin-proteasome pathways for protein proteolysis. Only MyoD2 showed significant differences in expression between the two diets, while no significant changes over the course of the experiment were determined for MyoD2 or the other muscle factors. For the degradation genes significant changes in expression were determined for calpain1 and calpastatin. Calpastatin also showed a significant increase in expression over the course of the experiment in the muscle of fish fed a fishmeal diet and significant decrease in expression in the liver of fish fed the fishmeal based diet. Differences in proteasome enzyme activity were found between diets in the liver and muscle of fish and for caspase-3 activity in muscle. Significant changes in activity over the course of the experiment were noted for proteasome and calpain activity in the liver and muscle. These findings suggest that diets replacing fishmeal with plant material can have some effects on protein turnover in muscle and that some degradation pathways are differentially regulated during the growth of rainbow trout.     

Molecular characterization of the MuRF genes in rainbow trout: Potential role in muscle degradation

Muscle growth is determined primarily by the balance between protein synthesis and degradation. When rates of protein synthesis are similar between individuals, protein degradation is critical in explaining differences in growth efficiency. Studies in mammals showed that muscle atrophy results from increased protein breakdown, and is associated with activation of the ubiquitin proteasome pathway, including induction of the muscle-specific ubiquitin protein ligase, MuRF1. Animals lacking MuRF1 are resistant to muscle atrophy. In fish, little is known about the role of the proteasome/MuRF pathway in muscle degradation. The objectives of this study were to: 1) clone and characterize MuRF genes in rainbow trout; and 2) determine expression of MuRF genes in association with starvation- and vitellogenesis-induced muscle atrophy in rainbow trout. We have identified full-length cDNA sequences for three MuRF genes (MuRF1, MuRF2, and MuRF3). These genes encode proteins with typical MuRF structural domains, including a RING-finger, a B-box and a Leucine-rich coiled-coil domain. RT-PCR analysis showed that MuRF genes are predominantly expressed in muscle and heart tissues. Real time PCR analysis revealed that expression of all MuRF genes is up-regulated during starvation and MuRF3 is up-regulated in vitellogenesis-associated muscle degradation. These results suggest that MuRF genes have an important role in fish muscle protein degradation. Further studies are warranted to assess the potential use of MuRF genes as tools to monitor fish muscle growth and degradation.     

Upregulation of MuRF1 and MAFbx participates to muscle wasting upon gentamicin-induced acute kidney injury

Acute Kidney Injury (AKI) is frequently encountered in hospitalized patients where it is associated with increased mortality and morbidity notably affecting muscle wasting. Increased protein degradation has been shown to be the main actor of AKI-induced muscle atrophy, but the proteolytic pathways involved are poorly known. The Ubiquitin Proteasome System (UPS) is almost systematically activated in various catabolic situations, and the E3 ligases MuRF1 and MAFbx are generally up regulated in atrophying muscles. We hypothesized that the UPS may be one of the main actors in catabolic skeletal muscles from AKI animals. We used gentamicin-induced acute kidney disease (G-AKI) in rats fed a high protein diet to promote acidosis. We first addressed the impact of G-AKI in the development of mild catabolic conditions. We found that both muscle atrophy and UPS activation were induced with the development of G-AKI. In addition, the phasic muscles were more sensitive to 7-days G-AKI (−11 to −17%, P < 0.05) than the antigravity soleus muscle (−11%, NS), indicating a differential impact of AKI in the musculature. We observed an increased expression of the muscle-specific E3 ligases MuRF1 and MAFbx in phasic muscles that was highly correlated to the G-AKI severity (R² = 0.64, P < 0.01 and R² = 0.71, P < 0.005 respectively). Conversely, we observed no variation in the expression of three other E3 ligases (Nedd4, Trim32 and Fbxo30/MUSA1). Altogether, our data indicate that MuRF1 and MAFbx are sensitive markers and potential targets to prevent muscle atrophy during G-AKI.     

Postprandial hepatic protein expression in trout Oncorhynchus mykiss a proteomics examination

Following a meal, a series of physiological changes occurs in animals as they digest, absorb and assimilate ingested nutrients, the kinetics of these responses depends on metabolic rate and nutrient quality. Here we investigated the hepatic proteome in the ectothermic teleost, the rainbow trout, following a single meal to define the postprandial expression of hepatic proteins. The fish were fed a high marine fishmeal/fish oil single meal following a period of 24 h without feeding. Liver protein profiles were examined by 2D gel electrophoresis just before feeding (time 0 h) and at 6 and 12 h after feeding. Of a total of 588 protein spots analysed in a temporal fashion, 49 differed significantly in abundance between the three time groups (ANOVA, p<0.05), before and after feeding, 15 were increased and 34 were decreased in abundance after feeding. Amino acid metabolism-regulated proteins such as phenylalanine-4-hydroxylase and proliferating cell antigen were increased in abundance 12 and 6 h following the meal, suggesting by this time that the fish were increasing their protein turnover to utilize efficiently their dietary protein consumption. Overall, these results highlight some specificity of the trout metabolism and identify postprandial response of metabolism-related proteins 6–12 h after feeding a single meal.     

Early-age changes in oxidative stress in brown trout,Salmo trutta

Fish are often used as models for studies investigating the ability of xenobiotics to induce oxidative stress, though age or developmental stage of the individuals studied has been given little attention. Oxidative stress in other organisms is associated with aging as well as with periods of rapid growth, which occurs in young brown trout. We measured protein carbonyls, 20S proteosome activity and glutathione (GSH) levels in farmed Salmo trutta in four different age groups from 5 months to 3 years. We found an increase in protein carbonyls and a decrease in 20S proteosome activity in both brain and liver tissues of the fish with increasing size and age. Total GSH levels in liver tissue declined as fish aged and the GSSG:GSH ratio increased. Five month and 1 year old trout were treated with paraquat (PQ) to induce oxidative stress. Five month old fish showed no changes in the measured parameters while 1 year old fish had both an increase in protein carbonylation in liver tissue and a decrease in 20S proteosome activity in brain tissue. These results indicate that oxidative stress biomarkers are affected by age or rapid growth in brown trout, and that individuals of different ages respond differently to oxidative stress induced by PQ.     

Ubiquitin-protein ligases in muscle wasting

Muscle wasting occurs when rates of protein degradation outstrip rates of protein synthesis. Accelerated rates of protein degradation develop in atrophying muscle largely through activation of the ubiquitin-proteasome pathway. The complexity of the ubiquitination process, however, has hampered our understanding of how this pathway is activated in atrophying muscles and which enzymes of the ubiquitin conjugation system are responsible. Recent studies demonstrate that two ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MuRF1 are critical in the development of muscle atrophy. Other experiments implicate E2(14k) and E3alpha, of the N-end rule pathway, as important players in the process. It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as to develop inhibitors of, these E3s.     

Temporal changes in human skeletal muscle and blood lipid composition with fish oil supplementation

The aim of this study was to examine changes in the lipid profile of red blood cells and muscle tissue along with the expression of anabolic signalling proteins in human skeletal muscle. Following a 2-week control period, 10 healthy male participants consumed 5 g d⁻¹ of fish oil (FO) for 4 weeks. Muscle biopsies and venous blood samples were collected in the fasted state 2 weeks prior (W-2) and immediately before (W0) the initiation of FO supplementation for internal control. Muscle biopsies and venous blood samples were again obtained at week 1 (W1), 2 (W2) and 4 (W4) during FO supplementation for assessment of changes in lipid composition and expression of anabolic signalling proteins. There was no change in the composition of any lipid class between W-2 and W0 confirming control. Following FO supplementation n-3 polyunsaturated fatty acid (n-3 PUFA) muscle lipid composition was increased from W0 to W2 and continued to rise at W4. n-3 PUFA blood lipid composition was increased from W0 to W1 and remained elevated for the remaining time points. Total protein content of focal adhesion kinase (FAK) increased from W0 to W4 whereas total mechanistic target of rapamycin (mTOR) was increased from W0 at W1 with no further significant increases at W2 and W4. These data show that FO supplementation results in discordant changes in the n-3 PUFA composition of skeletal muscle compared to blood that is associated with increases in total FAK content.     

Secretory expression and characterization of the recombinant myofibril-bound serine proteinase of crucian carp (Carassius auratus) in Pichia pastoris

The myofibril-bound serine proteinase (MBSP) is effective in the degradation of myofibrillar proteins, including myosin heavy chain (MHC), α-actinin, actin, and tropomyosin and was thus regarded as an important proteinase responsible for the metabolism of fish muscle in vivo. In order to better understand the characteristic differences between native MBSP and recombinant MBSP (rMBSP) and to obtain large quantity of MBSP for its application in protein science study, the crucian carp MBSP gene was cloned (669 bp) and expressed in Pichia pastoris (P. pastoris). The recombinant P. pastoris strain was cultured in shake flasks, and 66.85 mg rMBSP/L in the fermentation supernatant was obtained. SDS-polyacrylamide gel electrophoresis (PAGE) showed a main protein band with molecular weight of approximately 36 kDa. Substrate specificity analysis revealed that the rMBSP specifically cleaved substrates at the carboxyl side of lysine residue which differed from native MBSP that cleaved substrates at the carboxyl side of arginine and lysine residues. The optimum temperature and optimum pH range of the rMBSP were 55 °C and pH 7.5, respectively. Furthermore, similar to native MBSP, the rMBSP also revealed high thermostability and pH stability and is effective in degradation of myofibrillar proteins from the skeletal muscle of crucian carp.     

Cellular and molecular mechanisms of muscle atrophy

Skeletal muscle is a plastic organ that is maintained by multiple pathways regulating cell and protein turnover. During muscle atrophy, proteolytic systems are activated, and contractile proteins and organelles are removed, resulting in the shrinkage of muscle fibers. Excessive loss of muscle mass is associated with poor prognosis in several diseases, including myopathies and muscular dystrophies, as well as in systemic disorders such as cancer, diabetes, sepsis and heart failure. Muscle loss also occurs during aging. In this paper, we review the key mechanisms that regulate the turnover of contractile proteins and organelles in muscle tissue, and discuss how impairments in these mechanisms can contribute to muscle atrophy. We also discuss how protein synthesis and degradation are coordinately regulated by signaling pathways that are influenced by mechanical stress, physical activity, and the availability of nutrients and growth factors. Understanding how these pathways regulate muscle mass will provide new therapeutic targets for the prevention and treatment of muscle atrophy in metabolic and neuromuscular diseases.     

Chronic vitamin E deficiency promotes vitamin C deficiency in zebrafish leading to degenerative myopathy and impaired swimming behavior

We hypothesized that zebrafish (Danio rerio) undergoing long-term vitamin E deficiency with marginal vitamin C status would develop myopathy resulting in impaired swimming. Zebrafish were fed for 1 y a defined diet without (E ?) and with (E +) vitamin E (500 mg α-tocopherol/kg diet). For the last 150 days, dietary ascorbic acid concentrations were decreased from 3500 to 50 mg/kg diet and the fish sampled periodically to assess ascorbic acid concentrations. The ascorbic acid depletion curves were faster in the E ? compared with E + fish (P < 0.0001); the estimated half-life of depletion in the E ? fish was 34 days, while in it was 55 days in the E + fish. To assess swimming behavior, zebrafish were monitored individually following a “startle-response” stimulus, using computer and video technology. Muscle histopathology was assessed using hematoxylin and eosin staining on paramedian sections of fixed zebrafish. At study end, E ? fish contained 300-fold less α-tocopherol (p < 0.0001), half the ascorbic acid (p = 0.0001) and 3-fold more malondialdehyde (p = 0.0005) than did E + fish. During the first minute following a tap stimulus (p < 0.05), E + fish swam twice as far as did E ? fish. In the E ? fish, the sluggish behavior was associated with a multifocal, polyphasic, degenerative myopathy of the skeletal muscle. The myopathy severity ranged from scattered acute necrosis to widespread fibrosis and was accompanied by increased anti-hydroxynonenal staining. Thus, vitamin E deficiency in zebrafish causes increased oxidative stress and a secondary depletion of ascorbic acid, resulting in severe damage to muscle tissue and impaired muscle function.     

Serum profile in preeclampsia and intra-uterine growth restriction revealed by iTRAQ technology

In order to identify new protein markers modified in placental diseases, high-throughput analysis of proteins in the plasma of pregnant women was carried out for normal and pathological pregnancies (Preeclampsia and/or Intra-Uterine Growth Restriction) using iTRAQ technology. We could identify 166 proteins that were modified (p < 0.05) and the technique used allowed the detection of previously undetected factors, such as various members of the SERPINA clade. The modifications of two proteins (C reactive protein and antichymotrypsin, SERPINA3) were validated on individual samples. Complement and coagulation cascades proteins were significantly enriched among modified protein clusters in the case of intra-uterine growth restriction (p < 2.6 · 10^? 11). Several proteins were specifically enriched in isolated preeclampsia and depleted when preeclampsia was complicated by intra-uterine growth restriction. These findings suggest that the growth restricted foeto-placental unit is able to moderate some changes in maternal plasma composition. Overall, the use of iTRAQ technology, for the first time on this subject, enabled us to provide a new list of proteins modified in placental diseases, among which proteins expressed at a low level that were not accessible by other methods.     

Identification of serum proteins involved in pancreatic cancer cachexia

AimsTreatment of cachexia requires pharmacological intervention which, in turn, requires knowledge of the mediators and processes. Cachexia markers that are specifically expressed in pancreatic cancer and secreted into the blood circulation have yet to be identified. The aim of our study was to investigate the serum protein profiles and protein alterations associated with cachexia and to identify potential disease protein biomarkers indicative for this syndrome.Main methodsSerum samples from cachectic and non-cachectic patients undergoing pancreatic cancer (PaCa) surgery and controls were investigated by Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS). The identity of detected discriminatory markers was determined by a combination of protein fractionation, chromatographic purification steps, gel electrophoresis, and mass spectrometry.Key findingsUsing Cu-IMAC array and CM-10 array based SELDI-TOF-MS. we identified eleven up- and four down-regulated proteins associated with cachexia. CiphergenExpress analysis revealed four disease-associated protein features (38559 Da, 9138 Da, 8925 Da and 3358 Da) that were elevated by a factor of 2.3, 1.7, 1.4 and 1.4, respectively. Zinc-α2-glycoprotein (ZAG), apolipoproteins apo C-II and apo C-III and glucagon-like peptide-1 (GLP-1) were identified as markers for PaCa-associated cachexia syndrome. ZAG levels were additionally evaluated in serum and tissue samples by ELISA and immunohistochemistry and the obtained data confirmed the SELDI-TOF-MS results.SignificanceThe identified proteins could be routinely and reliably measured in the serum of patients and provide an elegant non-invasive approach for early diagnosis of cachectic pancreatic cancer patients. Controlling ZAG and GLP-1 activity could be beneficial in the management of cancers and cachexia-induced conditions.     

During muscle atrophy,thick, but not thin,filament components are degraded by MuRF1-dependent ubiquitylation

Loss of myofibrillar proteins is a hallmark of atrophying muscle. Expression of muscle RING-finger 1 (MuRF1), a ubiquitin ligase, is markedly induced during atrophy, and MuRF1 deletion attenuates muscle wasting. We generated mice expressing a Ring-deletion mutant MuRF1, which binds but cannot ubiquitylate substrates. Mass spectrometry of the bound proteins in denervated muscle identified many myofibrillar components. Upon denervation or fasting, atrophying muscles show a loss of myosin-binding protein C (MyBP-C) and myosin light chains 1 and 2 (MyLC1 and MyLC2) from the myofibril, before any measurable decrease in myosin heavy chain (MyHC). Their selective loss requires MuRF1. MyHC is protected from ubiquitylation in myofibrils by associated proteins, but eventually undergoes MuRF1-dependent degradation. In contrast, MuRF1 ubiquitylates MyBP-C, MyLC1, and MyLC2, even in myofibrils. Because these proteins stabilize the thick filament, their selective ubiquitylation may facilitate thick filament disassembly. However, the thin filament components decreased by a mechanism not requiring MuRF1.     

Induction of mitochondrial biogenesis protects against caspase-dependent and caspase-independent apoptosis in L6 myoblasts

Apoptotic signaling plays an important role in skeletal muscle degradation, atrophy, and dysfunction. Mitochondria are central executers of apoptosis by directly participating in caspase-dependent and caspase-independent cell death signaling. Given the important apoptotic role of mitochondria, altering mitochondrial content could influence apoptosis. Therefore, we examined the direct effect of modest, but physiological increases in mitochondrial biogenesis and content on skeletal muscle apoptosis using a cell culture approach. Treatment of L6 myoblasts with SNAP or AICAR (5 h/day for 5 days) significantly increased PGC-1, AIF, cytochrome c, and MnSOD protein content as well as MitoTracker staining. Following induction of mitochondrial biogenesis, L6 myoblasts displayed decreased sensitivity to apoptotic cell death as well as reduced caspase-3 and caspase-9 activation following exposure to staurosporine (STS) and C2-ceramide. L6 myoblasts with higher mitochondrial content also exhibited reduced apoptosis and AIF release following exposure to hydrogen peroxide (H₂O₂). Analysis of several key apoptosis regulatory proteins (ARC, Bax, Bcl-2, XIAP), antioxidant proteins (catalase, MnSOD, CuZnSOD), and reactive oxygen species (ROS) measures (DCF and MitoSOX fluorescence) revealed that these mechanisms were not responsible for the observed cellular protection. However, myoblasts with higher mitochondrial content were less sensitive to Ca^2 +-induced mitochondrial permeability transition pore formation (mPTP) and mitochondrial membrane depolarization. Collectively, these data demonstrate that increased mitochondrial content at physiological levels provides protection against apoptotic cell death by decreasing caspase-dependent and caspase-independent signaling through influencing mitochondrial Ca^2 +-mediated apoptotic events. Therefore, increasing mitochondrial biogenesis/content may represent a potential therapeutic approach in skeletal muscle disorders displaying increased apoptosis.     

Novel markers for tying-up in horses by proteomics analysis of equine muscle biopsies

The aim of the study was to identify new biomarkers for acute tying-up in horses. Skeletal muscle biopsies were taken from 3 horses suffering from acute tying-up and 3 healthy horses. We performed 2D gel electrophoresis and mass spectrometry for identification of proteins that are differentially expressed in tying-up. 2D gel electrophoresis of skeletal muscle sequential extracts yielded more than 350 protein spots on each gel, of which 14 were differentially expressed more than two-fold (p < 0.05). In-gel digestion followed by peptide mass fingerprinting enabled identification of three significantly increased proteins: alpha actin, tropomyosin alpha chain and creatine kinase M chain (CKM). CKM was represented by multiple spots probably due to posttranslational modification, one of which appeared to be unique for tying-up. Since changes in the rates of synthesis and degradation of proteins are likely to lead to pathological conditions, identification of differentially expressed proteins in acute tying-up might result in the finding of more specific diagnostic markers and in new hypotheses for the common mechanisms that result in this condition. Our findings point to a specific isoform of CKM as a novel biomarker for tying-up suggesting that altered energy distribution within muscle cells is part of the disease etiology.     

Wheat quality related differential expressions of albumins and globulins revealed by two-dimensional difference gel electrophoresis (2-D DIGE)

Comparative proteomics analysis offers a new approach to identify differential proteins among different wheat genotypes and developmental stages. In this study, the non-prolamin expression profiles during grain development of two common or bread wheat cultivars (Triticum aestivum L.), Jing 411 and Sunstate, with different quality properties were analyzed using two-dimensional difference gel electrophoresis (2-D DIGE). Five grain developmental stages during the post-anthesis period were sampled corresponding to the cumulative averages of daily temperatures (°C: 156 °C, 250 °C, 354 °C, 447 °C and 749.5 °C). More than 400 differential protein spots detected at one or more of the developmental stages of the two cultivars were monitored, among which 230 proteins were identified by MS. Of the identified proteins, more than 85% were enzymes possessing different physiological functions. A total of 36 differential proteins were characterized between the two varieties, which are likely to be related to wheat quality attributes. About one quarter of the proteins identified expressed in multiple spots with different pIs and molecular masses, implying certain post-translational modifications (PTMs) of proteins such as phosphorylations and glycosylations. The results provide new insights into biochemical mechanisms for grain development and quality.     

Combining suppressive subtractive hybridization and cDNA microarrays to identify dietary phosphorus-responsive genes of the rainbow trout (Oncorhynchus mykiss) kidney

Phosphorus (P)-responsive genes and how they regulate renal adaptation to phosphorous-deficient diets in animals, including fish, are not well understood. RNA abundance profiling using cDNA microarrays is an efficient approach to study nutrient–gene interactions and identify these dietary P-responsive genes. To test the hypothesis that dietary P-responsive genes are differentially expressed in fish fed varying P levels, rainbow trout were fed a practical high-P diet (R20: 0.96% P) or a low-P diet (R0: 0.38% P) for 7 weeks. The differentially-expressed genes between dietary groups were identified and compared from the kidney by combining suppressive subtractive hybridization (SSH) with cDNA microarray analysis. A number of genes were confirmed by real-time PCR, and correlated with plasma and bone P concentrations. Approximately 54 genes were identified as potential dietary P-responsive after 7 weeks on a diet deficient in P according to cDNA microarray analysis. Of 18 selected genes, 13 genes were confirmed to be P-responsive at 7 weeks by real-time PCR analysis, including: iNOS, cytochrome b, cytochrome c oxidase subunit II , α-globin I, β-globin, ATP synthase, hyperosmotic protein 21, COL1A3, Nkef, NDPK, glucose phosphate isomerase 1, Na+/H+ exchange protein and GDP dissociation inhibitor 2. Many of these dietary P-responsive genes responded in a moderate way (R0/R20 ratio: < 2–3 or > 0.5) and in a transient manner to dietary P limitation. In summary, renal adaptation to dietary P deficiency in trout involves changes in the expression of several genes, suggesting a profile of metabolic stress, since many of these differentially-expressed candidates are associated with the cellular adaptative responses.     

S-allyl cysteine inhibits TNFα-induced skeletal muscle wasting through suppressing proteolysis and expression of inflammatory molecules

Background

Elevated levels of inflammatory molecules are key players in muscle wasting/atrophy leading to human morbidity. TNFα is a well-known pro-inflammatory cytokine implicated in the pathogenesis of muscle wasting under diverse clinical settings. S-allyl cysteine (SAC), an active component of garlic (Allium sativum), has established anti-oxidant and anti-inflammatory effects in various cell types. However, the impact of SAC on skeletal muscle pathology remains unexplored. Owing to the known anti-inflammatory properties of SAC, we investigated whether pre-treatment with SAC has a protective role in TNFα-induced atrophy in cultured myotubes.

Strategies for maintaining Na⁺ balance in zebrafish (Danio rerio) during prolonged exposure to acidic water

Teleost fish store lipids among several tissues primarily as triacylglycerol (TG). Upon metabolic demand, stored TGs are hydrolyzed by hormone-sensitive lipase (HSL). In this study, two distinct cDNAs encoding HSL were isolated, cloned, and sequenced from adipose tissue of rainbow trout. The full-length cDNAs, designated HSL1 and HSL2, were 2562-bp and 2887-bp in length, respectively, and share 82% nucleotide identity. Phylogentic analysis suggests that the two HSLs derive from paralogous genes that may have arisen during a teleost-specific genome duplication event. Quantitative real-time PCR revealed that HSL1 and HSL2 were differentially expressed, both in terms of distribution among tissues as well as in terms of abundance within selected tissues of juvenile trout. HSL1 and HSL2 mRNAs were detected in the brain, spleen, pancreas, kidney, gill, intestine, heart, and white muscle, but were most abundant in the red muscle, liver, and adipose tissue. HSL1 mRNA was more abundant than HSL2 mRNA in the adipose tissue, whereas HSL2 mRNA was more abundant than HSL1 mRNA in the liver. Short term fasting (4 weeks) increased HSL1 and HSL2 mRNA expression in the adipose tissue, but only HSL1 mRNA levels increased in the liver and the red muscle. During a prolonged fast (6 weeks), there was continued elevation of HSL1 and HSL2 mRNA levels in the liver and muscle; HSL mRNA expression in mesenteric fat declined, coincident with depletion of mesenteric fat mass. Refeeding fish reduced HSL expression to levels seen in continuously fed fish. These findings indicate that the pattern of HSL expression is consistent with the diverse lipid storage pattern of fish and suggest that distinct mechanisms serve to regulate differential expression of the two HSLs in tissues and during a progressive fast.