Decreased Protein Degradation Research Articles

Targeted ablation of IKK2 improves skeletal muscle strength, maintains mass, and promotes regeneration

NF-kappaB is a major pleiotropic transcription factor modulating immune, inflammatory, cell survival, and proliferative responses, yet the relevance of NF-kappaB signaling in muscle physiology and disease is less well documented. Here we show that muscle-restricted NF-kappaB inhibition in mice, through targeted deletion of the activating kinase inhibitor of NF-kappaB kinase 2 (IKK2), shifted muscle fiber distribution and improved muscle force. In response to denervation, IKK2 depletion protected against atrophy, maintaining fiber type, size, and strength, increasing protein synthesis, and decreasing protein degradation. IKK2-depleted mice with a muscle-specific transgene expressing a local Igf-1 isoform (mIgf-1) showed enhanced protection against muscle atrophy. In response to muscle damage, IKK2 depletion facilitated skeletal muscle regeneration through enhanced satellite cell activation and reduced fibrosis. Our results establish IKK2/NF-kappaB signaling as an important modulator of muscle homeostasis and suggest a combined role for IKK inhibitors and growth factors in the therapy of muscle diseases.

Optimization of Culture Conditions for Determining Protein Degradation in Myoblasts Using Extracts of Adult Bovine Muscle Treated with Muscle Enhancing Compounds

AbstractThe objective of this study was to optimize a methodology in which muscle extracts from animals treated with growth agents are added to growth media and then applied to muscle cell cultures to determine effects of growth agents on indirect cellular protein degradation. Experiments were designed as a 2 × 2 × 2 × 2 factorial arrangement of cell type (C2C12 myoblasts or primary bovine muscle cell cultures), growth medium [(Dulbecco's Modified Eagle's Medium (DMEM)or skeletal muscle cell basal medium (SKBM)], muscle extract medium [potassium phosphate buffer (KPB) or prerigor extraction buffer], and β-adrenergic agonist treatments of the bovine (control or treated) to determine differences in cellular protein degradation. There was a treatment media × β-adrenergic agonist treatment × cell type interaction (P < 0.0001). C2C12 myoblasts with the β-adrenergic agonist in DMEM media had less protein degradation than controls in DMEM (P < 0.05). Primary bovine muscle cell cultures treated with the β-adrenergic agonist treatment in DMEM media had greater protein degradation than did controls in DMEM (P < 0.05). However, primary bovine muscle cell cultures treated with the β-adrenergic agonist treatment in SKBM media had reduced protein degradation than did controls in SKBM (P < 0.05). There was a treatment media × β-adrenergic agonist treatment × muscle cell extraction buffer interaction (P = 0.04). The β-agonist treatment decreased protein degradation when muscle samples were extracted with KPB or prerigor extraction buffer and made with SKBM culture media (P < 0.05). The results indicated SKBM and C2C12 myoblasts had the most consistent differences for showing relative β-adrenergic agonist treatment effects on indirect protein degradation.

Effect of nelfinavir on insulin metabolism, proteasome activity and protein degradation in HepG2 cells

HIV-1 protease inhibitors have revolutionized the treatment of HIV infection, but their use has been associated with lipodystrophy and insulin resistance. One suggestion for this has been the inhibition of insulin-degrading enzyme (IDE). We have previously demonstrated that insulin, through IDE, can inhibit the proteasome, thus decreasing cytosolic protein degradation. We examined whether the protease inhibitor nelfinavir inhibited IDE and its effect on protein degradation both in vitro and in whole cells. 125I-Insulin degradation was measured by trichloroacetic acid precipitation. Proteasome activities were measured using fluorogenic peptide substrates. Cellular protein degradation was measured by prelabelling cells with 3H-leucine and determining the release of TCA-soluble radioactivity. Nelfinavir inhibited IDE in a concentration-dependent manner with 50% inhibition at the maximal concentration tested, 100 microm. Similarly, the chymotrypsin-like and trypsin-like activities of the proteasome were decreased with an IC50 of approximately 3 microm. The ability of insulin to inhibit the proteasome was abrogated by nelfinavir. Treatment of HepG2 cells with 50 microm nelfinavir decreased 125I-insulin degradation and increased cell-associated radioactivity. Insulin alone maximally decreased protein degradation by 15%. Addition of 50 microm nelfinavir inhibited cellular protein degradation by 14% and blunted the effect of insulin. These data show that nelfinavir inhibits IDE, decreases insulin's ability to inhibit protein degradation via the proteasome and provides another possible mechanism for the insulin resistance seen in protease inhibitor-treated HIV patients.

Proteolytic Mechanisms, Not Malnutrition, Cause Loss of Muscle Mass in Kidney Failure

Hypoalbuminemia and muscle atrophy are frequently found in patients with chronic kidney disease (CKD) and patients being treated by dialysis. These abnormalities are usually attributed to malnutrition, meaning that they are caused by an inadequate diet. However, the evidence indicates that malnutrition is rarely the mechanism causing loss of protein stores. Instead, low values of serum albumin are closely related to the presence of inflammation and loss of muscle mass is attributable to activation of specific proteases. In uremic rodents and patients, the initial step in the loss of muscle protein is an activation of caspase-3. This cleaves the complex structure of muscle, and its action can be detected by the presence of a characteristic 14-kDa actin fragment in the insoluble fraction of muscle. The second step in uremia-induced loss of muscle protein is an activation of the ubiquitin-proteasome system, which rapidly degrades proteins released by caspase-3 cleavage of muscle proteins. Activation of both caspase-3 and the ubiquitin-proteasome system occur when there is suppression of the cellular signaling pathway activated by insulin/insulinlike growth factor 1, the phosphatidylinositol 3-kinase/Akt pathway. A potential therapeutic target for preventing loss of muscle protein is to stimulate activity of this signaling pathway.

Proteome analysis of yeast response to various nutrient limitations

We compared the response of Saccharomyces cerevisiae to carbon (glucose) and nitrogen (ammonia) limitation in chemostat cultivation at the proteome level. Protein levels were differentially quantified using unlabeled and 15N metabolically labeled yeast cultures. A total of 928 proteins covering a wide range of isoelectric points, molecular weights and subcellular localizations were identified. Stringent statistical analysis identified 51 proteins upregulated in response to glucose limitation and 51 upregulated in response to ammonia limitation. Under glucose limitation, typical glucose-repressed genes encoding proteins involved in alternative carbon source utilization, fatty acids β-oxidation and oxidative phosphorylation displayed an increased protein level. Proteins upregulated in response to nitrogen limitation were mostly involved in scavenging of alternative nitrogen sources and protein degradation. Comparison of transcript and protein levels clearly showed that upregulation in response to glucose limitation was mainly transcriptionally controlled, whereas upregulation in response to nitrogen limitation was essentially controlled at the post-transcriptional level by increased translational efficiency and/or decreased protein degradation. These observations underline the need for multilevel analysis in yeast systems biology.

Effect of Insulin with Concurrent Amino Acid Infusion on Protein Metabolism in Rapidly Growing Pubertal Children with Type 1 Diabetes

Insulin treatment of prepubertal children with insulin-dependent diabetes improves body protein balance by decreasing the rate of protein degradation without stimulating protein synthesis. However, insulin also causes hypoaminoacidemia, so the inability of insulin to stimulate protein synthesis may have been limited by substrate availability. We investigated the ability of insulin to stimulate protein synthesis in growing pubertal children who were given sufficient amino acids to counter insulin-induced hypoaminoacidemia. Protein metabolism in six pubertal children with type 1 diabetes was assessed from leucine kinetics during a primed, 6-h infusion of L-[1-(13)C]leucine. The children were studied in the postabsorptive state during a basal (insulin withdrawn) period and during the infusion of 0.83 mU * kg(-1) * min(-1) human regular insulin. Amino acids and glucose were given with insulin to prevent hypoaminoacidemia and hypoglycemia. Net leucine balance was significantly higher with insulin than in the basal state, the result of decreased protein degradation but also decreased protein synthesis. The data suggest that insulin alone does not increase protein synthesis in pubertal children with type 1 diabetes.

Acetoacetate Induces CYP2E1 Protein and Suppresses CYP2E1 mRNA in Primary Cultured Rat Hepatocytes

The ketone body acetoacetate (AA) in the absence of insulin or in the presence of diabetic insulin levels decreases CYP2E1 mRNA expression in a concentration- and time-dependent manner in primary cultured rat hepatocytes. AA activates p70 ribosomal S6 kinase (p70S6K) and protein kinase C (PKC) by approximately 2- to 2.5-fold, respectively, following 6-h treatment. The AA-mediated activation of p70S6K, but not PKC, was abolished by inhibition of PI 3-K with LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] or wortmannin, in agreement with p70S6K being downstream of phosphatidylinositol 3-kinase (PI 3-K). Inhibition of PI 3-K, mTOR with rapamycin, or PKC with bisindolylmaleimide ameliorated the AA-mediated down-regulation of CYP2E1 mRNA expression. Neither the mitogen-activated protein kinase kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone) nor the p38 mitogen-activated protein kinase inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] ameliorated the AA-mediated suppression of CYP2E1 mRNA expression. Heterogeneous nuclear RNA analysis revealed that AA suppressed CYP2E1 gene transcription by approximately 50% and that inhibition of PI 3-K and PKC diminished this AA-mediated effect on transcription. CYP2E1 mRNA half-life slightly increased from approximately 24 h in untreated hepatocytes to approximately 32 h in AA-treated cells. Interestingly, AA increased CYP2E1 protein levels by approximately 2- and 2.5-fold at 24 and 48 h, respectively. DL-beta-hydroxybutyrate was without effect. Polysomal distribution studies revealed that AA increased the proportion of RNA associated with the actively translated polysomal fractions versus the 40S to 60S untranslated fractions by approximately 40%. CYP2E1 protein half-life increased from approximately 8 h in untreated hepatocytes to approximately 24 in AA-treated cells. These data show that AA decreases CYP2E1 mRNA expression through inhibition of gene transcription while simultaneously elevating CYP2E1 protein levels through increased translation and decreased protein degradation.

Glycogen Synthase Kinase 3-Beta (GSK3-β) inhibitors decrease protein degradation in L6 myotubes

Introduction. Previous reports suggest that inhibition of GSK3-β results in muscle hypertrophy. Muscle hypertrophy may result from increased protein synthesis, decreased protein degradation, or a combination of both processes. The effect of GSK3-β inhibition on protein degradation in skeletal muscle has not been reported. We tested the hypothesis that the GSK3-β inhibitors lithium chloride (LiCl), SB216763, and SB415286 decrease protein degradation in cultured L6 myotubes. Methods. Fully-differentiated, cultured L6 myotubes, a rat skeletal muscle cell line, were treated with LiCl, SB216763, or SB415286 for 24 h. Protein degradation was assessed by measuring the release of TCA-soluble 3H-tyrosine from proteins that had been pre-labeled with the amino acid. Results. Treatment of cultured myotubes with the GSK3-β inhibitors resulted in a dose-dependent decrease in protein degradation. Results in the Table show the maximal inhibition (24–30%) caused by the GSK3-β inhibitors. Conclusions. This study provides evidence that inhibition of GSK3-β reduces protein degradation in skeletal muscle cells. The hypertrophy reported previously following inhibition of GSK3-β may, at least in part, be due to a decrease in protein breakdown. TABLE—ABSTRACT P26 − Inhibitor + Inhibitor LiCl 25 mM 13.7 ± 0.2 10.0 ± 0.1 ∗ SB216763 25 μM 11.5 ± 0.3 8.8 ± 0.5 ∗ SB415286 100 μM 12.8 ± 0.3 8.9 ± 0.8 ∗ Note. Protein degradation (%/24 hours) in myotubes treated with GSK3-β inhibitors. Results are means ± SEM with n = 6 per group. ∗ P < 0.001 versus no inhibitor by ANOVA.

Changes in Serum Metabolites and Growth Characteristics of Korean Native Steers Fed Alcohol-fermented Feeds

This study was carried out to assess whether feeding of alcohol-fermented feeds (AFF) affects the nutritional metabolism and growth characteristics of Korean native steers. Ten steers were randomly assigned to one of two treatment groups. The dietary treatments were AFF (50% commercial beef cattle feed+30% alcohol-fermented soybean curd dregs+20% rice straw) and control (80% commercial beef cattle feed+20% rice straw). The change of serum metabolites and growth characteristics were measured every two months during the whole twelve months experimental period and the relationships between serum metabolites and growth characteristics were simultaneously analyzed. Four hours after feeding AFF, serum alcohol concentration reached its peak with a significantly higher value than that after control feeding (11.9 and 4.9 mg/dl, respectively). Serum glucose and inorganic phosphorus (IP) concentrations (63.1 and 8.4 mg/dl, respectively) of steers fed AFF were higher than those (56.6 and 7.0 mg/dl) fed the control diet. In both treatments, the serum glucose concentration rapidly increased when body weight (BW) of the steer reached about 600kg, while IP concentrations were rapidly diminished at that BW. Lower concentrations of both blood urea nitrogen (BUN) and cholesterol were observed in steers fed AFF up to 450 kg of BW. The IP concentration was correlated with concentrations of BUN, cholesterol and glucose in AFF fed cattle but not in the cattle fed control diets. Average daily gain was higher in steers fed AFF than steers fed control, particularly during the growing stage of cattle. These findings indicated a capability of AFF to improve BW gain of Korean native steers by decreased protein degradation as well as increased fat synthesis.

Insulin inhibition of protein degradation in cells expressing wild-type and mutant insulin receptors.

The mechanism by which insulin decreases protein degradation is unknown. We examined insulin binding and degradation (125I[A14]insulin) and protein degradation (3H-leucine labeling) in Chinese hamster ovary (CHO) cells transfected with wild-type (WI) and mutant human insulin receptors. The deltaExon-16 mutant is missing the juxtamembrane domain that mediates endocytosis. The delta343 mutant receptor lacks the tyrosine kinase structural domain but retains the juxtamembrane internalization domain. The mutant deltaNPEY lacks the single NPEY sequence located 16 residues after the end of the transmembrane domain. Null transfected cells (NEO) not expressing human receptors were studied as controls. The WT and deltaNPEY cells equivalently internalized and degraded insulin; delta343 cells internalized and degraded insulin, but at a reduced rate; deltaExon-16 cells internalized and degraded significantly less insulin than the other mutants; NEO cells showed essentially no internalization and degradation. In contrast, all cell types showed the same efficacy at inhibition of protein degradation, albeit at different potencies. These results suggest insulin actions are mediated by multiple and redundant effector systems, but that receptor tyrosine kinase activity is not required for inhibition of protein degradation.

Potential environmental benefits of ionophores in ruminant diets.

A concern of the USEPA is the volatilization of NH3 from animal manure and CH4 produced from ruminal fermentation. Excess N in the environment has been associated with adverse effects on human health, and CH4 and N2O emissions are sources of greenhouse gases. The objectives of this paper are to summarize and quantify the benefits of ionophores, principally monensin, in decreasing NH3 and CH4 emissions to the environment and reducing resource utilization in cattle (Bos spp.) production. The data indicate that monensin in the diets of ruminants may decrease protein degradation in the rumen and may increase feed protein utilization by an average of 3.5 percentage units. These changes would have an effect in reducing N losses and decreasing fecal N and the amount of protein that must be fed to meet animal requirements. Additionally, CH4 is produced by enteric fermentation in ruminants, which is responsible for about 33 to 39% of CH4 emissions from agriculture. Ionophores can reduce CH4 production by 25% and decrease feed intake by 4% without affecting animal performance. The inclusion of monensin in beef and dairy cattle diets may benefit air quality by reducing CH4 and N emissions and water quality by reducing N in manure, which can potentially leave the farm through leaching into ground water and through runoff into surface water.

Osteopontin-dependent CD44v6 expression and cell adhesion in HepG2 cells.

The interaction of osteopontin (OPN) with CD44 and alphavbeta3-integrin has been implicated in numerous signal transduction pathways that may promote cancer metastasis. CD44v6 is a splice variant of CD44 which has been identified as a marker of cancer progression. In this study, immortalized liver carcinoma cells (HepG2) were used to examine the effect of OPN on two isoforms of CD44: CD44 standard (CD44 s) and CD44v6. Western blots demonstrated that OPN up-regulated plasma membrane CD44v6 protein expression in a concentration- and time-dependent fashion. CD44v6 levels returned to control levels when OPN-alphavbeta3-integrin binding was blocked by an RGD peptide or tyrosine kinase activity was inhibited. OPN significantly increased CD44v6 protein synthesis, while simultaneously decreasing protein degradation. Steady-state mRNA levels of both CD44s and CD44v6 were unaltered in the presence of OPN stimulation. OPN increased HepG2 in vitro adhesion to hyaluronate (HA); excess soluble HA extinguished OPN-mediated HepG2 adhesion, indicating CD44 dependence. In conclusion, OPN binds to the alphavbeta3-integrin to increase plasma membrane CD44v6 expression and augment in vitro adhesion to HA. This may contribute to the mechanism by which OPN enhances metastatic behavior in hepatocellular cancer cells.

Effects of time spent on pasture and the characteristics of the concentrate on milk yield, milk composition and body reserves in the Latxa dairy ewe

In the basque country, in the spring, dairy ewes can spend a limited number of hours between 4 and 8 hours on pasture (Oregui et al., 1997). Besides grazing, ewes receive a supplementation of concentrates and forages in parlour. Therefore the characteristics of this supplementation are very important. In this sense, there is evidence that an increase in rumen undegradable protein could lead to an increase in milk production (Santos et al., 1998). In recent years, it has been shown the potential of tannins to decrease protein degradation in the rumen (Hervas. 2001). However, there is a lack of knowledge concerning the effect of increasing the content of rumen undegradable protein of the concentrate in a production system like the one in the basque country.

Lysine Needs of Starting Chicks and Subsequent Effects During the Growing Period

The lysine requirement for broiler chicks has been studied extensively. Many factors, such as protein quality, experimental conditions, dietary nutrients, and genetic strain, affect lysine needs of chicks. Because lysine increases protein synthesis and decreases protein degradation in chicks, its requirement may vary as meat-type broiler genetics are improved. Two experiments were conducted to evaluate lysine needs in the starter period in a slow maturing commercial broiler (Ross × Ross 508) and to monitor subsequent effects of lysine level on quantities of salable meat.Lysine-deficient diets (0.88% total lysine or 80% of 1994 NRC specifications) were created by blending corn, soybean meal, corn gluten meal, wheat middlings, poultry fat, and crystalline amino acids. Dietary treatments represented graduations of lysine from 80 to 130% of NRC recommendations in 10% increments from Days 1 to 18. All broilers received a common diet from Days 19 to 41 and Days 19 to 42 in Experiments 1 and 2, respectively. Quadratic response curves were fitted to growth performance criteria of chicks. Broilers fed lysine levels below NRC specifications as chicks had reduced growth and carcass attributes at Day 41. The lysine level required (95% of the upper asymptote) for chick growth responses was 107% (1.18% total lysine of diet or 1.07% digestible lysine of diet) to 111% (1.22% total lysine of diet or 1.11% digestible lysine of diet) of NRC recommendations.

Higher skeletal muscle protein synthesis and lower breakdown after chemotherapy in cachectic mice.

The influence of cancer cachexia and chemotherapy and subsequent recovery of skeletal muscle protein mass and turnover was investigated in mice. Cancer cachexia was induced using colon 26 adenocarcinoma, which is characteristic of the human condition, and can be cured with 100% efficacy using an experimental nitrosourea, cystemustine (C(6)H(12)CIN(3)O(4)S). Reduced food intake was not a factor in these studies. Three days after cachexia began, healthy and tumor-bearing mice were given a single intraperitoneal injection of cystemustine (20 mg/kg). Skeletal muscle mass in tumor-bearing mice was 41% lower (P < 0.05) than in healthy mice 2 wk after cachexia began. Skeletal muscle wasting was mediated initially by decreased protein synthesis (-38%; P < 0.05) and increased degradation (+131%; P < 0.05); later wasting resulted solely from decreased synthesis (~-54 to -69%; P < 0.05). Acute cytotoxicity of chemotherapy did not appear to have an important effect on skeletal muscle protein metabolism in either healthy or tumor-bearing mice. Recovery began 2 days after treatment; skeletal muscle mass was only 11% lower than in healthy mice 11 days after chemotherapy. Recovery of skeletal muscle mass was affected initially by decreased protein degradation (-80%; P < 0.05) and later by increased protein synthesis (+46 to +73%; P < 0.05) in cured compared with healthy mice. This study showed that skeletal muscle wasted from cancer cachexia and after chemotherapeutic treatment is able to generate a strong anabolic response by making powerful changes to protein synthesis and degradation.

Effect of a tumour-produced lipid-mobilizing factor on protein synthesis and degradation.

Treatment of murine myoblasts, myotubes and tumour cells with a tumour-produced lipid mobilizing factor (LMF), caused a concentration-dependent stimulation of protein synthesis, within a 24 h period. There was no effect on cell number or [3H] thymidine incorporation, but a similar concentration-dependent stimulation of 2-deoxyglucose uptake. LMF produced an increase in intracellular cyclic AMP levels, which was linearly (r2= 0.973) related to the increase in protein synthesis. The effect of LMF was attenuated by the adenylate cyclase inhibitor MDL 12330A, and was additive with the stimulation produced by forskolin. Both propranolol (10 μM) and the specific β3-adrenergic receptor antagonist SR 59230A (10–5M), significantly reduced the stimulation of protein synthesis induced by LMF. Protein synthesis was also increased by 69% (P = 0.006) in soleus muscles of mice administered LMF, while there was a 26% decrease in protein degradation (P = 0.03). While LMF had no effect on the lysosomal enzymes, cathepsins B and L, there was a decrease in proteasome activity, as determined both by the ‘chymotrypsin-like’ enzyme activity, as well as expression of proteasome α-type subunits, determined by Western blotting. These results show that in addition to its lipid-mobilizing activity LMF also increases protein accumulation in skeletal muscle both by an increase in protein synthesis and a decrease in protein catabolism. © 2001 Cancer Research Campaign http://www.bjcancer.com

Muscle fibre growth in undernourished reindeer calves ( Rangifer tarandus tarandus L.) during winter

To study whether moderate under-nutrition causes muscle wasting, reindeer ( Rangifer tarandus tarandus L.) calves were fed either pelleted reindeer feed ad libitum ( n=8) or restricted amounts of lichens ( n=8). The restricted amount was 60% of ad libitum intake of lichens, and the feeding period was 6 weeks preceded by a 2-week adjustment period. Biopsy samples from the middle gluteal muscle ( M. gluteus medius) for the analysis of fibre composition and area, as well as for the activity of cathepsin B were taken before the restriction period in November and January, and after the restriction period in April. In all calves the muscle fibre composition remained unchanged during the winter. In the lichen group, the fibre size also remained unchanged, whereas in control calves the cross sectional area of type I and type IIA fibres increased significantly from November to April. Cathepsin B activity decreased in all calves from November to January and remained at that low level for the rest of the study period, which suggests an attenuated rate of protein degradation. These results can be taken as an indication that moderate under-nutrition causes no muscle wasting in reindeer calves, and the decreased availability of nitrogen is partially compensated for by adaptive decrease in protein degradation. Interestingly the adaptive changes in protein metabolism are equally well seen in the well-fed controls as in the undernourished lichen-fed reindeer.

Identification of a Novel Stretch-Responsive Skeletal Muscle Gene (Smpx)

Skeletal muscle is able to respond to a range of stimuli, including stretch and increased load, by increasing in diameter and length in the absence of myofiber division. This type of cellular growth (hypertrophy) is a highly complex process involving division of muscle precursor cells (myoblasts) and their fusion to existing muscle fibers as well as increased protein synthesis and decreased protein degradation. Underlying the alterations in protein levels are increases in a range of specific mRNAs including those coding for structural proteins and proteins that regulate the hypertrophic process. Seven days of passive stretch in vivo of tibialis anterior (TA) muscle has been shown to elicit muscle hypertrophy. We have identified a cDNA corresponding to an mRNA that exhibits increased expression in response to 7 days of passive stretch imposed on TA muscles in vivo. This 944-bp novel murine transcript is expressed primarily in cardiac and skeletal muscle and to a lesser extent in brain. Translation of the transcript revealed an open reading frame of 85 amino acids encoding a nuclear localization signal and two overlapping casein kinase II phosphorylation sites. This gene has been called “small muscle protein (X chromosome)” (Smpx; HGMW-approved human gene symbol SMPX) and we hypothesize that it plays a role in skeletal muscle hypertrophy.

Advanced glycation end products impair protein turnover in LLC-PK1: Amelioration by trypsin

Advanced glycation end products (AGEs) are assumed to play a key role in the pathogenesis of diabetic nephropathy (DN) and other diabetic complications. While AGEs have been shown to exert marked effects on mesangial and endothelial cells as well as on monocytes/macrophages, little is known about their effects on tubule cells. Therefore, we addressed the questions of (1) whether AGE-bovine serum albumin (AGE-BSA) impairs the protein metabolism in the tubule cells, and if so, (2) whether the AGE-induced effects are mediated via a protease sensitive mechanism. Arrested LLC-PK1 cells were exposed to a medium containing the vehicle (control, serum free), AGE-BSA (38 micromol/L), or BSA (38 micromol/L) in the presence or absence of trypsin (2.5 microg/mL) for 24 hours. We evaluated cell number, cell size, and cell protein content, as well as protein synthesis and protein degradation. After an incubation period of 24 hours, AGE-BSA decreased the cell number to 84.5 +/- 5.5% of control and 82.5 +/- 5.6% of BSA-treated cells (P < 0.05). [3H]-thymidine incorporation declined to 66% of control (P < 0.05), while BSA was without any effect. The same AGE-BSA dose reduced protein degradation (P < 0.05) and stimulated total protein synthesis slightly, as determined by L-[14C]Phe incorporation into acidic-insoluble proteins. These effects resulted in a rise in cell protein content (AGE-BSA vs. control, 21.9 +/- 6.7%; AGE-BSA vs. BSA, 11.1 +/- 6.0%, P < 0.05) and cell volume (AGE-BSA vs. control 9.4 +/- 3.2%, AGE-BSA vs. BSA 18.4 +/- 3.7%, P < 0.05). Coincubation with AGE-BSA and trypsin was associated with an amelioration of all investigated parameters concerning cell number, cell proliferation, raised cell protein content, decreased protein degradation, and enhanced protein synthesis. These data indicate that AGE-BSA impairs cell proliferation and protein turnover in LLC-PK1 cells with a consequent rise in cell protein. Since these alterations were abrogated by coincubation with trypsin, an interference of this serine protease with the AGE-binding proteins on cell surfaces is assumed.

The leucine metabolite 3‐hydroxy‐3‐methylbutyrate (HMB) modifies protein turnover in muscles of laboratory rats and domestic chickens in vitro

This study was conducted to assess the in vitro effects of leucine and 3‐hydroxy‐3‐methylbutyrate (HMB) on protein synthesis and degradation in isolated rat and chick muscles. Isolated muscles — extensor digitorum longus (EDL) and soleus (S) in rats and EDL and tibialis anterior (TA) in chicks — were taken out intact and incubated in Krebs—Henseleit buffer with [14C]phenylalanine. Protein synthesis was then determined by measuring [14C]phenylalanine incorporation into protein and protein breakdown was estimated by net release of tyrosine from muscle proteins. In all muscles investigated leucine stimulated protein synthesis (average 20%; p &lt; 0.05 versus control) but only slightly reduced proteolysis (average 8%, NS). In contrast, HMB had no significant effect on protein synthesis but consistently decreased protein degradation in the range from −29% (p &lt; 0.01 versus control) in rat extensor digitorus longus to −5% (NS) in rat soleus muscles. HMB appeared to suppress protein degradation more in white muscle fibres than in red muscle fibres (at least in the rat). These data support observations in humans indicating that HMB decreases muscle proteolysis. It is suggested that metabolism of leucine to HMB could be responsible for the reported suppressive effects of leucine on protein breakdown.