Danuta Huber

Local insulin-like growth factor I prevents sepsis-induced muscle atrophy

The present study tests the hypotheses that local bioavailability of insulin-like growth factor I (IGF-I) is capable of regulating muscle protein balance and that muscle-directed IGF-I can selectively maintain muscle mass during bacterial infection. Initial studies in C57BL/6 mice demonstrated that increasing or decreasing bioavailable IGF-I within muscle by local administration of either Leu(24) Ala(31) IGF-I or IGF binding protein 1, respectively, produced proportional changes in surrogate markers (eg, phosphorylation of 4E-BP1 and S6K1) of protein synthesis. We next examined the ability of a sustained local administration of IGF-I to prevent sepsis-induced muscle atrophy over a 5-day period. At the time of cecal ligation and puncture or sham surgery, mice had a time-release pellet containing IGF-I implanted next to the gastrocnemius and a placebo pellet placed in the contralateral limb. Data indicated that IGF-I released locally only affected the adjacent muscle and was not released into the circulation. Gastrocnemius from septic mice containing the placebo pellet was atrophied and had a reduced IGF-I protein content. In contrast, locally directed IGF-I increased IGF-I protein within adjacent muscle to basal control levels. This change was associated with a proportional increase in muscle weight and protein, as well as increased phosphorylation of 4E-BP1 and the redistribution of eIF4E from the inactive eIF4E4EBP1 complex to the active eIF4EeIF4G complex. Local IGF-I also prevented the sepsis-induced increase in atrogin-1 messenger RNA in the exposed muscle. Finally, local IGF-I prevented the sepsis-induced increase in muscle interleukin-6 messenger RNA. Thus, muscle-directed IGF-I attenuates the sepsis-induced atrophic response apparently by increasing muscle protein synthesis and potentially decreasing proteolysis. Collectively, our data suggest that agents that increase the bioavailability of IGF-I within muscle per se might be effective in ameliorating the sepsis-induced loss of muscle mass without having undesirable effects on metabolic processes in distant organs.

Alcohol regulates eukaryotic elongation factor 2 phosphorylation via an AMP-activated protein kinase-dependent mechanism in C2C12 skeletal myocytes.

Ethanol decreases protein synthesis in cells, although the underlying regulatory mechanisms of this process are not fully established. In the present study incubation of C2C12 myocytes with 100 mm EtOH decreased protein synthesis while markedly increasing the phosphorylation of eukaryotic elongation factor 2 (eEF2), a key component of the translation machinery. Both mTOR and MEK pathways were found to play a role in regulating the effect of EtOH on eEF2 phosphorylation. Rapamycin, an inhibitor of mammalian target of rapamycin, and the MEK inhibitor PD98059 blocked the EtOH-induced phosphorylation of eEF2, whereas the p38 MAPK inhibitor SB202190 had no effect. Unexpectedly, EtOH decreased the phosphorylation and activity of the eEF2 upstream regulator eEF2 kinase. Likewise, treatment of cells with the inhibitor rottlerin did not block the stimulatory effect of EtOH on eEF2, suggesting that eEF2 kinase (eEF2K) does not play a role in regulating eEF2. In contrast, increased eEF2 phosphorylation was correlated with an increase in AMP-activated protein kinase (AMPK) phosphorylation and activity. Compound C, an inhibitor of AMPK, suppressed the effects of EtOH on eEF2 phosphorylation but had no effect on eEF2K, indicating that AMPK regulates eEF2 independent of eEF2K. Finally, EtOH decreased protein phosphatase 2A activity when either eEF2 or AMPK was used as the substrate. Thus, this later action may partially account for the increased phosphorylation of eEF2 in response to EtOH and the observed sensitivity of AMPK to rapamycin and PD98059 treatments. Collectively, the induction of eEF2 phosphorylation by EtOH is controlled by an increase in AMPK and a decrease in protein phosphatase 2A activity.

Alcohol and PRAS40 Knockdown Decrease mTOR Activity and Protein Synthesis via AMPK Signaling and Changes in mTORC1 Interaction

The mTORC1 protein kinase complex consists of mTOR, raptor, mLST8/GβL and PRAS40. Previously, we reported that mTOR plays an important role in regulating protein synthesis in response to alcohol (EtOH). However, the mechanisms by which EtOH regulates mTORC1 activity have not been established. Here, we investigated the effect of EtOH on the phosphorylation and interaction of components of mTORC1 in C2C12 myocytes. We also examined the specific role that PRAS40 plays in this process. Incubation of myocytes with EtOH (100 mM, 24 h) increased raptor and PRAS40 phosphorylation. Likewise, there were increased levels of the PRAS40 upstream regulators Akt and IRS‐1. EtOH also caused changes in mTORC1 protein–protein interactions. EtOH enhanced the binding of raptor and PRAS40 with mTOR. These alterations occurred in concert with increased binding of 14‐3‐3 to raptor, while the PRAS40 and 14‐3‐3 interaction was not affected. The shRNA knockdown (KD) of PRAS40 decreased protein synthesis similarly to EtOH. PRAS40 KD increased raptor phosphorylation and its association with 14‐3‐3, whereas decreased GβL–mTOR binding. The effects of EtOH and PRAS40 KD were mediated by AMPK. Both factors increased in vitro AMPK activity towards the substrate raptor. In addition, KD enhanced the activity of AMPK towards TSC2. Collectively, our results indicate that EtOH stabilizes the association of raptor, PRAS40, and GβL with mTOR, while likewise increasing the interaction of raptor with 14‐3‐3. These data suggest a possible mechanism for the inhibitory effects of EtOH on mTOR kinase activity and protein synthesis in myocytes. J. Cell. Biochem. 109: 1172–1184, 2010.

Regulation of REDD1 by insulin‐like growth factor‐I in skeletal muscle and myotubes

Insulin‐like growth factor‐I (IGF‐I) is a major anabolic hormone for skeletal muscle and a potent stimulus for protein synthesis and translation initiation. Recent studies suggest that translation can be inhibited by over expression of the mammalian target of rapamycin (mTOR) repressor REDD1. The purpose of the present study was to determine whether IGF‐I alters the expression of REDD1 and whether this is associated with a concomitant change in protein synthesis in vitro. Subcutaneous injection of IGF‐I or intravenous delivery of insulin for 3–4 h increased REDD1 mRNA in skeletal muscle 7–10‐fold. A threefold increase in REDD1 was observed when C2C12 myotubes were treated with IGF‐I. REDD1 protein continued to be expressed for up to 24 h after addition of IGF‐I to cells. Withdrawal of IGF‐I from myotubes lead to a rapid loss of REDD1 protein content. IGF‐I‐induced REDD1 mRNA and protein expression were prevented by inhibitors of transcription and translation. IGF‐I had an additive effect with dexamethasone (Dex) on REDD1 protein content in myotubes. The PI3K inhibitor LY294002 blocked IGF‐I but not Dex induced REDD1. IGF‐I also stimulated REDD1 promoter activity. Although REDD1 protein was elevated 5–6 h after addition of IGF‐I to myotubes, protein synthesis measured during this 1 h window was paradoxically greater in myotubes expressing more REDD1. In contrast to the IGF‐I induced increase in REDD1 mRNA, REDD2 mRNA was decreased by IGF‐I. We conclude that IGF‐I stimulates REDD1 expression in skeletal muscle and myotubes but under these conditions the REDD1 response is not sufficient to repress protein synthesis. J. Cell. Biochem. 108: 1192–1202, 2009.

Castration Differentially Alters Basal and Leucine-Stimulated Tissue Protein Synthesis in Skeletal Muscle and Adipose Tissue

Reduced testosterone as a result of catabolic illness or aging is associated with loss of muscle and increased adiposity. We hypothesized that these changes in body composition occur because of altered rates of protein synthesis under basal and nutrient-stimulated conditions that are tissue specific. The present study investigated such mechanisms in castrated male rats (75% reduction in testosterone) with demonstrated glucose intolerance. Over 9 wk, castration impaired body weight gain, which resulted from a reduced lean body mass and preferential sparing of adipose tissue. Castration decreased gastrocnemius weight, but this atrophy was not associated with reduced basal muscle protein synthesis or differences in plasma IGF-I, insulin, or individual amino acids. However, oral leucine failed to normally stimulate muscle protein synthesis in castrated rats. In addition, castration-induced atrophy was associated with increased 3-methylhistidine excretion and in vitro-determined ubiquitin proteasome activity in skeletal muscle, changes that were associated with decreased atrogin-1 or MuRF1 mRNA expression. Castration decreased heart and kidney weight without reducing protein synthesis and did not alter either cardiac output or glomerular filtration. In contradistinction, the weight of the retroperitoneal fat depot was increased in castrated rats. This increase was associated with an elevated rate of basal protein synthesis, which was unresponsive to leucine stimulation. Castration also decreased whole body fat oxidation. Castration increased TNFα, IL-1α, IL-6, and NOS2 mRNA in fat but not muscle. In summary, the castration-induced muscle wasting results from an increased muscle protein breakdown and the inability of leucine to stimulate protein synthesis, whereas the expansion of the retroperitoneal fat depot appears mediated in part by an increased basal rate of protein synthesis-associated increased inflammatory cytokine expression.

Alcohol-Induced Modulation of Rictor and mTORC2 Activity in C2C12 Myoblasts

BACKGROUND The mammalian target of rapamycin (mTOR) kinase controls cell growth, proliferation, and metabolism through 2 distinct multiprotein complexes, mTORC1 and mTORC2. We reported that alcohol (EtOH) inhibits mTORC1 activity and protein synthesis in C2C12 myoblasts. However, the role that mTORC2 plays in this process has not been elucidated. In this study, we investigated whether mTORC2 functions as part of a feedback regulator in response to EtOH, acting to maintain the balance between the functions of Akt, mTORC2, and mTORC1. METHODS C2C12 myoblasts were incubated with EtOH for 18 to 24 hours. Levels of various mTORC2 proteins and mRNA were assessed by immunoblotting and real-time PCR, respectively, while protein-protein interactions were determined by immunoprecipitation and immunoblotting. An in vitro mTORC2 kinase activity assay was performed using Akt as a substrate. The rate of protein synthesis was determined by (35) S-methionine/cysteine incorporation into cellular protein. RESULTS EtOH (100 mM) increased the protein and mRNA levels of the mTORC2 components rictor, mSin1, proline-rich repeat protein 5, and Deptor. There was also an increased association of these proteins with mTOR. EtOH increased the in vitro kinase activity of mTORC2, and this was correlated with decreased binding of rictor with 14-3-3 and Deptor. Reduced rictor phosphorylation at T1135 by EtOH was most likely due to decreased S6K1 activity. Knockdown of rictor elevated mTORC1 activity, as indicated by increased S6K1 phosphorylation and protein synthesis. Likewise, there were decreased amounts and/or phosphorylation levels of various mTORC1 and mTORC2 components including raptor, proline-rich Akt substrate 40 kDa, mSin1, Deptor, and GβL. Activated PP2A was associated with decreased Akt and eukaryotic elongation factor 2 phosphorylation. Collectively, our results provide evidence of a homeostatic balance between the 2 mTOR complexes following EtOH treatments in myoblasts. CONCLUSIONS   EtOH increased the activity of mTORC2 by elevating levels of various components and their interaction with mTOR. Decreased rictor phosphorylation at T1135 acts as mTORC1-dependent feedback mechanisms, functioning in addition to the insulin receptor substrate-I/PI3K signaling pathway to regulate protein synthesis.

Lopinavir Impairs Protein Synthesis and Induces eEF2 Phosphorylation via the Activation of AMP-Activated Protein Kinase

HIV anti‐retroviral drugs decrease protein synthesis, although the underlying regulatory mechanisms of this process are not fully established. Therefore, we investigated the effects of the HIV protease inhibitor lopinavir (LPV) on protein metabolism. We also characterized the mechanisms that mediate the effects of this drug on elongation factor‐2 (eEF2), a key component of the translational machinery. Treatment of C2C12 myocytes with LPV produced a dose‐dependent inhibitory effect on protein synthesis. This effect was observed at 15 min and was maintained for at least 4 h. Mechanistically, LPV increased the phosphorylation of eEF2 and thereby decreased the activity of this protein. Increased phosphorylation of eEF2 was associated with increased activity of its upstream regulators AMP‐activated protein kinase (AMPK) and eEF2 kinase (eEF2K). Both AMPK and eEF2K directly phosphorylated eEF2 in an in vitro kinase assay suggesting two distinct paths lead to eEF2 phosphorylation. To verify this connection, myocytes were treated with the AMPK inhibitor compound C. Compound C blocked eEF2K and eEF2 phosphorylation, demonstrating that LPV affects eEF2 activity via an AMPK‐eEF2K dependent pathway. In contrast, incubation of myocytes with rottlerin suppressed eEF2K, but not eEF2 phosphorylation, suggesting that eEF2 can be regulated independent of eEF2K. Finally, LPV did not affect PP2A activity when either eEF2 or peptide was used as the substrate. Collectively, these results indicate that LPV decreases protein synthesis, at least in part, via inhibition of eEF2. This appears regulated by AMPK which can act directly on eEF2 or indirectly via the action of eEF2K. J. Cell. Biochem. 105: 814–823, 2008.