Dietary Protein and Amino Acid Deficiency Inhibit Pancreatic Digestive Enzyme mRNA Translation by Multiple Mechanisms.

BACKGROUND & AIMS: Chronic amino acid (AA) deficiency, as in kwashiorkor, reduces the size of the pancreas through an effect on mammalian target of rapamycin complex 1 (mTORC1). Because of the physiological importance of AAs and their role as a substrate, a stimulant of mTORC1, and protein synthesis, we studied the effect of acute protein and AA deficiency on the response to feeding. METHODS: ICR/CD-1 mice were fasted overnight and refed for 2 hours with 4 different isocaloric diets: control (20% Prot); Protein-free (0% Prot); control (AA-based diet), and a leucine-free (No Leu). Protein synthesis, polysomal profiling, and the activation of several protein translation factors were analyzed in pancreas samples. RESULTS: All diets stimulated the Protein Kinase-B (Akt)/mTORC1 pathway, increasing the phosphorylation of the kinase Akt, the ribosomal protein S6 (S6) and the formation of the eukaryotic initiation factor 4F (eIF4F) complex. Total protein synthesis and polysome formation were inhibited in the 0% Prot and No Leu groups to a similar extent, compared with the 20% Prot group. The 0% Prot diet partially reduced the Akt/mTORC1 pathway and the activity of the guanine nucleotide exchange factor eIF2B, without affecting eIF2α phosphorylation. The No Leu diet increased the phosphorylation of eIF2α and general control nonderepressible 2, and also inhibited eIF2B activity, without affecting mTORC1. Essential and nonessential AA levels in plasma and pancreas indicated a complex regulation of their cellular transport mechanisms and their specific effect on the synthesis of digestive enzymes. CONCLUSIONS: These studies show that dietary AAs are important regulators of postprandial digestive enzyme synthesis, and their deficiency could induce pancreatic insufficiency and malnutrition.

Effect of Midmorning Puree Snacks on Subjective Appetite, Food Intake, and Glycemic and Insulin Responses in Healthy Adults.

Objective: Dietary pattern changes, as a part of a healthy lifestyle, may improve weight management. The objective of this study was to examine the effect of midmorning puree snacks varying in macronutrient composition and energy content on subjective appetite, food intake, and glycemic and insulin responses in healthy adults.Method: In a randomized, repeated measures crossover design, 6 treatments (snack skipping and purees: control [186 kcal], maltodextrin [272 kcal], whey protein [201 kcal], oat [276 kcal], and coconut oil [276 kcal]) were administered to 23 normal weight adults (n = 14 males, n = 9 females). Subjective appetite, blood glucose, and insulin responses were measured at regular intervals for 2 hours immediately followed by an ad libitum pizza lunch. In vitro digestion experiments were conducted to corroborate results of the human trial.Results: Compared to snack skipping, all snack treatments similarly reduced subjective average appetite (net area under the curve), but only oat (p < 0.032) and coconut oil (p < 0.031) purees significantly decreased test meal food intake. However, caloric compensation did not differ among snack treatments (p < 0.73). Both blood glucose (incremental area under the curve [iAUC]; p < 0.0001) and serum insulin (iAUC; p < 0.0001) were affected by treatment. A positive correlation was found between blood glucose iAUC and in vitro glucose release (r = 0.993, p < 0.0001). The release of free fatty acids (FFAs) was sustained, and oats were difficult to disintegrate during in vitro digestion.Conclusions: Compared with snack skipping, coconut oil and oat puree snacks suppressed short-term food intake, which was likely due to the sustained release of FFA and slowly digestible oats, respectively. Our in vitro digestion model predicted the relative differences in the glycemic response in vivo.

Cacao seeds are a "Super Fruit": A comparative analysis of various fruit powders and products.

BACKGROUND: Numerous popular media sources have developed lists of "Super Foods" and, more recently, "Super Fruits". Such distinctions often are based on the antioxidant capacity and content of naturally occurring compounds such as polyphenols within those whole fruits or juices of the fruit which may be linked to potential health benefits. Cocoa powder and chocolate are made from an extract of the seeds of the fruit of the Theobroma cacao tree. In this study, we compared cocoa powder and cocoa products to powders and juices derived from fruits commonly considered "Super Fruits". RESULTS: Various fruit powders and retail fruit products were obtained and analyzed for antioxidant capacity (ORAC (µM TE/g)), total polyphenol content (TP (mg/g)), and total flavanol content (TF (mg/g)). Among the various powders that were tested, cocoa powder was the most concentrated source of ORAC and TF. Similarly, dark chocolate was a significantly more concentrated source of ORAC and TF than the fruit juices. CONCLUSIONS: Cocoa powder and dark chocolate had equivalent or significantly greater ORAC, TP, and TF values compared to the other fruit powders and juices tested, respectively. Cacao seeds thus provide nutritive value beyond that derived from their macronutrient composition and appear to meet the popular media's definition of a "Super Fruit".

CCK-independent mTORC1 activation during dietary protein-induced exocrine pancreas growth.

Dietary protein can stimulate pancreatic growth in the absence of CCK release, but there is little data on the regulation of CCK-independent growth. To identify mechanisms whereby protein stimulates pancreatic growth in the absence of CCK release, C57BL/6 control and CCK-null male mice were fed normal-protein (14% casein) or high-protein (75% casein) chow for 7 days. The weight of the pancreas increased by 32% in C57BL/6 mice and 26% in CCK-null mice fed high-protein chow. Changes in pancreatic weight in control mice were due to both cell hypertrophy and hyperplasia since there was an increase in protein-to-DNA ratio, total DNA content, and DNA synthesis. In CCK-null mice pancreatic growth was almost entirely due to hypertrophy with both protein-to-DNA ratio and cell size increasing without significant increases in DNA content or DNA synthesis. ERK, calcineurin, and mammalian target of rapamycin complex 1 (mTORC1) are activated in models of CCK-induced growth, but there were no differences in ERK or calcineurin activation between fasted and fed CCK-null mice. In contrast, mTORC1 activation was increased after feeding and the duration of activation was prolonged in mice fed high-protein chow compared with normal-protein chow. Changes in pancreatic weight and RNA content were completely inhibited, and changes in protein content were partially abated, when the mTORC1 inhibitor rapamycin was administered during high-protein chow feeding. Prolonged mTORC1 activation is thus required for dietary protein-induced pancreatic growth in the absence of CCK.

Regulator of calcineurin 1 controls growth plasticity of adult pancreas.

BACKGROUND & AIMS: Growth of exocrine pancreas is regulated by gastrointestinal hormones, notably cholecystokinin (CCK). CCK-driven pancreatic growth requires calcineurin (CN), which activates Nuclear Factor of Activated T cells (NFATs), but the genetic underpinnings and feedback mechanisms that regulate this response are not known. METHODS: Pancreatic growth was stimulated by protease inhibitor (PI)-containing chow, which induces secretion of endogenous CCK. Expression profiling of PI stimulation was performed on Affymetrix 430A chips, and CN was inhibited via FK506. Exocrine pancreas-specific overexpression of CN inhibitor Regulator of Calcineurin 1 (Rcan1) was achieved by breeding elastase-Cre(estrogen receptor [ER]) transgenics with "flox-on" Rcan1 mice. RESULTS: CN inhibitor FK506 blocked expression of 38 genes, as confirmed by quantitative polymerase chain reaction. The CN-dependent genes were linked to growth-related processes, whereas their promoters were enriched in NFAT and NFAT/AP1 sites. Multiple NFAT targets, including Rcan1, Rgs2, HB-EGF, Lif, and Gem, were validated by chromatin immunoprecipitation. One of these, a CN feedback inhibitor Rcan1, was induced >50 fold during 1-8 hours course of pancreatic growth and strongly inhibited (>99%) by FK506. To examine its role in pancreatic growth, we overexpressed Rcan1 in an inducible, acinar-specific fashion. Rcan1 overexpression inhibited CN-NFAT signaling, as shown using an NFAT-luciferase reporter and quantitative polymerase chain reaction. Most importantly, the increase in exocrine pancreas size, protein/DNA content, and acinar proliferation were all blocked in Rcan1 overexpressing mice. CONCLUSIONS: We profile adaptive pancreatic growth, identify Rcan1 as an important new feedback regulator, and firmly establish that CN-NFAT signaling is required for this response.

Molecular mechanisms of pancreatic dysfunction induced by protein malnutrition.

BACKGROUND & AIMS: Dietary protein deficiency results in diminished capacity of the pancreas to secrete enzymes needed for macronutrient digestion. Previous work has suggested that modulation of the mammalian target of rapamycin (mTOR) pathway by the hormone cholecystokinin (CCK) plays an important role in normal digestive enzyme synthesis after feeding. The purpose of this study was to elucidate the role of mTOR in protein deficiency-induced pancreatic dysfunction. METHODS: Wild-type and CCK-null mice were fed protein-deficient chow for 4 days and then allowed to recover on control chow in the presence or absence of the mTOR inhibitor rapamycin. RESULTS: The size and secretory capacity of the pancreas rapidly decreased after feeding protein-deficient chow. Refeeding protein-replete chow reversed these changes in both wild-type and CCK-null mice. Changes in the size of the pancreas were paralleled by changes in the content and secretion of digestive enzymes, as well as the phosphorylation of downstream targets of mTOR. Administration of the mTOR inhibitor rapamycin decreased regrowth of the pancreas but did not affect digestive enzyme content or secretory capacity. CONCLUSIONS: These studies demonstrate that dietary protein modulates pancreatic growth, but not digestive enzyme synthesis, via CCK-independent activation of the mTOR pathway.

AMPK represses TOP mRNA translation but not global protein synthesis in liver.

The AMP-activated protein kinase (AMPK) represses signaling through the mammalian target of rapamycin complex 1 (mTORC1). In muscle, repression of mTORC1 leads to a reduction in global protein synthesis. In contrast, repression of mTORC1 in the liver has no immediate effect on global protein synthesis. In the present study, signaling through mTORC1 and translation of specific mRNAs such as those bearing a 5'-terminal oligopyrimidine (TOP) tract and were examined in rat liver following activation of AMPK after treadmill running. Activation of AMPK repressed translation of the TOP mRNAs encoding rpS6, rpS8, and eEF1alpha. In contrast, neither global protein synthesis nor translation of mRNAs encoding GAPDH or beta-actin was changed. Basal phosphorylation of the mTORC1 target 4E-BP1, but not S6K1 or rpS6, was reduced following activation of AMPK. Thus, in liver, AMPK activation repressed translation of TOP mRNAs through a mechanism distinct from downregulated phosphorylation of S6K1 or rpS6.

CCK-induced pancreatic growth is not limited by mitogenic capacity in mice.

In mice fed trypsin inhibitor (camostat) to elevate endogenous CCK, pancreatic growth plateaus by 7 days. It is unknown whether this represents the maximum growth capacity of the pancreas. To test the ability of CCK to drive further growth, mice were fed chow containing camostat (0.1%) for 1 wk, then fed standard chow for 1 wk, and finally returned to the camostat diet for a week. Pancreatic mass increased to 245% of initial value (iv) following 1 wk of camostat feeding, decreased to 147% iv following a 1 wk return to normal chow, and increased to 257% iv with subsequent camostat feeding. Camostat feeding was associated with significant increases in circulating CCK and changes in pancreatic mass were paralleled by changes in protein and DNA content. Moreover, regression of the pancreas following camostat feeding was associated with changes in the expression of the autophagosome marker LC3. Pancreatic protein synthetic rates were 130% of control after 2 days on camostat but were equivalent to control after 7 days. Changes in the phosphorylation of 4E-BP1 and S6, downstream effectors of mammalian target of rapamycin (mTOR), paralleled changes in protein synthetic rates. Cellular content of Akt, an upstream activating kinase of mTOR, decreased after 7 days of camostat feeding whereas expression of the E3 ubiquitin-ligases and the cell cycle inhibitor p21 increased after 2 days. These results indicate that CCK-stimulated growth of the pancreas is not limited by acinar cell mitogenic capacity but is due, at least in part, to inhibition of promitogenic Akt signaling.

Activation of signaling pathways and regulatory mechanisms of mRNA translation following myocardial ischemia-reperfusion.

Protein expression in the heart is altered following periods of myocardial ischemia. The changes in protein expression are associated with increased cell size that can be maladaptive. There is little information regarding the regulation of protein expression through the process of mRNA translation during ischemia and reperfusion in the heart. Therefore, the purpose of this study was to identify changes in signaling pathways and downstream regulatory mechanisms of mRNA translation in an in vivo model of myocardial ischemia and reperfusion. Hearts were collected from rats whose left main coronary arteries had either been occluded for 25 min or reversibly occluded for 25 min and subsequently reperfused for 15 min. Following reperfusion, both the phosphoinositide 3-kinase and mitogen-activated protein kinase pathways were activated, as evidenced by increased phosphorylation of Akt (PKB), extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase. Activation of Akt stimulated signaling through the protein kinase mammalian target of rapamycin, as evidenced by increased phosphorylation of two of its effectors, the ribosomal protein S6 kinase and the eukaryotic initiation factor eIF4E binding protein 1. Ischemia and reperfusion also resulted in increased phosphorylation of eIF2 and eIF2B. These changes in protein phosphorylation suggest that control of mRNA translation following ischemia and reperfusion is modulated through a number of signaling pathways and regulatory mechanisms.

Activation of the mTOR signalling pathway is required for pancreatic growth in protease-inhibitor-fed mice.

Cholecystokinin (CCK)-induced pancreatic growth in mice involves parallel increases in DNA and protein. The mammalian target of rapamycin (mTOR) signalling pathway regulates mRNA translation and its activation is implicated in growth of various tissues. The aim of this study was to elucidate whether mTOR activation is required for pancreatic growth in a mouse model of increased endogenous CCK release. In mice fed chow containing the synthetic protease inhibitor camostat, protein synthetic rates and phosphorylation of two downstream targets of mTOR, eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and the ribosomal protein S6 (S6), increased in comparison with fasted controls. The camostat-induced increases in protein synthesis and 4E-BP1 and S6 phosphorylation were almost totally abolished by administration of the mTOR inhibitor rapamycin 1 h prior to camostat feeding. In contrast, the phosphorylation of ERK1/2 and JNK and the expression of the early response genes c-jun, c-fos, ATF3 and egr-1 induced by camostat feeding were not affected by rapamycin. In mice fed camostat for 7 days, the ratio of pancreatic to body weight increased by 143%, but when rapamycin was administered daily this was reduced to a 22% increase. Changes in pancreatic mass were paralleled by protein and DNA content following camostat feeding and rapamycin administration. Moreover, while BrdU incorporation, an indicator of DNA synthesis, was increased to 448% of control values after 2 days of camostat feeding, rapamycin administration completely inhibited this increase. We conclude that the mTOR signalling pathway is required for CCK-induced cell division and pancreatic growth.

Cellular energy status modulates translational control mechanisms in ischemic-reperfused rat hearts.

Mechanisms regulating ischemia and reperfusion (I/R)-induced changes in mRNA translation in the heart are poorly defined, as are the factors that initiate these changes. Because cellular energy status affects mRNA translation under physiological conditions, it is plausible that I/R-induced changes in translation may in part be a result of altered cellular energy status. Therefore, the purpose of the studies described herein was to compare the effects of I/R with those of altered energy substrate availability on biomarkers of mRNA translation in the heart. Isolated adult rat hearts were perfused with glucose or a combination of glucose plus palmitate, and effects of I/R on various biomarkers of translation were subsequently analyzed. When compared with hearts perfused with glucose plus palmitate, hearts perfused with glucose alone exhibited increased phosphorylation of eukaryotic elongation factor (eEF)2, the alpha-subunit of eukaryotic initiation factor (eIF)2, and AMP-activated protein kinase (AMPK), and these hearts also exhibited enhanced association of eIF4E with eIF4E binding protein (4E-BP)1. Regardless of the energy substrate composition of the buffer, phosphorylation of eEF2 and AMPK was greater than control values after ischemia. Phosphorylation of eIF2alpha and eIF4E and the association of eIF4E with 4E-BP1 were also greater than control values after ischemia but only in hearts perfused with glucose plus palmitate. Reperfusion reversed the ischemia-induced increase in eEF2 phosphorylation in hearts perfused with glucose and reversed ischemia-induced changes in eIF4E, eEF2, and AMPK phosphorylation in hearts perfused with glucose plus palmitate. Because many ischemia-induced changes in mRNA translation are mimicked by the removal of a metabolic substrate under normal perfusion conditions, the results suggest that cellular energy status represents an important modulator of I/R-induced changes in mRNA translation.

Meal feeding alters translational control of gene expression in rat liver.

Meal feeding after a period of food deprivation results in a subsequent increase in the protein and RNA content of the liver. To gain insight into the mechanisms involved in the response to food intake, changes in the association of selected mRNAs with polysomes were examined. On the day of the study, rat livers were collected at 0, 15, 60, and 180 min after the start of feeding and analyzed for biomarkers of the translational control of protein synthesis. Protein synthesis was increased within 60 min and was sustained for 180 min. Assembly of the active eukaryotic initiation factor (eIF) 4F complex was elevated within 15 min, as indicated by the relative association of eIF4E . eIF4G, but returned to the basal value within 180 min. Phosphorylation of the ribosomal protein (rp) S6 kinase S6K1 and its substrate rpS6 was increased within 15 min and was sustained for at least 180 min. Both eIF4F assembly and activation of S6K1 have been linked to upregulated translation of a subset of mRNAs. To identify translationally regulated mRNAs, polysomal (i.e., actively translated) and nonpolysomal (nontranslated) fractions were isolated and subjected to microarray analysis. The mRNAs encoding 78 proteins, including 42 proteins involved in protein synthesis, exhibited increased abundance in polysomes in response to feeding. Overall, the results demonstrate that protein synthesis as well as ribosomal protein mRNA translation undergo rapid and sustained stimulation in the liver after meal feeding and thus contribute to the previously observed increases in protein and RNA content.

Oral leucine administration stimulates protein synthesis in rat skeletal muscle.

Oral administration of a single bolus of leucine in an amount equivalent to the daily intake (1.35 g/kg body wt) enhances skeletal muscle protein synthesis in food-deprived rats. To elucidate whether smaller amounts of leucine can also stimulate protein synthesis, rats were administered the amino acid at concentrations ranging from 0.068 to 1.35 g/kg body wt by oral gavage. Thirty minutes following the administration of doses of leucine as low as 0.135 g/kg body wt, skeletal muscle protein synthesis was significantly greater than control values. The increase in protein synthesis was associated with changes in the regulation of biomarkers of mRNA translation initiation as evidenced by upregulated phosphorylation of the translational repressor, eukaryotic initiation factor (eIF)4E-binding protein 1 (4E-BP1), the association of eIF4G with the mRNA cap binding protein eIF4E, and the phosphorylation of the 70-kDa ribosomal protein S6 kinase. Alterations in the phosphorylation of eIF4G, as well as the association of 4E-BP1 with eIF4E, were observed following leucine administration; however, these changes appeared to be biphasic with maximal changes occurring when circulating insulin concentrations were elevated. Thus it appears that leucine administration affects mRNA translation and skeletal muscle protein synthesis through modulation of multiple biomarkers of mRNA translation. The ability of small doses of leucine to stimulate skeletal muscle protein synthesis suggests that future research on the regulation of skeletal muscle protein synthesis by orally administered leucine will be feasible in humans.

Repression of protein synthesis and mTOR signaling in rat liver mediated by the AMPK activator aminoimidazole carboxamide ribonucleoside.

The studies described herein were designed to investigate the effects of 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR), an activator of the AMP-activated protein kinase (AMPK), on the translational control of protein synthesis and signaling through the mammalian target of rapamycin (mTOR) in rat liver. Effects of AICAR observed in vivo were compared with those obtained in an in situ perfused liver preparation to investigate activation of AMPK in the absence of accompanying changes in hormones and nutrients. AMPK became hyperphosphorylated, as assessed by a gel-shift analysis, in response to AICAR both in vivo and in situ; however, increased relative phosphorylation at the Thr172 site on the kinase was observed only in perfused liver. Phosphorylation of AMPK either in vivo or in situ was associated with a repression of protein synthesis as well as decreased phosphorylation of a number of targets of mTOR signaling including ribosomal protein S6 kinase 1, eukaryotic initiation factor (eIF)4G, and eIF4E-binding protein (4E-BP)1. The phosphorylation changes in eIF4G and 4E-BP1 were accompanied by a reduction in the amount of eIF4E present in the active eIF4E.eIF4G complex and an increase in the amount present in the inactive eIF4E.4E-BP1 complex. Reduced insulin signaling as well as differences in nutrient availability may have contributed to the effects observed in vivo as AICAR caused a fall in the serum insulin concentration. Overall, however, the results from both experimental models support a scenario in which AICAR directly represses protein synthesis and mTOR signaling in the liver through an AMPK-dependent mechanism.

Assessment of biomarkers of protein anabolism in skeletal muscle during the life span of the rat: sarcopenia despite elevated protein synthesis.

Loss of muscle strength is a principal factor in the development of physical frailty, a condition clinically associated with increased risk of bone fractures, impairments in the activities of daily living, and loss of independence in older humans. A primary determinant in the decline in muscle strength that occurs during aging is a loss of muscle mass, which could occur through a reduction in the rate of protein synthesis, an elevation in protein degradation, or a combination of both. In the present study, rates of protein synthesis and the relative expression and function of various biomarkers involved in the initiation of mRNA translation in skeletal muscle were examined at different times throughout the life span of the rat. It was found that between 1 and 6 mo of age, body weight increased fourfold. However, by 6 mo, gastrocnemius protein synthesis and RNA content per gram of muscle were lower than values observed in 1-mo-old rats. Moreover, the relative expression of two proteins involved in the binding of initiator methionyl-tRNA to the 40S ribosomal subunit, eukaryotic initiation factors (eIF)2 and eIF2B, as well as the 70-kDa ribosomal protein S6 kinase, S6K1, was lower at 6 mo compared with 1 mo of age. Muscle mass, protein synthesis, and the aforementioned biomarkers remained unchanged until approximately 21 mo. Between 21 and 24 mo of age, muscle mass decreased precipitously. Surprisingly, during this period protein synthesis, relative RNA content, eIF2B activity, relative eIF2 expression, and S6K1 phosphorylation all increased. The results are consistent with a model wherein protein synthesis is enhanced during aging in a futile attempt to maintain muscle mass.

Immediate response of mammalian target of rapamycin (mTOR)-mediated signalling following acute resistance exercise in rat skeletal muscle.

The purpose of the present investigation was to determine whether mammalian target of rapamycin (mTOR)-mediated signalling and some key regulatory proteins of translation initiation are altered in skeletal muscle during the immediate phase of recovery following acute resistance exercise. Rats were operantly conditioned to reach an illuminated bar located high on a Plexiglass cage, such that the animals completed concentric and eccentric contractions involving the hindlimb musculature. Gastrocnemius muscle was extracted immediately after acute exercise and 5, 10, 15, 30 and 60 min of recovery. Phosphorylation of protein kinase B (PKB) on Ser-473 peaked at 10 min of recovery (282% of control, P < 0.05) with no significant changes noted for mTOR phosphorylation on Ser-2448. Eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) and S6 kinase-1 (S6K1), both downstream effectors of mTOR, were altered during recovery as well. 4E-BP1 phosphorylation was significantly elevated at 10 min (292%, P < 0.01) of recovery. S6K1 phosphorylation on Thr-389 demonstrated a trend for peak activation at 10 min following exercise (336%, P = 0.06) with ribosomal protein S6 phosphorylation being maximally activated at 15 min of recovery (647%, P < 0.05). Components of the eIF4F complex were enhanced during recovery as eIF4E association with eIF4G peaked at 10 min (292%, P < 0.05). Events regulating the binding of initiator methionyl-tRNA to the 40S ribosomal subunit were assessed through eIF2B activity and eIF2 alpha phosphorylation on Ser-51. No differences were noted with either eIF2B or eIF2 alpha. Collectively, these results provide strong evidence that mTOR-mediating signalling is transiently upregulated during the immediate period following resistance exercise and this response may constitute the most proximal growth response of the cell.

Tissue-specific regulation of protein synthesis by insulin and free fatty acids.

The purpose of the study described herein was to investigate how the mammalian target of rapamycin (mTOR)-signaling pathway and eukaryotic initiation factor 2B (eIF2B) activity, both having key roles in the translational control of protein synthesis in skeletal muscle, are regulated in cardiac muscle of rats in response to two different models of altered free fatty acid (FFA) and insulin availability. Protein synthetic rates were reduced in both gastrocnemius and heart of 3-day diabetic rats. The reduction was associated with diminished mTOR-mediated signaling and eIF2B activity in the gastrocnemius but only with diminished mTOR signaling in the heart. In response to the combination of acute hypoinsulinemia and hypolipidemia induced by administration of niacin, protein synthetic rates were also diminished in both gastrocnemius and heart. The niacin-induced changes were associated with diminished mTOR signaling and eIF2B activity in the heart but only with decreased mTOR signaling in the gastrocnemius. In the heart, mTOR signaling and eIF2B activity correlated with cellular energy status and/or redox potential. Thus FFAs may contribute to the translational control of protein synthesis in the heart but not in the gastrocnemius. In contrast, insulin, but not FFAs, is required for the maintenance of protein synthesis in the gastrocnemius.

Beta -oxidation of free fatty acids is required to maintain translational control of protein synthesis in heart.

The study described herein investigated the role of free fatty acids (FFAs) in the maintenance of protein synthesis in vivo in rat cardiac and skeletal muscle. Suppression of FFA beta-oxidation by methyl palmoxirate caused a marked reduction in protein synthesis in the heart. The effect on protein synthesis was mediated in part by changes in the function of eukaryotic initiation factors (eIFs) involved in the initiation of mRNA translation. The guanine nucleotide exchange activity of eIF2B was repressed, phosphorylation of the alpha-subunit of eIF2 was enhanced, and phosphorylation of eIF4E-binding protein-1 and ribosomal protein S6 kinase was reduced. Similar changes in protein synthesis and translation initiation were not observed in the gastrocnemius following treatment with methyl palmoxirate. In heart, repressed beta-oxidation of FFA correlated, as demarcated by changes in the ATP/AMP ratio and phosphorylation of AMP-activated kinase, with alterations in the energy status of the tissue. Therefore, the activation state of signal transduction pathways that are responsive to cellular energy stress represents one mechanism whereby translation initiation may be regulated in cardiac muscle.

AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling.

AMP-activated protein kinase (AMPK) is viewed as an energy sensor that acts to modulate glucose uptake and fatty acid oxidation in skeletal muscle. Given that protein synthesis is a high energy-consuming process, it may be transiently depressed during cellular energy stress. Thus, the intent of this investigation was to examine whether AMPK activation modulates the translational control of protein synthesis in skeletal muscle. Injections of 5-aminoimidazole-4-carboxamide 1-beta-d-ribonucleoside (AICAR) were used to activate AMPK in male rats. The activity of alpha1 AMPK remained unchanged in gastrocnemius muscle from AICAR-treated animals compared with controls, whereas alpha2 AMPK activity was significantly increased (51%). AICAR treatment resulted in a reduction in protein synthesis to 45% of the control value. This depression was associated with decreased activation of protein kinases in the mammalian target of rapamycin (mTOR) signal transduction pathway as evidenced by reduced phosphorylation of protein kinase B on Ser(473), mTOR on Ser(2448), ribosomal protein S6 kinase on Thr(389), and eukaryotic initiation factor eIF4E-binding protein on Thr(37). A reduction in eIF4E associated with eIF4G to 10% of the control value was also noted. In contrast, eIF2B activity remained unchanged in response to AICAR treatment and therefore would not appear to contribute to the depression in protein synthesis. This is the first investigation to demonstrate changes in translation initiation and skeletal muscle protein synthesis in response to AMPK activation.

Contribution of insulin to the translational control of protein synthesis in skeletal muscle by leucine.

Enhanced protein synthesis in skeletal muscle after ingestion of a balanced meal in postabsorptive rats is mimicked by oral leucine administration. To assess the contribution of insulin to the protein synthetic response to leucine, food-deprived (18 h) male rats (approximately 200 g) were intravenously administered a primed-constant infusion of somatostatin (60 microg + 3 microg.kg(-1).h(-1)) or vehicle beginning 1 h before administration of leucine (1.35 g L-leucine/kg) or saline (control). Rats were killed 15, 30, 45, 60, or 120 min after leucine administration. Compared with controls, serum insulin concentrations were elevated between 15 and 45 min after leucine administration but returned to basal values by 60 min. Somatostatin maintained insulin concentrations at basal levels throughout the time course. Protein synthesis was increased between 30 and 60 min, and this effect was blocked by somatostatin. Enhanced assembly of the mRNA cap-binding complex (composed of eukaryotic initiation factors eIF4E and eIF4G) and hyperphosphorylation of the eIF4E-binding protein 1 (4E-BP1), the 70-kDa ribosomal protein S6 kinase (S6K1), and the ribosomal protein S6 (rp S6) were observed as early as 15 min and persisted for at least 60 min. Somatostatin attenuated the leucine-induced changes in 4E-BP1 and S6K1 phosphorylation and completely blocked the change in rp S6 phosphorylation but had no effect on eIF4G small middle dot eIF4E assembly. Overall, the results suggest that the leucine-induced enhancement of protein synthesis and the phosphorylation states of 4E-BP1 and S6K1 are facilitated by the transient increase in serum insulin. In contrast, assembly of the mRNA cap-binding complex occurs independently of increases in insulin and, by itself, is insufficient to stimulate rates of protein synthesis in skeletal muscle after leucine administration.