[Show abstract][Hide abstract] ABSTRACT: Endoplasmic reticulum (ER) stress-induced responses are associated with the loss of insulin-producing β-cells in type 2 diabetes
mellitus. β-Cell survival during ER stress is believed to depend on decreased protein synthesis rates that are mediated via
phosphorylation of the translation initiation factor eIF2α. It is reported here that chronic ER stress correlated with increased
islet protein synthesis and apoptosis in β-cells in vivo. Paradoxically, chronic ER stress in β-cells induced an anabolic transcription program to overcome translational repression
by eIF2α phosphorylation. This program included expression of amino acid transporter and aminoacyl-tRNA synthetase genes downstream
of the stress-induced ATF4-mediated transcription program. The anabolic response was associated with increased amino acid
flux and charging of tRNAs for branched chain and aromatic amino acids (e.g. leucine and tryptophan), the levels of which are early serum indicators of diabetes. We conclude that regulation of amino
acid transport in β-cells during ER stress involves responses leading to increased protein synthesis, which can be protective
during acute stress but can lead to apoptosis during chronic stress. These studies suggest that the increased expression of
amino acid transporters in islets can serve as early diagnostic biomarkers for the development of diabetes.
Full-text · Article · May 2013 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Protein misfolding in the endoplasmic reticulum (ER) leads to cell death through PERK-mediated phosphorylation of eIF2α, although the mechanism is not understood. ChIP-seq and mRNA-seq of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), key transcription factors downstream of p-eIF2α, demonstrated that they interact to directly induce genes encoding protein synthesis and the unfolded protein response, but not apoptosis. Forced expression of ATF4 and CHOP increased protein synthesis and caused ATP depletion, oxidative stress and cell death. The increased protein synthesis and oxidative stress were necessary signals for cell death. We show that eIF2α-phosphorylation-attenuated protein synthesis, and not Atf4 mRNA translation, promotes cell survival. These results show that transcriptional induction through ATF4 and CHOP increases protein synthesis leading to oxidative stress and cell death. The findings suggest that limiting protein synthesis will be therapeutic for diseases caused by protein misfolding in the ER.
[Show abstract][Hide abstract] ABSTRACT: Protein synthesis across kingdoms involves the assembly of 70S (prokaryotes) or 80S (eukaryotes) ribosomes on the mRNAs to be translated. 70S ribosomes are protected from degradation in bacteria during stationary growth or stress conditions by forming dimers that migrate in polysome profiles as 100S complexes. Formation of ribosome dimers in Escherichia coli is mediated by proteins, namely the ribosome modulation factor (RMF), which is induced in the stationary phase of cell growth. It is reported here a similar ribosomal complex of 110S in eukaryotic cells, which forms during nutrient starvation. The dynamic nature of the 110S ribosomal complex (mammalian equivalent of the bacterial 100S) was supported by the rapid conversion into polysomes upon nutrient-refeeding via a mechanism sensitive to inhibitors of translation initiation. Several experiments were used to show that the 110S complex is a dimer of nontranslating ribosomes. Cryo-electron microscopy visualization of the 110S complex revealed that two 80S ribosomes are connected by a flexible, albeit localized, interaction. We conclude that, similarly to bacteria, rat cells contain stress-induced ribosomal dimers. The identification of ribosomal dimers in rat cells will bring new insights in our thinking of the ribosome structure and its function during the cellular response to stress conditions.
[Show abstract][Hide abstract] ABSTRACT: Induction of the transcription factor CHOP (CCAAT-binding homologous protein; GADD 153) is a critical cellular response for
the transcriptional control of endoplasmic reticulum (ER) stress-induced apoptosis. Upon nuclear translocation, CHOP upregulates
the transcription of proapoptotic factors and downregulates antiapoptotic genes. Transcriptional activation by CHOP involves
heterodimerization with other members of the basic leucine zipper transcription factor (bZIP) family. We show that the bZIP
protein C/EBPβ isoform LIP is required for nuclear translocation of CHOP during ER stress. In early ER stress, LIP undergoes
proteasomal degradation in the cytoplasmic compartment. During later ER stress, LIP binds CHOP in both cytoplasmic and nuclear
compartments and contributes to its nuclear import. By using CHOP-deficient cells and transfections of LIP-expressing vectors
in C/EBPβ−/− mouse embryonic fibroblasts (MEFs), we show that the LIP-CHOP interaction has a stabilizing role for LIP. At the same time,
CHOP uses LIP as a vehicle for nuclear import. LIP-expressing C/EBPβ−/− MEFs showed enhanced ER stress-induced apoptosis compared to C/EBPβ-null cells, a finding in agreement with the decreased
levels of Bcl-2, a known transcriptional control target of CHOP. In view of the positive effect of CHOP-LIP interaction in
mediating their proapoptotic functions, we propose this functional cooperativity as molecular symbiosis between proteins.
[Show abstract][Hide abstract] ABSTRACT: Regulation of cell volume is of great importance because persistent swelling or shrinkage leads to cell death. Tissues experience hypertonicity in both physiological (kidney medullar cells) and pathological states (hypernatremia). Hypertonicity induces an adaptive gene expression program that leads to cell volume recovery or apoptosis under persistent stress. We show that the commitment to apoptosis is controlled by phosphorylation of the translation initiation factor eIF2alpha, the master regulator of the stress response. Studies with cultured mouse fibroblasts and cortical neurons show that mutants deficient in eIF2alpha phosphorylation are protected from hypertonicity-induced apoptosis. A novel link is revealed between eIF2alpha phosphorylation and the subcellular distribution of the RNA-binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Stress-induced phosphorylation of eIF2alpha promotes apoptosis by inducing the cytoplasmic accumulation of hnRNP A1, which attenuates internal ribosome entry site-mediated translation of anti-apoptotic mRNAs, including Bcl-xL that was studied here. Hypertonic stress induced the eIF2alpha phosphorylation-independent formation of cytoplasmic stress granules (SGs, structures that harbor translationally arrested mRNAs) and the eIF2alpha phosphorylation-dependent accumulation of hnRNP A1 in SGs. The importance of hnRNP A1 was demonstrated by induction of apoptosis in eIF2alpha phosphorylation-deficient cells that express exogenous cytoplasmic hnRNP A1. We propose that eIF2alpha phosphorylation during hypertonic stress promotes apoptosis by sequestration of specific mRNAs in SGs in a process mediated by the cytoplasmic accumulation of hnRNP A1.
Full-text · Article · Mar 2010 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Whole body protein synthesis is reduced during the fed-to-fasted transition and in cases of chronic dietary restriction; however, less is known about tissue-specific alterations. We have assessed the extent to which protein synthesis in cardiac muscle responds to dietary perturbations compared with liver and skeletal muscle by applying a novel (2)H(2)O tracer method to quantify tissue-specific responses of protein synthesis in vivo. We hypothesized that protein synthesis in cardiac muscle would be unaffected by acute fasting or food restriction, whereas protein synthesis in the liver and gastrocnemius muscle would be reduced when there is a protein-energy deficit. We found that, although protein synthesis in liver and gastrocnemius muscle was significantly reduced by acute fasting, there were no changes in protein synthesis in the left ventricle of the heart for either the total protein pool or in isolated mitochondrial or cytosolic compartments. Likewise, a chronic reduction in calorie intake, induced by food restriction, did not affect protein synthesis in the heart, whereas protein synthesis in skeletal muscle and liver was decreased. The later observations are supported by changes in the phosphorylation state of two critical mediators of protein synthesis (4E-BP1 and eIF2alpha) in the respective tissues. We conclude that cardiac protein synthesis is maintained in cases of nutritional perturbations, in strong contrast to liver and gastrocnemius muscle, where protein synthesis is decreased by acute fasting or chronic food restriction.
Full-text · Article · Aug 2008 · AJP Endocrinology and Metabolism
[Show abstract][Hide abstract] ABSTRACT: Sugar consumption affects insulin release and, in hypertension, may stimulate cardiac signaling mechanisms that accelerate left ventricular hypertrophy and the development of heart failure. We investigated the effects of high-fructose or sucrose diets on ventricular function and mortality in hypertensive Dahl salt-sensitive rats.
Rats were fed chows that were either high starch (70% starch, 10% fat by energy), high fat (20% carbohydrates, 60% fat), high fructose (61% fructose, 9% starch, 10% fat), or high sucrose (61% sucrose, 9% starch, 10% fat). Hypertension was induced by adding 6% salt to the chow (n = 8-11/group).
After 8 weeks of treatment, systolic blood pressure and left ventricular mass were similarly increased in all rats that were fed high-salt diets. Hypertension caused a switch in mRNA myosin heavy chain isoform from alpha to beta, and this effect was greater in the high-salt sucrose and fructose groups than in starch and fat groups. The cardiac mRNA for atrial natriuretic factor was also increased in all high-salt groups compared to respective controls, with the increase being significantly greater in the hypertensive sucrose fed group. Mortality was greater in the sucrose group (44%) compared to all the other hypertensive groups (12-18%), as was cardiomyocyte apoptosis. Left ventricular ejection fraction was lower in the high-salt sucrose group, which was due to an increase in end-systolic volume, and not increased end-diastolic volume.
Diets high in sugar accelerated cardiac systolic dysfunction and mortality in hypertension compared to either a low-carbohydrate/high-fat or high-starch diet.
Full-text · Article · Aug 2008 · Journal of Hypertension
[Show abstract][Hide abstract] ABSTRACT: Inhibition of myocardial fatty acid oxidation can improve left ventricular (LV) mechanical efficiency by increasing LV power for a given rate of myocardial energy expenditure. This phenomenon has not been assessed at high workloads in nonischemic myocardium; therefore, we subjected in vivo pig hearts to a high workload for 5 min and assessed whether blocking mitochondrial fatty acid oxidation with the carnitine palmitoyltransferase-I inhibitor oxfenicine would improve LV mechanical efficiency. In addition, the cardiac content of malonyl-CoA (an endogenous inhibitor of carnitine palmitoyltransferase-I) and activity of acetyl-CoA carboxylase (which synthesizes malonyl-CoA) were assessed. Increased workload was induced by aortic constriction and dobutamine infusion, and LV efficiency was calculated from the LV pressure-volume loop and LV energy expenditure. In untreated pigs, the increase in LV power resulted in a 2.5-fold increase in fatty acid oxidation and cardiac malonyl-CoA content but did not affect the activation state of acetyl-CoA carboxylase. The activation state of the acetyl-CoA carboxylase inhibitory kinase AMP-activated protein kinase decreased by 40% with increased cardiac workload. Pretreatment with oxfenicine inhibited fatty acid oxidation by 75% and had no effect on cardiac energy expenditure but significantly increased LV power and LV efficiency (37 +/- 5% vs. 26 +/- 5%, P < 0.05) at high workload. In conclusion, 1) myocardial fatty acid oxidation increases with a short-term increase in cardiac workload, despite an increase in malonyl-CoA concentration, and 2) inhibition of fatty acid oxidation improves LV mechanical efficiency by increasing LV power without affecting cardiac energy expenditure.
Full-text · Article · Feb 2008 · AJP Heart and Circulatory Physiology
[Show abstract][Hide abstract] ABSTRACT: Chronic hypertension leads to cardiac hypertrophy, heart failure, and premature death. Little is known about the impact of dietary macronutrient composition on hypertension-induced cardiac hypertrophy and mortality. We investigated the effects of consuming either a high complex carbohydrate diet, a high simple sugar diet, or a high fat diet on cardiac hypertrophy and mortality in hypertensive Dahl salt-sensitive (DSS) rats.
Rats were assigned to four diets: complex carbohydrate (CC; 70% starch, 10% fat, 20% protein by energy), high fat (FAT; 20% carbohydrates, 60% fat, 20% protein), high fructose (FRU; 70% fructose, 10% fat, 20% protein), and "western" (WES; 35% fructose, 45% fat, 20% protein). Hypertension was initiated by adding 6% NaCl (+S) to the chow of half the animals within each diet (n = 10 to 13/group). Tail cuff blood pressure measurements were assessed after 5 and 11 weeks of treatment, and echocardiography were assessed after 12 weeks of treatment.
All rats fed a high salt diet had similar levels of hypertension (CC+S 220 +/-2 mm Hg, FAT+S 221 +/- 3 mm Hg, FRU+S 221 +/- 1 mm Hg, WES+S 226 +/- 3 mm Hg). Echocardiography results show that the addition of salt to FRU resulted in increased regional wall thickness that was not observed in other dietary groups. All rats fed a low salt diet (CC, FAT, FRU, WES) and the FAT+S group survived 90 days. On the other hand, there was 90-day mortality in the WES+S group (18% mortality) and the CC+S group (30% mortality). In addition, FRU+S rats started dying after 45 days of salt feeding, and only 15% survived the full 90 days.
These results demonstrate that a high fructose diet consumed during hypertension increases mortality and left ventricular (LV) wall thickness compared to either a high fat, high starch, or a "western" diet.
No preview · Article · May 2007 · American Journal of Hypertension
[Show abstract][Hide abstract] ABSTRACT: Steady state concentrations of ATP and ADP in vivo are similar at low and high cardiac workloads; however, the mechanisms that regulate the activation of substrate metabolism and oxidative phosphorylation that supports this stability are poorly understood. We tested the hypotheses that (1) there is parallel activation of mitochondrial and cytosolic dehydrogenases in the transition from low to high workload, which increases NADH/NAD+ ratio in both compartments, and (2) this response does not require an increase in fatty acid oxidation (FAO). Anaesthetized pigs were subjected to either sham treatment, or an abrupt increase in cardiac workload for 5 min with dobutamine infusion and aortic constriction. Myocardial oxygen consumption and FAO were increased 3- and 2-fold, respectively, but ATP and ADP concentrations did not change. NADH-generating pathways were rapidly activated in both the cytosol and mitochondria, as seen in a 40% depletion in glycogen stores, a 3.6-fold activation of pyruvate dehydrogenase, and a 50% increase in tissue NADH/NAD+. Simulations from a multicompartmental computational model of cardiac energy metabolism predicted that parallel activation of glycolysis and mitochondrial metabolism results in an increase in the NADH/NAD+ ratio in both cytosol and mitochondria. FAO was blocked by 75% in a third group of pigs, and a similar increase in and the NAHD/NAD+ ratio was observed. In conclusion, in the transition to a high cardiac workload there is rapid parallel activation of substrate oxidation that results in an increase in the NADH/NAD+ ratio.
Full-text · Article · Apr 2007 · The Journal of Physiology