J Appl Physiol. 2005 Sep.
Downregulation of diaphragm electron transport chain and glycolytic enzyme gene expression in sepsis.
Callahan LA, Supinski GS.
Div. of Pulmonary and Critical Care Medicine, 1120 15th St., Rm. BBR-5513, Medical College of Georgia, Augusta, GA 30912-3135, USA.
[ PubMed ] [ Free full text ]

Abstract: Cellular energy metabolism is altered in sepsis as a consequence of dysfunction of mitochondrial electron transport and glycolytic pathways. The purpose of the present study was to determine whether sepsis is associated with compensatory increases in gene expression of electron transport chain and glycolytic pathway proteins or, alternatively, whether gene expression decreases in sepsis, contributing to abnormalities in energy metabolism. Studies were performed using diaphragms from control and endotoxin-treated (8 mg x kg(-1) x day(-1)) rats; at 48 h after endotoxin administration, animals were killed. Microarrays and RNAse protection assays were used to assess the expression of several electron transport chain components (cytochrome-c oxidase subunits Cox 5A, Cox 5B, and Cox 6A, ATP synthase, and ATP synthase subunit 5B) and of the rate-limiting enzyme for glycolysis, phosphofructokinase (PFK). Western blotting was used to assess protein levels for these electron transport chain subunits and PFK. Activity assays were used to assess electron transport chain and phosphofructokinase function. We found that sepsis evoked 1) a downregulation of genes encoding all examined electron transport chain components (e.g., cytochrome-c oxidase 5A decreased 45 + 7%, P < 0.01) and PFK (P < 0.001), 2) reductions in protein levels for these electron transport chain subunits and PFK (P < 0.05 for each), and 3) decreases in mitochondrial state 3 respiration rates and phosphofructokinase enzyme activity (P < 0.01 for each comparison). We speculate that these sepsis-induced reductions in the expression of genes encoding critical electron transport and glycolytic proteins contribute to the development and persistence of sepsis-induced abnormalities in cellular energy metabolism.

Excerpts from the full text article:

The sepsis syndrome is commonly associated with the development of severe abnormalities in tissue oxygen utilization and acid-base balance (7, 24). Specifically, peripheral tissue oxygen extraction is often reduced, with the paradoxical finding of an elevated venous oxygen level in the presence of markedly increased tissue lactic acid generation. It has been argued that these disturbances are due, at least in part, to alterations in microvascular blood flow distribution, which result in hyperperfusion of some tissue areas and underperfusion of others (12). Recent work suggests, however, that intrinsic alterations in tissue metabolism may be the major factor limiting cellular oxygen utilization in this syndrome (2, 5, 10). These studies have suggested that alterations in metabolic processes that impact cellular ATP generation (i.e., mitochondrial dysfunction, alterations in glycolytic pathway activity) contribute to abnormalities in tissue oxygen utilization and tissue function in sepsis (1, 4, 13, 25, 28). In fact, one recent report found significant derangements in skeletal muscle mitochondrial function in many critically ill intensive care unit patients with sepsis, with poor mitochondrial function a strong predictor of multiorgan failure and death in this patient population (2). There has been little study of the effect of sepsis on the expression of genes encoding for enzymes involved in energy metabolism. In theory, altered metabolic pathway gene expression in this syndrome could have several effects. On the one hand, expression of genes encoding proteins inhibited, damaged, or depleted in the sepsis syndrome might increase, enabling compensatory production of new enzymatic proteins involved in electron transport and glycolysis, thereby assisting in recovery from sepsis. Alternatively, expression of genes encoding proteins involved in metabolism could decrease, reducing the functional activity of key metabolic enzymes.

The purpose of the present study was to examine the effect of sepsis on expression of the gene encoding for skeletal muscle phosphofructokinase (PFK), a rate-limiting enzyme of the glycolytic pathway, and representative genes expressing electron transport chain components. As a first step, we used microarrays to compare expression of these genes in diaphragm samples taken from control animals and animals receiving endotoxin for 48 h. We next used an RNAse protection assay to validate the microarray results. We then measured cellular levels of the proteins encoded by these particular genes. Finally, we assessed PFK activity and electron transport chain functional activity for diaphragm samples from control and endotoxin-treated animals and correlated alterations in mRNA levels with physiological capacity.

Methods

[...]

Results

Microarray determination of gene expression for samples form control and endotoxin-treated animals. Figure 1 displays typical Clontech microarrays generated with RNA isolated from diaphragm muscle samples from control and endotoxin-treated animals. Further analysis of array data was carried out by using AtlasImage 1.5 Software (Clontech) and was focused on genes encoding proteins involved in intermediary metabolism. This microarray includes assessment of five genes coding proteins that are components of the mitochondrial electron transport chain; results of the analysis of mRNA for these particular electron transport chain genes are shown in Fig. 2. Endotoxin administration induced reductions in the expression of all mitochondrial genes included in the microarray as follows: 1) 33 ± 10% reduction in Cox 5B (P < 0.05), 2) 23 ± 7% reduction in Cox 6A (P < 0.05), 3) 45 ± 7% reduction in Cox 5A (P < 0.005), 4) 56 ± 12% reduction in A5H (P < 0.01), and 5) 26 ± 8% reduction in A5B (P < 0.05).

We also assessed expression of PFK, the rate-limiting enzyme in glycolysis. Endotoxin administration resulted in large reductions in phosphofructokinase expression, as shown in Figs. 1 and 2 (expression of this gene was 77 ± 6% lower for samples from endotoxin-treated animals compared with the controls, P < 0.001).

RNAse protection assay results. To validate the findings of the microarray, we performed an RNAse protection assay examining PFK expression using this alternative methodology. As a control, 28S ribosomal RNA levels were also assessed when performing this assay. For this determination, we also examined the time course of expression of this particular gene to determine how soon after endotoxin administration expression declined. PFK expression remained relatively constant over the first 24 h but fell dramatically at the 36- and 48 -h time points after endotoxin administration, as shown for representative samples in Fig. 3. Mean RNAse expression data are shown in Fig. 4; on average, PFK expression decreased by 87 ± 11%, P < 0.001 (this correlated well with the 77% decrease noted in the microarray analysis). Levels of the control, 28S ribosomal RNA, were unchanged across the lanes, indicating equal loading of all lanes.

PFK and electron transport chain subunit protein levels. Representative Western blots for determination of protein levels of PFK and five electron transport chain subunits from control and endotoxin-treated diaphragm samples are presented in Fig. 5. Endotoxin administration was associated with an appreciable reduction in diaphragm protein levels for both PFK and all five electron transport chain protein subunits (Cox 5A, Cox 5B, Cox 6A, A5H, A5B) in these blots. Levels of a loading control protein (actin) were not different between control and endotoxin samples. Group mean data for protein determinations are displayed in Fig. 6. Mean protein levels declined significantly for both PFK and all electron transport chain proteins tested, falling 25–59% below levels observed for samples from control animals (P < 0.05 for comparison of control to endotoxin treated for each protein tested). Overall, endotoxin-induced reductions in protein levels for electron transport chain components (37%) were roughly comparable to reductions in mRNA levels for this group of genes (39%). On the other hand, the decrease in PFK protein level (36%) was smaller than the observed reduction in PFK mRNA level (77%).

Functional analysis of enzyme activities. Endotoxin administration elicited significant reductions in both mitochondrial respiration rate and PFK functional activity as shown in Table 1 and Fig. 7. Mitochondrial oxidative phosphorylation, assessed by measurement of state 3 respiration, declined by 48% in response to endotoxin administration (Table 1, P < 0.01). This decline in state 3 respiration rate was not associated with an increase in state 4 or a reduction in the amount of ADP utilized per nanoatom O consumed (ADP/O ratio). We also found that PFK functional activity decreased by an average of 27% in response to endotoxin administration (Fig. 7, P < 0.008).

Discussion

The sepsis syndrome is associated with the development of significant alterations in tissue metabolic function. Clinically, patients with this syndrome often demonstrate increased lactic acid generation and other evidence of tissue hypoxia despite the presence of a hyperdynamic circulation with a normal to increased level of cardiac output (7, 24). This contrasts with other clinical conditions, such as cardiogenic and hypovolemic shock, in which lactic acidosis develops as a consequence of reduced tissue perfusion secondary to reductions in cardiac output. This apparent paradox has lead to the concept that sepsis represents a state of "cytopathic hypoxia" in which the anaerobic pathways of ATP generation are recruited in tissues even though bulk delivery of blood and oxygen to these tissues remains adequate (2, 7, 8).

Several processes may contribute to the development of these metabolic derangements, including alterations in intermediary metabolism that limit ATP generation (1, 4, 12). A number of sepsis-induced alterations in cellular energy pathways have been described, with reports of sepsis-induced alterations in pyruvate dehydrogenase activity (13, 28), reductions in Krebs cycle enzyme function (27), inhibition of electron flow along the electron transport chain (4), uncoupling of oxidative phosphorylation (1), and impaired transport of ATP out of mitochondria by the sarcomeric creatine kinase shuttle (3). In keeping with this concept, one recent report found that the level of skeletal muscle mitochondrial impairment in critically ill patients with sepsis predicted survival, with higher degrees of mitochondrial dysfunction associated with a greater mortality (2). Various mechanisms have been proposed to explain these sepsis-induced alterations in energy pathway activities (4, 9, 13, 17). One process by which sepsis is thought to alter the function of proteins is via the generation of toxic species (e.g., free radicals, peroxynitrite, nitric oxide) that react with and alter the functional capacity of enzymes (4, 17). Sepsis also evokes cytokine-mediated activation of cell signaling pathways and resultant kinase-mediated phosphorylation of cellular enzyme systems (13).

All of the above mechanisms, however, act by altering the function of existing transport proteins and enzymes. Another manner in which metabolic function could be altered in sepsis is via an effect on gene transcription and translation, thereby increasing or decreasing synthesis of critical metabolic enzymes. There has been little work examining this issue in the past, however, with previous studies concentrating only on alterations in liver (25). Teleologically, one might expect sepsis to evoke a compensatory increase in genes encoding critical components of the metabolic machinery, thereby replacing proteins damaged by cellular toxins and allowing cells to adapt to the stress of sepsis by augmenting the cellular capacity to generate ATP aerobically (25). Alternatively, energy pathway gene expression in sepsis may decrease and may represent a pathological cellular response to cytokines. Such reductions in gene expression could make it difficult for cells to compensate for damage to energy pathway enzymes and, in the extreme, could directly cause or contribute to the energetic alterations seen in sepsis.

The present findings would suggest that the initial skeletal muscle response to sepsis is most consistent with the latter possibility, with our data indicating a significant sepsis-induced reduction in mRNA levels for genes encoding a number of important metabolic pathway enzymes. This was not part of a generalized downregulation of genes, as expression of a housekeeping gene (i.e., we examined HRPT in the present study) was not altered. Because our analysis was confined to measurement of mRNA levels for the genes examined, we cannot exclude the possibility that the effects of sepsis on mRNA levels of these genes was primarily the result of an effect to reduce mRNA stability rather than to reduce transcription rates for these genes. In either case, however, these sepsis-induced reductions in mRNA levels for PFK and several electron transport chain proteins would be expected, other factors being equal, to reduce translation rates for these particular proteins.

Reductions in PFK functional activity and electron transport chain capacity. To determine whether sepsis-induced reductions in PFK mRNA levels were associated with a physiological consequence, we also assessed PFK functional capacity using a well-described activity assay. We found the sepsis resulted in a statistically significant reduction in PFK functional activity, paralleling the sepsis-induced reductions in PFK mRNA and protein levels. To our knowledge, the present data provide the first demonstration that PFK gene expression and functional activity can acutely undergo major changes in response to a physiological stress. Chronic reductions in PFK activity are present, however, in Tarui's disease (22). PFK is the rate-limiting enzyme for glycolysis, and loss of activity of this enzyme reduces the capacity of cells to utilize glucose to generate pyruvate. Patients with Tarui's disease typically suffer from impaired exercise capacity and reduced muscle function (22). The functional reduction in PFK activity that we observed in sepsis would be expected to produce effects similar to those observed in Tarui's disease, reducing entry of glucose into metabolic pathways, ATP-generating capacity, and muscle function.

We also found reductions in state 3 respiration rates for mitochondria isolated at 48 h after the initiation of endotoxin administration. The state 3 respiration rate measures the maximal response of oxidative phosphorylation to ADP stimulation. We found that this index fell in parallel with concomitant reductions in mRNA and protein levels for several electron transport chain components. The present finding of a reduction in diaphragm skeletal muscle mitochondrial function during the development of sepsis is in keeping with several recent reports demonstrating sepsis-induced reductions in muscle mitochondrial capacity for generation of ATP (1, 4). The present report is the first, however, to show a sepsis-induced reduction in mRNA encoding skeletal muscle mitochondrial electron transport chain proteins. The fact that sepsis leads to a reduction in both the functional activity of the rate-limiting enzyme in glycolysis (PFK) and mitochondrial electron transport chain capacity indicates that sepsis is associated with a generalized reduction in the capacity of muscle to generate ATP by both aerobic and anaerobic pathways.

Potential effects of sepsis-induced metabolic alterations on diaphragm function. Numerous recent reports have indicated that significant diaphragmatic dysfunction occurs during the development of sepsis (23, 26). The magnitude of the reduction in diaphragm force-generating capacity evoked by this stress can be profound, and, in one animal study, it caused death due to respiratory failure if mechanical ventilation to support respiration was not provided (11). The mechanism by which sepsis producing alterations in the functional capacity of the diaphragm and other skeletal muscles has been the subject of much study, with a variety of subcellular sites of damage reported in animal models of sepsis. It has been suggested, moreover, that diaphragm mitochondrial dysfunction may play an important role in potentiating sepsis-induced muscle failure (1). According to this view, reductions in metabolic function may result in a shift in the equilibria between creatine phosphate, creatine, ATP, ADP, and phosphate such that higher levels of creatine, phosphate, and ADP result during periods of muscle activity. High phosphate levels have a direct and profound effect to reduce contractile protein function, shifting the force-pCa relationship of the contractile proteins (19). By this mechanism, sepsis-induced reductions in respiratory muscle high-energy phosphate compound generation could significantly reduce the functional capacity of these muscles. The present results are in keeping with this possibility, providing evidence that anaerobic as well as aerobic pathways of diaphragm energy metabolism may be compromised in sepsis and that sepsis-related alterations in gene expression may perpetuate these metabolic alterations.