Anne M. Karinch

Glutamine Metabolism in Sepsis and Infection

Severe infection causes marked derangements in the flow of glutamine among organs, and these changes are accompanied by significant alterations in regional cell membrane transport and intracellular glutamine metabolism. Skeletal muscle, the major repository of glutamine, exhibits a twofold increase in glutamine release during infection, which is associated with a significant increase in endogenous glutamine biosynthesis. Despite an increase in glutamine synthetase activity in skeletal muscle, the intracellular glutamine pool becomes depleted, indicating that release rates exceed rates of synthesis. Simultaneously, the circulating pool of glutamine does not increase, indicating accelerated uptake by other organs. The liver appears to be the major organ of glutamine uptake in severe infection; studies in endotoxemic rodents have shown net hepatic glutamine uptake to increase by as much as 8- to 10-fold. This increase is due partially to increases in liver blood flow, but also to a three- to fourfold increase in hepatocyte System N activity in the liver. Cytokines and glucocorticoids mediate the increased uptake of glutamine by the liver in septic states as well as other compounds. Sepsis does not appear to induce an increase in System N gene expression, indicating that the increase in hepatic glutamine transport observed during severe infection is probably regulated at the protein level. The bowel displays a decrease in glutamine utilization during sepsis, a response that may be related to the decrease in circulating insulin-like growth factor-1 (IGF-1) levels that is characteristic of sepsis. Recent studies suggest that IGF-1 has a direct effect on stimulating glutamine transport across the gut lumen and thus may represent a therapeutic avenue for improving gut nutrition during severe infection. The cells of the immune system (lymphocytes, macrophages) are also major glutamine consumers during inflammatory states in which cell proliferation is increased. Under these conditions, glutamine availability can become rate limiting for key cell functions, such as phagocytosis and antibody production.

Branched-Chain Amino Acid-Enriched Nutritional Support in Surgical and Cancer Patients

Prolonged surgical stress and advanced malignant disease lead to systemic catabolism characterized by depletion of muscle protein and oxidation of skeletal muscle BCAA. BCAA oxidation provides energy for muscle and other organs and is the precursor for amino acid synthesis to replenish alanine and glutamine depleted in catabolic states. Persistent excessive catabolism leads to skeletal muscle wasting, negative nitrogen balance, and immune compromise. BCAAs, especially leucine, stimulate protein synthesis, inhibit proteolysis (in cell culture models and in animals), and promote glutamine synthesis. A number of small and diverse clinical trials studied the effects of BCAA-enriched nutritional support in moderately to severely stressed surgical and cancer patients. The findings of these clinical trials have been inconsistent; some show improved nitrogen balance, increased skeletal muscle protein synthesis, and reduced skeletal muscle catabolism whereas others show no significant improvement. The value of these trials is compromised by small sample size, heterogeneous patients, poor study design, varying degrees of metabolic stress, and inappropriate endpoints. More recent trials that evaluate clinical outcomes in hepatocellular carcinoma patients show promising results; in addition to improving metabolic parameters, BCAA-enriched oral supplementation improved morbidity and quality of life in patients undergoing major liver resection and chemo-embolization. In summary, the role of BCAAs in the nutritional support of stressed surgical and cancer patients remains to be clearly defined, despite their potential beneficial biological properties.

Purification and characterization of eukaryotic translational initiation factor eIF-2B from liver.

Eukaryotic initiation factor (eIF)-2B was purified to greater than 95% homogeneity from both rat and bovine liver. The purified protein consisted of five nonidentical subunits with apparent molecular weights ranging from 30.9 to 89.1 kDa. The holoprotein was characterized in terms of its Stokes radius and frictional coefficient. The isoelectric points for the beta-, gamma-, and epsilon-subunits were found to be 6.4, 6.9, and approximately 6.0, respectively; the alpha- and delta-subunits did not focus well because their isoelectric points as predicted by the nucleotide sequences of cDNAs for the two proteins are greater than 8.5. The purified protein was used as antigen to generate monoclonal antibodies to the epsilon-subunit. The eIF-2B epsilon monoclonal antibodies and monoclonal antibodies to the alpha-subunit of eIF-2 were then used to directly quantitate the amounts of eIF-2B and eIF-2 in rat liver and rat reticulocytes. The ratio of eIF-2B to eIF-2 was found to be approx. 0.6 and 0.3 in liver and reticulocytes, respectively, supporting the proposition that phosphorylation of only part of the total cellular eIF-2 could potentially sequester all of the eIF-2B into an inactive eIF-2.eIF-2B complex. The purified protein was also used as substrate in protein kinase assays. Extracts of rat liver were shown to contain protein kinase activity directed toward the epsilon-subunit, but no other subunit of eIF-2B. Overall, the studies presented here are the first to show a direct quantitation of eIF-2 and eIF-2B in different tissues. They also provide evidence that the epsilon-subunit of eIF-2B is the only subunit of eIF-2B that is phosphorylated by protein kinase(s) present in extracts of rat liver.

Acute and chronic ethanol consumption differentially impact pathways limiting hepatic protein synthesis

This review identifies the various pathways responsible for modulating hepatic protein synthesis following acute and chronic alcohol intoxication and describes the mechanism(s) responsible for these changes. Alcohol intoxication induces a defect in global protein synthetic rates that is localized to impaired translation of mRNA at the level of peptide-chain initiation. Translation initiation is regulated at two steps: formation of the 43S preinitiation complex [controlled by eukaryotic initiation factors 2 (eIF2) and 2B (eIF2B)] and the binding of mRNA to the 40S ribosome (controlled by the eIF4F complex). To date, alcohol-induced alterations in eIF2 and eIF2B content and activity are best investigated. Ethanol decreases eIF2B activity when ingested either acutely or chronically. The reduced eIF2B activity most likely is a consequence of twofold increased phosphorylation of the alpha-subunit of eIF2 on Ser(51) following acute intoxication. The increase in eIF2alpha phosphorylation after chronic alcohol consumption is the same as that induced by acute ethanol intoxication, and protein synthesis is not further reduced by long-term alcohol ingestion despite additional reduced expression of initiation factors and elongation factors. eIF2alpha phosphorylation alone appears sufficient to maximally inhibit hepatic protein synthesis. Indeed, pretreatment with Salubrinal, an inhibitor of eIF2alpha(P) phosphatase, before ethanol treatment does not further inhibit protein synthesis or increase eIF2alpha phosphorylation, suggesting that acute ethanol intoxication causes maximal eIF2alpha phosphorylation elevation and hepatic protein synthesis inhibition. Ethanol-induced inhibition of hepatic protein synthesis is not rapidly reversed by cessation of ethanol consumption. In conclusion, sustained eIF2alpha phosphorylation is a hallmark of excessive alcohol intake leading to inhibition of protein synthesis. Enhanced phosphorylation of eIF2alpha represents a unique response of liver to alcohol intoxication, because the ethanol-induced elevation of eIF2alpha(P) is not observed in skeletal muscle or heart.

Epidermal growth factor activation of intestinal glutamine transport is mediated by mitogen-activated protein kinases

Glutamine is an essential nutrient for gut functions, but the regulation of its uptake by intestinal mucosal cells is poorly understood. Given the pivotal role of epidermal growth factor (EGF) in regulating gut metabolism, growth, and differentiation, this in vitro study was designed to investigate the intracellular signaling pathways involved in the regulation of EGF-mediated intestinal glutamine transport in intestinal epithelia. Continuous incubation with EGF (>30 hours, 100 ng/ml) stimulated glutamine transport activity across intestinal epithelial Caco-2 cell apical membrane. Exposure to EGF for 48 hours resulted in an increase in transport activity (50%) and glutamine transport system B gene ATB0 mRNA levels (ninefold). EGF stimulated glutamine transport activity by increasing the glutamine transporter maximal velocity (Vmax) without altering the transporter apparent affinity (Km). Furthermore, EGF stimulated both intracellular protein kinase C and mitogen-activated protein kinase MEK1/2 activities. The EGF-stimulated glutamine transport activity was attenuated individually by the specific protein kinase C inhibitor chelerythrine chloride and the mitogen-activated protein kinase MEK1 inhibitor PD 98059. These data suggest that EGF activates glutamine transport activity across intestinal epithelial membrane via a signaling mechanism that involves activation of protein kinase C and the mitogen-activated protein kinase MEK1/2 cascade. EGF activates glutamine transport via alterations in transporter mRNA levels and the number of functional copies of transporter units.

Activation of Intestinal Arginine Transport by Protein Kinase C Is Mediated by Mitogen-Activated Protein Kinases

L-Arginine uptake by the small intestine can play a pivotal role in regulating nitric oxide synthesis and immune functions in catabolic states. We previously showed that protein kinase C (PKC) activation stimulates intestinal brush-border membrane arginine transport. However, the signaling pathways implicated in this activation have not been studied. The purpose of this study was to investigate the intracellular signal transduction pathways involved in the protein kinase C stimulation of arginine transport across the apical membrane of intestinal epithelial Caco-2 cells. [3H]-L-arginine transport activity, Northern blot analysis of mRNA levels of the intestinal arginine transporter CAT1,and Western blot analysis of the mitogen-activated protein (MAP) kinases phospho-p44/42 activity and phospho-MEK1/2 were measured in cultured Caco-2 cells treated with phorbol ester (phorbol 12-myristate 13-acetate, TPA; 0 to 0.5_mol/L), and the MEK1 inhibitor PD 98059 (0 to 50_ mol/L). Phorbol ester stimulated intestinal arginine transport activity. Arginine transporter gene CAT1 mRNA, phospho-p44/42, and phospho-MEK1/2 levels were stimulated in phorbol ester-treated cells, compared with the control group. Phorbol ester stimulation of arginine transport activity and transporter CAT1 mRNA levels was blocked by PD 98059. These data suggest that phorbol ester stimulates arginine transport in Caco-2 cells via signaling pathways that lead to increased transcription and/or stabilization of CAT1 mRNA. Protein kinase C and MAP kinases MEK1/2 and p44/42 are key intracellular regulators involved in this signal transduction cascade.

Metabolic acidosis stimulates intestinal glutamine absorption.

Glutamine is an essential nutrient for cell integrity during acidotic states such as shock, but the effect of extracellular pH on intestinal mucosal cell glutamine uptake is poorly understood. The purpose of this in vitro study was to investigate the intracellular signaling pathways involved in controlling intestinal glutamine transport during acidosis. Lowering the pH in the cell culture medium resulted in an increase in glutamine transport activity in a time- and pH-dependent fashion. Chronic acidosis (pH 6.6 for 48 hours) resulted in a twofold increase in glutamine transport activity (1.63 ± 0.25 nmole/mg protein/ minute in acidosis vs. 0.78 ± 0.11 nmole/mg protein/minute in control) and a threefold increase in glutamine transport gene ATB messenger RNA levels. This acidosis-induced increase in glutamine transport activity was due to a stimulation of transporter maximal transport capacity (Vmax 13.6 ± 0.73 nmole/mg protein/minute in acidosis vs. 6.3 ± 0.46 nmole/mg protein/minute in control) rather than a change in transporter affinity (Km = 0.23 ± 0.02 mmol/L glutamine in acidosis vs. 0.19 ± 0.02 mmol/ L glutamine in control). This acidosis-stimulated glutamine transport activity was blocked by actinomycin-D or cycloheximide. Cellular mitogen-activated protein kinase (MAPK) MEK1/2 and p42/44 levels were elevated in acidotic cells, and the acidosis-induced glutamine transport activity was blocked by the MAPK MEK 1 inhibitor PD 98059. Acidosis stimulates glutamine transport in Caco-2 cells via signaling pathways that lead to transcription of the glutamine transporter gene and translation of functional transporters. Mitogen-activated protein kinases are key intracellular regulators involved in this signal transduction cascade. An increased availability of glutamine to cells subjected to redox stress may help in maintaining cellular integrity.

Mesolimbic expression of neurotensin and neurotensin receptor during stress-induced gastric mucosal injury

Neurotensin is a neurotransmitter present in the brain and gastrointestinal tract. Intracerebroventricular injection of neurotensin protects rats from gastric mucosal injury caused by cold water restraint (CWR). Direct injection of neurotensin into the nucleus accumbens (NACB), part of the mesolimbic dopamine system, reduces gastric mucosal injury, suggesting that neurotensin confers protection on the mucosa through interaction with the mesolimbic system. The hypothesis is that the concentration of neurotensin in the mesolimbic system decreases during CWR, affecting the expression of neurotensin and the neurotensin receptor. After 1 h of CWR, neurotensin concentration significantly decreased 41% in the NACB and returned toward control concentrations after 2 h of CWR. The concentration of neurotensin mRNA significantly decreased 46% after 1 h CWR and returned toward control after 2 h. In contrast, neurotensin binding sites in the NACB increased from 159 to 228 fmol/mg protein after 1 h of CWR and increased significantly to 280 fmol/mg protein after 2 h CWR, whereas the level of neurotensin receptor mRNA significantly decreased 51 and 50% at 1 and 2 h, respectively. These studies show that neurotensin concentration within the mesolimbic system is transiently reduced by CWR stress and that the number of neurotensin binding sites increases, presumably in response to the decrease in neurotensin.Neurotensin is a neurotransmitter present in the brain and gastrointestinal tract. Intracerebroventricular injection of neurotensin protects rats from gastric mucosal injury caused by cold water restraint (CWR). Direct injection of neurotensin into the nucleus accumbens (NACB), part of the mesolimbic dopamine system, reduces gastric mucosal injury, suggesting that neurotensin confers protection on the mucosa through interaction with the mesolimbic system. The hypothesis is that the concentration of neurotensin in the mesolimbic system decreases during CWR, affecting the expression of neurotensin and the neurotensin receptor. After 1 h of CWR, neurotensin concentration significantly decreased 41% in the NACB and returned toward control concentrations after 2 h of CWR. The concentration of neurotensin mRNA significantly decreased 46% after 1 h CWR and returned toward control after 2 h. In contrast, neurotensin binding sites in the NACB increased from 159 to 228 fmol/mg protein after 1 h of CWR and increased significantly to 280 fmol/mg protein after 2 h CWR, whereas the level of neurotensin receptor mRNA significantly decreased 51 and 50% at 1 and 2 h, respectively. These studies show that neurotensin concentration within the mesolimbic system is transiently reduced by CWR stress and that the number of neurotensin binding sites increases, presumably in response to the decrease in neurotensin.

Regulation of amino acid arginine transport by lipopolysaccharide and nitric oxide in intestinal epithelial IEC-6 cells

As a precursor for nitric oxide (NO) synthesis and an immune-enhancing nutrient, amino acid L-arginine plays a critical role in maintaining intestine mucosal integrity and immune functions in sepsis. However, the relationship between intestinal arginine transport and NO synthesis in sepsis remains unclear. In the present study, we investigated the effects of lipopolysaccharide (LPS) and NO on the arginine transport in cultured rat intestinal epithelial IEC-6 cell. Near-confluent IEC-6 cells were incubated with LPS (0-50 μg/ml) in serum-free Dulbecco’s modified Eagles’s medium, in the presence and absence of the NO donor sodium nitroprusside (SNP, 0–500 μmol/L) and the inducible nitric oxide synthase (iNOS) inhibitor N-ω-nitro-L-arginine (NNA, 0–1000 μmol/L) for various periods of time (0-48 hours). Arginine transport activity, arginine transporter CAT1 mRNA and protein levels were measured with transport assay, Northern blot analysis, and Western blot analysis, respectfully. LPS increased arginine transport activity in a time- and dose-dependent fashion. Prolonged incubation of LPS (24 hours, 25 μg/ml) resulted in a 3-fold increase of arginine transport activity (control: 28 ±5; LPS: 92 ±20 pmol/mg/ min, P < 0.05), with the System y+ as the predominant arginine transport system, and a 2-fold increase of System y+CAT1 mRNA and transporter protein levels (P < 0.05). LPS increased the arginine transport System y+ maximal velocity (Vmax, control: 1484 ±180; LPS: 2800 ±230 pmol/mg/min, P<0.05) without affecting the transport affinity (Km, control: 76 ±8; LPS: 84 ±14 μmol/L, p = NS). The LPSinduced arginine transport activity was blocked by sodium nitroprusside (SNP) (control: 25 ±6; LPS: 97 ±26*; SNP: 22 ±0.4+; LPS+SNP: 33 ±10.3+ pmole/mg/min, *P < 0.01 and +p = NS, compared with control). In contrary, the LPS-induced arginine transport activity was further augmented by NNA (control: 18 ±3.2; LPS: 59 ±2.7*; NNA: 26.3 ±5.8; LPS + NNA: 127 ±18+ pmol/mg/min; *P < 0.01 compared with control and +P < 0.01 compared with control or LPS). LPS-stimulates arginine transport activity in IEC-6 cells via a mechanism that involves increase of transport System y+ mRNA levels and transporter protein levels. The LPS-stimulated arginine transport activity is regulated by the availability of nitric oxide.

Epidermal growth factor regulation of system L alanine transport in undifferentiated and differentiated intestinal Caco-2 cells

Epidermal growth factor (EGF) in intestinal lumen regulates many gut epithelial cell functions. Influenced by growth factors at various differentiation stages, enterocytes execute the major task of absorbing nutrient amino acids. The purpose of this study was to investigate the effects of EGF on Na+-independent L-alanine transport in intestinal epithelial cells. Na+-independent [3H]-L-alanine transport was measured in the differentiating Caco-2 cells. In both the undifferentiated and differentiated states, L-alanine uptake occurred via a single saturable Na+-independent system L plus simple passive diffusion. System L activity decreased as the cells progressed from the undifferentiated to the differentiated state. Prolonged incubation with EGF (>30 hours) resulted in a 70% increase in system L activity in both undifferentiated and differentiated cells. EGF stimulated the system L Vmax without affecting Km. System L activity stimulation was inhibited by chelerythrine chloride, cycloheximide, or actinomycin D. These data suggest that intestinal epithelial cell differentiation is associated with a decrease in system L transport capacity. EGF activates system L transport activity through a signaling mechanism involving protein kinase C, independent of cell differentiation state. Both cell differentiation and EGF regulation of system L activity occur via alteration of functional copies of the system L transporter.