Thomas R. Austgen

The effects of endotoxin on the splanchnic metabolism of glutamine and related substrates

The effects of endotoxemia on glutamine metabolism by the splanchnic bed was studied in vivo in adult rats 12 hours following administration of E. coli LPS. Glutamine uptake by the portal-drained viscera fell in the endotoxin-treated animals while glucose uptake doubled. Simultaneously, hepatic glutamine uptake increased ten-fold owing to both an increase in hepatic blood flow and glutamine extraction from the bloodstream. Hepatic alanine uptake, oxygen consumption, and glucose release were also accelerated in endotoxemic rats. The increase in liver glutamine utilization was associated with increases in parenchymal DNA and glutathione levels and an increase in glutathione and urea release into the systemic circulation. This marked increase in metabolic activity occurred in animals with chemical evidence of hepatocellular injury and histologic evidence of hepatocyte necrosis. During endotoxemia the liver becomes the major organ of glutamine consumption. This accelerated utilization provides carbons for energy and gluconeogenesis, nitrogen for ureagenesis, and substrate for nucleotide and glutathione biosynthesis in order to support cell repair and detoxification reactions.

Adaptive Regulation in Skeletal Muscle Glutamine Metabolism in Endotoxin-Treated Rats

The effects of a single dose of endotoxin (7.5 mg/kg BW) on skeletal muscle glutamine metabolism were studied in vivo in rats to gain further understanding of the altered glutamine metabolism that characterizes sepsis and other catabolic diseases. In endotoxin-treated animals the arterial glutamine concentration fell early initially and then increased compared with control values. Twelve hours after treatment, the arteriovenous concentration difference for glutamine across the hindquarter doubled, resulting in a significant increase in net muscle glutamine release in endotoxin-treated rats. As a consequence, the muscle glutamine concentration fell in the endotoxin-treated animals by 25%-40%, an event that was apparent as early as two hours after endotoxin treatment. Skeletal muscle glutaminase activity, the major enzyme of glutamine breakdown, was unchanged by endotoxemia, but expression of glutamine synthetase mRNA and glutamine synthetase specific activity increased in a time-dependent fashion. The glutamine depletion that develops in skeletal muscle during endotoxemia is caused by accelerated muscle glutamine release rather than an increase in intracellular degradation or a fall in intracellular biosynthesis. The adaptive increase in glutamine synthetase expression that occurs requires de novo RNA and protein synthesis and may be designed to prevent complete depletion of the intracellular glutamine pool.

Cytokine regulation of intestinal glutamine utilization

The effects of cytokines on intestinal glutamine metabolism were studied to gain further insight into the regulation of altered glutamine metabolism that occurs during severe infection. One hundred thirteen adult rats were given a single dose of interleukin-1 (IL-1, 50 micrograms/kg), tumor necrosis factor (TNF, 50 micrograms/kg or 150 micrograms/kg), or saline (controls), and flux studies were performed 4 or 12 hours later. Intestinal blood flow was not different between control and cytokine-treated animals at either time point. At the 4-hour time point, arterial glutamine fell by 16% to 21% in the cytokine-treated animals (p less than 0.05); at the 12-hour time point, the arterial glutamine concentration had returned to normal. Intestinal glutamine extraction decreased in the animals treated with IL-1 at both time points (4 hours: 13% +/- 1.3% in IL-1 versus 20% +/- 1.6% in controls, p less than 0.05; and 12 hours: 9% +/- 2% in IL-1 versus 17% +/- 2% in controls, p less than 0.05). Consequently, net intestinal glutamine uptake fell in the animals treated with IL-1 at both time points (p less than 0.05). Similarly, the activity of mucosal glutaminase, the principal enzyme of glutamine hydrolysis in the gut, fell by 50% in the 4-hour study (6.1 +/- 0.6 mumol/h/mg protein in IL-1 versus 9.6 +/- 0.8 mumol/h/mg protein in controls, p less than 0.01) and by 40% in the 12-hour study (5.4 +/- 0.5 mumol/h/mg protein in IL-1 versus 8.8 +/- 0.4 mumol/h/mg protein in controls, p less than 0.05). Concomitant with the aforementioned decrease in gut glutamine metabolism was a 25% incidence of positive blood cultures for gram-negative organisms in IL-1 treated rats studied at the 12-hour time point (p = 0.05 versus controls). In the doses administered and at the time points studied, TNF had no effects on the parameters of gut glutamine metabolism examined. The results indicate that IL-1 is a potential mediator of the alterations in gut glutamine metabolism observed in sepsis and endotoxemia.

Tumor regulation of hepatic glutamine metabolism.

Fast-growing tumors are major glutamine consumers and may alter host glutamine metabolism to benefit the tumor. Previous studies from our laboratory have demonstrated that the liver switches from an organ of glutamine balance to one of glutamine release with progressive malignant growth. However, the regulation of this change is unclear. This study examined tumor modulation of hepatic glutamine metabolism by determining the activities of glutaminase, the principle enzyme of glutamine degradation, and glutamine synthetase, the principal enzyme of glutamine synthesis. Hepatic glutamine content was also determined. Rats with a fast-growing subcutaneous fibrosarcoma (TBR) and pair-fed controls were studied at 2 and 3 weeks after tumor or sham implantation, when the tumors comprised approximately 5% and 20% of total body weight. Arterial glutamine fell with progressive tumor growth (608 +/- 26 mumol/L in controls vs 494 +/- 15 in TBR, p less than 0.005) and was not attributable to a diminished food intake. Hepatic glutamine content was increased 45% (p less than 0.01) in tumor rats at 2 weeks due in part to a 35% fall in liver glutaminase activity. At 3 weeks, glutamine synthetase activity increased by 43% (0.58 +/- 0.07 mumol/mg of protein/hr in controls vs 0.83 +/- 0.04 in TBR, p less than 0.01) whereas glutaminase remained depressed (2.68 +/- 0.12 mumol/mg of protein/hr in controls vs 2.22 +/- 0.15 in TBR, p less than 0.05) and glutamine content fell compared to 2 week tumor-bearing rats, consistent with accelerated hepatic glutamine release. Tumors may alter liver glutamine metabolism by modulating hepatic enzyme activity in order to provide circulating glutamine for the growing malignancy.

Role of the lungs in maintaining amino acid homeostasis.

The relative contributions of skeletal muscle and the pulmonary bed in maintaining amino acid homeostasis were studied. Inasmuch as more than 60% of whole blood amino acid nitrogen is transported as glutamine and alanine, the flux of these two amino acids across the lungs (n = 20) and hindquarter (n = 20) was determined in the postabsorptive adult rat. Both skeletal muscle and the lungs released net amounts of glutamine and alanine in the postabsorptive state. Blood flow to the hindquarter was approximately 16% of cardiac output (3.8 +/- 0.3 cc/100 g BW/min), while pulmonary blood flow (cardiac output) was 23.7 +/- 1.7 cc/100 g BW/min. Thus, despite a lower glutamine concentration difference across the lungs (-32 +/- 6 mumol/liter) compared with the hindquarter (-59 +/- 10 mumol/liter (p less than 0.01), the lungs released significantly more glutamine (741 +/- 142 nmol/100 g BW/min) than the hindquarter (208 +/- 39 nmol/100 g BW/min) (p less than 0.01) because of the significantly higher pulmonary blood flow. Similarly, the concentration difference for alanine across the lungs was less than that of the hindquarter (-24 +/- 8 mumol/liter vs -60 +/- 12 mumol/liter, p less than 0.01) but the lungs released significantly more alanine than the hindquarter (553 +/- 159 nmol/100 g BW/min vs 221 +/- 41 nmol/100 g BW, p less than 0.01. Compositional studies demonstrated that the hindquarter comprises 40% of total body muscle mass in the rat; thus both total skeletal muscle mass and the lungs contribute approximately equally to the maintenance of blood glutamine and alanine levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of increased hepatic glutamine uptake in the endotoxin-treated rat

The mechanisms underlying the accelerated hepatic consumption of glutamine that occurs during endotoxemia were investigated in rats 12 hr after treatment with Escherichia coli lipopolysaccharide. Hepatic glutamine delivery and consumption were calculated from measurements of hepatic blood flow and blood glutamine levels. Hepatic glutaminase activity and glutamine and glutamate content were determined. Hepatocyte plasma membrane transport activity was evaluated employing isolated hepatic plasma membrane vesicles (HPMVs). Endotoxin treatment resulted in an 11-fold increase in hepatic glutamine consumption and a 2-fold increase in the delivered load of glutamine to the liver. Hepatic glutamate content doubled while glutamine content was unaffected, not withstanding a decrease in the specific activity of glutaminase. Studies employing HPMVs demonstrated that hepatic plasma membrane transport activity was unaffected by endotoxin treatment. The enhanced hepatic consumption of glutamine secondary to endotoxemia appears to be the result of both a mass-action effect and the concurrent activation of intracellular metabolism. Responses at the level of plasma membrane transport do not appear to play an active role in mediating this enhanced hepatic uptake.

Reoperation for Colorectal Carcinoma

In the management of the patient with intra-abdominal recurrence of colorectal carcinoma, surgery remains the primary mode of therapy when cure or significant palliation is anticipated. Appreciation of the importance of close follow-up after primary resection coupled with improved diagnostic modalities has allowed the surgeon not only to detect earlier recurrence but also to select the patients most likely to benefit from resection of recurrent disease. Improved surgical techniques with resultant decreases in the rates of morbidity and mortality have allowed safe hepatic resection of metastatic disease. In selected patients, this procedure produces 5-year survival rates approaching 50%. Although a clear consensus has not been reached, most studies agree that positive prognostic indicators include absence of extrahepatic disease, a small number of intrahepatic lesions, a low CEA level, and a better Dukes stage of the primary. Likewise, in the patient with recurrent disease locally, surgery provides the only means of cure and also plays a significant role in palliation. Aggressive resection with generous surgical margins in patients with contained disease may yield 5-year survival rates approaching 35%. In patients with unresectable disease and even in those with carcinomatosis, palliation can be obtained by surgical therapy. Judgment is necessary in treating these patients both preoperatively and intraoperatively. Surgical intervention for obstruction, perforation, or other anatomic or physiological compromise is often indicated and can improve the quality of life of the patient with intra-abdominal recurrence.

Simple Method of Determining Pulmonary Blood Flow in the Anesthetized Rat

We have developed a simple, accurate, and relatively inexpensive method of measuring pulmonary blood flow in the anesthetized rat using a modification of the dye dilution technique. The method is attractive because it allows for the measurement of pulmonary blood flow and also the determination of pulmonary substrate flux. Using rats with a catheter in the carotid artery and a doubly cannulated right ventricle (RV), a constant infusion of [14C]P-aminohippurate ([14C]PAH) is begun via the distal RV catheter. After steady state is obtained the infusion of [14C]PAH is then stopped and blood is immediately withdrawn from the proximal RV cannula. This maneuver insures that [14C]PAH from the infusate is not sampled through the proximal RV catheter. Obtaining the blood from the proximal RV catheter within 5 s after clearing the infusion catheter insures that no dilution of [14C]PAH in the right ventricle (from recirculating blood) occurs. Catheter position is verified at autopsy. Calculations are performed to determine pulmonary blood flow. In 20 normal rats studied, the pulmonary blood flow was 24 +/- 1 mL/100 g body wt min-1 and in 12 endotoxin-treated rats (10 mg/kg body wt) the pulmonary blood flow was 32 +/- 2 mL/100 g body wt. These values are similar to values obtained with other methods used to measure total pulmonary blood flow.