Retrieval and recycling of synaptic vesicle membrane in pinched-off nerve terminals (synaptosomes).

The morphological features of pinched-off presynaptic nerve terminals (synaptosomes) from rat brain were examined with electron microscope techniques; in many experiments, an extracellular marked (horseradish peroxidase or colloidal thorium dioxide) was included in the incubation media. When incubated in physiological saline, most terminals appeared approximately spherical, and were filled with small (approximately 400-A diameter) "synaptic vesicles"; mitochondria were also present in many of the terminals. In a number of instances the region of synaptic contact, with adhering portions of the postsynaptic cell membrane and postsynaptic density, could be readily discerned. Approximately 20--30% of the terminals in our preparations exhibited clear evidence of damage, as indicated by diffuse distribution of extracellular markers in the cytoplasm; the markers appeared to be excluded from the intraterminal vesicles under these circumstances. The markers were excluded from the cytoplasm in approximately 70--80% of the terminals, which may imply that these terminals have intact plasma membranes. When the terminals were treated with depolarizing agents (veratridine or K-rich media), in the presence of Ca, many new, large (600--900-A diameter) vesicles and some coated vesicles and new vacuoles appeared. When the media contained an extracellular marker, the newly formed structures frequently were labeled with the marker. If the veratridine-depolarized terminals were subsequently treated with tetrodotoxin (to repolarize the terminals) and allowed to "recover" for 60--90 min, most of the large marker-containing vesicles disappeared, and numerous small (approximately 400-A diameter) marker-containing vesicles appeared. These observations are consistent with the idea that pinched-off presynaptic terminals contain all of the machinery necessary for vesicular exocytosis and for the retrieval and recycling of synaptic vesicle membrane. The vesicle membrane appears to be retrieval primarily in the form of large diameter vesicles which are subsequently reprocessed to form new "typical" small-diameter synaptic vesicles.

Ablation of abnormal energy expenditure by curative tumor resection.

Resting energy expenditure is abnormal in most patients with cancer and may contribute to cancer cachexia. These metabolic abnormalities may be a direct measure of tumor metabolism, or represent alterations in the size or activity of the body cell mass, or both. To unravel this pathogenesis, we prospectively studied 68 preoperative patients with cancer about to undergo curative resection by measuring resting energy expenditure before and after tumor resection. The preoperative measured resting energy expenditure was compared with expected resting energy expenditure based on Harris-Benedict resting energy expenditure predictions: 10 patients were hypometabolic (less than 90% Harris-Benedict); 35 were normometabolic (90% to 110% Harris-Benedict); and 23 were hypermetabolic (greater than 110% Harris-Benedict). Using each patient as his or her own control, resting energy expenditure normalized or remained normal following curative resection. In contrast, after palliative resection, resting energy expenditure remained hypermetabolic or significantly increased toward hypermetabolism. Tumor induces an abnormal metabolic rate, since tumor removal results in prompt normalization of resting energy expenditure. The abnormal energy expenditure of patients with cancer cannot be solely attributed to abnormal host body composition.

Alterations in exogenous substrate metabolism in sepsis.

Metabolic rates and substrate utilization patterns were evaluated by using a rate cecal ligation and perforation model. Animals that survived for 48 hours after the induction of sepsis were hypermetabolic and responded appropriately to varying exogenous substrate infusions. In contrast, animals that did not survive to 48 hours were premorbidly hypometabolic and failed to adjust their oxidation patterns in response to the exogenous substrate supply. These findings suggest the benefit of individually tailoring the supply of exogenous nutrients in critically ill patients with sepsis and of frequent reassessment of metabolic parameters, including the resting energy expenditure and respiratory quotient.

Effects of nonglucose substrates (xylitol, medium-chain triglycerides, long-chain triglycerides) and carnitine on nitrogen metabolism in stressed rats.

To evaluate the efficacy of nonglucose energy substrates in promoting nitrogen retention and survival in stressed states, two series of studies were done. In study 1, 50 rats underwent cecal ligation/perforation and subsequent infusion for 24 hr with one of four isocaloric (220 kcal/kg/day), isonitrogenous (1.4 g/N/kg/day), isovolemic regimens which differed in caloric source: Glucose (GLU) + long-chain triglycerides (LCT) (50%:50%), GLU + LCT + medium-chain triglycerides (MCT) (50%:32%:18%), GLU + LCT/Carnitine (10 mg/dl) or GLU + LCT + Xylitol (XYL) (33%:33%:33%). The nitrogen-sparing effect of GLU + LCT was not enhanced by the addition of carnitine to facilitate LCT mitochondrial uptake or by MCT to bypass carnitine-dependent transport. In contrast, relative to GLU + LCT GLU + LCT + XYL decreased urinary 3-methylhistidine (3MH) excretion (p less than 0.01), and enhanced nitrogen retention (p less than 0.01 vs GLU + LCT). For study 2, 24 male rats were anesthetized, cannulated for TPN, and given a 25% burn. They were then randomized into three dietary groups. The diets were isocaloric (103 kcal/kg/day) and isonitrogenous (2.0 g N/kg/day) but differed in nonprotein calorie source: GLU + LCT (51%:49%), GLU + Glycerol (51%:49%) and XYL + LCT (51%:49%). As in the septic animals, N balance was best with the xylitol regimen (p less than 0.01). The polyol, xylitol, appears to have a significant nitrogen sparing effect in stressed animals.

Pentobarbital improves nitrogen retention in sepsis.

Pentobarbital therapy has been associated with decreased urinary nitrogen excretion and resting energy expenditure in stressed patients. The metabolic effects of pentobarbital in sepsis were investigated in 29 well-nourished rats who underwent superior vena caval cannulation, cecal ligation, and puncture. Animals were randomly assigned to receive either a continuous infusion of 20 mg/kg/day of pentobarbital combined with parenteral nutrition (n = 13) or parenteral nutrition alone (n = 16). Both groups received isocaloric, isonitrogenous parenteral nutrition postoperatively for 24 hr. Mean nitrogen balance (+/- SEM) was better in the pentobarbital group (+169 +/- 76 mg/kg/day vs -190 +/- 66 mg/kg/day, p less than 0.01). No significant differences between the pentobarbital and control groups were noted for urinary 3-methylhistidine excretion (9 +/- 0.7 micrograms/kg/day vs 11 +/- 0.6 micrograms/kg/day, respectively) or 24 hr survival (77% vs 69%, respectively). Pentobarbital improves nitrogen retention without decreasing urinary 3-methylhistidine excretion in septic rats.

The effects of glucose and amino acids on tumor and host DNA synthesis.

Tumor-bearing animals provided with intravenous glucose and amino acids (TPN) exhibit enhanced response to S-phase-specific chemotherapeutic agents (H. M. Reynolds, J. M. Daly, B. Rowlands, S. J. Dudrick, and E. M. Copeland. Cancer 45: 3069, 1980; M. H. Torosian, J. L. Mullen, E. E. Miller, et al. J. Parenter. Enteral Nutr. 7: 337, 1983). To determine the mechanism of this response, DNA synthesis rate during starvation or a 48-hr infusion of glucose/amino acids (Glu/AA) was evaluated in tumor, liver, and terminal ileal cells of 68 rats. Tumor cells exhibited a rapid increase in DNA synthesis following the initiation of an infusion of Glu/AA. This increase was most marked after 2 hr of infusion and returned to control levels within 24 hr. Liver DNA synthesis rate increased in both starved and Glu/AA animals over 48 hr with a larger increase in animals receiving Glu/AA. Ileal DNA synthesis decreased equally in both groups. Short pulse Glu/AA produced transient increases in tumor DNA synthesis. Changes in host tissues occurred but followed a different temporal sequence. This may indicate the existence of a period of time following initiation of metabolic manipulation when tumor susceptibility to phase-specific chemotherapeutic agents will be enhanced while host tissues will be spared from increased toxicity.

Effect of propranolol on nitrogen and energy metabolism in sepsis.

Pharmacologic therapy designed to block adrenergic activity or alter hormonal milieu may modulate energy and protein metabolism in stress. The metabolic effects of propranolol (beta adrenergic receptor blocker) in sepsis was investigated in 22 well-nourished rats that underwent superior vena caval cannulation, cecal ligation, and puncture. Animals were randomly assigned to receive either a continuous infusion of 0.7 mg/day of propranolol combined with parenteral nutrition (n = 11) or parenteral nutrition alone (n = 11). Both groups received isocaloric, isonitrogenous, isovolemic, parenteral nutrition post-operatively for 24 hr. Nitrogen balance was better for the propranolol group than for the control group (+743 +/- 84 mg/kg/day versus +300 +/- 63 mg/kg/day, respectively, P less than 0.05). A significant difference between the pharmacologic therapy and control groups was noted for urinary 3-methylhistidine excretion versus control (0.99 +/- 0.08 micrograms/kg/day versus 7.5 +/- 0.37 micrograms/kg/day, respectively, P less than 0.01). Measured energy expenditure was similar for both pharmacologic therapy and control groups (149 +/- 20 kcal/kg/day versus 134 +/- 11 kcal/kg/day, respectively, P = N.S.). No statistically significant difference was demonstrated for 24-hr survival between propranolol and control groups (73 and 64%, respectively). Continuous, low-dose propranolol promotes nitrogen retention and decreases 3-methylhistidine excretion without altering energy expenditure in parenterally fed septic rats.

Comparison of glucose, LCT, and LCT plus MCT as calorie sources for parenterally nourished septic rats.

Protein and energy metabolism were investigated in acutely septic rats. Rats were made septic by cecal puncture and ligation. For the next 24 h they were given one of five parenteral formulations differing only in the nonprotein calorie source. The five calorie sources were 1) glucose, 2) long-chain triglycerides (LCT), 3) a 27:73 mixture of medium- and long-chain triglycerides (M/LCT), 4) a 50:50 mixture of glucose plus LCT, and 5) a 50:50 mixture of glucose plus the M/LCT mixture. The formulations also contained amino acids at a calorie:ratio of 165:1. The results were that N retention was greater with the glucose plus LCT mixture than with glucose alone. With this mixture, N retention increased as calorie intake increased, whereas with glucose, N retention plateaued. In spite of the high glucose load given to the glucose-only groups, there was a significant reduction in endogenous fat in peripheral depots. Lipid loss was least with the glucose plus LCT combination. The M/LCT mixture given alone or with glucose resulted in significantly lower survival rates.

Barbiturate therapy reduces nitrogen excretion in acute head injury.

The effect of pentobarbital on nitrogen and energy metabolism was evaluated in seven severely head-injured patients (Glasgow Coma Scale 4.7 +/- 1.7) within the first week postinjury. Measured energy expenditure (% of predicted) was significantly lower in the pentobarbital group (n = 4) versus control (n = 3) (76 +/- 23% versus 132 +/- 28%, respectively, p less than 0.01). Similarly, 24-hour urinary nitrogen excretion was lower for the barbiturate group compared to control (11.2 +/- 4.0 gm versus 19.5 +/- 3.3 gm, respectively, p less than 0.01). No statistical difference was noted for urinary 3-methylhistidine excretion between the barbiturate and control groups (43 +/- 12 mcg/day versus 47 +/- 14 mcg/day, respectively, p = N.S.). Barbiturate therapy decreases measured energy expenditure and reduces nitrogen excretion without significantly altering 3-methylhistidine excretion in head-injured patients. The metabolic effects of pentobarbital may enable the ability to achieve energy and nitrogen equilibrium during metabolic support of acutely head-injured patients.