Harold A. Neufeld

Role of the Liver in Regulation of Ketone Body Production during Sepsis

During caloric deprivation, the septic host may fail to develop ketonemia as an adaptation to starvation. Because the plasma ketone body concentration is a function of the ratio of hepatic production and peripheral usage, a pneumococcal sepsis model was used in rats to measure the complex metabolic events that could account for this failure, including the effects of infection on lipolysis and esterification in adipose tissue, fatty acid transport in plasma and the rates of hepatic ketogenesis and whole body oxidation of ketones. Some of the studies were repeated with tularemia as the model infection. From these studies, it was concluded that during pneumococcal sepsis, the failure of rats to become ketonemic during caloric deprivation was the result of reduced ketogenic capacity of the liver and a possibly decreased hepatic supply of fatty acids. The latter appeared to be a secondary consequence of a severe reduction in circulating plasma albumin, the major transport protein for fatty acids, with no effect on the degree of saturation of the albumin with free fatty acids. Also, the infection had no significant effect on the rate of lipolysis or release of fatty acids from adipose tissue. Ketone body usage (oxidation) was either unaffected or reduced during pneumococcal sepsis in rats. Thus, a reduced rate of ketone production in the infected host was primarily responsible for the failure to develop starvation ketonemia under these conditions. The liver of the infected rat host appears to shuttle the fatty acids away from beta-oxidation and ketogenesis and toward triglyceride production, with resulting hepatocellular fatty metamorphosis.

The effect of bacterial infections on ketone concentrations in rat liver and blood and on free fatty acid concentrations in rat blood.

The concentrations of cytoplasmic lactate and pyruvate and the NAD+/NADH ratio and the concentrations of mitochondrial acetoacetate, beta-hydroxybutyrate, and the NAD+/NADH ratio were determined in normal, fed, and fasted rats, and in rats infected with Streptococcus pneumoniae, Francisella tularensis, and Salmonella typhimurium. The various infections were found to have little or no effect on the cytoplasmic parameters. In normal rats, fasting caused a marked increase in blood and hepatic ketone concentration and in serum free fatty acid content. Fasted infected rats, however, did not show the increase in ketone bodies or serum free fatty acids normally associated with fasting alone. The mitochondrial NAD+/NADH ratio increased as the infections progressed, reversing the normal trend. The introduction of an infection during the fasting state when ketone bodies and serum free fatty acids were elevated caused a marked depression in their concentration. These data have led to a postulation of decreased lipolysis in the infected host to account for the lowered hepatic and blood ketone bodies and the decreased level of serum free fatty acids.

Glucose and alanine metabolism during bacterial infections in rats and Rhesus monkeys.

To investigate the effects of bacterial infection on glucose and alanine metabolism, a variety of studies were carried out in rat and monkey models. These included glucose turnover by a pulse-dose technique in infected rats; alanine and glucose production and utilization in control and septic monkeys; in vivo measurement of gluconeogenesis in rats, with and without an alanine load; the in vitro rate of glucose formation from various substrates by isolated liver perfusion and hepatic cells; and in vivo rates of oxidation of glucose labeled with 14C at the 1 or 6 carbon position. In rats, glucose turnover was markedly accelerated 24 hr after inoculation of either 10(4) or 10(7) Streptococcus pneumoniae. Glucose utilization and production were also accelerated during illness and early recovery from a pneumococcal infection in monkeys. The rates of gluconeogenesis as measured by either a pulse technique in rats or continuous infusion of labeled alanine in monkey were significantly elevated during pneumococcal septicemia. During the agonal stages (10(7) of the pneumococcal infection in rat, an alanine load resulted in a decreased rate of labeled glucose production and an increase in plasma glucose when compared to values of fasted control rats. However, early illness caused similar or increased rates of glucose production from alanine in vivo. Similar reduced rates of glucose formation were observed when the isolated livers or hepatocytes from rats during the agonal stages of infection were perfused with excess quantities of gluconeogenic substrates. Thus, in the rat, gluconeogenic capacity (ability to form glucose from excess substrates) appears to decrease only during the agonal stages of pneumococcal infection. During acute pneumococcal sepsis in the rhesus monkey, alanine production and utilization were significantly elevated and it was estimated that over 90% of the newly produced alanine was utilized for glucose synthesis. When arterial--venous differences were measured across the hindquarters, significantly more alanine was released, presumably from skeletal muscle of the septic monkey, compared to the recovery period or in the control groups. Thus, the increase in glucose synthesis in both rat and monkey appears to be correlated with substrate availability and kinetic rate, rather than gluconeogenic capacity of the liver. The major increase in glucose utilization during both S. pneumoniae and Francisella tularensis live vaccine strain (LVS) infections in rat was a progressive elevation in the rate of oxidation via the pentose phosphate shunt in the rat. Further, the rate of oxidation appeared to be correlated with the magnitude of the bacteremia, which is an indication of the severity of the infection...

EFFECTS OF TRAINING ON METABOLIC RESPONSES AND PERFORMANCE CAPACITY IN STREPTOCOCCUS PNEUMONIAE INFECTED RATS

These experiments were designed to study whether endurance training prior to Streptococcus pneumoniae infection in rats (N = 15 in each group) alters lethality, performance capacity, and related energy metabolism. A 5-d.wk-1, 4-wk-long pre-infection training program with gradually increasing swim time caused no protection from lethality (48% at 72 h post-inoculation), but performance capacity increased by 68% (P less than 0.01). The catabolic responses as evidenced by changes in insulin and glucagon levels were less pronounced. Mobilization of free fatty acids increased twofold (P less than 0.01), and improved ketonemic adaptation (47%, P less than 0.01) occurred with concomitant saved carcass, liver, and skeletal muscle glycogen contents (P less than 0.01). This shift from carbohydrate toward fat metabolism during exercise as a result of training was also reflected by 21% lower (P less than 0.01) blood lactate levels. It was concluded that the improved metabolic status, characterizing the trained as compared with the untrained host, is partly preserved during ongoing acute gram-positive bacterial infection.

Does Exercise Stress Alter Susceptibility to Bacterial Infections

Swimming was used for evaluating alterations in performance capacity and as a means for studying the influence of exercise stress on susceptibility to Streptococcus pneumoniae and Francisella tularensis infections in two strains of rats, i.e. Fisher-Dunning (FD) and Sprague-Dawley (SD). The performance capacity was reduced by both diseases and was correlated to the dose of the given micro-organism. FD rats, however, were more susceptible to the infection and showed a greater deterioration than SD rats. The effects of exercise stress on disease lethality varied with the time that it was performed. Strenuous exercise immediately before infection drastically reduced susceptibility to either of the bacteria, while a similar bout of exercise performed after infection increased disease-related mortality in both diseases.

A rapid method for determining ATP by the firefly luciferin-luciferase system

Eine schnelle Methode zur Bestimmung von ATP in picogramm-Mengen wird beschrieben. Die Grundlage für die Methode ist eine Reaktion zwischen ATP und einem Inkubationsansatz, der Luciferin, Mg2+ und partiell gereinigte Luciferase enthält, und ist zur Bestimmung von ATP in biologischem Material von unterschiedlicher Beschaffenheit (Bakterien, Pflanzen-und Tiergewebe) geeignet.

Metabolic responses to swimming exercise in Streptococcus pneumoniae infected rats.

The present study was performed to determine whether alterations in fuel reserves or energy substrate utilization might explain the performance decrements that occur in bacterial infections. Male Fisher-Dunning rats were studied at 24, 48, and 72 h after inoculation with Streptococcus pneumoniae. Rats were either sedentary or subjected to a 2-h swimming session at these three time points (N = 10 in each group). A more than 60% reduction (P less than 0.01) in performance capacity was observed on day 3 of infection compared with that in noninfected controls. This infection in the rat is characterized by fever (P less than 0.01), depression of plasma zinc (P less than 0.01) and free fatty acid (FFA) levels (P less than 0.01), inhibition of the two- to threefold increase in fasting ketonemia, and a decreased (NS) insulin:glucagon ratio, indicating a catabolic state. Glycogen stores were reduced in the heart (47%), liver (43%), and skeletal muscles (39%) but not in the carcass. Superimposed exercise resulted in a further reduction but not depletion of liver, muscle, and carcass glycogen stores, a less pronounced lactic acid accumulation, and a lower oxygen debt. However, plasma FFA and ketone body levels were still maintained or even elevated, suggesting that fat is supplied as fuel during swimming exercise in this infection. Thus, results indicate that unavailability of energy substrates or lactacidosis is not limiting for performance capacity during this severe infection.

Effect of inflammatory and noninflammatory stress on plasma ketone bodies and free fatty acids and on glucagon and insulin in peripheral and portal blood

Inflammatory stress as characterized by infection withStreptococcus pneumoniae, administration of endotoxin, or the induction of a turpentine abscess is characterized by the inhibition of the ketosis associated with fasting and a decline in the level of free fatty acids in the plasma. Moreover, rats subjected to these inflammatory stresses demonstrate a significant rise in peripheral and portal insulin and glucagon. Rats subjected to noninflammatory stresses, screen-restraint, or noninvasive femoral fracture did not demonstrate the inhibition of ketosis but did show a decrease in plasma free fatty acids. The noninflammatory stresses did not show an abnormal elevation of plasma or portal insulin or glucagon.

Protein-sparing therapy during pneumococcal infection in rhesus monkeys.

A model was developed in the rhesus monkey to determine if the marked wasting of body proteins associated with sepsis could be prevented by an intravenous supply of various nutritional substrates. All monkeys were given a basic infusion of 0.5 gm of amino acid nitrogen/kg body weight via an indwelling catheter in the jugular vein. Three groups were given diets with no added calories, 85 calories/kg from dextrose or 85 calories from lipid. In each group, six monkeys were inoculated with 3 x 10(8) Streptococcus pneumoniae and four with heatkilled organisms. In the monkeys infused with the amino acids alone, pneumococcal sepsis resulted in a fourfold increase in loss of body proteins compared with calorie-restricted controls. Addition of 85 calories/kg/day of either dextrose or lipid reduced body wasting associated with infectious disease. The calories from lipid were utilized bythe septic host as a source of energy, with a slightly reduced efficiency when compared with the isocaloric infusion of dextrose. The nitrogen sparing of the fat emulsion could not be accounted for by its glycerol content. Therefore, the septic monkey seemed to utilize fatty acids as an energy substrate. It appears that the carbohydrate calories tend to favor the synthesis of peripheral proteins (associated mainly with skeletal muscle), while lipid calories favor synthesis of visceral proteins such as plasma albumin and acute-phase proteins.

Glucose-dependent insulin inhibition of ketone body formation from long-chain fatty acids in the perfused livers of fasted rats

The data presented in this report show a direct effect of insulin on impairment of ketone body production in perfused livers from fasted rats. The data also show that physiologic levels of insulin alone or glucose alone are not sufficient to cause an impairment in ketogenesis. Only when insulin and glucose are both present at levels seen in infected rats is ketone body production impaired.