Rates Of Ketone Body Production Research Articles

The changing therapeutic armamentarium for patients with type 1 diabetes.

This article evaluates the role of Sodium glucose co-transporter-2 (SGLT-2) inhibitor and Glucagon-like peptide-1 receptor agonist's (GLP-1 RA's)s in the treatment of type 1 diabetes (T1D). Both agents help to produce a small reduction in HbA1C levels with accompanying weight loss. Submaximal doses of SGLT-2 inhibitors may reduce the risk for hypoglycemia as well as limit glycemic variability. However, SGLT-2 inhibitors appear to increase rates of ketone body production and diabetic ketoacidosis, and therefore must only be used in the setting of appropriate risk mitigation. GLP-1 RA's increased the risk for hypoglycemia in the largest clinical trial done to date (with a small fall in A1C), which lead to a cessation of the development of liraglutide as a treatment for T1D. Off-label use of medications designed for individuals with type 2 diabetes in people with T1D has potential benefits as well as risks. If using an SGLT-2 inhibitor or a GLP-1 RA, it is important to have an informed, educated patient and follow a specific protocol. In the case of SGLT-2 inhibitors, clinical trials should help define how to use these agents more safely and effectively in T1D.

Ketone Body Turnover at Term and in Premature Newborns in the First 2 Weeks after Birth

Using the infusion of D-(-)-3-hydroxy-[1,2,3,4,-13C4]butyrate at tracer doses, we measured total ketone body turnover in 13 premature and 10 at term infants in the first 2 weeks after birth. The premature infants received parenteral and/or oral feeding. The normal newborns were either recently fed or briefly fasting. The premature and the fed at term infants had comparable concentrations of ketone body (476 +/- 86 and 406 +/- 78 mumol/l) and free fatty acids (FFA) (309 +/- 47 and 325 +/- 75 mumol/l). In the premature newborns, ketone body turnover rates (3.2 +/- 0.2 mumol kg-1 min-1) were 74% that of fed newborns at term (4.3 +/- 0.3 mumol kg-1 min-1, p < 0.05), and 18% that of normal newborns during a brief fast (17.3 +/- 1.3 mumol kg-1 min-1, p < 0.01). Ketone body production rates correlated with plasma FFA concentrations in both groups (r = 0.62 and 0.69, p < 0.05). However, for a similar plasma FFA content, ketone production was 2- to 3-fold lower in the premature, indicating an immature hepatic capacity to convert FFA into ketones. Our study therefore shows that ketogenesis is already active in infants born 10 weeks before normal term and continuously fed, but that daily ketone production is lower than at term.

Triacylglycerol and Fatty Acid Synthesis in Hepatocytes in Suspension Isolated from Rats Fed Soybean Phospholipid

The rates of triacylglycerol and fatty acid synthesis in hepatocytes in suspension isolated from rats fed dietary soybean phospholipid were compared with those from the animals fed soybean oil. The rats were fed the experimental diets containing either soybean phospholipid or soybean oil (the dietary levels correspond to 3% fatty acids, 5.1 and 3.2% on weight bases for the phospholipid and the oil, respectively) for 10 days. Dietary soybean phospholipid compared to the oil decreased the rate of incorporation of [2-3H]glycerol into triacylglycerol in isolated hepatocytes. However, the difference disappeared when the incubation mixture was fortified with oleate substrate (1 mm). The dietary phospholipid compared to the oil greatly decreased the rate of incorporation of [1-14C]acetate into fatty acids while it increased that of the tracer into cholesterol. The rate of ketone body production from oleate substrate was decreased in rats fed soybean phospholipid compared to those fed the oil. Thus, dietary soybe...

Increased ketogenesis related to insulin deficiency in isolated hepatocytes from NIDDM model rats.

To investigate the hepatic ketone body metabolism in NIDDM, we studied the ketone body production rates in hepatocytes from newly developed non-obese NIDDM model rats. NIDDM model rats were prepared by intraperitoneal injection of streptozotocin at 2 or 5 days of age (STZ2, STZ5 respectively). After 10-15 weeks, ketone body production rates in hepatocytes isolated from these rats were compared with those from control rats as well as ketotic rats made by intravenous injection of streptozotocin into adult rats. Basal ketone body production rates from 0.3 mM [U-14C] palmitate in hepatocytes from control, STZ 2, STZ 5 and ketotic rats were 11.7 +/- 0.98, 14.9 +/- 0.72, 16.0 +/- 0.45, 22.8 +/- 2.32 nmole.palmitate/mg.prot/hr, respectively. These rates were stimulated by 1 microgram/ml of glucagon in control, STZ 2 and STZ 5 rats (14.1 +/- 0.99, 18.6 +/- 1.36, 18.7 +/- 0.69 nmole.palmitate/mg.prot/hr, respectively), but not in ketotic rats (22.8 +/- 2.07 nmole.palmitate/mg.prot/hr). The similar effects were observed by 1 microgram/ml of epinephrine. The basal ketone body production rates were negatively correlated to both hepatic glycogen contents and plasma IRI levels. Considering these parameters together, the extent of metabolic derangement in STZ 2 and STZ 5 rats was between that in control and ketotic rats. These results indicate that the derangements of hepatic ketone body production are related to the severity of insulin deficiency and suggest that the enhanced hepatic ketogenesis contributes in part to the elevated plasma ketone body levels in non-obese NIDDM.

The effects of epinephrine on ketogenesis in the dog after a prolonged fast

The effects of a selective increase in epinephrine on ketogenesis and lipolysis were determined in the conscious dog following a prolonged fast (7 days). Plasma insulin and glucagon were fixed at basal levels by infusion of somatostatin (0.8 μg/kg/min) and basal intraportal replacement amounts of insulin (210 ± 20 μU/kg/min) and glucagon (0.65 ng/kg/min). Following a 40-minute control period, saline or epinephrine (0.04 μg/kg/min) was infused for 3 hours. Plasma insulin, glucagon, and norepinephrine levels did not change during saline (6 ± 1 μU/mL, 83 ± 17 pg/mL, and 137 ± 38 pg/mL, respectively) or epinephrine (10 ± 1 μU/mL, 73 ± 18 pg/ml, 98 ± 13 pg/mL, respectively) infusion. Plasma epinephrine levels increased from 80 ± 26 to 440 ± 47 pg/mL in response to infusion of the catecholamine, but remained unchanged during saline infusion. Glycerol levels (93 ± 10 μmol/L) remained unchanged during saline infusion, but increased in response to epinephrine (108 ± 9 to 170 ± 18 μmol/L by 30 minutes). The glycerol level had returned to baseline and to the value apparent in saline controls by 60 minutes. The nonesterified fatty acid (NEFA) level declined slowly during the 3-hour saline infusion, but was elevated in response to epinephrine infusion (1.27 ± 0.16 to 1.97 ± 0.25 mmol/L at 30 minutes). After the initial epinephrine-induced increase, the NEFA level declined so that by 3 hours it was not significantly different from the basal or saline values. Saline infusion was associated with a decline in ketone body levels and net hepatic ketone production (335 ± 43 to 239 ± 72 μmol/L and 10.99 ± 1.43 to 5.50 ± 1.81 μmol/kg/min). Epinephrine infusion caused an increase in the level and rate of ketone body production, which, when compared with the response in saline-infused animals, was significant at 60, 90, and 120 minutes. While saline infusion had no effect on plasma glucose (95 ± 3 to 89 ± 4 mg/dL) or blood lactate (0.89 ± 0.08 to 0.98 ± 0.19 mmol/L), epinephrine infusion caused both glucose (90 ± 4 to 130 ± 8 mg/dL) and lactate (0.60 ± 0.04 to 1.08 ± 0.17 mmol/L) to increase significantly. These data indicate that, contrary to observations in the 18 hour-fasted dog in which epinephrine had an inhibitory effect on ketogenesis, in a more prolonged fasted dog the catecholamine causes a stimulation of the process.

Metabolic and Neurologic Effects of an Intravenous Medium‐Chain Triglyceride Emulsion

These studies were undertaken to investigate the relationship between medium-chain fatty acid availability, medium-chain fatty acid oxidation, and central nervous system toxicity during infusion of medium-chain triglycerides in dogs. Six dogs received a sequential, stepwise infusion of trioctanoin at three different rates for 80 min each, providing calories below and equal to resting energy expenditure in the species. Ketone body production rates (using a 14C beta-hydroxybutyrate tracer) and plasma concentrations of lactate and octanoate were monitored. Three animals were infused with saline to serve as controls. Blood-brain barrier integrity was assessed with Evans blue dye, and brain samples were taken at the end of the study to quantify brain water. Three animals were studied under anesthesia to obtain good quality EEG and intracranial pressure measurements. Results were (1) plasma octanoate increased to 0.37 +/- 0.13, 0.78 +/- 0.2, and 1.44 +/- 0.41 mmol/liter during the three infusion intervals; (2) emesis, somnolence, and coma were observed at the two highest trioctanoin rates; (3) ketone body concentrations and production increased from 102 +/- 15 to 859 +/- 54 mumol/liter and 3.6 +/- 0.43 to 18.5 +/- 1.7 mumol/kg/min, respectively, at the highest trioctanoin infusion rate; and (4) plasma lactate also increased from 1.3 +/- 0.1 to 4.3 +/- 0.9 mmol/liter at the highest infusion rate. EEG changes were also observed, consisting of high amplitude slowing and reduction in amplitude of faster components. There was no extravasation of Evans blue dye, nor change in brain water or intracranial pressure. The conclusion--medium-chain triglycerides have significant dose-related central nervous system toxicity in dogs. Therefore, caution should be exercised in clinical studies with MCTs, including careful measurement of medium-chain fatty acid concentrations.

Systemic pH modifies ketone body production rates and lipolysis in humans.

To investigate whether changes in systemic pH influence ketone body production or utilization, total ketone body (TK) kinetics were measured with [3-14C]acetoacetate and D-beta-[1,3-13C2]hydroxybutyrate tracers in overnight fasted subjects during metabolic alkalosis (NaHCO3 infusion) or acidosis [NH4Cl ingestion or arginine (Arg)-HCl infusion]. Somatostatin, with insulin, glucagon, and growth hormone replacement, was infused in all studies. Blood pH and HCO3- (mM) increased from baseline (0-30 min) to 180-210 min by 0.08 +/- 0.02 and 7 +/- 1 with NaHCO3 and decreased by 0.08 +/- 0.2 and 7 +/- 1 or 5 +/- 1 with NH4Cl or Arg-HCl (all P less than 0.005). Over this period blood TK (microM) differed between the NaHCO3 (+198 +/- 65) and both NH4Cl (-90 +/- 53) and Arg-HCl (-154 +/- 55) (P less than 0.05). These changes resulted from parallel alterations in TK production rate of appearance (Ra TK, mumol.kg-1.min-1), because changes from baseline in Ra 14C TK also differed between NaHCO3 (+1.9 +/- 0.8) and NH4Cl (-1.0 +/- 0.6) and Arg-HCl (-2.0 +/- 0.5) (P less than 0.05). Ra TK calculated with single- or dual-tracer techniques were similar. Blood free fatty acids (FFA) increased with NaHCO3, and FFA and glycerol decreased with NH4Cl and Arg-HCl, suggesting that FFA availability mediated the pH effects on hepatic ketogenesis. These results demonstrate that modest changes in systemic pH modify FFA availability and TK production rates.

Interactions between plasticizers and fatty acid metabolism in the perfused rat liver and in vivo: Inhibition of ketogenesis by 2-ethylhexanol

Rates of ketone body (β-hydroxybutyrate plus acetoacetate) production by perfused livers from starved rats were decreased about 60% from 39 ± 2 to 17 ± 3 μmol/g/hr by 2-ethylhexanol (200,μM), a primary metabolite of the plasticizer diethylhexyl phthalate. Inhibition of ketogenesis by ethylhexanol was dose dependent (half-maximal inhibition occurred with 25 μM) in the presence or absence of 4-methylpyrazole, an inhibitor of alcohol dehydrogenase. Concentrations of β-hydroxbutyrate relative to acetoacetate (B/A) increased in a step-wise manner from 0.32 to 0.75 in the effluent perfusate when ethylhexanol was infused. In contrast, the B/A ratio decreased in parallel with inhibition of ketone body production when alcohol dehydrogenase was inhibited. Pretreatment of rats with phenobarbital, an inducer of ω and ω-1 hydroxylases, diminished inhibition of ketone body production by low (<50 μM) but not high concentrations (>50 μM) of ethylhexanol. Thus, ethylhexanol is oxidized via phenobarbitalinducible pathways to metabolites which do not inhibit ketogenesis. Studies were conducted to determine the site of inhibition of fatty acid oxidation by ethylhexanol. Rates of ketone body production in the presence of oleate (250 μM), which requires transport of the corresponding CoA compound into mitochondria, were reduced from 80 ± 6 to 58 ± 8 μmol/g/hr by ethylhexanol. In contrast, ketone body production from hexanoate, which is activated in the mitochondria, was not affected by ethylhexanol. Basal and oleate-stimulated rates of H 2O 2 production were not affected by ethylhexanol, indicating that peroxisomal β-oxidation was not altered by the compound. Based on these data it is concluded that 2-ethylhexanol inhibits β-oxidation of fatty acids in mitochondria but not in peroxisomes. Treatment of rats with ethylhexanol (0.32 g/kg, i.p.) decreased plasma ketone bodies from 1.6 to 0.8 mM, increased hepatic triglycerides and increased lipid predominantly in periportal regions of the liver lobule. These data indicate that alterations in hepatic fatty acid metabolism in periportal regions of the liver lobule may be early events in peroxisome proliferation.

Comparison of cultured human hepatocytes isolated from surgical biopsies or cold-stored organ donor livers

The purpose of this work was to compare yield, attachment rate and specific metabolic functions (stimulation of ketone body production by glucagon) of human hepatocytes isolated from surgical biopsies and from organ donor livers cold stored with a modified University of Wisconsin (MUW) solution. A significantly greater number of hepatocytes was isolated from MUW-stored livers than from surgical biopsies. On average, 60% of hepatocytes isolated from surgical biopsies attached to uncoated flask whereas the attachment rate of hepatocytes isolated from MUW-stored livers was inconsistent and always below 40%. Glucagon significantly enhanced the rate of ketone body production of hepatocytes isolated from surgical biopsies; in contrast, glucagon had marginal effects on the rate of ketone body production in hepatocytes isolated from MUW-stored livers. These results demonstrate that human hepatocytes isolated from surgical biopsies maintain liver-specific and non-specific functions better than hepatocytes isolated from MUW-stored livers. Human hepatocytes isolated from surgical biopsies should be preferentially used for the study of metabolism in human liver.

Octanoate metabolism in isolated hepatocytes and mitochondria from fetal, newborn and adult rabbit. Evidence for a high capacity for octanoate esterification in term fetal liver.

Ketogenesis from endogenous fatty acids or from exogenous octanoate has been studied in isolated hepatocytes from fetal. 24-h-old newborn and adult rabbit. In fed adult rabbits, endogenous ketogenesis is low and increases sixfold in the presence of 2 mM octanoate. At birth, endogenous ketogenesis is low and markedly increases 24 h after birth but, in both cases, the addition of 2 mM octanoate does not increase the rates of ketone body production. Hepatocytes isolated from 24-h-old newborn or fed adult rabbits and incubated with [1-14C]octanoate show a preferential channeling of fatty acid into oxidation (84-92% of octanoate metabolized). In contrast, esterification represents 43% of the amount of octanoate metabolized at birth. Chromatographic analysis of labelled triacylglycerols shows that 76 +/- 2% of labelled fatty acids are identified as octanoate and all of the radioactivity in the octanoate peak is due to the carboxyl carbon. In hepatocytes from term fetus, the low capacity for octanoate oxidation is associated with a high capacity for esterification, whatever the octanoate concentration in the medium. Octanoate activated to octanoyl-CoA in the cytosol of fetal hepatocyte is not oxidized in the mitochondria since carnitine acyltransferase I has a low activity at birth in the rabbit. This suggests that only a part of the octanoate pool is activated outside the mitochondria. Factors involved in the direct esterification of octanoate into triacylglycerols in term fetal hepatocytes are discussed.

Compartmentation of dicarboxylic acid β-oxidation in rat liver: importance of peroxisomes in the metabolism of dicarboxylic acids

Peroxisomal and mitochondrial beta-oxidation of dicarboxylic acids (DCAs) were investigated and compared. When isolated hepatocytes were incubated with DCAs of various chain lengths, H2O2 was derived from peroxisomal beta-oxidation, the rates of its generation being comparable to those seen with monocarboxylic acids (MCAs), whereas the rates of ketone body production, a measure of mitochondrial beta-oxidation, were much lower than those with MCAs. Peroxisomal beta-oxidation measured by cyanide-insensitive NAD reduction exhibited similar chain-length specificities for both dicarboxylyl-CoAs (DC-CoAs) and monocarboxylyl-CoAs (MC-CoAs), except that the activities for DC-CoAs with 10-16 carbon atoms were about half of those of the corresponding MC-CoAs. In contrast, mitochondrial beta-oxidation measured by antimycin A-sensitive O2 consumption had no activity for DCAs. In the study with purified enzymes, the reactivities of mitochondrial carnitine palmitoyltransferase and acyl-CoA dehydrogenase for DC-CoAs were much lower than those for MC-CoAs, while the reactivity of peroxisomal acyl-CoA oxidase for DC-CoAs was comparable to that for the corresponding MC-CoAs. Accordingly, the properties of carnitine palmitoyltransferase and acyl-CoA dehydrogenase must be the rate-limiting factors for mitochondrial beta-oxidation, with the result that DCAs might hardly be oxidized in mitochondria. Comparative study of beta-oxidation capacities of peroxisomes and mitochondria in the liver showed that DC12-CoA was hardly subjected to mitochondrial beta-oxidation, and that the beta-oxidation of DCAs in rat liver, therefore, must be carried out exclusively in peroxisomes.

Medium-Chain Triglyceride Feeding over the Long Term: the Metabolic Fate of [14C]Octanoate and [14C]Oleate in Isolated Rat Hepatocytes

Feeding medium-chain triglycerides (MCT) is reported to result in less weight gain as fat than feeding long-chain triglycerides (LCT). In this experiment, fatty acid metabolism was studied in hepatocytes isolated from rats fed high fat diets containing MCT (70.9% of total energy), LCT (72.3% total energy) or a low fat (LF) (10.8% total energy). In the MCT group, hepatocyte utilization of [1-14C]oleate or [1-14C]octanoate, unlabeled ketone body (KB) production and lipogenesis (LG) were greater than in the other groups. Esterified products (EST) from both fatty acids were greater in the MCT groups, and 5-tetradecycloxy-2-furoic acid, an effective inhibitor of LG, did not abolish this. Lactate/pyruvate increased EST from octanoate and total LG but had little effect on octanoate oxidation. These data suggest that the capacity of medium-chain fatty acid esterification is adaptable to MCT content of the diet and may help regulate their metabolic fate. Despite high rates of KB production in the MCT group, the NADH-NAD ratio was not elevated. Rapid lipogenesis may contribute to the utilization of the excess energy equivalents formed. High LG and EST in the presence of a glycerol precursor may limit the usefulness of MCT in dietetic products for weight control. The animal fed an MCT diet is unusual in that the antithetical pathways of fatty acid catabolism and anabolism are being carried out simultaneously and at rapid rates. The lesser food efficiency of MCT may be related to the energy losses necessarily associated with cycling between these processes.(ABSTRACT TRUNCATED AT 250 WORDS)

Premature appearance of gluconeogenesis and fatty acid oxidation in the liver of the postterm rabbit fetus.

The metabolic consequences of a prolonged gestation (35 vs 32 days) have been studied in the rabbit fetus. Gestation was prolonged by daily subcutaneous injections of progesterone (1.5 mg.kg-1) from day 28 to 34. In control animals, progesterone was injected from day 25 or 28 to day 31 of gestation. When the capacities for gluconeogenesis and fatty acid oxidation, measured on isolated hepatocytes, are normally low in the term control fetus and increase only within the first 24 h after birth, these capacities appear high in the postmature fetus. The rate of glucose production from lactate is 4-fold higher in the postmature fetus than in the normal term fetus. The rate of ketone body production from oleate is also 5-fold higher in the postmature fetus, which results from a switch on of the partition of oleate into esterification and oxidation: 8% of [1-14C]oleate is oxidized in term fetus hepatocytes, but 34% in postmature fetus hepatocytes. As a similar rate of lipogenesis takes place in both stages, this metabolic change could be explained by a 5-fold lower sensitivity of carnitine palmitoyltransferase I to the inhibition by malonyl-coenzyme A. Postmaturity decreases plasma insulin concentrations by 45% and increases plasma glucagon concentrations by 50% which, in turn, induces a 3-fold decrease in the plasma insulin:glucagon molar ratio. As previously shown in fasted or diabetic adult rat, this hormonal change might be a likely candidate for an enhancement of gluconeogenic and ketogenic capacity in the liver of the postterm rabbit fetus.

Participation of peroxisomes in the metabolism of xenobiotic acyl compounds: comparison between peroxisomal and mitochondrial β-oxidiation of ω-phenyl fatty acids in rat liver

The peroxisomal β-oxidation of ω-phenyl fatty acids (PFAs) as model compounds for xenobiotic acyl compounds was investigated. In isolated hepatocytes, ω-phenyllauric acid (PFA 12) was chain-shortened to PFAs having an even number of carbon atoms in the acyl side chain. Associated with this reaction, H 2O 2 generation was observed, the rate of which was markedly enhanced by clofibrate treatment of rats. Also when using isolated peroxisomes, such a chain-shortening of PFA 12 occurred, associated with stoichiometrical production of NADH and acetyl-CoA. The CoA-ester form of PFA 12 as a substrate and NAD as a cofactor were required in this reaction, indicating the participation of peroxisomal β-oxidation in the chain-shortening of PFA 12. When using PFAs with various chain lengths, the rates of H 2O 2 generation measured as the peroxisomal β-oxidation in isolated hepatocytes were similar to those with the corresponding fatty acids, whereas the rates of ketone body production measured as the mitochondrial β-oxidation were much lower than that with any fatty acid examined. From the study with isolated mitochondria and purified enzymes, it was found that the mitochondrial β-oxidation of PFAs was carnitine-dependent, and that the activities of carnitine palmitoyltransferase for PFA-CoAs are low. Moreover, the activities of acyl-CoA dehydrogenase for PFA-CoAs were lower than those for fatty acyl-CoAs, while the activities of acyl-CoA oxidase for PFA-CoAs were comparable to those for fatty acyl-CoAs. As a result, relatively long chain PFAs were hardly subjected to mitochondrial β-oxidation. Based on the maximum enzyme activities of the β-oxidation, which were measured by following acyl-CoA-dependent NAD reduction in isolated peroxisomes and O 2 consumption in isolated mitochondria, about 60% of the β-oxidation of PFA 12 in the rat liver was peroxisomal. In clofibrate-treated rats, the value reached about 85%. From these results it is concluded that the peroxisome is one of the important sites of degradation of xenobiotic acyl compounds.

Hepatic triglyceride hydrolysis and development of ketogenesis in rabbits.

Ketogenesis from endogenous fatty acids or exogenous oleate plus carnitine has been studied in isolated hepatocytes from fetal, newborn, and 70-day-old rabbits. During the first 48 h after birth, hepatic triacylglycerol stores decrease by 80%. The hydrolysis of hepatic triacylglycerol stores has been studied in isolated hepatocytes from 24-h-old fasting rabbits by using lysosomal acid lipase inhibitors and lysosomotropic agents. Their addition decreases the rates of ketone body production by 60-70%, suggesting that hepatic triacylglycerol hydrolysis proceeds via an acid lipase located in the lysosomes. Whereas the rates of ketogenesis from endogenous or exogenous fatty acids are very low in isolated hepatocytes from fetal rabbit, an eightfold increase in the rate of ketogenesis occurs between 6 and 24 h after birth; furthermore the hydrolysis of triacylglycerol stores is sufficient to support the ketogenic capacity in the hepatocytes isolated from 24-h-old rabbits. The emergence of ketogenesis in newborn rabbit hepatocytes is triggered by birth-associated factors rather than to an accurate stage of fetal maturation. Fatty acids are mainly oxidized in the mitochondria because peroxisomal oxidation does not exceed 10-15% of the overall beta-oxidation. Isolated hepatocytes incubated with [1-14C]oleate exhibit at birth a preferential channeling of fatty acid into esterification (93% of oleate metabolized) rather than into oxidation. Conversely oleate oxidation represents 50% of total oleate metabolized 24 h after birth. Factors involved in this switch on of the partition of oleate into esterification and oxidation during the 1st day after birth are discussed.

Effects of free fatty acid availability, glucagon excess, and insulin deficiency on ketone body production in postabsorptive man.

The present studies were undertaken to assess the relative effects of free fatty acid (FFA) availability, glucagon excess, and insulin deficiency on ketone body (KB) production in man. To determine whether an increase in FFA availability would augment KB production in the absence of insulin deficiency and glucagon excess, plasma insulin and glucagon were maintained at basal concentrations by infusion of somatostatin and exogenous insulin and glucagon, and plasma FFA were increased from 0.32 +/- 0.06 to 1.4 +/- 0.1 mM by a 2.5-h-infusion of a triglyceride emulsion plus heparin. KB production increased fivefold from 2.2 +/- 0.4 to 11.4 +/- 1.2 mumol . kg-1 . min-1, P less than 0.001. To determine whether insulin deficiency would further augment KB production, analogous experiments were performed but the replacement infusion of insulin was stopped. Despite a greater increase in plasma FFA (from 0.26 +/- 0.04 to 1.95 +/- 0.3 mM), KB production increased (from 1.5 +/- 0.3 to 11.1 +/- 1.8 mumol . kg-1 . min-1) to the same extent as in the absence of insulin deficiency. To determine whether hyperglucagonemia would augment KB production beyond that accompanying an increase in plasma FFA and, if so, whether this required insulin deficiency, similar experiments were performed in which the glucagon infusion rate was increased to produce plasma glucagon concentrations of 450-550 pg/ml with and without maintenance of the basal insulin infusion. When basal plasma insulin concentrations were maintained, hyperglucagonemia did not further increase KB production; however, when the basal insulin infusion was discontinued, hyperglucagonemia increased KB production significantly, whereas no change was observed in saline control experiments. These studies indicate that, in man, FFA availability is a major determinant of rates of KB production; insulin does not appear to influence ketogenesis rates by a direct hepatic effect, and glucagon can further augment KB production when FFA concentrations are increased but only in the setting of insulin deficiency.

Role of glucagon in enhancing ketone body production in ketotic diabetic man.

To assess the role of endogenous glucagon in regulating hepatic ketone body production in ketotic insulin-withdrawn diabetic subjects, ketone body kinetics were determined in two groups of C-peptide-negative diabetics 6 h after interruption of a s.c. insulin infusion. In group 1 (N = 5), glucagon levels were suppressed by infusion of somatostatin (SRIF), whereas in group 2 (N = 6) glucagon was replaced during SRIF by infusing glucagon at 2 ng/kg/min. Ketone body production rates as determined by primed-continuous infusion of [3-14C]acetoacetate declined from 19.5 +/- 0.8 to 16.4 +/- 0.4 mumol/kg/min (P less than 0.01) during 105 min of SRIF-induced glucagon suppression, whereas they remained unchanged (+0.2 +/- 0.4 mumol/kg/min, P less than 0.01 compared with SRIF) during glucagon replacement. Total ketone body concentrations remained unchanged during SRIF infusion but increased from 2.2 +/- 0.3 to 2.9 +/- 0.2 mmol/L (P less than 0.01) during glucagon replacement. The metabolic clearance rate of total ketone bodies declined significantly (P less than 0.01) by 27% and 21% in the two groups. Plasma free fatty acid and glycerol concentrations remained unchanged in both groups whereas plasma glucose decreased by 3.2 +/- 0.5 mmol/L during SRIF (P less than 0.01). Thus, endogenous glucagon contributed significantly to the maintenance of ketone body production rates in ketotic insulin-deficient diabetics. Since ketogenesis was altered in the absence of changes in free fatty acid levels, the results suggested that glucagon enhanced ketogenesis by an intrahepatic effect.

The effects of ketone bodies, bicarbonate, and calcium on hepatic mitochondrial ketogenesis

The effect of various factors on hepatic mitochondrial ketogenesis was investigated in the rat. A comparison of three different incubation media revealed that bicarbonate ion inhibited the rate of ketone body production and decreased the ratio of 3-hydroxybutyrate/acetoacetate. The addition of 0.8 m m calcium caused significant inhibition of ketogenesis from both octanoate (40–50%) and palmitate (25–30%) and no change in the ratio of 3-hydroxybutyrate/acetoacetate. In the presence of components of the malate/aspartate shuttle, the inhibition by calcium was 80% or more with both substrates. Experimental alteration of the respiratory state of the mitochondria from state 3 to state 4 was associated with an enhanced rate of ketogenesis. The addition of ketone bodies themselves had marked effects on the rate of ketone body production. Increasing amounts of exogenously added acetoacetate were accompanied by increasing rates of total ketone body production reflecting enhanced 3-hydroxybutyrate synthesis. In the presence of added 3-hydroxybutyrate, there was striking inhibition of ketogenesis. Rotenone, which prevents oxidation of NADH 2 via the electron transport chain, almost completely inhibited ketone body synthesis. This inhibition was partially overcome by the addition of acetoacetate which regenerates NAD + from NADH 2 during conversion to 3-hydroxybutyrate. These observations provide evidence for additional sites of metabolic control over hepatic ketogenesis.

Model of the kinetics of ketone bodies in humans.

The kinetics of ketone bodies was studied in normal humans by giving a combined bolus intravenous injection of labeled acetoacetate ([14C]AcAc) and D(--)-beta-hydroxybutyrate (beta-[14C]-OHB) to seven subjects after an overnight fast, on two different occasions, and by collecting frequent blood samples for 100 min. Kinetic data were analyzed with both noncompartmental and compartmental modeling techniques. A four-compartment model, representing AcAc and beta-OHB in blood and two equilibrating ketone body compartments, inside the liver and extrahepatic tissues, was chosen as the most reliable mathematical representation; it is physiologically plausible and was able to accurately fit the data. The model permitted evaluation of the in vivo rate of ketone body production in the liver, the individual plasma clearance rates of AcAc and beta-OHB, their initial volumes of distribution, and the transfer rate parameters among the four ketone body compartments. Moreover, the model provided estimates of the components of the rates of appearance of AcAc and beta-OHB in plasma due to newly synthesized ketone body from acetyl-CoA in the liver, and to interconversion and recycling in the liver and extrahepatic tissues. The model also was used to evaluate other methodologies currently employed in the analysis of ketone body turnover data: the conventional approach based on use of the combined specific activity of AcAc and beta-OHB required assumptions not satisfied in vivo, leading to substantial errors in key parameter estimates.

Oxidation and ketogenesis in hepatocytes of lean and obese Zucker rats

Ketone body production and oxidation of 14C fatty acids to CO 2 were measured in hepatocytes isolated from lean and obese Zucker rats. The oxidation of [1- 14C]octanoate, [1- 14C]palmitate and [1- 14C]palmitoyl carnitine to 14CO 2 was 50%–70% less in obese than in lean rats. Although ketone body production in hepatocytes from both lean and obese rats was increased by fasting, there was a significantly lower rate of ketone body production in hepatocytes from obese rats. Ketone body production was reduced to a comparable extent by increasing the glucose concentration in the incubation media of hepatocytes from both lean and obese rats. Glucagon and carnitine increased ketogenesis and the effects were additive and similar in lean and obese rats. These data suggest that β-oxidation and ketogenesis are suppressed in the obese Zucker rat, and further that ketone bodies can be modulated similarly in hepatocytes from lean and obese rats by nutritional and hormonal intervention. It is postulated that the decreased β-oxidation and ketone body production may play a role in the development or maintenance of obesity in the Zucker rat.