Robert J. Pawlosky

Dietary Linoleic Acid Elevates Endogenous 2‐AG and Anandamide and Induces Obesity

Suppressing hyperactive endocannabinoid tone is a critical target for reducing obesity. The backbone of both endocannabinoids 2‐arachidonoylglycerol (2‐AG) and anandamide (AEA) is the ω‐6 fatty acid arachidonic acid (AA). Here we posited that excessive dietary intake of linoleic acid (LA), the precursor of AA, would induce endocannabinoid hyperactivity and promote obesity. LA was isolated as an independent variable to reflect the dietary increase in LA from 1 percent of energy (en%) to 8 en% occurring in the United States during the 20th century. Mice were fed diets containing 1 en% LA, 8 en% LA, and 8 en% LA + 1 en% eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) in medium‐fat diets (35 en% fat) and high‐fat diets (60 en%) for 14 weeks from weaning. Increasing LA from 1 en% to 8 en% elevated AA‐phospholipids (PL) in liver and erythrocytes, tripled 2‐AG + 1‐AG and AEA associated with increased food intake, feed efficiency, and adiposity in mice. Reducing AA‐PL by adding 1 en% long‐chain ω‐3 fats to 8 en% LA diets resulted in metabolic patterns resembling 1 en% LA diets. Selectively reducing LA to 1 en% reversed the obesogenic properties of a 60 en% fat diet. These animal diets modeled 20th century increases of human LA consumption, changes that closely correlate with increasing prevalence rates of obesity. In summary, dietary LA increased tissue AA, and subsequently elevated 2‐AG + 1‐AG and AEA resulting in the development of diet‐induced obesity. The adipogenic effect of LA can be prevented by consuming sufficient EPA and DHA to reduce the AA‐PL pool and normalize endocannabinoid tone.

n-3 fatty acid metabolism in women.

The capacity for conversion of alpha-linolenic acid (ALNA) to n-3 long-chain polyunsaturated fatty acids was investigated in young men. Emulsified [U-13C]ALNA was administered orally with a mixed meal to six subjects consuming their habitual diet. Approximately 33 % of administered [13C]ALNA was recovered as 13CO2 on breath over the first 24 h. [13C]ALNA was mobilised from enterocytes primarily as chylomicron triacylglycerol (TAG), while [13C]ALNA incorporation into plasma phosphatidylcholine (PC) occurred later, probably by the liver. The time scale of conversion of [13C]ALNA to eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA) suggested that the liver was the principal site of ALNA desaturation and elongation, although there was some indication of EPA and DPA synthesis by enterocytes. [13C]EPA and [13C]DPA concentrations were greater in plasma PC than TAG, and were present in the circulation for up to 7 and 14 d, respectively. There was no apparent 13C enrichment of docosahexaenoic acid (DHA) in plasma PC, TAG or non-esterified fatty acids at any time point measured up to 21 d. This pattern of 13C n-3 fatty acid labelling suggests inhibition or restriction of DHA synthesis downstream of DPA. [13C]ALNA, [13C]EPA and [13C]DPA were incorporated into erythrocyte PC, but not phosphatidylethanolamine, suggesting uptake of intact plasma PC molecules from lipoproteins into erythrocyte membranes. Since the capacity of adult males to convert ALNA to DHA was either very low or absent, uptake of pre-formed DHA from the diet may be critical for maintaining adequate membrane DHA concentrations in these individuals.The extent to which women of reproductive age are able to convert the n-3 fatty acid alpha-linolenic acid (ALNA) to eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) was investigated in vivo by measuring the concentrations of labelled fatty acids in plasma for 21 d following the ingestion of [U-13C]ALNA (700 mg). [13C]ALNA excursion was greatest in cholesteryl ester (CE) (224 (sem 70) micromol/l over 21 d) compared with triacylglycerol (9-fold), non-esterified fatty acids (37-fold) and phosphatidylcholine (PC, 7-fold). EPA excursion was similar in both PC (42 (sem 8) micromol/l) and CE (42 (sem 9) micromol/l) over 21 d. In contrast both [13C]DPA and [13C]DHA were detected predominately in PC (18 (sem 4) and 27 (sem 7) micromol/l over 21 d, respectively). Estimated net fractional ALNA inter-conversion was EPA 21 %, DPA 6 % and DHA 9 %. Approximately 22 % of administered [13C]ALNA was recovered as 13CO2 on breath over the first 24 h of the study. These results suggest differential partitioning of ALNA, EPA and DHA between plasma lipid classes, which may facilitate targeting of individual n-3 fatty acids to specific tissues. Comparison with previous studies suggests that women may possess a greater capacity for ALNA conversion than men. Such metabolic capacity may be important for meeting the demands of the fetus and neonate for DHA during pregnancy and lactation. Differences in DHA status between women both in the non-pregnant state and in pregnancy may reflect variations in metabolic capacity for DHA synthesis.Using a stable-isotope tracer technique, Burdge & Wootton (2002) noted that women of child-bearing age (about 28 years) had a much greater capacity to convert a-linolenic acid (18 : 3n-3) to docosahexaenoic acid (22 : 6n-3) compared with men of a similar age (Burdge et al. 2002). Based on their analysis of the area under the curve of the time course plot of the plasma C-labelled fatty acids, they reported that there was approximately a 9 % excursion of C-labelled 18 : 3n-3 into C-labelled 22 : 6n-3 in women. In men, however, they found essentially no excursion of the label into plasma 22 : 6n-3 (0 % excursion). However, they did show excursion of the tracer into both eicosapentaenoic (20 : 5n-3) and docosapentaenoic (22 : 5n3) acids in both groups of subjects (Burdge et al. 2002). Using similar isotope tracer procedures, we previously reported that both men (n 4) and women (n 4) were capable of converting H5-labelled 18 : 3n-3 ethyl ester into C20 and C22 polyunsaturated fatty acids, including H5-labelled 22 : 6n-3 (Pawlosky et al. 2001). Moreover, we found that both male (n 5) and female (n 5) subjects were able to synthesize 22 : 6n-3 from 18 : 3n-3 when they subsisted on various diets (fishor beef-based, or ad libitum) that had different concentrations of long-chain polyunsaturated fatty acids (Pawlosky et al. 2003). We derived the in vivo rate constant coefficients for the individual transformations of n-3 fatty acids beginning with 18 : 3n-3 and calculated the percentage utilization of each precursor n-3 fatty acid for product formation (e.g. the percentage of 20 : 5n-3 converted to 22 : 5n-3 was calculated using the rate constant coefficients describing this transformation) using a compartmental modelling procedure. The differences between men and women in their capacities to utilize 18 : 3n-3 for 22 : 6n-3 production observed by Burdge & Wootton (2002) prompted us to analyse data from our dietary study (Pawlosky et al. 2003) in respect of gender. In evaluating the effects of the beefor fish-based or ad libitum diets on the kinetics of n-3 fatty acid metabolism, we observed that gender exerted a profound influence in the determination of a rate constant coefficient involved in one of the steps in the biosynthesis of 22 : 6n-3. During the period when subjects subsisted on a beef-based diet, the rate constant coefficient for the conversion of 22 : 5n-3 to 22 : 6n-3 was much greater (P1⁄40·001) in women (k 0·041 (SD 0·007)) compared with men (k 0·012 (SD 0·004)). The larger rate constant coefficient in women led to a nearly 3-fold greater amount of 22 : 5n-3 utilized for 22 : 6n-3 synthesis compared with men (Fig. 1). This was also observed during the ad libitum dietary phase of study, but did not reach statistical significance (P 1⁄4 0·08). Very interestingly, while subjects subsisted on the fish-based diet, both groups showed about equal capability in their utilization of 22 : 5n-3 for 22 : 6n-3 synthesis (Fig. 1). The effect of gender on the percentage excursion of labelled n-3 fatty acids observed by Burdge & Wootton (2002) in human subjects may be largely explained by the differences in the magnitude of the rate constant coefficient that describes the synthesis of 22 : 6n-3 from 22 : 5n-3 in male and female subjects. Gender appears to be a strong determinant that influences the synthesis of 22 : 6n-3 in human subjects. It is possible that Burdge & Wootton (2002) failed to detect excursion of the tracer into plasma 22 : 6n-3 in male subjects because of the lower conversion rate of 22 : 5n-3 to 22 : 6n-3, demanding highly sensitive analytical procedures. It is noteworthy that the beef-based diet did not appear to stimulate the synthesis of 22 : 6n-3 in male subjects compared with a fish-based diet (Fig. 1). This suggests that the regulation of n-3 fatty acid metabolism in women is more sensitive to dietary alterations and this may possibly be due to hormonal factors.

Kinetics, safety and tolerability of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate in healthy adult subjects.

Induction of mild states of hyperketonemia may improve physical and cognitive performance. In this study, we determined the kinetic parameters, safety and tolerability of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate, a ketone monoester administered in the form of a meal replacement drink to healthy human volunteers. Plasma levels of β-hydroxybutyrate and acetoacetate were elevated following administration of a single dose of the ketone monoester, whether at 140, 357, or 714 mg/kg body weight, while the intact ester was not detected. Maximum plasma levels of ketones were attained within 1-2h, reaching 3.30 mM and 1.19 mM for β-hydroxybutyrate and acetoacetate, respectively, at the highest dose tested. The elimination half-life ranged from 0.8-3.1h for β-hydroxybutyrate and 8-14 h for acetoacetate. The ketone monoester was also administered at 140, 357, and 714 mg/kg body weight, three times daily, over 5 days (equivalent to 0.42, 1.07, and 2.14 g/kg/d). The ketone ester was generally well-tolerated, although some gastrointestinal effects were reported, when large volumes of milk-based drink were consumed, at the highest ketone monoester dose. Together, these results suggest ingestion of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate is a safe and simple method to elevate blood ketone levels, compared with the inconvenience of preparing and consuming a ketogenic diet.

Compartmental analyses of plasma n-3 essential fatty acids among male and female smokers and nonsmokers.

The effects of cigarette smoking on n-3 essential FA metabolism were studied in male and female subjects by fitting the concentration-time curves of the d5-labeled plasma fatty acids (FAs) originating from a dose of d5-18:3n-3 to a compartmental model of n-3 FA metabolism. For 3 weeks, female (smokers, n = 5; nonsmokers, n = 5) and male (smokers, n = 5; nonsmokers, n = 5) subjects subsisted on a beef-based diet. Beginning in the third week, subjects received a dose of d5-18:3n-3 ethyl ester (1 g). Plasma FAs were analyzed using gas chromatography (GC) and GC-mass spectrometry, and the kinetic rate parameters were determined from the concentration-time curves for d5-18:3n-3, d5-20:5n-3, d5-22:5n-3, and d5-22:6n-3. Women smokers had a 2-fold greater percent of dose in plasma (5.8% vs. 2.9%; P < 0.01) and a higher fractional rate constant coefficient for formation of d5-22:6n-3 from d5-22:5n-3 (0.03 h−1 vs. 0.01 h−1; P < 0.01), compared with nonsmokers. Male smokers had elevated total plasma n-3 FAs, more-rapid turnover of 18:3n-3 (13.3 mg/day−1 vs. 4.3 mg/day−1; P < 0.001), a disappearance rate of d5-20:5n-3 that was both delayed and slower (0.001 h−1 vs. 0.012 h−1; P < 0.05), and a percentage of d5-20:5n-3 directed into formation of d5-22:5n-3 (99% vs. 61%; P < 0.03) that was greater compared with nonsmokers. Smoking increased the bioavailability of n-3 FAs from plasma, accelerated the fractional synthetic rates, and heightened the percent formation of some long-chain n-3 PUFAs in men and women.

Essential fatty acid uptake and metabolism in the developing rodent brain.

Studies were carried out to determine whether the brain takes up and metabolizes essential fatty acids during early postnatal development in rodents. Rats and mice were dosed with deuterium-labeled linoleic and linolenic acids either by intraperitoneal injection or by gavage. Animals were killed at different times thereafter, and organs were removed. Brains, livers, and blood were analyzed by gas chromatography— negative-ion-mass spectrometry for labeled fatty acids. To determine whether fatty acids were present in the brain apart from cerebral blood, a subset of animals was exsanguinated by perfusion with buffered saline, and the brain was then fractionated into subcellular components. Results demonstrated that the brain took up both labeled essential fatty acids within 8 h from the time of dosing. There was on average a greater uptake of linolenic acid into the cerebellum than into the cerebral cortex during the first 8 d of life in rats. The amount of linoleic acid taken into either region was similar, however. Docosahexaenoic acid intermediates, 20∶5n−3 and 22∶5n−3, were also found labeled in the brain. Time-course labeling experiments indicated that these intermediates may be converted to 22∶6n−3 within the brain. A rise of labeled 22∶6n−3 in the brain at 24 h appeared to be due to uptake of this fatty acid from the blood. The Amount of labeled 22∶6n−3 in the brain continued to increase beyond 24 h, and this did not appear to be correlated with its blood concentration. These results suggest that, during development in the rodent, different regions within the brain may vary in their capacity to synthesize 22∶6n−3, and this may be correlated with regional growth rates.

A Ketone Ester Diet Increases Brain Malonyl-CoA and Uncoupling Proteins 4 and 5 while Decreasing Food Intake in the Normal Wistar Rat

Three groups of male Wistar rats were pair fed NIH-31 diets for 14 days to which were added 30% of calories as corn starch, palm oil, or R-3-hydroxybutyrate-R-1,3-butanediol monoester (3HB-BD ester). On the 14th day, animal brains were removed by freeze-blowing, and brain metabolites measured. Animals fed the ketone ester diet had elevated mean blood ketone bodies of 3.5 mm and lowered plasma glucose, insulin, and leptin. Despite the decreased plasma leptin, feeding the ketone ester diet ad lib decreased voluntary food intake 2-fold for 6 days while brain malonyl-CoA was increased by about 25% in ketone-fed group but not in the palm oil fed group. Unlike the acute effects of ketone body metabolism in the perfused working heart, there was no increased reduction in brain free mitochondrial [NAD+]/[NADH] ratio nor in the free energy of ATP hydrolysis, which was compatible with the observed 1.5-fold increase in brain uncoupling proteins 4 and 5. Feeding ketone ester or palm oil supplemented diets decreased brain l-glutamate by 15–20% and GABA by about 34% supporting the view that fatty acids as well as ketone bodies can be metabolized by the brain.

Succinate-to-Fumarate Ratio as a New Metabolic Marker to Detect the Presence of SDHB/D-related Paraganglioma: Initial Experimental and Ex Vivo Findings

Pheochromocytomas (PHEOs) and paragangliomas (PGLs; extra-adrenal tumors) are rare neuroendocrine chromaffin cell tumors with a hereditary background in about 30%-35%. Those caused by succinate dehydrogenase subunit B (SDHB) germline mutations are associated with a high metastatic potential and ultimately higher patient mortality. Succinate dehydrogenase converts succinate to fumarate, uniquely linking the Krebs cycle and oxidative phosphorylation. SDH mutations result in the accumulation of succinate associated with various metabolic disturbances and the shift to aerobic glycolysis in tumor tissue. In the present study, we measured succinate and fumarate levels in mouse pheochromocytoma (MPC) and mouse tumor tissue (MTT) cells and in 10 apparently sporadic, 10 SDHB-, 5 SDHD-, and 2 neurofibromatosis 1-related PHEOs/PGLs and plasma samples using mass spectrometry. We found that the succinate-to-fumarate ratio was significantly higher in the SDHB- and SDHD-related PGLs than in apparently sporadic and neurofibromatosis 1-related PHEOs/PGLs (P = .0376). To further support our data, we silenced SDHB expression in MPC and MTT cells and evaluated the succinate and fumarate levels. Compared with control samples, SDHB-silenced MTT cells also showed an increase in the succinate-to-fumarate ratio (MTT cells: 2.45 vs 7.53), similar to the findings in SDHB-related PGLs. The present findings for the first time demonstrate a significantly increased succinate-to-fumarate ratio in SDHB/D-related PGLs and thus suggest this ratio may be used as a new metabolic marker for the detection of SDHB/D-related PHEOs/PGLs.

Novel ketone diet enhances physical and cognitive performance

Ketone bodies are the most energy‐efficient fuel and yield more ATP permole of substrate than pyruvate and increase the free energy released from ATP hydrolysis. Elevation of circulating ketones via high‐fat, low carbohydrate diets has been used for the treatment of drug‐refractory epilepsy and for neuro degenerative diseases, such as Parkinsons disease. Ketones may also be beneficial for muscle and brain in times of stress, such as endurance exercise. The challenge has been to raise circulating ketone levels by using a palatable diet without altering lipid levels. We found that blood ketone levels can be increased and cholesterol and triglycerides decreased by feeding rats a novel ketone ester diet: chow that is supplemented with (R)‐3‐hydroxybutyl (R)‐3‐hydroxybutyrate as 30% of calories. For 5 d, rats on the ketone diet ran 32% further on a tread mill than did control rats that ate an isocaloric diet that was supplemented with either corn starch or palmoil (P < 0.05). Ketone‐fed rats completed an 8‐ arm radial maze test 38% faster than did those on the other diets, making more correct decisions before making a mistake (P < 0.05). Isolated, perfused hearts fromrats that were fed the ketone diet had greater free energy available from ATP hydrolysis during increased work than did hearts from rats on the other diets as shown by using [31P]‐ NMRspectroscopy. The novelketone diet, therefore, improved physical performance and cognitive function in rats, and its energy‐sparing properties suggest that it may help to treat a range of human conditions with metabolic abnormalities.—Murray, A. J., Knight, N. S., Cole, M. A., Cochlin, L. E., Carter, E., Tchabanenko, K., Pichulik, T., Gulston, M.K., Atherton, H. J., Schroeder, M.A., Deacon, R.M. J., Kashiwaya, Y., King, M.T., Pawlosky, R., Rawlins, J. N. P., Tyler, D. J., Griffin, J. L., Robertson, J., Veech, R. L., Clarke, K. Novel ketone diet enhances physical and cognitive performance. FASEB J. 30, 4021–4032 (2016). www.fasebj.org

Oral 28-day and developmental toxicity studies of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate

(R)-3-Hydroxybutyl (R)-3-hydroxybutyrate (ketone monoester) has been developed as an oral source of ketones, which may be utilized for energy. In a 28-day toxicity study, Crl:WI (Wistar) rats received diets containing, as 30% of the calories, ketone monoester (12 and 15 g/kg body weight/day for male and female rats, respectively). Control groups received either carbohydrate- or fat-based diets. Rats in the test group consumed less feed and gained less weight than control animals; similar findings have been documented in studies of ketogenic diets. Between-group differences were noted in selected hematology, coagulation, and serum chemistry parameters; however, values were within normal physiological ranges and/or were not accompanied by other changes indicative of toxicity. Upon gross and microscopic evaluation, there were no findings associated with the ketone monoester. In a developmental toxicity study, pregnant Crl:WI (Han) rats were administered 2g/kg body weight/day ketone monoester or water (control) via gavage on days 6 through 20 of gestation. No Caesarean-sectioning or litter parameters were affected by the test article. The overall incidence of fetal alterations was higher in the test group; however, there were no specific alterations attributable to the test substance. The results of these studies support the safety of ketone monoester.

Compartmental analyses of 2H5-α-linolenic acid and C-U-eicosapentaenoic acid toward synthesis of plasma labeled 22:6n−3 in newborn term infants

BACKGROUND During early postnatal development, the nervous system accretes docosahexaenoic acid (DHA; 22:6n-3), a highly unsaturated n-3 (omega-3) fatty acid (FA) used in the formation of neural cell membranes. DHA, which is present in human breast milk, may also be biosynthesized from n-3 FAs such as 18:3n-3 [alpha-linolenic acid (ALA)] or 20:5n-3 [eicosapentaenoic acid (EPA)]. An important concern is to what extent these precursors can supply DHA to the developing infant. OBJECTIVE We analyzed measurements of fractional percentages of plasma (2)H(5)-ALA and (13)C-U-EPA directed toward the synthesis of labeled 22:6n-3 in 11 newborn infants by using compartmental modeling procedures. DESIGN One-week-old infants received doses of (2)H(5)-ALA and (13)C-U-EPA ethyl esters enterally. We drew blood from the infants periodically and analyzed the plasma for endogenous and labeled n-3 FAs. From the time-course concentrations of the labeled FAs, we determined rate constant coefficients, fractional synthetic rates, and plasma turnover rates of n-3 FAs. RESULTS In infants, approximately 0.04% of the (2)H(5)-ALA dose converted to plasma (2)H(5)-EPA. Plasma (2)H(5)-EPA and (2)H(5)-22:5n-3 [docosapentaenoic acid (DPA)] efficiently converted to (2)H(5)-DPA and (2)H(5)-DHA, respectively. The percentage of plasma (13)C-U-EPA directed toward the synthesis of (13)C-DHA was lower than the percentage of plasma (2)H(5)-EPA that originated from (2)H(5)-ALA. CONCLUSIONS Endogenously synthesized EPA was efficiently converted to DHA. In comparison, preformed EPA was less efficiently used for DHA biosynthesis, which suggests a differential metabolism of endogenous EPA compared with exogenous EPA. However, on a per mole basis, preformed EPA was 3.6 times more effective toward DHA synthesis than was ALA. Newborns required an intake of approximately 5 mg preformed DHA. kg(-1) x d(-1) to maintain plasma DHA homeostasis.