Dietary fat composition shapes bile acid metabolism and severity of liver injury in a pig model of pediatric NAFLD.

The objective of this study was to investigate the effect of dietary fatty acid (FA) composition on bile acid (BA) metabolism in a pig model of NAFLD, by using a multiomics approach combined with histology and serum biochemistry. Thirty 20-day-old Iberian pigs pair-housed in pens were randomly assigned to receive 1 of 3 hypercaloric diets for 10 wk: 1) lard-enriched (LAR; n = 5 pens), 2) olive oil-enriched (OLI; n = 5), and 3) coconut oil-enriched (COC; n = 5). Animals were euthanized on week 10 after blood sampling, and liver, colon, and distal ileum (DI) were collected for histology, metabolomics, and transcriptomics. Data were analyzed by multivariate and univariate statistics. Compared with OLI and LAR, COC increased primary and secondary BAs in liver, plasma, and colon. In addition, both COC and OLI reduced circulating fibroblast growth factor 19, increased hepatic necrosis, composite lesion score, and liver enzymes in serum, and upregulated genes involved in hepatocyte proliferation and DNA repair. The severity of liver disease in COC and OLI pigs was associated with increased levels of phosphatidylcholines, medium-chain triacylglycerides, trimethylamine-N-oxide, and long-chain acylcarnitines in the liver, and the expression of profibrotic markers in DI, but not with changes in the composition or size of BA pool. In conclusion, our results indicate a role of dietary FAs in the regulation of BA metabolism and progression of NAFLD. Interventions that aim to modify the composition of dietary FAs, rather than to regulate BA metabolism or signaling, may be more effective in the treatment of NAFLD.NEW & NOTEWORTHY Bile acid homeostasis and signaling is disrupted in NAFLD and may play a central role in the development of the disease. However, there are no studies addressing the impact of diet on bile acid metabolism in patients with NAFLD. In juvenile Iberian pigs, we show that fatty acid composition in high-fat high-fructose diets affects BA levels in liver, plasma, and colon but these changes were not associated with the severity of the disease.

High-Fructose, High-Fat Diet Alters Muscle Composition and Fuel Utilization in a Juvenile Iberian Pig Model of Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is a serious metabolic condition affecting millions of people worldwide. A "Western-style diet" has been shown to induce pediatric NAFLD with the potential disruption of skeletal muscle composition and metabolism. To determine the in vivo effect of a "Western-style diet" on pediatric skeletal muscle fiber type and fuel utilization, 28 juvenile Iberian pigs were fed either a control diet (CON) or a high-fructose, high-fat diet (HFF), with or without probiotic supplementation, for 10 weeks. The HFF diets increased the total triacylglycerol content of muscle tissue but decreased intramyocellular lipid (IMCL) content and the number of type I (slow oxidative) muscle fibers. HFF diets induced autophagy as assessed by LC3I and LC3II, and inflammation, as assessed by IL-1α. No differences in body composition were observed, and there was no change in insulin sensitivity, but HFF diets increased several plasma acylcarnitines and decreased expression of lipid oxidation regulators PGC1α and CPT1, suggesting disruption of skeletal muscle metabolism. Our results show that an HFF diet fed to juvenile Iberian pigs produces a less oxidative skeletal muscle phenotype, similar to a detraining effect, and reduces the capacity to use lipid as fuel, even in the absence of insulin resistance and obesity.

Turning crocodilian hearts into bird hearts: growth rates are similar for alligators with and without right-to-left cardiac shunt.

The functional and possible adaptive significance of non-avian reptiles' dual aortic arch system and the ability of all non-avian reptiles to perform central vascular cardiac shunts have been of great interest to comparative physiologists. The unique cardiac anatomy of crocodilians - a four-chambered heart with the dual aortic arch system - allows for only right-to-left (R-L; pulmonary bypass) cardiac shunt and for surgical elimination of this shunt. Surgical removal of the R-L shunt, by occluding the left aorta (LAo) upstream and downstream of the foramen of Panizza, results in a crocodilian with an obligatory, avian/mammalian central circulation. In this study, R-L cardiac shunt was eliminated in age-matched, female American alligators (Alligator mississippiensis; 5-7 months of age). We tested the hypothesis that surgical elimination of R-L cardiac shunt would impair growth (a readily measured proxy for fitness) compared with sham-operated, age-matched controls, especially in animals subjected to exhaustive exercise. While regular exercise caused a decrease in size (snout-to-vent length, head length and body mass), elimination of the capacity for R-L cardiac shunt did not greatly reduce animal growth, despite a chronic ventricular enlargement in surgically altered juvenile alligators. We speculate that, despite being slightly smaller, alligators with an occluded LAo would have reached sexual maturity in the same breeding season as control alligators. This study suggests that crocodilian R-L cardiac shunt does not provide an adaptive advantage for juvenile alligator growth and supports the logic that cardiac shunts persist in crocodilians because they have not been selected against.

Surgical removal of right-to-left cardiac shunt in the American alligator (Alligator mississippiensis) causes ventricular enlargement but does not alter apnoea or metabolism during diving.

Crocodilians have complete anatomical separation between the ventricles, similar to birds and mammals, but retain the dual aortic arch system found in all non-avian reptiles. This cardiac anatomy allows surgical modification that prevents right-to-left (R-L) cardiac shunt. A R-L shunt is a bypass of the pulmonary circulation and recirculation of oxygen-poor blood back to the systemic circulation and has often been observed during the frequent apnoeic periods of non-avian reptiles, particularly during diving in aquatic species. We eliminated R-L shunt in American alligators (Alligator mississippiensis) by surgically occluding the left aorta (LAo; arising from right ventricle) upstream and downstream of the foramen of Panizza (FoP), and we tested the hypotheses that this removal of R-L shunt would cause afterload-induced cardiac remodelling and adversely affect diving performance. Occlusion of the LAo both upstream and downstream of the FoP for approximately 21 months caused a doubling of RV pressure and significant ventricular enlargement (average approximately 65%) compared with age-matched, sham-operated animals. In a separate group of recovered, surgically altered alligators allowed to dive freely in a dive chamber at 23 degrees C, occlusion of the LAo did not alter oxygen consumption or voluntary apnoeic periods relative to sham animals. While surgical removal of R-L shunt causes considerable changes in cardiac morphology similar to aortic banding in mammals, its removal does not affect the respiratory pattern or metabolism of alligators. It appears probable that the low metabolic rate of reptiles, rather than pulmonary circulatory bypass, allows for normal aerobic dives.

Exhaustive exercise training enhances aerobic capacity in American alligator (Alligator mississippiensis).

The oxygen transport system in mammals is extensively remodelled in response to repeated bouts of activity, but many reptiles appear to be 'metabolically inflexible' in response to exercise training. A recent report showed that estuarine crocodiles (Crocodylus porosus) increase their maximum metabolic rate in response to exhaustive treadmill training, and in the present study, we confirm this response in another crocodilian, American alligator (Alligator mississippiensis). We further specify the nature of the crocodilian training response by analysing effects of training on aerobic [citrate synthase (CS)] and anaerobic [lactate dehydrogenase (LDH)] enzyme activities in selected skeletal muscles, ventricular and skeletal muscle masses and haematocrit. Compared to sedentary control animals, alligators regularly trained for 15 months on a treadmill (run group) or in a flume (swim group) exhibited peak oxygen consumption rates higher by 27 and 16%, respectively. Run and swim exercise training significantly increased ventricular mass (~11%) and haematocrit (~11%), but not the mass of skeletal muscles. However, exercise training did not alter CS or LDH activities of skeletal muscles. Similar to mammals, alligators respond to exercise training by increasing convective oxygen transport mechanisms, specifically heart size (potentially greater stroke volume) and haematocrit (increased oxygen carrying-capacity of the blood). Unlike mammals, but similar to squamate reptiles, alligators do not also increase citrate synthase activity of the skeletal muscles in response to exercise.

Temperature effects on metabolic rate of juvenile Pacific bluefin tuna Thunnus orientalis.

Pacific bluefin tuna inhabit a wide range of thermal environments across the Pacific ocean. To examine how metabolism varies across this thermal range, we studied the effect of ambient water temperature on metabolic rate of juvenile Pacific bluefin tuna, Thunnus thynnus, swimming in a swim tunnel. Rate of oxygen consumption (MO2) was measured at ambient temperatures of 8-25 degrees C and swimming speeds of 0.75-1.75 body lengths (BL) s(-1). Pacific bluefin swimming at 1 BL s(-1) per second exhibited a U-shaped curve of metabolic rate vs ambient temperature, with a thermal minimum zone between 15 degrees C to 20 degrees C. Minimum MO2 of 175+/-29 mg kg(-1) h(-1) was recorded at 15 degrees C, while both cold and warm temperatures resulted in increased metabolic rates of 331+/-62 mg kg(-1) h(-1) at 8 degrees C and 256+/-19 mg kg(-1) h(-1) at 25 degrees C. Tailbeat frequencies were negatively correlated with ambient temperature. Additional experiments indicated that the increase in MO2 at low temperature occurred only at low swimming speeds. Ambient water temperature data from electronic tags implanted in wild fish indicate that Pacific bluefin of similar size to the experimental fish used in the swim tunnel spend most of their time in ambient temperatures in the metabolic thermal minimum zone.

Influence of swimming speed on metabolic rates of juvenile pacific bluefin tuna and yellowfin tuna.

Bluefin tuna are endothermic and have higher temperatures, heart rates, and cardiac outputs than tropical tuna. We hypothesized that the increased cardiovascular capacity to deliver oxygen in bluefin may be associated with the evolution of higher metabolic rates. This study measured the oxygen consumption of juvenile Pacific bluefin Thunnus orientalis and yellowfin tuna Thunnus albacares swimming in a swim-tunnel respirometer at 20 degrees C. Oxygen consumption (Mo2) of bluefin (7.1-9.4 kg) ranged from 235+/-38 mg kg(-1) h(-1) at 0.85 body length (BL) s(-1) to 498+/-55 mg kg(-1) h(-1) at 1.80 BL s(-1). Minimal metabolic rates of swimming bluefin were 222+/-24 mg O(2) kg(-1) h(-1) at speeds of 0.75 to 1.0 BL s(-1). Mo2 of T. albacares (3.7-7.4 kg) ranged from 164+/-18 mg kg(-1) h(-1) at 0.65 BL s(-1) to 405+/-105 mg kg(-1) h(-1) at 1.8 BL s(-1). Bluefin tuna had higher metabolic rates than yellowfin tuna at all swimming speeds tested. At a given speed, bluefin had higher metabolic rates and swam with higher tailbeat frequencies and shorter stride lengths than yellowfin. The higher M dot o2 recorded in Pacific bluefin tuna is consistent with the elevated cardiac performance and enhanced capacity for excitation-contraction coupling in cardiac myocytes of these fish. These physiological traits may underlie thermal-niche expansion of bluefin tuna relative to tropical tuna species.

In situ cardiac performance of Pacific bluefin tuna hearts in response to acute temperature change.

This study reports the cardiovascular physiology of the Pacific bluefin tuna (Thunnus orientalis) in an in situ heart preparation. The performance of the Pacific bluefin tuna heart was examined at temperatures from 30 degrees C down to 2 degrees C. Heart rates ranged from 156 beats min(-1) at 30 degrees C to 13 beats min(-1) at 2 degrees C. Maximal stroke volumes were 1.1 ml x kg(-1) at 25 degrees C and 1.3 ml x kg(-1) at 2 degrees C. Maximal cardiac outputs were 18.1 ml x kg(-1) min(-1) at 2 degrees C and 106 ml x kg(-1) min(-1) at 25 degrees C. These data indicate that cardiovascular function in the Pacific bluefin tuna exhibits a strong temperature dependence, but cardiac function is retained at temperatures colder than those tolerated by tropical tunas. The Pacific bluefin tuna's cardiac performance in the cold may be a key adaptation supporting the broad thermal niche of the bluefin tuna group in the wild. In situ data from Pacific bluefin are compared to in situ measurements of cardiac performance in yellowfin tuna and preliminary results from albacore tuna.

Temperature dependence of the Ca2+-ATPase (SERCA2) in the ventricles of tuna and mackerel.

Recent physiological studies on the cardiovascular performance of tunas suggest that the elevated heart rates of these fish may rely on increased use of intracellular sarcoplasmic reticulum (SR) Ca2+ stores. In this study, we compare the cellular cardiac performance in endothermic tunas (bluefin, albacore, yellowfin) and their ectothermic sister taxa (mackerel) in response to acute temperature change. The cardiac sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) plays a major role during cardiac excitation-contraction (E-C) coupling, transporting Ca2+ from the cytosol into the lumen of the SR and thus promoting the relaxation of the muscle. Measurements of oxalate-supported Ca2+ uptake in SR-enriched ventricular vesicles indicated that tunas were capable of sustaining a rate of Ca2+ uptake that was significantly higher than the mackerel. Among tunas, the cold-tolerant bluefin had the highest rates of SR Ca2+ uptake and ATPase activity. The differences among Ca2+ uptake and ATP hydrolysis rates do not seem to result from intrinsic differences between the SERCA2 present in the different tunas, as shown by their similar temperature sensitivities and similar values for activation energy. Western blots reveal that increased SERCA2 protein content is associated with the higher Ca2+ uptake and ATPase activities seen in bluefin ventricles compared with albacore, yellowfin, and mackerel. We hypothesize that a key step in the evolution of high heart rate and high metabolic rate in tunas is increased activity of the SERCA2 enzyme. We also suggest that high levels of SERCA2 in bluefin tuna hearts may be important for retaining cardiac function at cold temperatures.

Effects of temperature, epinephrine and Ca(2+) on the hearts of yellowfin tuna (Thunnus albacares).

Tuna are endothermic fish with high metabolic rates, cardiac outputs and aerobic capacities. While tuna warm their skeletal muscle, viscera, brain and eyes, their hearts remain near ambient temperature, raising the possibility that cardiac performance may limit their thermal niches. We used an in situ perfused heart preparation to investigate the effects of acute temperature change and the effects of epinephrine and extracellular Ca(2+) on cardiac function in yellowfin tuna (Thunnus albacares). Heart rate showed a strong temperature-dependence, ranging from 20 beats min(-1) at 10 degrees C to 109 beats min(-1) at 25 degrees C. Maximal stroke volume showed an inverse temperature-dependence, ranging from 1.4 ml kg(-1) at 15 degrees C to 0.9 ml kg(-1) at 25 degrees C. Maximal cardiac outputs were 27 ml kg(-1) min(-1) at 10 degrees C and 98 ml kg(-1) min(-1) at 25 degrees C. There were no significant effects of perfusate epinephrine concentrations between 1 and 100 nmol l(-1) at 20 degrees C. Increasing extracellular Ca(2+) concentration from 1.84 to 7.36 mmol l(-1) at 20 degrees C produced significant increases in maximal stroke volume, cardiac output and myocardial power output. These data demonstrate that changes in heart rate and stroke volume are involved in maintaining cardiac output during temperature changes in tuna and support the hypothesis that cardiac performance may limit the thermal niches of yellowfin tuna.