Impaired ketogenesis in iron-deficient rat pups

Livers of Fe-deficient rat pups contain significantly less carnitine and more triacylglycerol (TG) than livers of control pups. Carnitine affects ketogenesis (KG), which is a vital adaptation in the neonate. To determine if KG is impaired by low carnitine in Fe-deficient pup liver, ketone body synthesis was measured in liver mitochondria from 15-d-old pups. Litters from Fe-deficient (-Fe) and Fe-adequate (+Fe) dams were orally supplemented with water (W) as a control, 18 mM ferrous sulfate, or 10 mM L-carnitine from d 8 through d 15 of lactation. The amount of ketone bodies (beta-hydroxybutyrate + acetoacetate) synthesized was 68% less (P less than 0.05) in -FeW pups than in +FeW pups. Iron or carnitine supplementation increased KG in -Fe pups to +Fe KG levels, but carnitine did not affect KG in +Fe pups. Liver TG in +Fe pups was not altered by supplementation, but liver TG was -FeW pups. The data support the hypothesis that in the Fe-deficient suckling rat -FeW pups. The data support the hypothesis that in the Fe-deficient suckling rat, low carnitine levels may contribute to impaired ketogenesis and increased lipids in liver.     

Carnitine levels in iron-deficient rat pups

Hypertriglyceridemia and fatty livers have been observed in pups of Fe-deficient rats. Lowered tissue carnitine level is proposed as a mechanism responsible for altered lipid metabolism. Two hydroxylases involved in carnitine synthesis have been shown to require Fe in vitro. To determine if dietary Fe deficiency reduces tissue carnitine levels, two groups of 12 rats were fed 6 ppm Fe (-Fe) or 250 ppm Fe (+Fe) ad libitum from d 1 gestation to d 16 lactation. Feeding -Fe diets to dams resulted in 15% lower hemoglobin levels in pups on d 2 (P less than 0.02) and 50% lower levels on d 16 (P less than 0.001). Total carnitine level (nanomoles/milligram noncollagen protein) and triacylglycerol were assayed in pup tissues on d 2 and 16. While tissue carnitine and triacyglycerol was similar on d 2, d 16 liver carnitine was lower (P less than 0.001), triacylglycerol was eightfold higher in -Fe pups than in controls. Fe deficiency did not alter either carnitine concentration in milk on d 2 or 16 or the concentration of amino acid precursors of carnitine in milk on d 16. Decreased carnitine levels in the -Fe rat pup are contribute to triacylglycerol accumulation in liver.     

Exercise training and supplementation with carnitine and antioxidants increases carnitine stores, triglyceride utilization, and endurance in exercising rats

This study evaluated the effects of supplementation of carnitine and antioxidants on lipids, carnitine concentrations, and exercise endurance time in both trained and untrained rats as compared to non-supplemented rats. Thirty-two male SD rats, age 7 wk were divided into four groups according to exercise training and modified AIN-76 diets: NTNS (non-trained non-supplemented), NTS (non-trained supplemented), LTNS (long-trained non-supplemented) and LTS (long-trained supplemented). The trained rats were run on a treadmill for 60 min per day (10(0) incline, 20 m/min for 8 wk). Carnitine (0.5%/diet) and vitamin E (0.5 mg/g b.w.) were supplemented in rat diets and vitamin C (0.5 mg/g b.w.) and melatonin (1 microg/g b.w.) were administered into the stomachs of the rats. LTNS and LTS rats had significantly lower serum total lipid, triglyceride, total cholesterol and liver triglycerides, but had higher serum HDL-cholesterol. There were no changes in exercise endurance time by supplementation in untrained animals, however endurance times were longer in LTS animals than in LTNS. The supplementation and training tended to increase carnitine palmitoyltransferase (CPT-I) activities, although the differences were not statistically significant. Likewise, CPT-I mRNA levels were higher in both supplemented and exercise trained rats. These results suggest that supplementation of carnitine and antioxidants may improve lipid profiles and exercise ability in exercise-trained rats.     

Development of iron deficiency decreases zinc requirement of rats

Effects of the development of Fe deficiency on changes in Fe and Zn metabolism and its possible interactions with dietary Zn were determined. Adequate (25 microg/g) and marginally deficient (5 microg/g) Zn diets containing a sufficient (40 microg/g) dietary Fe levels were fed for 2 wk. Thereafter, both dietary Zn groups were fed an Fe-deficient (2.2 microg/g) diet for 4 wk. It was found that the effects of an Fe-deficient diet began to occur 7 and 14 d after feeding the Fe-deficient diet. At this time, tissue Fe concentrations were depleted and rats were unable to maintain hemoglobin levels. The Fe-deficient diet also induced an immediate fall in plasma Fe concentration, transferrin saturation, and apparent Fe absorption, while the concentrations of liver cytochrome c increased as Fe deficiency developed. Decreases in liver and spleen Fe levels, as well as the activities of blood and bone marrow aminolevulinic acid dehydratase (ALA-D, EC 4.2.1.24) were observed 3, 7, and 14 d after feeding the Fe-deficient diet, and thereafter they were increased. On the other hand, the activity of plasma alkaline phosphatase (ALK-P, EC 3.1.3.1) decreased continuously as Fe deficiency progressed. With severe development of Fe deficiency, rats fed the Zn-adequate diet had increased levels of Zn concentration in the plasma, liver, spleen, kidney, and femur, whereas apparent Zn absorption was decreased. The decrease in apparent Zn absorption and the increase in tissue Zn concentration of rats might be related to the lowered Zn requirement, which is associated with the depressed Zn metabolism caused by feeding Fe-deficient diets.     

Necessity of carnitine supplementation in semistarved rats fed a high-fat diet.

We investigated the effects of carnitine supplementation on lipid metabolism in semistarved rats. The semistarved rats were fed a high-fat diet and half the normal energy intake for 2 wk. Carnitine was supplied daily at a dose of 250 mg/kg of body weight. The results showed that the concentration of plasma free carnitine increased significantly in semistarved and carnitine-supplemented rats compared with normal and semistarved rats. The activities of muscle carnitine palmitoyltransferase I and preheparin plasma lipoprotein lipase also were significantly increased in semistarved and carnitine-supplemented rats. The plasma triacylglycerol secretion rate was restored to normal by carnitine supplementation in semistarved rats. Urinary excretion of ketone bodies was reduced significantly after carnitine supplementation. We concluded that supplementation of carnitine can significantly increase the concentration of plasma free carnitine and improve lipid metabolism in semistarved rats fed a high-fat diet.     

Changes in kinetics of carnitine palmitoyltransferase in liver and skeletal muscle of dogs (Canis familiaris) throughout growth and development

This study was conducted to investigate developmental changes in the kinetics of carnitine palmitoyltransferase (CPT) within hepatic and skeletal muscle tissues of the canine species. Carnitine concentrations, CPT activity and the apparent K(m) for carnitine were measured in tissue homogenates from dogs in six age categories: newborn; 24-h-old; 3-, 6- and 9-wk-old; and adult. Hepatic CPT activity was low at birth, increased by 100% during the suckling period (P < 0.05) and then declined after weaning to adult levels. In contrast, CPT activity in muscle continued to increase with age, reaching adult levels after 9 wk. Congruent with CPT activity, nearly identical concentration profiles of liver and muscle acylcarnitines were observed. The apparent K(m) of hepatic CPT for carnitine also paralleled the increase in CPT activity during the suckling period; however, free and total liver carnitine concentrations declined by 50% during this time (P < 0.05). Beginning at 3 wk of age, the hepatic concentration of free carnitine was at or below the apparent K(m) of CPT for carnitine. A similar relationship existed in muscle of young dogs, but in adults, the free carnitine concentration was markedly increased and exceeded the apparent K(m) by 5-fold. Collectively, we infer that fatty acid oxidation capacity increases rapidly after birth in the canine, after ontogenic increases in CPT activity. Furthermore, based on the relatively low tissue carnitine concentrations when compared with the apparent carnitine K(m) of CPT, we suggest that carnitine may have an important role in the regulation of fatty acid oxidation and that increased dietary carnitine may improve fatty acid oxidative capacity in developing dogs.     

Effects of developmental iron deficiency and post-weaning iron repletion on the levels of iron transporter proteins in rats

BACKGROUND/OBJECTIVESIron deficiency in early life is associated with developmental problems, which may persist until later in life. The question of whether iron repletion after developmental iron deficiency could restore iron homeostasis is not well characterized. In the present study, we investigated the changes of iron transporters after iron depletion during the gestational-neonatal period and iron repletion during the post-weaning period.MATERIALS/METHODSPregnant rats were provided iron-deficient (< 6 ppm Fe) or control (36 ppm Fe) diets from gestational day 2. At weaning, pups from iron-deficient dams were fed either iron-deficient (ID group) or control (IDR group) diets for 4 week. Pups from control dams were continued to be fed with the control diet throughout the study period (CON).RESULTSCompared to the CON, ID rats had significantly lower hemoglobin and hematocrits in the blood and significantly lower tissue iron in the liver and spleen. Hepatic hepcidin and BMP6 mRNA levels were also strongly down-regulated in the ID group. Developmental iron deficiency significantly increased iron transporters divalent metal transporter 1 (DMT1) and ferroportin (FPN) in the duodenum, but decreased DMT1 in the liver. Dietary iron repletion restored the levels of hemoglobin and hematocrit to a normal range, but the tissue iron levels and hepatic hepcidin mRNA levels were significantly lower than those in the CON group. Both FPN and DMT1 protein levels in the liver and in the duodenum were not different between the IDR and the CON. By contrast, DMT1 in the spleen was significantly lower in the IDR, compared to the CON. The splenic FPN was also decreased in the IDR more than in the CON, although the difference did not reach statistical significance.CONCLUSIONSOur findings demonstrate that iron transporter proteins in the duodenum, liver and spleen are differentially regulated during developmental iron deficiency. Also, post-weaning iron repletion efficiently restores iron transporters in the duodenum and the liver but not in the spleen, which suggests that early-life iron deficiency may cause long term abnormalities in iron recycling from the spleen.     

Copper metabolism in iron-deficient maternal and neonatal rats

Groups of rats were fed diets providing 8 ppm iron (-Fe) and 250 ppm iron (+Fe) throughout pregnancy and lactation. In spite of the increase in apparent absorption of iron in pregnant -Fe dams, iron deficiency anemia developed, resulting in decreased iron levels in placenta, amniotic fluid and fetal liver. Copper concentration of amniotic fluid was elevated in -Fe dams. On day 17 of lactation, -Fe dams and their suckling pups had hematologic evidence of iron deficiency. While liver and spleen iron decreased in 17-day-old pups, levels of copper increased. Subcellularly, the greatest increase in hepatic copper in -Fe pups was found in the cytosol, thus the increased copper deposition is not similar to copper loading. Serum ceruloplasmin activity was significantly elevated in -Fe lactating dams and was slightly, but not significantly, increased in -Fe pregnant dams and suckling pups.     

Triiodothyronine (T3) and thyroxine (T4) levels in iron-deficient,hypertriglyceridemic rats

Two experiments were conducted to examine the role and status of thyroid hormone in iron-deficient hypertriglyceridemic rats. In Experiment I, male weanling rats were fed 6 diets containing 2 levels of dietary iron [6 ppm(?Fe) and 240 ppm(+Fe)] and 3 combinations of fats [14% beef tallow +1% safflower oil(B), 7.5% beef tallow +7.5% safflower oil(BS) and 15% safflower(S)]. After 3 weeks, rats receiving ?Fe diets had elevated triglyceride (TG) and depressed triiodothyronine(T3) and thyroxine (T4) levels in serum, regardless of the fat source. No difference in the post-heparin lipolytic activity could be detected after 6 weeks of dietary treatment between rats fed +Fe and ?Fe diets. Feeding the BS+Fe diet to the BS?Fe rats for 1 week brought their serum TG, T3, and T4 to levels similar to those of rats fed the BS+Fe diet for 7 weeks. In Experiment II, male weanling rats were fed B diets containing 6 levels of dietary iron (3, 6, 10, 15, 30, and 60 ppm) for 4 weeks. Weekly determinations of packed cell volume(PCV), serum T3, T4, and TG were made. PCV, serum T3 and T4 levels increased as dietary iron content increased. The relationship between serum TG level with dietary iron level or PCV was quadratic, curvilinear. Data also suggest that part of the iron effect on serum TG level is thyroid hormone related.     

In vivo evidence for a vitamin B-6 requirement in carnitine synthesis

The purpose of this study was to determine whether there is a requirement for vitamin B-6 (B6) in carnitine synthesis. Rats were fed a B6-deficient (-B6) (0.04 mg pyridoxine [PN]/kg) diet (ad libitum or meal-fed) or a control (+B6) (5.7 mg PN/kg) diet (ad libitum or pair-fed). These diets were fed for 6 wk, then some of the rats were repleted with the +B6 diet for 2 wk. Total acid-soluble carnitine (TCN) and free carnitine (FCN) levels were compared in the plasma, liver, skeletal muscle, heart muscle and urine and rats fed +B6 or -B6 diets. In -B6 rats vs. +B6 rats, TCN levels were significantly lower (P less than 0.05) in the plasma, skeletal muscle, heart muscle and urine, but not in the liver. However, if rats were fasted for 3 d, liver TCN concentration of -B6 rats was significantly lower than that of +B6 rats. After B6-deficient rats were repleted with the +B6 diet, the TCN level in the plasma, liver, skeletal muscle, heart muscle and urine returned to the levels of control rats. Thus, the decrease in TCN and FCN levels with a B6-deficient diet and the increase of these levels after B6 repletion provides evidence for the B6 requirement in the biosynthesis of carnitine.     

Dose-responsive alteration in hepatic lipid peroxidation and retinol metabolism with increasing dietary beta-carotene in iron deficient rats

Phosphatidylcholine hydroperoxide (PCOOH) levels are increased in the iron-deficient rat liver. We investigated the antioxidative effect of dietary beta-carotene and altered retinol metabolism in iron-deficient rats. Experiment 1: Male Wistar-strain rats were divided into six groups and fed a control diet, an iron-deficient diet, and iron-deficient diets with four different levels of dietary beta-carotene. The PCOOH concentration in the iron-deficient rat liver was decreased by supplementation with dietary beta-carotene. However, the beta-carotene dose response was not related to antioxidative potency. Hepatic and plasma beta-carotene concentrations were increased by iron deficiency. The hepatic retinol concentration was increased while the plasma retinol concentration was decreased in iron-deficient rats. Experiment 2: Male Wistar-strain rats were divided into two groups, with one group receiving a control diet with beta-carotene and the other an iron-deficient diet with beta-carotene. Intestinal iron was decreased and intestinal beta-carotene was unchanged in iron-deficient rats. The intestinal beta-carotene conversion ratio and beta-carotene cleavage enzyme activity were decreased in iron-deficient rats. Dietary beta-carotene played the role of an antioxidant in hepatic lipid peroxidation in the iron-deficient state, but there was no dose dependency. Moreover, intestinal beta-carotene cleavage and hepatic retinol release appear to be altered in iron-deficient rats.     

Sodium pivalate treatment reduces tissue carnitines and enhances ketosis in rats

Sodium pivalate, a compound conjugated to carnitine and excreted in the urine was used to induce a secondary carnitine deficiency. In the first series of experiments, rats received in their drinking water either 20 mmol/L sodium pivalate (experimental) or 20 mmol/L sodium bicarbonate (control) for 4 d, 2 wk, or 8 wk. Tissues and urine were collected, and carnitine concentrations in liver, skeletal muscle, heart, plasma and urine were determined. The total carnitine concentrations in tissues and plasma of pivalate-treated rats were significantly depressed (P less than 0.05) at all time points, except at 4 d for skeletal muscle and at 4 d and 2 wk for liver. The acylcarnitine:free carnitine ratios in urine and plasma of the pivalate-treated animals were significantly higher at all time points relative to the controls. In the second experiment, rats received either the pivalate or the bicarbonate treatments for 15 d followed by a 2-d fast. After fasting, the plasma beta-hydroxybutyrate of pivalate-treated rats was significantly higher relative to controls, but there was no significant difference in plasma glucose concentrations. The reduced plasma and tissue carnitine concentrations, increased acylcarnitine:free carnitine ratio in plasma and urine, and fasting ketosis found in pivalate-treated rats are findings also reported for human secondary carnitine deficiency due to organic acidurias.     

Effects of Iron Supplementation on Testicular Function and Spermatogenesis of Iron-Deficient Rats

Iron deficiency is the most common micronutrient deficiency in the world. Previous studies have shown that iron deficiency increases oxidative stress and decreases antioxidant enzymes, and studies of male infertility indicated that oxidative stress may affect male reproductive functions. The aim of this study was to investigate the effects of iron supplementation on spermatogenesis and testicular functions in iron-deficient rats. Three-week-old male Sprague Dawley (SD) rats were randomly divided into two groups: an iron-adequate control (AI group, 35 ppm FeSO₄) and an iron-deficient group (ID group, <5 ppm FeSO₄). After three weeks, the iron-deficient group was divided into an original iron-deficient group and five iron-supplemented groups, the latter fed diets containing different doses of FeSO₄ (6, 12, 18, 24, and 35 ppm). After five weeks, blood and testis tissue were analyzed. We presented as median (interquartile range, IQR) for continuous measurements and compared their differences using the Kruskal–Wallis test followed by the Mann–Whitney U test among groups. The results showed that as compared with the AI group, the ID group had significantly lower serum testosterone and poorer spermatogenesis (The medians (QR) were 187.4 (185.6–190.8) of AI group vs. 87.5 (85.7–90.4) of ID group in serum testosterone, p < 0.05; 9.3 (8.8–10.6) of AI group vs. 4.9 (3.4–5.4) of ID group in mean testicular biopsy score (MTBS], p < 0.05); iron supplementation reversed the impairment of testis tissue. In the testosterone biosynthesis pathway, iron supplementation improved the lowered protein expressions of hydroxysteroid dehydrogenases caused by iron deficiency. Additionally, decreased activities of glutathione peroxidase and catalase, and increased cleaved-caspase 8 and caspase 3 expression, were found in the iron-deficient rats. The iron-supplemented rats that received > 12 ppm FeSO₄ exhibited improvements in antioxidant levels. In conclusion, iron supplementation can abrogate testis dysfunction due to iron deficiency through regulation of the testicular antioxidant capacity.     

Iron deficiency alters DMBA-induced tumor burden and natural killer cell cytotoxicity in rats.

Natural killer (NK) cell activity is impaired in iron-deficient rats. Natural killer cells destroy tumor cells; therefore, iron-deficient rats may be less able to combat cancer growth. Natural killer cell cytotoxicity, both basal and interferon gamma (IFN gamma)-stimulated, was studied in moderately and severely iron-deficient rats challenged with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Female weanling rats were fed ad libitum semipurified diets containing 8, 13 or 42 mg Fe/kg. A pair-fed group was fed the 42 mg Fe/kg diet at the level consumed by the 8 mg Fe/kg group. Following 6 wk of dietary treatment, DMBA-treated rats received a single intragastric dose of DMBA. Dietary treatment was continued. Rats were killed at 1, 4, 8, 14 and 20 wk post-DMBA treatment. Natural killer cell cytotoxicity (both basal and IFN gamma-stimulated) was analyzed. Feeding the 13 mg Fe/kg diet resulted in lower NK cell activity (P = 0.006) and greater tumor burden (P = 0.045) and tumor incidence. Interferon gamma treatment relieved the lower NK cell cytotoxicity observed in moderate iron deficiency. Feeding the 8 mg Fe/kg diet impaired NK cell activity (P = 0.006), but tumor burden and incidence were less than in moderate iron deficiency. In this model, iron deficiency, particularly moderate iron deficiency, contributed to cancer development and compromised NK cell cytotoxicity.     

Iron deficiency: effect on plasma luteinizing hormone and testosterone levels in the adult male rat

We studied four groups of animals, all of which received an iron-deficient diet for 6 wk followed by a 4-wk recovery period during which all groups received Fe supplements. Group 1 (n = 12) and group 2 (n = 10) were intact male rats; group 1 received a dietary Fe supplement whereas group 2 received no Fe supplement. Group 3 (n = 12) and group 4 (n = 12) were castrated male rats; group 3 received a dietary Fe supplement whereas group 4 received no supplement. Analysis of circulating hormone values revealed that after 6 wk of dietary treatment, neither LH nor testosterone levels were affected by the Fe-deficient diet in either the castrated or intact groups. These observations suggest that neither testosterone secretion per se nor its feedback control by LH is affected by short-term Fe deficiency.     

Effect of selenium depletion and repletion on plasma glutathione and glutathione-dependent enzymes in the rat

Selenium deficiency has several known biochemical effects. In the rat, these effects include loss of glutathione peroxidase (GSH-Px) activity, increased plasma glutathione concentration and increased liver glutathione S-transferase (GSH S-Tr) activity. The time course of the development of these changes in rats fed selenium-deficient diets and the time course of reversal of these changes in selenium-deficient rats fed graded levels of selenium were determined. As selenium deficiency was produced, liver cytosolic and plasma GSH-Px activities decreased first and were less than 5% of control when plasma glutathione concentration and liver GSH S-Tr activity began to increase. Elevated liver GSH S-Tr activity in selenium-deficient rats was corrected by refeeding selenium at the lowest level of supplementation (0.015 ppm) for 4 wk. GSH-Px activity required a supplementation of 0.10 ppm selenium for correction to control levels in 4 wk. Based on these studies a classification of the severity of selenium deficiency into mild, moderate and severe categories is proposed. In addition, the effect of dietary sulfur amino acid supplementation on plasma glutathione concentration was studied.     

Alteration of tissue magnesium levels in rats by dietary vitamin B6 supplementation

Tissues from female Fisher rats fed varying levels of magnesium (100 ppm or 700 ppm) and pyridoxine hydrochloride (PN.HCl) (7, 35, or 1500 mg/kg diet for six weeks) were dry ashed and analyzed for magnesium using atomic absorption spectroscopy. In tissues from rats consuming magnesium diets, tissue magnesium levels increased as levels of dietary PN.HCl increased. Increases in plasma, liver, kidney, and brain magnesium levels were statistically significant (p less than 0.05). With the exception of liver, no statistically significant changes in tissue magnesium levels occurred with PN.HCl supplementation in rats fed diets with adequate magnesium. Dietary supplementation with PN.HCl produces alterations in tissue magnesium levels in the rat and these alterations are modulated by dietary magnesium and PN.HCl supplementation.     

Nicotinamide Riboside Supplementation to Suckling Male Mice Improves Lipid and Energy Metabolism in Skeletal Muscle and Liver in Adulthood

Nicotinamide riboside, an NAD⁺ precursor, has been attracting a lot of attention in recent years due to its potential benefits against multiple metabolic complications and age-related disorders related to NAD⁺ decline in tissues. The metabolic programming activity of NR supplementation in early-life stages is much less known. Here, we studied the long-term programming effects of mild NR supplementation during the suckling period on lipid and oxidative metabolism in skeletal muscle and liver tissues using an animal model. Suckling male mice received a daily oral dose of NR or vehicle (water) from day 2 to 20 of age, were weaned at day 21 onto a chow diet, and at day 90 were distributed to either a high-fat diet (HFD) or a normal-fat diet for 10 weeks. Compared to controls, NR-treated mice were protected against HFD-induced triacylglycerol accumulation in skeletal muscle and displayed lower triacylglycerol levels and steatosis degree in the liver and distinct capacities for fat oxidation and decreased lipogenesis in both tissues, paralleling signs of enhanced sirtuin 1 and AMP-dependent protein kinase signaling. These pre-clinical findings suggest that mild NR supplementation in early postnatal life beneficially impacts lipid and energy metabolism in skeletal muscle and liver in adulthood, serving as a potential preventive strategy against obesity-related disorders characterized by ectopic lipid accumulation.