Leptin Receptor Deficiency Results in Hyperphagia and Increased Fatty Acid Mobilization during Fasting in Rainbow Trout (Oncorhynchus mykiss).

Leptin is a pleiotropic hormone known for regulating appetite and metabolism. To characterize the role of leptin signaling in rainbow trout, we used CRISPR/Cas9 genome editing to disrupt the leptin receptor (LepR) genes, lepra1 and lepra2. We compared wildtype (WT) and mutant fish that were either fed to satiation or feed deprived for six weeks. The LepR mutants exhibited a hyperphagic phenotype, which led to heavier body weight, faster specific growth rate, increased viscero- and hepatosomatic indices, and greater condition factor. Muscle glycogen, plasma leptin, and leptin transcripts (lepa1) were also elevated in fed LepR mutant fish. Expression levels of several hypothalamic genes involved in feed regulation were analyzed (agrp, npy, orexin, cart-1, cart-2, pomc-a1, pomc-b). No differences were detected between fed WT and mutants except for pomc-b (proopiomelanocortin-b), where levels were 7.5-fold higher in LepR fed mutants, suggesting that pomc-b expression is regulated by leptin signaling. Fatty acid (FA) content did not statistically differ in muscle of fed mutant fish compared to WT. However, fasted mutants exhibited significantly lower muscle FA concentrations, suggesting that LepR mutants exhibit increased FA mobilization during fasting. These data demonstrate a key role for leptin signaling in lipid and energy mobilization in a teleost fish.

Time-restricted feeding mice a high-fat diet induces a unique lipidomic profile.

Time-restricted feeding (TRF) can reduce adiposity and lessen the co-morbidities of obesity. Mice consuming obesogenic high-fat (HF) diets develop insulin resistance and hepatic steatosis, but have elevated indices of long-chain polyunsaturated fatty acids (LCPUFA) that may be beneficial. While TRF impacts lipid metabolism, scant data exist regarding the impact of TRF upon lipidomic composition of tissues. We (1) tested the hypothesis that TRF of a HF diet elevates LCPUFA indices while preventing insulin resistance and hepatic steatosis and (2) determined the impact of TRF upon the lipidome in plasma, liver, and adipose tissue. For 12 weeks, male, adult mice were fed a control diet ad libitum, a HF diet ad libitum (HF-AL), or a HF diet with TRF, 12 hours during the dark phase (HF-TRF). HF-TRF prevented insulin resistance and hepatic steatosis resulting from by HF-AL treatment. TRF-blocked plasma increases in LCPUFA induced by HF-AL treatment but elevated concentrations of triacylglycerols and non-esterified saturated fatty acids. Analysis of the hepatic lipidome demonstrated that TRF did not elevate LCPUFA while reducing steatosis. However, TRF created (1) a separate hepatic lipid signature for triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine species and (2) modified gene and protein expression consistent with reduced fatty acid synthesis and restoration of diurnal gene signaling. TRF increased the saturated fatty acid content in visceral adipose tissue. In summary, TRF of a HF diet alters the lipidomic profile of plasma, liver, and adipose tissue, creating a third distinct lipid metabolic state indicative of positive metabolic adaptations following HF intake.

Dietary saturated fatty acid type impacts obesity-induced metabolic dysfunction and plasma lipidomic signatures in mice.

Saturated fatty acid (SFA) intake is associated with obesity, insulin resistance, and hepatic steatosis, but scant work examines the impact of SFA type upon these outcomes. We tested the hypothesis that an obesogenic diet prepared with medium chain SFA (MCSFA), mostly as lauric acid-derived from coconut oil, reduces obesity-induced outcomes compared to obesogenic diets prepared with increasing amounts long chain SFA (LCSFA), primarily palmitic acid. Mice were fed (16 weeks) a control, low fat diet or obesogenic diets prepared with differing content of MCSFA or LCSFA in which polyunsaturated and monounsaturated fatty acids (PUFA; MUFA) were kept constant. Inclusion of MCSFA in an obesogenic diet prevented hepatic lipid accumulation and lowered indices of insulin resistance. Obesogenic diets reduced hepatic levels of de novo lipogenesis proteins (SCD1 and FASN) but elevated the adipose levels of mRNA for the pro-inflammatory markers Mcp-1 and Tnfα. Lipidomic analysis of plasma indicated that MCSFA intake resulted in a different lipidomic signature than LCSFA intake, prevented elevation of pro-inflammatory ceramides, but elevated concentrations of some lipids associated with elevated cardiovascular disease risk. Intake of the obesogenic diets in an SFA-type dependent manner elevated plasma concentrations of several phosphatidylcholine (PC) lipids having the long chain PUFA (LCPUFA) arachidonic acid (ARA) and docosahexaenoic acid (DHA), altered phospholipid ethers, and changed the triacylglyceryl environments of these LCPUFA. Our data indicate that (1) MCSFA reduce the severity of some obesogenic co-morbidities, (2) SFA-type modulates lipidomic signatures associated with cardiovascular disease and diabetes, and (3) dietary SFA type impacts LCPUFA metabolism.

Comparative effects of high oleic acid vs high mixed saturated fatty acid obesogenic diets upon PUFA metabolism in mice.

Emerging evidence indicates that the fatty acid composition of obesogenic diets influences physiologic outcomes. There are scant data regarding how the content of non-essential fatty acids like monounsaturated fatty acids (MUFA) and saturated fatty acids (SFAs) impact the metabolism of polyunsaturated fatty acids (PUFAs). In this work, we tested the hypothesis that obesogenic diets enriched in oleic acid (OA; 18:1n-9) reduce polyunsaturated fatty acid (PUFA) levels vs an obesogenic diet enriched in SFAs. Adult male mice were fed for eight weeks either (1) a control 16% fat energy (en) diet with 5.7% en OA and 4.4% en SFA, (2) a 50% fat en diet with 33% en OA and 9.9% en SFA, or (3) a 50% en diet with a high SFA diet with 33% en SFA and 9.1% en OA. Dietary levels and intake of linoleic acid (LA; 18:2n-6) and α-linolenic acid (ALA; 18:3n-3) were constant between the experimental groups. Several peripheral organs (liver, heart, kidney, and adipose) were analyzed for lipid composition and oxylipin analysis was performed for liver and adipose. Our data demonstrate that a high OA diet reduced tissue content of LA and ALA (≥30%) in phospholipid and neutral lipid fractions, reduced the content of some LA-derived and ALA-derived oxylipins in liver and adipose, and conversely, elevated hepatic content of PGF2α. In all tissues examined, except for adipose, levels of arachidonic acid (ARA; 20:4n-6) and docosahexaenoic acid (DHA; 22:6n-3) were either elevated or unaffected by the obesogenic diets. Our data indicate that the non-essential fatty content of obesogenic diets impacts PUFA content in peripheral tissues and influences the levels of bioactive oxylipins.

PPAR mRNA Levels Are Modified by Dietary n-3 Fatty Acid Restriction and Energy Restriction in the Brain and Liver of Growing Rats.

BACKGROUND: Without dietary sources of n-3 (ω-3) long-chain polyunsaturated fatty acids (LCPUFAs), α-linolenic acid (ALA; 18:3n-3) is the precursor for docosahexaenoic acid (DHA; 22:6n-3). It is not known how energy restriction (ER) affects ALA conversion to DHA. OBJECTIVE: We tested the hypothesis that ER reduces n-3 LCPUFA concentrations in tissues of growing rats fed diets replete with and deficient in ALA. METHODS: Male Sprague-Dawley rats (23 d old) were provided AIN93G diets (4 wk) made with soybean oil (SO; ALA sufficient) or corn oil (CO; ALA deficient) providing 16% of energy as fat. For each dietary oil, ER rats were individually pair-fed 75% of another rat's ad libitum (AL) intake. Fatty acid (FA) concentrations in brain regions, liver, and plasma were analyzed. Expression of peroxisome proliferator-activated receptors (PPARs), uncoupling proteins (UCPs), and mitochondrial DNA was analyzed in the brain and liver. RESULTS: AL rats consuming CO had a 65% lower concentration of n-3 docosapentaenoic acid (22:5n-3) and a 10% lower DHA concentration in the cerebral cortex and cerebellum than did the SO-AL group. ER did not alter cerebral n-3 LCPUFA status. Liver n-3 LCPUFA concentrations were reduced in rats fed CO compared with SO. ER reduced hepatic linoleic acid (18:2n-6), ALA, and arachidonic acid (20:4n-6) regardless of oil. ER and n-3 FA deficiency had independent effects on the mRNA levels of Pparα, Pparß/δ, and Pparγ in the liver, cerebral cortex, and cerebellum. ER reduced Ucp3 mRNA by nearly 50% in the cerebral cortex, cerebellum, and liver, and Ucp5 mRNA was 30% lower in the cerebellum of rats receiving the CO diet. CONCLUSIONS: Small perturbations in PUFA concentration and ER modify the mRNA levels of Ppar and Ucp in the juvenile rat brain. More research is needed to identify the long-term physiologic and behavioral impacts of ER and PUFA restriction in the juvenile brain.

S-Glutathionylation of hepatic and visceral adipose proteins decreases in obese rats.

UNLABELLED: A number of clinical and biochemical studies demonstrate that obesity and insulin resistance are associated with increases in oxidative stress and inflammation. Paradoxically, insulin sensitivity can be enhanced by oxidative inactivation of cysteine residues of phosphatases, and inflammation can be reduced by S-glutathionylation with formation of protein-glutathione mixed disulfides (PSSG). Although oxidation of protein-bound thiols (PSH) is increased in multiple diseases, it is not known whether there are changes in PSH oxidation species in obesity. OBJECTIVE: In this work, the hypothesis that obesity is associated with decreased levels of proteins containing oxidized protein thiols was tested. DESIGN AND METHODS: The tissue levels of protein sulfenic acids (PSOH) and PSSG in liver, visceral adipose tissue, and skeletal muscle derived from glucose intolerant, obese-prone Sprague-Dawley rats were examined. RESULTS: The data in this study indicate that decreases in PSSG content occurred in liver (44%) and adipose (26%) but not skeletal muscle in obese rats that were fed a 45% fat-calorie diet versus lean rats that were fed a 10% fat-calorie diet. PSOH content did not change in the tissue between the two groups. The activity of the enzyme glutaredoxin (GLRX) responsible for reversal of PSSG formation did not change in muscle and liver between the two groups. However, levels of GLRX1 were elevated 70% in the adipose tissue of the obese, 45% fat calorie-fed rats. CONCLUSION: These are the first data to link changes in S-glutathionylation and GLRX1 to adipose tissue in the obese and demonstrate that redox changes in thiol status occur in adipose tissue as a result of obesity.

Differential responses to selenomethionine supplementation by sex and genotype in healthy adults.

A year-long intervention trial was conducted to characterise the responses of multiple biomarkers of Se status in healthy American adults to supplemental selenomethionine (SeMet) and to identify factors affecting those responses. A total of 261 men and women were randomised to four doses of Se (0, 50, 100 or 200 µg/d as L-SeMet) for 12 months. Responses of several biomarkers of Se status (plasma Se, serum selenoprotein P (SEPP1), plasma glutathione peroxidase activity (GPX3), buccal cell Se, urinary Se) were determined relative to genotype of four selenoproteins (GPX1, GPX3, SEPP1, selenoprotein 15), dietary Se intake and parameters of single-carbon metabolism. Results showed that supplemental SeMet did not affect GPX3 activity or SEPP1 concentration, but produced significant, dose-dependent increases in the Se contents of plasma, urine and buccal cells, each of which plateaued by 9-12 months and was linearly related to effective Se dose (µg/d per kg0·75). The increase in urinary Se excretion was greater for women than men, and for individuals of the GPX1 679 T/T genotype than for those of the GPX1 679 C/C genotype. It is concluded that the most responsive Se-biomarkers in this non-deficient cohort were those related to body Se pools: plasma, buccal cell and urinary Se concentrations. Changes in plasma Se resulted from increases in its non-specific component and were affected by both sex and GPX1 genotype. In a cohort of relatively high Se status, the Se intake (as SeMet) required to support plasma Se concentration at a target level (Se(pl-target)) is: Se(in) = [(Se(pl - target) - Se(pl))/(18.2ng d kg°.75/ml per mu g)] .

Calcium requirements of growing rats based on bone mass, structure, or biomechanical strength are similar.

Although calcium (Ca) supplementation increases bone density, the increase is small and the effect on bone strength and fracture risk is uncertain. To investigate if bone mass, morphology, and biomechanical properties are affected by deficient to copious dietary Ca concentrations, the long bones (tibia and femur) of growing female Sprague-Dawley rats (8/group) were assessed after 13 wk of consuming 1, 2, 3, 4, 5, 6, or 7 g Ca/kg of a modified AIN-93G diet. Dietary phosphorous (P) and vitamin D remained constant at recommended concentrations. The assessment included mineralization, density, biomechanical properties of breaking by a 3-point flexure test, and morphological properties by microcomputed topography scanning of trabecular bone of the proximal tibia metaphysis. Dietary treatment did not affect food intake, weight gain, renal and muscle Ca concentrations, and bone hydroxyproline. All bone parameters measured were significantly impaired by Ca deficiency in rats fed the diet containing 1 g Ca/kg. Modest impairments occurred with some parameters (bone density, biomechanical bending moment, modulus of elasticity, and stress) in rats fed 2 g Ca/kg, but all parameters stabilized between 2 and 3 g/kg diet, with no differences between 3 and 7 g/kg. The results suggest that a threshold response in bone Ca retention or bone mass at approximately 2.5 g Ca/kg diet is associated with similar threshold responses in bone breaking strength and related biomechanics as well as trabecular structural properties. There was no evidence of a relative P deficiency or of improved or impaired bone strength and structure as Ca intakes increased beyond those needed to maximize bone density.

The selenium metabolite methylselenol inhibits the migration and invasion potential of HT1080 tumor cells.

There is increasing evidence for the efficacy of certain forms of selenium as cancer-chemopreventive compounds. Methylselenol has been hypothesized to be a critical selenium metabolite for anticancer activity in vivo. To determine whether tumor cell migration, invasion, and cell cycle characteristics are inhibited by methylselenol, we exposed HT1080 cells to methylselenol. Methylselenol was generated with seleno-L-methionine (a substrate for methioninase). Submicromolar methylselenol exposure led to an increase in the G1 and G2 fractions with a concomitant drop in the S-phase, indicating slower cell growth. Furthermore, methylselenol inhibited the migration and invasion rate of the tumor cells by up to 53 and 76%, respectively, when compared with the control tumor cells. Although all cells had increased matrix metalloproteinase (MMP) enzyme activities of pro-MMP-2 and pro-MMP-9, the active form of MMP-2 was decreased in HT1080 cells cultured with methylselenol. In addition, methylselenol increased the protein levels of antimetastasic tissue inhibitor metalloproteinase (TIMP)-1 and TIMP-2. Collectively, these results demonstrate that submicromolar concentrations of methylselenol increase both prometastasis MMP-2 and MMP-9 and antimetastasis TIMP-1 and TIMP-2 expression. The apparent net effect of these changes is the inhibition of pro-MMP-2 activation and carcinogenic potential or activity.

Contrasting and cooperative effects of copper and iron deficiencies in male rats fed different concentrations of manganese and different sources of sulfur amino acids in an AIN-93G-based diet.

Dietary nutrient interactions are important factors to consider in the study of nutrient status and requirements. Here, the effects of dietary interactions among copper (Cu), iron (Fe), manganese (Mn) and sulfur amino acids (SAA) on blood cell characteristics and enzyme activities were observed. Male rats (n = 8) were used in a 2 x 2 x 2 x 2 factorial design and fed an AIN-93G-based diet containing dietary Cu (<1 and 5 mg/kg), Fe (10 and 35 mg/kg), Mn (10 and 50 mg/kg) and either L-cystine (LCys) or DL-methionine (DLMet). Blood was analyzed by automated hematology cell counting and by flow cytometry. Severe Cu deficiency was verified by reductions in the activities of serum ceruloplasmin (1% of control), RBC superoxide dismutase (SOD1) (14% of control), liver cytochrome c oxidase activity (25% of control) and serum extracellular SOD (SOD3) activity (20% of controls). Because Cu is required for Fe utilization, many physiologic responses that require Fe were affected by both deficiencies, including lowered blood hemoglobin (Hgb), lower RBC volume and Hgb concentration, and an increased number of reticulocytes. Cu and Fe deficiencies together worsened some conditions, i.e., lower Hgb, lower RBC Hgb, increased RBC distribution width, increased number of reticulocytes and nucleated RBC, and a higher platelet count. Increasing dietary Mn had little effect on most variables, except to reduce serum Cu when dietary Cu was adequate but not when it was low, and to reduce RBC SOD1 activity when dietary Fe was low but not when it was adequate. Hgb concentrations were higher (P < 0.002) in Cu-deficient rats fed LCys than in those fed DLMet. There was no effect in Cu-adequate rats. Hgb was higher (P < 0.004) in Fe-adequate rats fed LCys than in those fed DLMet, with no effect in Fe-deficient rats. Although the anemia of Cu deficiency in AIN-93G-fed rats was not as pronounced as that reported in rats fed the AIN-76A-based diet, other manifestations of the deficiency were prominent.