Supplementation-induced change in muscle carnosine is paralleled by changes in muscle metabolism, protein glycation and reactive carbonyl species sequestering.

Carnosine is a performance-enhancing food supplement with a potential to modulate muscle energy metabolism and toxic metabolites disposal. In this study we explored interrelations between carnosine supplementation (2 g/day, 12 weeks) induced effects on carnosine muscle loading and parallel changes in (i) muscle energy metabolism, (ii) serum albumin glycation and (iii) reactive carbonyl species sequestering in twelve (M/F=10/2) sedentary, overweight-to-obese (BMI: 30.0+/-2.7 kg/m2) adults (40.1+/-6.2 years). Muscle carnosine concentration (Proton Magnetic Resonance Spectroscopy; 1H-MRS), dynamics of muscle energy metabolism (Phosphorus Magnetic Resonance Spectroscopy; 31P-MRS), body composition (Magnetic Resonance Imaging; MRI), resting energy expenditure (indirect calorimetry), glucose tolerance (oGTT), habitual physical activity (accelerometers), serum carnosine and carnosinase-1 content/activity (ELISA), albumin glycation, urinary carnosine and carnosine-propanal concentration (mass spectrometry) were measured. Supplementation-induced increase in muscle carnosine was paralleled by improved dynamics of muscle post-exercise phosphocreatine recovery, decreased serum albumin glycation and enhanced urinary carnosine-propanal excretion (all p<0.05). Magnitude of supplementation-induced muscle carnosine accumulation was higher in individuals with lower baseline muscle carnosine, who had lower BMI, higher physical activity level, lower resting intramuscular pH, but similar muscle mass and dietary protein preference. Level of supplementation-induced increase in muscle carnosine correlated with reduction of protein glycation, increase in reactive carbonyl species sequestering, and acceleration of muscle post-exercise phosphocreatine recovery.

Altered dynamics of lipid metabolism in muscle cells from patients with idiopathic inflammatory myopathy is ameliorated by 6 months of training.

KEY POINTS: Regular exercise improves muscle functional capacity and clinical state of patients with idiopathic inflammatory myopathy (IIM). In our study, we used an in vitro model of human primary muscle cell cultures, derived from IIM patients before and after a 6-month intensive supervised training intervention to assess the impact of disease and exercise on lipid metabolism dynamics. We provide evidence that muscle cells from IIM patients display altered dynamics of lipid metabolism and impaired adaptive response to saturated fatty acid load compared to healthy controls. A 6-month intensive supervised exercise training intervention in patients with IIM mitigated disease effects in their cultured muscle cells, improving or normalizing their capacity to handle lipids. These findings highlight the putative role of intrinsic metabolic defects of skeletal muscle in the pathogenesis of IIM and the positive impact of exercise, maintained in vitro by yet unknown epigenetic mechanisms. ABSTRACT: Exercise improves skeletal muscle function, clinical state and quality of life in patients with idiopathic inflammatory myopathy (IIM). Our aim was to identify disease-related metabolic perturbations and the impact of exercise in skeletal muscle cells of IIM patients. Patients underwent a 6-month intensive supervised training intervention. Muscle function, anthropometric and metabolic parameters were examined and muscle cell cultures were established (m. vastus lateralis; Bergström needle biopsy) before and after training from patients and sedentary age/sex/body mass index-matched controls. [14 C]Palmitate was used to determine fat oxidation and lipid synthesis (thin layer chromatography). Cells were exposed to a chronic (3 days) and acute (3 h) metabolic challenge (the saturated fatty acid palmitate, 100 µm). Reduced oxidative (intermediate metabolites, -49%, P = 0.034) and non-oxidative (diglycerides, -38%, P = 0.013) lipid metabolism was identified in palmitate-treated muscle cells from IIM patients compared to controls. Three days of palmitate exposure elicited distinct regulation of oxidative phosphorylation (OxPHOS) complex IV and complex V/ATP synthase (P = 0.012/0.005) and adipose triglyceride lipase in patients compared to controls (P = 0.045) (immunoblotting). Importantly, 6 months of training in IIM patients improved lipid metabolism (CO2 , P = 0.010; intermediate metabolites, P = 0.041) and activation of AMP kinase (P = 0.007), and nearly normalized palmitate-induced changes in OxPHOS proteins in myotubes from IIM patients, in parallel with improvements of patients' clinical state. Myotubes from IIM patients displayed altered dynamics of lipid metabolism and impaired response to metabolic challenge with saturated fatty acid. Our observations suggest that metabolic defects intrinsic to skeletal muscle could represent non-immune pathomechanisms, which can contribute to muscle weakness in IIM. A 6-month training intervention mitigated disease effects in muscle cells in vitro, indicating the existence of epigenetic regulatory mechanisms.

Effects of running on adiponectin, insulin and cytokines in cerebrospinal fluid in healthy young individuals.

Exercise can prevent the sedentary lifestyle-related risk of metabolic and cognitive decline, but mechanisms and mediators of exercise effects on human brain are relatively unexplored. We measured acute exercise-induced changes in adiponectin, insulin and other bioactive molecules in cerebrospinal fluid (CSF) and serum from young lean individuals. Samples of serum and CSF were obtained before and 1-h after the 90-min run (75-80% HRmax; maximal heart rate), additional serum was taken at finish-line. Body composition, physical fitness, metabolic rate, cognitive functions, food preference, glucose, insulin and albumin were measured. The spectrum of 174 cytokines was assessed by protein arrays, adiponectin was also determined by ELISA and immunoblotting. CSF adiponectin decreased post-exercise by 21.3% (arrays) and 25.8% (ELISA) (p < 0.009). Immunoblotting revealed reduction in a low-molecular-weight-adiponectin (p < 0.005). CSF adiponectin positively correlated with CSF/serum albumin ratio (p < 0.022), an indicator of blood-brain-barrier permeability. CSF and serum adiponectin were positively associated with memory and running-induced changes in insulinemia and CSF insulin. Additionally, running modulated CSF levels of 16 other cytokines. Acute running reduced CSF adiponectin and modulated insulin and albumin in CSF and serum. Associations of adiponectin with memory and metabolism indicate the potential role of this bioactive molecule in mediating exercise-induced adaptive response in human brain.

Prior Repeated Stress Attenuates Cold-Induced Immunomodulation Associated with "Browning" in Mesenteric Fat of Rats.

Continuous exposure to cold leads to activation of adaptive thermogenesis in brown adipose tissue but also to induction of brown/beige cell phenotype in white adipose tissue. The aim of this work was to investigate whether prior exposure to immobilization (IMO) stress may affect immune response associated with adipocyte "browning" in mesenteric adipose tissue (mWAT). In the first experiment, Sprague-Dawley rats were exposed to acute (3 h) or prolonged (7 days) cold exposure (4 ± 1 °C). 7-day cold stimulated gene expression of uncoupling protein 1 and other "browning"-associated factors. In the second experiment, rats were immobilized for 7 days (2 h daily) followed by exposure to continuous cold for 1 or 7 days. Prior IMO exaggerated cold-induced sympathetic response manifested by elevated tyrosine hydroxylase (TH) protein and norepinephrine in mWAT. Induction of non-sympathetic catecholamine production demonstrated by elevated TH and PNMT (phenylethanolamine N-methyltransferase) mRNAs was observed after 7-day cold; however, prior IMO attenuated this response. 7-day cold-induced gene expression of anti-inflammatory mediators (IL-4, IL-13, IL-10, adiponectin), markers of M2 macrophages (Arg1, Retnlα), and eosinophil-associated molecules (eotaxin, IL-5), while inhibited expression of pro-inflammatory cytokines (IFNγ, IL-1b, IL-6, IL-17) and monocytes (MCP-1, Ly6C). This immune response was accompanied by elevated expression of uncoupling protein-1 and other thermogenic factors. Rats exposed to prior IMO exhibited inhibition of cold-induced immune and "browning"-related expression pattern. Overall, we demonstrated that 7-day cold-induced browning"-associated changes in rat mWAT, while prior history of repeated stress prevented this response.

Lipopolysaccharide induces catecholamine production in mesenteric adipose tissue of rats previously exposed to immobilization stress.

Catecholamines (CAs) are mainly produced by sympathoadrenal system but their de novo production has been also observed in adipose tissue cells. The aim of this work was to investigate whether immune challenge induced by lipopolysaccharide (LPS) modulates biosynthesis of CAs in mesenteric adipose tissue (MWAT), as well as whether previous exposure to immobilization (IMO) stress could modulate this process. Sprague-Dawley rats were exposed to single (2 h) or repeated (2 h/7 days) IMO and afterwards injected with LPS (i.p., 100 µg/kg body weight) and sacrificed 3 h later. LPS did not alter CA biosynthesis in MWAT in control rats. Single and repeated IMO elevated CAs and expression of CA biosynthetic enzymes in MWAT, including adipocyte and stromal/vascular fractions (SVF). Repeated IMO followed by LPS treatment led to the up-regulation of CA-biosynthetic enzymes expression, elevation of CAs in SVF but depletion of norepinephrine and epinephrine in adipocyte fraction. Prior IMO caused a marked LPS-induced macrophage infiltration in MWAT as evaluated by F4/80 expression. A positive correlation between expression of tyrosine hydroxylase and F4/80 suggests macrophages as the main source of LPS-induced CA production in MWAT. Furthermore, prior exposure to the single or repeated IMO differently affected immune responses following LPS treatment by modulation of inflammatory cytokine expression. These data suggest that stress might be a significant modulator of immune response in MWAT via stimulation of the macrophage infiltration associated with cytokine response and de novo production of CAs.

BCR-ABL1 mutation development during first-line treatment with dasatinib or imatinib for chronic myeloid leukemia in chronic phase.

BCR-ABL1 mutations are a common, well-characterized mechanism of resistance to imatinib as first-line treatment of chronic myeloid leukemia in chronic phase (CML-CP). Less is known about mutation development during first-line treatment with dasatinib and nilotinib, despite increased use because of higher response rates compared with imatinib. Retrospective analyses were conducted to characterize mutation development in patients with newly diagnosed CML-CP treated with dasatinib (n=259) or imatinib (n=260) in DASISION (Dasatinib versus Imatinib Study in Treatment-Naive CML-CP), with 3-year minimum follow-up. Mutation screening, including patients who discontinued treatment and patients who had a clinically relevant on-treatment event (no confirmed complete cytogenetic response (cCCyR) and no major molecular response (MMR) within 12 months; fivefold increase in BCR-ABL1 with loss of MMR; loss of CCyR), yielded a small number of patients with mutations (dasatinib, n=17; imatinib, n=18). Dasatinib patients had a narrower spectrum of mutations (4 vs 12 sites for dasatinib vs imatinib), fewer phosphate-binding loop mutations (1 vs 9 mutations), fewer multiple mutations (1 vs 6 patients) and greater occurrence of T315I (11 vs 0 patients). This trial was registered at www.clinicaltrials.gov as NCT00481247.

Obesogenic and diabetogenic impact of high organochlorine levels (HCB, p,p'-DDE, PCBs) on inhabitants in the highly polluted Eastern Slovakia.

OBJECTIVE: This study was aimed to evaluate possible obesogenic and diabetogenic impact of highly increased serum level of persistent organochlorinated pollutants POPs, such as polychlorinated biphenyls (PCBs), dichlorodiethyl-dichloroethylene (p,p'-DDE), and hexachlorobenzene (HCB), on the level of obesity markers (cholesterol and triglyceride level in serum, and body mass index [BMI]) and diabetes markers (fasting glucose and fasting insulin in serum) in inhabitants of Eastern Slovakia. METHODS: In young (21-40 years) males (n=248) and females (n=330) as well as in old (41-75 years) males (n=586) and females (n=889), the serum levels of 15 polychlorinated biphenyl congeners (Σ15PCBs), p,p'-DDE and HCB, and serum insulin, testosterone, total cholesterol, triglycerides and glucose levels have been estimated by high resolution gas chromatography/mass spectrometry and by the appropriate electrochemiluminiscent immunoassay or chemical methods, respectively. RESULTS: In both age groups of males and females, the levels of Σ15PCBs, p,p'-DDE, and HCB were very high and their mutual interrelations were highly significant (p<0.01). However, it should be noted that no significant changes were found in individual variables related to very high level of Σ15PCBs, except of increased BMI (p>0.05) in females.In all ages and gender groups, defined above general as related to increasing level of individual OCPs in individual age and gender groups, significant increase in cholesterol and triglyceride levels as well as BMI values, supported their obesogenic effect, while significant increase in fasting glucose and insulin in serum, supported their diabetogenic effect. Finally, highly significant decrease in testosterone level, as found in both young and old males, supported the antiandrogenic effect, namely of HCB. However, somewhat less of p,p'-DDE, while PCBs did not show any such effect in spite of their very high level. CONCLUSIONS: Highly increased blood levels of diabetes (fasting glucose and insulin) and obesity markers (cholesterol, triglyceride and BMI) were found in large groups of males and females in highly polluted area of Slovakia. Significant decrease in testosterone level was also observed in males.

Long-term liquid nutrition intake and development of obesity: differences between young and adult rats.

OBJECTIVE: Obesity is particularly associated with an increased consumption of palatable fat and sugar dense food and beverages. Therefore, we examined the effect of a normocaloric liquid diet (Fresubin) with increased carbohydrate content (constituting 55% of calories) on development of obesity in different developmental periods in male Wistar rats. METHODS: Fresubin was provided to 3 groups of rats: the first group received Fresubin immediately after weaning (21st day of age) to the end of experiment (150th day of age) for 5 months; the second group was fed with Fresubin from weaning to adulthood (90th day of age) for 3 months; and the third group received Fresubin only in adulthood (from 90th to 150th day of age) for 2 months. The control group was fed with standard pellet chow from weaning to the end of the experiment. Body weight, food and water intake were periodically measured. After terminating the experiment, the adiposity index was determined. RESULTS: Rats fed with liquid nutrition showed increased energy intake and body weight in comparison with the control rats. Interestingly, while obesity in the juvenile rats developed as late as of 13 weeks after the Fresubin intake, the adult rats fed with liquid nutrition had significantly elevated the body weight already 2 weeks after starting the treatment. Increased adiposity index was observed in both groups of rats fed with Fresubin during the whole study as well as the adulthood. CONCLUSIONS: Our data indicate that feeding of male Wistar rats with a high carbohydrate normocaloric diet results in a substantial development of obesity. Moreover, exposure of juvenile individuals to obesogenic environment leads, after a certain "latent period", to the development of obesity that may reflect low protein content of used liquid diet or higher resistance of juvenile organism to the obesogenic factors. Finally, based on the data obtained we suggest that Fresubin, with respect to its properties, may serve as a diet for the development of obesity which may exemplify an "obesity model" applicable in small laboratory animals.

Effects of hypoxia on adipose tissue expression of NFκB, IκBα, IKKγ and IKAP in patients with chronic obstructive pulmonary disease.

Recently we observed increased adipose tissue (AT) expression of CD40-related signaling proteins but no activation of tumor necrosis factor-α or CD68 in patients with chronic sustained hypoxia resulting from chronic obstructive pulmonary disease (COPD). Transcription factor nuclear factor-κB (NFκB) is involved in cellular responses to hypoxia and activates the proinflammatory gene expression with concomitant upregulation of its own repressors--inhibitors of κB (IκB) in an auto feedback loop. Inhibitor of kappaB kinase (IKK)-γ and inhibitor of kappaB kinase complex-associated protein (IKAP) are further regulatory proteins involved in NFκB signaling. In this study, we hypothesized that chronic sustained hypoxia significantly relates to IκBα, IKKγ and IKAP within the AT in COPD patients. In 20 patients with stable disease, samples of subcutaneous AT were analyzed using real-time PCR. Although no significant differences were observed between two groups categorized by median PaO2 in NFκB (p = 0.065), gene expressions of IκBα, IKKγ and IKAP were all higher in hypoxemic patients (p = 0.033; p = 0.050; p = 0.010, respectively). In multivariate analyses, PaO2 independently predicted AT IκBα, IKKγ and IKAP (R (2) = 0.490, p = 0.012; R (2) = 0.586, p = 0.002; R (2) = 0.504, p = 0.009, respectively). In conclusion, our data suggest significant AT upregulation of IκBα, IKKγ and IKAP by chronic sustained hypoxia in COPD patients.

Circulatory and adipose tissue leptin and adiponectin in relationship to resting energy expenditure in patients with chronic obstructive pulmonary disease.

Increases in resting energy expenditure (REE) likely contribute to weight loss in various chronic diseases. In chronic obstructive pulmonary disease (COPD), relationships between the ventilatory impairment and increased REE, and between disturbances in adipokines and weight loss were previously described. Therefore, we investigated serum levels and adipose tissue expression of leptin and adiponectin, and their relationships to REE in patients with COPD. In 44 patients with stable COPD (38 male; age 62.3+/-7.2 years), REE was assessed using indirect calorimetry. Subcutaneous adipose tissue samples were analyzed using real-time PCR. From underweight [n=9; body mass index (BMI) <20.0 kg.m(-2)], to normal weight-overweight (n=24, BMI=20.0-29.9 kg.m(-2)) and obese patients (n=11; BMI>/=30 kg.m(-2)), REE adjusted for body weight decreased (32.9+/-6.1 vs. 26.2+/-5.8 vs. 23.9+/-6.6 kcal.kg(-1).24 h(-1), p=0.006), serum levels and adipose tissue expression of leptin increased (p<0.001 for both), and serum and adipose tissue adiponectin decreased (p<0.001; p=0.004, respectively). REE was inversely related to serum and adipose tissue leptin (R=-0.547, p<0.001; R=-0.458, p=0.002), and directly to serum adiponectin (R=0.316, p=0.039). Underweight patients had increased REE compared to normal weight-overweight patients, in association with reductions in serum and adipose tissue leptin, and increased serum adiponectin, suggesting a role of adipokines in energy imbalance in COPD-related cachexia.

Stress stimulates production of catecholamines in rat adipocytes.

The sympathoadrenal system is the main source of catecholamines (CAs) in adipose tissues and therefore plays the key role in the regulation of adipose tissue metabolism. We recently reported existence of an alternative CA-producing system directly in adipose tissue cells, and here we investigated effect of various stressors-physical (cold) and emotional stress (immobilization) on dynamics of this system. Acute or chronic cold exposure increased intracellular norepinephrine (NE) and epinephrine (EPI) concentration in isolated rat mesenteric adipocytes. Gene expression of CA biosynthetic enzymes did not change in adipocytes but was increased in stromal vascular fraction (SVF) after 28 day cold. Exposure of rats to a single IMO stress caused increases in NE and EPI levels, and also gene expression of CA biosynthetic enzymes in adipocytes. In SVF changes were similar but more pronounced. Animals adapted to a long-term cold exposure (28 days, 4°C) did not show those responses found after a single IMO stress either in adipocytes or SVF. Our data indicate that gene machinery accommodated in adipocytes, which is able to synthesize NE and EPI de novo, is significantly activated by stress. Cold-adapted animals keep their adaptation even after an exposure to a novel stressor. These findings suggest the functionality of CAs produced endogenously in adipocytes. Taken together, the newly discovered CA synthesizing system in adipocytes is activated in stress situations and might significantly contribute to regulation of lipolysis and other metabolic or thermogenetic processes.

Alpha-fetoprotein, carcinoembryonic antigen and beta2-microglobulin in adult population highly exposed to organochlorinated pollutants (PCB, DDE and HCB).

OBJECTIVE: It is aimed to obtain some general information about the prevalence of certain biomarkers in highly exposed population and on the interrelations between their serum level as related to that of some major organochlorines (OCs). METHODS: The level of alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and beta2-microglobulin (beta2-MG) as well as that of polychlorinated biphenyls (Σ15PCBs), dichlorodiphenyl-dichloroethylene (DDE) and hexachlorobenzene (HCB) was estimated in 2046 adults (834 males and 1212 females) from highly polluted Eastern Slovakia. RESULTS: Great majority of blood levels was lower than two specific units used for individual markers, while the prevalence of values higher than two specific units of appropriate markers. At the same time, the prevalence of all markers level higher than 2 specific units was highly significantly increasing with of stratified PCBs level quintiles which were also positively related to these of DDE and HCB. Some significant correlations between biomarkers level and age were also observed. CONCLUSIONS: Although from the data obtained within this multipurpose field survey any notable interrelations between AFP, CEA and beta2-MG and some specific diseases and/or malignant processes could not be retrospectively specified, from the data obtained it appears that some of such interrelations cannot be definitely excluded.

High prevalence of prediabetes and diabetes in a population exposed to high levels of an organochlorine cocktail.

AIMS/HYPOTHESIS: A heavily polluted area of Eastern Slovakia was targeted by the PCBRISK cross-sectional survey to search for possible links between environmental pollution and both prediabetes and diabetes. METHODS: Associations of serum levels of five persistent organic pollutants (POPs), namely polychlorinated biphenyls (PCBs), 2,2'-bis(4-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE), 2,2'-bis(4-chlorophenyl)-1,1,1-trichloro-ethane (p,p'-DDT), hexachlorobenzene (HCB) and beta-hexachlorocyclohexane (beta-HCH), with prediabetes and diabetes were investigated in 2,047 adults. Diabetes and prediabetes were diagnosed by fasting plasma glucose in all participants and by OGTT in 1,220 compliant participants. RESULTS: Our population was stratified in terms of individual POPs quintiles and associations between environmental pollution, prediabetes and diabetes were investigated. Prevalence of prediabetes and diabetes increased in a dose-dependent manner, with individuals in upper quintiles of individual POPs showing striking increases in prevalence of prediabetes as shown by OR and 95% CI for PCBs (2.74; 1.92-3.90), DDE (1.86; 1.17-2.95), DDT (2.48; 1.77-3.48), HCB (1.86; 1.7-2.95) and beta-HCH (1.97; 1.28-3.04). Interestingly, unlike PCBs, DDT and DDE, increased levels of HCB and beta-HCH seemed not to be associated with increased prevalence of diabetes. Nevertheless, individuals in the 5th quintile of the variable expressing the cumulative effect of all five POPs (sum of orders) had a more than tripled prevalence of prediabetes and more than six times higher prevalence of diabetes when compared with the 1st referent quintile. CONCLUSIONS/INTERPRETATION: Increasing serum concentrations of individual POPs considerably increased prevalence of prediabetes and diabetes in a dose-dependent manner. Interaction of industrial and agricultural pollutants in increasing prevalence of prediabetes or diabetes is likely.

Concerted action of leptin in regulation of fatty acid oxidation in skeletal muscle and liver.

Central action of leptin on food intake and energy expenditure is integrated with leptin's peripheral action modulating the fatty acid and glucose metabolism and preventing the accumulation of lipids in nonadipose tissues. However, exact mechanism(s) of the leptin's action in the peripheral tissues has not yet been fully elucidated. Therefore, we investigated the effect of a single intravenous injection of leptin on palmitoyl-CoA and palmitoyl-carnitine oxidation rate in liver and skeletal muscle followed by measurements of the carnitine-palmitoyl transferase 1 (CPT1) activity and activities of ss-oxidation enzymes in mitochondria (acyl-CoA dehydrogenase) and in peroxisomes (acyl-CoA oxidase) of rats. Animals were euthanized and tissues and serum harvested 15 min, 1 hour, 3 hours and 6 hours after leptin administration. Intravenous leptin injection increased mitochondrial palmitoyl-CoA oxidation rate in both liver (95%; P<0.025) and skeletal muscle (2.7-fold; P<0.05). This was paralleled by lowering hepatic (-156%; P<0.001) and skeletal muscle (-191%; P<0.001) triglyceride content. Leptin-induced elevation of palmitoyl-CoA oxidation rate in liver was paralleled by increased CPT1 activity (52%; P<0.05) and ss-oxidation capacity (52%; P<0.05). Lack of the leptin's effect on the CPT1-activity in muscle (20%; p=0.09) suggests the existence of an alternative pathway for increasing the palmitoyl-CoA-oxidation rate bypassing the CPT1 regulatory step. Interestingly, leptin stimulated the overall ss-oxidation capacity in muscle by 69% (P=0.027). This may indicate to an involvement of mitochondrial acyl-CoA dehydrogenases as well as of peroxisomal fat catabolism. Taken together, we showed that leptin acutely increases palmitoyl-CoA oxidation rate in liver and in skeletal muscle, which was associated with tissue specific effect on the CPT1 activity as well as on the downstream enzymes of fatty acid oxidation pathways in rat mitochondria and peroxisomes. Tangible evidence for the leptin-induced increase of fatty acid catabolism was provided by a lowered skeletal muscle and hepatic lipid deposition.

The hypotriglyceridemic effect of dietary n-3 FA is associated with increased beta-oxidation and reduced leptin expression.

To study the mechanisms responsible for the hypotriglyceridemic effect of marine oils, we monitored the effects of high dietary intake of n-3 PUFA on hepatic and muscular beta-oxidation, plasma leptin concentration, leptin receptor gene expression, and in vivo insulin action. Two groups of male Wistar rats were fed either a high-fat diet [28% (w/w) of saturated fat] or a high-fat diet containing 10% n-3 PUFA and 18% saturated fat for 3 wk. The hypotriglyceridemic effect of n-3 PUFA was accompanied by increased hepatic oxidation of palmitoyl-CoA (125%, P < 0.005) and palmitoyl-L-carnitine (480%, P < 0.005). These findings were corroborated by raised carnitine palmitoyltransferase-2 activity (154%, P < 0.001) and mRNA levels (91%, P < 0.01) as well as by simultaneous elevation of hepatic peroxisomal acyl-CoA oxidase activity (144%, P < 0.01) and mRNA content (82%, P < 0.05). In contrast, hepatic carnitine palmitoyltransferase-1 activity remained unchanged despite a twofold increased mRNA level after n-3 PUFA feeding. Skeletal muscle FA oxidation was less affected by dietary n-3 PUFA, and the stimulatory effect was found only in peroxisomes. Dietary intake of n-3 PUFA was followed by increased acyl-CoA oxidase activity (48%, P < 0.05) and mRNA level (83%, P < 0.05) in skeletal muscle. The increased FA oxidation after n-3 PUFA supplementation of the high-fat diet was accompanied by lower plasma leptin concentration (-38%, P < 0.05) and leptin mRNA expression (-66%, P < 0.05) in retroperitoneal adipose tissue, and elevated hepatic mRNA level for the leptin receptor Ob-Ra (140%, P < 0.05). Supplementation of the high-fat diet with n-3 PUFA enhanced in vivo insulin sensitivity, as shown by normalization of the glucose infusion rate during euglycemic hyperinsulinemic clamp. Our results indicate that the hypotriglyceridemic effect of dietary n-3 PUFA is associated with stimulation of FA oxidation in the liver and to a smaller extent in skeletal muscle. This may ameliorate dyslipidemia, tissue lipid accumulation, and insulin action, in spite of decreased plasma leptin level and leptin mRNA in adipose tissue.

Comparison of the extrapancreatic action of gamma-linolenic acid and n-3 PUFAs in the high fat diet-induced insulin resistance [corrected].

OBJECTIVE: The effect of dietary borage oil (rich in the gamma-linolenic acid [GLA]) on insulin sensitivity and lipid metabolism was compared with that of fish oil (rich in n-3 polyunsaturated fatty acids [PUFAs]) in high fat (HF) diet-induced insulin resistance (IR) of rats. METHODS: Male Wistar rats were fed ad libitum for 3 weeks a standard laboratory chow (Controls) or high fat diet consisting of 70-cal % fat. In addition, a group of rats was fed high fat (HF) diet where a part of saturated fat was replaced with fish oil as a source of n-3 PUFAs (HF+FO), or borage oil as a source of GLA (HF+GLA). In vivo insulin action was assessed by the euglycemic hyperinsulinemic clamp. Glucose, insulin, free fatty acids (FFA), triglycerides (Tg) and glycerol levels in blood and tissue depots were also measured. RESULTS: Increased levels of Tg, FFA and glycerol in circulation after HF diet were accompanied by their raised accumulation in insulin sensitive tissues. FO feeding lowered the concentration of all lipids in serum and prevented their accumulation in both tissues. On the other hand GLA supplementation into the high fat diet did not suppress increased levels of Tg, FFA and glycerol in circulation and tissue depots as well. FO feeding significantly reduced HF diet-induced in vivo IR, while GLA supplementation did not improve the in vivo insulin sensitivity in HF diet induced insulin resistance. CONCLUSIONS: 1. Substitution of FO into the high fat diet led to an improvement of in vivo insulin action; 2. this insulin sensitizing effect of FO was accompanied by a decrease of circulating Tg, FFA and glycerol levels in the postprandial state and by a lower lipid content in liver and skeletal muscle. 3. on the opposite, GLA treatment failed to improve in vivo insulin action; and 4. was associated with an adverse effect on lipid levels both in circulation and tissue depots.

An increase in peroxisomal fatty acid oxidation is not sufficient to prevent tissue lipid accumulation in hHTg rats.

We observed earlier that increased skeletal muscle lipid content in the hereditary hypertriglyceridemic (hHTg) rat is accompanied by a decline in plasma leptin. Leptin has recently been shown to enhance peripheral insulin sensitivity by decreasing the tissue triglyceride accumulation, possibly through regulation of fatty acid oxidation and lipogenesis. Thus, to test the hypothesis that insulin resistance and increased skeletal muscle lipid accumulation in hHTg rats are due to a defect in lipid catabolism, we measured mitochondrial and peroxisomal fatty acid oxidation and malonyl-CoA and acetyl-CoA carboxylase-2 content in skeletal muscles of these animals. In addition, we investigated possible molecular mechanisms responsible for the lower leptin levels in hHTg rats by measuring leptin and leptin-receptor (Ob-Ra) mRNA levels. We found the following: (1) in spite of a higher skeletal muscle malonyl-CoA content and an increased sensitivity of carnitine palmitoyltransferase-1 to malonyl-CoA, carnitine palmitoyltransferase-1 activity in muscle of hHTg rats was normal; (2) increased peroxisomal fatty acid oxidation did not seem to be sufficient to prevent the tissue lipid accumulation in these animals; (3) both lower leptin production by white adipose tissue and increased leptin uptake seem to be responsible for lower circulating leptin levels and therefore lower fatty acid catabolism.

Insulin resistance in the hereditary hypertriglyceridemic rat is associated with an impairment of delta-6 desaturase expression in liver.

Our previous studies have shown that insulin resistance (IR) in the hereditary hypertriglyceridemic (hHTg) rat is accompanied by a specific fatty acid (FA) profile in insulin target tissues, possibly due to a defect in the desaturation pathway. Increased dietary intake of n-3 polyunsaturated fatty acids (PUFAs) was shown to shape FA composition and to improve insulin sensitivity in this animal strain. Thus, the aim of this study is twofold: (1) to evaluate a defect in the FA desaturation by direct measurement of enzyme activity and gene expression for Delta-6 desaturase (Delta-6 D) in liver of hHTg rats and (2) to investigate the effect of dietary n-3 PUFAs on hepatic Delta-6 D in relation to tissue FA composition. Male Wistar or hHTg rats were fed ad libitum for 21 days either the basal or fish oil (FO)-supplemented diets. Triglyceride (Tg) levels in serum and tissue lipid extracts were measured with the aid of a commercially available enzymatic set. Hepatic activity of the Delta-6 D was determined radiometrically in a microsomal fraction using 1-(14)C-linoleic acid as a substrate. The Delta-6 D mRNA levels were measured using the Northern blot technique. Tissue FA composition was determined by gas chromatography in the total phospholipid fraction after TLC separation. Increased levels of Tg in hHTg rat circulation were accompanied by raised accumulation of Tg in skeletal muscles. FO feeding lowered the concentration of Tg in serum and prevented their accumulation in skeletal muscles of hHTg rats. A pronounced decrease in the hepatic Delta-6 D activity in hHTg rats (by about 80%) was not further diminished by FO feeding. On the other hand, the activity of Delta-6 D in liver of control rats was reduced by about 40% after FO supplementation. These changes were paralleled by a decrease in the Delta-6 D index as calculated from the liver phospholipid FA profile. In particular, an increase in the amount of 18:2 n-6 and a decrease in arachidonic acid and PUFA n-6 metabolites were found. The results indicate that a decrease of insulin action in hHTg rats is accompanied by an impairment of the hepatic Delta-6 D activity already at the gene level, which is not further affected by n-3 PUFA supplementation.

Nutrient sensing, leptin and insulin action.

The glucose-fatty acid cycle as proposed four decades ago by Randle suggests that insulin resistance develops in consequence of alterations of the metabolic pressure of lipids. The more recently published 'hexosamine pathway theory' and the 'malonyl-CoA hypothesis' depict insulin resistance as a consequence of an imbalance between utilization of lipids and carbohydrates. The latter is finely tuned by entry of fatty acids into the mitochondria and/or by entry of glucose to the hexosamine pathway. A significant body of evidence has also been accumulated which points to the complex effects of leptin, an adipocyte-derived signal of lipid stores, on the storage and metabolism of fats and carbohydrates. These are mediated either directly, through actions on specific tissues, or indirectly, via CNS, endocrine and neural mechanisms. The available literature also provides good evidence that leptin orchestrates the metabolic changes in a number of organs and tissues, and alters nutrient fluxes to favor energy expenditure over energy storage. In this article, the proposed lipopenic effects of leptin as studied in various animal models of diet-induced insulin resistance, and possible regulations of leptin production and action by marine fish oil feeding are reviewed.

Delta-6 desaturase activity and gene expression, tissue fatty acid profile and glucose turnover rate in hereditary hypertriglyceridemic rats.

OBJECTIVE: We have shown previously that the impaired insulin action in hereditary hypertriglyceridemic (hHTg) rat is accompanied by a specific fatty acid (FA) profile in the insulin target tissues, possibly due to a desaturation defect. Thus, the aim of this study was to measure the enzymatic activity and gene expression of delta-6 desaturase in liver of hHTg rats and the tissue FA composition in relation to insulin action. METHODS: Glucose, triglycerides and insulin in plasma were measured using commercially available enzymatic sets. The hepatic delta-6 desaturase activity in hHTg rats was determined radiometrically in a microsomal fraction using the 1-14C-linoleic acid as substrate. delta-6 Desaturase gene expression was measured by the Northern blot technique using a specific cDNA probe. Tissue FA profile was determined by gas chromatography in the total lipid fraction extracted to chloroform. The glucose turnover rate was measured in conscious freely moving animals with the aid of euglycemic hyperinsulinic clamp method. RESULTS: Tissue triglycerides showed a high accumulation in skeletal muscle of hHTg rats. In the liver of these animals, a defect in delta-6 desaturase enzymatic activity was found, while the gene expression for delta-6 desaturase was not changed. Such decreased delta-6 desaturase activity in the liver was linked to a decrease of delta-6 desaturase index as calculated from the liver FA composition. Also the concentration of arachidonic acid (a final metabolite in the biosynthesis of polyunsaturated fatty acids of the n-6 series) was significantly decreased in hHTg rat liver. These changes in FA metabolism were accompanied by a decreased glucose infusion rate (a measure of in vivo insulin action) required to maintain euglycemia at hyperinsulinemia in hHTg rats, and correlated with the hepatic delta-6 desaturase activity. CONCLUSIONS: 1. hHTg rats showed a reduced activity of the delta-6 desaturase in liver without any changes in gene expression for this enzyme; 2. such impairment is accompanied by a lower delta-6 desaturase index (18:2n-6/18:3n-6) found in the liver of these animals and by specific FA profiles in the tissues, particularly regarding the amount of long-chain PUFAs and 18:2n-6 metabolites; and (4) these alterations seem to be related to the impaired insulin action of hHTg rats.