Lipid peroxidation is not a prerequisite for the development of obesity and diabetes in high-fat-fed mice.

The mechanism, by which a high-fat (HF) diet could impair glucose metabolism, is not completely understood but could be related to inflammation, lipotoxicity and oxidative stress. Lipid peroxides have been proposed as key mediators of intracellular metabolic response. The purpose of the present study was to analyse, in mice fed with a HF diet, the possible association between obesity and glucose tolerance on the one hand, and between oxidative stress and lipid peroxidation on the other hand. The present results show that a HF diet (70 % energy as fat), v. a high-carbohydrate chow diet (control), increases body weight and fat mass development, and impairs glycaemia and insulinaemia within 4 weeks. It also promotes the expression of NADPH oxidase in the liver--signing both oxidative and inflammatory stress--but decreases thiobarbituric acid-reactive substances content in the liver as well as in epididymal, subcutaneous and visceral adipose tissues. HF diet, with elevated vitamin E content, induces high concentration of alpha-tocopherol in liver and adipose tissues, which contributes to the protection against lipid peroxidation. Thus, lipid peroxidation in key organs is not necessarily related to the development of metabolic disorders associated with diabetes and obesity.

The metabolic syndrome of omega3-depleted rats. III. Brain phospholipids.

Rats exposed from 7 weeks after birth and for the ensuing 3 to 7 months to a diet depleted in long-chain polyunsaturated omega3 fatty acids were recently proposed as a new animal model for the metabolic syndrome. The present study aimed mainly at investigating whether, in this new model, the perturbation of the fatty acid total content and pattern of brain phospholipids simulates that previously documented in second-generation omega3-depleted rats. Such was indeed the case, with the apparent exception of changes in the C18:1omega9, C20:0, C22:0 and C24:0 relative content of brain phospholipids. Moreover, the C22:5omega3 content of such phospholipids was unexpectedly lower in the present model than in the second-generation omega3-depleted rats. The changes in brain phospholipids were also monitored when the rats deprived of omega3 fatty acids for 7 months were given access for 2 to 4-5 weeks to a flaxseed oil-enriched diet. Most phospholipid variables were rapidly normalized under the latter experimental conditions. The results obtained under these conditions suggest that an increase in the brain phospholipid C22:5omega3 content may play a key role in the orexigenic effects of exogenous omega3 fatty acids supplied to omega3-depleted animals.

The metabolic syndrome of omega3-depleted rats. I. Liver data.

Second-generation rats depleted in long-chain polyunsaturated omega3 fatty acids were recently proposed as a novel animal model for the metabolic syndrome. In the present study, a dietary deprivation of omega3 acids for 3-7 months was found sufficient to provoke in 6-week-old normal rats the same alteration of the fatty acid content and profile of liver phospholipids and triglycerides as that otherwise prevailing in the second-generation omega3-depleted rats, with emphasis on a severe decrease in their omega3 fatty acid content, alterations in the relative contribution of and ratio between selected long-chain polyunsaturated omega6 fatty acids, saturated and monodesaturated fatty acids and precursors of nervonic acid, and liver steatosis. When the omega3-depleted rats were exposed, after the first 7 months of the present experiments and for 2-4 weeks to a diet supplemented with 5% (w/w) flaxseed oil, most of these hepatic variables returned towards or beyond control values. In both the omega3-depleted rats and control animals, however, the eventual exposure to the flaxseed oil-enriched diet failed to suppress liver steatosis and, on the contrary, provoked a further increase in liver triglyceride content. It is proposed, therefore, that the present approach represents a simple and realistic animal model to study the consequences of omega3-depletion. Moreover, the results suggest that to oppose such consequences, e.g. liver steatosis, it may be necessary to combine the dietary supply of omega3 acids with a suitable control of food intake, in both qualitative and quantitative terms.

Alteration of lipid fatty acid profile and cationic fluxes in ventricular cardiomyocytes from omega3-depleted rats.

The bolus intravenous injection of a medium-chain triglyceride:fish oil emulsion was recently found to increase within 60 min the cell phospholipid content in long-chain polyunsaturated omega3 fatty acids and, hence, proposed as a potential tool to prevent cardiac arrhythmia in subjects with a decreased dietary intake of such fatty acids. In the present study, ventricular cardiomyocytes from second generation rats depleted in omega3 fatty acids were found to display the same changes in the phospholipid fatty acid pattern as that previously documented in the cardiac muscle and endothelium of such rats, altered 86Rb and 45Ca fluxes with emphasis on a decrease in both K+ inflow and K+ content and an increase in both Ca2+ inflow and content. The alteration of K+ inflow could not be attributed to a decrease in ouabain-sensitive Na+,K+-ATPase activity as measured in cell homogenates. The cationic alterations were corrected, in part at least, by the prior intravenous injection of the medium-chain triglyceride:fish oil emulsion 60 min before sacrifice of the omega3-depleted rats.

Preferential enrichment of liver phospholipids in docosahexaenoate relative to eicosapentaenoate in omega-3-depleted rats injected with a medium-chain triglyceride: fish oil emulsion.

The bolus intravenous injection of a novel medium-chain triglyceride:fish oil (FO) emulsion was recently proposed as a tool to provoke a rapid enrichment of cell phospholipids in long-chain polyunsaturated omega-3 fatty acids. In the present study, the enrichment of liver phospholipids and triglycerides in C20:5omega-3, C22:5omega-3 and C22:6omega-3 was assessed 60min after the intravenous administration of FO (1.0ml) to second-generation omega-3-depleted rats. When compared to uninjected rats, or animals injected with a control omega-3 fatty acid-poor medium-chain triglyceride:olive oil (OO) emulsion, the enrichment of liver phospholipids, and to a lesser extent liver triglycerides, attributable to the injection of the FO emulsion was more pronounced for C22:6omega-3 than C20:5omega-3, despite the presence of equal amounts of these two omega-3 fatty acids in the injected diglycerides and triglycerides. The possible determinants and potential beneficial effects of such a difference are briefly discussed.

Rapid lipid enrichment in omega3 fatty acids: plasma data.

The bolus intravenous injection of a novel medium-chain triglyceride:fish oil emulsion to normal subjects was recently reported to enrich within 60 min the phospholipid content of leucocytes and platelets in long-chain polyunsaturated omega3 fatty acids. The present study, conducted in second generation omega3-depleted rats, aimed at investigating whether such a procedure may also increase within 60 min the phospholipid content of omega3 fatty acids in cells located outwards the bloodstream, in this case liver cells, and whether this coincides with correction of the perturbation in the liver triglyceride fatty acid content and profile otherwise prevailing in these rats. This first report deals mainly with the fatty acid pattern of plasma lipids in male omega3-depleted rats that were non-injected or injected with either the omega3-rich emulsion or a control medium-chain triglyceride:olive oil emulsion. The results provide information on the fate of the exogenous lipids present in the lipid emulsions and injected intravenously 60 min before sacrifice. Moreover, in the uninjected omega3-depleted rats the comparison between individual plasma and liver measurements indicated positive correlations in the fatty acid profile of phospholipids and triglycerides.

Rapid lipid enrichment in omega3 fatty acids: cause-to-effect relationships.

The bolus intravenous administration of a novel medium-chain triglyceride:fish oil emulsion to second generation rats depleted in long-chain polyunsaturated omega3 fatty acids was recently found to enrich within 60 min the content of both plasma and liver lipids in such omega3 fatty acids, this coinciding with correction of the perturbation in liver triglyceride fatty acid content and profile otherwise prevailing in these rats. The present report draws attention to cause-to-effect relationships between changes in liver phospholipid and triglyceride fatty acid content and/or pattern operative under these experimental conditions.

Rapid lipid enrichment in omega3 fatty acids: liver data.

The bolus intravenous injection of a novel medium-chain triglyceride:fish oil emulsion to normal subjects was recently reported to enrich within 60 min the phospholipid content of leucocytes and platelets in long-chain polyunsaturated omega3 fatty acids. The present study, conducted in second generation omega3-depleted rats, aims at investigating whether such a procedure may also increase within 60 min the phospholipid content of omega3 fatty acids in cells located outwards of the bloodstream, in this case liver cells, and whether this coincides with correction of the perturbation in the liver triglyceride fatty acid content and profile otherwise prevailing in these rats. The results indicate that such is indeed the case and further suggest a cause-to-effect relationship between the two events.

Hemostatic safety of the bolus intravenous injection of a novel medium-chain triglyceride:fish oil emulsion.

The bolus intravenous injection of a novel 8:2 medium-chain triglyceride:fish oil emulsion was recently found to increase within 60 min and for the subsequent 24-48 h the long-chain polyunsaturated omega3 fatty acid content of both leucocyte and platelet phospholipids in 12 normal subjects. The aim of the present report is to document the hemostatic safety of such a procedure in the same 12 subjects. No adverse effect was found when comparing the results obtained after administration of either the fish oil-containing emulsion or a control 5:5 medium-chain triglyceride:soybean triglyceride emulsion, whether in terms of the occlusion time in either an ADP or epinephrine test or in terms of the [CD]42b, [CD]62p, fibrinogen and PAC-1 response to ADP, collagen or thrombin receptor analog peptide 6 in platelets examined by fluorescence activated cell sorting. In conclusion, this novel procedure for the rapid enrichment of cell phospholipid in long-chain polyunsaturated omega3 fatty acids presents the required safety in a hemostatic perspective.

Fatty acid profile of plasma and liver lipids in mice depleted in long-chain polyunsaturated (n-3) fatty acids.

Considering the numerous features of the metabolic syndrome found in rats depleted in long-chain polyunsaturated (n-3) fatty acids and in the perspective of further work conducted in (n-3)-depleted mice, the fatty acid profile of plasma and liver lipids was assessed in both male and female control and second-generation (n-3)-depleted mice. In addition to gender differences, the major alteration found in the (n-3)-depleted animals consisted in the expected severe depletion of plasma triacylglycerols and phospholipids, as well as liver phospholipids, in C20:5(n-3), C22:5(n-3) and C22:6(n-3). In plasma triacylglycerols, the weight percentages of C18:2(n-6) and C18:3(n-6) were lower in (n-3)-depleted mice than in control animals. In both plasma and liver phospholipids, however, the weight percentages of long-chain polyunsaturated (n-6) fatty acids (C20:4(n-6) and C22:4(n-6)) were higher in (n-3)-depleted mice than in control animals. The C16:1(n-7)/C16:0 and C18:1(n-9)/C18:0 ratio in both plasma and liver phospholipids were also increased in female (n-3)-depleted mice but not so in male animals. Highly significant correlations were found between the weight percentage of each fatty acid in liver versus plasma phospholipids. Taken as a whole, these findings indicate that second-generation mice depleted in (n-3) fatty acids represent a suitable model, in terms of the remodelling of the fatty acid profile in plasma and liver lipids, to investigate the metabolic and functional consequences of such a depletion.

Correlation between liver and plasma fatty acid profile of phospholipids and triglycerides in rats.

Considering the changes in the fatty acid profile of liver lipids related to age, gender and nutritional status or occurring in pathological situations, this study aimed at investigating whether such changes could be judged from measurements conducted in plasma lipids. The fatty acid profile of both liver and plasma phospholipids and triglycerides was measured in 16 control animals and 26 rats depleted in long-chain polyunsaturated (n-3) fatty acids. Within each group of rats, significant correlations prevailed between the percentage of each fatty acid in liver versus plasma phospholipids or triglycerides. However, the plasma/liver ratio for the relative content of C20:5(n-3), C22:5(n-3) and C22:6(n-3) in triglycerides displayed abnormally high values in 2 control animals. The fatty acid profile of liver phospholipids and triglycerides can, as a rule, be judged from measurements made in the corresponding plasma lipids. For instance, measurements in plasma phospholipids could help to identify subjects deficient in (n-3) fatty acids and to assess the dietary correction of this defect.

Perturbation of 11-eicosenoate metabolism in female diabetic rats.

Considering the proposed preventive effect of nervonic acid on obesity- and diabetes-related coronary risk factors, the content of its precursors (oleic, 11-eicosenoic and 13-docosenoic acids) was measured in liver and plasma phospholipids and triglycerides, brain and spleen phospholipids, and adipose tissue lipids of fed or overnight fasted control and hereditarily diabetic Goto-Kakizaki female rats, as well as fed streptozotocin-induced diabetic female rats. In liver and brain phospholipids, the 11-eicosenoate/oleate ratio was significantly higher in diabetic rats than in control animals. Such was not the case in either spleen phospholipids or liver triglycerides and adipose tissue lipids. The increase in the liver phospholipid 11-eicosenoate/oleate ratio found in female diabetic rats represents a mirror image of the situation recently documented, in the same animal models of diabetes, in male rats. These contrasting findings may be relevant to the higher coronary heart disease risk prevailing in female, as compared to male, diabetic subjects.

Phospholipid and triacylglycerol fatty acid content and pattern in the cardiac endothelium of rats depleted in long-chain polyunsaturated omega 3 fatty acids.

Rats depleted in long-chain polyunsaturated omega3 fatty acids (omega3-depleted rats) display several features of the metabolic syndrome including hypertension and cardiac hypertrophy. This coincides with alteration of the cardiac muscle phospholipid and triacylglycerol fatty acid content and/or pattern. In the present study, the latter variables were measured in the cardiac endothelium of normal and omega3-depleted rats. Samples derived from four rats each were obtained from 16 female normal fed rats and three groups of 36-40 female fed omega3-depleted rats each aged 8-9, 15-16 and 22-23 weeks. At comparable mean age, the ratio between the square root of the total fatty acid content of phospholipids and cubic root of the total fatty acid content of triacylglycerols was lower in omega3-depleted rats than in control animals. The total fatty acid content of triacylglycerols was inversely related to their relative content in C20:4omega6. Other differences between omega3-depleted rats and control animals consisted in a lower content of long-chain polyunsaturated omega3 fatty acids in both phospholipids and triacylglycerols, higher content of long-chain polyunsaturated omega6 fatty acids in phospholipids, higher activity of delta9-desaturase (C16:0/C16:1omega7 and C18:0/C18:1omega9 ratios) and elongase [(C16:0 + C16:1omega7)/(C18:0 + C18:1omega9) and C20:4omega6/C22:4omega6 ratios], but impaired generation of C22:6omega3 from C22:5omega3 in the former rats. These findings support the view that cardiovascular perturbations previously documented in the omega3-depleted rats may involve impaired heart endothelial function.

Phospholipid fatty acid pattern and D-glucose metabolism in muscles from omega3 fatty acid-depleted rats.

A depletion in long-chain polyunsaturated omega3 fatty acids may affect fuel homeostasis. In such a perspective, the present study deals mainly with the in vitro fate of D-[U-(14)C]glucose in hemidiaphragms, stretched soleus and plantaris muscle pieces obtained from normal and omega3-depleted rats (second generation) and incubated in the absence or presence of insulin. When so required, the omega3-depleted rats were injected 120 min before sacrifice with either a omega3 fatty acid-rich medium-chain triglyceride:fish oil emulsion (FO) or a control medium-chain triglyceride:olive oil emulsion (OO). The content of the soleus muscle in long-chain polyunsaturated omega3 fatty acids was severely decreased in the omega3-depleted rats, and modestly albeit significantly increased after injection of FO to these animals. In stretched soleus muscles from OO-injected omega3-depleted rats, the absolute values for glycogen synthesis measured in the absence or presence of insulin were about twice higher than in normal animals. In the OO-injected omega3-depleted rats, insulin augmented the output of (14)C-labelled amino acids, whilst such was not the case in normal animals. These and other findings suggest a lower catabolism of D-glucose relative to the anabolic process of glycogen synthesis and a lower availability of endogenous amino acids in the muscles of omega3-depleted rats, as compared to those of control animals. The prior injection of FO to the omega3-depleted rats restored a normal value for the paired ratio between the output of (14)C-labelled amino acids and acidic metabolites, but further increased glycogen net synthesis. It is proposed, therefore, that the perturbation of d-glucose metabolism in muscles from omega3-depleted rats involves a multifactorial determinism, only some of the concerned factors being susceptible to rapid correction after enrichment of cell phospholipids in long-chain polyunsaturated omega3 fatty acids.

Fatty acid content and pattern of spleen phospholipids and triglycerides in normal and either type-1 or type-2 diabetic rats.

The fatty acid pattern of spleen phospholipids and triglycerides was examined in fed or overnight fasted normal rats, streptozotocin-induced diabetic animals (type-1 diabetes) and Goto-Kakizaki rats (type-2 diabetes). In both phospholipids and triglycerides, differences were observed in the relative contribution of several fatty acids, as well as in the ratio between distinct fatty acids, when comparing fed to fasted rats, normal to diabetic animals and male to female Goto-Kakizaki rats. Diabetes increased to a greater extent the C22:6omega3 content of phospholipids in the spleen than in either the liver or the brain. However, the diabetes-induced changes in the C22:6omega3 content of triglycerides was closely comparable in the spleen, liver and brain. These findings suggest that the incorporation of fatty acids into triglycerides is controlled by comparable regulatory factor(s), e.g. insulinemia, in the spleen, liver and brain. In the case of phospholipids, however, an apparent adaptation to diabetic stress was more marked in the spleen than in the liver, and virtually absent in the brain. The proposed dichotomy in the environmental regulation of fatty acid synthesis and incorporation into phospholipids and triglycerides was further supported by distinct diabetes-related changes in the apparent activity of Delta9-desaturase in these two classes of lipids.

Perturbation and age-related changes in the fatty acid pattern of soleus muscle phospholipids and triglycerides in rats depleted in long-chain polyunsaturated omega3 fatty acids.

Altered D-glucose metabolism prevails in the soleus muscle of rats depleted in long-chain polyunsaturated omega3 fatty acids (omega3). In these animals, the prior intravenous injection of an omega3-rich medium-chain triglyceride:fish oil emulsion (omega3-FO rats), as compared to that of an omega3-poor medium-chain triglyceride:olive oil emulsion (omega3-OO rats), may either correct or aggravate selected metabolic variables. This study deals with the fatty acid pattern of soleus phospholipids and triglycerides in control animals versus omega3-depleted rats not injected with any lipid emulsion (omega3-NI rats) and in omega3-OO versus omega3-FO rats. In each group of omega3-depleted rats, age-related changes were also monitored. The omega3-depleted rats displayed low long-chain polyunsaturated omega3 fatty acid content, facilitated metabolism of long-chain polyunsaturated omega6 fatty acids, and increased Delta9-desaturase activity. Both the age-related changes in lipid variables and those attributable to the prior intravenous injection of the omega3-rich lipid emulsion consisted either in a move towards normalization or in the opposite direction, i.e. towards aggravation of the defect found in the omega3-depleted rats. Emphasis is placed, therefore, on the unusual situation found in the soleus muscle of omega3-depleted rats, in which both lipid and metabolic variables may be either favourably or adversely affected by the same environmental factor(s).

n-3 fatty acids and the metabolic syndrome.

The metabolic syndrome is defined as the coexistence of 3 or more components, some of which indicate alterations in glucose and lipid metabolism. The prevalence of the metabolic syndrome is rapidly increasing in relation to obesity, and it is considered to be an important predictor of cardiovascular disease. Increased intakes or supplements of n-3 marine fatty acids may improve defects in insulin signaling and prevent alterations in glucose homeostasis and the further development of type 2 diabetes. This is largely mediated through a reduction in fatty acid accumulation in muscle and liver. n-3 Polyunsaturated fatty acids (n-3 PUFAs) reduce plasma triacylglycerols and improve the lipoprotein profile by decreasing the fraction of atherogenic small, dense LDL. However, n-3 PUFAs do not lower LDL cholesterol. These effects are likely mediated through the activity of transcription factors relating to expression of genes involved in lipid oxidation and synthesis. Other pleiotrophic effects of n-3 PUFAs may contribute to decreasing the burden of the metabolic syndrome, such as modulating inflammation, platelet activation, endothelial function, and blood pressure. Although studies comparing the effect of both major n-3 PUFAs are limited, docosahexaenoic acid appears at least as efficient as eicosapentaenoic acid in correcting several risk factors. The use of n-3 PUFAs should be considered in more global strategies including changes in lifestyle, such as adhering to a healthy Mediterranean type of diet and practicing regular physical exercise.

Glucose activation of the glucagon receptor gene: functional dissimilarity with several other glucose response elements.

The glucagon receptor (GLR) expression is positively regulated by glucose. This regulation is allowed by the presence, in the promotor of the rat GLR gene, of a sequence feature similar to the two E-boxes motifs constituting the carbohydrate response elements (ChoRE) described for several glycolytic and lipogenic enzyme genes. Using reporter gene assays, we demonstrated here that, despite structural homologies with these ChoREs, the GLR gene glucose response element presents various functional dissimilarities. Testing glucose analogs, we demonstrated that, as for other genes, the glucose must be first phosphorylated. However, at variance with others homologue genes, our data showed the implication of the nonoxidative branch of the pentose phosphate pathway in the transmission of the glucose signal and lack of inhibition by adenosine monophosphate (AMP)-kinase. Furthermore, the activity of our reporter gene was strongly stimulated by butyrate, propionate, and acetate. This observation contrasts with fatty-acid-induced inhibition of the glucose activation, observed for all other genes containing homolog ChoREs. We also showed that glucose and butyrate influence the reporter gene expression via different features.

Brain phospholipid and triglyceride fatty acid content and pattern in Type 1 and Type 2 diabetic rats.

The liver phospholipid and triglyceride content and/or fatty acid pattern differ(s) not solely in normal versus diabetic rats, but also in distinct rat models of diabetes mellitus. The present study reveals that a comparable situation prevails in the brain. Fed and overnight fasted female normal rats (N) and Goto-Kakizaki rats (GK), as well as fed rats rendered diabetic by a prior injection 3 days before sacrifice of streptozotocin (STZ) were examined. The brain phospholipid content, expressed as milligrams of fatty acids per gram wet weight, was comparable in all groups of rats, with an overall mean value of 31.2+/-0.8 (n=22). The GK rats differed from N and STZ rats by lower C18:0/C18:1omega9 and C18:2omega6/C18:3omega6 ratios and a lower C20:5omega3 content of brain phospholipids. The total amount of fatty acids in triglycerides was 7-8 times higher in GK than N and STZ rats. The GK rats differed from N and STZ rats by lower C16:0/C16:1omega7, C18:0/C18:1omega9 and (C16:0+C16:1omega7)/(C18:0+C18:1omega9) ratios in triglycerides. These findings extend to the brain, the knowledge of alterations in phospholipid and triglyceride content and/or fatty acid pattern in GK rats, as compared to N or STZ rats. The former rats indeed displayed: (i) an apparently increased activity of Delta9- and Delta6-desaturases, as suggested by the phospholipid measurements, and a decreased C20:5omega3 content in such phospholipids; (ii) a dramatic increase in brain triglyceride content; and (iii) an increased activity of Delta9-desaturase, as well as elongase, as judged from the triglyceride data.

Preclinical investigations of a medium-chain triglyceride:fish oil emulsion. I. Effects of bovine milk lipoprotein lipase on lipid composition.

The bolus intravenous injection of a novel medium-chain triglyceride:fish oil emulsion (MCT:FO, 8:2, w:w) was recently found to increase within 60 min the leucocyte and platelet phospholipid content of long-chain polyunsaturated omega3 fatty acids. The present report deals with the effects of bovine milk lipoprotein lipase on the lipid composition of this emulsion. The results are compared to those obtained with either a pure fish oil emulsion or a medium-chain triglyceride: long-chain triglyceride:fish oil emulsion (MLF, 5:4:1). Emphasis is placed on i) differences in the fate of distinct fatty acids initially present in the triglycerides, di glycerides and phospholipids, ii) the generation of unesterified fatty acids relative to their initial content in each emulsion, and iii) the time course for these various events. The comparison between the three emulsions under consideration also provides information relevant to their respective sensitivity to lipoprotein lipase and suitability in terms of the generation of distinct unesterified fatty acids, including long-chain polyunsaturated omega3 fatty acids. Furthermore, attention is drawn to the greater efficiency for the hydrolysis of fatty acids from diglycerides as compared to triglycerides and a transient increase in the paired C8:0/C10:0 ratio in the diglycerides generated from the MCT:FO or MLF emulsion. The present study thus affords novel information relevant to the possible use of the MCT:FO emulsion in human subjects.