Minimizing Visceral Fat Delocalization on Tissue Sections with Porous Aluminum Oxide Slides for Imaging Mass Spectrometry.

A high correlation of bioanalytes with their corresponding histologies is the landmark feature of matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS). Lipids are one of the most studied classes of biomolecules, and monitoring lipid distribution and abundance in tissue samples can lead to major inputs in the understanding of disease. Lipid delocalization and ion suppression are two major effects that can lead to misinterpretation of the IMS results to an unaware analyst. We and others have observed that tissue specimens containing high amounts of visceral fat are challenging to analyze because of fat delocalization on and off section leading to significant triacylglyceride and phospholipid delocalization and major ion suppression effects. In this work, we introduce a novel and easy to produce reusable porous aluminum oxide sample slide that minimizes visceral fat delocalization after thaw-mounting of tissue sections. Using fatty mouse kidneys and other tissues, we demonstrate its efficacy in minimizing delocalization of triacylglycerides, the primary constituents of fat, and the resulting beneficial effects on phospholipid MALDI IMS.

Brain-specific natriuretic peptide receptor-B deletion attenuates high-fat diet-induced visceral and hepatic lipid deposition in mice.

C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor-B (NPR-B), are abundantly distributed in the hypothalamus. To explore the role of central CNP/NPR-B signaling in energy regulation, we generated mice with brain-specific NPR-B deletion (BND mice) by crossing Nestin-Cre transgenic mice and mice with a loxP-flanked NPR-B locus. Brain-specific NPR-B deletion prevented body weight gain induced by a high-fat diet (HFD), and the mesenteric fat and liver weights were significantly decreased in BND mice fed an HFD. The decreased liver weight in BND mice was attributed to decreased lipid accumulation in the liver, which was confirmed by histologic findings and lipid content. Gene expression analysis revealed a significant decrease in the mRNA expression levels of CD36, Fsp27, and Mogat1 in the liver of BND mice, and uncoupling protein 2 mRNA expression was significantly lower in the mesenteric fat of BND mice fed an HFD than in that of control mice. This difference was not observed in the epididymal or subcutaneous fat. Although previous studies reported that CNP/NPR-B signaling inhibits SNS activity in rodents, SNS is unlikely to be the underlying mechanism of the metabolic phenotype observed in BND mice. Taken together, CNP/NPR-B signaling in the brain could be a central factor that regulates visceral lipid accumulation and hepatic steatosis under HFD conditions. Further analyses of the precise mechanisms will enhance our understanding of the contribution of the CNP/NPR-B system to energy regulation.

Melatonin reduces obesity and restores adipokine patterns and metabolism in obese (ob/ob) mice.

The increasing incidence of obesity, leading to metabolic complications, is now recognized as a major public health problem. The adipocytes are not merely energy-storing cells, but they play crucial roles in the development of the so-called metabolic syndrome due to the adipocyte-derived bioactive factors such as adipokines, cytokines, and growth factors. The dysregulated production and secretion of adipokines seen in obesity is linked to the pathogenesis of the metabolic disease processes. In this study, we hypothesized that dietary melatonin administration would support an anti-inflammatory response and play an important role in energy metabolism in subcutaneous and visceral adipose tissues of obese mice and so may counteract some of the disruptive effects of obesity. Lean and obese mice (ob/ob) received melatonin or vehicle in drinking water for 8 weeks. Thereafter, they were evaluated for morphologic alteration, inflammatory cell infiltration, and the adipokine patterns in visceral and subcutaneous white fat depots. In obese mice treated with vehicle, we observed a significant increase in fat depots, inflammation, and a dysregulation of the adipokine network. In particular, we measured a significant reduction of adiponectin and an increase of tumor necrosis factor α, resistin, and visfatin in adipose tissue deposits. These changes were partially reversed when melatonin was supplemented to obese mice. Melatonin supplementation by regulating inflammatory infiltration ameliorates obesity-induced adipokine alteration, whereas melatonin administration in lean mice was unaffected. Thus, it is likely that melatonin would be provided in supplement form to control some of the disruptive effects on the basis of obesity pathogenic process.

Quantification of the triglyceride fatty acid composition with 3.0 T MRI.

The aim of this work was to validate a sequential method for quantifying the triglyceride fatty acid composition with 3.0 T MRI. The image acquisition was performed with a 3D spoiled gradient multiple echo sequence. A specific phase correction algorithm was implemented to correct the native phase images for wrap, zero- and first-order phase and rebuild the real part images. Then, using a model of a fat (1)H MR spectrum integrating nine components, the number of double bonds (ndb) and the number of methylene-interrupted double bonds (nmidb) were derived. The chain length (CL) was obtained from these parameters using heuristic approximation. Validations were performed on different vegetable oils whose theoretical fatty acid composition was used as reference and in five human subjects. In vivo measurements were made in the liver and in the subcutaneous and visceral adipose tissues. Linear regressions showed strong correlations between ndb and nmidb quantified with MRI and the theoretical values calculated using oil composition. Mean ndb/nmidb/CL were 1.80 ± 0.25/0.51 ± 0.21/17.43 ± 0.07, 2.72 ± 0.31/0.94 ± 0.16/17.47 ± 0.08 and 2.53 ± 0.21/0.84 ± 0.14/17.43 ± 0.07 in the liver, subcutaneous and visceral adipose tissues respectively. The results suggest that the triglyceride fatty acid composition can be assessed in human fatty liver and adipose tissues with a clinically relevant MRI method at 3.0 T.

Influence of level of barley supplementation on plasma carotenoid content and fat spectrocolorimetric characteristics in lambs fed a carotenoid-rich diet.

This study investigated changes in plasma carotenoid concentration and fat reflectance spectrum characteristics and color in lambs fed a carotenoid-rich diet with low-level (L, 100 g/lamb/day) or high-level (H, 400 g/lamb/day) of barley supplementation for 75 days before slaughter. Each treatment used 24 Romane male lambs that were individually penned indoors. Plasma carotenoid concentration at slaughter was 16% lower in H lambs than in L lambs. H lambs had heavier and fatter carcasses than L lambs. Yellowness and redness of perirenal fat were slightly lower in H lambs than in L lambs. The absolute value of the mean integral (AVMI) calculated from the reflectance spectrum of the fat in the 450-510 nm band was not affected by the treatment. Yellowness, chroma and AVMI of subcutaneous fat were not affected by the treatment but decreased with initial animal's liveweight.

Influence of betaine and conjugated linoleic acid on development of carcass cuts of Iberian pigs growing from 20 to 50 kg body weight.

Twenty Iberian gilts (20 kg body weight, BW) were fed diets containing no betaine or conjugated linoleic acid (CLA) (Control), 0.5% betaine, 1% CLA, or 0.5% betaine+1% CLA. Additionally, 5 pigs were killed at 20 kg BW for the initial points of the allometric equations. At 50 kg BW, left semicarcasses were cut into primal cuts, hams and shoulders trimmed and dissected. CLA alone did not affect any analyzed parameter. Betaine increased (23 and 21%, respectively) the yield of shoulder butt and spine and decreased allometric growth coefficient of belly and backfat, compared to Control diet. Tenderloins and trimmed hams of pigs fed CLA+betaine diet developed later and were heavier (22 and 5%, respectively) than Control pigs. Also, leaf fat developed earlier and had lighter weight (32%). Furthermore, pigs fed CLA+betaine diet had heavier lean (5%) and fat free lean (6%) of shoulders compared to Control pigs.

Effects of vitamin E and flaxseed on rumen-derived fatty acid intermediates in beef intramuscular fat.

To elucidate the effects of dietary vitamin E with or without flaxseed on beef fatty acid composition, 80 feedlot steers were fed 4 diets: Control-E (451 IU dl-α-tocopheryl acetate/head/day), Control+E (1051 IU dl-α-tocopheryl acetate/head/day), Flax-E (10% ground) and Flax+E. Vitamin E had no effect on animal growth or carcass weight (p>0.05), while flaxseed-fed steers had greater average daily gain (p=0.007), final live weight (p=0.005) and heavier carcasses (p=0.012). Feeding flaxseed increased the total n-3 fatty acid content of beef and this response was further accentuated by the inclusion of high levels of vitamin E in the diet. Feeding flax increased levels of some 18:3n-3 partial hydrogenation products including c15- and t13/14-18:1 and several 18:2 isomers (p<0.001) but decreased t10-18:1 (p<0.001). Vitamin E enhanced intramuscular levels of 18:3n-3 and its biohydrogenation products leading to greater accumulations of total n-3 fatty acids in lean ground beef. The consequences of increasing the concentrations of partially hydrogenated products on human health have yet to be investigated.

Compositional and instrumental firmness variations within fresh pork bellies.

Fresh pork bellies (n=24) were cut into 15 sections to measure the intra-belly variation in compositional and mechanical firmness characteristics. Length and width of each belly was measured before the belly was divided into 3 rows (D = dorsal; C = central; and V = ventral) and 5 columns (labeled 1, 2, 3, 4, and 5 from cranial to caudal), resulting in 15 belly sections of equal dimensions. The belly section with the greatest compression value was D-1, whereas the lowest compression value was found in the V-4 section (column×row, P<0.001). Conversely, the greatest and least puncture values were observed in the C-2 and V-5 locations, respectively (column×row, P=0.016). The D-3 section had the lowest proportion of lean and the greatest proportion of fat, but the greatest lean and lowest fat percentages were found in the V-1 and C-4 sections, respectively (column×row, P<0.001). The greatest proportions of saturated fatty acids (SFA) were found in the V-4 and V-5, and the lowest proportions of SFA were in D-1 (column×row, P<0.001). Moreover, C-4 and V-1 had the greatest percentages of monounsaturated fatty acids (MUFA), whereas the lowest MUFA content was observed in D-1, D-2, and D-3 (column×row, P<0.001). The D row (columns 1, 2, 3, and 5) also had the greatest proportion of polyunsaturated fatty acids (PUFA), but the lowest proportions of PUFA were located in C-4, V-4, and V-5 (column×row, P<0.001). Consequently, the iodine value was greatest in D-1 and lowest in V-4, V-5, and C-5 (column×row, P<0.001). It is apparent from these results that there is an obvious fatty acid composition gradient within bellies, which results in considerable intra-belly variation in composition and firmness.

Effect of feeding growing-fattening rabbits a diet supplemented with whole white lupin (Lupinus albus cv. Amiga) seeds on fatty acid composition and indexes related to human health in hind leg meat and perirenal fat.

A total of 20 weaned rabbits (33 days old) (10 per treatment) were fed one of two diets that included 150 g of sunflower meal (SF)/kg of diet or 120 g of whole white lupin (WL)/kg of diet for 42 days. The WL diet contained less saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) but more monounsaturated fatty acids (MUFA) than the SF diet. The WL diet significantly decreased SFA and PUFA content, as well as the PUFA n-6/PUFA n-3 ratio and saturation, atherogenic and thrombogenic indexes in hind leg meat. The fatty acid composition in perirenal fat was similar to that of hind leg meat; however, significantly higher MUFA levels were observed in rabbits fed the WL diet. Thus, feeding rabbits the WL diet affected the fatty acid profile of hind leg meat and perirenal fat in a favourable manner.

Fatty acid composition of minced meat, longissimus muscle and omental fat from Small East African goats finished on different levels of concentrate supplementation.

Effects of supplementing Small East African (SEA) goats with concentrate diets on fatty acids composition of minced meat, M. longissimus dorsi (LD) and omental fat were assessed using 23 animals (14.5 months old and 20.1 kg body weight). Goats were subjected to four levels of concentrate supplementation: ad libitum concentrate allowance (T100), 66% (T66), 33% (T33) and 0% (T0) of ad libitum concentrate allowance. All goats were slaughtered after 90 days of experimental period. Minced meat from concentrate-supplemented goats had higher (P<0.05) proportions of unsaturated fatty acids (UFA), monounsaturated fatty acids (MUFA) and desirable fatty acid (DFA) than that of non-supplemented ones (T0). Minced meat from T00 and T66 goats had similar proportions of polyunsaturated fatty acids (PUFA) and n-6 PUFA that were higher (P<0.05) than that of other dietary groups. There was limited variation in fatty acids composition of LD attributable to concentrate supplementation. Trans-vaccenic and linoleic acids were in higher (P<0.05) proportion in omental fat from concentrate-supplemented goats whereas margaric and arachidonic acids were in higher (P<0.05) proportion in omental fat from non-supplemented goats. Overall, LD was associated with PUFA, omental fat with saturated fatty acids (SFA), minced meat with MUFA. It is concluded that finishing SEA goats on concentrate diets will increase the proportion of DFA in meat from them. In addition, the proportion of PUFA in meat from such goats will peak at concentrate supplementation equivalent to 66% of their ad libitum intake. Consumers should avoid high intake of internal fat due to their richness in SFA.

Serum adipokine profile and fatty acid composition of adipose tissues are affected by conjugated linoleic acid and saturated fat diets in obese Zucker rats.

Conjugated linoleic acid (CLA) has been reported as having body fat lowering properties and the ability to modulate the inflammatory system in several models. In the present study, the effects of CLA added to saturated fat diets, from vegetable and animal origins, on the serum adipokine profile of obese Zucker rats were assessed. In addition, the fatty acid composition of epididymal and retroperitoneal adipose tissues was determined and a principal component analysis (PCA) was used to assess possible relationships between fatty acids and serum metabolites. Atherogenic diets (2 % cholesterol) were formulated with palm oil and ovine fat and supplemented or not with 1 % of a mixture (1:1) of cis-9, trans-11 and trans-10, cis-12-CLA isomers. CLA-fed animals exhibited lower daily feed intake, final body and liver weights, and hepatic lipids content. Total and LDL-cholesterol levels were increased in CLA-supplemented groups. CLA also promoted higher adiponectin and lower plasminogen activator inhibitor-1 (PAI-1) serum concentrations. In contrast to palm oil diets, ovine fat increased insulin resistance and serum levels of leptin, TNF-alpha and IL-1beta. Epididymal and retroperitoneal adipose tissues had similar deposition of individual fatty acids. The PCA analysis showed that the trans-10, cis-12-CLA isomer was highly associated with adiponectin and PAI-1 levels. Summing up, CLA added to vegetable saturated enriched diets, relative to those from animal origin, seems to improve the serum profile of adipokines and inflammatory markers in obese Zucker rats due to a more favourable fatty acid composition.

Fat and water 1H MRI to investigate effects of leptin in obese mice.

Leptin is known to be associated with regulation of body weight and fat content. The effects of exogenous leptin on abdominal visceral (VS) and subcutaneous (SC) fat volume and hepatic fat-to-water ratio in leptin-deficient obese mice were investigated by (1)H magnetic resonance imaging (MRI). Chemical shift-selected fat and water (1)H MRI of control and leptin-treated mice were obtained 1 day before treatment and after 7 days of treatment (0.3 mg/kg/day). Hepatic fat-to-water ratio and VS fat volume decreased significantly with treatment, whereas SC fat volume did not change. Noninvasive measurement of fat and water content in different body regions using MRI should prove useful for evaluating new drugs for the treatment of obesity and other metabolic disorders.

Influence of herring (Clupea harengus) and herring fractions on metabolic status in rats fed a high energy diet.

AIM: Few dietary studies have looked beyond fish oil to explain the beneficial metabolic effects of a fish-containing diet. Our aim was to study whether addition of herring, or sub-fractions of herring, could counteract negative metabolic effects known to be induced by a high-fat, high-sugar diet. METHODS: Rats were given six different diets: standard pellets; high energy diet with chicken mince (HiE control); high energy diet with herring mince (HiE herring); and high energy diet with chicken mince and either herring oil (HiE herring oil), herring press juice, PJ (HiE PJ) or herring low molecular weight PJ (HiE LMW-PJ). Factors associated with the metabolic syndrome were measured. RESULTS: There were no differences in energy intake or body weight between the groups, but animals fed high energy diets had a higher body fat content compared with the pellet group, although not statistically significant in all groups. Mesenteric adipocyte size was smaller in the HiE herring oil group compared with the HiE control. Glucose clamp studies showed that, compared with the pellet group, the HiE control and HiE herring diets, but not the HiE herring oil diet, induced insulin resistance. Addition of herring or herring oil to the high energy diet decreased total cholesterol levels, triacylglycerols and the atherogenic index compared with the HiE control group. CONCLUSIONS: The results suggest that addition of herring or herring oil counteracts negative effects on blood lipids induced by a high energy diet. The lipid component of herring thus seems to be responsible for these beneficial effects.

N-3 HUFAs affect fat deposition, susceptibility to oxidative stress, and apoptosis in Atlantic salmon visceral adipose tissue.

We have investigated how n-3 highly unsaturated fatty acids (HUFAs) in the diet affect fatty acid (FA) utilization, fat storage and oxidative stress (OS) in Atlantic salmon (Salmo salar) white adipose tissue (WAT). Four groups of Atlantic salmon were fed for 21 weeks on one of the four diets supplemented with 23% (of dry matter) lipid. Docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3) levels increased from 10% of total FAs in the rapeseed oil (RO) diet, to 20% in the fish oil (FO) diet, and to 50% and 55% in the DHA-enriched and EPA-enriched diets, respectively. Increased dietary levels of n-3 HUFAs resulted in lower fat percentage in WAT. Furthermore, mitochondrial FA beta-oxidation activity was higher in the FO group than it was in the RO group. The relative levels of DHA and EPA in phospholipids (PLs) from WAT and mitochondrial membranes increased with the increasing dietary levels of these HUFAs. In general, the mitochondrial membrane PLs were characterised by lower relative levels of n-3 HUFAs and higher relative levels of linoleic acid (LA; 18:2 n-6) than WAT membrane PLs. The predominance of LA relative to n-3 HUFAs in mitochondrial membrane PLs may help to protect these PLs from peroxidation. Cytochrome c oxidase measurements revealed higher incidence of disrupted mitochondrial membranes in the DHA and EPA dietary groups than in the FO and RO dietary groups. This disruption further affected the mitochondrial function, resulting in a marked reduction in FA beta-oxidation capacities. The reduction in mitochondrial function and the increase in the activity of superoxide dismutase (SOD) in the DHA and EPA groups showed that high dietary dose of DHA and EPA resulted in oxidative stress (OS). The increased activity of caspase 3 in the high n-3 HUFA groups suggested the induction of apoptosis and increased incidence of cell death in WAT, which may be one of the factors explaining the lower fat percentage found in these groups.

Relationship between fat cell size and number and fatty acid composition in adipose tissue from different fat depots in overweight/obese humans.

OBJECTIVE: To evaluate the body fat distribution and fat cell size and number in an overweight/obese population from both genders, and to determine the possible relationship between fat cell data from three different adipose tissue localizations (subcutaneous (SA), perivisceral and omental) and adipose tissue composition and dietary fatty acid. DESIGN: The sample consisted of 84 overweight/obese patients (29 men and 55 women) who have undergone abdominal surgery. The adipocyte size and total fat cell number was studied. Fat cell data were related with anthropometric, adipose tissue and subject's habitual diet fatty acid composition. MEASUREMENTS: Fat cell size was measured according to a Sjöström method from the three adipose depots. Total fat cell number was also calculated. The fatty acid composition of adipose tissue was examined by gas chromatography. The subjects diet was studied by a 7 days dietary record. RESULTS: Our data showed a negative relationship between the adipocyte size and the n-6 and n-3 fatty acids content of the SA adipose tissue (r=-0.286, P=0,040; r=-0.300, P=0.030) respectively, and the n-6 in the omental depots (r=-0.407, P=0.049) in the total population. Positive associations with the total of saturated (r=0.357, P=0.045) and negative (r=-0.544, P=0.001) with the n-9 fatty acids were observed when the relationship between the adipocyte number and the fatty acid composition of the different anatomical fat regions was studied. Dietary fatty acids composition positively correlated with fat cell size for the myristic acid (14:0) in men in the visceral depot (r=0.822, P=0.023), and for the saturated fatty acids (SFAs) in women in the omental depot (r=0.486, P=0.035). CONCLUSION: In the present study, for the first time in humans we found that n-3 and n-6 fatty acids are related to a reduced adipocyte size according to the depot localization. In contrast, adipose tissue and dietary SFAs significantly correlated with an increase in fat cell size and number. No significant associations were found between n-9 acids content and adipocyte size. However, n-9 adipose tissue fatty acids content was inversely associated with fat cell number showing that this type of fatty acid could limit hyperplasia in obese populations. The differences observed in the three different regions, perivisceral, omental and SA fat, indicate that this population adipose tissue have depot-specific differences.

Dietary coconut oil increases conjugated linoleic acid-induced body fat loss in mice independent of essential fatty acid deficiency.

Conjugated linoleic acid (CLA) induces a body fat loss that is enhanced in mice fed coconut oil (CO), which lacks essential fatty acids (EFA). Our objective was to determine if CO enhancement of CLA-induced body fat loss is due to the lack of EFA. The CLA-EFA interaction was tested by feeding CO and fat free (FF) diets for varying times with and without replenishment of individual EFA. Mice fed CO during only the 2-week CLA-feeding period did not differ from control mice in their adipose EFA content but still tended (P=0.06) to be leaner than mice fed soy oil (SO). Mice raised on CO or FF diets and fed CLA were leaner than the SO+CLA-fed mice (P<0.01). Mice raised on CO and then replenished with linoleic, linolenic, or arachidonic acid were leaner when fed CLA than mice raised on SO (P<0.001). Body fat of CO+CLA-fed mice was not affected by EFA addition. In summary, CO-fed mice not lacking in tissue EFA responded more to CLA than SO-fed mice. Also, EFA addition to CO diets did not alter the enhanced response to CLA. Therefore, the increased response to CLA in mice raised on CO or FF diets appears to be independent of a dietary EFA deficiency.