Effects of triacylglycerol structure and solid fat content on fasting responses of mice.

PURPOSE: Fat randomization and interesterification change triacylglycerol (TAG) structure and its solid fat content profile. It has not been thoroughly investigated whether these changes affect lipid metabolism. METHODS: Two experiments were conducted to investigate the effects of TAG structure and solid fat content on feed intake, body weight change, and serum metabolite concentrations in mice. An experiment used two fats rich in 1,2-dipalmitoyl-3-oleoylglycerol (PPO) and 1,3-dipalmitoyl-2-oleoylglycerol (POP) as comparative pair of fats to assess the effect of TAG structure since PPO and POP have the same fatty acid composition and solid fat content at 37 °C. Another experiment used a fat rich in 1-palmitoyl-2,3-dioleoylglycerol (POO) with solid fat content of zero at 37 °C and a mixture of fats that had the same general fatty acid composition and palmitic acid positional distribution, but with solid fat content of 22 % at 37 °C. This pair of fats was used to examine the effect of solid fat content on blood lipid profile. RESULTS: After 6-week feeding, the pair of fats with different solid fat contents did not significantly affect the concentrations of total serum cholesterol, HDL cholesterol, TAG, non-esterified fatty acid (NEFA), or blood glucose. However, the PPO fat significantly reduced feed intake, body weight, and serum glucose concentration as compared to POP. CONCLUSION: These results suggest that the presence of solid fat at the level examined does not affect lipid metabolism and lipemia, but PPO diet significantly affects NEFA and glucose concentrations. Palmitic acid at the sn-2 position of the TAG may have significant effect on appetite, which may be mediated via the gut receptors.

Early lesion formation in colorectal carcinogenesis is associated with adiponectin status whereas neoplastic lesions are associated with diet and sex in C57BL/6J mice.

Adiponectin is an antiinflammatory and insulin-sensitizing hormone that is decreased in obesity. Although controversial, it has been suggested that decreased adiponectin contributes to colorectal cancer risk in obesity. To further investigate the role of adiponectin in obesity-linked colorectal carcinogenesis, we used male and female adiponectin knockout (KO) and wild-type (Wt) C57BL/6J mice. Tumorigenesis was induced in all mice with the combined treatment of azoxymethane (AOM) and dextran sodium sulfate (DSS). Following AOM/DSS treatment, mice were fed a low-fat control (LFC), or high-fat lard (HFL) diet for 7 1/2 wk. KO mice developed fewer total lesions than Wt mice, males developed fewer lesions than females, and mice fed the HFL diet developed fewer lesions than those fed the LFC diet. Early lesion multiplicity was influenced by genotype, whereas advanced lesion development was influenced by sex and diet. Moreover, lesion types were differentially correlated with serum adipokines and colon gene expression of adiponectin receptors, insulin receptor, and toll-like receptor 4. These data suggest that in the AOM/DSS model of carcinogenesis, adiponectin functions to promote early lesion development whereas sex and diet are important regulators of advanced lesion development through pathways involved in inflammation and insulin signaling.

Low-dose dietary resveratrol has differential effects on colorectal tumorigenesis in adiponectin knockout and wild-type mice.

Obesity is associated with a decrease in the antiinflammatory hormone, adiponectin, and increases in the circulating concentrations of multiple proinflammatory cytokines. These changes contribute to colon tumorigenesis. Resveratrol increases adiponectin production in adipocytes and attenuates the development of colon cancer. Thus, we hypothesized that adiponectin is an integral component of the mechanism by which resveratrol antagonizes colorectal tumorigenesis. To investigate this, we induced tumorigenesis in adiponectin knockout (KO) and wild-type (Wt) C57BL/6 mice through combined azoxymethane and dextran sodium sulfate treatment during which mice were fed a high-fat, lard-based diet, or the same diet containing 20 mg/kg resveratrol. After 14 wk on diet, Wt mice gained more weight and, on a percentage basis, had higher fat mass and lower lean mass than KO mice. Resveratrol tended to attenuate this response in male Wt mice. Resveratrol also tended to reduce aberrant crypt foci development and decrease circulating interleukin 6 and insulin concentrations in male but not female Wt mice. Taken together, resveratrol improved overall health of obese Wt but not KO mice as hypothesized with a differential sex response.

Effect of a mitochondria-targeted vitamin E derivative on mitochondrial alteration and systemic oxidative stress in mice.

The objective of the present study was to determine whether a mitochondria-targeted vitamin E derivative (MitoVit E) would affect certain mitochondrial parameters, as well as systemic oxidative stress. A total of sixty-four mice were fed a high-fat (HF) diet for 5 weeks. They were then switched to either a low-fat (LF) or a medium-fat (MF) diet, and administered orally with MitoVit E (40 mg MitoVit E/kg body weight) or drug vehicle (10 % (v/v) ethanol in 0·9 % (w/v) NaCl solution), every other day for 5 weeks. Mitochondrial ATP and H(2)O(2) production rates in both the liver and the gastrocnemius were not affected by MitoVit E administration in either LF or MF diet-fed mice. However, the number and average size of the subsarcolemmal mitochondria, but not the intermyofibrillar mitochondria, from the soleus muscle were significantly higher in the MF group receiving MitoVit E (MF-E) than in the MF group receiving vehicle only (MF-C). After the mice were switched from the HF diet to the four dietary treatments (LF-C, LF-E, MF-C and MF-E), the decrease in urinary isoprostane concentration was significantly greater in the LF-E group than in the other three groups during the whole study (weeks 6-10). In addition, MitoVit E significantly increased plasma superoxide dismutase (SOD) activity in the MF diet-fed group without affecting plasma glutathione peroxidase activity or H(2)O(2) levels. Overall, these data suggest that MitoVit E affects subsarcolemmal mitochondrial density and systemic oxidative stress parameters such as plasma SOD activity and urinary isoprostane concentration.

Characterization of ß-adrenergic receptors in bovine intramuscular and subcutaneous adipose tissue: comparison of lubabegron fumarate with ß-adrenergic receptor agonists and antagonists.

Chinese hamster ovary cell constructs expressing either the ß 1-, ß 2- or ß 3-adrenergic receptor (AR) were used to determine whether a novel ß-AR modulator, lubabegron fumarate (LUB; Experior, Elanco Animal Health) might exert greater potency for a specific ß-AR subtype. EC50 values calculated based on cAMP accumulation in dose response curves indicate that LUB is highly selective for the ß 3-AR subtype, with an EC50 of 6 × 10-9 M, with no detectible agonistic activity at the ß 2-AR. We hypothesized that the accumulation of lipolytic markers would reflect the agonist activity at each of the ß-receptor subtypes of the specific ligand; additionally, there would be differences in receptor subtype expression in subcutaneous (s.c.) and intrmuscular (i.m.) adipose tissues. Total RNA was extracted from adipose tissue samples and relative mRNA levels for ß 1-, ß2-, and ß 3-AR were measured using real-time quantitative polymerase chain reaction. Fresh s.c. and i.m. adipose tissue explants were incubated with isoproterenol hydrochloride (ISO; ß-AR pan-agonist), dobutamine hydrochloride (DOB; specific ß 1-AA), salbutamol sulfate (SAL; specific ß 2-AA), ractopamine hydrochloride (RAC), zilpaterol hydrochloride (ZIL), BRL-37344 (specific ß 3-agonist), or LUB for 30 min following preincubation with theophylline (inhibitor of phosphodiesterase). Relative mRNA amounts for ß 1-, ß 2-, and ß 3-AR were greater (P < 0.05) in s.c. than in i.m. adipose tissue. The most abundant ß-AR mRNA in both adipose tissues was the ß 2-AR (P < 0.05), with the ß 1- and ß 3-AR subtypes being minimally expressed in i.m. adipose tissue. ISO, RH, and ZH stimulated the release of glycerol and nonesterified fatty acid (NEFA) from s.c. adipose tissue, but these ß-AR ligands did not alter concentrations of these lipolytic markers in i.m. adipose tissue. LUB did not affect glycerol or NEFA concentrations in s.c. or i.m. adipose tissue, but attenuated (P < 0.05) the accumulation of cAMP mediated by the ß 1- and ß 2-AR ligands DOB and SAL in s.c. adipose tissue. Collectively, these data indicate that bovine i.m. adipose tissue is less responsive than s.c. adipose tissue to ß-adrenergic ligands, especially those that are agonists at the ß 1- and ß3-receptor subtypes. The minimal mRNA expression of the ß 1- and ß 3 subtypes in i.m. adipose tissue likely limits the response potential to agonists for these ß-AR subtypes.

A mitochondria-targeted vitamin E derivative decreases hepatic oxidative stress and inhibits fat deposition in mice.

Our objective in this study was to determine whether a mitochondria-targeted vitamin E derivative (MitoVit E) would decrease oxidative stress and associated obesity by preventing a previously proposed aconitase inhibition cascade. Sixty-four mice were fed a high-fat (HF) diet for 5 wk. They were then switched to either a low-fat (LF) or a medium-fat (MF) diet and gavaged with MitoVit E (40 mg MitoVit E x kg body weight(-1)) or drug vehicle (10% ethanol in 0.9% NaCl solution) every other day for 5 wk. Epididymal fat weight, as well as liver lipid and remaining carcass lipid, were significantly lower in the MF group receiving MitoVit E (MF-E) than in the MF group receiving vehicle only (MF-C). Liver mitochondrial H(2)O(2) production and the protein carbonyl level were also significantly lower in MF-E than in MF-C mice. In contrast, none of the biochemical variables (aconitase activity, ATP and H(2)O(2) production, and protein carbonyl level) in the muscle mitochondria were modified by MitoVit E in either MF or LF groups. Expression of acetyl-CoA carboxylase and fatty acid synthase in both liver and adipose tissue of MF groups was not affected by MitoVit E. However, expression of carnitine palmitoyltransferase 1a in the liver and uncoupling protein 2 in adipose tissue were significantly enhanced by MitoVit E in both LF and MF groups. In conclusion, MitoVit E attenuates hepatic oxidative stress and inhibits fat deposition in mice but not through alleviation of the aconitase inhibition cascade.

Feeding long-chain n-3 polyunsaturated fatty acids during gestation increases intestinal glucose absorption potentially via the acute activation of AMPK.

The current study utilized Ussing chambers to examine the impact of supplementing maternal gestation and/or lactation diets with n-3 polyunsaturated fatty acids (PUFA) provided via a protected fish oil (PFO) product on intestinal fatty acid profiles and ex vivo glucose uptake in the jejunum of weanling piglets. Jejunum tissues were enriched with n-3 PUFA as a result of feeding the sows the PFO during gestation and/or lactation (P<.05). Glucose uptake improved by twofold (P<.042) in intestinal preparations obtained from the offspring of sows fed PFO during gestation or throughout gestation/lactation versus lactation alone. This was also reflected in the jejunum protein expressions of glucose transporter 2 (GLUT2) and sodium-dependent glucose transporter 1 (SGLT1). Furthermore, adding docosahexaenoic acid (DHA) or an AMP-activated protein kinase (AMPK) agonist to the chamber buffer improved glucose uptake (P<.05) in intestinal preparations obtained from the offspring fed the control diet, devoid of the PFO product and containing minimal concentrations of n-3 PUFA. Collectively, these data indicate two important points. First, long-term exposure to n-3 PUFA via the maternal gestation diet effectively enhances glucose uptake in the weanling piglet, and the underlying mechanism may be associated with changes in the intestinal fatty acid profile. Secondly, there is an apparent direct and acute effect of DHA that is achieved within a time frame that precludes substantial changes in the intestinal fatty acid profile. Additionally, both mechanisms may involve activation of AMPK. Thus, n-3 PUFA delivered in utero and postnatally via the maternal diet may help the offspring adapt quickly to rapidly changing diets early in life and allow optimal nutrient uptake.

The development of porcine models of obesity and the metabolic syndrome.

Despite aggressive research aimed at understanding the myriad biochemical factors that are integrated to balance energy intake and expenditure to maintain normal body weight, obesity is increasing at an alarming rate, and the long-term success of prevention and intervention strategies is minimal. Because much of the scientific literature addressing obesity has originated with rodent models, there is considerable interest among researchers and funding agencies in the development of comparative animal models. Furthermore, numerous disparate results between rodent models and humans (i.e., adipsin, leptin, resistin, tumor necrosis factor-alpha, and other adipokines) have hindered the translation of rodent data into actionable technologies for humans. The pig is an exceptional restenosis model, and is emerging rapidly as a biomedical model for energy metabolism and obesity in humans because it is devoid of brown fat postnatally and because of their similar metabolic features, cardiovascular systems, and proportional organ sizes. This article highlights the current literature devoted to the development of porcine models for obesity and the metabolic syndrome, with a particular emphasis on the role of adipose tissue and adipokines in the regulation of energy balance and the inflammation associated with obesity.

n-3 PUFA attenuate lipopolysaccharide-induced down-regulation of toll-like receptor 4 expression in porcine adipose tissue but does not alter the expression of other immune modulators.

The objective of this study was to test the hypothesis that the inflammatory response to lipopolysaccharide (LPS) in vivo is accompanied by down-regulation of toll-like receptor (TLR) 4 in adipose tissue, and a source of protected n-3 polyunsaturated fatty acid (PUFA) attenuates this response. Seventy-two castrated male pigs were individually fed either a control (CONT) diet, or the CONT diet containing 1.87% (LF) or 7.50% (HF) protected n-3 PUFA on a weight basis for 7 weeks. Adipose and muscle tissue biopsy samples were taken at Weeks 1, 2, 3, 4 and 7 to assess gene expression and/or confirm tissue enrichment with eicosapentaenoic acid and docosahexaenoic acid and reflected the n-3 PUFA contained in the diet. The LPS challenge was performed at week 7 and consisted of sequential injections of 10 and 2.5 mug LPS per kilogram of body weight 23 h apart. The LPS challenge resulted in a marked down-regulation (P=.004) of TLR4 at the protein level in the adipose tissue of challenged vs. control pigs, but LF and HF clearly blocked this response at the mRNA level. Although LF and HF also attenuated (P<.001) the LPS-induced acute febrile response and lowered (P<.002) serum concentrations of tumour necrosis factor alpha. Cyclooxygenase 2 and 12-lipoxygenase were readily expressed in porcine adipose tissue, but there was no effect of LF, HF or LPS on expression levels of these inflammatory mediators, or that of TNF and interleukin 6, at the conclusion of the challenge period. These findings indicate that adipose tissue responds to LPS administration in vivo by reducing TLR4 mRNA and protein abundance and that the anti-inflammatory effects of n-3 PUFA do not include down-regulation of TLR4 in adipose tissue.

Chronic leptin administration increases serum NEFA in the pig and differentially regulates PPAR expression in adipose tissue.

Two in vivo studies were conducted with pigs to determine the effects of exogenous leptin on the expression of peroxisome proliferator activated receptors (PPAR), and on serum concentrations of selected metabolites and hormones. Initially, leptin was administered i.m. to young pigs for 15 days at 0 (control), 0.003 (low), 0.01 (medium) and 0.03 (high) mg. kg(-1). day(-1). There was no leptin effect on serum glucose (P > 0.84), triglycerides (P > 0.69), non-esterified fatty acids (NEFA, P > 0.53), or glycerol (P > 0.33). Leptin at the intermediate and high doses depressed adipose expression of both PPARgamma1 (P < 0.06) and PPARgamma2 (P < 0.01). In a second study, we used a paired-feeding experimental design to determine the effects of a higher dose of leptin (0.05 mg. kg(-1). day(-1)) on serum metabolites and PPAR expression in selected tissues. At this dose, leptin increased (P < 0.0001) serum NEFA concentrations relative to both the ad libitum and pair-fed control groups. However, in this study, there was no difference in the expression of PPARgamma1 in adipose tissue, but PPARgamma2 mRNA was upregulated by leptin (P < 0.08). In contrast, leptin had no impact on the expression of PPARalpha in liver, skeletal muscle or adipose tissue. Adipose tissue explants were also incubated with leptin to assess the effect on PPARgamma expression, in vitro. The abundance of PPARgamma1 mRNA (P < 0.05) was increased after 24 hr of exposure, but the effect of leptin on gamma2 was not significant (P > 0.24). The lipolytic effect of leptin was also evaluated in vitro using isolated adipocytes. In keeping with the increase in serum NEFA concentrations in vivo, leptin stimulated lipolysis in vitro, increasing glycerol concentrations in the medium to about 219% of that in basal (non-treated) culture medium after 8 hr of incubation. Collectively, the data presented herein indicate that leptin modulates lipid metabolism in the pig, but that PPARalpha expression is not a parallel target of leptin as it is in rodent models. The regulation of PPARgamma by leptin seems complex in that it varied in relation to dose in vivo, and may be impacted by in vitro vs. in vivo circumstances.

The regulation of IGF-1 by leptin in the pig is tissue specific and independent of changes in growth hormone.

A combination of in vivo and in vitro experiments were performed to determine the extent to which exogenous leptin regulates serum growth hormone (GH) and insulin-like growth factor I (IGF-1) concentrations, and the abundance of IGF-1 mRNA in major peripheral tissues. Initially (Experiment 1), a recombinant human leptin analog was administered i.m. to young growing pigs (approximately 27 kg body weight) for 15 days at 0 (control), 0.003, 0.01 and 0.03 mg. kg(-1). day(-1). Although there was no sustained effect of leptin on serum GH, there was a reduction (P < 0.02) in serum IGF-1 at the intermediate dose that paralleled a decrease (P < 0.09) in hepatic IGF-1 expression. Leptin, at these doses, did not reduce feed intake (P > 0.57), nor was there an effect of leptin on dietary nitrogen retention (P > 0.97). In a second experiment, pigs were injected with vehicle or a higher dose of leptin (0.05 mg. kg(-1). day(-1)) for 14 days. A third treatment group was injected with vehicle and pair-fed to the intake of the group treated with leptin. In this study, exogenous leptin resulted in a sustained increase in serum leptin (P < 0.0001) and reduction in feed intake of approximately 30% (P < 0.0001). Serum IGF-1 was depressed in both the leptin-treated and pair-fed groups, relative to the group allowed ad-libitum intake (P < 0.01). Furthermore, there was no difference among treatments in the relative abundance of IGF-1 mRNA in skeletal muscle (P > 0.42) or adipose tissue (P > 0.26), and liver mRNA abundance was actually increased (P < 0.01) by leptin, despite the lower feed intake. Finally, to determine whether leptin altered the secretion of IGF-1 by isolated pig hepatocytes, primary cultures were incubated with leptin for 24 to 48 hr (Experiment 3). Leptin (100 nM) caused a sharp reduction (P < 0.0001) in dexamethasone-induced IGF-1 secretion at 24 hr (47% reduction) and at 48 hr (40% reduction). Collectively, these data indicate that leptin may regulate hepatic IGF-1 production in the pig, independent of GH, but that hepatocyte sensitivity to leptin may be depend on dose and in vitro vs. in vivo conditions.

Leptin regulation of lipid homeostasis: dietary and metabolic implications.

Leptin is a 16 kDa protein synthesized and secreted primarily from adipocytes. Leptin acts centrally to regulate appetite and energy expenditure and has peripheral effects to coordinate whole-body energy metabolism. Leptin acts as an energy balance and nutrient sensor; its expression and function are regulated by endocrine and dietary factors. Leptin regulates lipid metabolism, specifically lipid storage in adipocytes as well as in skeletal muscle, liver and the pancreas. Effects of leptin on tissue lipid metabolism include regulation of lipogenesis and fatty acid oxidation. Leptin resistance is a hallmark of rodent and human obesity and appears to be due to defects in leptin-receptor signalling (proximal and perhaps distal) as well as impaired leptin transport into the brain. Dietary composition, especially dietary fatty acid intake and profile, impacts on the expression of genes encoding leptin and leptin receptors as well as downstream leptin effectors. Dietary fat consumption, especially consumption of saturated fatty acids, can induce leptin resistance. Leptin has been implicated in nutritional programming during fetal and neonatal growth with long-term effects on susceptibility to obesity, diabetes and CHD in adults.

Inflammation in response to n3 fatty acids in a porcine obesity model.

Fatty acids have distinct cellular effects related to inflammation and insulin sensitivity. Dietary saturated fat activates toll-like receptor 4, which in turn can lead to chronic inflammation, insulin resistance, and adipose tissue macrophage infiltration. Conversely, n3 fatty acids are generally antiinflammatory and promote insulin sensitivity, in part via peroxisome proliferator-activated receptor γ. Ossabaw swine are a useful biomedical model of obesity. We fed Ossabaw pigs either a low-fat control diet or a diet containing high-fat palm oil with or without additional n3 fatty acids for 30 wk to investigate the effect of saturated fats and n3 fatty acids on obesity-linked inflammatory markers. The diet did not influence the inflammatory markers C-reactive protein, TNFα, IL6, or IL12. In addition, n3 fatty acids attenuated the increase in inflammatory adipose tissue CD16(-)CD14(+) macrophages induced by high palm oil. High-fat diets with and without n3 fatty acids both induced hyperglycemia without hyperinsulinemia. The high-fat only group but not the high-fat group with n3 fatty acids showed reduced insulin sensitivity in response to insulin challenge. This effect was not mediated by decreased phosphorylation of protein kinase B. Therefore, in obese Ossabaw swine, n3 fatty acids partially attenuate insulin resistance but only marginally change inflammatory status and macrophage phenotype in adipose tissue.

Absence of Tlr2 protects against high-fat diet-induced inflammation and results in greater insulin-stimulated glucose transport in cultured adipocytes.

We have previously shown that toll-like receptor-4 (Tlr4) is involved in obesity-induced inflammation in adipose tissue (AT). However, less is known about the role of Tlr2 in this process. To determine the involvement of this receptor in obesity-induced inflammation, we utilized male Tlr2(-/-) mice that were backcrossed onto a mouse model of diet-induced obesity (DIO). Mice were fed either low-fat control (LFD) or high-fat diet (HFD) ad libitum for 16 weeks. Despite negligible differences in body weight or energy intake, Tlr2(-/-) mice were protected from HFD-induced adiposity as was evident by reduced epididymal fat pad weight and carcass lipid content. Corresponding with these effects was a blunted accumulation of F4/80-positive macrophages in AT of Tlr2(-/-) mice. Furthermore, transcript abundance of proinflammatory mediators, including monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNFα) and nitric oxide synthase-2 (NOS2) in AT of Tlr2(-/-) mice, was lower or less responsive to DIO. There were no significant differences in serum markers of insulin sensitivity (data not shown). However, adipocytes derived from stromal vascular cells (SVCs) isolated from AT of Tlr2(-/-) mice had considerably greater basal and insulin-stimulated glucose uptake as compared with those obtained from Tlr2(+/+) mice. Furthermore, the absence of Tlr2(-/-) precluded the induction of insulin resistance by zymosan A (ZymA) but not by palmitate. These data indicate that Tlr2 may be directly involved in HFD-induced inflammation and may also regulate basal and insulin-stimulated glucose uptake in adipocytes.

Intracoronary adiponectin at reperfusion reduces infarct size in a porcine myocardial infarction model.

Reperfusion injury (RI) remains an important limitation of myocardial revascularization. The aim of the present study was to evaluate the influence of the intracoronary injection of adiponectin on RI and cardiomyocyte death in a porcine myocardial infarction model. Acute infarction in 14 Polish domestic pigs was induced by inflation of an over the wire balloon (OTW) catheter in the medial left anterior descending artery for 60 min. The study group consisted of 7 pigs in which intracoronary adiponectin (50 µg) was infused through the OTW catheter immediately before reperfusion. The control group (n=7) was administered placebo. Animals were sacrificed after two days of follow-up. The infarct area (IA) was stained with tetrazoline and the area at risk (AAR) with intracoronary administration of Evans Blue dye before euthanasia. Hearts in each group had similar AARs (46.2±9.9% vs. 48.4±6.2% of the whole myocardium, p=ns). The IA/AAR% and IA were smaller in the study group when compared to the control (24.7±4.0% vs. 45.3±22.5%, p=0.005; and 11.7±4.9% vs. 20.5±5.6%, p=0.01, respectively). These outcomes corresponded well with the peak troponin levels after 12 h (109.9±60.9 ng/ml vs. 185.5±39.4 ng/ml, p=0.017). After two days there was a significantly higher LVEF in the study group (51.4±8.5% vs. 33.9±8.6%, p=0.002). There was also a trend toward lower apoptosis enhancement in the viable myocardium in the study group (3.11±2.3 vs. 8.92±6.3; p=0.07). The administration of adiponectin into the infarct- related artery is safe and feasible. The treatment significantly reduced the infarct size.

Myostatin null mice respond differently to dietary-induced and genetic obesity.

Our objective was to determine sensitivity of myostatin null (MN) mice to obesity induction by dietary or genetic means. To induce dietary obesity, 3-week-old wild type (WT) and MN mice were fed diets with 60% calories (HF) or 10% calories from fat (LF) for 4 weeks. MN mice did gain body fat on the HF diet but to a lesser extent than WT mice. Body weight and fat content was similar in MN mice fed HF and LF diets. To induce genetic obesity, the MN mutation was incorporated into leptin db/db (DB) mice generating mice homozygous for each mutation (MNDB). Nine-week-old MNDB mice were obese, similar to DB mice. Body weight, body fat content, fat pad weight and adipocyte size were all increased in MNDB mice compared to MN and WT mice and were quite similar to DB mice. However, fasting blood glucose, an indicator of insulin resistance and diabetes, was reduced in MNDB mice compared to DB mice. These results indicate that MN mice gain less body fat than WT on a HF diet, but the MN mutation does not alter fat accumulation caused by DB mutation. Thus, MN mice are not always resistant to obesity development.

Tlr-4 deficiency selectively protects against obesity induced by diets high in saturated fat.

Toll-like receptor-4 (Tlr-4), a key pattern recognition receptor involved in innate immune response, is activated by saturated fatty acids (SFAs). To investigate the involvement of this receptor in obesity caused by consumption of diets high in fat, we utilized male Tlr-4-deficient 10ScN mice and 10J controls. Mice were fed either low fat (low-fat control (LFC)), high unsaturated fat (high-fat control (HFC)), or high saturated fat + palmitate (HFP) diets ad libitum for 16 weeks. Relative to the LFC diet, the HFC diet resulted in greater epididymal fat pad weights and adipocyte hypertrophy in both Tlr-4-deficient and normal mice. However, the 10ScN mice were completely protected against the obesigenic effects of the HFP diet. Moreover, macrophage infiltration and monocyte chemotactic protein-1 (MCP-1) transcript abundance were lower in adipose tissue of 10ScN mice fed the HFP diet, and the hyperinsulinemic response was negated. Tlr-4-deficient mice also had markedly lower circulating concentrations of MCP-1 and much less nuclear factor-kappaB (NFkappaB) protein in nuclear extracts prepared from adipose tissue, irrespective of diet. In contrast, Tlr-4 deficiency did not attenuate the induction of tumor necrosis factor-alpha (TNF-alpha) or interleukin-6 (IL-6) expression in adipose tissue. These data indicate that Tlr-4 deficiency selectively protects against the obesigenic effects of SFA and alters obesity-related inflammatory responses in adipose tissue.

In utero and postnatal exposure to long chain (n-3) PUFA enhances intestinal glucose absorption and energy stores in weanling pigs.

The aim of this research was to determine whether feeding gestating and lactating sows (n-3) PUFA [eicosapentaenoic acid (EPA) and/or docosahexenoic acid (DHA)] or coconut fat (saturated fat) influences ex vivo glucose absorption in the proximal jejunum and glucose and glycogen concentration of liver and muscle of their offspring at weaning. Sows were fed 1 of 4 diets for 150 d, which included the entire gestation and lactation periods. The diets consisted of basal corn/soybean meal (CONT), CONT + protected EPA and DHA-rich fish oil (PFO), CONT + DHA Gold fat (DHAGF), and CONT + coconut fat (COCO). All tissues were collected from piglets (n = 4 per treatment) following a 24-h period of food deprivation, which was initiated at weaning. Proximal jejunum samples were mounted in modified Ussing chambers for transport determinations. Relative to the CONT (7 muA/cm(2)), active glucose transport was greater (P = 0.013) in piglets from sows fed the PFO (30 microA/cm(2)) and DHAGF (40 microA/cm(2)) diets, but not the COCO diet (19 microA/cm(2); pooled SEM = 5). Likewise, jejunum expression of glucose transporter 2 and sodium glucose transporter 1 protein tended (P < 0.10) to be greater in piglets from dams fed the PFO and DHAGF diets, as did AMP-activated protein kinase activity. Piglets' muscle glycogen was greater than in CONT (34 +/- 5.2 mg/g wet tissue) only in piglets from dams fed the DHAGF (46 +/- 5.2 mg/g wet tissue; P < 0.05). These results indicate that (n-3) PUFA, particularly DHA, improves intestinal glucose absorption and muscle glycogen concentrations in newly weaned pigs. These findings may also have important implications for human mothers and infants.

Adiponectin inhibits LPS-induced NF-kappaB activation and IL-6 production and increases PPARgamma2 expression in adipocytes.

Obesity and insulin resistance are often associated with lower circulating adiponectin concentrations and elevated serum interleukin-6 (IL-6) and/or tumor necrosis factor-alpha (TNF-alpha). Adiponectin suppresses activation of nuclear factor-kappaB (NF-kappaB) in aortic endothelial cells and porcine macrophages. Accordingly, we hypothesized that adiponectin is an anti-inflammatory hormone and suppresses activation of NF-kappaB in adipocytes. Because peroxisome proliferator-activated receptor gamma2 (PPARgamma2) antagonizes the transcriptional activity of NF-kappaB, we determined whether adiponectin alters PPARgamma2 expression in pig adipocytes. In addition, we determined whether interferon-gamma alters the expression of PPARgamma2 in the presence or absence of adiponectin. Primary adipocytes from pig subcutaneous adipose tissue were treated with or without lipopolysaccharide (LPS; 10 microg/ml) and adiponectin (30 microg/ml), and nuclear extracts were obtained for gel shift assays to assess nuclear localization of NF-kappaB. Whereas LPS induced an increase in NF-kappaB activation, adiponectin suppressed both NF-kappaB activation and the induction of IL-6 expression by LPS (P<0.05). Similar results were obtained in 3T3-L1 adipocytes. In addition, adiponectin antagonized LPS-induced increase in TNF-alpha mRNA expression (P<0.05) and tended (P<0.065) to diminish its accumulation in the culture media in 3T3-L1 adipocytes. Adiponectin also induced an upregulation of PPARgamma2 mRNA (P<0.05). Although IFN-gamma did not reduce the basal expression of PPARgamma2, it suppressed PPARgamma2 induction by adiponectin (P<0.05). These findings indicate that adiponectin may be a local regulator of inflammation in the adipocyte and adipose tissue via its regulation of the NF-kappaB and PPARgamma2 transcription factors.

Direct regulation of lipolysis by interleukin-15 in primary pig adipocytes.

We recently provided evidence that interleukin-15 (IL-15) is expressed lowly in the pig adipocyte and that interferon-gamma (IFN-gamma) markedly increases this expression through a pathway regulated in part by protein kinase C. In the present study, we tested the hypothesis that IL-15 acts directly on the adipocyte to regulate lipid accretion by enhancing lipolysis or suppressing lipogenesis. Using recombinant porcine IL-15, we determined that this cytokine stimulates lipolysis in a dose-dependent manner (P < 0.001). Furthermore, comparative studies with other cytokines showed that IL-15 is more potent in its acute stimulation of lipolysis than either TNF-alpha, IL-6, or LPS (P < 0.001). When specific inhibitors of protein kinase A or Janus kinase are present, the lipolytic effect of IL-15 is attenuated (P < 0.01). These data indicate that, in addition to its regulation of muscle protein accretion and T-cell growth and development, IL-15 also targets the adipocyte directly to alter stimulate lipolysis. Thus, when induced by IFN-gamma or other inflammatory mediators, IL-15 may be a significant homeorhetic factor that mobilizes and directs energy away from the adipocyte to other cells during the acute phase of the inflammatory response.