Effects of Anthocyanin and Flavanol Compounds on Lipid Metabolism and Adipose Tissue Associated Systemic Inflammation in Diet-Induced Obesity.

Background. Naturally occurring substances from the flavanol and anthocyanin family of polyphenols have been proposed to exert beneficial effects in the course of obesity. We hypothesized that their effects on attenuating obesity-induced dyslipidemia as well as the associated inflammatory sequelae especially have health-promoting potential. Methods. Male C57BL/6J mice (n = 52) received a control low-fat diet (LFD; 10 kcal% fat) for 6 weeks followed by 24 weeks of either LFD (n = 13) or high-fat diet (HFD; 45 kcal% fat; n = 13) or HFD supplemented with 0.1% w/w of the flavanol compound epicatechin (HFD+E; n = 13) or an anthocyanin-rich bilberry extract (HFD+B; n = 13). Energy substrate utilization was determined by indirect calorimetry in a subset of mice following the dietary switch and at the end of the experiment. Blood samples were collected at baseline and at 3 days and 4, 12, and 20 weeks after dietary switch and analyzed for systemic lipids and proinflammatory cytokines. Adipose tissue (AT) histopathology and inflammatory gene expression as well as hepatic lipid content were analyzed after sacrifice. Results. The switch from a LFD to a HFD lowered the respiratory exchange ratio and increased plasma cholesterol and hepatic lipid content. These changes were not attenuated by HFD+E or HFD+B. Furthermore, the polyphenol compounds could not prevent HFD-induced systemic rise of TNF-α levels. Interestingly, a significant reduction in Tnf gene expression in HFD+B mice was observed in the AT. Furthermore, HFD+B, but not HFD+E, significantly prevented the early upregulation of circulating neutrophil chemoattractant mKC. However, no differences in AT histopathology were observed between the HFD types. Conclusion. Supplementation of HFD with an anthocyanin-rich bilberry extract but not with the flavanol epicatechin may exert beneficial effects on the systemic early inflammatory response associated with diet-induced obesity. These systemic effects were transient and not observed after prolongation of HFD-feeding (24 weeks). On the tissue level, long-term treatment with bilberry attenuated TNF-α expression in adipose tissue.

Tumor necrosis factor receptor 1 gain-of-function mutation aggravates nonalcoholic fatty liver disease but does not cause insulin resistance in a murine model.

UNLABELLED: Ectodomain shedding of tumor necrosis factor receptor 1 (TNFR1) provides negative feedback to the inflammatory loop induced by TNFα. As the significance of this mechanism in obesity-associated pathologies is unclear, we aimed to unravel how much TNFR1 ectodomain shedding controls the development of nonalcoholic fatty liver disease (NAFLD), as well as its role in the development of insulin resistance. We used knockin mice expressing a mutated TNFR1 ectodomain (p55(Δns)), incapable of shedding and dampen the inflammatory response. Our data show that persistent TNFα signaling through this inability of TNFR1 ectodomain shedding contributes to chronic low-grade inflammation, which is confined to the liver. In spite of this, hepatic lipid levels were not affected by the nonshedding mutation in mice fed a chow diet, nor were they worse off following 12 weeks of high-fat diet (HFD) than controls (p55(+/+)) fed an HFD. We detected inflammatory infiltrates, hepatocellular necrosis, and apoptosis in livers of p55(Δns/Δns) mice fed an HFD, suggesting advanced progression of NAFLD toward nonalcoholic steatohepatitis (NASH). Indeed, fibrosis was present in p55(Δns/Δns) mice, but absent in wildtype mice, confirming that the p55(Δns/Δns) mice had a more severe NASH phenotype. Despite low-grade hepatic inflammation, insulin resistance was not observed in p55(Δns/Δns) mice fed a chow diet, and HFD-induced insulin resistance was no worse in p55(Δns/Δns) mice than p55(+/+) mice. CONCLUSION: TNFR1 ectodomain shedding is not an essential feedback mechanism in preventing the development of hepatic steatosis or insulin resistance. It is, however, pivotal in attenuating the progression from "simple steatosis" towards a more serious phenotype with many NASH features. Targeting TNFR1 could therefore be beneficial in attenuating NASH.

Palmitate-induced skeletal muscle insulin resistance does not require NF-κB activation.

Palmitate activates the NF-κB pathway, and induces accumulation of lipid metabolites and insulin resistance in skeletal muscle cells. Little information is available whether and how these processes are causally related. Therefore, the objectives were to investigate whether intra-cellular lipid metabolites are involved in FA-induced NF-κB activation and/or insulin resistance in skeletal muscle and to investigate whether FA-induced insulin resistance and NF-κB activation are causally related. Inhibiting DGAT or CPT-1 by using, respectively, amidepsine or etomoxir increased DAG accumulation and sensitized myotubes to palmitate-induced insulin resistance. While co-incubation of palmitate with etomoxir increased NF-κB transactivation, co-incubation with amidepsine did not, indicating that DAG accumulation is associated with insulin resistance but not with NF-κB activation. Furthermore, pharmacological or genetic inhibition of the NF-κB pathway could not prevent palmitate-induced insulin resistance. In conclusion, we have demonstrated that activation of the NF-κB pathway is not required for palmitate-induced insulin resistance in skeletal muscle cells.

Inflammatory signaling in skeletal muscle insulin resistance: green signal for nutritional intervention?

PURPOSE OF REVIEW: To review the evidence implying a role of inflammatory signaling pathways, specifically nuclear factor-κB and c-Jun NH2-terminal kinase, in fatty acid-induced skeletal muscle insulin resistance and to discuss the potential of dietary interventions to interfere with these processes. RECENT FINDINGS: Fatty acids can induce skeletal muscle insulin resistance via inflammatory signaling after binding Toll-like receptors at the cell membrane of muscle cells or after accumulating as intramyocellular lipid metabolites. In both processes, activation of intracellular inflammatory signaling is involved. The majority of literature addressing the causality of muscle nuclear factor-κB activation in skeletal muscle insulin resistance suggests that insulin resistance does not require muscle nuclear factor-κB activation. Recently, strong evidence was given that c-Jun NH2-terminal kinase signaling is an important inflammatory pathway involved in skeletal muscle insulin resistance. Furthermore, it is well established that proinflammatory cytokines originating from the enlarged adipose tissue or from activated adipose tissue macrophages can cause muscle insulin resistance. Recently, also macrophages resided in the muscle have been proposed to play an important role in muscle insulin resistance. Because of their anti-inflammatory characteristics, several dietary components like polyphenols may be interesting candidates for manipulating skeletal muscle insulin resistance. SUMMARY: Several dietary components, like polyphenols, have been reported to interfere with inflammatory signaling. To test whether these compounds can be used to prevent or reverse insulin resistance, well controlled human intervention studies have to be designed.

High-fat diet induced obesity primes inflammation in adipose tissue prior to liver in C57BL/6j mice.

Metabolic inflammation in adipose tissue and the liver is frequently observed as a result of diet-induced obesity in human and rodent studies. Although the adipose tissue and the liver are both prone to become chronically inflamed with prolonged obesity, their individual contribution to the development of metabolic inflammation remains speculative. Thus, we aimed to elucidate the sequence of inflammatory events in adipose and hepatic tissues to determine their contribution to the development of metabolic inflammation and insulin resistance (IR) in diet-induced obesity. To confirm our hypothesis that adipose tissue (AT) inflammation is initiated prior to hepatic inflammation, C57BL/6J male mice were fed a low-fat diet (LFD; 10% kcal fat) or high-fat diet (HFD; 45% kcal fat) for either 24, 40 or 52 weeks. Lipid accumulation and inflammation was measured in AT and liver. Glucose tolerance was assessed and plasma levels of glucose, insulin, leptin and adiponectin were measured at various time points throughout the study. With HFD, C57BL/6j mice developed a progressive obese phenotype, accompanied by IR at 24 and 40 weeks of HFD, but IR was attenuated after 52 weeks of HFD. AT inflammation was present after 24 weeks of HFD, as indicated by the increased presence of crown-like structures and up-regulation of pro-inflammatory genes Tnf, Il1ß, Mcp1 and F4/80. As hepatic inflammation was not detected until 40 weeks of HFD, we show that AT inflammation is established prior to the development of hepatic inflammation. Thus, AT inflammation is likely to have a greater contribution to the development of IR compared to hepatic inflammation.

Trans fatty acid-induced NF-kappaB activation does not induce insulin resistance in cultured murine skeletal muscle cells.

Long-chain saturated fatty acids such as palmitic acid induce insulin resistance and NF-kappaB activation in skeletal muscle cells. Here we investigated the effects of long-chain fatty acid (FA) saturation and configuration on NF-kappaB activity and insulin sensitivity in cultured skeletal muscle cells. Of all tested unsaturated FAs, only elaidic acid (3-fold), cis9,trans11-CLA (3-fold) and trans10,cis12-CLA (13-fold) increased NF-kappaB transactivation in myotubes. This was not accompanied by decreased insulin sensitivity (measured as insulin-induced glucose uptake and GLUT4 translocation). We therefore conclude that FA-induced NF-kappaB activation is not sufficient for the induction of insulin resistance in skeletal muscle cells.

Trans-10, cis-12 conjugated linoleic acid inhibits skeletal muscle differentiation and GLUT4 expression independently from NF-κB activation.

SCOPE: The capacity of skeletal muscle to contribute to glucose homeostasis depends on muscular insulin sensitivity. The expression of glucose transporter (GLUT)-4 is increased during myoblast differentiation, a process essential in maintenance of adult muscle. Therefore, processes that affect muscle differentiation may influence insulin dependent glucose homeostasis. Conjugated linoleic acids, and in particular trans-10, cis-12 CLA (t10, c12-CLA), are potent inducers of NF-kB in cultured skeletal myotubes, and NF-kB activation inhibits muscle differentiation. The aims of this study were to evaluate whether CLAs inhibit myogenic differentiation and lower GLUT4 mRNA expression and to address the involvement of NF-kB activation in potential effects of CLA on these processes. METHODS AND RESULTS: Incubation of C2C12 cells with t10, c12-CLA blocked the formation of myotubes, which was accompanied by reduced expression of the muscle specific genes creatine kinase, myogenin, myosin heavy chain perinatal and myosin heavy chain IIB, as well as decreased GLUT4 mRNA levels. However, genetic blockade of NF-kB was not sufficient to restore reduced myosin heavy chain protein expression following t10, c12-CLA treatment. Surprisingly, in contrast to myotubes, t10, c12-CLA was not able to activate NF-kB transcriptional activity in myoblasts. CONCLUSION: In conclusion, t10, c12-CLA inhibits myogenic differentiation and GLUT4 expression, independently from NF-kB activation.

Fatty acid-induced NF-kappaB activation and insulin resistance in skeletal muscle are chain length dependent.

The saturated fatty acid (SFA) palmitate induces insulin resistance in cultured skeletal muscle cells, which may be related to NF-kappaB activation. The aim of this study was to evaluate whether other SFAs also exert these effects on skeletal muscle and whether these relate to chain length. Therefore, we incubated L6 and C(2)C(12) skeletal muscle cells with four different fatty acids, caprylate (C8:0), laurate (C12:0), palmitate (C16:0), and stearate (C18:0), to study effects on GLUT4 translocation, deoxyglucose uptake, and NF-kappaB activation. Incubation of L6 cells with the long-chain FAs C16:0 and C18:0 reduced insulin-stimulated GLUT4 translocation and deoxyglucose uptake, whereas L6 cells incubated with the medium-chain FAs C8:0 and C12:0 remained insulin sensitive. Besides increasing NF-kappaB DNA binding activity in both L6 and C(2)C(12) cells, C16:0 also induced NF-kappaB transcriptional activity. C18:0 showed comparable effects, whereas the SFAs with shorter chain lengths were not able to elevate NF-kappaB transcriptional activity. Collectively, these results demonstrate that SFA-induced NF-kappaB activation coincides with insulin resistance and depends on FA chain length.