B Cell Activity Is Impaired in Human and Mouse Obesity and Is Responsive to an Essential Fatty Acid upon Murine Influenza Infection.

Obesity is associated with increased risk for infections and poor responses to vaccinations, which may be due to compromised B cell function. However, there is limited information about the influence of obesity on B cell function and underlying factors that modulate B cell responses. Therefore, we studied B cell cytokine secretion and/or Ab production across obesity models. In obese humans, B cell IL-6 secretion was lowered and IgM levels were elevated upon ex vivo anti-BCR/TLR9 stimulation. In murine obesity induced by a high fat diet, ex vivo IgM and IgG were elevated with unstimulated B cells. Furthermore, the high fat diet lowered bone marrow B cell frequency accompanied by diminished transcripts of early lymphoid commitment markers. Murine B cell responses were subsequently investigated upon influenza A/Puerto Rico/8/34 infection using a Western diet model in the absence or presence of docosahexaenoic acid (DHA). DHA, an essential fatty acid with immunomodulatory properties, was tested because its plasma levels are lowered in obesity. Relative to controls, mice consuming the Western diet had diminished Ab titers whereas the Western diet plus DHA improved titers. Mechanistically, DHA did not directly target B cells to elevate Ab levels. Instead, DHA increased the concentration of the downstream specialized proresolving lipid mediators (SPMs) 14-hydroxydocosahexaenoic acid, 17-hydroxydocosahexaenoic acid, and protectin DX. All three SPMs were found to be effective in elevating murine Ab levels upon influenza infection. Collectively, the results demonstrate that B cell responses are impaired across human and mouse obesity models and show that essential fatty acid status is a factor influencing humoral immunity, potentially through an SPM-mediated mechanism.

A Direct Comparison of Metabolic Responses to High-Fat Diet in C57BL/6J and C57BL/6NJ Mice.

Although nicotinamide nucleotide transhydrogenase (NNT)-deficient C57BL/6J (6J) mice are known to be highly susceptible to diet-induced metabolic disease, this notion stems primarily from comparisons of 6J mice to other inbred strains. To date, very few studies have directly compared metabolic disease susceptibility between NNT-deficient 6J mice and NNT-competent C57BL/6 substrains. In this study, comprehensive profiling of the metabolic response to a high-fat/high-sucrose diet (HFD) were compared across time in 6J and C57BL/6NJ (6N) mice. Given that increased peroxide exposure drives insulin resistance, coupled with the fact that NNT regulates peroxide detoxification, it was hypothesized that 6J mice would experience greater derangements in redox homeostasis/metabolic disease upon HFD exposure. Contrary to this, both lines were found to be highly susceptible to diet-induced metabolic disease, as evidenced by impairments in glucose tolerance as early as 24 h into the HFD. Moreover, various markers of the metabolic syndrome, as well as peroxide stress, were actually blunted, rather than exacerbated, in the 6J mice, likely reflecting compensatory increases in alterative redox-buffering pathways. Together, these data provide evidence that the susceptibility to HFD-induced metabolic disease is similar in the 6J and 6N substrains. Given the numerous genetic variances in the 6J stain, including loss of NNT function, these findings suggest that the 6N substrain is the more logical and representative genetic background model for metabolic studies.

Targeted overexpression of mitochondrial catalase protects against cancer chemotherapy-induced skeletal muscle dysfunction.

The loss of strength in combination with constant fatigue is a burden on cancer patients undergoing chemotherapy. Doxorubicin, a standard chemotherapy drug used in the clinic, causes skeletal muscle dysfunction and increases mitochondrial H2O2 We hypothesized that the combined effect of cancer and chemotherapy in an immunocompetent breast cancer mouse model (E0771) would compromise skeletal muscle mitochondrial respiratory function, leading to an increase in H2O2-emitting potential and impaired muscle function. Here, we demonstrate that cancer chemotherapy decreases mitochondrial respiratory capacity supported with complex I (pyruvate/glutamate/malate) and complex II (succinate) substrates. Mitochondrial H2O2-emitting potential was altered in skeletal muscle, and global protein oxidation was elevated with cancer chemotherapy. Muscle contractile function was impaired following exposure to cancer chemotherapy. Genetically engineering the overexpression of catalase in mitochondria of muscle attenuated mitochondrial H2O2 emission and protein oxidation, preserving mitochondrial and whole muscle function despite cancer chemotherapy. These findings suggest mitochondrial oxidants as a mediator of cancer chemotherapy-induced skeletal muscle dysfunction.

Fuzhuan tea consumption imparts hepatoprotective effects and alters intestinal microbiota in high saturated fat diet-fed rats.

SCOPE: Nonalcoholic fatty liver disease is an obesity-related disorder characterized by lipid infiltration of the liver. Management is limited to lifestyle modifications, highlighting the need for alternative therapeutic options. The objective of this study was to examine if fermented Fuzhuan tea prevents metabolic impairments associated with development of hepatic steatosis. METHODS AND RESULTS: Rats consumed control (CON) or high saturated fat (SAT) diets with or without Fuzhuan tea for 8 weeks. Outcomes included enzymatic and gene expression measures of metabolic dysregulation in liver and adipose tissue. Pyrosequencing was used to assess intestinal microbiota adaptations. Fuzhuan tea prevented diet-induced inflammation in the liver. Liver triglycerides of ∼18 mg/g were observed in SAT-fed animals, but remained similar to CON diet levels (∼12 mg/g) when supplemented with Fuzhuan tea. In adipose tissue, tea treatment prevented SAT-induced inflammation and reduced plasma leptin approximately twofold. Fuzhuan tea also altered intestinal function and was associated with a threefold increase in two Lactobacillus spp. CONCLUSIONS: These data suggest that Fuzhuan tea protects against liver and adipose tissue stress induced by a high SAT diet and positively influences intestinal function. Further investigation of the molecular targets of Fuzhuan tea is warranted.

Protein Kinase A Governs Oxidative Phosphorylation Kinetics and Oxidant Emitting Potential at Complex I.

The mitochondrial electron transport system (ETS) is responsible for setting and maintaining both the energy and redox charges throughout the cell. Reversible phosphorylation of mitochondrial proteins, particularly via the soluble adenylyl cyclase (sAC)/cyclic AMP (cAMP)/Protein kinase A (PKA) axis, has recently been revealed as a potential mechanism regulating the ETS. However, the governance of cAMP/PKA signaling and its implications on ETS function are incompletely understood. In contrast to prior reports using exogenous bicarbonate, we provide evidence that endogenous CO2 produced by increased tricarboxylic acid (TCA) cycle flux is insufficient to increase mitochondrial cAMP levels, and that exogenous addition of membrane permeant 8Br-cAMP does not enhance mitochondrial respiratory capacity. We also report important non-specific effects of commonly used inhibitors of sAC which preclude their use in studies of mitochondrial function. In isolated liver mitochondria, inhibition of PKA reduced complex I-, but not complex II-supported respiratory capacity. In permeabilized myofibers, inhibition of PKA lowered both the K m and V max for complex I-supported respiration as well as succinate-supported H2O2 emitting potential. In summary, the data provided here improve our understanding of how mitochondrial cAMP production is regulated, illustrate a need for better tools to examine the impact of sAC activity on mitochondrial biology, and suggest that cAMP/PKA signaling contributes to the governance of electron flow through complex I of the ETS.

Pyruvate dehydrogenase complex and nicotinamide nucleotide transhydrogenase constitute an energy-consuming redox circuit.

Cellular proteins rely on reversible redox reactions to establish and maintain biological structure and function. How redox catabolic (NAD+/NADH) and anabolic (NADP+/NADPH) processes integrate during metabolism to maintain cellular redox homoeostasis, however, is unknown. The present work identifies a continuously cycling mitochondrial membrane potential (ΔΨm)-dependent redox circuit between the pyruvate dehydrogenase complex (PDHC) and nicotinamide nucleotide transhydrogenase (NNT). PDHC is shown to produce H2O2 in relation to reducing pressure within the complex. The H2O2 produced, however, is effectively masked by a continuously cycling redox circuit that links, via glutathione/thioredoxin, to NNT, which catalyses the regeneration of NADPH from NADH at the expense of ΔΨm. The net effect is an automatic fine-tuning of NNT-mediated energy expenditure to metabolic balance at the level of PDHC. In mitochondria, genetic or pharmacological disruptions in the PDHC-NNT redox circuit negate counterbalance changes in energy expenditure. At the whole animal level, mice lacking functional NNT (C57BL/6J) are characterized by lower energy-expenditure rates, consistent with their well-known susceptibility to diet-induced obesity. These findings suggest the integration of redox sensing of metabolic balance with compensatory changes in energy expenditure provides a potential mechanism by which cellular redox homoeostasis is maintained and body weight is defended during periods of positive and negative energy balance.

Oral nitrite therapy improves vascular function in diabetic mice.

AIM: We tested the hypothesis that short-term oral sodium nitrite supplementation would improve vascular dysfunction in obese, diabetic mice. METHODS AND RESULTS: Vascular function was determined in control mice and in db/db mice receiving drinking water with or without sodium nitrite (50 mg/L) for 5 weeks. Nitrite supplementation increased plasma nitrite concentrations in db/db mice (0.19±0.02 µM vs 0.80±0.26 µM; p < 0.05). Db/db mice had lower endothelium-dependent dilation (EDD) in response to increasing doses of acetylcholine versus heterozygous control mice (71.2% ± 14.3% vs 93% ± 7.0%; p < 0.05), and sodium nitrite supplementation restored endothelium-dependent dilation to control levels (92.9% ± 2.3% vs 93% ± 7.0%; p < 0.05). The improvement in endothelial function was accompanied by a reduction in intrinsic stiffness, but not by alterations in plasma or vascular markers of inflammation. CONCLUSION: These data suggest that sodium nitrite may be a novel therapy for treating diabetes-related vascular dysfunction; however, the mechanisms of improvement are unknown.

Activation of the unfolded protein response in vascular endothelial cells of nondiabetic obese adults.

CONTEXT: Activation of the unfolded protein response (UPR) is emerging as an important molecular signature of cardiometabolic diseases associated with obesity. However, despite the well-established role of the vascular endothelium in obesity-related cardiometabolic dysfunction, it is unclear whether the UPR is activated in endothelial cells of obese adults. OBJECTIVE: The objective of the study was to determine whether markers of UPR activation are increased in endothelial cells (ECs) of nondiabetic obese adults with impaired endothelial function. DESIGN, SETTING, AND PARTICIPANTS: Endothelial cells were obtained from antecubital veins of the nondiabetic obese adults [body mass index (BMI) ≥ 30 kg/m(2), n = 12] with impaired endothelial function and from their nonobese peers (BMI < 30 kg/m(2), n = 14). MAIN OUTCOME VARIABLES: UPR activation via expression (quantitative immunofluorescence) of the proximal UPR sensors, inositol-requiring endoplasmic reticulum (ER)-to-nucleus signaling protein 1 (IRE1), RNA-dependent protein kinase-like ER eukaryotic initiation factor-2α kinase (PERK), and activating transcription factor 6 (ATF6), were the main outcome variables. RESULTS: IRE1 expression was greater in obese vs nonobese individuals (0.84 ± 0.09 vs 0.47 ± 0.02 IRE1 intensity/human umbilical vein EC (HUVEC) intensity (n = 10/8, P < .01). Obese individuals also had greater EC activation of UPR stress sensors PERK and ATF6, indicated by increased expression of phosphorylated PERK [p-PERK; 0.49 ± 0.05 vs 0.36 ± 0.03, p-PERK (threonine 981) intensity/HUVEC intensity, n = 10 men, 13 women, P < .05] and nuclear localization of ATF6 (0.38 ± 0.05 vs 0.23 ± 0.02, nuclear ATF6 intensity/HUVEC intensity, n = 5 men, 9 women, P < .01), respectively. Stepwise linear regression analysis revealed that indices of body fat (BMI and waist circumference) were the strongest independent predictors of all 3 UPR mediators, explaining between 18% and 59% of the variance in endothelial cell expression of IRE1, p-PERK, and nuclear ATF6 localization. CONCLUSION: These results provide novel evidence for UPR activation in the endothelial cells of nondiabetic obese adults with vascular endothelial dysfunction.

Fatty acid-mediated endoplasmic reticulum stress in vivo: differential response to the infusion of Soybean and Lard Oil in rats.

BACKGROUND: In cell systems, saturated fatty acids, compared to unsaturated fatty acids, induce a greater degree of ER stress and inflammatory signaling in a number of cell types, including hepatocytes and adipocytes. The aim of the present study was to determine the effects of infusions of lard oil (enriched in saturated fatty acids) and soybean oil (enriched in unsaturated fatty acids) on liver and adipose tissue ER stress and inflammatory signaling in vivo. METHODS: Lipid emulsions containing glycerol, phosphatidylcholine, antibiotics (Control, n=7) and either soybean oil (Soybean, n=7) or lard oil (Lard, n=7) were infused intravenously into rats over a 4 h period. RESULTS: Plasma free fatty acid levels were 0.5±0.1 mmol/L (mean±SD) in Control and were increased to 1.0±0.3 mmol/L and 1.1±0.3 mmol/L in Soybean and Lard, respectively. Glucose and insulin levels were not different among groups. Markers of endoplasmic reticulum (ER) stress and activation of inflammatory pathway signaling were increased in liver and adipose tissue from Soybean and Lard compared to Control, but were increased to a greater extent in Lard compared to Soybean. CONCLUSIONS: These data suggest that elevated plasma free fatty acids can induce hepatic and adipose tissue ER stress and inflammation in vivo. In addition, saturated fatty acids appear to be more cytotoxic than unsaturated fatty acids in vivo.

Rapamycin inhibits postprandial-mediated X-box-binding protein-1 splicing in rat liver.

Recent studies have linked the unfolded protein response (UPR), in particular the inositol-requiring, endoplasmic reticulum-to-nucleus signaling protein 1alpha (IRE1alpha)-X-box-binding protein-1 (XBP1) branch of the UPR, to the regulation of lipogenesis and hepatic steatosis. In this study, we examined the hypothesis that the postprandial environment can activate the IRE1alpha-XBP1 branch of the UPR in the liver via a mammalian target of rapamycin complex 1 (mTORC1)-dependent mechanism. Toward this end, rats were fed a high-carbohydrate diet (68% of energy from corn starch) for 3 h in the absence or presence of rapamycin (intraperitoneal injection of 1 mg/kg) and liver tissue was taken 1 or 7 h following the feeding period. Feeding activated the mTORC1 pathway and IRE1alpha, induced XBP1 splicing, and increased the expression of XBP1 target genes and lipogenic genes in the liver. The presence of rapamycin prevented the activation of mTORC1 and IRE1alpha, XBP1 splicing, and the increased expression of XBP1 target genes and lipogenic genes. Rapamycin also prevented the feeding-induced increase in nuclear sterol regulatory element binding protein 1c. These data suggest that the postprandial environment promotes activation of the IRE1-XBP1 branch of the UPR in the liver. This activation appears to be mediated in part by mTORC1.