Male Mice Lacking NLRX1 Are Partially Protected From High-Fat Diet-Induced Hyperglycemia.

Nod-like receptor (NLR)X1 is an NLR family protein that localizes to the mitochondrial matrix and modulates reactive oxygen species production, possibly by directly interacting with the electron transport chain. Recent work demonstrated that cells lacking NLRX1 have higher oxygen consumption but lower levels of adenosine triphosphate, suggesting that NLRX1 might prevent uncoupling of oxidative phosphorylation. We therefore hypothesized that NLRX1 might regulate whole-body energy metabolism through its effect on mitochondria. Male NLRX1 whole-body knockout (KO) mice and wild-type (WT) C57BL/6N controls were fed a low-fat or a high-fat (HF) diet for 16 weeks from weaning. Contrary to this hypothesis, there were no differences in body weight, adiposity, energy intake, or energy expenditure between HF-fed KO and WT mice, but instead HF KO mice were partially protected from the development of diet-induced hyperglycemia. Additionally, HF KO mice did not present with hyperinsulinemia during the glucose tolerance test, as did HF WT mice. There were no genotype differences in insulin tolerance, which led us to consider a pancreatic phenotype. Histology revealed that KO mice were protected from HF-induced pancreatic lipid accumulation, suggesting a potential role for NLRX1 in pancreatic dysfunction during the development diet-induced type 2 diabetes mellitus. Hence, NLRX1 depletion partially protects against postabsorptive hyperglycemia in obesity that may be linked to the prevention of pancreatic lipid accumulation. Although the actual mechanisms restoring glucose and insulin dynamics remain unknown, NLRX1 emerges as a potentially interesting target to inhibit for the prevention of type 2 diabetes mellitus.

Circulating NOD1 Activators and Hematopoietic NOD1 Contribute to Metabolic Inflammation and Insulin Resistance.

Insulin resistance is a chronic inflammatory condition accompanying obesity or high fat diets that leads to type 2 diabetes. It is hypothesized that lipids and gut bacterial compounds in particular contribute to metabolic inflammation by activating the immune system; however, the receptors detecting these "instigators" of inflammation remain largely undefined. Here, we show that circulating activators of NOD1, a receptor for bacterial peptidoglycan, increase with high fat feeding in mice, suggesting that NOD1 could be a critical sensor leading to metabolic inflammation. Hematopoietic depletion of NOD1 did not prevent weight gain but protected chimeric mice against diet-induced glucose and insulin intolerance. Mechanistically, while macrophage infiltration of adipose tissue persisted, notably these cells were less pro-inflammatory, had lower CXCL1 production, and consequently, lower neutrophil chemoattraction into the tissue. These findings reveal macrophage NOD1 as a cell-specific target to combat diet-induced inflammation past the step of macrophage infiltration, leading to insulin resistance.

Effects of 12 Months of Caloric Restriction on Muscle Mitochondrial Function in Healthy Individuals.

Context: The effects of caloric restriction (CR) on in vivo muscle mitochondrial function in humans are controversial. Objective: We evaluated muscle mitochondrial function and associated transcriptional profiles in nonobese humans after 12 months of CR. Design: Individuals from an ancillary study of the CALERIE 2 randomized controlled trial were assessed at baseline and 12 months after a 25% CR or ad libitum (control) diet. Setting: The study was performed at Pennington Biomedical Research Center in Baton Rouge, LA. Participants: Study participants included 51 (34 female subjects, 25 to 50 years of age) healthy nonobese individuals randomized to 1 of 2 groups (CR or control). Intervention: This study included 12 months of a 25% CR or ad libitum (control) diet. Main Outcomes: In vivo mitochondrial function [maximal ATP synthesis rate (ATPmax), ATPflux/O2 (P/O)] was determined by 31P-magnetic resonance spectroscopy and optical spectroscopy, and body composition was determined by dual-energy X-ray absorptiometry. In a subset of individuals, a muscle biopsy was performed for transcriptional profiling via quantitative reverse transcription polymerase chain reaction and microarrays. Results: Weight, body mass index (BMI), fat, and fat-free mass (P < 0.001 for all) significantly decreased at month 12 after CR vs control. In vivo ATPmax and P/O were unaffected by 12 months of CR. Targeted transcriptional profiling showed no effects on pathways involved in mitochondrial biogenesis, function, or oxidative stress. A subgroup analysis according to baseline P/O demonstrated that a higher (vs lower) P/O was associated with notable improvements in ATPmax and P/O after CR. Conclusions: In healthy nonobese humans, CR has no effect on muscle mitochondrial function; however, having a "more coupled" (versus "less coupled") phenotype enables CR-induced improvements in muscle mitochondrial function.

Differences in Mitochondrial Coupling Reveal a Novel Signature of Mitohormesis in Muscle of Healthy Individuals.

CONTEXT: Reduced mitochondrial coupling (ATP/O2 [P/O]) is associated with sedentariness and insulin resistance. Interpreting the physiological relevance of P/O measured in vitro is challenging. OBJECTIVE: To evaluate muscle mitochondrial function and associated transcriptional profiles in nonobese healthy individuals distinguished by their in vivo P/O. DESIGN: Individuals from an ancillary study of Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy phase 2 were assessed at baseline. SETTING: The study was performed at Pennington Biomedical Research Center. PARTICIPANTS: Forty-seven (18 males, 26-50 y of age) sedentary, healthy nonobese individuals were divided into 2 groups based on their in vivo P/O. INTERVENTION: None. Main Outcome(s): Body composition by dual-energy x-ray absorptiometry, in vivo mitochondrial function (P/O and maximal ATP synthetic capacity) by 31P-magnetic resonance spectroscopy and optical spectroscopy were measured. A muscle biopsy was performed to measure fiber type, transcriptional profiling (microarray), and protein expressions. RESULTS: No differences in body composition, peak aerobic capacity, type I fiber content, or mitochondrial DNA copy number were observed between the 2 groups. Compared with the uncoupled group (lower P/O), the coupled group (higher P/O) had higher rates of maximal ATP synthetic capacity (maximal ATP synthetic capacity, P < .01). Transcriptomics analyses revealed higher expressions of genes involved in mitochondrial remodeling and the oxidative stress response in the coupled group. A trend for higher mitonuclear protein imbalance (P = .06) and an elevated mitochondrial unfolded protein response (heat shock protein 60 protein; P = .004) were also identified in the coupled group. CONCLUSIONS: Higher muscle mitochondrial coupling is accompanied by an overall elevation in mitochondrial function, a novel transcriptional signature of oxidative stress and mitochondrial remodeling and indications of an mitochondrial unfolded protein response.

Palmitoleate Reverses High Fat-induced Proinflammatory Macrophage Polarization via AMP-activated Protein Kinase (AMPK).

A rise in tissue-embedded macrophages displaying "M1-like" proinflammatory polarization is a hallmark of metabolic inflammation during a high fat diet or obesity. Here we show that bone marrow-derived macrophages (BMDM) from high fat-fed mice retain a memory of their dietary environment in vivo (displaying the elevated proinflammatory genes Cxcl1, Il6, Tnf, Nos2) despite 7-day differentiation and proliferation ex vivo. Notably, 6-h incubation with palmitoleate (PO) reversed the proinflammatory gene expression and cytokine secretion seen in BMDM from high fat-fed mice. BMDM from low fat-fed mice exposed to palmitate (PA) for 18 h ex vivo also showed elevated expression of proinflammatory genes (Cxcl1, Il6, Tnf, Nos2, and Il12b) associated with M1 polarization. Conversely, PO treatment increased anti-inflammatory genes (Mrc1, Tgfb1, Il10, Mgl2) and oxidative metabolism, characteristic of M2 macrophages. Therefore, saturated and unsaturated fatty acids bring about opposite macrophage polarization states. Coincubation of BMDM with both fatty acids counteracted the PA-induced Nos2 expression in a PO dose-dependent fashion. PO also prevented PA-induced IκBα degradation, RelA nuclear translocation, NO production, and cytokine secretion. Mechanistically, PO exerted its anti-inflammatory function through AMP-activated protein kinase as AMP kinase knockout or inhibition by Compound C offset the PO-dependent prevention of PA-induced inflammation. These results demonstrate a nutritional memory of BMDM ex vivo, highlight the plasticity of BMDM polarization in response to saturated and unsaturated fatty acids, and identify the potential to reverse diet- and saturated fat-induced M1-like polarization by administering palmitoleate. These findings could have applicability to reverse obesity-linked inflammation in metabolically relevant tissues.

Mice lacking NOX2 are hyperphagic and store fat preferentially in the liver.

Chronic low-grade inflammation is an important contributor to the development of insulin resistance, a hallmark of type 2 diabetes mellitus (T2DM). Obesity and high-fat feeding lead to infiltration of immune cells into metabolic tissues, promoting inflammation and insulin resistance. We hypothesized that macrophages from mice lacking NOX2 (Cybb), an essential component of the NADPH oxidase complex highly expressed in immune cells and associated with their inflammatory response, would be less inflammatory and that these mice would be protected from the development of high-fat-induced insulin resistance. Bone marrow-derived macrophages from NOX2 knockout (NOX2-KO) mice expressed lower levels of inflammatory markers (Nos2, Il6); however, NOX2-KO mice were hyperphagic and gained more weight than wild-type (WT) mice when fed either a chow or a high-fat (HF) diet. Surprisingly, NOX2-KO mice stored less lipid in epididymal white adipose tissue but more lipid in liver and had higher indexes of liver inflammation and macrophage infiltration than WT mice. Contrary to our hypothesis, HF-fed NOX2-KO mice were hyperinsulinemic and more insulin resistant than HF-fed WT mice, likely as a result of their higher hepatic steatosis and inflammation. In summary, NOX2 depletion promoted hyperphagia, hepatic steatosis, and inflammation with either normal or high-fat feeding, exacerbating insulin resistance. We propose that NOX2 participates in food intake control and lipid distribution in mice.

Pro-inflammatory macrophages increase in skeletal muscle of high fat-fed mice and correlate with metabolic risk markers in humans.

OBJECTIVE: In obesity, immune cells infiltrate adipose tissue. Skeletal muscle is the major tissue of insulin-dependent glucose disposal, and indices of muscle inflammation arise during obesity, but whether and which immune cells increase in muscle remain unclear. METHODS: Immune cell presence in quadriceps muscle of wild type mice fed high-fat diet (HFD) was studied for 3 days to 10 weeks, in CCL2-KO mice fed HFD for 1 week, and in human muscle. Leukocyte presence was assessed by gene expression of lineage markers, cyto/chemokines and receptors; immunohistochemistry; and flow cytometry. RESULTS: After 1 week HFD, concomitantly with glucose intolerance, muscle gene expression of Ly6b, Emr1 (F4/80), Tnf, Ccl2, and Ccr2 rose, as did pro- and anti-inflammatory markers Itgax (CD11c) and Mgl2. CD11c+ proinflammatory macrophages in muscle increased by 76%. After 10 weeks HFD, macrophages in muscle increased by 47%. Quadriceps from CCL2-KO mice on HFD did not gain macrophages and maintained insulin sensitivity. Muscle of obese, glucose-intolerant humans showed elevated CD68 (macrophage marker) and ITGAX, correlating with poor glucose disposal and adiposity. CONCLUSION: Mouse and human skeletal muscles gain a distinct population of inflammatory macrophages upon HFD or obesity, linked to insulin resistance in humans and CCL2 availability in mice.

TAK-242, a small-molecule inhibitor of Toll-like receptor 4 signalling, unveils similarities and differences in lipopolysaccharide- and lipid-induced inflammation and insulin resistance in muscle cells.

Emerging evidence suggests that TLR (Toll-like receptor) 4 and downstream pathways [MAPKs (mitogen-activated protein kinases) and NF-κB (nuclear factor κB)] play an important role in the pathogenesis of insulin resistance. LPS (lipopolysaccharide) and saturated NEFA (non-esterified fatty acids) activate TLR4, and plasma concentrations of these TLR4 ligands are elevated in obesity and Type 2 diabetes. Our goals were to define the role of TLR4 on the insulin resistance caused by LPS and saturated NEFA, and to dissect the independent contribution of LPS and NEFA to the activation of TLR4-driven pathways by employing TAK-242, a specific inhibitor of TLR4. LPS caused robust activation of the MAPK and NF-κB pathways in L6 myotubes, along with impaired insulin signalling and glucose transport. TAK-242 completely prevented the inflammatory response (MAPK and NF-κB activation) caused by LPS, and, in turn, improved LPS-induced insulin resistance. Similar to LPS, stearate strongly activated MAPKs, although stimulation of the NF-κB axis was modest. As seen with LPS, the inflammatory response caused by stearate was accompanied by impaired insulin action. TAK-242 also blunted stearate-induced inflammation; yet, the protective effect conferred by TAK-242 was partial and observed only on MAPKs. Consequently, the insulin resistance caused by stearate was only partially improved by TAK-242. In summary, TAK-242 provides complete and partial protection against LPS- and NEFA-induced inflammation and insulin resistance, respectively. Thus, LPS-induced insulin resistance depends entirely on TLR4, whereas NEFA works through TLR4-dependent and -independent mechanisms to impair insulin action.

Skeletal muscle NAMPT is induced by exercise in humans.

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is responsible for the first and rate-limiting step in the conversion of nicotinamide to nicotinamide adenine dinucleotide (NAD+). NAD+ is an obligate cosubstrate for mammalian sirtuin-1 (SIRT1), a deacetylase that activates peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), which in turn can activate mitochondrial biogenesis. Given that mitochondrial biogenesis is activated by exercise, we hypothesized that exercise would increase NAMPT expression, as a potential mechanism leading to increased mitochondrial content in muscle. A cross-sectional analysis of human subjects showed that athletes had about a twofold higher skeletal muscle NAMPT protein expression compared with sedentary obese, nonobese, and type 2 diabetic subjects (P < 0.05). NAMPT protein correlated with mitochondrial content as estimated by complex III protein content (R(2) = 0.28, P < 0.01), MRS-measured maximal ATP synthesis (R(2) = 0.37, P = 0.002), and Vo(2max) (R(2) = 0.63, P < 0.0001). In an exercise intervention study, NAMPT protein increased by 127% in sedentary nonobese subjects after 3 wk of exercise training (P < 0.01). Treatment of primary human myotubes with forskolin, a cAMP signaling pathway activator, resulted in an approximately 2.5-fold increase in NAMPT protein expression, whereas treatment with ionomycin had no effect. Activation of AMPK via AICAR resulted in an approximately 3.4-fold increase in NAMPT mRNA (P < 0.05) as well as modest increases in NAMPT protein (P < 0.05) and mitochondrial content (P < 0.05). These results demonstrate that exercise increases skeletal muscle NAMPT expression and that NAMPT correlates with mitochondrial content. Further studies are necessary to elucidate the pathways regulating NAMPT as well as its downstream effects.