C-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentameric protein in plasma. Recently, a potential dissociation mechanism from the disc-shaped pentameric CRP (pCRP) into single monomers (monomeric or mCRP) has been described. It has been shown that mCRP has strong pro-inflammatory effects on monocytes. To further define the role of mCRP in determining monocyte phenotype, the effects of CRP isoforms on THP-1 protein expression profiles were determined. The hypothesis to be tested was that mCRP induces specific changes in the protein expression profile of THP-1 cells that differ from that of pCRP.

The development of skeletal muscle insulin resistance is an early physiological defect, yet the intracellular mechanisms accounting for this metabolic defect remained unresolved. Here, we have examined the role of glucose-6-phosphate dehydrogenase (G6PDH) activity in the pathogenesis of insulin resistance in skeletal muscle.

Macrophages are abundant within adipose tissue depots where they are exposed to fatty acids, leading to lipid accumulation. Herein, we have determined the effects of various fatty acids on the macrophage lipidome. Using targeted mass-spectrometry, we were able to detect 641 individual lipid species in primary murine macrophages treated with a variety of saturated fatty acids and an un-saturated fatty acid, either alone or in combination. The most pronounced effects were observed for the long-chain saturated fatty acid palmitate, which increased the total abundance of numerous classes of lipids. While other medium- and long-chain saturated fatty acids, as well as the long-chain unsaturated fatty acid, had less pronounced effects on the total abundance of specific lipid classes, all fatty acids induced marked alterations in the abundance of numerous lipid species within given lipid classes. Fatty acid treatment markedly altered overall phospholipid saturation status; these effects were most pronounced for phosphatidylcholine and ether-phosphatidylcholine lipid species. Finally, treatment of macrophages with either palmitate or stearate in combination with oleate prevented many of the changes that were observed in macrophages treated with palmitate or stearate alone. Collectively, our results reveal substantial and specific remodelling of the macrophage lipidome following treatment with fatty acids.

The gp130 receptor cytokines IL-6 and CNTF improve metabolic homeostasis but have limited therapeutic use for the treatment of type 2 diabetes. Accordingly, we engineered the gp130 ligand IC7Fc, in which one gp130-binding site is removed from IL-6 and replaced with the LIF-receptor-binding site from CNTF, fused with the Fc domain of immunoglobulin G, creating a cytokine with CNTF-like, but IL-6-receptor-dependent, signalling. Here we show that IC7Fc improves glucose tolerance and hyperglycaemia and prevents weight gain and liver steatosis in mice. In addition, IC7Fc either increases, or prevents the loss of, skeletal muscle mass by activation of the transcriptional regulator YAP1. In human-cell-based assays, and in non-human primates, IC7Fc treatment results in no signs of inflammation or immunogenicity. Thus, IC7Fc is a realistic next-generation biological agent for the treatment of type 2 diabetes and muscle atrophy, disorders that are currently pandemic.

Plasmalogens or alkenylphospholipids are a sub-class of glycerophospholipids with numerous biological functions and are thought to have protective effects against metabolic disease. Dietary supplementation with alkylglycerols (AKGs) has been shown to increase endogenous plasmalogen levels, however effective modulation of different molecular plasmalogen species has not yet been demonstrated. In this study, the effects of an orally-administered AKG mix (a mixture of chimyl, batyl and selachyl alcohol at a 1:1:1 ratio) on plasma and tissue lipids, including plasmalogens, was evaluated. Mice on a Western-type diet were treated with either an AKG mix or vehicle (lecithin) for 1, 2, 4, 8 and 12 weeks. Treatment with the AKG mix significantly increased the total plasmalogen content of plasma, liver and adipose tissue as a result of elevations in multiple plasmalogen species with different alkenyl chains. Alkylphospholipids, the endogenous precursors of plasmalogens, showed a rapid and significant increase in plasma, adipose tissue, liver and skeletal muscle. A significant accumulation of alkyl-diacylglycerol and lyso-ether phospholipids was also observed in plasma and tissues. Additionally, the dynamics of plasmalogen-level changes following AKG mix supplementation differed between tissues. These findings indicate that oral supplementation with an AKG mix is capable of upregulating and maintaining stable expression of multiple molecular plasmalogen species in circulation and tissues.

Alterations in the immune cell profile and the induction of inflammation within adipose tissue are a hallmark of obesity in mice and humans. Dual-specificity phosphatase 2 (DUSP2) is widely expressed within the immune system and plays a key role promoting immune and inflammatory responses dependent on mitogen-activated protein kinase (MAPK) activity. We hypothesised that the absence of DUSP2 would protect mice against obesity-associated inflammation and insulin resistance. Accordingly, male and female littermate mice that are either wild-type (wt) or homozygous for a germ-line null mutation of the dusp2 gene (dusp2-/-) were fed either a standard chow diet (SCD) or high fat diet (HFD) for 12 weeks prior to metabolic phenotyping. Compared with mice fed the SCD, all mice consuming the HFD became obese, developed glucose intolerance and insulin resistance, and displayed increased macrophage recruitment and markers of inflammation in epididymal white adipose tissue. The absence of DUSP2, however, had no effect on the development of obesity or adipose tissue inflammation. Whole body insulin sensitivity in male mice was unaffected by an absence of DUSP2 in response to either the SCD or HFD; however, HFD-induced insulin resistance was slightly, but significantly, reduced in female dusp2-/- mice. In conclusion, DUSP2 plays no role in regulating obesity-associated inflammation and only a minor role in controlling insulin sensitivity following HFD in female, but not male, mice. These data indicate that rather than DUSP2 being a pan regulator of MAPK dependent immune cell mediated inflammation, it appears to differentially regulate inflammatory responses that have a MAPK component.

Monocytes in humans consist of 3 subsets; CD14+CD16- (classical), CD14+CD16+ (intermediate) and CD14dimCD16+ (non-classical), which exhibit distinct and heterogeneous responses to activation. During acute inflammation CD14+CD16- monocytes are significantly elevated and migrate to the sites of injury via the adhesion cascade. The field of immunometabolism has begun to elucidate the importance of the engagement of specific metabolic pathways in immune cell function. Yet, little is known about monocyte metabolism and the role of metabolism in mediating monocyte activation and adherence to vessels. Accordingly, we aimed to determine whether manipulating the metabolism of CD14+CD16- monocytes alters their ability to become activated and adhere. We discovered that LPS stimulation increased the rate of glycolysis in human CD14+CD16- monocytes. Inhibition of glycolysis with 2-deoxy-D-glucose blunted LPS-induced activation and adhesion of monocytes. Mechanistically, we found that increased glycolysis was regulated by mTOR-induced glucose transporter (GLUT)-1. Furthermore, enhanced glycolysis increased accumulation of reactive oxygen species (ROS) and activation of p38 MAPK, which lead to activation and adhesion of monocytes. These findings reveal that glycolytic metabolism is critical for the activation of CD14+CD16- monocytes and contributes to our understanding of the interplay between metabolic substrate preference and immune cell function.

Cardiovascular disease (CVD) is the leading cause of death in patients with diabetes, and tight glycemic control fails to reduce the risk of developing CVD. Thiazolidinediones (TZDs), a class of peroxisome proliferator-activated receptor γ (PPARγ) agonists, are potent insulin sensitizers with antiatherogenic properties, but their clinical use is limited by side effects. PPARγ deacetylation on two lysine residues (K268 and K293) induces brown remodeling of white adipose tissue and uncouples the adverse effects of TZDs from insulin sensitization. Here we show that PPARγ deacetylation confers antiatherogenic properties and retains the insulin-sensitizing effects of TZD while circumventing its detriments. We generated mice homozygous with mice with deacetylation-mimetic PPARγ mutations K268R/K293R (2KR) on an LDL-receptor knockout (Ldlr -/- ) background. 2KR:Ldlr -/- mice showed smaller atherosclerotic lesion areas than Ldlr -/- mice, particularly in aortic arches. With rosiglitazone treatment, 2KR:Ldlr -/- mice demonstrated a residual antiatherogenic response and substantial protection against bone loss and fluid retention. The antiatherosclerotic effect of 2KR was attributed to the protection of endothelium, indicated by improved endothelium-dependent vasorelaxation and repressed expression of proatherogenic factors including inducible nitric oxide synthase, interleukin-6, and NADPH oxidase 2. Therefore, manipulating PPARγ acetylation is a promising therapeutic strategy to control risk of CVD in diabetes treatment.

The intrinsic apoptotic pathway is controlled by the BCL-2 family of proteins, which exhibit either a pro-death or pro-survival function. Gene knockout studies revealed that different pro-survival BCL-2 proteins are critical for the survival of distinct cell types, although overlapping functions amongst such proteins have also been identified. In the process of studying mice lacking single alleles of Mcl-1 (Mcl-1+/-), Bcl-2 (Bcl-2+/-), or both in combination (Mcl-1+/-Bcl-2+/-), we observed that Mcl-1+/-Bcl-2+/- mice weighed less when compared with their wild-type littermates as they aged. Body composition analysis demonstrated that while fat mass was similar to wild-type controls, lean mass was significantly reduced in Mcl-1+/-, Bcl-2+/-, and, most strikingly in Mcl-1+/-Bcl-2+/- mice. The weights of several tissues including the heart, tibialis anterior, and kidney were likewise reduced in Mcl-1+/-Bcl-2+/- mice. When lean mass and specific tissue weights were expressed relative to body weight, these differences were no longer significant, indicating that that Mcl-1+/-Bcl-2+/- mice, and to a lesser extent Mcl-1+/- and Bcl-2+/- mice, are smaller than their wild-type counterparts. Consistently, the anal-naso length was reduced in Mcl-1+/-Bcl-2+/- mice. While minor reductions in size were observed in female Mcl-1+/-Bcl-2+/- mice, these effects were most prominent in males. Notably, Mcl-1+/-Bcl-2+/- males had markedly smaller testes even after accounting for differences in body weight. Collectively, these data reveal that combined loss of a single allele of Mcl-1 and Bcl-2, while not overtly impairing organismal development, leads to a reduction in animal size.

Multiple beneficial cardiovascular effects of HDL depend on sphingosine-1-phosphate (S1P). S1P associates with HDL by binding to apolipoprotein M (ApoM). Insulin resistance is a major driver of dyslipidemia and cardiovascular risk. However, the mechanisms linking alterations in insulin signaling with plasma lipoprotein metabolism are incompletely understood. The insulin-repressible FoxO transcription factors mediate key effects of hepatic insulin action on glucose and lipoprotein metabolism. This work tested whether hepatic insulin signaling regulates HDL-S1P and aimed to identify the underlying molecular mechanisms. We report that insulin-resistant, nondiabetic individuals had decreased HDL-S1P levels, but no change in total plasma S1P. This also occurred in insulin-resistant db/db mice, which had low ApoM and a specific reduction of S1P in the HDL fraction, with no change in total plasma S1P levels. Using mice lacking hepatic FoxOs (L-FoxO1,3,4), we found that hepatic FoxOs were required for ApoM expression. Total plasma S1P levels were similar to those in controls, but S1P was nearly absent from HDL and was instead increased in the lipoprotein-depleted plasma fraction. This phenotype was restored to normal by rescuing ApoM in L-FoxO1,3,4 mice. Our findings show that insulin resistance in humans and mice is associated with decreased HDL-associated S1P. Our study shows that hepatic FoxO transcription factors are regulators of the ApoM/S1P pathway.

The fatty acid translocase and scavenger receptor CD36 is important in the recognition and uptake of lipids. Accordingly, we hypothesized that it plays a role in saturated fatty acid-induced macrophage lipid accumulation and proinflammatory activation.

Follistatin is essential for skeletal muscle development and growth, but the intracellular signaling networks that regulate follistatin-mediated effects are not well defined. We show here that the administration of an adeno-associated viral vector expressing follistatin-288aa (rAAV6:Fst-288) markedly increased muscle mass and force-producing capacity concomitant with increased protein synthesis and mammalian target of rapamycin (mTOR) activation. These effects were attenuated by inhibition of mTOR or deletion of S6K1/2. Furthermore, we identify Smad3 as the critical intracellular link that mediates the effects of follistatin on mTOR signaling. Expression of constitutively active Smad3 not only markedly prevented skeletal muscle growth induced by follistatin but also potently suppressed follistatin-induced Akt/mTOR/S6K signaling. Importantly, the regulation of Smad3- and mTOR-dependent events by follistatin occurred independently of overexpression or knockout of myostatin, a key repressor of muscle development that can regulate Smad3 and mTOR signaling and that is itself inhibited by follistatin. These findings identify a critical role of Smad3/Akt/mTOR/S6K/S6RP signaling in follistatin-mediated muscle growth that operates independently of myostatin-driven mechanisms.

Plasmalogens are membrane glycerophospholipids with diverse biological functions. Reduced plasmalogen levels have been observed in metabolic diseases; hence, increasing their levels might be beneficial in ameliorating these conditions. Shark liver oil (SLO) is a rich source of alkylglycerols that can be metabolized into plasmalogens. This study was designed to evaluate the impact of SLO supplementation on endogenous plasmalogen levels in individuals with features of metabolic disease. In this randomized, double-blind, placebo-controlled cross-over study, the participants (10 overweight or obese males) received 4-g Alkyrol® (purified SLO) or placebo (methylcellulose) per day for 3 weeks followed by a 3-week washout phase and were then crossed over to 3 weeks of the alternate placebo/Alkyrol® treatment. SLO supplementation led to significant changes in plasma and circulatory white blood cell lipidomes, notably increased levels of plasmalogens and other ether lipids. In addition, SLO supplementation significantly decreased the plasma levels of total free cholesterol, triglycerides, and C-reactive protein. These findings suggest that SLO supplementation can enrich plasma and cellular plasmalogens and this enrichment may provide protection against obesity-related dyslipidemia and inflammation.

Weight is defended so that increases or decreases in body mass elicit responses that favor restoration of one's previous weight. While much is known about the signals that respond to weight loss and the central role that leptin plays, the lack of experimental systems studying the overfed state has meant little is known about pathways defending against weight gain. We developed a system to study this physiology and found that overfed mice defend against increased weight gain with graded anorexia but, unlike weight loss, this response is independent of circulating leptin concentration. In overfed mice that are unresponsive to orexigenic stimuli, adipose tissue is transcriptionally and immunologically distinct from fat of ad libitum-fed obese animals. These findings provide evidence that overfeeding-induced obesity alters adipose tissue and central responses in ways that are distinct from ad libitum obesity and activates a non-leptin system to defend against weight gain.

The leading cause of mortality in patients with rheumatoid arthritis is atherosclerotic cardiovascular disease (CVD). We have shown that murine arthritis impairs atherosclerotic lesion regression, because of cellular cholesterol efflux defects in haematopoietic stem and progenitor cells (HSPCs), causing monocytosis and impaired atherosclerotic regression. Therefore, we hypothesised that improving cholesterol efflux using a Liver X Receptor (LXR) agonist would improve cholesterol efflux and improve atherosclerotic lesion regression in arthritis.

Activation of the receptor for advanced glycation end products (RAGE) in diabetic vasculature is considered to be a key mediator of atherogenesis. This study examines the effects of deletion of RAGE on the development of atherosclerosis in the diabetic apoE(-/-) model of accelerated atherosclerosis.

Tumors are composed of phenotypically heterogeneous cancer cells that often resemble various differentiation states of their lineage of origin. Within this hierarchy, it is thought that an immature subpopulation of tumor-propagating cancer stem cells (CSCs) differentiates into non-tumorigenic progeny, providing a rationale for therapeutic strategies that specifically eradicate CSCs or induce their differentiation. The clinical success of these approaches depends on CSC differentiation being unidirectional rather than reversible, yet this question remains unresolved even in prototypically hierarchical malignancies, such as acute myeloid leukemia (AML). Here, we show in murine and human models of AML that, upon perturbation of endogenous expression of the lineage-determining transcription factor PU.1 or withdrawal of established differentiation therapies, some mature leukemia cells can de-differentiate and reacquire clonogenic and leukemogenic properties. Our results reveal plasticity of CSC maturation in AML, highlighting the need to therapeutically eradicate cancer cells across a range of differentiation states.

Diabetes is characterized by pancreatic β-cell dedifferentiation. Dedifferentiating β cells inappropriately metabolize lipids over carbohydrates and exhibit impaired mitochondrial oxidative phosphorylation. However, the mechanism linking the β-cell's response to an adverse metabolic environment with impaired mitochondrial function remains unclear.

Chronic inflammation is a hallmark of obesity and is linked to the development of numerous diseases. The activation of toll-like receptor 4 (TLR4) by long-chain saturated fatty acids (lcSFAs) is an important process in understanding how obesity initiates inflammation. While experimental evidence supports an important role for TLR4 in obesity-induced inflammation in vivo, via a mechanism thought to involve direct binding to and activation of TLR4 by lcSFAs, several lines of evidence argue against lcSFAs being direct TLR4 agonists. Using multiple orthogonal approaches, we herein provide evidence that while loss-of-function models confirm that TLR4 does, indeed, regulate lcSFA-induced inflammation, TLR4 is not a receptor for lcSFAs. Rather, we show that TLR4-dependent priming alters cellular metabolism, gene expression, lipid metabolic pathways, and membrane lipid composition, changes that are necessary for lcSFA-induced inflammation. These results reconcile previous discordant observations and challenge the prevailing view of TLR4's role in initiating obesity-induced inflammation.

Rheumatoid arthritis (RA), an inflammatory joint disorder, independently increases the risk of cardiovascular disease (CVD). IL-1β contributes to both RA and CVD. We hypothesised that inhibiting IL-1 signalling with the IL-1R antagonist, anakinra, would dampen inflammation and promote resolution of atherosclerosis in arthritic mice.