Altered expression of Raet1e, a major histocompatibility complex class 1-like molecule, underlies the atherosclerosis modifier locus Ath11 10b.

RATIONALE: Quantitative trait locus mapping of an intercross between C57.Apoe⁻/⁻ and FVB.Apoe⁻/⁻ mice revealed an atherosclerosis locus controlling aortic root lesion area on proximal chromosome 10, Ath11. In a previous work, subcongenic analysis showed Ath11 to be complex with proximal (10a) and distal (10b) regions. OBJECTIVE: To identify the causative genetic variation underlying the atherosclerosis modifier locus Ath11 10b. METHODS AND RESULTS: We now report subcongenic J, which narrows the 10b region to 5 genes, Myb, Hbs1L, Aldh8a1, Sgk1, and Raet1e. Sequence analysis of these genes revealed no amino acid coding differences between the parental strains. However, comparing aortic expression of these genes between F1.Apoe⁻/⁻ Chr10SubJ((B/F)) and F1.Apoe⁻/⁻ Chr10SubJ((F/F)) uncovered a consistent difference only for Raet1e, with decreased, virtually background, expression associated with increased atherosclerosis in the latter. The key role of Raet1e was confirmed by showing that transgene-induced aortic overexpression of Raet1e in F1.Apoe⁻/⁻ Chr10SubJ((F/F)) mice decreased atherosclerosis. Promoter reporter constructs comparing C57 and FVB sequences identified an FVB mutation in the core of the major aortic transcription start site abrogating activity. CONCLUSIONS: This nonbiased approach has revealed Raet1e, a major histocompatibility complex class 1-like molecule expressed in lesional aortic endothelial cells and macrophage-rich regions, as a novel atherosclerosis gene and represents one of the few successes of the quantitative trait locus strategy in complex diseases.

Trans-10, cis-12 CLA dose-dependently inhibits milk fat synthesis without disruption of lactation in C57BL/6J mice.

BACKGROUND: Trans-10, cis-12 conjugated linoleic acid (10,12 CLA) is a potent inhibitor of milk fat synthesis in mammals. In the cow, 10 g/d of 10,12 CLA specifically and reversibly inhibits mammary lipogenesis, whereas substantially higher doses are not specific and cause a generalized inhibition of milk synthesis. OBJECTIVE: The objective of this study was to validate a lactating mouse model by establishing the dose response, specificity, and reversibility of the inhibition of milk fat synthesis by 10,12 CLA. METHODS: Lactating mice (C57BL/6J) received daily doses of 0 (control), 7, 20, or 60 mg of 10,12 CLA for 5 d during established lactation. A second group of lactating mice was treated with 20 mg/d of 10,12 CLA for 4 d and followed post-treatment to evaluate reversibility. RESULTS: CLA decreased pup growth with a 49% decrease occurring with 60 mg/d of CLA. Milk fat percentage was decreased 11% and 20% with the 7 and 20 mg/d dose, respectively, and all CLA treatments had a decreased concentration of de novo synthesized fatty acids (FAs) in milk fat. In agreement, 20 mg/d of 10,12 CLA decreased the lipogenic capacity of mammary tissue by 30% and mammary expression of FA synthase (Fasn), sterol response element binding protein 1 (Srebf1), and thyroid hormone responsive spot 14 (Thrsp) by 30-60%, whereas milk protein percentage and mammary expression of α-lactalbumin (Lalba) were unaltered. This dose of CLA reduced pup growth by nearly 20% and milk de novo synthesized FAs by >35%, and these effects were completely reversed 5 d after 10,12 CLA treatment was terminated. CONCLUSION: Inhibition of mammary lipogenesis by 10,12 CLA is dose-dependent in the mouse, with a specific and reversible reduction in milk fat synthesis at the 20 mg/d dose and additional nonspecific effects on milk synthesis at higher CLA doses.

Obesity-associated microglial inflammatory activation paradoxically improves glucose tolerance.

Hypothalamic gliosis associated with high-fat diet (HFD) feeding increases susceptibility to hyperphagia and weight gain. However, the body-weight-independent contribution of microglia to glucose regulation has not been determined. Here, we show that reducing microglial nuclear factor κB (NF-κB) signaling via cell-specific IKKß deletion exacerbates HFD-induced glucose intolerance despite reducing body weight and adiposity. Conversely, two genetic approaches to increase microglial pro-inflammatory signaling (deletion of an NF-κB pathway inhibitor and chemogenetic activation through a modified Gq-coupled muscarinic receptor) improved glucose tolerance independently of diet in both lean and obese rodents. Microglial regulation of glucose homeostasis involves a tumor necrosis factor alpha (TNF-α)-dependent mechanism that increases activation of pro-opiomelanocortin (POMC) and other hypothalamic glucose-sensing neurons, ultimately leading to a marked amplification of first-phase insulin secretion via a parasympathetic pathway. Overall, these data indicate that microglia regulate glucose homeostasis in a body-weight-independent manner, an unexpected mechanism that limits the deterioration of glucose tolerance associated with obesity.

Dietary Fat Does Not Overcome trans-10, cis-12 Conjugated Linoleic Acid Inhibition of Milk Fat Synthesis in Lactating mice.

Trans-10, cis-12 conjugated linoleic acid (CLA) is a potent inhibitor of milk fat synthesis in the cow and similarly reduces milk fat in rodents. The objective of this study was to determine whether dietary fat can overcome CLA inhibition of milk fat concentration in lactating mice. Wild type C57Bl/6J mice (n = 31) were fed semipurified diets containing either low fat (LF; 4% fat) or high fat (HF; 23.6% fat) starting 4-6 days postpartum. Dietary fat was increased by inclusion of high oleic sunflower oil. After 2 days on the experimental diets, lactating dams were orally dosed with either water (control) or trans-10, cis-12 CLA (20 mg/day) for 5 days. CLA treatment decreased pup growth similarly in both HF and LF diets. Milk fat percent was increased over 16% by the HF diet and decreased over 12% by CLA, but there was no interaction of dietary fat and CLA. Both CLA and the HF diet reduced the proportion of short- and medium-chain fatty acids that originate from de novo synthesis, and there was no interaction of diet and CLA. CLA had no effect on the percent of preformed fatty acids, but the HF diet increased their abundance. Dietary fat and CLA both modified mammary expression of lipogenic enzymes and regulators, but no interactions were observed. In conclusion, CLA reduced milk fat concentration and litter growth, but these effects were not overcome by increased dietary fat from high oleic sunflower oil. CLA inhibition of milk fat in the mammary gland is not substrate dependent, and the mechanism is independent from dietary supply of oleic acid.

Microglial Inflammatory Signaling Orchestrates the Hypothalamic Immune Response to Dietary Excess and Mediates Obesity Susceptibility.

Dietary excess triggers accumulation of pro-inflammatory microglia in the mediobasal hypothalamus (MBH), but the components of this microgliosis and its metabolic consequences remain uncertain. Here, we show that microglial inflammatory signaling determines the immunologic response of the MBH to dietary excess and regulates hypothalamic control of energy homeostasis in mice. Either pharmacologically depleting microglia or selectively restraining microglial NF-κB-dependent signaling sharply reduced microgliosis, an effect that includes prevention of MBH entry by bone-marrow-derived myeloid cells, and greatly limited diet-induced hyperphagia and weight gain. Conversely, forcing microglial activation through cell-specific deletion of the negative NF-κB regulator A20 induced spontaneous MBH microgliosis and cellular infiltration, reduced energy expenditure, and increased both food intake and weight gain even in absence of a dietary challenge. Thus, microglial inflammatory activation, stimulated by dietary excess, orchestrates a multicellular hypothalamic response that mediates obesity susceptibility, providing a mechanistic rationale for non-neuronal approaches to treat metabolic diseases.

Saturated Fatty Acids Engage an IRE1α-Dependent Pathway to Activate the NLRP3 Inflammasome in Myeloid Cells.

Diets rich in saturated fatty acids (SFAs) produce a form of tissue inflammation driven by "metabolically activated" macrophages. We show that SFAs, when in excess, induce a unique transcriptional signature in both mouse and human macrophages that is enriched by a subset of ER stress markers, particularly IRE1α and many adaptive downstream target genes. SFAs also activate the NLRP3 inflammasome in macrophages, resulting in IL-1ß secretion. We found that IRE1α mediates SFA-induced IL-1ß secretion by macrophages and that its activation by SFAs does not rely on unfolded protein sensing. We show instead that the ability of SFAs to stimulate either IRE1α activation or IL-1ß secretion can be specifically reduced by preventing their flux into phosphatidylcholine (PC) or by increasing unsaturated PC levels. Thus, IRE1α is an unrecognized intracellular PC sensor critical to the process by which SFAs stimulate macrophages to secrete IL-1ß, a driver of diet-induced tissue inflammation.

Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function.

Diets rich in saturated fat produce inflammation, gliosis, and neuronal stress in the mediobasal hypothalamus (MBH). Here, we show that microglia mediate this process and its functional impact. Although microglia and astrocytes accumulate in the MBH of mice fed a diet rich in saturated fatty acids (SFAs), only the microglia undergo inflammatory activation, along with a buildup of hypothalamic SFAs. Enteric gavage specifically with SFAs reproduces microglial activation and neuronal stress in the MBH, and SFA treatment activates murine microglia, but not astrocytes, in culture. Moreover, depleting microglia abrogates SFA-induced inflammation in hypothalamic slices. Remarkably, depleting microglia from the MBH of mice abolishes inflammation and neuronal stress induced by excess SFA consumption, and in this context, microglial depletion enhances leptin signaling and reduces food intake. We thus show that microglia sense SFAs and orchestrate an inflammatory process in the MBH that alters neuronal function when SFA consumption is high.

Trib1 is a lipid- and myocardial infarction-associated gene that regulates hepatic lipogenesis and VLDL production in mice.

Recent genome-wide association studies have identified a genetic locus at human chromosome 8q24 as having minor alleles associated with lower levels of plasma triglyceride (TG) and LDL cholesterol (LDL-C), higher levels of HDL-C, as well as decreased risk for myocardial infarction. This locus contains only one annotated gene, tribbles homolog 1 (TRIB1), which has not previously been implicated in lipoprotein metabolism. Here we demonstrate a role for Trib1 as a regulator of lipoprotein metabolism in mice. Hepatic-specific overexpression of Trib1 reduced levels of plasma TG and cholesterol by reducing VLDL production; conversely, Trib1-knockout mice showed elevated levels of plasma TG and cholesterol due to increased VLDL production. Hepatic Trib1 expression was inversely associated with the expression of key lipogenic genes and measures of lipogenesis. Thus, we provide functional evidence for what we believe to be a novel gene regulating hepatic lipogenesis and VLDL production in mice that influences plasma lipids and risk for myocardial infarction in humans.