Marnie L. Gruen

Diet-Induced Increases in Adiposity, but Not Plasma Lipids, Promote Macrophage Infiltration Into White Adipose Tissue

Obesity, hyperlipidemia, and insulin resistance are cardinal features of the metabolic syndrome and individually increase the risk for developing diabetes and cardiovascular disease, a risk that is amplified when they are simultaneously present. It is becoming increasingly clear that macrophages can infiltrate white adipose tissue (WAT) in the obese state, and their presence is associated with pathophysiological consequences of obesity, such as inflammation and insulin resistance. To determine whether hyperlipidemia could potentiate macrophage infiltration into WAT in the presence of obesity, obesity-prone agouti yellow mice (Ay/a) on a hyperlipidemia-prone LDL receptor (LDLR)–deficient (LDLR−/−) background were placed on chow or Western diet. In addition, Ay/a mice that were LDLR sufficient were also placed on Western diet. Both genetics and diet increased the degree of adiposity; however, plasma lipids were elevated only in the Western diet–fed LDLR−/− mice. The extent of macrophage accumulation in WAT correlated with the degree of adiposity. However, hyperlipidemia did not impact macrophage recruitment to WAT or the downstream metabolic consequences of macrophage accumulation in WAT, such as inflammation and insulin resistance. These data have important implications for the pathogenesis of diet-induced obesity in humans, even when plasma lipid abnormalities are not present.

Impact of macrophage toll-like receptor 4 deficiency on macrophage infiltration into adipose tissue and the artery wall in mice

Aims/hypothesisToll-like receptor 4 (TLR4) is a receptor for saturated fatty acids (SFAs), global deficiency of which has been shown to protect against inflammation, insulin resistance and atherosclerotic lesion formation. Because macrophages express Tlr4 and are important in insulin resistance and atherosclerotic lesion formation due to their infiltration of white adipose tissue (WAT) and the artery wall, respectively, we hypothesised that deficiency of macrophage TLR4 could protect against these disorders.MethodsBone marrow transplantation of agouti, LDL-receptor deficient (Ay/a; Ldlr−/−) mice with marrow from either C57BL/6 or Tlr4−/− mice was performed. Recipient mice with Tlr4+/+ marrow (MθTLR4+/+) or with Tlr4−/− marrow (MθTLR4−/−) were then placed on one of four diets: (1) low fat; (2) high fat; (3) high fat rich in SFAs (HFSFA); and (4) HFSFA supplemented with fish oil.ResultsThere were no differences in body composition or plasma lipids between MθTLR4+/+ and MθTLR4−/− mice on any of the diets. However, we observed a decrease in some macrophage and inflammatory markers in WAT of female low fat-fed MθTLR4−/− mice compared with MθTLR4+/+ mice. MθTLR4−/− mice fed low-fat diet also displayed decreased atherosclerotic lesion area. There were no differences in macrophage accrual in WAT or atherosclerosis between MθTLR4+/+ and MθTLR4−/− mice fed any of the high-fat diets. Finally, no difference was seen in insulin sensitivity between MθTLR4+/+ and MθTLR4−/− mice fed the HFSFA diet.Conclusions/interpretationThese data suggest that under certain dietary conditions, macrophage expression of Tlr4 can be an important mediator of macrophage accumulation in WAT and the artery wall.

Impact of Macrophage Inflammatory Protein-1α Deficiency on Atherosclerotic Lesion Formation, Hepatic Steatosis, and Adipose Tissue Expansion

Macrophage inflammatory protein-1α (CCL3) plays a well-known role in infectious and viral diseases; however, its contribution to atherosclerotic lesion formation and lipid metabolism has not been determined. Low density lipoprotein receptor deficient (LDLR−/−) mice were transplanted with bone marrow from CCL3−/− or C57BL/6 wild type donors. After 6 and 12 weeks on western diet (WD), recipients of CCL3−/− marrow demonstrated lower plasma cholesterol and triglyceride concentrations compared to recipients of C57BL/6 marrow. Atherosclerotic lesion area was significantly lower in female CCL3−/− recipients after 6 weeks and in male CCL3−/− recipients after 12 weeks of WD feeding (P<0.05). Surprisingly, male CCL3−/− recipients had a 50% decrease in adipose tissue mass after WD-feeding, and plasma insulin, and leptin levels were also significantly lower. These results were specific to CCL3, as LDLR−/− recipients of monocyte chemoattractant protein−/− (CCL2) marrow were not protected from the metabolic consequences of high fat feeding. Despite these improvements in LDLR−/− recipients of CCL3−/− marrow in the bone marrow transplantation (BMT) model, double knockout mice, globally deficient in both proteins, did not have decreased body weight, plasma lipids, or atherosclerosis compared with LDLR−/− controls. Finally, there were no differences in myeloid progenitors or leukocyte populations, indicating that changes in body weight and plasma lipids in CCL3−/− recipients was not due to differences in hematopoiesis. Taken together, these data implicate a role for CCL3 in lipid metabolism in hyperlipidemic mice following hematopoietic reconstitution.

Loss of CCR5 results in glucose intolerance in diet-induced obese mice

Macrophage and T cell infiltration into metabolic tissues contributes to obesity-associated inflammation and insulin resistance (IR). C-C chemokine receptor 5 (CCR5), expressed on macrophages and T cells, plays a critical role in the recruitment and activation of proinflammatory M1 and TH1 immune cells to tissues and is elevated in adipose tissue (AT) and liver of obese humans and mice. Thus, we hypothesized that deficiency of CCR5 would protect against diet-induced inflammation and IR. CCR5-deficient (CCR5(-/-)) mice and C57BL/6 (WT) controls were fed 10% low-fat (LF) or 60% high-fat (HF) diets for 16 wk. HF feeding increased adiposity, blood glucose, and plasma insulin levels equally in both genotypes. Opposing our hypothesis, HF-fed CCR5(-/-) mice were significantly more glucose intolerant than WT mice. In AT, there was a significant reduction in the M1-associated gene CD11c, whereas M2 associated genes were not different between genotypes. In addition, HF feeding caused a twofold increase in CD4(+) T cells in the AT of CCR5(-/-) compared with WT mice. In liver and muscle, no differences in immune cell infiltration or inflammatory cytokine expression were detected. However, in AT and muscle, there was a mild reduction in insulin-induced phosphorylation of AKT and IRβ in CCR5(-/-) compared with WT mice. These findings suggest that whereas CCR5 plays a minor role in regulating immune cell infiltration and inflammation in metabolic tissues, deficiency of CCR5 impairs systemic glucose tolerance as well as AT and muscle insulin signaling.

The role of macrophage leptin receptor in aortic root lesion formation

Plasma leptin is often elevated in obese individuals, and previous studies have suggested leptin as a factor that links obesity and atherosclerosis. Because macrophages play a key role in atherogenesis and are responsive to leptin, we hypothesized that leptin increases aortic root lesion formation, in part, through macrophage leptin receptor (LepR). Three different bone marrow transplantation studies were conducted in which bone marrow, with or without LepR, was transplanted into lethally irradiated 1) LDL receptor-deficient (LDLR(-/-)) mice with moderate hyperleptinemia due to Western diet (WD) feeding, 2) LDLR(-/-) mice with WD feeding plus pharmacologically induced hyperleptinemia (daily injection of 125 microg leptin), or 3) obese, hyperleptinemic, LepR-deficient LDLR(-/-) (LepR(db/db);LDLR(-/-)) mice. Minor differences in plasma parameters such as cholesterol, triglycerides, and insulin were observed in some groups; however, a consistent trend for the role of LepR on these parameters was not detected. In each of the studies, macrophage LepR expression did not have an effect on aortic root atherosclerotic lesion formation. These results suggest that nonhematopoietic cells may have a more significant role than macrophages in leptin-mediated effects on aortic root lesion formation.

Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

Objective Obesity is a metabolic disorder that has reached epidemic proportions worldwide and leads to increased risk for diabetes, cardiovascular disease, asthma, certain cancers, and various other diseases. Obesity and its comorbidities are associated with impaired adipose tissue (AT) function. In the last decade, eosinophils have been identified as regulators of proper AT function. Our study aimed to determine whether normalizing the number of AT eosinophils in obese mice, to those of lean healthy mice, would reduce obesity and/or improve metabolic fitness. Methods C57BL/6J mice fed a high fat diet (HFD) were simultaneously given recombinant interleukin-5 (rIL5) for 8 weeks to increase AT eosinophils. Metabolic fitness was tested by evaluating weight gain, AT inflammation, glucose, lipid, and mixed-meal tolerance, AT insulin signaling, energy substrate utilization, energy expenditure, and white AT beiging capacity. Results Eosinophils were increased ∼3-fold in AT of obese HFD-fed mice treated with rIL5, and thus were restored to levels observed in lean healthy mice. However, there were no significant differences in rIL5-treated mice among the above listed comprehensive set of metabolic assays, despite the increased AT eosinophils. Conclusions We have shown that restoring obese AT eosinophils to lean healthy levels is not sufficient to allow for improvement in any of a range of metabolic features otherwise impaired in obesity. Thus, the mechanisms that identified eosinophils as positive regulators of AT function, and therefore systemic health, are more complex than initially understood and will require further study to fully elucidate.

Fat-water MRI of a diet-induced obesity mouse model at 15.2T.

Abstract. Quantitative fat-water MRI (FWMRI) methods provide valuable information about the distribution, volume, and composition of adipose tissue (AT). Ultra high field FWMRI of animal models may have the potential to provide insights into the progression of obesity and its comorbidities. Here, we present quantitative FWMRI with all known confounder corrections on a 15.2T preclinical scanner for noninvasive in vivo monitoring of an established diet-induced obesity mouse model. Male C57BL/6J mice were placed on a low-fat (LFD) or a high-fat diet (HFD). Three-dimensional (3-D) multiple gradient echo MRI at 15.2T was performed at baseline, 4, 8, 12, and 16 weeks after diet onset. A 3-D fat-water separation algorithm and additional processing were used to generate proton-density fat fraction (PDFF), local magnetic field offset, and R2* maps. We examined these parameters in perirenal AT ROIs from LFD and HFD mice. The data suggest that PDFF, local field offset, and R2* have different time course behaviors between LFD and HFD mice over 16 weeks. This work suggests FWMRI at 15.2T may be a useful tool for longitudinal studies of adiposity due to the advantages of ultra high field although further investigation is needed to understand the observed time course behavior.

Fat-water MRI is sensitive to local adipose tissue inflammatory changes in a diet-induced obesity mouse model at 15T

In obesity, fat-water MRI (FWMRI) methods provide valuable information about adipose tissue (AT) distribution. AT is known to undergo complex metabolic and endocrine changes in association with chronic inflammation including iron overloading. Here, we investigate the potential for FWMRI parameters (fat signal fraction (FSF), local magnetic field offset, and T2*) to be sensitive to AT inflammatory changes in an established diet-induced obesity mouse model. Male C57BL/6J mice were placed on a low fat (LFD) or a high fat diet (HFD). 3D multi- gradient-echo MRI at 15.2T was performed at baseline, 4, 8, 12, and 16 weeks after diet onset. A 3D fat-water separation algorithm and additional processing was used to generate FSF, local field offset, and T2* maps. We examined these parameters in perirenal AT ROIs from HFD and LFD mice. Results: The data suggest that FSF, local field offset, and T2* can differentiate time course behavior between inflamed and control AT (increasing FSF, decreasing local field offset, increasing followed by decreasing T2*). The biophysical mechanisms of these observed changes are not well understood and require further study. To the best of our knowledge, we report the first evidence that FWMRI can provide biomarkers sensitive to AT inflammation, and that FWMRI has the potential for longitudinal non-invasive assessment of AT inflammation in obesity.