Plasma sphingosine-1-phosphate is elevated in obesity

Background: Dysfunctional lipid metabolism is a hallmark of obesity and insulin resistance and a risk factor for various cardiovascular and metabolic complications. In addition to the well known increase in plasma triglycerides and free fatty acids, recent work in humans and rodents has shown that obesity is associated with elevations in the bioactive class of sphingolipids known as ceramides. However, in obesity little is known about the plasma concentrations of sphinogsine-1-phosphate (S1P), the breakdown product of ceramide, which is an important signaling molecule in mammalian biology. Therefore, the purpose of this study was to examine the impact of obesity on circulating S1P concentration and its relationship with markers of glucose metabolism and insulin sensitivity. Methodology/Principal Findings: Plasma S1P levels were determined in high-fat diet (HFD)-induced and genetically obese (ob/ob) mice along with obese humans. Circulating S1P was elevated in both obese mouse models and in obese humans compared with lean healthy controls. Furthermore, in humans, plasma S1P positively correlated with total body fat percentage, body mass index (BMI), waist circumference, fasting insulin, HOMA-IR, HbA1c (%), total and LDL cholesterol. In addition, fasting increased plasma S1P levels in lean healthy mice. Conclusion: We show that elevations in plasma S1P are a feature of both human and rodent obesity and correlate with metabolic abnormalities such as adiposity and insulin resistance

The role of natural killer cells in atherosclerosis.

Atherosclerosis is a progressive multifactorial disease of large elastic and muscular arteries in which lesions are characterized by the deposition of cholesterol, leukocyte influx, smooth muscle cell proliferation, cell death and collagen accumulation. Inflammatory cells of the innate and adaptive immune systems contribute to lesion development and progression. NK cells have been detected in human atherosclerotic lesions in low numbers and also detected in atherosclerotic lesions of LDLR-deficient mice fed a high fat diet. However their significance for atherosclerotic lesion development is still unclear. The studies presented in this thesis unequivocally demonstrate an important role for NK cells in the development of atherosclerosis and provide novel insights as to how these cells contribute to atherosclerosis. Chapter 3 examines the role of NK cell in the development of atherosclerosis in ApoE-deficient mice fed a high fat diet. Despite being very minor lymphocyte population in developing atherosclerotic lesions, the study has definitely demonstrated that NK cells are proatherogenic in mice. Specific depletion of NK cells by more than 90% attenuated lesion size and macrophage accumulation. Whilst not specifically examined the reduction in macrophage accumulation could be due to lack of production of NK cell derived cytokines and chemokines in the lesions. Chapter 4 examines whether the activation of NK cells further augments development of atherosclerosis. Activation of NK cells with Poly IC exacerbates the development of atherosclerosis. Since Poly IC can also activate T cells, the effects of Poly IC were determined in T and B cells deficient ApoE-/- Rag2-/- mice. Poly IC augmented atherosclerosis in these mice which suggest a strong dependency on NK cells. To confirm the dependency of Poly IC effects on atherosclerosis on NK cells, the effects of Poly IC in NK cell depleted mice were also determined. The depletion of NK cells reduced atherosclerotic lesion development. Therefore the study indicates a dependency of Poly IC’s proatherogenic effects on NK cells. Chapter 5 examines the mechanisms by which NK cells are activated during development of atherosclerosis. NK cells can be activated by via NKT cells and/or via their activating receptor NKG2D. Activation of NKT cells has been shown to stimulate NK cell proliferation and cytotoxicity. This study demonstrates that NKT cells contribute to NK cell activation during development of atherosclerosis since the atherosclerotic lesions were similar in mice deficient in invariant NKT cells in ApoE-/- Jalpha18-/- mice and in NK cell depleted ApoE-/- Jalpha18-/- mice. As to the role of NKG2D receptors in activating NK cells during the development of atherosclerosis, the study indicate that these receptors are not involved despite the high expression of its activating ligands Rae-1δ, Rae-1ε and MULT-1. Chapter 6 examines the mechanisms by which NK cells promote the development of atherosclerosis. Activated NK cells produce cytokines and cytotoxicity. This study examined whether NK cells participate in atherosclerosis via production of cytokines implicated in atherosclerosis, or through secretion of cytotoxic molecules. IL-2 activated NK cells from mice deficient in cytokines interferon-gamma (IFN-γ) were adoptively transferred to NK-, NKT-, T- and B- cell-deficient ApoE-/- Rag2-/- γ-/- mice. Interferon-gamma was found to be unnecessary for the proatherogenic actions of NK cells, as NK cells deficient in IFN-γ were capable of exacerbating atherosclerosis to the same extent as wild-type IL-2 activated NK cells. Conversely, adoptive transfer of IL-2 activated NK cells deficient in cytotoxic molecules perforin or granzyme B did not increase atherosclerosis in ApoE-/- Rag2-/- γ-/- mice. Therefore the atherosclerosis-promoting effects of NK cells appear to be dependent on their cytotoxic properties. Increased cytotoxicity in atherosclerotic lesions can lead to increased necrosis and inflammation via activation of inflammasomes. Lesions from mice that received perforin- or granzyme B-deficient NKT cells had smaller necrotic areas and lower expression of pro-IL-1β and caspase-1 mRNA. In summary, the studies indicate that NK cell within the developing lesions augment atherosclerosis by secreting cytotoxic molecules perforin and granzyme B. The magnitude of effects of NK cells on lesion development is dependent not only on NK cell numbers but also on their activation status. Increased understanding of the mechanisms of NK cell actions during atherogenesis may lead to new strategies to develop immunotherapeutic treatments to attenuate atherosclerosis

The role of natural killer cells in atherosclerosis.

Atherosclerosis is a progressive multifactorial disease of large elastic and muscular arteries in which lesions are characterized by the deposition of cholesterol, leukocyte influx, smooth muscle cell proliferation, cell death and collagen accumulation. Inflammatory cells of the innate and adaptive immune systems contribute to lesion development and progression. NK cells have been detected in human atherosclerotic lesions in low numbers and also detected in atherosclerotic lesions of LDLR-deficient mice fed a high fat diet. However their significance for atherosclerotic lesion development is still unclear. The studies presented in this thesis unequivocally demonstrate an important role for NK cells in the development of atherosclerosis and provide novel insights as to how these cells contribute to atherosclerosis. Chapter 3 examines the role of NK cell in the development of atherosclerosis in ApoE-deficient mice fed a high fat diet. Despite being very minor lymphocyte population in developing atherosclerotic lesions, the study has definitely demonstrated that NK cells are proatherogenic in mice. Specific depletion of NK cells by more than 90% attenuated lesion size and macrophage accumulation. Whilst not specifically examined the reduction in macrophage accumulation could be due to lack of production of NK cell derived cytokines and chemokines in the lesions. Chapter 4 examines whether the activation of NK cells further augments development of atherosclerosis. Activation of NK cells with Poly IC exacerbates the development of atherosclerosis. Since Poly IC can also activate T cells, the effects of Poly IC were determined in T and B cells deficient ApoE-/- Rag2-/- mice. Poly IC augmented atherosclerosis in these mice which suggest a strong dependency on NK cells. To confirm the dependency of Poly IC effects on atherosclerosis on NK cells, the effects of Poly IC in NK cell depleted mice were also determined. The depletion of NK cells reduced atherosclerotic lesion development. Therefore the study indicates a dependency of Poly IC’s proatherogenic effects on NK cells. Chapter 5 examines the mechanisms by which NK cells are activated during development of atherosclerosis. NK cells can be activated by via NKT cells and/or via their activating receptor NKG2D. Activation of NKT cells has been shown to stimulate NK cell proliferation and cytotoxicity. This study demonstrates that NKT cells contribute to NK cell activation during development of atherosclerosis since the atherosclerotic lesions were similar in mice deficient in invariant NKT cells in ApoE-/- Jalpha18-/- mice and in NK cell depleted ApoE-/- Jalpha18-/- mice. As to the role of NKG2D receptors in activating NK cells during the development of atherosclerosis, the study indicate that these receptors are not involved despite the high expression of its activating ligands Rae-1δ, Rae-1ε and MULT-1. Chapter 6 examines the mechanisms by which NK cells promote the development of atherosclerosis. Activated NK cells produce cytokines and cytotoxicity. This study examined whether NK cells participate in atherosclerosis via production of cytokines implicated in atherosclerosis, or through secretion of cytotoxic molecules. IL-2 activated NK cells from mice deficient in cytokines interferon-gamma (IFN-γ) were adoptively transferred to NK-, NKT-, T- and B- cell-deficient ApoE-/- Rag2-/- γ-/- mice. Interferon-gamma was found to be unnecessary for the proatherogenic actions of NK cells, as NK cells deficient in IFN-γ were capable of exacerbating atherosclerosis to the same extent as wild-type IL-2 activated NK cells. Conversely, adoptive transfer of IL-2 activated NK cells deficient in cytotoxic molecules perforin or granzyme B did not increase atherosclerosis in ApoE-/- Rag2-/- γ-/- mice. Therefore the atherosclerosis-promoting effects of NK cells appear to be dependent on their cytotoxic properties. Increased cytotoxicity in atherosclerotic lesions can lead to increased necrosis and inflammation via activation of inflammasomes. Lesions from mice that received perforin- or granzyme B-deficient NKT cells had smaller necrotic areas and lower expression of pro-IL-1β and caspase-1 mRNA. In summary, the studies indicate that NK cell within the developing lesions augment atherosclerosis by secreting cytotoxic molecules perforin and granzyme B. The magnitude of effects of NK cells on lesion development is dependent not only on NK cell numbers but also on their activation status. Increased understanding of the mechanisms of NK cell actions during atherogenesis may lead to new strategies to develop immunotherapeutic treatments to attenuate atherosclerosis

Plasma S1P concentrations are elevated in obesity.

<p>Plasma S1P concentrations in chow (N = 13) and HFD (N = 14) fed mice (A); plasma S1P levels in lean (N = 6) and <i>ob/ob</i> (N = 8) mice (B) and circulating S1P in lean (N = 15) and obese (N = 10) humans (C). Data are mean ± SEM. **P&lt;0.01.</p

Plasma S1P concentrations correlate with clinical indices of metabolic dysfunction in humans.

<p>Plasma S1P concentrations correlate with percent body fat (A), BMI (B), waist circumference (C), fasting plasma insulin (D), HOMA-IR (E), HbA1c (F), total (G) and LDL (H) cholesterol.</p

Plasma S1P concentrations in mice are elevated in response to fasting.

<p>Changes in body mass (A), blood glucose (B), plasma NEFA (C) and plasma S1P concentrations (D) in response to fasting. Data are mean ± SEM. *P&lt;0.05; **P&lt;0.01, N = 11–12.</p

Application of dynamic metabolomics to examine in vivo skeletal muscle glucose metabolism in the chronically high-fat fed mouse.

RATIONALE: Defects in muscle glucose metabolism are linked to type 2 diabetes. Mechanistic studies examining these defects rely on the use of high fat-fed rodent models and typically involve the determination of muscle glucose uptake under insulin-stimulated conditions. While insightful, they do not necessarily reflect the physiology of the postprandial state. In addition, most studies do not examine aspects of glucose metabolism beyond the uptake process. Here we present an approach to study rodent muscle glucose and intermediary metabolism under the dynamic and physiologically relevant setting of the oral glucose tolerance test (OGTT). METHODS AND RESULTS: In&nbsp;vivo muscle glucose and intermediary metabolism was investigated following oral administration of [U-(13)C] glucose. Quadriceps muscles were collected 15 and 60&nbsp;min after glucose administration and metabolite flux profiling was determined by measuring (13)C mass isotopomers in glycolytic and tricarboxylic acid (TCA) cycle intermediates via gas chromatography-mass spectrometry. While no dietary effects were noted in&nbsp;the glycolytic pathway, muscle from mice fed a high fat diet (HFD) exhibited a reduction in labelling in TCA intermediates. Interestingly, this appeared to be independent of alterations in flux through pyruvate dehydrogenase. In addition, our findings suggest that TCA cycle anaplerosis is negligible in muscle during an OGTT. CONCLUSIONS: Under the dynamic physiologically relevant conditions of the OGTT, skeletal muscle from HFD fed mice exhibits alterations in glucose metabolism at the level of the TCA cycle

In vivo cardiac glucose metabolism in the high-fat fed mouse: comparison of euglycemic-hyperinsulinemic clamp derived measures of glucose uptake with a dynamic metabolomic flux profiling approach

Rationale Cardiac metabolism is thought to be altered in insulin resistance and type 2 diabetes (T2D). Our understanding of the regulation of cardiac substrate metabolism and insulin sensitivity has largely been derived from ex vivo preparations which are not subject to the same metabolic regulation as in the intact heart in vivo. Studies are therefore required to examine in vivo cardiac glucose metabolism under physiologically relevant conditions. Objective To determine the temporal pattern of the development of cardiac insulin resistance and to compare with dynamic approaches to interrogate cardiac glucose and intermediary metabolism in vivo. Methods and results Studies were conducted to determine the evolution of cardiac insulin resistance in C57Bl/6 mice fed a high-fat diet (HFD) for between 1 and 16 weeks. Dynamic in vivo cardiac glucose metabolism was determined following oral administration of [U-13C] glucose. Hearts were collected after 15 and 60 min and flux profiling was determined by measuring 13C mass isotopomers in glycolytic and tricarboxylic acid (TCA) cycle intermediates. Cardiac insulin resistance, determined by euglycemic-hyperinsulinemic clamp, was evident after 3 weeks of HFD. Despite the presence of insulin resistance, in vivo cardiac glucose metabolism following oral glucose administration was not compromised in HFD mice. This contrasts our recent findings in skeletal muscle, where TCA cycle activity was reduced in mice fed a HFD. Similar to our report in muscle, glucose derived pyruvate entry into the TCA cycle in the heart was almost exclusively via pyruvate dehydrogenase, with pyruvate carboxylase mediated anaplerosis being negligible after oral glucose administration. Conclusions Under experimental conditions which closely mimic the postprandial state, the insulin resistant mouse heart retains the ability to stimulate glucose metabolism