Clodronate liposomes improve metabolic profile and reduce visceral adipose macrophage content in diet-induced obese mice

Background

Obesity-related adipose inflammation has been thought to be a causal factor for the development of insulin resistance and type 2 diabetes. Infiltrated macrophages in adipose tissue of obese animals and humans are an important source for inflammatory cytokines. Clodronate liposomes can ablate macrophages by inducing apoptosis. In this study, we aim to determine whether peritoneal injection of clodronate liposomes has any beneficial effect on systemic glucose homeostasis/insulin sensitivity and whether macrophage content in visceral adipose tissue will be reduced in diet-induced obese (DIO) mice.

Methodology/Principal Findings

Clodronate liposomes were used to deplete macrophages in lean and DIO mice. Macrophage content in visceral adipose tissue, metabolic parameters, glucose and insulin tolerance, adipose and liver histology, adipokine and cytokine production were examined. Hyperinsulinemic-euglycemic clamp study was also performed to assess systemic insulin sensitivity. Peritoneal injection of clodronate liposomes significantly reduced blood glucose and insulin levels in DIO mice. Systemic glucose tolerance and insulin sensitivity were mildly improved in both lean and DIO mice treated with clodronate liposomes by intraperitoneal (ip) injection. Hepatosteatosis was dramatically alleviated and suppression of hepatic glucose output was markedly increased in DIO mice treated with clodronate liposomes. Macrophage content in visceral adipose tissue of DIO mice was effectively decreased without affecting subcutaneous adipose tissue. Interestingly, levels of insulin sensitizing hormone adiponectin, including the high molecular weight form, were significantly elevated in circulation.

Conclusions/Significance

Intraperitoneal injection of clodronate liposomes reduces visceral adipose tissue macrophages, improves systemic glucose homeostasis and insulin sensitivity in DIO mice, which can be partially attributable to increased adiponectin levels.     

Dynamics of macrophage polarization reveal new mechanism to inhibit IL-1β release through pyrophosphates

In acute inflammation, extracellular ATP activates P2X₇ ion channel receptors (P2X₇R) on M1 polarized macrophages to release pro-inflammatory IL-1β through activation of the caspase-1/nucleotide-binding domain and leucine-rich repeat receptor containing pyrin domain 3 (NLRP3) inflammasome. In contrast, M2 polarized macrophages are critical to the resolution of inflammation but neither actions of P2X₇R on these macrophages nor mechanisms by which macrophages switch from pro-inflammatory to anti-inflammatory phenotypes are known. Here, we investigated extracellular ATP signalling over a dynamic macrophage polarity gradient from M1 through M2 phenotypes. In macrophages polarized towards, but not at, M2 phenotype, in which intracellular IL-1β remains high and the inflammasome is intact, P2X₇R activation selectively uncouples to the NLRP3-inflammasome activation but not to upstream ion channel activation. In these intermediate M1/M2 polarized macrophages, extracellular ATP now acts through its pyrophosphate chains, independently of other purine receptors, to inhibit IL-1β release by other stimuli through two independent mechanisms: inhibition of ROS production and trapping of the inflammasome complex through intracellular clustering of actin filaments.     

MCTR1 enhances the resolution of lipopolysaccharide‐induced lung injury through STAT6‐mediated resident M2 alveolar macrophage polarization in mice

Acute respiratory distress syndrome (ARDS) is a fatal disease characterized by excessive infiltration of inflammatory cells. MCTR1 is an endogenously pro‐resolution lipid mediator. We tested the hypothesis that MCTR1 accelerates inflammation resolution through resident M2 alveolar macrophage polarization. The mice received MCTR1 via intraperitoneal administration 3 days after LPS stimulation, and then, the bronchoalveolar lavage (BAL) fluid was collected 24 hours later to measure the neutrophil numbers. Flow cytometry was used to sort the resident and recruited macrophages. Post‐treatment with MCTR1 offered dramatic benefits in the resolution phase of LPS‐induced lung injury, including decreased neutrophil numbers, reduced BAL fluid protein and albumin concentrations and reduced histological injury. In addition, the expression of the M2 markers Arg1, FIZZ1, Remlα, CD206 and Dectin‐1 was increased on resident macrophages in the LPS + MCTR1 group. Resident macrophage depletion abrogated the therapeutic effects of MCTR1, and reinjection of the sorted resident macrophages into the lung decreased neutrophil numbers. Finally, treatment with MCTR1 increased STAT6 phosphorylation. The STAT6 inhibitor AS1517499 abolished the beneficial effects of MCTR1. In conclusion, MCTR1 promotes resident M2 alveolar macrophage polarization via the STAT6 pathway to accelerate resolution of LPS‐induced lung injury.     

Effect of linseed oil and macadamia oil on metabolic changes induced by high‐fat diet in mice

The effects of linseed oil (LO) and macadamia oil (MO) on the metabolic changes induced by a high‐fat diet (HFD) rich in saturated fatty acid were investigated. For the purpose of this study, the vegetable oil present in the HFD, i.e. soybean oil (SO) was replaced with LO (HFD‐LO) or MO (HFD‐MO). For comparative purposes, a group was included, which received a normal fat diet (NFD). Male Swiss mice (6‐week old) were used. After 14 days under the dietary conditions, the mice were fasted for 18 h, and experiments were then performed. The HFD‐SO, HFD‐LO and HFD‐MO groups showed higher glycaemia (p < 0.05 versus NFD). However, no significant effect was observed on glycaemia, liver gluconeogenesis and liver ketogenesis when SO was replaced by either LO or MO. The body weight and the sum of epididymal, mesenteric, retroperitoneal and inguinal fat weights were higher (p < 0.05) in the HFD‐SO and HFD‐MO groups as compared with the NFD group. However, there was no significant difference in these parameters between the NFD and HFD‐LO groups. Thus, the protective role of LO on lipid accumulation induced by an HFD rich in saturated fatty acid is potentially mediated by the high content of ?‐3 polyunsaturated fatty acid in LO. Copyright © 2013 John Wiley & Sons, Ltd.     

Myometrial‐derived CXCL12 promotes lipopolysaccharide induced preterm labour by regulating macrophage migration,polarization and function in mice

Preterm birth is a major contributor to neonatal mortality and morbidity. Infection results in elevation of inflammation‐related cytokines followed by infiltration of immune cells into gestational tissue. CXCL12 levels are elevated in preterm birth indicating it may have a role in preterm labour (PTL); however, the pathophysiological correlations between CXCL12/CXCR4 signalling and premature labour are poorly understood. In this study, PTL was induced using lipopolysaccharide (LPS) in a murine model. LPS induced CXCL12 RNA and protein levels significantly and specifically in myometrium compared with controls (3‐fold and 3.5‐fold respectively). Highest levels were found just before the start of labour. LPS also enhanced the infiltration of neutrophils, macrophages and T cells, and induced macrophage M1 polarization. In vitro studies showed that condition medium from LPS‐treated primary smooth muscle cells (SMC) induced macrophage migration, M1 polarization and upregulated inflammation‐related cytokines such as interleukin (IL)‐1, IL‐6 and tumor necrosis factor alpha (TNF‐α). AMD3100 treatment in pregnant mice led to a significant decrease in the rate of PTL (70%), prolonged pregnancy duration and suppressed macrophage infiltration into gestation tissue by 2.5‐fold. Further, in‐vitro treatment of SMC by AMD3100 suppressed the macrophage migration, decreased polarization and downregulated IL‐1, IL‐6 and TNF‐α expression. LPS treatment in pregnant mice induced PTL by increasing myometrial CXCL12, which recruits immune cells that in turn produce inflammation‐related cytokines. These effects stimulated by LPS were completely reversed by AMD3100 through blocking of CXCL12/CXCR4 signalling. Thus, the CXCL12/CXCR4 axis presents an excellent target for preventing infection and inflammation‐related PTL.     

Lack of UCP1 stimulates fatty liver but mediates UCP1-independent action of beige fat to improve hyperlipidemia in Apoe knockout mice

Brown adipose tissue (BAT) plays a critical role in lipid metabolism and may protect from hyperlipidemia; however, its beneficial effect appears to depend on the ambient temperature of the environment. In this study, we investigated the effects of uncoupling protein 1 (UCP1) deficiency on lipid metabolism, including the pathophysiology of hyperlipidemia, in apolipoprotein E knockout (APOE-KO) mice at a normal (23 °C) and thermoneutral (30 °C) temperature. Unexpectedly, UCP1 deficiency caused improvements in hyperlipidemia, atherosclerosis, and glucose metabolism, regardless of an increase in hepatic lipid deposition, in Ucp1/Apoe double-knockout (DKO) mice fed a high-fat diet at 23 °C, with BAT hyperplasia and robust browning of inguinal white adipose tissue (IWAT) observed. Proteomics and gene expression analyses revealed significant increases in many proteins involved in energy metabolism and strong upregulation of brown/beige adipocyte-related genes and fatty acid metabolism-related genes in browned IWAT, suggesting an induction of beige fat formation and stimulation of lipid metabolism in DKO mice at 23 °C. Conversely, mRNA levels of fatty acid oxidation-related genes decreased in the liver of DKO mice. The favorable phenotypic changes were lost at 30 °C, with BAT whitening and disappearance of IWAT browning, while fatty liver further deteriorated in DKO mice compared with that in APOE-KO mice. Finally, longevity analysis revealed a significant lifespan extension of DKO mice compared with that of APOE-KO mice at 23 °C. Irrespective of the fundamental role of UCP1 thermogenesis, our results highlight the importance of beige fat for the improvement of hyperlipidemia and longevity under the atherogenic status at normal room temperature.     

Long non‐coding RNA expressed in macrophage co‐varies with the inflammatory phenotype during macrophage development and polarization

Advances in microarray, RNA‐seq and omics techniques, thousands of long non‐coding RNAs (lncRNAs) with unknown functions have been discovered. LncRNAs have presented a diverse perspective on gene regulation in diverse biological processes, especially in human immune response. Macrophages participate in the whole phase of immune inflammatory response. They are able to shape their phenotype and arouse extensive functional activation after receiving physiological and pathological stimuli. Emerging studies indicated that lncRNAs participated in the gene regulatory network during complex biological processes of macrophage, including macrophage‐induced inflammatory responses. Here, we reviewed the existing knowledges of lncRNAs in the processes of macrophage development and polarization, and their roles in several different inflammatory diseases. Specifically, we focused on how lncRNAs function in macrophage, which might help to discover some potential therapeutic targets and diagnostic biomarkers.     

Deletion of pancreatic β‐cell adenosine kinase improves glucose homeostasis in young mice and ameliorates streptozotocin‐induced hyperglycaemia

Severe reduction in the β‐cell number (collectively known as the β‐cell mass) contributes to the development of both type 1 and type 2 diabetes. Recent pharmacological studies have suggested that increased pancreatic β‐cell proliferation could be due to specific inhibition of adenosine kinase (ADK). However, genetic evidence for the function of pancreatic β‐cell ADK under physiological conditions or in a pathological context is still lacking. In this study, we crossed mice carrying LoxP‐flanked Adk gene with Ins2‐Cre mice to acquire pancreatic β ‐cell ADK deficiency (Ins2‐Cre^±Adk^fl/fl) mice. Our results revealed that Ins2‐Cre^+/‐Adk^fl/fl mice showed improved glucose metabolism and β‐cell mass in younger mice, but showed normal activity in adult mice. Moreover, Ins2‐Cre^±Adk^fl/fl mice were more resistant to streptozotocin (STZ) induced hyperglycaemia and pancreatic β‐cell damage in adult mice. In conclusion, we found that ADK negatively regulates β‐cell replication in young mice as well as under pathological conditions, such as STZ induced pancreatic β‐cell damage. Our study provided genetic evidence that specific inhibition of pancreatic β‐cell ADK has potential for anti‐diabetic therapy.     

Tetrahydro iso-alpha acids from hops improve glucose homeostasis and reduce body weight gain and metabolic endotoxemia in high-fat diet-fed mice

Obesity and related metabolic disorders such as insulin resistance and type 2 diabetes are associated with a low-grade inflammatory state possibly through changes in gut microbiota composition and the development of higher plasma lipopolysaccharide (LPS) levels, i.e. metabolic endotoxemia. Various phytochemical compounds have been investigated as potential tools to regulate these metabolic features. Humulus lupulus L. (hops) contains several classes of compounds with anti-inflammatory potential. Recent evidence suggests that hops-derived compounds positively impact adipocyte metabolism and glucose tolerance in obese and diabetic rodents via undefined mechanisms. In this study, we found that administration of tetrahydro iso-alpha acids (termed META060) to high-fat diet (HFD)-fed obese and diabetic mice for 8 weeks reduced body weight gain, the development of fat mass, glucose intolerance, and fasted hyperinsulinemia, and normalized insulin sensitivity markers. This was associated with reduced portal plasma LPS levels, gut permeability, and higher intestinal tight junction proteins Zonula occludens-1 and occludin. Moreover, META060 treatment increased the plasma level of the anti-inflammatory cytokine interleukin-10 and decreased the plasma level of the pro-inflammatory cytokine granulocyte colony-stimulating factor. In conclusion, this research allows us to decipher a novel mechanism contributing to the positive effects of META060 treatment, and supports the need to investigate such compounds in obese and type 2 diabetic patients.     

Effect of (R)‐salbutamol on the switch of phenotype and metabolic pattern in LPS‐induced macrophage cells

Evidence demonstrates that M1 macrophage polarization promotes inflammatory disease. Here, we discovered that (R)‐salbutamol, a β₂ receptor agonist, inhibits and reprograms the cellular metabolism of RAW264.7 macrophages. (R)‐salbutamol significantly inhibited LPS‐induced M1 macrophage polarization and downregulated expressions of typical M1 macrophage cytokines, including monocyte chemotactic protein‐1 (MCP‐1), interleukin‐1β (IL‐1β) and tumour necrosis factor α (TNF‐α). Also, (R)‐salbutamol significantly decreased the production of inducible nitric oxide synthase (iNOS), nitric oxide (NO) and reactive oxygen species (ROS), while increasing the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio. In contrast, (S)‐salbutamol increased the production of NO and ROS. Bioenergetic profiles showed that (R)‐salbutamol significantly reduced aerobic glycolysis and enhanced mitochondrial respiration. Untargeted metabolomics analysis demonstrated that (R)‐salbutamol modulated metabolic pathways, of which three metabolic pathways, namely, (a) phenylalanine metabolism, (b) the pentose phosphate pathway and (c) glycerophospholipid metabolism were the most noticeably impacted pathways. The effects of (R)‐salbutamol on M1 polarization were inhibited by a specific β₂ receptor antagonist, ICI‐118551. These findings demonstrated that (R)‐salbutamol inhibits the M1 phenotype by downregulating aerobic glycolysis and glycerophospholipid metabolism, which may propose (R)‐salbutamol as the major pharmacologically active component of racemic salbutamol for the treatment of inflammatory diseases and highlight the medicinal value of (R)‐salbutamol.     

Dehydrozingerone exerts beneficial metabolic effects in high‐fat diet‐induced obese mice via AMPK activation in skeletal muscle

Dehydrozingerone (DHZ) exerts beneficial effects on human health; however, its mechanism of action remains unclear. Here, we found that DHZ suppressed high‐fat diet‐induced weight gain, lipid accumulation and hyperglycaemia in C57BL/6 mice and increased AMP‐activated protein kinase (AMPK) phosphorylation and stimulated glucose uptake in C2C12 skeletal muscle cells. DHZ activated p38 mitogen‐activated protein kinase (MAPK) signalling in an AMPK‐dependent manner. Inhibiting AMPK or p38 MAPK blocked DHZ‐induced glucose uptake. DHZ increased GLUT4 (major transporter for glucose uptake) expression in skeletal muscle. Glucose clearance and insulin‐induced glucose uptake increased in DHZ‐fed animals, suggesting that DHZ increases systemic insulin sensitivity in vivo. Thus, the beneficial health effects of DHZ could possibly be explained by its ability to activate the AMPK pathway in skeletal muscle.     

Effects of neonatal sympathectomy on brown fat development and susceptibility to high fat diet induced obesity in mice.

Injections of 6-hydroxydopamine in mouse neonates caused extensive and long lasting damage to the sympathetic nervous system and impaired brown fat development. Brown adipose tissue (BAT) thermogenic capacity of sympathectomized mice (up to 120 days old) was reduced because of marked reductions in the tissue mitochondrial protein content and the mitochondrial concentration of uncoupling protein, as assessed by [3H]GDP binding and immunoassay. Neonatal sympathectomy did not affect BAT DNA content. Sympathectomized mice also had reduced epinephrine-stimulated rates of oxygen consumption. BAT of sympathectomized mice failed to respond by increases in [3H]GDP binding to isolated mitochondria and uncoupling protein concentration when animals were offered a palatable high-fat dietary supplement that increased calorie intake of both normal and sympathectomized mice. The high-fat diet caused increases in body weight, carcass fat, and gonadal white fat pad weights in sympathectomized animals that were similar to those of control mice. These results show that inactivation of BAT metabolism did not accentuate the development of obesity caused by a dietary supplement rich in fat and suggest that stimulation of BAT metabolism was not very effective in counteracting the obesity-inducing effect of this diet.     

In vivo study of metabolic dynamics and heterogeneity in brown and beige fat by label‐free multiphoton redox and fluorescence lifetime microscopy

In this work, the metabolic characteristics of adipose tissues in live mouse model were investigated using a multiphoton redox ratio and fluorescence lifetime imaging technology. By analyzing the intrinsic fluorescence of metabolic coenzymes, we measured the optical redox ratios of adipocytes in vivo and studied their responses to thermogenesis. The fluorescence lifetime imaging further revealed changes in protein bindings of metabolic coenzymes in the adipocytes during thermogenesis. Our study uncovered significant heterogeneity in the cellular structures and metabolic characteristics of thermogenic adipocytes in brown and beige fat. Subgroups of brown and beige adipocytes were identified based on the distinct lipid size distributions, redox ratios, fluorescence lifetimes and thermogenic capacities. The results of our study show that this label‐free imaging technique can shed new light on in vivo study of metabolic dynamics and heterogeneity of adipose tissues in live organisms.