Hepatic glycerol shunt and glycerol-3-phosphate phosphatase control liver metabolism and glucodetoxification under hyperglycemia.

OBJECTIVE: Glycerol-3-phosphate (Gro3P) phosphatase (G3PP) hydrolyzes Gro3P to glycerol that exits the cell, thereby operating a "glycerol shunt", a metabolic pathway that we identified recently in mammalian cells. We have investigated the role of G3PP and the glycerol shunt in the regulation of glucose metabolism and lipogenesis in mouse liver. METHODS: We generated hepatocyte-specific G3PP-KO mice (LKO), by injecting AAV8-TBG-iCre to male G3PPfl/fl mice. Controls received AAV8-TBG-eGFP. Both groups were fed chow diet for 10 weeks. Hyperglycemia (16-20 mM) was induced by glucose infusion for 55 h. Hepatocytes were isolated from normoglycemic mice for ex vivo studies and targeted metabolomics were measured in mice liver after glucose infusion. RESULTS: LKO mice showed no change in body weight, food intake, fed and fasted glycemia but had increased fed plasma triglycerides. Hepatic glucose production from glycerol was increased in fasted LKO mice. LKO mouse hepatocytes displayed reduced glycerol production, elevated triglyceride and lactate production at high glucose concentration. Hyperglycemia in LKO mice led to increased liver weight and accumulation of triglycerides, glycogen and cholesterol together with elevated levels of Gro3P, dihydroxyacetone phosphate, acetyl-CoA and some Krebs cycle intermediates in liver. Hyperglycemic LKO mouse liver showed elevated expression of proinflammatory cytokines and M1-macrophage markers accompanied by increased plasma triglycerides, LDL/VLDL, urea and uric acid and myocardial triglycerides. CONCLUSIONS: The glycerol shunt orchestrated by G3PP acts as a glucose excess detoxification pathway in hepatocytes by preventing metabolic disturbances that contribute to enhanced liver fat, glycogen storage, inflammation and lipid build-up in the heart. We propose G3PP as a novel therapeutic target for hepatic disorders linked to nutrient excess.

Glycerol-3-phosphate phosphatase operates a glycerol shunt in pancreatic ß-cells that controls insulin secretion and metabolic stress.

OBJECTIVE: The recently identified glycerol-3-phosphate (Gro3P) phosphatase (G3PP) in mammalian cells, encoded by the PGP gene, was shown to regulate glucose, lipid and energy metabolism by hydrolyzing Gro3P and to control glucose-stimulated insulin secretion (GSIS) in ß-cells, in vitro. However, whether G3PP regulates ß-cell function and insulin secretion in vivo is not known. METHODS: We now examined the role of G3PP in the control of insulin secretion in vivo, ß-cell function and glucotoxicity in inducible ß-cell specific G3PP-KO (BKO) mice. Inducible BKO mice were generated by crossing floxed-G3PP mice with Mip-Cre-ERT (MCre) mice. All the in vivo studies were done using BKO and control mice fed normal diet and the ex vivo studies were done using pancreatic islets from these mice. RESULTS: BKO mice, compared to MCre controls, showed increased body weight, adiposity, fed insulinemia, enhanced in vivo GSIS, reduced plasma triglycerides and mild glucose intolerance. Isolated BKO mouse islets incubated at high (16.7 mM), but not at low or intermediate glucose (3 and 8 mM), showed elevated GSIS, Gro3P content as well as increased levels of metabolites and signaling coupling factors known to reflect ß-cell activation for insulin secretion. BKO islets also showed reduced glycerol release and increased O2 consumption and ATP production at high glucose only. BKO islets chronically exposed to elevated glucose levels showed increased apoptosis, reduced insulin content and decreased mRNA expression of ß-cell differentiation markers, Pdx-1, MafA and Ins-2. CONCLUSIONS: The results demonstrate that ß-cells are endowed with a "glycerol shunt", operated by G3PP that regulates ß-cell metabolism, signaling and insulin secretion in vivo, primarily at elevated glucose concentrations. We propose that the glycerol shunt plays a role in preventing insulin hypersecretion and excess body weight gain and contributes to ß-cell mass preservation in the face of hyperglycemia.

Adipose ABHD6 regulates tolerance to cold and thermogenic programs.

Enhanced energy expenditure in brown (BAT) and white adipose tissues (WAT) can be therapeutic against metabolic diseases. We examined the thermogenic role of adipose α/ß-hydrolase domain 6 (ABHD6), which hydrolyzes monoacylglycerol (MAG), by employing adipose-specific ABHD6-KO mice. Control and KO mice showed similar phenotypes at room temperature and thermoneutral conditions. However, KO mice were resistant to hypothermia, which can be accounted for by the simultaneously increased lipolysis and lipogenesis of the thermogenic glycerolipid/free fatty acid (GL/FFA) cycle in visceral fat, despite unaltered uncoupling protein 1 expression. Upon cold stress, nuclear 2-MAG levels increased in visceral WAT of the KO mice. Evidence is provided that 2-MAG causes activation of PPARα in white adipocytes, leading to elevated expression and activity of GL/FFA cycle enzymes. In the ABHD6-ablated BAT, glucose and oxidative metabolism were elevated upon cold induction, without changes in GL/FFA cycle and lipid turnover. Moreover, response to in vivo ß3-adrenergic stimulation was comparable between KO and control mice. Our data reveal a MAG/PPARα/GL/FFA cycling metabolic signaling network in visceral adipose tissue, which contributes to cold tolerance, and that adipose ABHD6 is a negative modulator of adaptive thermogenesis.

Monoacylglycerol signalling and ABHD6 in health and disease.

Lipid metabolism dysregulation underlies chronic pathologies such as obesity, diabetes and cancer. Besides their role in structure and energy storage, lipids are also important signalling molecules regulating multiple biological functions. Thus, understanding the precise lipid metabolism enzymatic steps that are altered in some pathological conditions is helpful for designing better treatment strategies. Several monoacylglycerol (MAG) species are only recently being recognized as signalling lipid molecules in different tissues. Recent studies indicated the importance of the ubiquitously expressed serine hydrolase α/ß-hydrolase domain 6 (ABHD6), which is a MAG hydrolase, in regulating signalling competent MAG in both central and peripheral tissues. The central and peripheral function of the endocannabinoid 2-arachidonoylglycerol, which is a 2-MAG, and its breakdown by both ABHD6 and classical MAG lipase has been well documented. ABHD6 and its substrate MAG appear to be involved in the regulation of various physiological and pathological processes including insulin secretion, adipose browning, food intake, neurotransmission, autoimmune disorders, neurological and metabolic diseases as well as cancer. Diverse cellular targets such as mammalian unc13-1 (Munc13-1), PPARs, GPR119 and CB1/2 receptors, for MAG-mediated signalling processes have been proposed in different cell types. The purpose of this review is to provide a comprehensive summary of the current state of knowledge regarding ABHD6/MAG signalling and its possible therapeutic implications.

A beta cell ATGL-lipolysis/adipose tissue axis controls energy homeostasis and body weight via insulin secretion in mice.

AIMS/HYPOTHESIS: To directly assess the role of beta cell lipolysis in insulin secretion and whole-body energy homeostasis, inducible beta cell-specific adipose triglyceride lipase (ATGL)-deficient (B-Atgl-KO) mice were studied under normal diet (ND) and high-fat diet (HFD) conditions. METHODS: Atgl flox/flox mice were cross-bred with Mip-Cre-ERT mice to generate Mip-Cre-ERT/+;Atgl flox/flox mice. At 8 weeks of age, these mice were injected with tamoxifen to induce deletion of beta cell-specific Atgl (also known as Pnpla2), and the mice were fed an ND or HFD. RESULTS: ND-fed male B-Atgl-KO mice showed decreased insulinaemia and glucose-induced insulin secretion (GSIS) in vivo. Changes in GSIS correlated with the islet content of long-chain saturated monoacylglycerol (MAG) species that have been proposed to be metabolic coupling factors for insulin secretion. Exogenous MAGs restored GSIS in B-Atgl-KO islets. B-Atgl-KO male mice fed an HFD showed reduced insulinaemia, glycaemia in the fasted and fed states and after glucose challenge, as well as enhanced insulin sensitivity. Moreover, decreased insulinaemia in B-Atgl-KO mice was associated with increased energy expenditure, and lipid metabolism in brown (BAT) and white (WAT) adipose tissues, leading to reduced fat mass and body weight. CONCLUSIONS/INTERPRETATION: ATGL in beta cells regulates insulin secretion via the production of signalling MAGs. Decreased insulinaemia due to lowered GSIS protects B-Atgl-KO mice from diet-induced obesity, improves insulin sensitivity, increases lipid mobilisation from WAT and causes BAT activation. The results support the concept that fuel excess can drive obesity and diabetes via hyperinsulinaemia, and that an islet beta cell ATGL-lipolysis/adipose tissue axis controls energy homeostasis and body weight via insulin secretion.

α/ß-Hydrolase Domain 6 Deletion Induces Adipose Browning and Prevents Obesity and Type 2 Diabetes.

Suppression of α/ß-domain hydrolase-6 (ABHD6), a monoacylglycerol (MAG) hydrolase, promotes glucose-stimulated insulin secretion by pancreatic ß cells. We report here that high-fat-diet-fed ABHD6-KO mice show modestly reduced food intake, decreased body weight gain and glycemia, improved glucose tolerance and insulin sensitivity, and enhanced locomotor activity. ABHD6-KO mice also show increased energy expenditure, cold-induced thermogenesis, brown adipose UCP1 expression, fatty acid oxidation, and white adipose browning. Adipose browning and cold-induced thermogenesis are replicated by the ABHD6 inhibitor WWL70 and by antisense oligonucleotides targeting ABHD6. Our evidence suggests that one mechanism by which the lipolysis derived 1-MAG signals intrinsic and cell-autonomous adipose browning is via PPARα and PPARγ activation, and that ABHD6 regulates adipose browning by controlling signal competent 1-MAG levels. Thus, ABHD6 regulates energy homeostasis, brown adipose function, and white adipose browning and is a potential therapeutic target for obesity and type 2 diabetes.

Identification of a mammalian glycerol-3-phosphate phosphatase: Role in metabolism and signaling in pancreatic ß-cells and hepatocytes.

Obesity, and the associated disturbed glycerolipid/fatty acid (GL/FA) cycle, contribute to insulin resistance, islet ß-cell failure, and type 2 diabetes. Flux through the GL/FA cycle is regulated by the availability of glycerol-3-phosphate (Gro3P) and fatty acyl-CoA. We describe here a mammalian Gro3P phosphatase (G3PP), which was not known to exist in mammalian cells, that can directly hydrolyze Gro3P to glycerol. We identified that mammalian phosphoglycolate phosphatase, with an uncertain function, acts in fact as a G3PP. We found that G3PP, by controlling Gro3P levels, regulates glycolysis and glucose oxidation, cellular redox and ATP production, gluconeogenesis, glycerolipid synthesis, and fatty acid oxidation in pancreatic islet ß-cells and hepatocytes, and that glucose stimulated insulin secretion and the response to metabolic stress, e.g., glucolipotoxicity, in ß-cells. In vivo overexpression of G3PP in rat liver lowers body weight gain and hepatic glucose production from glycerol and elevates plasma HDL levels. G3PP is expressed at various levels in different tissues, and its expression varies according to the nutritional state in some tissues. As Gro3P lies at the crossroads of glucose, lipid, and energy metabolism, control of its availability by G3PP adds a key level of metabolic regulation in mammalian cells, and G3PP offers a potential target for type 2 diabetes and cardiometabolic disorders.

Acute effects of protein composition and fibre enrichment of yogurt consumed as snacks on appetite sensations and subsequent ad libitum energy intake in healthy men.

The objective of the study was to assess the impact of protein composition and/or fibre enrichment of yogurt on appetite sensations and subsequent energy intake. In this double-blind crossover study, 20 healthy men (aged 32.4 ± 9.1 years) were submitted to 5 randomized testing sessions, during which they had to consume 5 isocaloric and isonproteinemic yogurt snacks (120-g servings, ∼230 kJ, ∼4.5 g protein) differing by their casein-to-whey protein ratio (C:W) or dietary fibre content: (i) control C:W = 2.8:1; (ii) high whey (HW) C:W = 1.5:1, and fibre-enriched formulations using control; (iii) 2.4 g of inulin; (iv) 1.9 g of inulin and 0.5 g of ß-glucan (+IN-ßG); and (v) 0.5 g of ß-glucan. Appetite sensations were assessed using 150-mm visual analog scales. Plasma variables (glucose, insulin, ghrelin) were measured at 30-min intervals post-yogurt consumption for 2 h. Finally, energy intakes during ad libitum lunches offered 2 h after yogurt snacks were recorded. None of the yogurts impacted appetite sensations. Ad libitum energy intake was significantly different only between HW and control yogurts (-812 kJ; p = 0.03). Regarding post-yogurt plasma variables, a significant difference was found only between ghrelin area under the curve of the +IN-ßG and the HW yogurts (-15 510 pmol/L per 120 min, p = 0.04). In conclusion, although appetite sensations were not influenced by variations in yogurts' protein compositions, a reduced energy intake was observed during the ad libitum lunch after the HW yogurt that may be attributable to its lower C:W. Surprisingly, the fibre enrichments studied did not exert effect on appetite sensations and energy intake.

α/ß-Hydrolase domain-6 and saturated long chain monoacylglycerol regulate insulin secretion promoted by both fuel and non-fuel stimuli.

OBJECTIVE: α/ß-Hydrolase domain-6 (ABHD6) is a newly identified monoacylglycerol (MAG) lipase. We recently reported that it negatively regulates glucose stimulated insulin secretion (GSIS) in the ß cells by hydrolyzing lipolysis-derived MAG that acts as a metabolic coupling factor and signaling molecule via exocytotic regulator Munc13-1. Whether ABHD6 and MAG play a role in response to all classes of insulin secretagogues, in particular various fuel and non-fuel stimuli, is unknown. METHODS: Insulin secretion in response to various classes of secretagogues, exogenous MAG and pharmacological agents was measured in islets of mice deficient in ABHD6 specifically in the ß cell (BKO). Islet perifusion experiments and determinations of glucose and fatty acid metabolism, cytosolic Ca(2+) and MAG species levels were carried out. RESULTS: Deletion of ABHD6 potentiated insulin secretion in response to the fuels glutamine plus leucine and α-ketoisocaproate and to the non-fuel stimuli glucagon-like peptide 1, carbamylcholine and elevated KCl. Fatty acids amplified GSIS in control and BKO mice to the same extent. Exogenous 1-MAG amplified insulin secretion in response to fuel and non-fuel stimuli. MAG hydrolysis activity was greatly reduced in BKO islets without changes in total diacylglycerol and triacylglycerol lipase activity. ABHD6 deletion induced insulin secretion independently from KATP channels and did not alter the glucose induced rise in intracellular Ca(2+). Perifusion studies showed elevated insulin secretion during second phase of GSIS in BKO islets that was not due to altered cytosolic Ca(2+) signaling or because of changes in glucose and fatty acid metabolism. Glucose increased islet saturated long chain 1-MAG species and ABHD6 deletion caused accumulation of these 1-MAG species at both low and elevated glucose. CONCLUSION: ABHD6 regulates insulin secretion in response to fuel stimuli at large and some non-fuel stimuli by controlling long chain saturated 1-MAG levels that synergize with other signaling pathways for secretion.

Cross-sectional associations of acylation stimulating protein (ASP) and adipose tissue gene expression with estradiol and progesterone in pre- and postmenopausal women.

OBJECTIVE: Sex steroid hormones play an important regulatory role in fat metabolism and obesity. We hypothesized involvement of interactions between ovarian hormones with acylation stimulating protein (ASP). DESIGN, PATIENTS AND MEASUREMENTS: In 392 women with wide age (18-69 years) and body size (BMI: 17 to 90 kg/m(2) ) ranges, fasting plasma levels of ASP, ovarian hormones, glucose, adiponectin and lipids/apolipoproteins were assessed, along with determination of metabolic syndrome (MS) features. Gene expression of C3 (ASP precursor) and related receptors C5L2, C3aR and C5aR in subcutaneous and omental adipose tissues was measured in a subset. RESULTS: Acylation stimulating protein correlated negatively with concentrations of estradiol (P < 0·0001), adiponectin (P < 0·001) and apolipoprotein A1 (P < 0·001) and positively with apolipoprotein B levels (P < 0·001), systolic blood pressure (P < 0·001), waist circumference (P < 0·001), and triglyceride concentrations (P < 0·01). In age-matched groups of lean, overweight, metabolically healthy obese (MHO) and obese with metabolic syndrome (MSO), there was a stepwise increase in ASP levels (P < 0·001) while concentrations of adiponectin (P < 0·0001) and estradiol (P < 0·001) but not those of progesterone decreased. Progesterone but not estradiol levels correlated positively with C3 gene expression in omental adipose tissue (P < 0·05) and negatively with C5L2 expression in both omental (P < 0·01) and subcutaneous (P < 0·05) adipose tissues. CONCLUSION: Our results are consistent with the concept that sex hormones differentially influence circulating ASP and adipose tissue gene expression of its related proteins in a depot-specific manner. ASP may play a role in the regulation of regional fat metabolism through interactions with sex hormones in women.

Downregulation of complement C3 and C3aR expression in subcutaneous adipose tissue in obese women.

BACKGROUND: The central component of the complement system, C3, is associated with obesity, metabolic syndrome and cardiovascular disease however the underlying reasons are unknown. In the present study we evaluated gene expression of C3, the cleavage product C3a/C3adesArg and its cognate receptor C3aR in subcutaneous and omental adipose tissue in women. METHODS: Women (n = 140, 21-69 years, BMI 19.5-79 kg/m2) were evaluated for anthropometric and blood parameters, and adipose tissue gene expression. RESULTS: Subjects were separated into groups (n = 34-36) according to obesity: normal/overweight (≤30 kg/m2), obese I (≤45 kg/m2), obese II (≤51 kg/m2), and obese III (≤80 kg/m2). Overall, while omental expression remained unchanged, subcutaneous C3 and C3aR gene expression decreased with increasing adiposity (2-way ANOVA, p<0.01), with a concomitant decrease in SC/OM ratio (p<0.001). In subcutaneous adipose, both C3 and C3aR expression correlated with apoB, and apoA1 and inversely with waist circumference and blood pressure, while C3aR also correlated with glucose (p<0.05-0.0001). While omental C3aR expression did not correlate with any factor, omental C3 correlated with waist circumference, glucose and apoB (all p<0.05). Further, while plasma C3a/C3adesArg increased and adiponectin decreased with increasing BMI, both correlated (C3a negatively and adiponectin positively) with subcutaneous C3 and C3aR expression (p<0.05-0.001) or less). CONCLUSIONS: The obesity-induced down-regulation of complement C3 and C3aR which is specific to subcutaneous adipose tissue, coupled to the strong correlations with multiple anthropometric, plasma and adipokine variables support a potential role for complement in immunometabolism.

Association of immune and metabolic receptors C5aR and C5L2 with adiposity in women.

Adipose tissue receptors C5aR and C5L2 and their heterodimerization/functionality and interaction with ligands C5a and acylation stimulating protein (ASP) have been evaluated in cell and rodent studies. Their contribution to obesity factors in humans remains unclear. We hypothesized that C5a receptors, classically required for host defense, are also associated with adiposity. Anthropometry and fasting blood parameters were measured in 136 women divided by body mass index (BMI): normal/overweight (≤30 kg/m(2); n = 34), obese I (≤45 kg/m(2); n = 33), obese II (≤51 kg/m(2); n = 33), and obese III (≤80 kg/m(2); n = 36). Subcutaneous and omental adipose tissue C5aR and C5L2 expression were analysed. C5L2 expression was comparable between subcutaneous and omental across all BMI groups. Plasma ASP and ASP/omental C5L2 expression increased with BMI (P < 0.001 and P < 0.01, resp.). While plasma C5a was unchanged, C5aR expression decreased with increasing BMI in subcutaneous and omental tissues (P < 0.01 and P < 0.05, resp.), with subcutaneous omental depots. Omental C5L2/C5aR ratio increased with BMI (P < 0.01) with correlations between C5L2/C5aR and waist circumference, HDL-C, and adiponectin. Tissue and BMI differences in receptors and ligands, particularly in omental, suggest relationship to metabolic disturbances and highlight adipose-immune interactions.

C5aR and C5L2 act in concert to balance immunometabolism in adipose tissue.

Recent studies suggested that the immunometabolic receptors; C5aR and C5L2, constitutively self-associate into homo-/heterodimers and that acylation stimulating protein (ASP/C3adesArg) or C5a treatment of adipocytes increased their colocalization. The present study evaluates the C5aR contribution in adipocytes to the metabolic and immune responses elicited by ligand stimulation. The effects of C5a, ASP, and insulin on cytokine production, triglyceride synthesis (TGS), and key signaling pathways were evaluated in isolated primary adipocytes and cultured 3T3-L1 differentiated adipocytes. In addition, mRNA expression of IRS1 and PGC1α was compared in adipose tissue samples from WT vs. C5aRKO mice. Both C5a and ASP directly increased MCP-1 (238±4%; P<0.001, and 377±2% vs. basal 100%; P<0.001, respectively) and KC (413±11%; P<0.001, and 529±16%; P<0.001 vs. basal 100%, respectively) secretion, TGS (131±1%; P<0.001, and 152±6%; P<0.001, vs. basal 100% respectively), and Akt/NFκB phosphorylation pathways in adipocytes. However, in C5aRKO adipocytes, C5a effects were disrupted, while stimulatory effects of ASP were mostly maintained. Addition of C5a completely blocked ASP signaling and activity in both C5aRKO and WT adipocytes as well as 3T3-L1 adipocytes. Furthermore, C5aRKO adipocytes revealed impaired insulin stimulation of cytokine production, with partial impairment of signaling and TGS stimulation, consistent with decreased IRS1 and PGC1α mRNA expression in adipose tissue. These observations indicate the importance of C5aR in adipose tissue metabolism and immunity, which may be regulated through heterodimerization with C5L2.

Paradoxical glucose-sensitizing yet proinflammatory effects of acute ASP administration in mice.

Acylation stimulating protein (ASP) is an adipokine derived from the immune complement system, which stimulates fat storage and is typically increased in obesity, type 2 diabetes, and cardiovascular disease. Using a diet-induced obesity (DIO) mouse model, the acute effects of ASP on energy metabolism and inflammatory processes in vivo were evaluated. We hypothesized that ASP would specifically exert proinflammatory effects. C57Bl/6 wild-type mice were put on a high-fat-high-sucrose diet for 12 weeks. Mice were then subjected to both glucose and insulin tolerance tests, each manipulation being preceded by recombinant ASP or vehicle (control) bolus injection. ASP supplementation increased whole-body glucose excursion, and this was accomplished with reduced concomitant insulin levels. However, ASP did not directly alter insulin sensitivity. ASP supplementation induced a proinflammatory phenotype, with higher levels of cytokines including IL-6 and TNF-α in plasma and in adipose tissue, liver, and skeletal muscle mRNA. Additionally, ASP increased M1 macrophage content of these tissues. ASP exerted a direct concentration-dependent role in the migration and M1 activation of cultured macrophages. Altogether, the in vivo and in vitro experiments demonstrate that ASP plays a role in both energy metabolism and inflammation, with paradoxical whole-body glucose-sensitizing yet proinflammatory effects.

C5a receptor deficiency alters energy utilization and fat storage.

OBJECTIVE: To investigate the impact of whole body C5a receptor (C5aR) deficiency on energy metabolism and fat storage. DESIGN: Male wildtype (WT) and C5aR knockout (C5aRKO) mice were fed a low fat (CHOW) or a high fat high sucrose diet-induced obesity (DIO) diet for 14 weeks. Body weight and food intake were measured weekly. Indirect calorimetry, dietary fatload clearance, insulin and glucose tolerance tests were also evaluated. Liver, muscle and adipose tissue mRNA gene expression were measured by RT-PCR. RESULTS: At week one and 12, C5aRKO mice on DIO had increased oxygen consumption. After 12 weeks, although food intake was comparable, C5aRKO mice had lower body weight (-7% CHOW, -12% DIO) as well as smaller gonadal (-38% CHOW, -36% DIO) and inguinal (-29% CHOW, -30% DIO) fat pads than their WT counterparts. Conversely, in WT mice, C5aR was upregulated in DIO vs CHOW diets in gonadal adipose tissue, muscle and liver, while C5L2 mRNA expression was lower in C5aRKO on both diet. Furthermore, blood analysis showed lower plasma triglyceride and non-esterified fatty acid levels in both C5aRKO groups, with faster postprandial triglyceride clearance after a fatload. Additionally, C5aRKO mice showed lower CD36 expression in gonadal and muscle on both diets, while DGAT1 expression was higher in gonadal (CHOW) and liver (CHOW and DIO) and PPARγ was increased in muscle and liver. CONCLUSION: These observations point towards a role (either direct or indirect) for C5aR in energy expenditure and fat storage, suggesting a dual role for C5aR in metabolism as well as in immunity.

C5L2 and C5aR interaction in adipocytes and macrophages: insights into adipoimmunology.

Obesity is associated with inflammation characterized by increased infiltration of macrophages into adipose tissue. C5aR-like receptor 2 (C5L2) has been identified as a receptor for acylation-stimulating protein (ASP) and the inflammatory factor C5a, which also binds C5aR. The present study examines the effects of ligands ASP and C5a on interactions between the receptors C5L2 and C5aR in 3T3-L1 adipocytes and J774 macrophages. BRET experiments indicate that C5L2 and C5aR form homo- and heterodimers in transfected HEK 293 cells, which were stable in the presence of ligand. Cell surface receptor levels of C5L2 and C5aR increased during 3T3-L1 adipocyte differentiation; both receptors are also highly expressed in J774 macrophages. Using confocal microscopy to evaluate endogenous receptors in adipocytes following stimulation with ASP or C5a, C5L2 is internalized with increasing perinuclear colocalization with C5aR. There is little C5a-dependent colocalization in macrophages. While adipocyte-conditioned medium (ACM) increased C5L2-C5aR colocalization in macrophages, this was blocked by C5a. ASP stimulation increased Akt (Ser(473)) phosphorylation in both cell types; C5a induced slight Akt phosphorylation in adipocytes with less effect in macrophages. ASP, but not C5a, increased fatty acid uptake/esterification in adipocytes. C5L2-C5aR homodimerization versus heterodimerization may thus contribute to differential responses obtained following ASP vs C5a stimulation of adipocytes and macrophages, providing new insights into the complex interaction between these two cell types within adipose tissue. Studying the mechanisms involved in the differential responses of C5L2-C5aR activation based on cell type will further our understanding of inflammatory processes in obesity.

Substance P decreases fat storage and increases adipocytokine production in 3T3-L1 adipocytes.

Obesity, inflammation, and insulin resistance are closely linked. Substance P (SP), via its neurokinin 1 receptor (NK1R), mediates inflammatory and, possibly, neuroendocrine processes. We examined SP effects on lipid storage and cytokine production in 3T3-L1 adipocytes and adipose tissues. 3T3-L1 adipocytes and preadipocytes express NK1R, and 8 days of SP supplementation during differentiation to 3T3-L1 preadipocytes decreased lipid droplet accumulation. SP (10 nM, 24 h) increased lipolysis in primary adipocytes (138 ± 7%, P < 0.05) and reduced fatty acid uptake (-31 ± 7%, P < 0.05) and mRNA expression of the differentiation-related transcription factors peroxisome proliferator-activated receptor-γ type 2 (-64 ± 2%, P < 0.001) and CCAAT enhancer-binding protein (CEBP)-α (-65 ± 2%, P < 0.001) and the lipid storage genes fatty acid-binding protein type 4 (-59 ± 2%, P < 0.001) and diacylglycerol O-acyltransferase-1 (-45 ± 2%, P < 0.01) in 3T3-L1 adipocytes, while CD36, a fatty acid transporter (+82 ± 19%, P < 0.01), was augmented. SP increased secretion of complement C3 (148 ± 15%, P < 0.04), monocyte chemoattractant protein-1 (156 ± 16%, P < 0.03), and keratinocyte-derived chemokine (148 ± 18%, P = 0.045) in 3T3-L1 adipocytes and monocyte chemoattractant protein-1 (496 ± 142%, P < 0.02) and complement C3 (152 ± 25%, P < 0.04) in adipose tissue and primary adipocytes, respectively. These SP effects were accompanied by downregulation of insulin receptor substrate 1 (-82 ± 2%, P < 0.01) and GLUT4 (-76 ± 2%, P < 0.01) mRNA expression, and SP acutely blocked insulin-mediated stimulation of fatty acid uptake and Akt phosphorylation. Although adiponectin secretion was unchanged, mRNA expression was decreased (-86 ± 8%, P < 0.001). In humans, NK1R expression correlates positively with plasma insulin, fatty acid, and complement C3 and negatively with adiponectin, CEBPα, CEBPß, and peroxisome proliferator-activated receptor-γ mRNA expression in omental, but not subcutaneous, adipose tissue. Our results suggest that, beyond its neuroendocrine and inflammatory effects, SP could also be involved in targeting adipose tissue and influencing insulin resistance.

Differential chemoattractant response in adipocytes and macrophages to the action of acylation stimulating protein.

Obesity is characterized by chronic low-grade inflammation with increased adipose tissue pro-inflammatory cytokine production. Acylation stimulating protein (ASP) stimulates triglyceride synthesis and glucose transport via its receptor C5L2. Circulating ASP is increased in obesity, insulin resistance and metabolic syndrome. The present study examines the effects of normal (50 nM), high physiological (200 nM) and pathological (600 nM) levels of ASP on inflammatory changes in 3T3-L1 adipocytes and J774 macrophages and the underlying mechanisms involved. Treatment with ASP for 24h increased monocyte chemoattractant protein-1 (MCP1, 800%, P<0.001) and keratinocyte-derived chemokine (KC, >150%, P<0.01) secretion in adipocytes in a dose-dependent manner, with no effect on IL-6 or adiponectin. In macrophages, ASP had no effect on these cytokines. C5a, a ligand for C5L2 and C5aR receptors, differed from ASP. Macrophage-adipocyte coculture increased MCP-1 and adiponectin secretion, and ASP further enhanced secretion (P<0.001 and P<0.05, respectively) at doses of 50 nM and 200 nM. ASP increased Ser(468) and Ser(536) phosphorylation of p65 NFκB in a time- and concentration-dependent manner (P<0.05) as well as phosphorylation of Akt Ser(473) (p=0.02). ASP and insulin stimulations of Ser(536) p65 NFκB phosphorylation were comparable (both p<0.05) but not additive. Both inhibition of PI3kinase (with wortmannin) and NFκB (with BAY11-7085) prevented ASP stimulation of MCP-1 and KC secretion in adipocytes. These findings suggest that ASP, especially at high physiologic doses, may stimulate specific inflammatory cytokines in adipocytes through PI3kinase- and NFκB-dependant pathways, thus further promoting macrophage infiltration and local inflammation in obese adipose tissue.

Serum adenosine deaminase activity in patients with systemic lupus erythematosus: a study based on ADA1 and ADA2 isoenzymes pattern.

Adenosine deaminase (ADA) plays a crucial role in the development and maintenance of normal immune system. So, any immunological imbalances could associate with its altered activity in serum. This study evaluated the activity of total ADA and its isoenzymes in serum of 45 patients with systemic lupus erythematosus (SLE). Included were 23 patients with active SLE, 22 during the inactive phase of the disease, and 45 healthy subjects. Our results provided evidence that the significantly elevated total ADA activity in serum of SLE patients is correlated mainly with the increased ADA2 level. The highest mean ADA2 activity during the relapse phase of the disease could be an indication to the macrophages, the main source of ADA2. It might be concluded that ADA and its isoenzymes analysis in serum of patients could be used as a useful and non-invasive diagnostic tool in evaluation of SLE active phase and the disease severity.

Adenosine deaminase in patients with primary immunodeficiency syndromes: the analysis of serum ADA1 and ADA2 activities.

OBJECTIVES: We aimed to investigate the activity of ADA and its isoenzymes in serum of patients with various primary immunodeficiency (PID) syndromes. DESIGN AND METHODS: Total ADA (tADA) and its isoenzymes were measured in 76 children with PID syndromes and 30 healthy controls using the Ellis method. RESULTS: Our results indicated that tADA and ADA2 levels were higher in patients with Chronic Granulomatous Disease (CGD), Leukocyte Adhesion Deficiency (LAD), hyper IgM (HIM) and Wiskott-Aldrich Syndrome (WAS) than those of corresponding controls (P<0.01). There was a significant elevation of tADA and ADA1 activities in IgA deficiency patients as compared to healthy individuals (P<0.01). CONCLUSIONS: Our results hypothesized that altered ADA activity may be associated with altered immunity. Therefore, serum ADA level could be used as an indicator along with other parameters in follow up of patients with CGD, LAD, IgA deficiency, HIM and WAS.