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Diacylglycerol kinase α deficiency alters inflammation markers in adipose tissue in response to a high-fat diet

2017; Elsevier BV; Volume: 59; Issue: 2 Linguagem: Inglês

10.1194/jlr.m079517

1539-7262

Emmani B. M. Nascimento, Louise Mannerås-Holm, Alexander Chibalin, Marie Björnholm, Juleen R. Zierath,

Metabolism, Diabetes, and Cancer

Conversion of diacylglycerol to phosphatidic acid is mediated by diacylglycerol kinases (DGKs), with DGKα specifically linked to adaptive immune responses. We determined the role of DGKα in obesity and inflammatory responses to a high-fat diet (HFD). DGKα KO and WT littermates were either a) chow-fed, b) HFD-fed for 12 weeks (Long-Term HFD), or c) HFD-fed for 3 days (Acute HFD). Body weight/composition, oxygen consumption, food intake, and glucose tolerance was unaltered between chow-fed DGKα KO and WT mice. Insulin concentration during the intraperitoneal glucose tolerance (IPGT) test was elevated in chow-fed DGKα KO mice, suggesting mild insulin resistance. Insulin concentration during the IPGT test was reduced in Long-Term HFD-fed DGKα KO mice, suggesting a mild enhancement in insulin sensitivity. Acute HFD increased hormone sensitive lipase protein abundance and altered expression of interleukin 1β mRNA, an inflammatory marker in perigonadal adipose tissue of DGKα KO mice. In conclusion, DGKα ablation is associated with mild alterations in insulin sensitivity. However, DGKα is dispensable for whole body insulin-mediated glucose uptake, hepatic glucose production, and energy homeostasis. Our results suggest DGKα aids in modulating the early immune response of adipose tissue following an acute exposure to HFD, possibly through modulation of acute T-cell action. Conversion of diacylglycerol to phosphatidic acid is mediated by diacylglycerol kinases (DGKs), with DGKα specifically linked to adaptive immune responses. We determined the role of DGKα in obesity and inflammatory responses to a high-fat diet (HFD). DGKα KO and WT littermates were either a) chow-fed, b) HFD-fed for 12 weeks (Long-Term HFD), or c) HFD-fed for 3 days (Acute HFD). Body weight/composition, oxygen consumption, food intake, and glucose tolerance was unaltered between chow-fed DGKα KO and WT mice. Insulin concentration during the intraperitoneal glucose tolerance (IPGT) test was elevated in chow-fed DGKα KO mice, suggesting mild insulin resistance. Insulin concentration during the IPGT test was reduced in Long-Term HFD-fed DGKα KO mice, suggesting a mild enhancement in insulin sensitivity. Acute HFD increased hormone sensitive lipase protein abundance and altered expression of interleukin 1β mRNA, an inflammatory marker in perigonadal adipose tissue of DGKα KO mice. In conclusion, DGKα ablation is associated with mild alterations in insulin sensitivity. However, DGKα is dispensable for whole body insulin-mediated glucose uptake, hepatic glucose production, and energy homeostasis. Our results suggest DGKα aids in modulating the early immune response of adipose tissue following an acute exposure to HFD, possibly through modulation of acute T-cell action. Diacylglycerol (DAG) is a precursor for triglycerides and phospholipids and acts as a second messenger. Infusion of free fatty acids in healthy humans increases DAG concentration and causes insulin resistance (1.Itani S.I. Ruderman N.B. Schmieder F. Boden G. Lipid-induced insulin resistance in human muscle is associated with changes in diacylglycerol, protein kinase C, and IkappaB-alpha.Diabetes. 2002; 51: 2005-2011Crossref PubMed Scopus (1111) Google Scholar). As a second messenger, conversion of DAG to phosphatidic acid (PA) marks inactivation of DAG-sensitive targets; however, DAG to PA conversion can also mark activation of PA-sensitive targets. Diacylglycerol kinase (DGK) is responsible for the conversion of DAG to PA. DGKs constitute a family of 10 different enzymes that are classified into five subfamilies. Various DGK family members can be expressed in the same tissue (2.Topham M.K. Epand R.M. Mammalian diacylglycerol kinases: molecular interactions and biological functions of selected isoforms.Biochim. Biophys. Acta. 2009; 1790: 416-424Crossref PubMed Scopus (132) Google Scholar, 3.Topham M.K. Prescott S.M. Mammalian diacylglycerol kinases, a family of lipid kinases with signaling functions.J. Biol. Chem. 1999; 274: 11447-11450Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Specific DGK isoforms have been linked to growth and metabolism. For example, DGKζ plays a role in skeletal muscle growth and differentiation (4.Evangelisti C. Riccio M. Faenza I. Zini N. Hozumi Y. Goto K. Cocco L. Martelli A.M. Subnuclear localization and differentiation-dependent increased expression of DGK-zeta in C2C12 mouse myoblasts.J. Cell. Physiol. 2006; 209: 370-378Crossref PubMed Scopus (32) Google Scholar, 5.Evangelisti C. Tazzari P.L. Riccio M. Fiume R. Hozumi Y. Fala F. Goto K. Manzoli L. Cocco L. Martelli A.M. Nuclear diacylglycerol kinase-zeta is a negative regulator of cell cycle progression in C2C12 mouse myoblasts.FASEB J. 2007; 21: 3297-3307Crossref PubMed Scopus (42) Google Scholar) and cardiac hypertrophy (6.Bilim O. Takeishi Y. Kitahara T. Arimoto T. Niizeki T. Sasaki T. Goto K. Kubota I. Diacylglycerol kinase zeta inhibits myocardial atrophy and restores cardiac dysfunction in streptozotocin-induced diabetes mellitus.Cardiovasc. Diabetol. 2008; 7: 2Crossref PubMed Scopus (30) Google Scholar, 7.Harada M. Takeishi Y. Arimoto T. Niizeki T. Kitahara T. Goto K. Walsh R.A. Kubota I. Diacylglycerol kinase zeta attenuates pressure overload-induced cardiac hypertrophy.Circ. J. 2007; 71: 276-282Crossref PubMed Scopus (34) Google Scholar). DGKδ has been associated with the development of insulin resistance and type 2 diabetes (8.Chibalin A.V. Leng Y. Vieira E. Krook A. Bjornholm M. Long Y.C. Kotova O. Zhong Z. Sakane F. Steiler T. et al.Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance.Cell. 2008; 132: 375-386Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Partial ablation of DGKδ in mice resulted in decreased DAG content in skeletal muscle, insulin resistance, and increased weight gain with age (8.Chibalin A.V. Leng Y. Vieira E. Krook A. Bjornholm M. Long Y.C. Kotova O. Zhong Z. Sakane F. Steiler T. et al.Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance.Cell. 2008; 132: 375-386Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Whether other DGKs influence growth and metabolic processes, including insulin sensitivity and obesity, remains to be determined. DGKα is a member of the type I subfamily of DGKs (9.Raben D.M. Wattenberg B.W. Signaling at the membrane interface by the DGK/SK enzyme family.J. Lipid Res. 2009; 50: S35-S39Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). The type I DGK isoforms, including α, β, and γ, contain the DAG-binding C1 domains and catalytic domain, typical of all subfamilies, as well as calcium-binding EF hand motifs, that render these members more active in the presence of calcium (10.Yamada K. Sakane F. Matsushima N. Kanoh H. EF-hand motifs of alpha, beta and gamma isoforms of diacylglycerol kinase bind calcium with different affinities and conformational changes.Biochem. J. 1997; 321: 59-64Crossref PubMed Scopus (75) Google Scholar). DGKα has been studied with respect to immunology and T-cell responses. DGKα is expressed in mouse spleen, skeletal muscle, lung, and testis (11.Sanjuán M.A. Pradet-Balade B. Jones D.R. Martinez A.C. Stone J.C. Garcia-Sanz J.A. Merida I. T cell activation in vivo targets diacylglycerol kinase alpha to the membrane: a novel mechanism for Ras attenuation.J. Immunol. 2003; 170: 2877-2883Crossref PubMed Scopus (93) Google Scholar). DGKα and DGKζ are the predominant DGK family members expressed in T-cells, and ablation of these DGK family members modulates T-cell responses (12.Gorentla B.K. Wan C.K. Zhong X.P. Negative regulation of mTOR activation by diacylglycerol kinases.Blood. 2011; 117: 4022-4031Crossref PubMed Scopus (82) Google Scholar, 13.Guo R. Wan C.K. Carpenter J.H. Mousallem T. Boustany R.M. Kuan C.T. Burks A.W. Zhong X.P. Synergistic control of T cell development and tumor suppression by diacylglycerol kinase alpha and zeta.Proc. Natl. Acad. Sci. USA. 2008; 105: 11909-11914Crossref PubMed Scopus (73) Google Scholar). Thus, T-cells lacking DGKα show impaired anergy in vitro, as demonstrated by interleukin (IL) 2 secretion and proliferation of T-cells following restimulation with antigen staphylococcal enterotoxin B (14.Olenchock B.A. Guo R. Carpenter J.H. Jordan M. Topham M.K. Koretzky G.A. Zhong X.P. Disruption of diacylglycerol metabolism impairs the induction of T cell anergy.Nat. Immunol. 2006; 7: 1174-1181Crossref PubMed Scopus (228) Google Scholar). These findings highlight a prominent role for DGKα in modulating T-cell responses. Furthermore, DGKα overexpression protects cancer cells from TNF α-induced apoptosis (15.Yanagisawa K. Yasuda S. Kai M. Imai S. Yamada K. Yamashita T. Jimbow K. Kanoh H. Sakane F. Diacylglycerol kinase alpha suppresses tumor necrosis factor-alpha-induced apoptosis of human melanoma cells through NF-kappaB activation.Biochim. Biophys. Acta. 2007; 1771: 462-474Crossref PubMed Scopus (63) Google Scholar). TNFα is a potent inducer of insulin resistance (16.Nielsen S.T. Lehrskov-Schmidt L. Krogh-Madsen R. Solomon T.P. Lehrskov-Schmidt L. Holst J.J. Moller K. Tumour necrosis factor-alpha infusion produced insulin resistance but no change in the incretin effect in healthy volunteers.Diabetes Metab. Res. Rev. 2013; 29: 655-663Crossref PubMed Scopus (21) Google Scholar) and transcription of TNFα is increased in white adipose tissue (WAT) derived from obese and diabetic rodent models (17.Hotamisligil G.S. Shargill N.S. Spiegelman B.M. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance.Science. 1993; 259: 87-91Crossref PubMed Scopus (6138) Google Scholar). This provides further evidence that DGKα modulates T-cell responses and TNFα-mediated signaling. Excess caloric intake combined with unaltered energy expenditure increases WAT mass and leads to obesity. Obesity is a major prodrome of insulin resistance and type 2 diabetes. Increased adiposity is accompanied by a low-grade inflammatory state in WAT (18.Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante Jr, A.W. Obesity is associated with macrophage accumulation in adipose tissue.J. Clin. Invest. 2003; 112: 1796-1808Crossref PubMed Scopus (7458) Google Scholar), which can further exacerbate metabolic abnormalities in obese individuals, including insulin resistance, type 2 diabetes, fatty liver disease, hypertension, dyslipidemia, atherosclerosis, and some cancers (19.Blüher M. Adipose tissue dysfunction in obesity.Exp. Clin. Endocrinol. Diabetes. 2009; 117: 241-250Crossref PubMed Scopus (445) Google Scholar). The inflammatory state is visible by macrophages and T-cells that are recruited to WAT in obese individuals and ob/ob mice (20.Kintscher U. Hartge M. Hess K. Foryst-Ludwig A. Clemenz M. Wabitsch M. Fischer-Posovszky P. Barth T.F. Dragun D. Skurk T. et al.T-lymphocyte infiltration in visceral adipose tissue: a primary event in adipose tissue inflammation and the development of obesity-mediated insulin resistance.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 1304-1310Crossref PubMed Scopus (558) Google Scholar, 21.Pettersson U.S. Walden T.B. Carlsson P.O. Jansson L. Phillipson M. Female mice are protected against high-fat diet induced metabolic syndrome and increase the regulatory T cell population in adipose tissue.PLoS One. 2012; 7: e46057Crossref PubMed Scopus (293) Google Scholar, 22.Rausch M.E. Weisberg S. Vardhana P. Tortoriello D.V. Obesity in C57BL/6J mice is characterized by adipose tissue hypoxia and cytotoxic T-cell infiltration.Int. J. Obes. (Lond.). 2008; 32: 451-463Crossref PubMed Scopus (431) Google Scholar). The recruitment of these cells is facilitated by inflammatory cytokines that are secreted from WAT, including TNFα, IL6, and IL1β, which are associated with insulin resistance and obesity (23.Steinberg G.R. Michell B.J. van Denderen B.J. Watt M.J. Carey A.L. Fam B.C. Andrikopoulos S. Proietto J. Gorgun C.Z. Carling D. et al.Tumor necrosis factor alpha-induced skeletal muscle insulin resistance involves suppression of AMP-kinase signaling.Cell Metab. 2006; 4: 465-474Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 24.Deiuliis J. Shah Z. Shah N. Needleman B. Mikami D. Narula V. Perry K. Hazey J. Kampfrath T. Kollengode M. et al.Visceral adipose inflammation in obesity is associated with critical alterations in tregulatory cell numbers.PLoS One. 2011; 6: e16376Crossref PubMed Scopus (225) Google Scholar, 25.Ehses J.A. Lacraz G. Giroix M.H. Schmidlin F. Coulaud J. Kassis N. Irminger J.C. Kergoat M. Portha B. Homo-Delarche F. et al.IL-1 antagonism reduces hyperglycemia and tissue inflammation in the type 2 diabetic GK rat.Proc. Natl. Acad. Sci. USA. 2009; 106: 13998-14003Crossref PubMed Scopus (293) Google Scholar). Excessive intracellular lipid metabolites in peripheral tissues in obesity can also induce chronic inflammation (26.Zierath J.R. The path to insulin resistance: paved with ceramides?.Cell Metab. 2007; 5: 161-163Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Thus, lipid metabolizing enzymes may also influence the immune tone of organs that control glucose and energy homeostasis. As DGKα plays a role in inflammatory responses, we determined whether this lipid metabolizing enzyme is associated with obesity and insulin resistance. Specifically, we determined the role of DGKα in whole-body glucose and energy homeostasis as well as WAT inflammation. We report that DGKα deficiency is associated with reduced expression of IL1β in WAT in mice fed a high-fat diet (HFD) for 3 days. However, with prolonged (3 months) HFD, inflammatory responses and glucose homeostasis were unaltered. Our results suggest DGKα is involved in the early inflammatory responses to an Acute HFD but is dispensable for the Long-Term maintenance of glucose and energy homeostasis. Animals were housed in a temperature- and light-controlled environment. Experiments were performed in female DGKα KO mice and WT littermates. Generation of the whole-body DGKα KO mouse model is described elsewhere (14.Olenchock B.A. Guo R. Carpenter J.H. Jordan M. Topham M.K. Koretzky G.A. Zhong X.P. Disruption of diacylglycerol metabolism impairs the induction of T cell anergy.Nat. Immunol. 2006; 7: 1174-1181Crossref PubMed Scopus (228) Google Scholar). The expression of DGK δ, η, ε, and ζ isoforms known to be expressed in adipose tissue (27.Mannerås-Holm L. Kirchner H. Bjornholm M. Chibalin A.V. Zierath J.R. mRNA expression of diacylglycerol kinase isoforms in insulin-sensitive tissues: effects of obesity and insulin resistance.Physiol. Rep. 2015; 3Crossref PubMed Scopus (15) Google Scholar) was unaltered between DGKα KO and WT littermates (Data not shown). Thus, other DGK isoforms do not appear to compensate for the loss of DGKα in this model. Animals were maintained at a 12 h light-dark cycle and had ad libitum access to water and a standard rodent chow (Lantmännen, Stockholm, Sweden) or 55%; adjusted fat HFD (TD.93075, Harlan Teklad, Indianapolis, IN). Three groups of mice were studied: a) 12-month-old mice fed a chow diet, b) 6-week-old mice fed HFD for 12 weeks (Long-Term HFD), and c) 10-week-old mice fed HFD for 3 days (Acute HFD). All animal experiments were approved by the Regional Ethical Committee on Animal Research, Stockholm North, Sweden. Glucose tolerance was assessed by an intraperitoneal injection of glucose (2 g/kg body weight). Mice were fasted 4 h prior to the intraperitoneal injection. Plasma glucose was determined directly after fasting and 15, 30, 60, and 120 min after injection with glucose. Blood samples were obtained prior to and 15 min after the glucose administration to assess plasma insulin levels using the ultra-sensitive insulin ELISA (Crystal Chem Inc. Downers Grove, IL). Whole-body composition was measured using an EchoMRI-100 Analyzer from EchoMRI LLC (Houston, TX). Fat and lean mass were measured. Whole-body energy homeostasis was determined in DGKα KO and WT mice using the Comprehensive Lab Animal Monitoring System (Columbus Instruments, Columbus, OH). Mice were individually housed with ad libitum access to water and standard chow or HFD. After a 24-h acclimatization period, oxygen consumption was measured for 48 h. Thereafter, mice were fasted overnight and refed in the morning. Food intake was determined for the dark and light phase during 24 h before the fasting. Whole body insulin-mediated glucose uptake and hepatic glucose production was determined in conscious DGKα KO and WT mice by means of the euglycemic-hyperinsulinemic clamp as described (8.Chibalin A.V. Leng Y. Vieira E. Krook A. Bjornholm M. Long Y.C. Kotova O. Zhong Z. Sakane F. Steiler T. et al.Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance.Cell. 2008; 132: 375-386Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). A catheter was placed in the left jugular vein and mice were allowed to recover for at least 3 days. On the day of the experiment, mice were fasted for 5 h and placed in individual plastic containers and blood was sampled from the tail. Glucose turnover rate was measured in the basal state and during the euglycemic-hyperinsulinemic state, using a constant infusion of [3-3H]glucose (NET331C001MC, PerkinElmer, Waltham, MA). Basal glucose production and utilization were assessed at 65–75 min after the start of the tracer infusion. After 75 min, the euglycemic-hyperinsulinemic clamp was started. A priming dose of insulin (22 mU/kg; Actrapid, Novo Nordisk, Bagsvaerd, Denmark) was administered, followed by a constant infusion rate of 2.5 mU/kg/min. Glucose (30%) was infused to maintain euglycemia. When the glucose infusion rate reached a steady state, blood samples were drawn to determine whole-body glucose utilization and hepatic glucose production. Hepatic glucose production was determined by subtracting the average glucose infusion rate from whole-body glucose uptake. Total DGK activity was measured via ATP dependent conversion of DAG to PA. Homogenates of perigonadal adipose tissue were prepared in ice-cold buffer containing 20 mM Tris.HCl (pH 7.5), 250 mM sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, protease inhibitor cocktail set 1 (Calbiochem, Darmstadt, Germany). Samples were subjected to centrifugation (800 g, 15 min, 4°C) and protein concentration was determined in supernatant. An octyl glucoside/phosphatidylserine mixed-micelle assay for DGK activity was performed using [γ-32P]ATP as described (28.Lee I.K. Koya D. Ishi H. Kanoh H. King G.L. d-Alpha-tocopherol prevents the hyperglycemia induced activation of diacylglycerol (DAG)-protein kinase C (PKC) pathway in vascular smooth muscle cell by an increase of DAG kinase activity.Diabetes Res. Clin. Pract. 1999; 45: 183-190Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Samples were subsequently separated by TLC and plates were developed. Regions of interest containing the phospholipid product were quantified by phospho-imaging and densitometry. Mice were fasted for 4 h, anesthetized with 2,2,2-tribromoethanol via intraperitoneal injection, and perigonadal WAT was collected. Tissue (∼20 mg) was incubated in the absence or presence of isoprenaline (10−5 M, 10−6 M) for 90 min at 37°C in D-PBS supplemented with 2% RIA-grade BSA. The tissue was removed and the glycerol concentration in the medium was determined. Glycerol release into the medium was measured as an index of lipolysis. Glycerol was measured using a Zenbio Glycerol Analysis Kit (Research Triangle Park, NC). RNA from perigonadal WAT was extracted using a Trizol reagent and RNeasy kit from Qiagen (Hilden, Germany). RNA was DNase treated (Qiagen) prior to the cDNA synthesis. cDNA was created by using the High Capacity Reverse Transcription Kit from Applied Biosystems (Foster City, CA). Quality and yield of RNA was assessed using a NanoDrop spectrophotometer from Thermo Fisher Scientific (Waltham, MA). Gene expression was determined using a StepOnePlus quantitative real time PCR machine from Applied Biosystems. Comparison of relative gene expression data was accomplished using the 2-ΔΔCt method. Gene expression data was normalized against peptidyl-prolyl isomerase A and hypoxanthine guanine phosphoribosyl transferase 1. Primers were from Applied Biosystems (Foster City, CA): TNFα (Mm00443260_g1), IFNγ (Mm01168134_m1), IL1β (Mm00434228_m1), IL2 (Mm00434256_m1), IL6 (Mm00446190_m1), F4/80 (Mm00802529_m1), hormone sensitive lipase (HSL) (Mm00495359_m1), peptidyl-prolyl isomerase A (Mm02342430_g1) and hypoxanthine guanine phosphoribosyl transferase 1 (Mm00446968_m1). Perigonadal WAT was homogenized in an ice-cold homogenization buffer (137 mM NaCl, 20 mM Tris.HCl pH 7.8, 2.7 mM KCl, 10% glycerol, 5 mM sodium pyrophosphate, 1% Triton X-100, 1 mM MgCl2, 10 mM NaF, 1 mM EDTA, 0.2 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 0.5 mM sodium orthovandate). From the resulting homogenate, the supernatant was collected and 15 μg protein was separated by SDS-PAGE and transferred to polyvinyl difluoride membranes. Membranes were incubated with primary antibodies, followed by incubation with an HRP tagged secondary antibody (Bio-Rad). Antibodies were visualized by enhanced chemiluminescence and quantified using ImageJ software. The antibodies against total HSL protein (#4107) and phosphorylated (p)-HSLSer565 (#4137) were from Cell Signaling (Danvers, MA). The IgG2c antibody (A90-136AP) was purchased from Bethyl Laboratories (Montgomery, TX). Equal loading was verified by Ponceau S staining of the polyvinyl difluoride membrane (see Fig. 6C) (29.Romero-Calvo I. Ocon B. Martinez-Moya P. Suarez M.D. Zarzuelo A. Martinez-Augustin O. de Medina F.S. Reversible Ponceau staining as a loading control alternative to actin in Western blots.Anal. Biochem. 2010; 401: 318-320Crossref PubMed Scopus (554) Google Scholar). Statistical differences were analyzed using a 2-way ANOVA. Student's t-test was used for comparison of two parameters. Significance was accepted at P < 0.05. The role of DGKα on whole-body glucose metabolism and WAT was studied in chow-fed mice. Body weight and fat mass were unaltered in 12-month-old DGKα KO versus WT mice (Fig. 1A, B). Although glucose tolerance was unaltered between DGKα KO and WT mice (Fig. 1C), the insulin concentration measured at 15 min was elevated in DGKα KO mice (Fig. 1D), suggesting mild insulin resistance. Furthermore, the glucose-to-insulin ratio tended to be decreased in the DGKα KO mice (WT 8.7 ± 1.3 versus KO 5.5 ± 0.6; P = 0.07), suggesting mild insulin resistance. Whole-body energy expenditure was measured via indirect calorimetry. Changes in oxygen consumption (VO2) during the light/dark cycle, as well as with fasting, were similar between DGKα KO versus WT mice (Fig. 1E). In addition, food intake was not altered between WT and DGKα KO mice (Fig. 1F). To further study the effects of DGKα ablation on insulin sensitivity, we performed a euglycemic-hyperinsulinemic clamp in conscious mice. Whole body insulin-mediated glucose utilization and hepatic insulin sensitivity were similar between DGKα KO and WT mice (Fig. 2A–C). Insulin infusion was verified by measuring the insulin concentrations under basal- and insulin-stimulated (clamp state) conditions. We found that the insulin concentration during the euglycemic-hyperinsulinemic clamp was similar between DGKα KO and WT mice (Fig. 2D).Fig. 2Whole-body insulin-mediated glucose utilization and hepatic glucose production in chow-fed DGKα KO and WT mice (group a). Whole-body glucose utilization was assessed in conscious DGKα KO (closed bar) and WT (open bar) mice. A: Basal and insulin-stimulated whole-body glucose utilization. B: Suppression of hepatic glucose production (HGP). C: Glucose infusion rate during the euglycemic-hyperinsulinemic clamp. D: Plasma insulin concentration during basal and insulin-stimulated conditions of the euglycemic-hyperinsulinemic clamp. Results are mean ± SEM (n = 7–10 mice).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The role of DGKα was determined on lipolysis in perigonadal WAT ex vivo as measured by isoprenaline-stimulated glycerol release. Isoprenaline increased glycerol release in a concentration-dependent manner; however, glycerol release was unaltered between DGKα KO and WT mice (Fig. 3A), concomitant with unchanged HSL mRNA expression in adipose tissue (Fig. 3B). Expression of inflammatory markers IL1β, IL2, IL6, IFNγ, and TNFα were unaltered between DGKα KO and WT mice (Fig. 3C–G). Total DGK activity in perigonadal WAT was unchanged in WT and DGKα KO mice when determining concentrations of PA (Fig. 3H). DGKα KO and WT mice were fed HFD for 12 weeks and measures of body weight, body composition, glucose tolerance and energy homeostasis were determined. Final body weight was unaltered between Long-Term HFD-fed DGKα KO and WT mice (Fig. 4A, B). Lean muscle mass and fat mass was unaltered between DGKα KO mice and WT mice (Fig. 4C). Although glucose tolerance was unaltered between Long-Term HFD-fed DGKα KO and WT mice (Fig. 4D), the insulin concentration measured at 15 min was reduced in DGKα KO mice (Fig. 4E), suggesting a modest enhancement of insulin sensitivity. The glucose to insulin ratio was however similar between WT and DGKα KO mice (WT 7.5 ± 1.1 versus KO 7.4 ± 0.9). VO2 was calculated in Long-Term HFD-fed DGKα KO and WT mice by indirect calorimetry. VO2 was similar between DGKα KO and WT mice during the light/dark cycle, as well with fasting (Fig. 4F), suggesting energy homeostasis is unaltered between genotypes. Lipolysis in perigonadal WAT was examined ex vivo as measured by isoprenaline-stimulated glycerol release. Isoprenaline increased glycerol release in a concentration dependent manner with similar effects noted between DGKα KO and WT mice (Fig. 5A). In addition, HSL mRNA expression in perigonadal WAT was unaltered between genotypes (Fig. 5B). Markers of inflammatory status were assessed in Long-Term HFD-fed DGKα KO and WT mice. mRNA expression of IL1β, IL2, IL6, IFNγ, and F4/80 was unaltered between genotypes (Fig. 5C–G). Moreover, conversion of DAG to PA was similar between DGKα KO and WT mice (Fig. 5H). We next assessed the effects of Acute HFD on inflammatory markers in perigonadal WAT of DGKα KO and WT mice. This acute protocol affords the opportunity to assess the role of DGKα on inflammatory markers prior to the onset of weight gain that typically accompanies Long-Term HFD. Body weight was unaltered by Acute HFD in DGKα KO and WT mice (Fig. 6A). The inflammatory status of WAT was assessed by determining expression of markers associated with activation of T-cells (e.g., IL2, IFNγ), B-cells (e.g., IgG2c), macrophages (e.g., IL1β, F4/80), and secreted factors (i.e., IL6) from WAT. Acute HFD tended to decrease IgG2c protein abundance in perigonadal WAT of DGKα KO mice (P = 0.12; Fig. 6B). Acute HFD did not alter p-HSLSer565 (P = 0.22; Fig. 6D) whereas HSL protein abundance (Fig. 6E) was increased in perigonadal WAT from DGKα KO mice. HSL mRNA expression (P = 0.14; Fig. 6G) was unaltered in perigonadal WAT of DGKα KO mice. Conversely, Acute HFD decreased IL1β mRNA expression in perigonadal WAT of DGKα KO mice (Fig. 6H). Finally, Acute HFD tended to increase IFNγ mRNA expression in perigonadal WAT of DGKα KO mice (P = 0.08; Fig. 6K), whereas IL2, IL6, and F4/80 were unchanged between genotypes (Fig. 6I, J, L). DGKs form a diverse family of enzymes that play a role in signal transduction and lipid homeostasis. DGKs modulate numerous biological processes by controlling the balance of DAG and PA at discrete cellular locations. The presence of multiple DGKs suggest isoform-specific physiological roles for individual DGK isoforms. For example, DGKδ deficiency is associated with peripheral insulin resistance and obesity (8.Chibalin A.V. Leng Y. Vieira E. Krook A. Bjornholm M. Long Y.C. Kotova O. Zhong Z. Sakane F. Steiler T. et al.Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance.Cell. 2008; 132: 375-386Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), whereas DGKε ablation preserves glucose tolerance and modulates lipid metabolism (30.Mannerås-Holm L. Schonke M. Brozinick J.T. Vetterli L. Bui H.H. Sanders P. Nascimento E.B.M. Bjornholm M. Chibalin A.V. Zierath J.R. Diacylglycerol kinase epsilon deficiency preserves glucose tolerance and modulates lipid metabolism in obese mice.J. Lipid Res. 2017; 58: 907-915Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The opposing metabolic phenotypes observed between these KO models further reinforce that notion that isoform-specific actions of DGK may influence metabolism. Here, we report DGKα, a ubiquitously expressed isoform that modulates adaptive immune cell function (13.Guo R. Wan C.K. Carpenter J.H. Mousallem T. Boustany R.M. Kuan C.T. Burks A.W. Zhong X.P. Synergistic control of T cell development and tumor suppression by diacylglycerol kinase alpha and zeta.Proc. Natl. Acad. Sci. USA. 2008; 105: 11909-11914Crossref PubMed Scopus (73) Google Scholar, 14.Olenchock B.A. Guo R. Carpenter J.H. Jordan M. Topham M.K. Koretzky G.A. Zhong X.P. Disruption of diacylglycerol metabolism impairs the induction of T cell anergy.Nat. Immunol. 2006; 7: 1174-1181Crossref PubMed Scopus (228) Google Scholar), is associated with alterations of insulin levels during a glucose tolerance test in chow-fed and Long-Term HFD fed mice. Moreover, DGKα appears to modulate the early immune response of perigonadal WAT following Acute HFD, possibly through modulation of T-cell action. With obesity, T-cells infil