AMPK regulates macrophage polarization in adipose tissue inflammation and NASH

COMMENTARY ON: Hematopoietic AMPK beta1 reduces mouse adipose tissue macrophage inflammation and insulin resistance in obesity. Galic S, Fullerton MD, Schertzer JD, Sikkema S, Marcinko K, Walkley CR, Izon D, Honeyman J, Chen ZP, van Denderen BJ, Kemp BE, Steinberg GR. J Clin Invest 2011;121(12):4903–15. Copyright 2011. Reprinted with permission of American Society for Clinical Investigation. http://www.ncbi.nlm.nih.gov/pubmed/22080866 Abstract: Individuals who are obese are frequently insulin resistant, putting them at increased risk of developing type 2 diabetes and its associated adverse health conditions. The accumulation in adipose tissue of macrophages in an inflammatory state is a hallmark of obesity-induced insulin resistance. Here, we reveal a role for AMPK β1 in protecting macrophages from inflammation under high lipid exposure. Genetic deletion of the AMPK β1 subunit in mice (referred to herein as β1(−/−) mice) reduced macrophage AMPK activity, acetyl-CoA carboxylase phosphorylation, and mitochondrial content, resulting in reduced rates of fatty acid oxidation. β1(−/−) macrophages displayed increased levels of diacylglycerol and markers of inflammation, effects that were reproduced in WT macrophages by inhibiting fatty acid oxidation and, conversely, prevented by pharmacological activation of AMPK β1-containing complexes. The effect of AMPK β1 loss in macrophages was tested in vivo by transplantation of bone marrow from WT or β1(−/−) mice into WT recipients. When challenged with a high-fat diet, mice that received β1(−/−) bone marrow displayed enhanced adipose tissue macrophage inflammation and liver insulin resistance compared with animals that received WT bone marrow. Thus, activation of AMPK β1 and increasing fatty acid oxidation in macrophages may represent a new therapeutic approach for the treatment of insulin resistance. Obesity has long been linked to type 2 diabetes (T2D), but the involved metabolic dysregulation is incompletely understood. Galic et al. [[1]Galic S. Fullerton M.D. Schertzer J.D. Sikkema S. Marcinko K. Walkley C.R. et al.Hematopoietic AMPK beta1 reduces mouse adipose tissue macrophage inflammation and insulin resistance in obesity.J Clin Invest. 2011; 121: 4903-4915Crossref PubMed Scopus (249) Google Scholar] recently reported that adenosine monophosphate kinase (AMPK) β1 plays a hitherto unknown role in modulating obesity-induced insulin resistance. AMPK is a central regulator of energy balance. Decreased cellular energy status, as reflected by accumulation of AMP, activates AMPK which turns on expression of catabolic enzymes that permit ATP production and shuts down energy consuming biosynthetic pathways. Thus, AMPK is a central regulator of fatty acid, cholesterol, and glucose homeostasis through phosphorylation of metabolism-regulating enzymes including acetyl-CoA carboxylase (ACC), glycogen synthase (GS), glucose transporter 4 (GLUT4), HMG-CoA reductase, hormone-sensitive lipase (HSL), and mammalian target of rapamycin (mTOR). AMPK is a heterotrimer of a catalytic α-chain and regulatory β- and γ-chains. There exist several trimers, with α1β1γ1 and α2β1γ1 being prominent in the liver, and α2β2γ1 and α2β2γ3 being found in skeletal and cardiac muscle [[2]Viollet B. Athea Y. Mounier R. Guigas B. Zarrinpashneh E. Horman S. et al.AMPK: lessons from transgenic and knockout animals.Front Biosci. 2009; 14: 19-44Crossref PubMed Scopus (228) Google Scholar]. Moreover, trimers containing the β1 subunit are prevalent in pancreas and brown fat, whereas the β2 subunit predominates in brain or gastric tissue [[3]Dasgupta B.J.J. Sasaki Y. Liu X. Jung S.R. Higashida K. Lindquist D. et al.The AMPK beta2 subunit is required for energy homeostasis during metabolic stress.Mol Cell Biol. 2012; PubMed Google Scholar]. Although AMPK is a ubiquitous regulator of mitochondrial activity, little has been known about its role in metabolic regulation of inflammatory cells, and studies in non-alcoholic steatohepatitis (NASH) have been linked to hepatocyte AMPK [[4]Browning J.D. Horton J.D. Molecular mediators of hepatic steatosis and liver injury.J Clin Invest. 2004; 114: 147-152Crossref PubMed Scopus (1713) Google Scholar]. Therefore, investigations into the role of macrophage AMPK are timely, since these cells have been identified as central players in adipose tissue inflammation, T2D and NASH [[5]Yang Z. Kahn B.B. Shi H. Xue B.Z. Macrophage alpha1 AMP-activated protein kinase (alpha1AMPK) antagonizes fatty acid-induced inflammation through SIRT1.J Biol Chem. 2010; 285: 19051-19059Crossref PubMed Scopus (320) Google Scholar]. The report by Galic et al. partly fills this gap in by showing that macrophage AMPK regulates fat-induced inflammation in both peripheral adipose tissue and fatty liver [[1]Galic S. Fullerton M.D. Schertzer J.D. Sikkema S. Marcinko K. Walkley C.R. et al.Hematopoietic AMPK beta1 reduces mouse adipose tissue macrophage inflammation and insulin resistance in obesity.J Clin Invest. 2011; 121: 4903-4915Crossref PubMed Scopus (249) Google Scholar]. The authors first confirmed reduced activity of α1 subunit containing AMPK trimers and increased expression of inflammatory cytokines in peritoneal macrophages of leptin deficient ob/ob mice, which spontaneously develop insulin resistance. In these mice, downregulated AMPK is likely due to a reduction of adiponectin receptors which are upstream of AMPK [[6]Nawrocki A.R. Rajala M.W. Tomas E. Pajvani U.B. Saha A.K. Trumbauer M.E. et al.Mice lacking adiponectin show decreased hepatic insulin sensitivity and reduced responsiveness to peroxisome proliferator-activated receptor gamma agonists.J Biol Chem. 2006; 281: 2654-2660Crossref PubMed Scopus (514) Google Scholar]. In addition, they showed that loss of AMPKβ1 causes the concomitant reduction of AMPKα1 in bone marrow-derived mononuclear cells (BMDM). In the presence of palmitate, these mutant BMDM displayed reduced fatty acid oxidation and upregulated expression of the inflammatory cytokines TNFα, IL-6, and IL-1β. Moreover, the increased ratio of iNOS/Arg1 demonstrated M1-polarization of macrophages in these BMDM. The relevance of these in vitro data was then proven in chimeric mice fed a high-fat diet (HFD), whose bone marrow cells were deficient in AMPK β1, demonstrating enhanced systemic inflammation, insulin resistance and M1 macrophage infiltration in liver and adipose tissues. Since there was no difference in T-cell infiltration between bone marrow chimeras and wild type mice, these data suggest that the observed liver and adipose tissue inflammation and insulin resistance were caused by macrophages turned towards M1 polarization by their deficiency in AMPK trimers containing the β1 chain. Moreover, treatment with A769662, a specific activator of AMPK β1, shut off fatty acid synthesis via phosphorylation and inactivation of ACC, enhanced fatty acid oxidation and decreased activation of inflammation inducing Jun-N-terminal kinase (JNK) in wild type but not in AMPK β1 deficient BMDM. No agonistic effect was found when A769662 treatment was combined with the mitochondrial pathway inhibitors etomoxir or rotenone, confirming that AMPK β1 regulates fatty acid oxidation via the mitochondrial pathway (Fig. 1). Notably, constitutive genetic deletion of components of the AMPK-heterotrimer can either promote [[3]Dasgupta B.J.J. Sasaki Y. Liu X. Jung S.R. Higashida K. Lindquist D. et al.The AMPK beta2 subunit is required for energy homeostasis during metabolic stress.Mol Cell Biol. 2012; PubMed Google Scholar] or prevent [[7]Dzamko N. van Denderen B.J. Hevener A.L. Jorgensen S.B. Honeyman J. Galic S. et al.AMPK beta1 deletion reduces appetite, preventing obesity and hepatic insulin resistance.J Biol Chem. 2010; 285: 115-122Crossref PubMed Scopus (133) Google Scholar] development of insulin resistance and the metabolic syndrome under metabolic stress. With global deletion of AMPKβ1, mice lose appetite and body mass due to a decrease of AMPK activity in the hypothalamus. Thus, even with the associated reduction of hepatic fatty acid oxidation in vivo and in hepatocytes in vitro, these mutant mice fed a HFD did not develop hepatic steatosis and insulin resistance [[7]Dzamko N. van Denderen B.J. Hevener A.L. Jorgensen S.B. Honeyman J. Galic S. et al.AMPK beta1 deletion reduces appetite, preventing obesity and hepatic insulin resistance.J Biol Chem. 2010; 285: 115-122Crossref PubMed Scopus (133) Google Scholar]. However, globally β2-deficient mice fed a HFD gained more weight and peripheral as well as central fat mass, and developed higher insulin resistance compared to their wild type littermates [[3]Dasgupta B.J.J. Sasaki Y. Liu X. Jung S.R. Higashida K. Lindquist D. et al.The AMPK beta2 subunit is required for energy homeostasis during metabolic stress.Mol Cell Biol. 2012; PubMed Google Scholar]. The data with the global AMPKβ2 knockout (mainly affecting muscle and cardiac tissue) share similarities with those of the chimeric mice with AMPKβ1 deficient BMDM, since both displayed higher insulin resistance in adipose tissue and liver, as measured by the hyperinsulinemic-euglycemic clamp assay. Therefore, AMPK variants in different tissues and especially different cells fulfill divergent tasks in energy homeostasis and fatty acid metabolism. Specifically, in fat-laden macrophages, AMPKβ1 appears to mitigate fatty acid-induced inflammation and subsequent insulin resistance. These results are in line with a recent study demonstrating that AMPKα1 deficient mice fed a HFD also gained more weight and showed systemic insulin resistance [[8]Zhang W. Zhang X. Wang H. Guo X. Li H. Wang Y. et al.AMP-activated protein kinase alpha1 protects against diet-induced insulin resistance and obesity.Diabetes. 2012; Google Scholar]. Moreover, irradiated WT mice transplanted with AMPKα1 deficient BMDM developed insulin resistance but no obesity. However, in their BM transplantation models both studies did not address the possible contribution of (immune) cells other than monocytes and macrophages. The novelty of the study by Galic et al. [[1]Galic S. Fullerton M.D. Schertzer J.D. Sikkema S. Marcinko K. Walkley C.R. et al.Hematopoietic AMPK beta1 reduces mouse adipose tissue macrophage inflammation and insulin resistance in obesity.J Clin Invest. 2011; 121: 4903-4915Crossref PubMed Scopus (249) Google Scholar] is the connection of macrophage AMPK activity (as represented by the β1 chain containing complex) with obesity, insulin resistance and NAFLD/NASH. Another central metabolic regulator, PPARγ, has already been shown to promote peripheral adipogenesis and prominently repress inflammatory genes in macrophages, since its deletion in macrophages alone drives inflammation and insulin resistance in the liver and peripheral adipose tissue in mice on a normal diet, generating an even more severe inflammatory phenotype than the AMPK β1 deficient BM chimera [[9]Hevener A.L. Olefsky J.M. Reichart D. Nguyen M.T. Bandyopadyhay G. Leung H.Y. et al.Macrophage PPAR gamma is required for normal skeletal muscle and hepatic insulin sensitivity and full antidiabetic effects of thiazolidinediones.J Clin Invest. 2007; 117: 1658-1669Crossref PubMed Scopus (396) Google Scholar]. A possible explanation is that PPARγ locates further upstream of AMPK and regulates its activity through adiponectin [[6]Nawrocki A.R. Rajala M.W. Tomas E. Pajvani U.B. Saha A.K. Trumbauer M.E. et al.Mice lacking adiponectin show decreased hepatic insulin sensitivity and reduced responsiveness to peroxisome proliferator-activated receptor gamma agonists.J Biol Chem. 2006; 281: 2654-2660Crossref PubMed Scopus (514) Google Scholar]. An important issue addressed in this paper is the M1–M2 polarization of macrophages regulated through AMPK. Correspondingly, upon stimulation of macrophages with lipopolysaccharide (LPS), deletion of AMPKα1 induced expression of the macrophage TNFα and IL-6, cytokines that are characteristic of M1 polarization and central to fatty tissue inflammation and NASH [[10]Sag D. Carling D. Stout R.D. Suttles J. Adenosine 5′-monophosphate-activated protein kinase promotes macrophage polarization to an anti-inflammatory functional phenotype.J Immunol. 2008; 181: 8633-8641Crossref PubMed Scopus (529) Google Scholar]. Moreover, a specific activator of AMPK inhibits LPS-induced TNFα expression in murine macrophages [[11]Jhun B.S. Jin Q. Oh Y.T. Kim S.S. Kong Y. Cho Y.H. et al.5-Aminoimidazole-4-carboxamide riboside suppresses lipopolysaccharide-induced TNF-alpha production through inhibition of phosphatidylinositol 3-kinase/Akt activation in RAW 264.7 murine macrophages.Biochem Biophys Res Commun. 2004; 318: 372-380Crossref PubMed Scopus (99) Google Scholar]. On the other hand, macrophage AMPK expression and activity are suppressed by LPS, which is an important contributor to NAFLD/NASH. One recent report mentioned that even the lipid content of food modulates macrophage polarization [[12]Lumeng C.N. Bodzin J.L. Saltiel A.R. Obesity induces a phenotypic switch in adipose tissue macrophage polarization.J Clin Invest. 2007; 117: 175-184Crossref PubMed Scopus (3241) Google Scholar]. Thus, more M2 macrophages are found in adipose tissues of lean mice fed a normal vs. high-fat diet. These M2 macrophages in adipose tissues are protective due to secretion of anti-inflammatory cytokines such as IL-10 [[12]Lumeng C.N. Bodzin J.L. Saltiel A.R. Obesity induces a phenotypic switch in adipose tissue macrophage polarization.J Clin Invest. 2007; 117: 175-184Crossref PubMed Scopus (3241) Google Scholar]. However, there is a yet ill explored connection between M2 polarization with a fibrogenic cytokine milieu linked to Th2 T-cell activation [[13]Wynn T.A. Fibrotic disease and the T(H)1/T(H)2 paradigm.Nat Rev Immunol. 2004; 4: 583-594Crossref PubMed Scopus (1187) Google Scholar]. Therefore, induction of M2 macrophage polarization to combat fatty tissue inflammation and obesity-induced insulin resistance needs to take into account potential adverse effects on fibrosis progression. This leaves us with intriguing options, but also novel challenges when developing macrophage-based therapies of T2D and NAFLD/NASH. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.