Obesity is characterized by an accumulation of macrophages in adipose, some of which form distinct crown-like structures (CLS) around fat cells. While multiple discrete adipose tissue macrophage (ATM) subsets are thought to exist, their respective effects on adipose tissue, and the transcriptional mechanisms that underlie the functional differences between ATM subsets, are not well understood. We report that obese fat tissue of mice and humans contain multiple distinct populations of ATMs with unique tissue distributions, transcriptomes, chromatin landscapes, and functions. Mouse Ly6c ATMs reside outside of CLS and are adipogenic, while CD9 ATMs reside within CLS, are lipid-laden, and are proinflammatory. Adoptive transfer of Ly6c ATMs into lean mice activates gene programs typical of normal adipocyte physiology. By contrast, adoptive transfer of CD9 ATMs drives gene expression that is characteristic of obesity. Importantly, human adipose tissue contains similar ATM populations, including lipid-laden CD9 ATMs that increase with body mass. These results provide a higher resolution of the cellular and functional heterogeneity within ATMs and provide a framework within which to develop new immune-directed therapies for the treatment of obesity and related sequela.

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is known to regulate lipid metabolism in many tissues, including macrophages. Here we report that peritoneal macrophage respiration is enhanced by rosiglitazone, an activating PPARγ ligand, in a PPARγ-dependent manner. Moreover, PPARγ is required for macrophage respiration even in the absence of exogenous ligand. Unexpectedly, the absence of PPARγ dramatically affects the oxidation of glutamine. Both glutamine and PPARγ have been implicated in alternative activation (AA) of macrophages, and PPARγ was required for interleukin 4 (IL4)-dependent gene expression and stimulation of macrophage respiration. Indeed, unstimulated macrophages lacking PPARγ contained elevated levels of the inflammation-associated metabolite itaconate and express a proinflammatory transcriptome that, remarkably, phenocopied that of macrophages depleted of glutamine. Thus, PPARγ functions as a checkpoint, guarding against inflammation, and is permissive for AA by facilitating glutamine metabolism. However, PPARγ expression is itself markedly increased by IL4. This suggests that PPARγ functions at the center of a feed-forward loop that is central to AA of macrophages.

The histone deacetylase HDAC3 is a critical mediator of hepatic lipid metabolism, and liver-specific deletion of HDAC3 leads to fatty liver. To elucidate the underlying mechanism, here we report a method of cross-linking followed by mass spectrometry to define a high-confidence HDAC3 interactome in vivo that includes the canonical NCoR–HDAC3 complex as well as Prospero-related homeobox 1 protein (PROX1). HDAC3 and PROX1 co-localize extensively on the mouse liver genome, and are co-recruited by hepatocyte nuclear factor 4α (HNF4α). The HDAC3–PROX1 module controls the expression of a gene program regulating lipid homeostasis, and hepatic-specific ablation of either component increases triglyceride content in liver. These findings underscore the importance of specific combinations of transcription factors and coregulators in the fine tuning of organismal metabolism.

G protein-coupled receptor (GPCR) kinases (GRKs) were discovered by virtue of their ability to phosphorylate activated GPCRs. They constitute a branch of the AGC kinase superfamily, but their mechanism of activation is largely unknown. To initiate a study of GRK2 activation, we sought to identify sites on GRK2 remote from the active site that are involved in interactions with their substrate receptors. Using the atomic structure of GRK2 in complex with Gβγ as a guide, we predicted that residues on the surface of the kinase domain that face the cell membrane would interact with the intracellular loops and carboxyl (C) terminal tail of the GPCR. Our study focused on two regions: the kinase large lobe and an extension of the kinase domain known as the C-tail. Conserved residues in the GRK2 large lobe whose side-chains are solvent exposed and facing the membrane were targeted for mutagenesis. Residues in the C-tail of GRK2, although not ordered in the crystal structure, were also targeted because this region has been implicated in receptor binding and in the regulation of AGC kinase activity. Four substitutions out of twenty, all within or adjacent to the C-tail, resulted in significant deficiencies in the ability of the enzyme to phosphorylate activated GPCRs, rhodopsin and the β 2 -adrenergic receptor (β 2 AR). The mutant exhibiting the most dramatic impairment, V477D, also showed significant defects in phosphorylation of non-receptor substrates. Interestingly, Michaelis-Menten kinetics suggested that V477D had 12-fold lower k cat , but no changes in K M , suggesting a defect in acquisition or stabilization of the closed state of the kinase domain. V477D was also resistant to activation by agonist-treated β 2 AR. Therefore, Val477 and other residues in the C-tail are expected to play a role in the activation of GRK2 by GPCRs.G protein-coupled receptors (GPCRs), distinguished by their seven transmembrane helices and their ability to couple to heterotrimeric G proteins (Gαβγ), are activated by hormones, neurotransmitters and sensory signals, and regulate many diverse processes. Receptor stimulation catalyzes the binding of GTP to the Gα subunit, allowing Gα and Gβγ to directly † This work was funded by the National Science Foundation grants MCB0315888 and MCB0744739 (to RSM), National Institutes of Health grants HL086865 and HL071818 and American Heart Association Scientist Development Grant 0235273N (to JJGT), and a summer undergraduate research fellowship from the Northeast Affiliate of the American Heart Association (to EDB). *To whom correspondence should be addressed. Biology Department, Siena College, Morrell Science Center, 515 Loudon Road, Loudonville, NY 12211, Tel: (518) 783-2462; Fax: (518) ; email: sternemarr@siena.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 May 26. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript modulate effectors such as adenylyl cyclase, phospholipase Cβ, cGMP phosphodiesterase, ion channels and ...

ObjectiveHistone deacetylases are epigenetic regulators known to control gene transcription in various tissues. A member of this family, histone deacetylase 3 (HDAC3), has been shown to regulate metabolic genes. Cell culture studies with HDAC-specific inhibitors and siRNA suggest that HDAC3 plays a role in pancreatic β-cell function, but a recent genetic study in mice has been contradictory. Here we address the functional role of HDAC3 in β-cells of adult mice.MethodsAn HDAC3 β-cell specific knockout was generated in adult MIP-CreERT transgenic mice using the Cre-loxP system. Induction of HDAC3 deletion was initiated at 8 weeks of age with administration of tamoxifen in corn oil (2 mg/day for 5 days). Mice were assayed for glucose tolerance, glucose-stimulated insulin secretion, and islet function 2 weeks after induction of the knockout. Transcriptional functions of HDAC3 were assessed by ChIP-seq as well as RNA-seq comparing control and β-cell knockout islets.ResultsHDAC3 β-cell specific knockout (HDAC3βKO) did not increase total pancreatic insulin content or β-cell mass. However, HDAC3βKO mice demonstrated markedly improved glucose tolerance. This improved glucose metabolism coincided with increased basal and glucose-stimulated insulin secretion in vivo as well as in isolated islets. Cistromic and transcriptomic analyses of pancreatic islets revealed that HDAC3 regulates multiple genes that contribute to glucose-stimulated insulin secretion.ConclusionsHDAC3 plays an important role in regulating insulin secretion in vivo, and therapeutic intervention may improve glucose homeostasis.