Spatial mapping of hepatic ER and mitochondria architecture reveals zonated remodeling in fasting and obesity.

The hepatocytes within the liver present an immense capacity to adapt to changes in nutrient availability. Here, by using high resolution volume electron microscopy, we map how hepatic subcellular spatial organization is regulated during nutritional fluctuations and as a function of liver zonation. We identify that fasting leads to remodeling of endoplasmic reticulum (ER) architecture in hepatocytes, characterized by the induction of single rough ER sheet around the mitochondria, which becomes larger and flatter. These alterations are enriched in periportal and mid-lobular hepatocytes but not in pericentral hepatocytes. Gain- and loss-of-function in vivo models demonstrate that the Ribosome receptor binding protein1 (RRBP1) is required to enable fasting-induced ER sheet-mitochondria interactions and to regulate hepatic fatty acid oxidation. Endogenous RRBP1 is enriched around periportal and mid-lobular regions of the liver. In obesity, ER-mitochondria interactions are distinct and fasting fails to induce rough ER sheet-mitochondrion interactions. These findings illustrate the importance of a regulated molecular architecture for hepatocyte metabolic flexibility.

Sympathetic tone dictates the impact of lipolysis on FABP4 secretion.

Levels of circulating fatty acid binding protein 4 (FABP4) protein are strongly associated with obesity and metabolic disease in both mice and humans, and secretion is stimulated by ß-adrenergic stimulation both in vivo and in vitro. Previously, lipolysis-induced FABP4 secretion was found to be significantly reduced upon pharmacological inhibition of adipose triglyceride lipase (ATGL) and was absent from adipose tissue explants from mice specifically lacking ATGL in their adipocytes (ATGLAdpKO). Here, we find that upon activation of ß-adrenergic receptors in vivo, ATGLAdpKO mice unexpectedly exhibited significantly higher levels of circulating FABP4 as compared with ATGLfl/fl controls, despite no corresponding induction of lipolysis. We generated an additional model with adipocyte-specific deletion of both FABP4 and ATGL (ATGL/FABP4AdpKO) to evaluate the cellular source of this circulating FABP4. In these animals, there was no evidence of lipolysis-induced FABP4 secretion, indicating that the source of elevated FABP4 levels in ATGLAdpKO mice was indeed from the adipocytes. ATGLAdpKO mice exhibited significantly elevated corticosterone levels, which positively correlated with plasma FABP4 levels. Pharmacological inhibition of sympathetic signaling during lipolysis using hexamethonium or housing mice at thermoneutrality to chronically reduce sympathetic tone significantly reduced FABP4 secretion in ATGLAdpKO mice compared with controls. Therefore, activity of a key enzymatic step of lipolysis mediated by ATGL, per se, is not required for in vivo stimulation of FABP4 secretion from adipocytes, which can be induced through sympathetic signaling.

Ubiquinone deficiency drives reverse electron transport to disrupt hepatic metabolic homeostasis in obesity.

Mitochondrial reactive oxygen species (mROS) are central to physiology. While excess mROS production has been associated with several disease states, its precise sources, regulation, and mechanism of generation in vivo remain unknown, limiting translational efforts. Here we show that in obesity, hepatic ubiquinone (Q) synthesis is impaired, which raises the QH 2 /Q ratio, driving excessive mROS production via reverse electron transport (RET) from site I Q in complex I. Using multiple complementary genetic and pharmacological models in vivo we demonstrated that RET is critical for metabolic health. In patients with steatosis, the hepatic Q biosynthetic program is also suppressed, and the QH 2 /Q ratio positively correlates with disease severity. Our data identify a highly selective mechanism for pathological mROS production in obesity, which can be targeted to protect metabolic homeostasis.

Regulation of liver subcellular architecture controls metabolic homeostasis.

Cells display complex intracellular organization by compartmentalization of metabolic processes into organelles, yet the resolution of these structures in the native tissue context and their functional consequences are not well understood. Here we resolved the three-dimensional structural organization of organelles in large (more than 2.8 × 105 µm3) volumes of intact liver tissue (15 partial or full hepatocytes per condition) at high resolution (8 nm isotropic pixel size) using enhanced focused ion beam scanning electron microscopy1,2 imaging followed by deep-learning-based automated image segmentation and 3D reconstruction. We also performed a comparative analysis of subcellular structures in liver tissue of lean and obese mice and found substantial alterations, particularly in hepatic endoplasmic reticulum (ER), which undergoes massive structural reorganization characterized by marked disorganization of stacks of ER sheets3 and predominance of ER tubules. Finally, we demonstrated the functional importance of these structural changes by monitoring the effects of experimental recovery of the subcellular organization on cellular and systemic metabolism. We conclude that the hepatic subcellular organization of the ER architecture are highly dynamic, integrated with the metabolic state and critical for adaptive homeostasis and tissue health.

Aberrant Ca2+ signaling by IP3Rs in adipocytes links inflammation to metabolic dysregulation in obesity.

Chronic metabolic inflammation is a key feature of obesity, insulin resistance, and diabetes. Here, we showed that altered regulation of the Ca2+ channel inositol trisphosphate receptor (IP3R) was an adipocyte-intrinsic event involved in the emergence and propagation of inflammatory signaling and the resulting insulin resistance. Inflammation induced by cytokine exposure in vitro or by obesity in vivo led to increases in the abundance and activity of IP3Rs and in the phosphorylation of the Ca2+-dependent kinase CaMKII in adipocytes in a manner dependent on the kinase JNK. In mice, adipocyte-specific loss of IP3R1/2 protected against adipose tissue inflammation and insulin resistance, despite the mice exhibiting substantial diet-induced weight gain. Thus, this work suggests that increased IP3R activity is a key link between obesity, inflammation, and insulin resistance. These data also suggest that approaches to target IP3R-mediated Ca2+ homeostasis in adipocytes may offer new therapeutic opportunities against metabolic diseases, especially because GWAS studies also implicate this locus in human obesity.

Defective STIM-mediated store operated Ca2+ entry in hepatocytes leads to metabolic dysfunction in obesity.

Defective Ca2+ handling is a key mechanism underlying hepatic endoplasmic reticulum (ER) dysfunction in obesity. ER Ca2+ level is in part monitored by the store-operated Ca2+ entry (SOCE) system, an adaptive mechanism that senses ER luminal Ca2+ concentrations through the STIM proteins and facilitates import of the ion from the extracellular space. Here, we show that hepatocytes from obese mice displayed significantly diminished SOCE as a result of impaired STIM1 translocation, which was associated with aberrant STIM1 O-GlycNAcylation. Primary hepatocytes deficient in STIM1 exhibited elevated cellular stress as well as impaired insulin action, increased glucose production and lipid droplet accumulation. Additionally, mice with acute liver deletion of STIM1 displayed systemic glucose intolerance. Conversely, over-expression of STIM1 in obese mice led to increased SOCE, which was sufficient to improve systemic glucose tolerance. These findings demonstrate that SOCE is an important mechanism for healthy hepatic Ca2+ balance and systemic metabolic control.