Annexin A6 Is Critical to Maintain Glucose Homeostasis and Survival During Liver Regeneration in Mice.

BACKGROUND AND AIMS: Liver regeneration requires the organized and sequential activation of events that lead to restoration of hepatic mass. During this process, other vital liver functions need to be preserved, such as maintenance of blood glucose homeostasis, balancing the degradation of hepatic glycogen stores, and gluconeogenesis (GNG). Under metabolic stress, alanine is the main hepatic gluconeogenic substrate, and its availability is the rate-limiting step in this pathway. Na+ -coupled neutral amino acid transporters (SNATs) 2 and 4 are believed to facilitate hepatic alanine uptake. In previous studies, we demonstrated that a member of the Ca2+ -dependent phospholipid binding annexins, Annexin A6 (AnxA6), regulates membrane trafficking along endo- and exocytic pathways. Yet, although AnxA6 is abundantly expressed in the liver, its function in hepatic physiology remains unknown. In this study, we investigated the potential contribution of AnxA6 in liver regeneration. APPROACH AND RESULTS: Utilizing AnxA6 knockout mice (AnxA6-/- ), we challenged liver function after partial hepatectomy (PHx), inducing acute proliferative and metabolic stress. Biochemical and immunofluorescent approaches were used to dissect AnxA6-/- mice liver proliferation and energetic metabolism. Most strikingly, AnxA6-/- mice exhibited low survival after PHx. This was associated with an irreversible and progressive drop of blood glucose levels. Whereas exogenous glucose administration or restoration of hepatic AnxA6 expression rescued AnxA6-/- mice survival after PHx, the sustained hypoglycemia in partially hepatectomized AnxA6-/- mice was the consequence of an impaired alanine-dependent GNG in AnxA6-/- hepatocytes. Mechanistically, cytoplasmic SNAT4 failed to recycle to the sinusoidal plasma membrane of AnxA6-/- hepatocytes 48 hours after PHx, impairing alanine uptake and, consequently, glucose production. CONCLUSIONS: We conclude that the lack of AnxA6 compromises alanine-dependent GNG and liver regeneration in mice.

Annexin A6 modulates TBC1D15/Rab7/StARD3 axis to control endosomal cholesterol export in NPC1 cells.

Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.

Annexins-Coordinators of Cholesterol Homeostasis in Endocytic Pathways.

The spatiotemporal regulation of calcium (Ca2+) storage in late endosomes (LE) and lysosomes (Lys) is increasingly recognized to influence a variety of membrane trafficking events, including endocytosis, exocytosis, and autophagy. Alterations in Ca2+ homeostasis within the LE/Lys compartment are implicated in human diseases, ranging from lysosomal storage diseases (LSDs) to neurodegeneration and cancer, and they correlate with changes in the membrane binding behaviour of Ca2+-binding proteins. This also includes Annexins (AnxA), which is a family of Ca2+-binding proteins participating in membrane traffic and tethering, microdomain organization, cytoskeleton interactions, Ca2+ signalling, and LE/Lys positioning. Although our knowledge regarding the way Annexins contribute to LE/Lys functions is still incomplete, recruitment of Annexins to LE/Lys is greatly influenced by the availability of Annexin bindings sites, including acidic phospholipids, such as phosphatidylserine (PS) and phosphatidic acid (PA), cholesterol, and phosphatidylinositol (4,5)-bisphosphate (PIP2). Moreover, the cytosolic portion of LE/Lys membrane proteins may also, directly or indirectly, determine the recruitment of Annexins to LE. Strikingly, within LE/Lys, AnxA1, A2, A6, and A8 differentially contribute to cholesterol transport along the endocytic route, in particular, cholesterol transfer between LE and other compartments, positioning Annexins at the centre of major pathways mediating cellular cholesterol homeostasis. Underlying mechanisms include the formation of membrane contact sites (MCS) and intraluminal vesicles (ILV), as well as the modulation of LE-cholesterol transporter activity. In this review, we will summarize the current understanding how Annexins contribute to influence LE/Lys membrane transport and associated functions.

Selective Degradation Permits a Feedback Loop Controlling Annexin A6 and Cholesterol Levels in Endolysosomes of NPC1 Mutant Cells.

We recently identified elevated annexin A6 (AnxA6) protein levels in Niemann-Pick-type C1 (NPC1) mutant cells. In these cells, AnxA6 depletion rescued the cholesterol accumulation associated with NPC1 deficiency. Here, we demonstrate that elevated AnxA6 protein levels in NPC1 mutants or upon pharmacological NPC1 inhibition, using U18666A, were not due to upregulated AnxA6 mRNA expression, but caused by defects in AnxA6 protein degradation. Two KFERQ-motifs are believed to target AnxA6 to lysosomes for chaperone-mediated autophagy (CMA), and we hypothesized that the cholesterol accumulation in endolysosomes (LE/Lys) triggered by the NPC1 inhibition could interfere with the CMA pathway. Therefore, AnxA6 protein amounts and cholesterol levels in the LE/Lys (LE-Chol) compartment were analyzed in NPC1 mutant cells ectopically expressing lysosome-associated membrane protein 2A (Lamp2A), which is well known to induce the CMA pathway. Strikingly, AnxA6 protein amounts were strongly decreased and coincided with significantly reduced LE-Chol levels in NPC1 mutant cells upon Lamp2A overexpression. Therefore, these findings suggest Lamp2A-mediated restoration of CMA in NPC1 mutant cells to lower LE-Chol levels with concomitant lysosomal AnxA6 degradation. Collectively, we propose CMA to permit a feedback loop between AnxA6 and cholesterol levels in LE/Lys, encompassing a novel mechanism for regulating cholesterol homeostasis in NPC1 disease.

Annexin A6 improves anti-migratory and anti-invasive properties of tyrosine kinase inhibitors in EGFR overexpressing human squamous epithelial cells.

Annexin A6 (AnxA6), a member of the calcium (Ca2+ ) and membrane binding annexins, is known to stabilize and establish the formation of multifactorial signaling complexes. At the plasma membrane, AnxA6 is a scaffold for protein kinase Cα (PKCα) and GTPase-activating protein p120GAP to promote downregulation of epidermal growth factor receptor (EGFR) and Ras/mitogen-activated protein kinase (MAPK) signaling. In human squamous A431 epithelial carcinoma cells, which overexpress EGFR, but lack endogenous AnxA6, restoration of AnxA6 expression (A431-A6) promotes PKCα-mediated threonine 654 (T654)-EGFR phosphorylation, which inhibits EGFR tyrosine kinase activity. This is associated with reduced A431-A6 cell growth, but also decreased migration and invasion in wound healing, matrigel, and organotypic matrices. Here, we show that A431-A6 cells display reduced EGFR activity in vivo, with xenograft analysis identifying increased pT654-EGFR levels, but reduced tyrosine EGFR phosphorylation compared to controls. In contrast, PKCα depletion in A431-A6 tumors is associated with strongly reduced pT654 EGFR levels, yet increased EGFR tyrosine phosphorylation and MAPK activity. Moreover, tyrosine kinase inhibitors (TKIs; gefitinib, erlotinib) more effectively inhibit cell viability, clonogenic growth, and wound healing of A431-A6 cells compared to controls. Likewise, the ability of AnxA6 to inhibit A431 motility and invasiveness strongly improves TKI efficacy in matrigel invasion assays. This correlates with a greatly reduced invasion of the surrounding matrix of TKI-treated A431-A6 when cultured in 3D spheroids. Altogether, these findings implicate that elevated AnxA6 scaffold levels contribute to improve TKI-mediated inhibition of growth and migration, but also invasive properties in EGFR overexpressing human squamous epithelial carcinoma.

Altered hepatic glucose homeostasis in AnxA6-KO mice fed a high-fat diet.

Annexin A6 (AnxA6) controls cholesterol and membrane transport in endo- and exocytosis, and modulates triglyceride accumulation and storage. In addition, AnxA6 acts as a scaffolding protein for negative regulators of growth factor receptors and their effector pathways in many different cell types. Here we investigated the role of AnxA6 in the regulation of whole body lipid metabolism and insulin-regulated glucose homeostasis. Therefore, wildtype (WT) and AnxA6-knockout (KO) mice were fed a high-fat diet (HFD) for 17 weeks. During the course of HFD feeding, AnxA6-KO mice gained less weight compared to controls, which correlated with reduced adiposity. Systemic triglyceride and cholesterol levels of HFD-fed control and AnxA6-KO mice were comparable, with slightly elevated high density lipoprotein (HDL) and reduced triglyceride-rich lipoprotein (TRL) levels in AnxA6-KO mice. AnxA6-KO mice displayed a trend towards improved insulin sensitivity in oral glucose and insulin tolerance tests (OGTT, ITT), which correlated with increased insulin-inducible phosphorylation of protein kinase B (Akt) and ribosomal protein S6 kinase (S6) in liver extracts. However, HFD-fed AnxA6-KO mice failed to downregulate hepatic gluconeogenesis, despite similar insulin levels and insulin signaling activity, as well as expression profiles of insulin-sensitive transcription factors to controls. In addition, increased glycogen storage in livers of HFD- and chow-fed AnxA6-KO animals was observed. Together with an inability to reduce glucose production upon insulin exposure in AnxA6-depleted HuH7 hepatocytes, this implicates AnxA6 contributing to the fine-tuning of hepatic glucose metabolism with potential consequences for the systemic control of glucose in health and disease.