Coding variants identified in patients with diabetes alter PICK1 BAR domain function in insulin granule biogenesis

Bin/amphiphysin/Rvs (BAR) domains are positively charged crescent-shaped modules that mediate curvature of negatively charged lipid membranes during remodeling processes. The BAR domain proteins PICK1, ICA69, and the arfaptins have recently been demonstrated to coordinate the budding and formation of immature secretory granules (ISGs) at the trans-Golgi network. Here, we identify 4 coding variants in the PICK1 gene from a whole-exome screening of Danish patients with diabetes that each involve a change in positively charged residues in the PICK1 BAR domain. All 4 coding variants failed to rescue insulin content in INS-1E cells upon knock down of endogenous PICK1. Moreover, 2 variants showed dominant-negative properties. In vitro assays addressing BAR domain function suggested that the coding variants compromised BAR domain function but increased the capacity to cause fission of liposomes. Live confocal microscopy and super-resolution microscopy further revealed that PICK1 resides transiently on ISGs before egress via vesicular budding events. Interestingly, this egress of PICK1 was accelerated in the coding variants. We propose that PICK1 assists in or complements the removal of excess membrane and generic membrane trafficking proteins, and possibly also insulin, from ISGs during the maturation process; and that the coding variants may cause premature budding, possibly explaining their dominant-negative function.     

Cyclooxygenase-2 in adipose tissue macrophages limits adipose tissue dysfunction in obese mice

Obesity-associated complications are causing increasing morbidity and mortality worldwide. Expansion of adipose tissue in obesity leads to a state of low-grade chronic inflammation and dysregulated metabolism, resulting in insulin resistance and metabolic syndrome. Adipose tissue macrophages (ATMs) accumulate in obesity and are a source of proinflammatory cytokines that further aggravate adipocyte dysfunction. Macrophages are rich sources of cyclooxygenase (COX), the rate limiting enzyme for prostaglandin E2 (PGE2) production. When mice were fed a high-fat diet (HFD), ATMs increased expression of COX-2. Selective myeloid cell COX-2 deletion resulted in increased monocyte recruitment and proliferation of ATMs, leading to increased proinflammatory ATMs with decreased phagocytic ability. There were increased weight gain and adiposity, decreased peripheral insulin sensitivity and glucose utilization, increased adipose tissue inflammation and fibrosis, and abnormal adipose tissue angiogenesis. HFD pair-feeding led to similar increases in body weight, but mice with selective myeloid cell COX-2 still exhibited decreased peripheral insulin sensitivity and glucose utilization. Selective myeloid deletion of the macrophage PGE2 receptor subtype, EP4, produced a similar phenotype, and a selective EP4 agonist ameliorated the metabolic abnormalities seen with ATM COX-2 deletion. Therefore, these studies demonstrated that an ATM COX-2/PGE2/EP4 axis plays an important role in inhibiting adipose tissue dysfunction.     

Integration of ER protein quality control mechanisms defines β cell function and ER architecture

Three principal ER quality-control mechanisms, namely, the unfolded protein response, ER-associated degradation (ERAD), and ER-phagy are each important for the maintenance of ER homeostasis, yet how they are integrated to regulate ER homeostasis and organellar architecture in vivo is largely unclear. Here we report intricate crosstalk among the 3 pathways, centered around the SEL1L-HRD1 protein complex of ERAD, in the regulation of organellar organization in β cells. SEL1L-HRD1 ERAD deficiency in β cells triggers activation of autophagy, at least in part, via IRE1α (an endogenous ERAD substrate). In the absence of functional SEL1L-HRD1 ERAD, proinsulin is retained in the ER as high molecular weight conformers, which are subsequently cleared via ER-phagy. A combined loss of both SEL1L and autophagy in β cells leads to diabetes in mice shortly after weaning, with premature death by approximately 11 weeks of age, associated with marked ER retention of proinsulin and β cell loss. Using focused ion beam scanning electron microscopy powered by deep-learning automated image segmentation and 3D reconstruction, our data demonstrate a profound organellar restructuring with a massive expansion of ER volume and network in β cells lacking both SEL1L and autophagy. These data reveal at an unprecedented detail the intimate crosstalk among the 3 ER quality-control mechanisms in the dynamic regulation of organellar architecture and β cell function.     

Sec13 promotes oligodendrocyte differentiation and myelin repair through autocrine pleiotrophin signaling

Dysfunction of protein trafficking has been intensively associated with neurological diseases, including neurodegeneration, but whether and how protein transport contributes to oligodendrocyte (OL) maturation and myelin repair in white matter injury remains unclear. ER-to-Golgi trafficking of newly synthesized proteins is mediated by coat protein complex II (COPII). Here, we demonstrate that the COPII component Sec13 was essential for OL differentiation and postnatal myelination. Ablation of Sec13 in the OL lineage prevented OPC differentiation and inhibited myelination and remyelination after demyelinating injury in the central nervous system (CNS), while improving protein trafficking by tauroursodeoxycholic acid (TUDCA) or ectopic expression of COPII components accelerated myelination. COPII components were upregulated in OL lineage cells after demyelinating injury. Loss of Sec13 altered the secretome of OLs and inhibited the secretion of pleiotrophin (PTN), which was found to function as an autocrine factor to promote OL differentiation and myelin repair. These data suggest that Sec13-dependent protein transport is essential for OL differentiation and that Sec13-mediated PTN autocrine signaling is required for proper myelination and remyelination.     

A sublethal ATP11A mutation associated with neurological deterioration causes aberrant phosphatidylcholine flipping in plasma membranes

ATP11A translocates phosphatidylserine (PtdSer), but not phosphatidylcholine (PtdCho), from the outer to the inner leaflet of plasma membranes, thereby maintaining the asymmetric distribution of PtdSer. Here, we detected a de novo heterozygous point mutation of ATP11A in a patient with developmental delays and neurological deterioration. Mice carrying the corresponding mutation died perinatally of neurological disorders. This mutation caused an amino acid substitution (Q84E) in the first transmembrane segment of ATP11A, and mutant ATP11A flipped PtdCho. Molecular dynamics simulations revealed that the mutation allowed PtdCho binding at the substrate entry site. Aberrant PtdCho flipping markedly decreased the concentration of PtdCho in the outer leaflet of plasma membranes, whereas sphingomyelin (SM) concentrations in the outer leaflet increased. This change in the distribution of phospholipids altered cell characteristics, including cell growth, cholesterol homeostasis, and sensitivity to sphingomyelinase. Matrix-assisted laser desorption ionization–imaging mass spectrometry (MALDI-IMS) showed a marked increase of SM levels in the brains of Q84E-knockin mouse embryos. These results provide insights into the physiological importance of the substrate specificity of plasma membrane flippases for the proper distribution of PtdCho and SM.     

Embryonic osteocalcin signaling determines lifelong adrenal steroidogenesis and homeostasis in the mouse

Through their ability to regulate gene expression in most organs, glucocorticoid (GC) hormones influence numerous physiological processes and are therefore key regulators of organismal homeostasis. In bone, GC hormones inhibit expression of the hormone Osteocalcin for poorly understood reasons. Here, we show that in a classical endocrine feedback loop, osteocalcin in return enhanced the biosynthesis of GC as well as mineralocorticoid hormones (adrenal steroidogenesis) in rodents and primates. Conversely, inactivation of osteocalcin signaling in adrenal glands significantly impaired adrenal growth and steroidogenesis in mice. Embryo-made osteocalcin was necessary for normal Sf1 expression in fetal adrenal cells and adrenal cell steroidogenic differentiation and therefore determined the number of steroidogenic cells present in the adrenal glands of adult animals. Embryonic, not postnatal, osteocalcin also governed adrenal growth, adrenal steroidogenesis, blood pressure, electrolyte equilibrium, and the rise in circulating corticosterone levels during the acute stress response in adult offspring. This osteocalcin-dependent regulation of adrenal development and steroidogenesis occurred even in the absence of a functional hypothalamus/pituitary/adrenal axis and explains why osteocalcin administration during pregnancy promoted adrenal growth and steroidogenesis and improved the survival of adrenocorticotropic hormone signaling–deficient animals. This study reveals that a bone-derived embryonic hormone influences lifelong adrenal functions and organismal homeostasis in the mouse.     

Endothelial FGF signaling is protective in hypoxia-induced pulmonary hypertension

Hypoxia-induced pulmonary hypertension (PH) is one of the most common and deadliest forms of PH. Fibroblast growth factor receptors 1 and 2 (FGFR1/2) are elevated in patients with PH and in mice exposed to chronic hypoxia. Endothelial FGFR1/2 signaling is important for the adaptive response to several injury types and we hypothesized that endothelial FGFR1/2 signaling would protect against hypoxia-induced PH. Mice lacking endothelial FGFR1/2, mice with activated endothelial FGFR signaling, and human pulmonary artery endothelial cells (HPAECs) were challenged with hypoxia. We assessed the effect of FGFR activation and inhibition on right ventricular pressure, vascular remodeling, and endothelial-mesenchymal transition (EndMT), a known pathologic change seen in patients with PH. Hypoxia-exposed mice lacking endothelial FGFRs developed increased PH, while mice overexpressing a constitutively active FGFR in endothelial cells did not develop PH. Mechanistically, lack of endothelial FGFRs or inhibition of FGFRs in HPAECs led to increased TGF-β signaling and increased EndMT in response to hypoxia. These phenotypes were reversed in mice with activated endothelial FGFR signaling, suggesting that FGFR signaling inhibits TGF-β pathway–mediated EndMT during chronic hypoxia. Consistent with these observations, lung tissue from patients with PH showed activation of FGFR and TGF-β signaling. Collectively, these data suggest that activation of endothelial FGFR signaling could be therapeutic for hypoxia-induced PH.     

New insights into adipose tissue dysfunction in insulin resistance

In a state of caloric excess, adipose tissue plays an essential role by storing lipids. Its expandability determines the onset of metabolic syndrome (central obesity, dyslipidemia, glucose intolerance and hypertension). When the adipocyte endoplasmic reticulum is no longer capable of processing the excess nutrients, the so-called "endoplasmic reticulum stress" develops. This triggers efflux of free fatty acids from adipocytes into the circulation and causes triglyceride overload in skeletal muscle, liver and pancreas. Adipose tissue hypoxia then develops, due to the failure of vasculature to expand with adipocyte hypertrophy. Increased catabolism in mitochondria leads there to oxidative stress. Both phenomena cause deranged adipokine secretion and low-grade inflammation. Inflammatory cytokines, reactive oxygen species and ectopic lipid deposition are the main mediators of insulin resistance and vascular impairment, which both lead finally to diabetes type 2 and cardiovascular disease. Recently, fibrosis of adipose tissue was also demonstrated in obesity, contributing to the interplay of deleterious factors forcing inflammation. The present paper reviews recent evidence for adipose tissue dysfunction, trying to define causes and consequences. In conclusion, insulin resistance and associated complications originate from excess lipids, which cannot be stored without limit in adipose tissue, thus affecting its integrity and adipokine secretion.     

Lysophospholipid acylation modulates plasma membrane lipid organization and insulin sensitivity in skeletal muscle

Aberrant lipid metabolism promotes the development of skeletal muscle insulin resistance, but the exact identity of lipid-mediated mechanisms relevant to human obesity remains unclear. A comprehensive lipidomic analysis of primary myocytes from individuals who were insulin-sensitive and lean (LN) or insulin-resistant with obesity (OB) revealed several species of lysophospholipids (lyso-PLs) that were differentially abundant. These changes coincided with greater expression of lysophosphatidylcholine acyltransferase 3 (LPCAT3), an enzyme involved in phospholipid transacylation (Lands cycle). Strikingly, mice with skeletal muscle–specific knockout of LPCAT3 (LPCAT3-MKO) exhibited greater muscle lysophosphatidylcholine/phosphatidylcholine, concomitant with improved skeletal muscle insulin sensitivity. Conversely, skeletal muscle–specific overexpression of LPCAT3 (LPCAT3-MKI) promoted glucose intolerance. The absence of LPCAT3 reduced phospholipid packing of cellular membranes and increased plasma membrane lipid clustering, suggesting that LPCAT3 affects insulin receptor phosphorylation by modulating plasma membrane lipid organization. In conclusion, obesity accelerates the skeletal muscle Lands cycle, whose consequence might induce the disruption of plasma membrane organization that suppresses muscle insulin action.     

RSPO2 and RANKL signal through LGR4 to regulate osteoclastic premetastatic niche formation and bone metastasis

Therapeutics targeting osteoclasts are commonly used treatments for bone metastasis; however, whether and how osteoclasts regulate premetastatic niche and bone tropism are largely unknown. In this study, we report that osteoclast precursors (OPs) can function as a premetastatic niche component that facilitates breast cancer (BCa) bone metastasis at early stages. At the molecular level, unbiased GPCR ligand/agonist screening in BCa cells suggested that R-spondin 2 (RSPO2) and RANKL, through interaction with their receptor LGR4, promoted osteoclastic premetastatic niche formation and enhanced BCa bone metastasis. This was achieved by RSPO2/RANKL-LGR4 signal modulating the WNT inhibitor DKK1 through Gα_q and β-catenin signaling. DKK1 directly facilitated OP recruitment through suppression of its receptor LDL receptor–related protein 5 (LRP5) but not LRP6, upregulating Rnasek expression via inhibition of canonical WNT signaling. In clinical samples, RSPO2, LGR4, and DKK1 expression showed a positive correlation with BCa bone metastasis. Furthermore, soluble LGR4 extracellular domain (ECD) protein, acting as a decoy receptor for RSPO2 and RANKL, significantly alleviated bone metastasis and osteolytic lesions in a mouse bone metastasis model. These findings provide unique insights into the functional role of OPs as key components of the premetastatic niche for BCa bone metastasis and identify RSPO2/RANKL-LGR4 signaling as a promising target for inhibiting BCa bone metastasis.     

Natural killer T cells in adipose tissue prevent insulin resistance

Lipid overload and adipocyte dysfunction are key to the development of insulin resistance and can be induced by a high-fat diet. CD1d-restricted invariant natural killer T (iNKT) cells have been proposed as mediators between lipid overload and insulin resistance, but recent studies found decreased iNKT cell numbers and marginal effects of iNKT cell depletion on insulin resistance under high-fat diet conditions. Here, we focused on the role of iNKT cells under normal conditions. We showed that iNKT cell-deficient mice on a low-fat diet, considered a normal diet for mice, displayed a distinctive insulin resistance phenotype without overt adipose tissue inflammation. Insulin resistance was characterized by adipocyte dysfunction, including adipocyte hypertrophy, increased leptin, and decreased adiponectin levels. The lack of liver abnormalities in CD1d-null mice together with the enrichment of CD1d-restricted iNKT cells in both mouse and human adipose tissue indicated a specific role for adipose tissue-resident iNKT cells in the development of insulin resistance. Strikingly, iNKT cell function was directly modulated by adipocytes, which acted as lipid antigen-presenting cells in a CD1d-mediated fashion. Based on these findings, we propose that, especially under low-fat diet conditions, adipose tissue-resident iNKT cells maintain healthy adipose tissue through direct interplay with adipocytes and prevent insulin resistance.     

ICOS signaling limits regulatory T cell accumulation and function in visceral adipose tissue

A unique population of Foxp3⁺ regulatory T cells (T_Rs) resides in visceral adipose tissue (VAT) that regulates adipose inflammation and helps preserve insulin sensitivity. Inducible T cell co-stimulator (ICOS) is highly expressed on effector (e)T_Rs that migrate to nonlymphoid tissues, and contributes to their maintenance and function in models of autoimmunity. In this study, we report an unexpected cell-intrinsic role for ICOS expression and downstream phosphoinositide 3-kinase (PI3K) signaling in limiting the abundance, VAT-associated phenotype, and function of T_Rs specifically in VAT. Icos^−/− mice and mice expressing a knock-in form of ICOS that cannot activate PI3K had increased VAT-T_R abundance and elevated expression of canonical VAT-T_R markers. Loss of ICOS signaling facilitated enhanced accumulation of T_Rs to VAT associated with elevated CCR3 expression, and resulted in reduced adipose inflammation and heightened insulin sensitivity in the context of a high-fat diet. Thus, we have uncovered a new and surprising molecular pathway that regulates VAT-T_R accumulation and function.     

PD-L1 translocation to the plasma membrane enables tumor immune evasion through MIB2 ubiquitination

Programmed death-ligand 1 (PD-L1), a critical immune checkpoint ligand, is a transmembrane protein synthesized in the endoplasmic reticulum of tumor cells and transported to the plasma membrane to interact with programmed death 1 (PD-1) expressed on T cell surface. This interaction delivers coinhibitory signals to T cells, thereby suppressing their function and allowing evasion of antitumor immunity. Most companion or complementary diagnostic devices for assessing PD-L1 expression levels in tumor cells used in the clinic or in clinical trials require membranous staining. However, the mechanism driving PD-L1 translocation to the plasma membrane after de novo synthesis is poorly understood. Herein, we showed that mind bomb homolog 2 (MIB2) is required for PD-L1 transportation from the trans-Golgi network (TGN) to the plasma membrane of cancer cells. MIB2 deficiency led to fewer PD-L1 proteins on the tumor cell surface and promoted antitumor immunity in mice. Mechanistically, MIB2 catalyzed nonproteolytic K63-linked ubiquitination of PD-L1, facilitating PD-L1 trafficking through Ras-associated binding 8–mediated (RAB8-mediated) exocytosis from the TGN to the plasma membrane, where it bound PD-1 extrinsically to prevent tumor cell killing by T cells. Our findings demonstrate that nonproteolytic ubiquitination of PD-L1 by MIB2 is required for its transportation to the plasma membrane and tumor cell immune evasion.     

FOXA1 overexpression suppresses interferon signaling and immune response in cancer

Androgen receptor–positive prostate cancer (PCa) and estrogen receptor–positive luminal breast cancer (BCa) are generally less responsive to immunotherapy compared with certain tumor types such as melanoma. However, the underlying mechanisms are not fully elucidated. In this study, we found that FOXA1 overexpression inversely correlated with interferon (IFN) signature and antigen presentation gene expression in PCa and BCa patients. FOXA1 bound the STAT2 DNA-binding domain and suppressed STAT2 DNA-binding activity, IFN signaling gene expression, and cancer immune response independently of the transactivation activity of FOXA1 and its mutations detected in PCa and BCa. Increased FOXA1 expression promoted cancer immuno- and chemotherapy resistance in mice and PCa and BCa patients. These findings were also validated in bladder cancer expressing high levels of FOXA1. FOXA1 overexpression could be a prognostic factor to predict therapy resistance and a viable target to sensitize luminal PCa, BCa, and bladder cancer to immuno- and chemotherapy.