The architecture of Cidec-mediated interfaces between lipid droplets.

Lipid droplets (LDs) are intracellular organelles responsible for storing surplus energy as neutral lipids. Their size and number vary enormously. In white adipocytes, LDs can reach 100 μm in diameter, occupying >90% of the cell. Cidec, which is strictly required for the formation of large LDs, is concentrated at interfaces between adjacent LDs and facilitates directional flux of neutral lipids from the smaller to the larger LD. The mechanism of lipid transfer is unclear, in part because the architecture of interfaces between LDs remains elusive. Here we visualize interfaces between LDs by electron cryo-tomography and analyze the kinetics of lipid transfer by quantitative live fluorescence microscopy. We show that transfer occurs through closely apposed monolayers, is slowed down by increasing the distance between the monolayers, and follows exponential kinetics. Our data corroborate the notion that Cidec facilitates pressure-driven transfer of neutral lipids through two "leaky" monolayers between LDs

Compartmentalized Synthesis of Triacylglycerol at the Inner Nuclear Membrane Regulates Nuclear Organization.

Cells dynamically adjust organelle organization in response to growth and environmental cues. This requires regulation of synthesis of phospholipids, the building blocks of organelle membranes, or remodeling of their fatty-acyl (FA) composition. FAs are also the main components of triacyglycerols (TGs), which enable energy storage in lipid droplets. How cells coordinate FA metabolism with organelle biogenesis during cell growth remains unclear. Here, we show that Lro1, an acyltransferase that generates TGs from phospholipid-derived FAs in yeast, relocates from the endoplasmic reticulum to a subdomain of the inner nuclear membrane. Lro1 nuclear targeting is regulated by cell cycle and nutrient starvation signals and is inhibited when the nucleus expands. Lro1 is active at this nuclear subdomain, and its compartmentalization is critical for nuclear integrity. These data suggest that Lro1 nuclear targeting provides a site of TG synthesis, which is coupled with nuclear membrane remodeling

Compartmentalized Synthesis of Triacylglycerol at the Inner Nuclear Membrane Regulates Nuclear Organization

Cells dynamically adjust organelle organization in response to growth and environmental cues. This requires regulation of synthesis of phospholipids, the building blocks of organelle membranes, or remodeling of their fatty-acyl (FA) composition. FAs are also the main components of triacyglycerols (TGs), which enable energy storage in lipid droplets. How cells coordinate FA metabolism with organelle biogenesis during cell growth remains unclear. Here, we show that Lro1, an acyltransferase that generates TGs from phospholipid-derived FAs in yeast, relocates from the endoplasmic reticulum to a subdomain of the inner nuclear membrane. Lro1 nuclear targeting is regulated by cell cycle and nutrient starvation signals and is inhibited when the nucleus expands. Lro1 is active at this nuclear subdomain, and its compartmentalization is critical for nuclear integrity. These data suggest that Lro1 nuclear targeting provides a site of TG synthesis, which is coupled with nuclear membrane remodeling

PCYT1A Regulates Phosphatidylcholine Homeostasis from the Inner Nuclear Membrane in Response to Membrane Stored Curvature Elastic Stress.

Cell and organelle membranes consist of a complex mixture of phospholipids (PLs) that determine their size, shape, and function. Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic membranes, yet how cells sense and regulate its levels in vivo remains unclear. Here we show that PCYT1A, the rate-limiting enzyme of PC synthesis, is intranuclear and re-locates to the nuclear membrane in response to the need for membrane PL synthesis in yeast, fly, and mammalian cells. By aligning imaging with lipidomic analysis and data-driven modeling, we demonstrate that yeast PCYT1A membrane association correlates with membrane stored curvature elastic stress estimates. Furthermore, this process occurs inside the nucleus, although nuclear localization signal mutants can compensate for the loss of endogenous PCYT1A in yeast and in fly photoreceptors. These data suggest an ancient mechanism by which nucleoplasmic PCYT1A senses surface PL packing defects on the inner nuclear membrane to control PC homeostasis

The architecture of Cidec-mediated interfaces between lipid droplets.

Lipid droplets (LDs) are intracellular organelles responsible for storing surplus energy as neutral lipids. Their size and number vary enormously. In white adipocytes, LDs can reach 100 μm in diameter, occupying >90% of the cell. Cidec, which is strictly required for the formation of large LDs, is concentrated at interfaces between adjacent LDs and facilitates directional flux of neutral lipids from the smaller to the larger LD. The mechanism of lipid transfer is unclear, in part because the architecture of interfaces between LDs remains elusive. Here we visualize interfaces between LDs by electron cryo-tomography and analyze the kinetics of lipid transfer by quantitative live fluorescence microscopy. We show that transfer occurs through closely apposed monolayers, is slowed down by increasing the distance between the monolayers, and follows exponential kinetics. Our data corroborate the notion that Cidec facilitates pressure-driven transfer of neutral lipids through two "leaky" monolayers between LDs

PPARγΔ5, a Naturally Occurring Dominant-Negative Splice Isoform, Impairs PPARγ Function and Adipocyte Differentiation

Summary: Peroxisome-proliferator-activated receptor γ (PPARγ) regulates glucose and lipid homeostasis, insulin signaling, and adipocyte differentiation. Here, we report the skipping of exon 5 as a legitimate splicing event generating PPARγΔ5, a previously unidentified naturally occurring truncated isoform of PPARγ, which lacks the entire ligand-binding domain. PPARγΔ5 is endogenously expressed in human adipose tissue and, during adipocyte differentiation, lacks ligand-dependent transactivation ability and acts as a dominant-negative isoform reducing PPARγ activity. Ligand-mediated PPARγ activation induces exon 5 skipping in a negative feedback loop, suggesting alternative splicing as a mechanism regulating PPARγ activity. PPARγΔ5 overexpression modifies the PPARγ-induced transcriptional network, significantly impairing the differentiation ability of adipocyte precursor cells. Additionally, PPARγΔ5 expression in subcutaneous adipose tissue positively correlates with BMI in two independent cohorts of overweight or obese and type 2 diabetic patients. From a functional perspective, PPARγΔ5 mimics PPARG dominant-negative mutated receptors, possibly contributing to adipose tissue dysfunction. These findings open an unexplored scenario in PPARG regulation and PPARγ-related diseases. : Aprile et al. report the identification of PPARγΔ5, a naturally occurring splicing isoform of PPARγ lacking the ligand-binding domain. PPARγΔ5 reduces the adipogenic potential of precursor cells through dominant-negative inhibition of PPARγ transactivation. PPARγΔ5 is highly expressed in adipose tissue and positively correlates with BMI in obese and diabetic patients. Keywords: PPARγ, alternative splicing, adipocyte differentiation, adipose tissue, obesity, insulin resistance, dominant-negative isofor

Phenotypic characterization of Adig null mice suggests roles for adipogenin in the regulation of fat mass accrual and leptin secretion.

Adipogenin (Adig) is an adipocyte-enriched transmembrane protein. Its expression is induced during adipogenesis in rodent cells, and a recent genome-wide association study associated body mass index (BMI)-adjusted leptin levels with the ADIG locus. In order to begin to understand the biological function of Adig, we studied adipogenesis in Adig-deficient cultured adipocytes and phenotyped Adig null (Adig-/-) mice. Data from Adig-deficient cells suggest that Adig is required for adipogenesis. In vivo, Adig-/- mice are leaner than wild-type mice when fed a high-fat diet and when crossed with Ob/Ob hyperphagic mice. In addition to the impact on fat mass accrual, Adig deficiency also reduces fat-mass-adjusted plasma leptin levels and impairs leptin secretion from adipose explants, suggesting an additional impact on the regulation of leptin secretion

A mouse model of human mitofusin-2-related lipodystrophy exhibits adipose-specific mitochondrial stress and reduced leptin secretion.

Funder: Swedish Research CouncilFunder: Ramón Areces FoundationMitochondrial dysfunction has been reported in obesity and insulin resistance, but primary genetic mitochondrial dysfunction is generally not associated with these, arguing against a straightforward causal relationship. A rare exception, recently identified in humans, is a syndrome of lower body adipose loss, leptin-deficient severe upper body adipose overgrowth, and insulin resistance caused by the p.Arg707Trp mutation in MFN2, encoding mitofusin 2. How the resulting selective form of mitochondrial dysfunction leads to tissue- and adipose depot-specific growth abnormalities and systemic biochemical perturbation is unknown. To address this, Mfn2R707W/R707W knock-in mice were generated and phenotyped on chow and high fat diets. Electron microscopy revealed adipose-specific mitochondrial morphological abnormalities. Oxidative phosphorylation measured in isolated mitochondria was unperturbed, but the cellular integrated stress response was activated in adipose tissue. Fat mass and distribution, body weight, and systemic glucose and lipid metabolism were unchanged, however serum leptin and adiponectin concentrations, and their secretion from adipose explants were reduced. Pharmacological induction of the integrated stress response in wild-type adipocytes also reduced secretion of leptin and adiponectin, suggesting an explanation for the in vivo findings. These data suggest that the p.Arg707Trp MFN2 mutation selectively perturbs mitochondrial morphology and activates the integrated stress response in adipose tissue. In mice, this does not disrupt most adipocyte functions or systemic metabolism, whereas in humans it is associated with pathological adipose remodelling and metabolic disease. In both species, disproportionate effects on leptin secretion may relate to cell autonomous induction of the integrated stress response