Coordinated control of adiposity and growth by anti-anabolic kinase ERK7.

Glucosylceramide synthase (GlcT-1) catalyzes the synthesis of glucosylceramide (GlcCer), the core structure of major glycosphingolipids (GSLs). Obesity is a metabolic disorder caused by an imbalance between energy uptake and expenditure, resulting in excess stored body fat. Recent studies have shown that GSL levels are increased in obese rodents and that pharmacologically reducing GSL levels by inhibiting GlcCer synthesis improves adipocyte function. However, the molecular mechanism underlying these processes is still not clearly understood. Using Drosophila as a model animal, we report that GlcT-1 expression in the fat body, which is equivalent to mammalian adipose tissue, regulates energy metabolism. Overexpression of GlcT-1 increases stored nutrition (triacylglycerol and carbohydrate) levels. Conversely, reduced expression of GlcT-1 in the fat body causes a reduction of fat storage. This regulation occurs, at least in part, through the activation of p38-ATF2 signaling. Furthermore, we found that GlcCer is the sole GSL of the fat body, indicating that regulation of GlcCer synthesis by GlcT-1 in the fat body is responsible for regulating energy homeostasis. Both GlcT-1 and p38-ATF2 signaling are evolutionarily conserved, leading us to propose an evolutionary perspective in which GlcT-1 appears to be one of the key factors that control fat metabolism.

Regulation of lipid homeostasis by the TBC protein dTBC1D22 via modulation of the small GTPase Rab40 to facilitate lipophagy.

This work analyzed the process of lipid storage in fat body of larval Manduca sexta, focusing on the role of lipid transfer particle (LTP). Incubation of fat bodies with [(3)H]diacylglycerol-labeled lipophorin resulted in a significant accumulation of diacylglycerol (DAG) and triacylglycerol (TAG) in the tissue. Transfer of DAG to fat body and its storage as TAG was significantly inhibited (60%) by preincubating the tissue with anti-LTP antibody. Lipid transfer was restored to control values by adding LTP to fat body. Incubation of fat body with dual-labeled DAG lipophorin or its treatment with ammonium chloride showed that neither a membrane-bound lipoprotein lipase nor lipophorin endocytosis is a relevant pathway to transfer or to storage lipids into fat body, respectively. Treatment of fat body with suramin caused a 50% inhibition in [(3)H]DAG transfer from lipophorin. Treatment of [(3)H]DAG-labeled fat body with lipase significantly reduced the amount of [(3)H]DAG associated with the tissue, suggesting that the lipid is still on the external surface of the membrane. Whether this lipid represents irreversibly adsorbed lipophorin or a DAG lipase-sensitive pool is unknown. Nevertheless, these results indicate that the main pathway for DAG transfer from lipophorin to fat body is via LTP and receptor-mediated processes.

A dynamic cycle of O-linked GlcNAc (O-GlcNAc) addition and removal is catalyzed by O-GlcNAc transferase and O-GlcNAcase, respectively, in a process that serves as the final step in a nutrient-driven "hexosamine-signaling pathway." Evidence points to a role for O-GlcNAc cycling in diabetes and insulin resistance. We have used Drosophila melanogaster to determine whether O-GlcNAc metabolism plays a role in modulating Drosophila insulin-like peptide (dilp) production and insulin signaling. We employed transgenesis to either overexpress or knock down Drosophila Ogt(sxc) and Oga in insulin-producing cells (IPCs) or fat bodies using the GAL4-UAS system. Knockdown of Ogt decreased Dilp2, Dilp3, and Dilp5 production, with reduced body size and decreased phosphorylation of Akt in vivo. In contrast, knockdown of Oga increased Dilp2, Dilp3, and Dilp5 production, increased body size, and enhanced phosphorylation of Akt in vivo. However, knockdown of either Ogt(sxc) or Oga in the IPCs increased the hemolymph carbohydrate concentration. Furthermore, phosphorylation of Akt stimulated by extraneous insulin in an ex vivo cultured fat body of third instar larvae was diminished in strains subjected to IPC knockdown of Ogt or Oga. Knockdown of O-GlcNAc cycling enzymes in the fat body dramatically reduced neutral lipid stores. These results demonstrate that altered O-GlcNAc cycling in Drosophila IPCs modulates insulin production and influences the insulin responsiveness of peripheral tissues. The observed phenotypes in O-GlcNAc cycling mimic pancreatic β-cell dysfunction and glucose toxicity related to sustained hyperglycemia in mammals.

Type 2 diabetes is characterized by excessive lipid storage in skeletal muscle. Excessive intramyocellular lipid (IMCL) storage exceeds intracellular needs and induces lipotoxic events, ultimately contributing to the development of insulin resistance. Lipid droplet (LD)–coating proteins may control proper lipid storage in skeletal muscle. Perilipin 2 (PLIN2/adipose differentiation–related protein [ADRP]) is one of the most abundantly expressed LD-coating proteins in skeletal muscle. Here we examined the role of PLIN2 in myocellular lipid handling and insulin sensitivity by investigating the effects of in vitro PLIN2 knockdown and in vitro and in vivo overexpression. PLIN2 knockdown decreased LD formation and triacylglycerol (TAG) storage, marginally increased fatty-acid (FA) oxidation, and increased incorporation of palmitate into diacylglycerols and phospholipids. PLIN2 overexpression in vitro increased intramyocellular TAG storage paralleled with improved insulin sensitivity. In vivo muscle-specific PLIN2 overexpression resulted in increased LD accumulation and blunted the high-fat diet–induced increase in protein content of the subunits of the oxidative phosphorylation (OXPHOS) chain. Diacylglycerol levels were unchanged, whereas ceramide levels were increased. Despite the increased IMCL accumulation, PLIN2 overexpression improved skeletal muscle insulin sensitivity. We conclude that PLIN2 is essential for lipid storage in skeletal muscle by enhancing the partitioning of excess FAs toward TAG storage in LDs, thereby blunting lipotoxicity-associated insulin resistance.

Lipid storage in the form of triacylglycerol (TAG)is essential for insect life, as it enables flight, development, and reproduction. The activity of the lipase brummer (bmm) has been shown to be essential to insects' homeostasis. The objective of this study was to evaluate how bmm expression occurs in Aedes aegypti larvae and adults, and to observe TAG levels during fasting in adult females. The bmm sequence was identified in A. aegypti and exhibited a patatin-like phospholipase domain reinforced by the presence of a catalytic dyad with serine and aspartate residues, revealing a high degree of similarity with other organisms. Bmm expression was differentiated in the larvae and adult fat body (FB)following TAG reserve dynamics. Bmm was expressed three times in larval stages L3, L4, and pupae compared with L1 and L2, which could indicate its role in the maturation of these insects. In the postemergence (PE)and post-blood meal (PBM)FB of adult insects, bmm expression varied over several days. PE adults showed a pronounced bmm increase from the third day onward compared with those not subjected to fasting. This was accompanied by a decrease in TAG from the third day onward, suggesting the participation of bmm. Six hours after blood feeding, TAG levels increased in mosquitos reared in the absence of sucrose, suggesting lipid accumulation to guarantee reproduction. Bmm responded positively to fasting, followed by TAG mobilization in adult FB. During the previtellogenic period, bmm levels responded to low TAG levels, unlike the PBM period.

Lipid droplets (LDs) are organelles of cellular lipid storage with fundamental roles in energy metabolism and cell membrane homeostasis. There has been an explosion of research into the biology of LDs, in part due to their relevance in diseases of lipid storage, such as atherosclerosis, obesity, type 2 diabetes, and hepatic steatosis. Consequently, there is an increasing need for a resource that combines datasets from systematic analyses of LD biology. Here, we integrate high-confidence, systematically generated human, mouse, and fly data from studies on LDs in the framework of an online platform named the "Lipid Droplet Knowledge Portal" (https://lipiddroplet.org/). This scalable and interactive portal includes comprehensive datasets, across a variety of cell types, for LD biology, including transcriptional profiles of induced lipid storage, organellar proteomics, genome-wide screen phenotypes, and ties to human genetics. This resource is a powerful platform that can be utilized to identify determinants of lipid storage.

The fat-specific protein 27 (Fsp27), a protein localized to lipid droplets (LDs), plays an important role in controlling lipid storage and mitochondrial activity in adipocytes. Fsp27-null mice display increased energy expenditure and are resistant to high fat diet-induced obesity and diabetes. However, little is known about how the Fsp27 protein is regulated. Here, we show that Fsp27 stability is controlled by the ubiquitin-dependent proteasomal degradation pathway in adipocytes. The ubiquitination of Fsp27 is regulated by three lysine residues located in the C-terminal region. Substitution of these lysine residues with alanines greatly increased Fsp27 stability and enhanced lipid storage in adipocytes. Furthermore, Fsp27 was stabilized and rapidly accumulated following treatment with beta-agonists that induce lipolysis and fatty acid re-esterification in adipocytes. More importantly, Fsp27 stabilization was dependent on triacylglycerol synthesis and LD formation, because knockdown of diacylglycerol acyltransferase in adipocytes significantly reduced Fsp27 accumulation in adipocytes. Finally, we observed that increased Fsp27 during beta-agonist treatment preferentially associated with LDs. Taken together, our data revealed that Fsp27 can be stabilized by free fatty acid availability, triacylglycerol synthesis, and LD formation. The stabilization of Fsp27 when free fatty acids are abundant further enhances lipid storage, providing positive feedback to regulate lipid storage in adipocytes.

Silencing of ATG6 and ATG8 promotes increased levels of triacylglycerol (TAG) in the fat body during prolonged starvation periods in the Chagas disease vector Rhodnius prolixus

Lipid storage must be efficiently mobilized to sustain the energy demands during processes of exercise or starvation. In insects, adipokinetic hormone (AKH) and brummer lipase are well-known regulators of lipid mobilization. We recently demonstrated that brummer-dependent lipolysis regulates starvation resistance in the brown planthopper, Nilaparvata lugens, one of the most destructive rice pests. The present work investigated the roles of the AKH signaling system in lipid mobilization during the starvation process in N. lugens. NlAKHR is a typical G protein-coupled receptor (GPCR) and possesses high structure and sequence similarity to other insect AKHRs. Spatial and developmental expression profiles suggested that NlAKH is released from the corpora cardiaca to activate NlAKHR mainly expressed in the fat body. Starvation significantly induced the expression of NlAKH and NlAKHR, indicating a potential role of the AKH signaling system in starvation resistance. To reveal the functions of the AKH signaling system, a double-stranded RNA (dsRNA)-mediated knockdown of NlAKHR and NlAKH peptide injection was performed. The results show NlAKHR silencing decreased the levels of 1,2-diacylglycerol (DAG) in the hemolymph and increased triacylglycerol (TAG) levels in the fat body, whereas NlAKH injection led to a critical accumulation of DAG in the hemolymph and a severe reduction of TAG content in the fat body. Knockdown of NlAKHR resulted in prolonged lifespan and high levels of whole-body TAG, indicating an inability to mobilize TAG reserves during starvation. Conversely, the NlAKH injection reduced the survival and accelerated TAG mobilization during starvation, which further confirms the role of NlAKH in lipolysis. Moreover, NlAKHR silencing caused obesity in N. lugens, whereas NlAKH injection depleted organismal TAG reserves in vivo and produced a slim phenotype. These results indicate that lipid mobilization is regulated by the AKH signaling system, which is essential for adjusting body lipid homeostasis and ensuring energy supplement during starvation in N. lugens.

The Wnt pathway, which controls crucial steps of the development and differentiation programs, has been proposed to influence lipid storage and homeostasis. In this paper, using an unbiased strategy based on high-content genome-wide RNAi screens that monitored lipid distribution and amounts, we find that Wnt3a regulates cellular cholesterol. We show that Wnt3a stimulates the production of lipid droplets and that this stimulation strictly depends on endocytosed, LDL-derived cholesterol and on functional early and late endosomes. We also show that Wnt signaling itself controls cholesterol endocytosis and flux along the endosomal pathway, which in turn modulates cellular lipid homeostasis. These results underscore the importance of endosome functions for LD formation and reveal a previously unknown regulatory mechanism of the cellular programs controlling lipid storage and endosome transport under the control of Wnt signaling.

Lipid droplet (LD)-associated hydrolase (LDAH) is a newly identified LD protein abundantly expressed in tissues that predominantly store triacylglycerol (TAG). However, how LDAH regulates TAG metabolism remains unknown. We found that upon oleic acid loading LDAH translocalizes from the ER to newly formed LDs, and induces LD coalescence in a tubulin-dependent manner. LDAH overexpression and downregulation in HEK293 cells increase and decrease, respectively, TAG levels. Pulse and chase experiments show that LDAH enhances TAG biogenesis, but also decreases TAG turnover and fatty acid release from cells. Mutations in predicted catalytic and acyltransferase motifs do not influence TAG levels, suggesting that the effect is independent of LDAH’s enzymatic activity. However, a LDAH alternative-splicing variant missing 90 amino acids at C-terminus does not promote LD fusion or TAG accumulation, while it still localizes to LDs. Interestingly, LDAH enhances polyubiquitination and proteasomal degradation of adipose triglyceride lipase (ATGL), a rate limiting enzyme of TAG hydrolysis. Co-expression of ATGL reverses the changes in LD phenotype induced by LDAH, and both proteins counterbalance their effects on TAG stores. Together, these studies support that under conditions of TAG storage in LDs LDAH plays a primarily lipogenic role, inducing LD growth and enhancing degradation of ATGL.

Obesity has increased dramatically worldwide and leads to numerous disorders including cardiovascular diseases, type 2 diabetes, dyslipidemia and cancers. These pathological states are closely associated with hyperinsulinemia or insulin resistance. Obesity itself is characterized by excessive triacylglycerol (TAG) storage as lipid droplets (LDs) in adipose tissue due to an increase in nutrient intake and insufficient energy expenditure. Excessive TAG storage in adipocytes causes adipose tissue inflammation and deregulation of adipokines, both of which induce systemic insulin resistance. At the same time, a hallmark of obesity is TAG accumulation in the liver and skeletal muscle, which is also important in inducing insulin resistance in these tissues. These findings underline the importance of lipid accumulation in insulin-sensitive organs in the development of insulin resistance and type 2 diabetes. Lipid droplets are comprised of a central core of neutral lipids containing TAG and cholesterol ester and surrounded by a phospholipid monolayer. This monolayer of LDs is decorated with a variety of proteins, which contribute to the formation of the droplets, the synthesis and hydrolysis of lipids, and the movement of theses lipids to specific intracellular and secretory pathways. The ability to store TAG in LDs is evolutionarily conserved in yeast, plants, invertebrates and vertebrates. In mammals, excess energy is primarily stored as TAG in LDs in adipose tissue. Especially white adipocytes are specifically differentiated cells for storing TAG as the large unilocular LDs that occupy most of the cell volume. The LD size can be in the 100 lm range. Such TAG storage in LDs serves a vital role as the body’s major stored energy supply. In the case of energy demand, such as starvation and exercise, TAG reserves are hydrolyzed by lipolysis to supply free fatty acid (FFA) to a variety of tissues. LDs are also recognized in non-adipose cells; however, non-adipocyte LDs are usually much smaller than those of adipocytes. Such TAG storage in liver, skeletal muscle and heart is thought to serve as a local energy supply. Recently, a fat-specific protein of 27 kDa (FSP27) was found to be the determining factor for LD size and critical for large unilocular LD formation in white adipocytes [1–3]. FSP27 was first identified in 1992 as a fat-specific protein whose expression was regulated by C/EBP, although its function in adipocytes remained elusive then [4]. Subsequently, FSP27 was reported to be a member of the CIDE family of proteins, comprised of CIDE-A, CIDEB and FSP27, which had homology with the amino-terminal domain of DFF-45 (45-kDa subunit of the DNA fragmentation factor) [5]. Later, CIDE-A was shown to be expressed in brown adipose tissue in mice and to inhibit energy expenditure [6]. CIDE-B is expressed mainly in the liver and kidney and is important for the maturation of VLDL as apolipoprotein B binding protein in the liver [7]. As for FSP27, functional analysis of FSP27 has robustly progressed since knockout (KO) mice were generated in 2008 [1, 3], although FSP27 was already found to be a LD Y. Tamori (&) Division of Metabolism and Endocrinology, Department of Internal Medicine, Chibune Hospital, 2-2-45 Tsukuda, Nishiyodogawa-ku, Osaka 555-0001, Japan e-mail: tamori@med.kobe-u.ac.jp

Cells store lipids in droplets. Studies addressing how mammals control lipid-based energy homeostasis have implicated proteins of the PAT domain family, such as perilipin that surrounds the lipid droplets. Perilipin knock-out mice are lean and resistant to obesity. Factors that mediate lipid storage in fungi are still unknown. Here we describe a gene (Mpl1) in the economically important insect fungal pathogen Metarhizium anisopliae that has structural similarities to mammalian perilipins. Consistent with a role in lipid storage, Mpl1 is predominantly expressed when M. anisopliae is engaged in accumulating lipids and ectopically expressed green fluorescent protein-tagged MPL1 (Metarhizium perilipin-like protein) localized to lipid droplets. Mutant M. anisopliae lacking MPL1 have thinner hyphae, fewer lipid droplets, particularly in appressoria (specialized infection structures at the end of germ tubes), and a decrease in total lipids. Mpl1 therefore acts in a perilipin-like manner suggesting an evolutionary conserved function in lipid metabolism. However, reflecting general differences between animal and fungal lineages, these proteins have also been selected to cope with different tasks. Thus, turgor generation by DeltaMpl1 appressoria is dramatically reduced indicating that lipid droplets are required for solute accumulation. This was linked with the reduced ability to breach insect cuticle so that Mpl1 is a pathogenicity determinant. Blast searches of fungal genomes revealed that perilipin homologs are found only in pezizomycotinal ascomycetes and occur as single copy genes. Expression of Mpl1 in yeast cells, a fungus that lacks a perilipin-like gene, blocked their ability to mobilize lipids during starvation conditions.

Lipid droplets (LDs) are lipid storage depots as well as metabolically dynamic organelles. LD-associated hydrolase (LDAH) is a newly identified protein that is highly expressed in liver and adipose tissues, where triacylglycerol (TAG) is the main form of lipid stored in LDs. However, to date the role of LDAH in TAG metabolism has not been reported. In this study we show that upon oleic acid loading LDAH translocalizes from the endoplasmic reticulum (ER) to the LD surface, and increases the size of LDs by promoting fusion in a tubulin-dependent fashion. LDAH overexpression in HEK 293 cells increased the net TAG content, while LDAH knockdown decreased TAG levels. Mutations in two predicted acyltransferase motifs aiming to assess if changes in TAG content is due to LDAH’s direct enzymatic activity did not affect TAG levels. However, an LDAH splicing variant missing 90 amino acid at C-terminus also localized to LDs, but failed to increase both LD fusion and TAG levels. Consistent with the increase in LD size and area, LDAH increased the level of Perilipin3, a major LD associated protein in HEK293 cells, but decreased the level of adipose triglyceride lipase (ATGL) that regulates rate-limiting step of TAG hydrolysis. ATGL ubiquitination, which ultimately leads to protein degradation, was higher in LDAH-overexpressing cells, and treatment with MG132 to block the activity of the 26S proteasome complex attenuated the reduction in ATGL. Coexpression of ATGL with LDAH reduced LDAH-mediated increase in LD size, total area of LDs, and TAG content. Alternatively, LDAH delayed TAG hydrolysis by ATGL upon treatment with β-adrenergic agonists. In conclusion, the data support a role of LDAH in facilitating LD growth and TAG accumulation, and uncover a mutually antagonistic interaction between LDAH and the catabolic enzyme ATGL.