Growth Retardation, Impaired Triacylglycerol Catabolism, Hepatic Steatosis, and Lethal Skin Barrier Defect in Mice Lacking Comparative Gene Identification-58 (CGI-58)

Comparative gene identification-58 (CGI-58), also designated as α/β-hydrolase domain containing-5 (ABHD-5), is a lipid droplet-associated protein that activates adipose triglyceride lipase (ATGL) and acylates lysophosphatidic acid. Activation of ATGL initiates the hydrolytic catabolism of cellular triacylglycerol (TG) stores to glycerol and nonesterified fatty acids. Mutations in both ATGL and CGI-58 cause “neutral lipid storage disease” characterized by massive accumulation of TG in various tissues. The analysis of CGI-58-deficient (Cgi-58^−/−) mice, presented in this study, reveals a dual function of CGI-58 in lipid metabolism. First, systemic TG accumulation and severe hepatic steatosis in newborn Cgi-58^−/− mice establish a limiting role for CGI-58 in ATGL-mediated TG hydrolysis and supply of nonesterified fatty acids as energy substrate. Second, a severe skin permeability barrier defect uncovers an essential ATGL-independent role of CGI-58 in skin lipid metabolism. The neonatal lethal skin barrier defect is linked to an impaired hydrolysis of epidermal TG. As a consequence, sequestration of fatty acids in TG prevents the synthesis of acylceramides, which are essential lipid precursors for the formation of a functional skin permeability barrier. This mechanism may also underlie the pathogenesis of ichthyosis in neutral lipid storage disease patients lacking functional CGI-58.     

Deletion of CGI-58 or adipose triglyceride lipase differently affects macrophage function and atherosclerosis

Cellular TG stores are efficiently hydrolyzed by adipose TG lipase (ATGL). Its coactivator comparative gene identification-58 (CGI-58) strongly increases ATGL-mediated TG catabolism in cell culture experiments. To investigate the consequences of CGI-58 deficiency in murine macrophages, we generated mice with a targeted deletion of CGI-58 in myeloid cells (macCGI-58^−/− mice). CGI-58^−/− macrophages accumulate intracellular TG-rich lipid droplets and have decreased phagocytic capacity, comparable to ATGL^−/− macrophages. In contrast to ATGL^−/− macrophages, however, CGI-58^−/− macrophages have intact mitochondria and show no indications of mitochondrial apoptosis and endoplasmic reticulum stress, suggesting that TG accumulation per se lacks a significant role in processes leading to mitochondrial dysfunction. Another notable difference is the fact that CGI-58^−/− macrophages adopt an M1-like phenotype in vitro. Finally, we investigated atherosclerosis susceptibility in macCGI-58/ApoE-double KO (DKO) animals. In response to high-fat/high-cholesterol diet feeding, DKO animals showed comparable plaque formation as observed in ApoE^−/− mice. In agreement, antisense oligonucleotide-mediated knockdown of CGI-58 in LDL receptor^−/− mice did not alter atherosclerosis burden in the aortic root. These results suggest that macrophage function and atherosclerosis susceptibility differ fundamentally in these two animal models with disturbed TG catabolism, showing a more severe phenotype by ATGL deficiency.     

Global deletion of MGL in mice delays lipid absorption and alters energy homeostasis and diet-induced obesity

Monoacylglycerol lipase (MGL) is a ubiquitously expressed enzyme that catalyzes the hydrolysis of monoacylglycerols (MGs) to yield FFAs and glycerol. MGL contributes to energy homeostasis through the mobilization of fat stores and also via the degradation of the endocannabinoid 2-arachidonoyl glycerol. To further examine the role of MG metabolism in energy homeostasis, MGL^−/− mice were fed either a 10% (kilocalories) low-fat diet (LFD) or a 45% (kilocalories) high-fat diet (HFD) for 12 weeks. Profound increases of MG species in the MGL^−/− mice compared with WT control mice were found. Weight gain over the 12 weeks was blunted in both diet groups. MGL^−/− mice were leaner than WT mice at both baseline and after 12 weeks of LFD feeding. Circulating lipids were decreased in HFD-fed MGL^−/− mice, as were the levels of several plasma peptides involved in glucose homeostasis and energy balance. Interestingly, MGL^−/− mice had markedly reduced intestinal TG secretion following an oral fat challenge, suggesting delayed lipid absorption. Overall, the results indicate that global MGL deletion leads to systemic changes that produce a leaner phenotype and an improved serum metabolic profile.     

Adipose Triglyceride Lipase Is Implicated in Fuel- and Non-fuel-stimulated Insulin Secretion

Reduced lipolysis in hormone-sensitive lipase-deficient mice is associated with impaired glucose-stimulated insulin secretion (GSIS), suggesting that endogenous β-cell lipid stores provide signaling molecules for insulin release. Measurements of lipolysis and triglyceride (TG) lipase activity in islets from HSL^−/− mice indicated the presence of other TG lipase(s) in the β-cell. Using real time-quantitative PCR, adipose triglyceride lipase (ATGL) was found to be the most abundant TG lipase in rat islets and INS832/13 cells. To assess its role in insulin secretion, ATGL expression was decreased in INS832/13 cells (ATGL-knockdown (KD)) by small hairpin RNA. ATGL-KD increased the esterification of free fatty acid (FFA) into TG. ATGL-KD cells showed decreased glucose- or Gln + Leu-induced insulin release, as well as reduced response to KCl or palmitate at high, but not low, glucose. The K_ATP-independent/amplification pathway of GSIS was considerably reduced in ATGL-KD cells. ATGL^−/− mice were hypoinsulinemic and hypoglycemic and showed decreased plasma TG and FFAs. A hyperglycemic clamp revealed increased insulin sensitivity and decreased GSIS and arginine-induced insulin secretion in ATGL^−/− mice. Accordingly, isolated islets from ATGL^−/− mice showed reduced insulin secretion in response to glucose, glucose + palmitate, and KCl. Islet TG content and FFA esterification into TG were increased by 2-fold in ATGL^−/− islets, but glucose usage and oxidation were unaltered. The results demonstrate the importance of ATGL and intracellular lipid signaling for fuel- and non-fuel-induced insulin secretion.Free fatty acids (FFA)⁵ and other lipid molecules are important for proper glucose-stimulated insulin secretion (GSIS) by β-cells. Thus, deprivation of fatty acids (FA) in vivo (1) diminishes GSIS, whereas a short term exposure to FFA enhances it (1–3). In contrast, a sustained provision of FA, particularly in the presence of high glucose in vitro, is detrimental to β-cells in that it reduces insulin gene expression (4) and secretion (5) and induces β-cell apoptosis (6). The FA supply to the β-cells can be from exogenous sources, such as plasma FFAs and lipoproteins, or endogenous sources, such as intracellular triglyceride (TG) stores. Studies from our laboratory (7–10) and others (11, 12) support the concept that the hydrolysis of endogenous TG plays an important role in fuel-induced insulin secretion because TG depletion with leptin (13) or inhibition of TG lipolysis by lipase inhibitors such as 3,5-dimethylpyrazole (7) or orlistat (11, 12) markedly curtail GSIS in rat islets. Furthermore, mice with β-cell-specific knock-out of hormone-sensitive lipase (HSL), which hydrolyzes both TG and diacylglycerol (DAG), show defective first phase GSIS in vivo and in vitro (14).Lipolysis is an integral part of an essential metabolic pathway, the TG/FFA cycle, in which FFA esterification onto a glycerol backbone leading to the synthesis of TG is followed by its hydrolysis with the release of the FFA that can then be re-esterified. Intracellular TG/FFA cycling is known to occur in adipose tissue of rats and humans (15, 16) and also in liver and skeletal muscle (17). It is generally described as a “futile cycle” as it leads to the net hydrolysis of ATP with the generation of heat (18). However, several studies have shown that this cycle has important functions in the cell. For instance, in brown adipose tissue, it contributes to overall thermogenesis (17, 19). In islets from the normoglycemic, hyperinsulinemic, obese Zucker fatty rat, increased GSIS is associated with increased glucose-stimulated lipolysis and FA esterification, indicating enhanced TG/FFA cycling (10). Stimulation of lipolysis by glucose has also been observed in isolated islets from normal rats (12) and HSL^−/− mice (8) indicating the presence of glucose-responsive TG/FFA cycling in pancreatic β-cells.The identity of the key lipases involved in the TG/FFA cycle in pancreatic islets is uncertain. HSL is expressed in islets (20), is up-regulated by long term treatment with elevated glucose (21), and is associated with insulin secretory granules (22). In addition, our earlier results suggested that elevated HSL expression correlates with augmented TG/FFA cycling in islets of Zucker fatty rats (10). However, it appears that other lipases may contribute to lipolysis and the regulation of GSIS in islet tissue. Thus, results from studies using HSL^−/− mice showed unaltered GSIS (8, 23), except in fasted male mice (8, 9) in which lipolysis was decreased but not abolished. Furthermore, HSL^−/− mice show residual TG lipase activity (8) indicating the presence of other TG lipases.Recently, adipocyte triglyceride lipase (ATGL; also known as Desnutrin, TTS-2, iPLA₂-ζ, and PNPLA2) (24–26) was found to account for most if not all of the residual lipolysis in HSL^−/− mice (26, 27). Two homologues of ATGL, Adiponutrin and GS2, have been described in adipocytes (24). All three enzymes contain a patatin-like domain with broad lipid acyl-hydrolase activity. However, it is not known if adiponutrin and GS2 are actually TG hydrolases. An additional lipase, TG hydrolase or carboxylesterase-3, has been identified in rat adipose tissue (28, 29). Although the hydrolysis of TG is catalyzed by all these lipases, HSL can hydrolyze both TG and DAG, the latter being a better substrate (30).In this study, we observed that besides HSL, ATGL (31), adiponutrin, and GS2 are expressed in rat islets and INS832/13 cells, with ATGL being the most abundant. We then focused on the role of ATGL in fuel-stimulated insulin secretion in two models, INS832/13 β-cells in which ATGL expression was reduced by RNA interference-knockdown (ATGL-KD) and ATGL^−/− mice.     

CD36-deficient mice are resistant to alcohol- and high-carbohydrate-induced hepatic steatosis

CD36 is a scavenger receptor with multiple ligands and cellular functions, including facilitating cellular uptake of free fatty acids (FFAs). Chronic alcohol consumption increases hepatic CD36 expression, leading to the hypothesis that this promotes uptake of circulating FFAs, which then serve as a substrate for triglyceride (TG) synthesis and the development of alcoholic steatosis. We investigated this hypothesis in alcohol-fed wild-type and Cd36-deficient (Cd36^−/−) mice using low-fat/high-carbohydrate Lieber-DeCarli liquid diets, positing that Cd36^−/− mice would be resistant to alcoholic steatosis. Our data show that the livers of Cd36^−/− mice are resistant to the lipogenic effect of consuming high-carbohydrate liquid diets. These mice also do not further develop alcoholic steatosis when chronically fed alcohol. Surprisingly, we did not detect an effect of alcohol or CD36 deficiency on hepatic FFA uptake; however, the lower baseline levels of hepatic TG in Cd36^−/− mice fed a liquid diet were associated with decreased expression of genes in the de novo lipogenesis pathway and a lower rate of hepatic de novo lipogenesis. In conclusion, Cd36^−/− mice are resistant to hepatic steatosis when fed a high-carbohydrate liquid diet, and they are also resistant to alcoholic steatosis. These studies highlight an important role for CD36 in hepatic lipid homeostasis that is not associated with hepatic fatty acid uptake.     

The Hepatitis C Virus Core Protein Inhibits Adipose Triglyceride Lipase (ATGL)-mediated Lipid Mobilization and Enhances the ATGL Interaction with Comparative Gene Identification 58 (CGI-58) and Lipid Droplets

Liver steatosis is a common health problem associated with hepatitis C virus (HCV) and an important risk factor for the development of liver fibrosis and cancer. Steatosis is caused by triglycerides (TG) accumulating in lipid droplets (LDs), cellular organelles composed of neutral lipids surrounded by a monolayer of phospholipids. The HCV nucleocapsid core localizes to the surface of LDs and induces steatosis in cultured cells and mouse livers by decreasing intracellular TG degradation (lipolysis). Here we report that core at the surface of LDs interferes with the activity of adipose triglyceride lipase (ATGL), the key lipolytic enzyme in the first step of TG breakdown. Expressing core in livers or mouse embryonic fibroblasts of ATGL^−/− mice no longer decreases TG degradation as observed in LDs from wild-type mice, supporting the model that core reduces lipolysis by engaging ATGL. Core must localize at LDs to inhibit lipolysis, as ex vivo TG hydrolysis is impaired in purified LDs coated with core but not when free core is added to LDs. Coimmunoprecipitation experiments revealed that core does not directly interact with the ATGL complex but, unexpectedly, increased the interaction between ATGL and its activator CGI-58 as well as the recruitment of both proteins to LDs. These data link the anti-lipolytic activity of the HCV core protein with altered ATGL binding to CGI-58 and the enhanced association of both proteins with LDs.     

Enterocyte-specific ATGL overexpression affects intestinal and systemic cholesterol homeostasis

Enterocytes of the small intestine (SI) play an important role in maintaining systemic lipid levels by regulating dietary lipid absorption and postprandial lipoprotein secretion. An excessive amount of dietary-derived triglycerides (TGs) taken up by the apical side of enterocytes or basolaterally internalized lipoprotein remnants can be transiently stored in cytosolic lipid droplets (cLDs). As mice lacking adipose TG lipase (ATGL) in the SI display massive accumulation of cLDs but also delayed cholesterol absorption, we hypothesized that SI-specific overexpression of ATGL (Atgl iTg) might have beneficial effects on lipid homeostasis in the gut and possibly throughout the body. Here, we demonstrate that Atgl iTg mice had only modestly increased enzymatic activity despite drastically elevated Atgl mRNA levels (up to 120-fold) on chow diet, and was highly induced upon high-fat/high-cholesterol diet (HF/HCD) feeding. Atgl iTg mice showed markedly reduced intestinal TG concentrations after acute and chronic lipid challenge without affecting chylomicron TG secretion. Circulating plasma cholesterol levels were significantly lower in Atgl iTg mice under different feeding conditions, contrasting the accelerated uptake of dietary cholesterol into the circulation after HF/HCD feeding. In the fasted state, gene expression analysis revealed modulation of PPARα and liver X receptor (LXR) target genes by an increased fatty acid release, whereas the decreased plasma cholesterol concentrations in refed mice were more likely due to changes in HDL synthesis and secretion. We conclude that ATGL, in addition to its role in TG catabolism, plays a critical role in whole-body cholesterol homeostasis by modulating PPARα and LXR signaling in intestinal enterocytes.     

The Triggering Receptor Expressed on Myeloid Cells 2 Inhibits Complement Component 1q Effector Mechanisms and Exerts Detrimental Effects during Pneumococcal Pneumonia

Phagocytosis and inflammation within the lungs is crucial for host defense during bacterial pneumonia. Triggering receptor expressed on myeloid cells (TREM)-2 was proposed to negatively regulate TLR-mediated responses and enhance phagocytosis by macrophages, but the role of TREM-2 in respiratory tract infections is unknown. Here, we established the presence of TREM-2 on alveolar macrophages (AM) and explored the function of TREM-2 in the innate immune response to pneumococcal infection in vivo. Unexpectedly, we found Trem-2 ^−/− AM to display augmented bacterial phagocytosis in vitro and in vivo compared to WT AM. Mechanistically, we detected that in the absence of TREM-2, pulmonary macrophages selectively produced elevated complement component 1q (C1q) levels. We found that these increased C1q levels depended on peroxisome proliferator-activated receptor-δ (PPAR-δ) activity and were responsible for the enhanced phagocytosis of bacteria. Upon infection with S. pneumoniae, Trem-2 ^−/− mice exhibited an augmented bacterial clearance from lungs, decreased bacteremia and improved survival compared to their WT counterparts. This work is the first to disclose a role for TREM-2 in clinically relevant respiratory tract infections and demonstrates a previously unknown link between TREM-2 and opsonin production within the lungs.     

The Pivotal Role of 5-Lipoxygenase-Derived LTB4 in Controlling Pulmonary Paracoccidioidomycosis

Leukotrienes (LTs) produced from arachidonic acid by the action of 5-lipoxygenase (5-LO) are classical mediators of inflammatory responses. However, studies published in the literature regarding these mediators are contradictory and it remains uncertain whether these lipid mediators play a role in host defense against the fungal pathogen Paracoccidioides brasiliensis. To determine the involvement of LTs in the host response to pulmonary infection, wild-type and LT-deficient mice by targeted disruption of the 5-lipoxygenase gene (knockout mice) were studied following intratracheal challenge with P. brasiliensis yeasts. The results showed that infection is uniformly fatal in 5-LO-deficient mice and the mechanisms that account for this phenotype are an exacerbated lung injury and higher fungal pulmonary burden. Genetic ablation or pharmacological inhibition of LTs resulted in lower phagocytosis and fungicidal activity of macrophages in vitro, suggesting that deficiency in fungal clearance seems to be secondary to the absence of activation in 5-LO^−/− macrophages. Exogenous LTB₄ restored phagocytosis and fungicidal activity of 5-LO^−/− macrophages. Moreover, P. brasiliensis killing promoted by LTB₄ was dependent on nitric oxide (NO) production by macrophages. Taken together, these results reveal a fundamental role for 5-LO-derived LTB₄ in the protective response to P. brasiliensis infection and identify relevant mechanisms for the control of fungal infection during the early stages of the host immune response.     

Cholesteryl ester hydrolase activity is abolished in HSL macrophages but unchanged in macrophages lacking KIAA1363

Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363^−/− and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL^−/− mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363^−/− but unchanged in HSL^−/− mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL^−/− macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages.     

Adipose triglyceride lipase is a TG hydrolase of the small intestine and regulates intestinal PPARα signaling

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme mediating triglyceride (TG) hydrolysis. The lack of ATGL results in TG accumulation in multiple tissues, underscoring the critical role of ATGL in maintaining lipid homeostasis. Recent evidence suggests that ATGL affects TG metabolism via activation of peroxisome proliferator-activated receptor α (PPARα). To investigate specific effects of intestinal ATGL on lipid metabolism we generated mice lacking ATGL exclusively in the intestine (ATGLiKO). We found decreased TG hydrolase activity and increased intracellular TG content in ATGLiKO small intestines. Intragastric administration of [³H]trioleate resulted in the accumulation of radioactive TG in the intestine, whereas absorption into the systemic circulation was unchanged. Intraperitoneally injected [³H]oleate also accumulated within TG in ATGLiKO intestines, indicating that ATGL mobilizes fatty acids from the systemic circulation absorbed by the basolateral side from the blood. Down-regulation of PPARα target genes suggested modulation of cholesterol absorption by intestinal ATGL. Accordingly, ATGL deficiency in the intestine resulted in delayed cholesterol absorption. Importantly, this study provides evidence that ATGL has no impact on intestinal TG absorption but hydrolyzes TGs taken up from the intestinal lumen and systemic circulation. Our data support the role of ATGL in modulating PPARα-dependent processes also in the small intestine.     

Phosphatidylserine exposure by Toxoplasma gondii is fundamental to balance the immune response granting survival of the parasite and of the host

Phosphatidylserine (PS) exposure on the cell surface indicates apoptosis, but has also been related to evasion mechanisms of parasites, a concept known as apoptotic mimicry. Toxoplasma gondii mimics apoptotic cells by exposing PS, inducing secretion of TGF-beta1 by infected activated macrophages leading to degradation of inducible nitric oxide (NO) synthase, NO production inhibition and consequently persisting in these cells. Here PS⁺ and PS⁻ subpopulation of tachyzoites were separated and the entrance mechanism, growth and NO inhibition in murine macrophages, and mice survival and pathology were analyzed. Infection index in resident macrophages was similar for both PS subpopulations but lower when compared to the total T. gondii population. Growth in resident macrophages was higher for the total T. gondii population, intermediate for the PS⁺ and lower for the PS⁻ subpopulation. Production of NO by activated macrophages was inhibited after infection with the PS⁺ subpopulation and the total populations of tachyzoites. However, the PS⁻ subpopulation was not able to inhibit NO production. PS⁺ subpopulation invaded macrophages by active penetration as indicated by tight-fitting vacuoles, but the PS⁻ subpopulation entered macrophages by phagocytosis as suggested by loose-fitting vacuoles containing these tachyzoites. The entrance mechanism of both subpopulations was confirmed in a non-professional phagocytic cell line where only the PS⁺ tachyzoites were found inside these cells in tight-fitting vacuoles. Both subpopulations of T. gondii killed mice faster than the total population. Clear signs of inflammation and no tachyzoites were seen in the peritoneal cavity of mice infected with the PS⁻ subpopulation. Moreover, mice infected with the PS⁺ subpopulation had no sign of inflammation and the parasite burden was intense. These results show that PS⁺ and PS⁻ subpopulations of T. gondii are necessary for a successful toxoplasma infection indicating that both subpopulations are required to maintain the balance between inflammation and parasite growth.     

Glucose-dependent insulinotropic polypeptide prevents the progression of macrophage-driven atherosclerosis in diabetic apolipoprotein E-null mice

Aim

We recently reported that glucose-dependent insulinotropic polypeptide (GIP) prevents the development of atherosclerosis in apolipoprotein E-null (Apoe ^−/−) mice. GIP receptors (GIPRs) are found to be severely down-regulated in diabetic animals. We examined whether GIP can exert anti-atherogenic effects in diabetes.

Methods

Nondiabetic Apoe ^−/− mice, streptozotocin-induced diabetic Apoe ^−/− mice, and db/db mice were administered GIP (25 nmol/kg/day) or saline (vehicle) through osmotic mini-pumps for 4 weeks. The animals were assessed for aortic atherosclerosis and for oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages.

Results

Diabetic Apoe ^−/− mice of 21 weeks of age exhibited more advanced atherosclerosis than nondiabetic Apoe ^−/− mice of the same age. GIP infusion in diabetic Apoe ^−/− mice increased plasma total GIP levels by 4-fold without improving plasma insulin, glucose, or lipid profiles. GIP infusion significantly suppressed macrophage-driven atherosclerotic lesions, but this effect was abolished by co-infusions with [Pro³]GIP, a GIPR antagonist. Foam cell formation was stimulated by 3-fold in diabetic Apoe ^−/− mice compared with their nondiabetic counterparts, but this effect was halved by GIP infusion. GIP infusion also attenuated the foam cell formation in db/db mice. In vitro treatment with GIP (1 nM) reduced foam cell formation by 15% in macrophages from diabetic Apoe ^−/− mice, and this attenuating effect was weaker than that attained by the same treatment of macrophages from nondiabetic counterparts (35%). While GIPR expression was reduced by only about a half in macrophages from diabetic mice, it was reduced much more dramatically in pancreatic islets from the same animals. Incubation with high glucose (500 mg/dl) for 9–10 days markedly reduced GIPR expression in pancreatic islet cells, but not in macrophages.

Conclusions

Long-term infusion of GIP conferred significant anti-atherogenic effects in diabetic mice even though the GIPR expression in macrophages was mildly down-regulated in the diabetic state.     

Functional Cardiac Lipolysis in Mice Critically Depends on Comparative Gene Identification-58

Efficient catabolism of cellular triacylglycerol (TG) stores requires the TG hydrolytic activity of adipose triglyceride lipase (ATGL). The presence of comparative gene identification-58 (CGI-58) strongly increased ATGL-mediated TG catabolism in cell culture experiments. Mutations in the genes coding for ATGL or CGI-58 in humans cause neutral lipid storage disease characterized by TG accumulation in multiple tissues. ATGL gene mutations cause a severe phenotype especially in cardiac muscle leading to cardiomyopathy that can be lethal. In contrast, CGI-58 gene mutations provoke severe ichthyosis and hepatosteatosis in humans and mice, whereas the role of CGI-58 in muscle energy metabolism is less understood. Here we show that mice lacking CGI-58 exclusively in muscle (CGI-58KOM) developed severe cardiac steatosis and cardiomyopathy linked to impaired TG catabolism and mitochondrial fatty acid oxidation. The marked increase in ATGL protein levels in cardiac muscle of CGI-58KOM mice was unable to compensate the lack of CGI-58. The addition of recombinant CGI-58 to cardiac lysates of CGI-58KOM mice completely reconstituted TG hydrolytic activities. In skeletal muscle, the lack of CGI-58 similarly provoked TG accumulation. The addition of recombinant CGI-58 increased TG hydrolytic activities in control and CGI-58KOM tissue lysates, elucidating the limiting role of CGI-58 in skeletal muscle TG catabolism. Finally, muscle CGI-58 deficiency affected whole body energy homeostasis, which is caused by impaired muscle TG catabolism and increased cardiac glucose uptake. In summary, this study demonstrates that functional muscle lipolysis depends on both CGI-58 and ATGL.     

Differential expression and function of ABCG1 and ABCG4 during development and aging

ABCG1 and ABCG4 are highly homologous members of the ATP binding cassette (ABC) transporter family that regulate cellular cholesterol homeostasis. In adult mice, ABCG1 is known to be expressed in numerous cell types and tissues, whereas ABCG4 expression is limited to the central nervous system (CNS). Here, we show significant differences in expression of these two transporters during development. Examination of β-galactosidase-stained tissue sections from Abcg1^−/−LacZ and Abcg4^−/−LacZ knockin mice shows that ABCG4 is highly but transiently expressed both in hematopoietic cells and in enterocytes during development. In contrast, ABCG1 is expressed in macrophages and in endothelial cells of both embryonic and adult liver. We also show that ABCG1 and ABCG4 are both expressed as early as E12.5 in the embryonic eye and developing CNS. Loss of both ABCG1 and ABCG4 results in accumulation in the retina and/or brain of oxysterols, in altered expression of liver X receptor and sterol-regulatory element binding protein-2 target genes, and in a stress response gene. Finally, behavioral tests show that Abcg4^−/− mice have a general deficit in associative fear memory. Together, these data indicate that loss of ABCG1 and/or ABCG4 from the CNS results in changes in metabolic pathways and in behavior.     

Adipose Triglyceride Lipase Deficiency Causes Tissue-specific Changes in Insulin Signaling

Triacylglycerol accumulation in insulin target tissues is associated with insulin resistance. Paradoxically, mice with global targeted deletion of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in triacylglycerol hydrolysis, display improved glucose tolerance and insulin sensitivity despite triacylglycerol accumulation in multiple tissues. To determine the molecular mechanisms for this phenotype, ATGL-deficient (ATGL^−/−) and wild-type mice were injected with saline or insulin (10 units/kg, intraperitoneally), and then phosphorylation and activities of key insulin-signaling proteins were determined in insulin target tissues (liver, adipose tissue, and muscle). Insulin signaling and/or glucose transport was also evaluated in isolated adipocytes and skeletal muscle ex vivo. In ATGL^−/− mice, insulin-stimulated phosphatidylinositol 3-kinase and Akt activities as well as phosphorylation of critical residues of IRS1 (Tyr(P)-612) and Akt (Ser(P)-473) were increased in skeletal muscle in vivo. Insulin-stimulated phosphatidylinositol 3-kinase activity and total insulin receptor and insulin receptor substrate 1, but not other parameters, were also increased in white adipose tissue in vivo. In contrast, in vivo measures of insulin signaling were decreased in brown adipose tissue and liver. Interestingly, the enhanced components of insulin signaling identified in skeletal muscle and white adipose tissue in vivo and their expected downstream effects on glucose transport were not present ex vivo. ATGL deficiency altered intramyocellular lipids as well as serum factors known to influence insulin sensitivity. Thus, skeletal muscle, rather than other tissues, primarily contributes to enhanced insulin sensitivity in ATGL^−/− mice in vivo despite triacylglycerol accumulation, and both local and systemic factors contribute to tissue-specific effects of global ATGL deficiency on insulin action.Triacylglycerols (TAGs)⁴ are the predominant form of energy storage in animals. The ability to store and release this energy in response to variable energy availability requires a carefully regulated balance between TAG synthesis and hydrolysis. In the setting of chronic energy excess, however, TAGs and other lipid metabolites accumulate in adipose tissue as well as in metabolically relevant non-adipose tissues where they have been proposed to contribute to cellular dysfunction via a process known as lipotoxicity (1–3). Indeed, intracellular TAG accumulation has been repeatedly associated with metabolic dysfunction, a relationship that is particularly strong for insulin resistance (1–3). Despite this strong association, however, intracellular TAG accumulation is not always associated with insulin resistance (4) and may even be associated with insulin sensitivity, as is the case with highly trained endurance athletes (the so-called “athlete paradox”) (5). Thus, the contribution of intracellular TAGs and TAG metabolism per se to lipotoxicity remains controversial. What is clear is that lipid-induced insulin resistance is a major risk factor for morbidity and mortality from a variety of causes, including overt diabetes mellitus, nonalcoholic fatty liver disease, and cardiovascular disease. Hence, understanding the mechanisms by which dysregulated TAG metabolism contributes to steatosis, lipotoxicity, and insulin resistance is essential to understanding and treating these increasingly prevalent disorders.Although no mechanistic data have been identified directly linking intracellular TAGs per se to insulin resistance, lipotoxicity may occur when the capacity of the lipid droplets to effectively store TAGs is exceeded. Several other lipid metabolites that are products of TAG hydrolysis (i.e. diacylglyerols (DAGs), fatty acids (FAs), fatty acyl-CoAs (FA-CoAs), and ceramides) have been shown to directly or indirectly interfere with insulin signaling and glucose transport via a variety of mechanisms (6–9). Under normal physiological circumstances, insulin binds to the insulin receptor (IR), thereby triggering its intrinsic protein-tyrosine kinase activity. The subsequent autophosphorylation of several IR tyrosine residues promotes the recruitment and tyrosine phosphorylation of IR substrates (IRSs) followed by activation of phosphatidylinositol 3-kinase (PI3K) and Akt, which in turn promote the pleiotrophic downstream effects of insulin. The above lipid metabolites have been shown to increase serine/threonine phosphorylation and decrease tyrosine phosphorylation of IRS1, decrease serine/threonine phosphorylation of Akt, decrease IRS1-associated PI3K activity and Akt activity, and decrease Glut4 translocation (6–9). Possible mechanisms by which these lipid metabolites may influence glucose homeostasis and insulin action include competition for substrate oxidation, interference with cellular energy sensing, regulation of gene expression, promotion of oxidative stress and mitochondrial dysfunction, and activation of inflammatory and apoptotic pathways (6–9). However, most studies evaluating the role of lipotoxicity in insulin resistance have focused on cellular lipid uptake or oxidation, both of which produce unidirectional changes in intracellular TAGs and other intracellular lipid metabolites and hence do not adequately address the role of intracellular TAGs and TAG metabolism per se to this process.Understanding the role of TAG metabolism in lipotoxicity and insulin resistance has been further complicated by the fact that the rate-limiting enzyme for TAG hydrolysis, adipose triglyceride lipase (ATGL), has only recently been identified (10–12). ATGL has been most extensively studied in adipose tissue where it mediates the hydrolysis of long chain fatty acyl TAGs (10). ATGL is also expressed in other tissues, including liver, muscle, and pancreas (13), where its contribution to tissue-specific and systemic metabolism is less well understood. Mice with global targeted deletion of ATGL (ATGL^−/− mice) have severe defects in TAG hydrolysis, leading to TAG accumulation in virtually all tissues (14). Surprisingly, despite increased adiposity and “ectopic” TAG accumulation, which are characteristically associated with insulin resistance, ATGL^−/− mice paradoxically exhibit enhanced glucose tolerance and insulin sensitivity (14). This finding has largely been attributed to the effect of reduced systemic FA delivery on energy substrate availability (14). However, the contribution of altered tissue-specific insulin action to this phenotype has not been evaluated.ATGL^−/− mice represent a unique model for examining the contribution of intracellular TAG accumulation to glucose homeostasis and insulin action because intracellular TAG accumulation is dissociated from systemic FA delivery, and presumably also from the production/accumulation of other intracellular lipid metabolites. In addition, ATGL^−/− mice differ from the other models in which increased adiposity is paradoxically associated with insulin sensitivity in that enhanced expansion of adipose tissue mass and reduced systemic FA delivery do not protect against ectopic lipid deposition in ATGL^−/− mice (15, 16). The aims of this study were to evaluate the mechanisms by which impaired TAG hydrolysis and intracellular TAG accumulation because of global ATGL deficiency promote whole-body glucose tolerance and insulin sensitivity and to define the contribution of tissue-specific changes in insulin action to this phenotype. Here we demonstrate that global ATGL deficiency in mice not only reduces energy substrate availability but also produces tissue-specific changes in insulin action.     

Depletion of Vitamin E Increases Amyloid �� Accumulation by Decreasing Its Clearances from Brain and Blood in a Mouse Model of Alzheimer Disease

Increased oxidative damage is a prominent and early feature in Alzheimer disease. We previously crossed Alzheimer disease transgenic (APPsw) model mice with α-tocopherol transfer protein knock-out (Ttpa^−/−) mice in which lipid peroxidation in the brain was significantly increased. The resulting double-mutant (Ttpa^−/−APPsw) mice showed increased amyloid β (Aβ) deposits in the brain, which was ameliorated with α-tocopherol supplementation. To investigate the mechanism of the increased Aβ accumulation, we here studied generation, degradation, aggregation, and efflux of Aβ in the mice. The clearance of intracerebral-microinjected ¹²⁵I-Aβ_1–40 from brain was decreased in Ttpa^−/− mice to be compared with wild-type mice, whereas the generation of Aβ was not increased in Ttpa^−/−APPsw mice. The activity of an Aβ-degrading enzyme, neprilysin, did not decrease, but the expression level of insulin-degrading enzyme was markedly decreased in Ttpa^−/− mouse brain. In contrast, Aβ aggregation was accelerated in Ttpa^−/− mouse brains compared with wild-type brains, and well known molecules involved in Aβ transport from brain to blood, low density lipoprotein receptor-related protein-1 (LRP-1) and p-glycoprotein, were up-regulated in the small vascular fraction of Ttpa^−/− mouse brains. Moreover, the disappearance of intravenously administered ¹²⁵I-Aβ_1–40 was decreased in Ttpa^−/− mice with reduced translocation of LRP-1 in the hepatocytes. These results suggest that lipid peroxidation due to depletion of α-tocopherol impairs Aβ clearances from the brain and from the blood, possibly causing increased Aβ accumulation in Ttpa^−/−APPsw mouse brain and plasma.