Angptl3 regulates lipid metabolism in mice.

The KK obese mouse is moderately obese and has abnormally high levels of plasma insulin (hyperinsulinemia), glucose (hyperglycemia) and lipids (hyperlipidemia). In one strain (KK/San), we observed abnormally low plasma lipid levels (hypolipidemia). This mutant phenotype is inherited recessively as a mendelian trait. Here we report the mapping of the hypolipidemia (hypl) locus to the middle of chromosome 4 and positional cloning of the autosomal recessive mutation responsible for the hypolipidemia. The hypl locus encodes a unique angiopoietin-like lipoprotein modulator, which we named Allm1. It is identical to angiopoietin-like protein 3, encoded by Angptl3, and has a highly conserved counterpart in humans. Overexpression of Angptl3 or intravenous injection of the purified protein in KK/San mice elicited an increase in circulating plasma lipid levels. This increase was also observed in C57BL/6J normal mice. Taken together, these data suggest that Angptl3 regulates lipid metabolism in animals.

Raldh3 expression in diabetic islets reciprocally regulates secretion of insulin and glucagon from pancreatic islets.

We have previously reported that obesity-induced diabetes developed in high-fat diet (HFD)-fed BDF1 mice. This is caused by insufficient insulin response to an excess glucose load. In this study, we have shown that the enhanced expression of retinaldehyde dehydrogenase 3 (Raldh3) causes functional disorders of pancreatic islets in diabetic mouse models. In the pancreatic islets of HFD-induced diabetic BDF1 mice and spontaneously diabetic C57BL/KsJ(db/db) mice, gene expression analysis with oligonucleotide microarray revealed a significant increase in Raldh3 expression. Exposure to a culture medium containing a higher glucose concentration (25 mM) significantly increased Raldh3 expression in murine MIN6 and alphaTC1 clone 9 cells, which derived from the α and ß-cells of pancreatic islets, respectively. Overexpression of Raldh3 reduced the insulin secretion in MIN6 cells, and surprisingly, increased the glucagon secretion in alphaTC1 clone 9 cells. Furthermore, the knockdown of Raldh3 expression with siRNA decreased the glucagon secretion in alphaTC1 clone 9 cells. Raldh3 catalyzes the conversion of 13-cis retinal to 13-cis retinoic acid and we revealed that 13-cis retinoic acid significantly reduces cell viability in MIN6 and alphaTC1 clone 9 cells, but not in cells of H4IIEC3, 3T3-L1, and COS-1 cell lines. These findings suggest that an increasing expression of Raldh3 deregulates the balanced mechanisms of insulin and glucagon secretion in the pancreatic islets and may induce ß-cell dysfunction leading to the development of type 2 diabetes.

Angiopoietin-like protein3 regulates plasma HDL cholesterol through suppression of endothelial lipase.

OBJECTIVE: A low level of high-density lipoprotein (HDL) in plasma has been recognized as an aspect of metabolic syndrome and as a crucial risk factor of cardiovascular events. However, the physiological regulation of plasma HDL levels has not been completely defined. Current studies aim to reveal the contribution of angiopoietin-like protein3 (angptl3), previously known as a plasma suppressor of lipoprotein lipase, to HDL metabolism. METHODS AND RESULTS: Angptl3-deficient mice showed low plasma HDL cholesterol and HDL phospholipid (PL), and which were increased by ANGPTL3 supplementation via adenovirus. In vitro, ANGPTL3 inhibited the phospholipase activity of endothelial lipase (EL), which hydrolyzes HDL-PL and hence decreases plasma HDL levels, through a putative heparin-binding site in the N-terminal domain of ANGPTL3. Post-heparin plasma in Angptl3-knockout mice had higher phospholipase activity than did that in wild-type mice, suggesting that the activity of endogenous EL is elevated in Angptl3-deficient mice. Furthermore, we established an ELISA system for human ANGPTL3 and found that plasma ANGPTL3 levels significantly correlated with plasma HDL cholesterol and HDL-PL levels in human subjects. CONCLUSIONS: Angptl3 acts as an inhibitor of EL and may be involved in the regulation of plasma HDL cholesterol and HDL-PL levels in humans and rodents.

Biofabricated Structures Reconstruct Functional Urinary Bladders in Radiation-Injured Rat Bladders.

The ability to repair damaged urinary bladders through the application of bone marrow-derived cells is in the earliest stages of development. We investigated the application of bone marrow-derived cells to repair radiation-injured bladders. We used a three-dimensional bioprinting robot system to biofabricate bone marrow-derived cell structures. We then determined if the biofabricated structures could restore the tissues and functions of radiation-injured bladders. The bladders of female 10-week-old Sprague-Dawley (SD) rats were irradiated with 2-Gy once a week for 5 weeks. Adherent and proliferating bone marrow-derived cells harvested from the femurs of male 17-week-old green fluorescence protein-transfected Tg-SD rats were cultured in collagen-coated flasks. Bone marrow-derived cell spheroids were formed in 96-well plates. Three layers of spheroids were assembled by the bioprinter onto a 9 × 9 microneedle array. The assembled spheroids were perfusion cultured for 7 days, and then the microneedle array was removed. Two weeks after the last radiation treatment, the biofabricated structures were transplanted into an incision on the anterior wall of the bladders (n = 10). Control rats received the same surgery but without the biofabricated structures (sham-structure, n = 12). At 2 and 4 weeks after surgery, the sham-structure control bladder tissues exhibited disorganized smooth muscle layers, decreased nerve cells, and significant fibrosis with increased presence of fibrosis-marker P4HB-positive cells and hypoxia-marker hypoxia-induced factor 1α (HIF1α)-positive cells. The transplanted structures survived within the recipient tissues, and blood vessels extended within them from the recipient tissues. The bone marrow-derived cells in the structures differentiated into smooth muscle cells and formed smooth muscle clusters. The recipient tissues near the transplanted structures had distinct smooth muscle layers and reconstructed nerve cells, and only minimal fibrosis with decreased presence of P4HB- and HIF1α-positive cells. At 4 weeks after surgery, the sham-structure control rats exhibited significant urinary frequency symptoms with irregular and short voiding intervals, and low micturition volumes. In contrast, the structure-transplanted rats had regular micturition with longer voiding intervals and higher micturition volumes compared with the control rats. Furthermore, the residual volume of the structure-transplanted rats was lower than for the controls. Therefore, transplantation of biofabricated bone marrow-derived cell structures reconstructed functional bladders.

Scaffold-free 3D bio-printed human liver tissue stably maintains metabolic functions useful for drug discovery.

The liver plays a central role in metabolism. Although many studies have described in vitro liver models for drug discovery, to date, no model has been described that can stably maintain liver function. Here, we used a unique, scaffold-free 3D bio-printing technology to construct a small portion of liver tissue that could stably maintain drug, glucose, and lipid metabolism, in addition to bile acid secretion. This bio-printed normal human liver tissue maintained expression of several kinds of hepatic drug transporters and metabolic enzymes that functioned for several weeks. The bio-printed liver tissue displayed glucose production via cAMP/protein kinase A signaling, which could be suppressed with insulin. Bile acid secretion was also observed from the printed liver tissue, and it accumulated in the culture medium over time. We observed both bile duct and sinusoid-like structures in the bio-printed liver tissue, which suggested that bile acid secretion occurred via a sinusoid-hepatocyte-bile duct route. These results demonstrated that our bio-printed liver tissue was unique, because it exerted diverse liver metabolic functions for several weeks. In future, we expect our bio-printed liver tissue to be applied to developing new models that can be used to improve preclinical predictions of long-term toxicity in humans, generate novel targets for metabolic liver disease, and evaluate biliary excretion in drug development.

A decreased expression of angiopoietin-like 3 is protective against atherosclerosis in apoE-deficient mice.

KK/Snk mice (previously KK/San) possessing a recessive mutation (hypl) of the angiopoietin-like 3 (Angptl3) gene homozygously exhibit a marked reduction of VLDL due to the decreased Angptl3 expression. Recently, we proposed that Angptl3 is a new class of lipid metabolism modulator regulating VLDL triglyceride (TG) levels through the inhibition of lipoprotein lipase (LPL) activity. In this study, to elucidate the role of Angptl3 in atherogenesis, we investigated the effects of hypl mutation against hyperlipidemia and atherosclerosis in apolipoprotein E knockout (apoEKO) mice. ApoEKO mice with hypl mutation (apoEKO-hypl) exhibited a significant reduction of VLDL TG, VLDL cholesterol, and plasma apoB levels compared with apoEKO mice. Hepatic VLDL TG secretion was comparable between both apoE-deficient mice. Turnover studies revealed that the clearance of both [3H]TG-labeled and 125I-labeled VLDL was significantly enhanced in apoEKO-hypl mice. Postprandial plasma TG levels also decreased in apoEKO-hypl mice. Both LPL and hepatic lipase activities in the postheparin plasma increased significantly in apoEKO-hypl mice, explaining the enhanced lipid metabolism. Furthermore, apoEKO-hypl mice developed 3-fold smaller atherogenic lesions in the aortic sinus compared with apoEKO mice. Taken together, the reduction of Angptl3 expression is protective against hyperlipidemia and atherosclerosis, even in the absence of apoE, owing to the enhanced catabolism and clearance of TG-rich lipoproteins.

Leptin and insulin down-regulate angiopoietin-like protein 3, a plasma triglyceride-increasing factor.

We reported previously that angiopoietin-like protein3 (ANGPTL3), a liver-specific secretory factor, increased plasma triglyceride (TG) via inhibition of lipoprotein lipase and free fatty acid (FFA) by activating adipose-lipolysis. The current study examined the regulation of Angptl3 by leptin and insulin, both of which are key players in the metabolic syndrome. Angptl3 expression and plasma ANGPTL3 levels were increased in leptin-resistant C57BL/6J(db/db) and -deficient C57BL/6J(ob/ob) mice, relative to the control. Leptin supplements decreased Angptl3 gene expression and plasma ANGPTL3 in C57BL/6J(ob/ob) mice. The changes of Angptl3 were associated with alterations of plasma TG and FFA levels. Leptin treatment directly suppressed Angptl3 gene expression in hepatocytes. Angptl3 gene expression and plasma protein levels were also increased in insulin-deficient streptozotocin-treated mice. Insulin treatment of hepatocytes decreased Angptl3 gene expression and protein secretion. Our results suggest that elevated ANGPTL3 by leptin- or insulin-resistance is attributed to increased plasma TG and FFA concentrations in obesity.

Angiopoietin-like protein 3, a hepatic secretory factor, activates lipolysis in adipocytes.

Our previous work identified a genetic mutation in the gene encoding angiopoietin-like protein 3 (Angptl3) in KK/Snk mice (previously KK/San), a mutant strain of KK obese mice. KK/Snk had significantly lower plasma triglyceride and free fatty acid (FFA) than KK mice. Human ANGPTL3 treatment increased both plasma triglyceride and FFA. ANGPTL3 inhibited the activity of lipoprotein lipase, which accounted for the increase of plasma triglyceride. The mechanism how ANGPTL3 affects plasma FFA has not been known. The current study reveals that ANGPTL3 targets on adipose cells and induces lipolysis. Both plasma FFA and glycerol decreased in KK/Snk and increased by the treatment of human ANGPTL3. Specific bindings of ANGPTL3 to adipose cells were shown using fluorescence-labeled protein visually and 125I-labeled protein by the binding analysis. Furthermore, ANGPTL3 activated the lipolysis to stimulate the release of FFA and glycerol from adipocytes. We conclude that ANGPTL3 is a liver-derived lipolytic factor targeting on adipocyte.

Protein region important for regulation of lipid metabolism in angiopoietin-like 3 (ANGPTL3): ANGPTL3 is cleaved and activated in vivo.

Angiopoietin-like 3 (ANGPTL3) is a secreted protein that is mainly expressed in the liver and regulates lipid metabolism by inhibiting the lipolysis of triglyceriderich lipoproteins. Using deletion mutants of human ANGPTL3, we demonstrated that the N-terminal coiled-coil domain-containing fragment-(17-207) and not the C-terminal fibrinogen-like domain-containing fragment-(207-460) increased the plasma triglyceride levels in mice. We also found that the N-terminal region 17-165 was required to increase plasma triglyceride levels in mice and that a substitution of basic amino acid residues in the region 61-66 of the fragment showed no increase in the plasma triglyceride levels and no inhibition of lipolysis by lipoprotein lipase. In addition, when we analyzed ANGPTL3 in human plasma, we detected cleaved fragments of ANGPTL3. By analyzing recombinant ANGPTL3 in mouse plasma, we found that it was cleaved at two sites, Arg221 downward arrow Ala222 and Arg224 downward arrow Thr225, which are located in the linker region between the coiled-coil domain and the fibrinogen-like domain. Furthermore, a cleavage-resistant mutant of ANGPTL3 was determined to be less active than wild-type ANGPTL3 in increasing mouse plasma triglyceride levels but not in inhibiting lipoprotein lipase activity. These findings suggest that the cleavage of ANGPTL3 is important for the activation of ANGPTL3 in vivo.

Angiopoietin-like protein 3 mediates hypertriglyceridemia induced by the liver X receptor.

The KK/San obese and diabetic mouse, a mutant strain from KK obese mice, exhibits significantly low plasma triglyceride levels. In KK/San mice, genetic analysis identified a mutation in the gene encoding angiopoietinlike protein 3 (Angptl3), a liver-specific secretory protein, which had suppressive effect on lipoprotein lipase activity. In the current study, LXR ligands augmented Angptl3 mRNA expression and protein production in hepatoma cells. LXR ligands and LXR.retinoid X receptor (RXR) complex increased the promoter activity of Angptl3 gene. Serial deletion and point mutation of Angptl3 promoter identified an LXR response element (LXRE). Gel mobility shift assay showed the direct binding of LXR.RXR complex to the LXRE of the Angptl3 promoter. Furthermore, treatment of mice with synthetic LXR ligand caused triglyceride accumulation in the liver and plasma, which was accompanied by induction of hepatic mRNAs of several LXR target genes, including sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), and Angptl3. In Angptl3-deficient C57BL/6J mice, LXR ligand did not cause hypertriglyceridemia but accumulation of triglyceride in the liver. Our results demonstrate that Angptl3 is a direct target of LXR and that induction of hepatic Angptl3 accounts for hypertriglyceridemia associated with the treatment of LXR ligand.

ANGPTL3 decreases very low density lipoprotein triglyceride clearance by inhibition of lipoprotein lipase.

KK/San is a mutant mouse strain established in our laboratory from KK obese mice. KK/San mice show low plasma lipid levels compared with wild-type KK mice despite showing signs of hyperglycemia and hyperinsulinemia. Recently, we identified a mutation in the gene encoding angiopoietin-like protein 3 (Angptl3) in KK/San mice, and injection of adenoviruses encoding Angptl3 or recombinant ANGPTL3 protein to mutant KK/San mice raised plasma lipid levels. To elucidate the regulatory mechanism of ANGPTL3 on lipid metabolism, we focused on the metabolic pathways of triglyceride in the present study. Overexpression of Angptl3 in KK/San mice resulted in a marked increase of triglyceride-enriched very low density lipoprotein (VLDL). In vivo studies using Triton WR1339 revealed that there is no significant difference between mutant and wild-type KK mice in the hepatic VLDL triglyceride secretion rate. However, turnover studies using radiolabeled VLDL revealed that the clearance of (3)H-triglyceride-labeled VLDL was significantly enhanced in KK/San mice, whereas the clearance of (125)I-labeled VLDL was only slightly enhanced. In vitro analysis of recombinant protein revealed that ANGPTL3 directly inhibits LPL activity. These data strongly support the hypothesis that ANGPTL3 is a new class of lipid metabolism modulator, which regulates VLDL triglyceride levels through the inhibition of LPL activity.