Ectopic clotting factor VIII expression and misfolding in hepatocytes as a cause for hepatocellular carcinoma.

Hemophilia A gene therapy targets hepatocytes to express B domain deleted (BDD) clotting factor VIII (FVIII) to permit viral encapsidation. Since BDD is prone to misfolding in the endoplasmic reticulum (ER) and ER protein misfolding in hepatocytes followed by high-fat diet (HFD) can cause hepatocellular carcinoma (HCC), we studied how FVIII misfolding impacts HCC development using hepatocyte DNA delivery to express three proteins from the same parental vector: (1) well-folded cytosolic dihydrofolate reductase (DHFR); (2) BDD-FVIII, which is prone to misfolding in the ER; and (3) N6-FVIII, which folds more efficiently than BDD-FVIII. One week after DNA delivery, when FVIII expression was undetectable, mice were fed HFD for 65 weeks. Remarkably, all mice that received BDD-FVIII vector developed liver tumors, whereas only 58% of mice that received N6 and no mice that received DHFR vector developed liver tumors, suggesting that the degree of protein misfolding in the ER increases predisposition to HCC in the context of an HFD and in the absence of viral transduction. Our findings raise concerns of ectopic BDD-FVIII expression in hepatocytes in the clinic, which poses risks independent of viral vector integration. Limited expression per hepatocyte and/or use of proteins that avoid misfolding may enhance safety.

Factor VIII exhibits chaperone-dependent and glucose-regulated reversible amyloid formation in the endoplasmic reticulum.

Hemophilia A, an X-linked bleeding disorder caused by deficiency of factor VIII (FVIII), is treated by protein replacement. Unfortunately, this regimen is costly due to the expense of producing recombinant FVIII as a consequence of its low-level secretion from mammalian host cells. FVIII expression activates the endoplasmic reticulum (ER) stress response, causes oxidative stress, and induces apoptosis. Importantly, little is known about the factors that cause protein misfolding and aggregation in metazoans. Here, we identified intrinsic and extrinsic factors that cause FVIII to form aggregates. We show that FVIII forms amyloid-like fibrils within the ER lumen upon increased FVIII synthesis or inhibition of glucose metabolism. Significantly, FVIII amyloids can be dissolved upon restoration of glucose metabolism to produce functional secreted FVIII. Two ER chaperone families and their cochaperones, immunoglobulin binding protein (BiP) and calnexin/calreticulin, promote FVIII solubility in the ER, where the former is also required for disaggregation. A short aggregation motif in the FVIII A1 domain (termed Aggron) is necessary and sufficient to seed ß-sheet polymerization, and BiP binding to this Aggron prevents amyloidogenesis. Our findings provide novel insight into mechanisms that limit FVIII secretion and ER protein aggregation in general and have implication for ongoing hemophilia A gene-therapy clinical trials.

Loss of lipin 1-mediated phosphatidic acid phosphohydrolase activity in muscle leads to skeletal myopathy in mice.

Lipin 1 regulates glycerolipid homeostasis by acting as a phosphatidic acid phosphohydrolase (PAP) enzyme in the triglyceride-synthesis pathway and by regulating transcription factor activity. Mutations in human lipin 1 are a common cause of recurrent rhabdomyolysis in children. Mice with constitutive whole-body lipin 1 deficiency have been used to examine mechanisms connecting lipin 1 deficiency to myocyte injury. However, that mouse model is confounded by lipodystrophy not phenocopied in people. Herein, 2 muscle-specific mouse models were studied: 1) Lpin1 exon 3 and 4 deletion, resulting in a hypomorphic protein without PAP activity, but which preserved transcriptional coregulatory function; and 2) Lpin1 exon 7 deletion, resulting in total protein loss. In both models, skeletal muscles exhibited a chronic myopathy with ongoing muscle fiber necrosis and regeneration and accumulation of phosphatidic acid and, paradoxically, diacylglycerol. Additionally, lipin 1-deficient mice had abundant, but abnormal, mitochondria likely because of impaired autophagy. Finally, these mice exhibited increased plasma creatine kinase following exhaustive exercise when unfed. These data suggest that mice lacking lipin 1-mediated PAP activity in skeletal muscle may serve as a model for determining the mechanisms by which lipin 1 deficiency leads to myocyte injury and for testing potential therapeutic approaches.-Schweitzer, G. G., Collier, S. L., Chen, Z., McCommis, K. S., Pittman, S. K., Yoshino, J., Matkovich, S. J., Hsu, F.-F., Chrast, R., Eaton, J. M., Harris, T. E., Weihl, C. C., Finck, B. N. Loss of lipin 1-mediated phosphatidic acid phosphohydrolase activity in muscle leads to skeletal myopathy in mice.

A murine Niemann-Pick C1 I1061T knock-in model recapitulates the pathological features of the most prevalent human disease allele.

Niemann-Pick Type C1 (NPC1) disease is a rare neurovisceral, cholesterol-sphingolipid lysosomal storage disorder characterized by ataxia, motor impairment, progressive intellectual decline, and dementia. The most prevalent mutation, NPC1(I1061T), encodes a misfolded protein with a reduced half-life caused by ER-associated degradation. Therapies directed at stabilization of the mutant NPC1 protein reduce cholesterol storage in fibroblasts but have not been tested in vivo because of lack of a suitable animal model. Whereas the prominent features of human NPC1 disease are replicated in the null Npc1(-/-) mouse, this model is not amenable to examining proteostatic therapies. The objective of the present study was to develop an NPC1 I1061T knock-in mouse in which to test proteostatic therapies. Compared with the Npc1(-/-) mouse, this Npc1(tm(I1061T)Dso) model displays a less severe, delayed form of NPC1 disease with respect to weight loss, decreased motor coordination, Purkinje cell death, lipid storage, and premature death. The murine NPC1(I1061T) protein has a reduced half-life in vivo, consistent with protein misfolding and rapid ER-associated degradation, and can be stabilized by histone deacetylase inhibition. This novel mouse model faithfully recapitulates human NPC1 disease and provides a powerful tool for preclinical evaluation of therapies targeting NPC1 protein variants with compromised stability.

Mice with an adipocyte-specific lipin 1 separation-of-function allele reveal unexpected roles for phosphatidic acid in metabolic regulation.

Lipin 1 is a coregulator of DNA-bound transcription factors and a phosphatidic acid (PA) phosphatase (PAP) enzyme that catalyzes a critical step in the synthesis of glycerophospholipids. Lipin 1 is highly expressed in adipocytes, and constitutive loss of lipin 1 blocks adipocyte differentiation; however, the effects of Lpin1 deficiency in differentiated adipocytes are unknown. Here we report that adipocyte-specific Lpin1 gene recombination unexpectedly resulted in expression of a truncated lipin 1 protein lacking PAP activity but retaining transcriptional regulatory function. Loss of lipin 1-mediated PAP activity in adipocytes led to reduced glyceride synthesis and increased PA content. Characterization of the deficient mice also revealed that lipin 1 normally modulates cAMP-dependent signaling through protein kinase A to control lipolysis by metabolizing PA, which is an allosteric activator of phosphodiesterase 4 and the molecular target of rapamycin. Consistent with these findings, lipin 1 expression was significantly related to adipose tissue lipolytic rates and protein kinase A signaling in adipose tissue of obese human subjects. Taken together, our findings identify lipin 1 as a reciprocal regulator of triglyceride synthesis and hydrolysis in adipocytes, and suggest that regulation of lipolysis by lipin 1 is mediated by PA-dependent modulation of phosphodiesterase 4.

Liver-specific loss of lipin-1-mediated phosphatidic acid phosphatase activity does not mitigate intrahepatic TG accumulation in mice.

Lipin proteins (lipin 1, 2, and 3) regulate glycerolipid homeostasis by acting as phosphatidic acid phosphohydrolase (PAP) enzymes in the TG synthesis pathway and by regulating DNA-bound transcription factors to control gene transcription. Hepatic PAP activity could contribute to hepatic fat accumulation in response to physiological and pathophysiological stimuli. To examine the role of lipin 1 in regulating hepatic lipid metabolism, we generated mice that are deficient in lipin-1-encoded PAP activity in a liver-specific manner (Alb-Lpin1(-/-) mice). This allele of lipin 1 was still able to transcriptionally regulate the expression of its target genes encoding fatty acid oxidation enzymes, and the expression of these genes was not affected in Alb-Lpin1(-/-) mouse liver. Hepatic PAP activity was significantly reduced in mice with liver-specific lipin 1 deficiency. However, hepatocytes from Alb-Lpin1(-/-) mice had normal rates of TG synthesis, and steady-state hepatic TG levels were unaffected under fed and fasted conditions. Furthermore, Alb-Lpin1(-/-) mice were not protected from intrahepatic accumulation of diacylglycerol and TG after chronic feeding of a diet rich in fat and fructose. Collectively, these data demonstrate that marked deficits in hepatic PAP activity do not impair TG synthesis and accumulation under acute or chronic conditions of lipid overload.

Glucose transporter 8 (GLUT8) mediates fructose-induced de novo lipogenesis and macrosteatosis.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world, and it is thought to be the hepatic manifestation of the metabolic syndrome. Excess dietary fructose causes both metabolic syndrome and NAFLD in rodents and humans, but the pathogenic mechanisms of fructose-induced metabolic syndrome and NAFLD are poorly understood. GLUT8 (Slc2A8) is a facilitative glucose and fructose transporter that is highly expressed in liver, heart, and other oxidative tissues. We previously demonstrated that female mice lacking GLUT8 exhibit impaired first-pass hepatic fructose metabolism, suggesting that fructose transport into the hepatocyte, the primary site of fructose metabolism, is in part mediated by GLUT8. Here, we tested the hypothesis that GLUT8 is required for hepatocyte fructose uptake and for the development of fructose-induced NAFLD. We demonstrate that GLUT8 is a cell surface-localized transporter and that GLUT8 overexpression or GLUT8 shRNA-mediated gene silencing significantly induces and blocks radiolabeled fructose uptake in cultured hepatocytes. We further show diminished fructose uptake and de novo lipogenesis in fructose-challenged GLUT8-deficient hepatocytes. Finally, livers from long term high-fructose diet-fed GLUT8-deficient mice exhibited attenuated fructose-induced hepatic triglyceride and cholesterol accumulation without changes in hepatocyte insulin-stimulated Akt phosphorylation. GLUT8 is thus essential for hepatocyte fructose transport and fructose-induced macrosteatosis. Fructose delivery across the hepatocyte membrane is thus a proximal, modifiable disease mechanism that may be exploited to prevent NAFLD.

Inhibiting monoacylglycerol acyltransferase 1 ameliorates hepatic metabolic abnormalities but not inflammation and injury in mice.

Abnormalities in hepatic lipid metabolism and insulin action are believed to play a critical role in the etiology of nonalcoholic steatohepatitis. Monoacylglycerol acyltransferase (MGAT) enzymes convert monoacylglycerol to diacylglycerol, which is the penultimate step in one pathway for triacylglycerol synthesis. Hepatic expression of Mogat1, which encodes an MGAT enzyme, is increased in the livers of mice with hepatic steatosis, and knocking down Mogat1 improves glucose metabolism and hepatic insulin signaling, but whether increased MGAT activity plays a role in the etiology of nonalcoholic steatohepatitis is unclear. To examine this issue, mice were placed on a diet containing high levels of trans fatty acids, fructose, and cholesterol (HTF-C diet) or a low fat control diet for 4 weeks. Mice were injected with antisense oligonucleotides (ASOs) to knockdown Mogat1 or a scrambled ASO control for 12 weeks while remaining on diet. The HTF-C diet caused glucose intolerance, hepatic steatosis, and induced hepatic gene expression markers of inflammation, macrophage infiltration, and stellate cell activation. Mogat1 ASO treatment, which suppressed Mogat1 expression in liver and adipose tissue, attenuated weight gain, improved glucose tolerance, improved hepatic insulin signaling, and decreased hepatic triacylglycerol content compared with control ASO-treated mice on HTF-C chow. However, Mogat1 ASO treatment did not reduce hepatic diacylglycerol, cholesterol, or free fatty acid content; improve histologic measures of liver injury; or reduce expression of markers of stellate cell activation, liver inflammation, and injury. In conclusion, inhibition of hepatic Mogat1 in HTF-C diet-fed mice improves hepatic metabolic abnormalities without attenuating liver inflammation and injury.

Association between specific adipose tissue CD4+ T-cell populations and insulin resistance in obese individuals.

BACKGROUND & AIMS: An increased number of macrophages in adipose tissue is associated with insulin resistance and metabolic dysfunction in obese people. However, little is known about other immune cells in adipose tissue from obese people, and whether they contribute to insulin resistance. We investigated the characteristics of T cells in adipose tissue from metabolically abnormal insulin-resistant obese (MAO) subjects, metabolically normal insulin-sensitive obese (MNO) subjects, and lean subjects. Insulin sensitivity was determined by using the hyperinsulinemic euglycemic clamp procedure. METHODS: We assessed plasma cytokine concentrations and subcutaneous adipose tissue CD4(+) T-cell populations in 9 lean, 12 MNO, and 13 MAO subjects. Skeletal muscle and liver samples were collected from 19 additional obese patients undergoing bariatric surgery to determine the presence of selected cytokine receptors. RESULTS: Adipose tissue from MAO subjects had 3- to 10-fold increases in numbers of CD4(+) T cells that produce interleukin (IL)-22 and IL-17 (a T-helper [Th] 17 and Th22 phenotype) compared with MNO and lean subjects. MAO subjects also had increased plasma concentrations of IL-22 and IL-6. Receptors for IL-17 and IL-22 were expressed in human liver and skeletal muscle samples. IL-17 and IL-22 inhibited uptake of glucose in skeletal muscle isolated from rats and reduced insulin sensitivity in cultured human hepatocytes. CONCLUSIONS: Adipose tissue from MAO individuals contains increased numbers of Th17 and Th22 cells, which produce cytokines that cause metabolic dysfunction in liver and muscle in vitro. Additional studies are needed to determine whether these alterations in adipose tissue T cells contribute to the pathogenesis of insulin resistance in obese people.

Conditional hepatocyte ablation of PDIA1 uncovers indispensable roles in both APOB and MTTP folding to support VLDL secretion.

OBJECTIVES: The assembly and secretion of hepatic very low-density lipoprotein (VLDL) plays pivotal roles in hepatic and plasma lipid homeostasis. Protein disulfide isomerase A1 (PDIA1/P4HB) is a molecular chaperone whose functions are essential for protein folding in the endoplasmic reticulum. Here we investigated the physiological requirement in vivo for PDIA1 in maintaining VLDL assembly and secretion. METHODS: Pdia1/P4hb was conditionally deleted in adult mouse hepatocytes and the phenotypes characterized. Mechanistic analyses in primary hepatocytes determined how PDIA1 ablation alters MTTP synthesis and degradation as well as altering synthesis and secretion of Apolipoprotein B (APOB), along with complementary expression of intact PDIA1 vs a catalytically inactivated PDIA1 mutant. RESULTS: Hepatocyte-specific deletion of Pdia1/P4hb inhibited hepatic MTTP expression and dramatically reduced VLDL production, leading to severe hepatic steatosis and hypolipidemia. Pdia1-deletion did not affect mRNA expression or protein stability of MTTP but rather prevented Mttp mRNA translation. We demonstrate an essential role for PDIA1 in MTTP synthesis and function and show that PDIA1 interacts with APOB in an MTTP-independent manner via its molecular chaperone function to support APOB folding and secretion. CONCLUSIONS: PDIA1 plays indispensable roles in APOB folding, MTTP synthesis and activity to support VLDL assembly. Thus, like APOB and MTTP, PDIA1 is an obligatory component of hepatic VLDL production.

Insulin resistance and metabolic derangements in obese mice are ameliorated by a novel peroxisome proliferator-activated receptor γ-sparing thiazolidinedione.

Currently approved thiazolidinediones (TZDs) are effective insulin-sensitizing drugs that may have efficacy for treatment of a variety of metabolic and inflammatory diseases, but their use is limited by side effects that are mediated through ectopic activation of the peroxisome proliferator-activated receptor γ (PPARγ). Emerging evidence suggests that the potent anti-diabetic efficacy of TZDs can be separated from the ability to serve as ligands for PPARγ. A novel TZD analog (MSDC-0602) with very low affinity for binding and activation of PPARγ was evaluated for its effects on insulin resistance in obese mice. MSDC-0602 treatment markedly improved several measures of multiorgan insulin sensitivity, adipose tissue inflammation, and hepatic metabolic derangements, including suppressing hepatic lipogenesis and gluconeogenesis. These beneficial effects were mediated at least in part via direct actions on hepatocytes and were preserved in hepatocytes from liver-specific PPARγ(-/-) mice, indicating that PPARγ was not required to suppress these pathways. In conclusion, the beneficial pharmacology exhibited by MSDC-0602 on insulin sensitivity suggests that PPARγ-sparing TZDs are effective for treatment of type 2 diabetes with reduced risk of PPARγ-mediated side effects.

Cellular stress and coagulation factor production: when more is not necessarily better.

Remarkably, it has been 40 years since the isolation of the 2 genes involved in hemophilia A (HA) and hemophilia B (HB), encoding clotting factor (F) VIII (FVIII) and FIX, respectively. Over the years, these advances led to the development of purified recombinant protein factors that are free of contaminating viruses from human pooled plasma for hemophilia treatments, reducing the morbidity and mortality previously associated with human plasma-derived clotting factors. These discoveries also paved the way for modified factors that have increased plasma half-lives. Importantly, more recent advances have led to the development and Food and Drug Administration approval of a hepatocyte-targeted, adeno-associated viral vector-mediated gene transfer approach for HA and HB. However, major concerns regarding the durability and safety of HA gene therapy remain to be resolved. Compared with FIX, FVIII is a much larger protein that is prone to misfolding and aggregation in the endoplasmic reticulum and is poorly secreted by the mammalian cells. Due to the constraint of the packaging capacity of adeno-associated viral vector, B-domain deleted FVIII rather than the full-length protein is used for HA gene therapy. Like full-length FVIII, B-domain deleted FVIII misfolds and is inefficiently secreted. Its expression in hepatocytes activates the cellular unfolded protein response, which is deleterious for hepatocyte function and survival and has the potential to drive hepatocellular carcinoma. This review is focused on our current understanding of factors limiting FVIII secretion and the potential pathophysiological consequences upon expression in hepatocytes.

Evidence for regulated monoacylglycerol acyltransferase expression and activity in human liver.

Intrahepatic lipid accumulation is extremely common in obese subjects and is associated with the development of insulin resistance and diabetes. Hepatic diacylglycerol and triacylglycerol synthesis predominantly occurs through acylation of glycerol-3-phosphate. However, an alternative pathway for synthesizing diacylglycerol from monoacylglycerol acyltransferases (MGAT) could also contribute to hepatic glyceride pools. MGAT activity and the expression of the three genes encoding MGAT enzymes (MOGAT1, MOGAT2, and MOGAT3) were determined in liver biopsies from obese human subjects before and after gastric bypass surgery. MOGAT expression was also assessed in liver of subjects with nonalcoholic fatty liver disease (NAFLD) or control livers. All MOGAT genes were expressed in liver, and hepatic MGAT activity was readily detectable in liver lysates. The hepatic expression of MOGAT3 was highly correlated with MGAT activity, whereas MOGAT1 and MOGAT2 expression was not, and knockdown of MOGAT3 expression attenuated MGAT activity in a liver-derived cell line. Marked weight loss following gastric bypass surgery was associated with a significant reduction in MOGAT2 and MOGAT3 expression, which were also overexpressed in NAFLD subjects. These data suggest that the MGAT pathway is active and dynamically regulated in human liver and could be an important target for pharmacologic intervention for the treatment of obesity-related insulin resistance and NAFLD.

Lipin 1 is an inducible amplifier of the hepatic PGC-1alpha/PPARalpha regulatory pathway.

Perturbations in hepatic lipid homeostasis are linked to the development of obesity-related steatohepatitis. Mutations in the gene encoding lipin 1 cause hepatic steatosis in fld mice, a genetic model of lipodystrophy. However, the molecular function of lipin 1 is unclear. Herein, we demonstrate that the expression of lipin 1 is induced by peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator 1alpha (PGC-1alpha), a transcriptional coactivator controlling several key hepatic metabolic pathways. Gain-of-function and loss-of-function strategies demonstrated that lipin selectively activates a subset of PGC-1alpha target pathways, including fatty acid oxidation and mitochondrial oxidative phosphorylation, while suppressing the lipogenic program and lowering circulating lipid levels. Lipin activates mitochondrial fatty acid oxidative metabolism by inducing expression of the nuclear receptor PPARalpha, a known PGC-1alpha target, and via direct physical interactions with PPARalpha and PGC-1alpha. These results identify lipin 1 as a selective physiological amplifier of the PGC-1alpha/PPARalpha-mediated control of hepatic lipid metabolism.

Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) stimulates VLDL assembly through activation of cell death-inducing DFFA-like effector B (CideB).

Abnormalities in very low density lipoprotein (VLDL) assembly and secretion impact intrahepatic lipid homeostasis, plasma lipoprotein profile, and energy metabolism of distal peripheral tissues. We have evaluated the role of the transcriptional coactivator, the peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), in VLDL assembly and secretion. PGC-1alpha overexpression in HepG2 cells led to diminished rates of triglyceride (TG) synthesis but strongly stimulated VLDL-TG secretion, markedly increasing the efficiency of secretion of newly synthesized TG. PGC-1alpha overexpression increased the rate of secretion of apoB100 and promoted secretion of larger, less dense VLDL particles. PGC-1alpha overexpression in intact mouse liver also stimulated rates of VLDL TG secretion and attenuated hepatic TG accumulation resulting from high fat diet feeding. To determine the molecular mechanisms mediating the effect of PGC-1alpha on VLDL assembly, we evaluated the expression of several candidate mediators known to be involved in VLDL assembly or hepatic lipid homeostasis. Cell death-inducing DFFA-like effector B (CideB) expression was greatly induced by PGC-1alpha, and siRNA against CideB reversed the effects of PGC-1alpha on the secretion of TG and VLDL-sized particles by HepG2 cells, indicating that CideB is a critical mediator of stimulatory effects of PGC-1alpha on VLDL secretion. Collectively, these data suggest that PGC-1alpha plays an important role in partitioning cytoplasmic TG toward the VLDL secretory compartments and promoting VLDL secretion via transcriptional induction of CideB.

Chop/Ddit3 depletion in ß cells alleviates ER stress and corrects hepatic steatosis in mice.

Type 2 diabetes (T2D) is a metabolic disorder characterized by hyperglycemia, hyperinsulinemia, and insulin resistance (IR). During the early phase of T2D, insulin synthesis and secretion by pancreatic ß cells is enhanced, which can lead to proinsulin misfolding that aggravates endoplasmic reticulum (ER) protein homeostasis in ß cells. Moreover, increased circulating insulin may contribute to fatty liver disease. Medical interventions aimed at alleviating ER stress in ß cells while maintaining optimal insulin secretion are therefore an attractive therapeutic strategy for T2D. Previously, we demonstrated that germline Chop gene deletion preserved ß cells in high-fat diet (HFD)-fed mice and in leptin receptor-deficient db/db mice. In the current study, we further investigated whether targeting Chop/Ddit3 specifically in murine ß cells conferred therapeutic benefits. First, we showed that Chop deletion in ß cells alleviated ß cell ER stress and delayed glucose-stimulated insulin secretion (GSIS) in HFD-fed mice. Second, ß cell-specific Chop deletion prevented liver steatosis and hepatomegaly in aged HFD-fed mice without affecting basal glucose homeostasis. Third, we provide mechanistic evidence that Chop depletion reduces ER Ca2+ buffering capacity and modulates glucose-induced islet Ca2+ oscillations, leading to transcriptional changes of ER chaperone profile ("ER remodeling"). Last, we demonstrated that a GLP1-conjugated Chop antisense oligonucleotide strategy recapitulated the reduction in liver triglycerides and pancreatic insulin content. In summary, our results demonstrate that Chop depletion in ß cells provides a therapeutic strategy to alleviate dysregulated insulin secretion and consequent fatty liver disease in T2D.

Dynamic and differential regulation of proteins that coat lipid droplets in fatty liver dystrophic mice.

Lipid droplet proteins (LDPs) coat the surface of triglyceride-rich lipid droplets and regulate their formation and lipolysis. We profiled hepatic LDP expression in fatty liver dystrophic (fld) mice, a unique model of neonatal hepatic steatosis that predictably resolves between postnatal day 14 (P14) and P17. Western blotting revealed that perilipin-2/ADRP and perilipin-5/OXPAT were markedly increased in steatotic fld liver but returned to normal by P17. However, the changes in perilipin-2 and perilipin-5 protein content in fld mice were exaggerated compared with relatively modest increases in corresponding mRNAs encoding these proteins, a phenomenon likely mediated by increased protein stability. Conversely, cell death-inducing DFFA-like effector (Cide) family genes were strongly induced at the level of mRNA expression in steatotic fld mouse liver. Surprisingly, levels of peroxisome proliferator-activated receptor gamma, which is known to regulate Cide expression, were unchanged in fld mice. However, sterol-regulatory element binding protein 1 (SREBP-1) was activated in fld liver and CideA was revealed as a new direct target gene of SREBP-1. In summary, LDP content is markedly increased in liver of fld mice. However, whereas perilipin-2 and perilipin-5 levels are primarily regulated posttranslationally, Cide family mRNA expression is induced, suggesting that these families of LDP are controlled at different regulatory checkpoints.

1,25(OH)2 vitamin d inhibits foam cell formation and suppresses macrophage cholesterol uptake in patients with type 2 diabetes mellitus.

BACKGROUND: Cardiovascular disease is the leading cause of death among those with diabetes mellitus. Vitamin D deficiency is associated with an increased risk of cardiovascular disease in this population. To determine the mechanism by which vitamin D deficiency mediates accelerated cardiovascular disease in patients with diabetes mellitus, we investigated the effects of active vitamin D on macrophage cholesterol deposition. METHODS AND RESULTS: We obtained macrophages from 76 obese, diabetic, hypertensive patients with vitamin D deficiency (25-hydroxyvitamin D <80 nmol/L; group A) and 4 control groups: obese, diabetic, hypertensive patients with normal vitamin D (group B; n=15); obese, nondiabetic, hypertensive patients with vitamin D deficiency (group C; n=25); and nonobese, nondiabetic, nonhypertensive patients with vitamin D deficiency (group D; n=10) or sufficiency (group E; n=10). Macrophages from the same patients in all groups were cultured in vitamin D-deficient or 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] -supplemented media and exposed to modified low-density lipoprotein cholesterol. 1,25(OH)(2)D(3) suppressed foam cell formation by reducing acetylated or oxidized low-density lipoprotein cholesterol uptake in diabetic subjects only. Conversely, deletion of the vitamin D receptor in macrophages from diabetic patients accelerated foam cell formation induced by modified LDL. 1,25(OH)(2)D(3) downregulation of c-Jun N-terminal kinase activation reduced peroxisome proliferated-activated receptor-gamma expression, suppressed CD36 expression, and prevented oxidized low-density lipoprotein-derived cholesterol uptake. In addition, 1,25(OH)(2)D(3) suppression of macrophage endoplasmic reticulum stress improved insulin signaling, downregulated SR-A1 expression, and prevented oxidized and acetylated low-density lipoprotein-derived cholesterol uptake. CONCLUSIONS: These results identify reduced vitamin D receptor signaling as a potential mechanism underlying increased foam cell formation and accelerated cardiovascular disease in diabetic subjects.

Alterations in hepatic metabolism in fld mice reveal a role for lipin 1 in regulating VLDL-triacylglyceride secretion.

OBJECTIVE: Lipin 1 controls fatty acid metabolism in the nucleus as a transcriptional regulator and in the cytosol as an enzyme catalyzing the penultimate step in phosphoglycerol triacylglyceride (TAG) synthesis. We sought to evaluate the effects of lipin 1 on hepatic TAG synthesis and secretion by gain-of-function and loss-of-function approaches. METHODS AND RESULTS: Rates of TAG synthesis were not impaired in hepatocytes isolated from adult lipin 1-deficient (fld) mice and were actually increased in 14-day-old fld mice. Additionally, compared to littermate controls, VLDL-TAG secretion rates were markedly increased in fld mice of both ages. Lipin 1 overexpression did not alter TAG synthesis rates but significantly suppressed VLDL-TAG secretion. The lipin 1-mediated suppression of VLDL-TAG secretion was linked to the peptide motif mediating its transcriptional-regulatory effects. However, the expression of candidate genes required for VLDL assembly and secretion was unaltered by lipin 1 activation or deficiency. Finally, the hepatic expression of lipin 1 was diminished in obese insulin-resistant mice, whereas adenoviral-mediated overexpression of lipin 1 in liver of these mice inhibits VLDL-TAG secretion and improves hepatic insulin signaling. CONCLUSIONS: Collectively, these studies reveal new and unexpected effects of lipin 1 on hepatic TAG metabolism and obesity-related hepatic insulin resistance.

PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity.

Regulated proinsulin biosynthesis, disulfide bond formation and ER redox homeostasis are essential to prevent Type two diabetes. In ß cells, protein disulfide isomerase A1 (PDIA1/P4HB), the most abundant ER oxidoreductase of over 17 members, can interact with proinsulin to influence disulfide maturation. Here we find Pdia1 is required for optimal insulin production under metabolic stress in vivo. ß cell-specific Pdia1 deletion in young high-fat diet fed mice or aged mice exacerbated glucose intolerance with inadequate insulinemia and increased the proinsulin/insulin ratio in both serum and islets compared to wildtype mice. Ultrastructural abnormalities in Pdia1-null ß cells include diminished insulin granule content, ER vesiculation and distention, mitochondrial swelling and nuclear condensation. Furthermore, Pdia1 deletion increased accumulation of disulfide-linked high molecular weight proinsulin complexes and islet vulnerability to oxidative stress. These findings demonstrate that PDIA1 contributes to oxidative maturation of proinsulin in the ER to support insulin production and ß cell health.