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The β-isoform of protein kinase C (PKC) has paradoxically been suggested to be important for both insulin action and insulin resistance as well as for contributing to the pathogenesis of diabetic complications. Presently, we evaluated the effects of knockout of the PKCβ gene on overall glucose homeostasis and insulin regulation of glucose transport. To evaluate subtle differences in glucose homeostasis in vivo, knockout mice were extensively backcrossed in C57BL/6 mice to diminish genetic differences other than the absence of the PKCβ gene. PKCβ−/− knockout offspring obtained through this backcrossing had 10% lower blood glucose levels than those observed in PKCβ+/+ wild-type offspring in both the fasting state and 30 min after ip injection of glucose despite having similar or slightly lower serum insulin levels. Also, compared with commercially obtained C57BL/6–129/SV hybrid control mice, serum glucose levels were similar, and serum insulin levels were similar or slightly lower, in C57BL/6–129/SV hybrid ...

The present investigation was designed to determine if atrial natriuretic peptides (ANPs) are increased in a spontaneous model of non-obese type 2 diabetes, the Goto-Kakizaki (GK) rat. Four peptide hormones originating from the ANP prohormone were increased twofold (P < .05) to sixfold (P < .01) in the circulation of GK rats compared with nondiabetic Wistar rats from which the GK colony was originally derived. Thus, ANP, long-acting natriuretic peptide (LANP), vessel dilator, and kaliuretic peptide were (mean +/- SE) 497 +/- 78, 1,285 +/- 105, 457 +/- 45, and 385 +/- 87 pg/mL in GK rats, versus 78 +/- 23, 542 +/- 77, 137 +/- 26, and 134 +/- 33 pg/mL, respectively, in Wistar rats. In evaluating the cause of the increased ANPs, the blood volume of GK rats (16.2 +/- 0.4 mL) was significantly (P < .01) increased compared with Wistar rats (9.5 +/- 0.3 mL). The ventricles of GK rats were not dilated when examined by transthoracic echocardiography, but the venous system was markedly distended. GK rats had a 48% to 79% decrease in renal function (ie, increased serum creatinine and blood urea nitrogen [BUN]) compared with Wistar rats. These results indicate that circulating ANPs are increased in the GK spontaneously diabetic rat secondary to (1) increased blood volume, which leads to increased synthesis and release of ANPs, and (2) renal failure, which results in a delayed metabolic processing of these peptides. The early combined increases of the four atrial peptides collectively may contribute to the hyperfiltration that occurs in early diabetes mellitus.

The activation of c-Jun N-terminal kinase (JNK) by insulin and anisomycin has been reported to result in increases in glycogen synthase (GS) activity in rat skeletal muscle (Moxham et al., J Biol Chem, 1996, 271:30765-30773). In addition, the protein kinase C (PKC) inhibitor, RO 31-8220, has been reported to activate JNK in rat-1 fibroblasts (Beltman et al., J Biol Chem, 1996, 271:27018-27024). Presently, we found that the RO 31-8220, as well as insulin, activated JNK and GS and stimulated glucose incorporation into glycogen in rat adipocytes and L6 myotubes. In contrast to activation of JNK, RO 31-8220 inhibited extracellular response kinases 1 and 2 (ERK1/2) and had no significant effects on protein kinase B (PKB). Stimulatory effects of RO 31-8220 on JNK and glycogen metabolism were not explained by PKC inhibition, as other PKC inhibitors were without effect on glucose incorporation into glycogen and have no effect on JNK (Beltman et al., J Biol Chem, 1996, 271:27018). Insulin, on the other hand, activated JNK, as well as PKB and ERK1/2. However, stimulatory effects of insulin on GS and glucose incorporation into glycogen appeared to be fully intact and additive to those of RO 31-8220, despite the fact that insulin did not provoke additive increases in JNK activity above those observed with RO 31-8220 alone. Our findings suggest that JNK serves to activate GS during the action of RO 31-8220 in rat adipocytes and L6 myotubes; insulin, on the other hand, appears to activate GS largely independently of JNK.

Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, is known to provoke insulin-like effects on GLUT4 translocation and glucose transport, but the underlying mechanism is obscure. Presently, we found in both rat adipocytes and 3T3/L1 adipocytes that okadaic acid provoked partial insulin-like increases in glucose transport, which were inhibited by phosphatidylinositol (PI) 3-kinase inhibitors, wortmannin and LY294002, and inhibitors of atypical protein kinase C (PKC) isoforms, zeta and lambda. Moreover, in both cell types, okadaic acid provoked increases in the activity of immunoprecipitable PKC-zeta/lambda by a PI 3-kinase-dependent mechanism. In keeping with apparent PI 3-kinase dependence of stimulatory effects of okadaic acid on glucose transport and PKC-zeta/lambda activity, okadaic acid provoked insulin-like increases in membrane PI 3-kinase activity in rat adipocytes; the mechanism for PI 3-kinase activation was uncertain, however, because it was not apparent in phosphotyrosine immunoprecipitates. Of further note, okadaic acid provoked partial insulin-like increases in the translocation of hemagglutinin antigen-tagged GLUT4 to the plasma membrane in transiently transfected rat adipocytes, and these stimulatory effects on hemagglutinin antigen-tagged GLUT4 translocation were inhibited by co-expression of kinase-inactive forms of PKC-zeta and PKC-lambda but not by a double mutant (T308A, S473A), activation-resistant form of protein kinase B. Our findings suggest that, as with insulin, PI 3-kinase-dependent atypical PKCs, zeta and lambda, are required for okadaic acid-induced increases in GLUT4 translocation and glucose transport in rat adipocytes and 3T3/L1 adipocytes.

The synthesis of cholesterol esters by acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) is an important component of cellular cholesterol homeostasis. Cholesterol ester formation also is hypothesized to be important in several physiologic processes, including intestinal cholesterol absorption, hepatic lipoprotein production, and macrophage foam cell formation in atherosclerotic lesions. Mouse tissue expression studies and the disruption of the mouse ACAT gene (Acact) have indicated that more than one ACAT exists in mammals and specifically that another enzyme is important in mouse liver and intestine. We now describe a second mammalian ACAT enzyme, designated ACAT-2, that is 44% identical to the first cloned mouse ACAT (henceforth designated ACAT-1). Infection of H5 insect cells with an ACAT-2 recombinant baculovirus resulted in expression of a ∼46-kDa protein in cell membranes that was associated with high levels of cholesterol esterification activity. Both ACAT-1 and ACAT-2 also catalyzed the esterification of the 3β-hydroxyl group of a variety of oxysterols. Cholesterol esterification activities for ACAT-1 and ACAT-2 exhibited different IC50 values when assayed in the presence of several ACAT-specific inhibitors, demonstrating that ACAT inhibitors can selectively target specific forms of ACAT. ACAT-2 was expressed primarily in mouse liver and small intestine, supporting the hypothesis that ACAT-2 contributes to cholesterol esterification in these tissues. The mouse ACAT-2 gene (Acact2) maps to chromosome 15 in a region containing a quantitative trait locus influencing plasma cholesterol levels. The identification and cloning of ACAT-2 will facilitate molecular approaches to understanding the role of ACAT enzymes in mammalian biology.

Recent experiments have identified LXR α , a nuclear hormone receptor that binds oxysterols, as a key regulator of hepatic cholesterol homeostasis.

Acyl coenzyme A:cholesterol acyltransferase (ACAT) (EC 2.3.1.26) is an enzyme, located in the endoplasmic reticulum of many types of cells, that catalyzes cholesterol ester formation from cholesterol and fatty acyl CoA substrates. Sterol esterification by ACAT or homologous enzymes is conserved in evolution dating back to yeast. The recent cloning of a human cDNA for ACAT, together with genome sequencing projects, has led to the identification of an ACAT gene family and provided molecular tools for determining ACAT's functions in vivo. Summarized here is the current knowledge concerning the molecular genetics of ACAT.

Electroporation of rat adipocytes with guanosine 5′-3-O-(thio)triphosphate (GTPγS) elicited sizable insulin-like increases in glucose transport and GLUT4 translocation. Like insulin, GTPγS activated membrane phosphatidylinositol (PI) 3-kinase in rat adipocytes, but, unlike insulin, this activation was blocked by Clostridium botulinum C3 transferase, suggesting a requirement for the small G-protein, RhoA. Also suggesting that Rho may operate upstream of PI 3-kinase during GTPγS action, the stable overexpression of Rho in 3T3/L1 adipocytes provoked increases in membrane PI 3-kinase activity. As with insulin treatment, GTPγS stimulation of glucose transport in rat adipocytes was blocked by C3 transferase, wortmannin, LY294002, and RO 31-8220; accordingly, the activation of glucose transport by GTPγS, as well as insulin, appeared to require Rho, PI 3-kinase, and another downstream kinase, e.g. protein kinase C-ζ (PKC-ζ) and/or protein kinase N (PKN). Whereas insulin activated both PKN and PKC-ζ, GTPγS activated PKN but not PKC-ζ. In transfection studies in 3T3/L1 cells, stable expression of wild-type Rho and PKN activated glucose transport, and dominant-negative forms of Rho and PKN inhibited insulin-stimulated glucose transport. In transfection studies in rat adipocytes, transient expression of wild-type and constitutive Rho and wild-type PKN provoked increases in the translocation of hemagglutinin (HA)-tagged GLUT4 to the plasma membrane; in contrast, transient expression of dominant-negative forms of Rho and PKN inhibited the effects of both insulin and GTPγS on HA-GLUT4 translocation. Our findings suggest that (a) GTPγS and insulin activate Rho, PI 3-kinase, and PKN, albeit by different mechanisms; (b) each of these signaling substances appears to be required for, and may contribute to, increases in glucose transport; and (c) PKC-ζ may contribute to increases in glucose transport during insulin, but not GTPγS, action.

Although cholesterol has long been known to be an essential component of cell membranes in vertebrate organisms, recent studies have suggested that cholesterol plays a crucial role in specific processes during embryonic development, including the covalent modification of Hedgehog proteins. Here we review the overlapping developmental phenotypes associated with pharmacologically or genetically induced defects in cholesterol biosynthesis, embryonic cholesterol transport and Hedgehog proteins. Shared aspects of these phenotypes suggest that common mechanisms underlie impaired central nervous system development associated with cholesterol deficiency.

ald, a recessive allele in AKR inbred mice, is responsible for complete adrenocortical lipid depletion in postpubertal males, which appears to be androgen dependent. Two recent observations (adrenocortical lipid depletion in acyl-CoA:cholesterol acyltransferase-deficient (Acact-/-) mice and the mapping of Acact to a region of chromosome 1 containing the ald locus) prompted us to ask whether adrenocortical lipid depletion in AKR mice results from an Acact mutation. Refined genetic mapping of Acact and ald was consistent with colocalization of these loci. Crossing Acact-/- with AKR (ald/ald) mice yielded postpubertal male offspring characterized by adrenocortical lipid depletion, indicating that these loci are not complementational and are therefore allelic. Immunoblotting of preputial gland homogenates demonstrated that AKR mice had an ACAT protein with a lower molecular mass than other mouse strains. Analysis of Acact cDNA from AKR mice revealed a deletion of the first coding exon and two missense mutations. Despite these coding sequence differences, the ACAT protein from the ald allele catalyzed cholesterol esterification activity at levels similar to that of wild-type protein. We speculate that the adrenocortical lipid depletion resulting from the ald mutation is caused by an altered susceptibility of the mutant protein to modifying factors, such as androgen production at puberty, in an as yet undetermined manner.

Atherosclerosis is the product of excessive lipid accumulation and inflammation in the artery wall. The lipid that tops every list of suspects is cholesterol, which is the primary lipid in low-density lipoproteins (LDL). Cholesterol exists either as a simple molecule or as cholesterol esters, in which the hydroxyl group is linked to a fatty acyl moiety. In cells, cholesterol esters are synthesized in an intracellular reaction catalyzed by acyl coenzyme A (CoA):cholesterol acyltransferase (ACAT) enzymes.1,2 Owing to the discoveries of cholesterol esters in arterial lesions in 19103 and of ACAT activity in the mid 1900s,4 inhibiting ACAT has been considered as a strategy for preventing or treating atherosclerosis. Over the past 25 years, interest in ACAT inhibitors has waxed and waned as new studies advance knowledge in the field. Prominently reported and disappointing results from a recent human trial of an ACAT inhibitor5 dampened enthusiasm for this potential therapy. However, a study by Bell et al in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology 6 revives the idea of targeting ACAT enzymes and highlights a key unanswered question: Can ACAT2-specific inhibitors lower plasma cholesterol and treat or prevent atherosclerosis? See page 1814 After the discovery of the ACAT reaction, two rationales for inhibiting ACAT emerged. One, in retrospect, was perhaps overly simplistic: blocking cholesterol esterification in macrophages would diminish macrophage “foam cell” formation and thereby decrease atherosclerotic lesion development. The other was to decrease hepatic and intestinal cholesterol ester formation, resulting in decreases in plasma levels …

Monocyte chemoattractant protein-1 (MCP-1) is a potent agonist for mononuclear leukocytes and has been implicated in the pathogenesis of atherosclerosis and granulomatous lung disease. To determine the role of MCP-1 and related family members in vivo, we used homologous recombination in embryonic stem cells to generate mice with a targeted disruption of C-C chemokine receptor 2 (CCR2), the receptor for MCP-1. CCR2-/- mice were born at the expected Mendelian ratios and developed normally. In response to thioglycollate, the recruitment of peritoneal macrophages decreased selectively. In in vitro chemotaxis assays, CCR2-/- leukocytes failed to migrate in response to MCP-1. Granulomatous lung disease was induced in presensitized mice by embolization with beads coupled to purified protein derivative (PPD) of Mycobacterium bovis. As compared with wild-type littermates, CCR2-/- mice had a decrease in granuloma size accompanied by a dramatic decrease in the level of interferon gamma in the draining lymph nodes. Production of interferon gamma was also decreased in PPD-sensitized splenocytes from CCR2-/- mice and in naive splenocytes activated by concanavalin A. We conclude that CCR2-/- mice have significant defects in both delayed-type hypersensitivity responses and production of Th1-type cytokines. These data suggest an important and unexpected role for CCR2 activation in modulating the immune response, as well as in recruiting monocytes/macrophages to sites of inflammation.

Cholesterol esterification is involved in the regulation of cellular cholesterol content and has been hypothesized to play a role in important physiologic processes including intestinal cholesterol absorption, hepatic lipoprotein production, and macrophage foam cell formation in atherosclerotic lesions. Although initial studies of the mouse acyl CoA:cholesterol acyltransferase gene (Acact) suggested that its gene product was responsible for cholesterol esterification in most tissues, we observed recently that Acact-/- mice have only tissue-specific reductions in cholesterol esterification. To better understand the role of Acact in cholesterol esterification, we used in situ hybridization and immunoblotting to perform tissue expression studies in wild-type mice. We found high levels of Acact expression in steroidogenic tissues, sebaceous glands, and atherosclerotic lesions, but not in the liver or the small intestine. These data support the hypothesis that multiple cholesterol esterification enzymes exist in mammals and that another enzyme is likely to be responsible for cholesterol esterification activity in mouse liver and intestine.

Insulin reportedly (Standaert, M. L., Avignon, A., Yamada, K., Bandyopadhyay, G., and Farese, R. V. (1996) Biochem. J. 313, 1039-1046) activates phospholipase D (PLD)-dependent hydrolysis of phosphatidylcholine (PC) in plasma membranes of rat adipocytes by a mechanism that may involve wortmannin-sensitive phosphatidylinositol (PI) 3-kinase. Because Rho and ADP ribosylation factor (ARF) activate PC-PLD, we questioned whether these small G-proteins are regulated by insulin and PI 3-kinase. We found that insulin provoked a rapid translocation of both Rho and ARF to the plasma membrane and increased GTP loading of Rho. Wortmannin and LY294002 inhibited Rho translocation in intact adipocytes, and the polyphosphoinositide, PI 4,5-(PO4)2, stimulated Rho translocation in adipocyte homogenates. On the other hand, wortmannin did not block GTP loading of Rho. Guanosine 5′-3-O-(thio)triphosphate stimulated both Rho and ARF translocation and activated PC-PLD in homogenates. C3 transferase, which inhibits and depletes Rho, inhibited PC-PLD activation by insulin in intact adipocytes. C3 transferase also inhibited insulin stimulation of [3H]2-deoxyglucose uptake. Our findings suggest that: (a) insulin translocates Rho by a PI 3-kinase-dependent mechanism, but another factor is responsible for GTP loading of Rho; (b) both Rho and ARF may contribute to PC-PLD activation during insulin action; and (c) Rho may be required for insulin stimulation of glucose transport.

We presently studied (a) insulin effects on protein kinase C (PKC) and (b) effects of transfection-induced, stable expression of PKC isoforms on glucose transport in 3T3/L1 cells. In both fibroblasts and adipocytes, insulin provoked increases in membrane PKC enzyme activity and membrane levels of PKC-alpha and PKC-beta. However, insulin-induced increases in PKC enzyme activity were apparent in both non-down-regulated adipocytes and adipocytes that were down-regulated by overnight treatment with 5 microM phorbol ester, which largely depletes PKC-alpha, PKC-beta, and PKC-epsilon, but not PKC-zeta. Moreover, insulin provoked increases in the enzyme activity of immunoprecipitable PKC-zeta. In transfection studies, stable overexpression of wild-type or constitutively active forms of PKC-alpha, PKC-beta1, and PKC-beta2 failed to influence basal or insulin-stimulated glucose transport (2-deoxyglucose uptake) in fibroblasts and adipocytes, despite inhibiting insulin effects on glycogen synthesis. In contrast, stable overexpression of wild-type PKC-zeta increased, and a dominant-negative mutant form of PKC-zeta decreased, basal and insulin-stimulated glucose transport in fibroblasts and adipocytes. These findings suggested that: (a) insulin activates PKC-zeta, as well as PKC-alpha and beta; and (b) PKC-zeta is required for, and may contribute to, insulin effects on glucose transport in 3T3/L1 cells.

Transactive response DNA-binding protein 43 (TDP-43) is a major pathological protein in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). There are many disease-associated mutations in TDP-43, and several cellular and animal models with ectopic overexpression of mutant TDP-43 have been established. Here we sought to study altered molecular events in FTD and ALS by using induced pluripotent stem cell (iPSC) derived patient neurons. We generated multiple iPSC lines from an FTD/ALS patient with the TARDBP A90V mutation and from an unaffected family member who lacked the mutation. After extensive characterization, two to three iPSC lines from each subject were selected, differentiated into postmitotic neurons, and screened for relevant cell-autonomous phenotypes. Patient-derived neurons were more sensitive than control neurons to 100 nM straurosporine but not to other inducers of cellular stress. Three disease-relevant cellular phenotypes were revealed under staurosporine-induced stress. First, TDP-43 was localized in the cytoplasm of a higher percentage of patient neurons than control neurons. Second, the total TDP-43 level was lower in patient neurons with the A90V mutation. Third, the levels of microRNA-9 (miR-9) and its precursor pri-miR-9-2 decreased in patient neurons but not in control neurons. The latter is likely because of reduced TDP-43, as shRNA-mediated TDP-43 knockdown in rodent primary neurons also decreased the pri-miR-9-2 level. The reduction in miR-9 expression was confirmed in human neurons derived from iPSC lines containing the more pathogenic TARDBP M337V mutation, suggesting miR-9 downregulation might be a common pathogenic event in FTD/ALS. These results show that iPSC models of FTD/ALS are useful for revealing stress-dependent cellular defects of human patient neurons containing rare TDP-43 mutations in their native genetic contexts.

Atypical protein kinase C (aPKC) levels and activity are elevated in hepatocytes of individuals with type 2 diabetes and cause excessive increases in the levels of lipogenic and gluconeogenic enzymes; aPKC inhibitors largely correct these aberrations. Metformin improves hepatic gluconeogenesis by activating 5'-AMP-activated protein kinase (AMPK). However, metformin also activates aPKC in certain tissues; in the liver, this activation could amplify diabetic aberrations and offset the positive effects of AMPK. In this study, we examined whether metformin activates aPKC in human hepatocytes and the metabolic consequences of any such activation.We compared protein kinase activities and alterations in lipogenic and gluconeogenic enzyme levels during activity of the AMPK activators metformin and AICAR, relative to those of an aPKC-ι inhibitor, in hepatocytes from non-diabetic and type 2 diabetic human organ donors.Metformin and 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) activated aPKC at concentrations comparable with those required for AMPK activation. Moreover, both agents increased lipogenic enzyme levels by an aPKC-dependent mechanism. Thus, whereas insulin- and diabetes-dependent increases in lipogenic enzyme levels were reversed by aPKC inhibition, such levels were increased in hepatocytes from non-diabetic donors and remained elevated in hepatocytes from diabetic donors following metformin and AICAR treatment. In addition, whereas aPKC inhibition diminished gluconeogenic enzyme levels in the absence and presence of insulin in hepatocytes from both non-diabetic and diabetic donors, metformin and AICAR increased gluconeogenic enzyme levels in hepatocytes from non-diabetic individuals, but nevertheless diminished gluconeogenic enzyme levels in insulin-treated hepatocytes from diabetic donors.Metformin and AICAR activate aPKC together with AMPK in human hepatocytes. Activation of aPKC increases lipogenic enzyme levels and alters gluconeogenic enzyme levels, and therefore appears to offset the positive effects of AMPK.

Cancer cells commonly have characteristic changes in metabolism. Cellular proliferation, a common feature of all cancers, requires fatty acids for synthesis of membranes and signaling molecules. Here, we provide a view of cancer cell metabolism from a lipid perspective, and we summarize evidence that limiting fatty acid availability can control cancer cell proliferation.

Progranulin (PGRN) is a secreted glycoprotein expressed in neurons and glia that is implicated in neuronal survival on the basis that mutations in theGRNgene causing haploinsufficiency result in a familial form of frontotemporal dementia (FTD). Recently, a direct interaction between PGRN and tumor necrosis factor receptors (TNFR I/II) was reported and proposed to be a mechanism by which PGRN exerts anti-inflammatory activity, raising the possibility that aberrant PGRN–TNFR interactions underlie the molecular basis for neuroinflammation in frontotemporal lobar degeneration pathogenesis. Here, we report that we find no evidence for a direct physical or functional interaction between PGRN and TNFRs. Using coimmunoprecipitation and surface plasmon resonance (SPR) we replicated the interaction between PGRN and sortilin and that between TNF and TNFRI/II, but not the interaction between PGRN and TNFRs. Recombinant PGRN or transfection of a cDNA encoding PGRN did not antagonize TNF-dependent NFκB, Akt, and Erk1/2 pathway activation; inflammatory gene expression; or secretion of inflammatory factors in BV2 microglia and bone marrow-derived macrophages (BMDMs). Moreover, PGRN did not antagonize TNF-induced cytotoxicity on dopaminergic neuroblastoma cells. Last, co-addition or pre-incubation with various N- or C-terminal-tagged recombinant PGRNs did not alter lipopolysaccharide-induced inflammatory gene expression or cytokine secretion in any cell type examined, including BMDMs fromGrn+/− orGrn−/− mice. Therefore, the neuroinflammatory phenotype associated with PGRN deficiency in the CNS is not a direct consequence of the loss of TNF antagonism by PGRN, but may be a secondary response by glia to disrupted interactions between PGRN and Sortilin and/or other binding partners yet to be identified.

Frontotemporal dementia (FTD) is a neurodegenerative disease with hallmark deficits in social and emotional function. Heterozygous loss-of-function mutations inGRN, the progranulin gene, are a common genetic cause of the disorder, but the mechanisms by which progranulin haploinsufficiency causes neuronal dysfunction in FTD are unclear. Homozygous progranulin knock-out (Grn−/−) mice have been studied as a model of this disorder and show behavioral deficits and a neuroinflammatory phenotype with robust microglial activation. However, homozygousGRNmutations causing complete progranulin deficiency were recently shown to cause a different neurological disorder, neuronal ceroid lipofuscinosis, suggesting that the total absence of progranulin may have effects distinct from those of haploinsufficiency. Here, we studied progranulin heterozygous (Grn+/−) mice, which model progranulin haploinsufficiency. We found thatGrn+/−mice developed age-dependent social and emotional deficits potentially relevant to FTD. However, unlikeGrn−/−mice, behavioral deficits inGrn+/−mice occurred in the absence of gliosis or increased expression of tumor necrosis factor-α. Instead, we found neuronal abnormalities in the amygdala, an area of selective vulnerability in FTD, inGrn+/−mice. Our findings indicate that FTD-related deficits resulting from progranulin haploinsufficiency can develop in the absence of detectable gliosis and neuroinflammation, thereby dissociating microglial activation from functional deficits and suggesting an important effect of progranulin deficiency on neurons.

Frontotemporal degeneration (FTD) is a common cause of dementia for which there are currently no approved therapies. Over the past decade, there has been an explosion of knowledge about the biology and clinical features of FTD that has identified a number of promising therapeutic targets as well as animal models in which to develop drugs. The close association of some forms of FTD with neuropathological accumulation of tau protein or increased neuroinflammation due to progranulin protein deficiency suggests that a drug's success in treating FTD may predict efficacy in more common diseases such as Alzheimer's disease. A variety of regulatory incentives, clinical features of FTD such as rapid disease progression, and relatively pure molecular pathology suggest that there are advantages to developing drugs for FTD as compared with other more common neurodegenerative diseases such as Alzheimer's disease. In March 2011, the Frontotemporal Degeneration Treatment Study Group sponsored a conference entitled "FTD, the Next Therapeutic Frontier," which focused on preclinical aspects of FTD drug development. The goal of the meeting was to promote collaborations between academic researchers and biotechnology and pharmaceutical researchers to accelerate the development of new treatments for FTD. Here we report the key findings from the conference, including the rationale for FTD drug development; epidemiological, genetic, and neuropathological features of FTD; FTD animal models and how best to use them; and examples of successful drug development collaborations in other neurodegenerative diseases.

The triglyceride-synthesizing enzyme acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) plays a critical role in hepatitis C virus (HCV) infection by recruiting the HCV capsid protein core onto the surface of cellular lipid droplets (LDs). Here we find a new interaction between the non-structural protein NS5A and DGAT1 and show that the trafficking of NS5A to LDs depends on DGAT1 activity. DGAT1 forms a complex with NS5A and core and facilitates the interaction between both viral proteins. A catalytically inactive mutant of DGAT1 (H426A) blocks the localization of NS5A, but not core, to LDs in a dominant-negative manner and impairs the release of infectious viral particles, underscoring the importance of DGAT1-mediated translocation of NS5A to LDs in viral particle production. We propose a model whereby DGAT1 serves as a cellular hub for HCV core and NS5A proteins, guiding both onto the surface of the same subset of LDs, those generated by DGAT1. These results highlight the critical role of DGAT1 as a host factor for HCV infection and as a potential drug target for antiviral therapy.

Progranulin is a secreted glycoprotein, and the GRN gene is mutated in some cases of frontotemporal dementia. Progranulin has also been implicated in cell growth, wound healing, inflammation, and cancer. We investigated the molecular nature of secreted progranulin and provide evidence that progranulin exists as a homodimer. Although recombinant progranulin has a molecular mass of ∼85 kDa by SDS-PAGE, it elutes in fractions corresponding to ∼170–180 kDa by gel-filtration chromatography. Additionally, recombinant progranulin can be intermolecularly cross-linked, yielding a complex corresponding to a dimer (∼180 kDa), and progranulins containing different epitope tags physically interact. In plasma, progranulin similarly forms complexes of ∼180–190 kDa. Although progranulin partially co-fractionated with high density lipoproteins (HDL) by gel-filtration chromatography, we found no evidence that progranulin in mouse or human plasma is a component of HDL either by ultracentrifugation or by lipid binding assays. We conclude that circulating progranulin exists as a dimer and is not likely a component of HDL.

Lipid droplets (LDs) are dynamic, cytosolic lipid-storage organelles found in nearly all cell types. Too many or too few LDs during excess or deficient fat storage lead to many different human diseases. Recent insights into LD biology and LD protein functions shed new light on mechanisms underlying those metabolic pathologies. These findings will likely provide opportunities for treatment of diseases associated with too much or too little fat.

Biological membranes are complex, and the mechanisms underlying their homeostasis are incompletely understood. Here, we present a quantitative genetic interaction map (E-MAP) focused on various aspects of lipid biology, including lipid metabolism, sorting, and trafficking. This E-MAP contains ∼250,000 negative and positive genetic interaction scores and identifies a molecular crosstalk of protein quality control pathways with lipid bilayer homeostasis. Ubx2p, a component of the endoplasmic-reticulum-associated degradation pathway, surfaces as a key upstream regulator of the essential fatty acid (FA) desaturase Ole1p. Loss of Ubx2p affects the transcriptional control of OLE1, resulting in impaired FA desaturation and a severe shift toward more saturated membrane lipids. Both the induction of the unfolded protein response and aberrant nuclear membrane morphologies observed in cells lacking UBX2 are suppressed by the supplementation of unsaturated FAs. Our results point toward the existence of dedicated bilayer stress responses for membrane homeostasis.

Insulin provokes rapid changes in phospholipid metabolism and thereby generates biologically active lipids that serve as intracellular signaling factors that regulate glucose transport and glycogen synthesis. These changes include: (i) activation of phosphatidylinositol 3-kinase (PI3K) and production of PIP3; (ii) PIP3-dependent activation of atypical protein kinase Cs (PKCs); (iii) PIP3-dependent activation of PKB; (iv) PI3K-dependent activation of phospholipase D and hydrolysis of phosphatidylcholine with subsequent increases in phosphatidic acid (PA) and diacylglycerol (DAG); (v) PI3K-independent activation of glycerol-3-phosphate acylytansferase and increases in de novo synthesis of PA and DAG; and (vi) activation of DAG-sensitive PKCs. Recent findings suggest that atypical PKCs and PKB serve as important positive regulators of insulin-stimulated glucose metabolism, whereas mechanisms that result in the activation of DAG-sensitive PKCs serve mainly as negative regulators of insulin signaling through PI3K. Atypical PKCs and PKB are rapidly activated by insulin in adipocytes, liver, skeletal muscles, and other cell types by a mechanism requiring PI3K and its downstream effector, 3-phosphoinositide-dependent protein kinase-1 (PDK-1), which, in conjunction with PIP3, phosphorylates critical threonine residues in the activation loops of atypical PKCs and PKB. PIP3 also promotes increases in autophosphorylation and allosteric activation of atypical PKCs. Atypical PKCs and perhaps PKB appear to be required for insulin-induced translocation of the GLUT 4 glucose transporter to the plasma membrane and subsequent glucose transport. PKB also appears to be the major regulator of glycogen synthase. Together, atypical PKCs and PKB serve as a potent, integrated PI3K/PDK-1-directed signaling system that is used by insulin to regulate glucose metabolism.

Apolipoprotein (apo)-B is found in two forms in mammals: apo-B100, which is made in the liver and the yolk sac, and apo-B48, a truncated protein made in the intestine. To provide models for understanding the physiologic purpose for the two forms of apo-B, we used targeted mutagenesis of the apo-B gene to generate mice that synthesize exclusively apo-B48 (apo-B48-only mice) and mice that synthesize exclusively apo-B100 (apo-B100-only mice). Both the apo-B48-only mice and apo-B100-only mice developed normally, were healthy, and were fertile. Thus, apo-B48 synthesis was sufficient for normal embryonic development, and the synthesis of apo-B100 in the intestines of adult mice caused no readily apparent adverse effects on intestinal function or nutrition. Compared with wild-type mice fed a chow diet, the levels of low density lipoprotein (LDL)-cholesterol and very low density lipoprotein- and LDL-triacylglycerols were lower in apo-B48-only mice and higher in the apo-B100-only mice. In the setting of apo-E-deficiency, the apo-B100-only mutation lowered cholesterol levels, consistent with the fact that apo-B100-lipoproteins can be cleared from the plasma via the LDL receptor, whereas apo-B48-lipoproteins lacking apo-E cannot. The apo-B48-only and apo-B100-only mice should prove to be valuable models for experiments designed to understand the purpose for the two forms of apo-B in mammalian metabolism.

Initial studies suggested that insulin increases diacylglycerol and activates protein kinase C (PKC) in BC3H-1 myocytes. In these earlier studies, insulin was found to translocate PKC-β, but the presence of PKC-ϵ was not appreciated. More recently, the presence of PKC-ϵ was documented, but PKC-β was not detected, and it was questioned whether insulin activates PKC in BC3H-1 myocytes [Stumpo, D.J., Haupt, D.M. and Blackshear, P.J. (1994)J. Biol. Chem. 269:21184–21190]. We questioned whether insulin translocates PKC-ϵ in BC3H-1 myocytes, and re-evaluated the question of whether myocytes truly contain a PKC-β isoform whose existence can be verified by its response to phorbol ester treatment. We found that PKC-ϵ was acutely translocated by insulin and phorbol esters from the cytosol to the membrane fraction in BC3H-1 myocytes; in addition, PKC-ϵ, like PKC-α, was depleted by chronic phorbol ester treatment. We also found that BC3H-I myocytes containing a 76,000 Mr PKC-β isoform that is acutely translocated and subsequently depleted by phorbol esters. Moreover, chronic phorbol ester treatment induced an 84,000 Mr PKC-β2 isoform that appeared to be persistently translocated and activated, as suggested by studies of myristoylated arginine-rich C kinase substrate (MARCKS) phosphorylation. We conclude that: (1) insulin acutely translocates PKC-ϵ, as well as PKC-β, in BC3H-1 myocytes; and (2) PKC-β is not truly downregulated by phorbol esters in BC3H-1 myocytes.

The integrin alpha v beta 6 is only expressed in epithelial cells. In healthy adult epithelia, this receptor is barely detectable, but expression is rapidly induced following epithelial injury. Mice homozygous for a null mutation in the gene encoding the beta 6 subunit had juvenile baldness associated with infiltration of macrophages into the skin, and accumulated activated lymphocytes around conducting airways in the lungs. Beta 6-/- mice also demonstrated airway hyperresponsiveness to acetylcholine, a hallmark feature of asthma. These results suggest that the epithelial integrin alpha v beta 6 participates in the modulation of epithelial inflammation. Genetic or acquired alterations in this integrin could thus contribute to the development of inflammatory diseases of epithelial organs, such as the lungs and skin.

Apolipoprotein (apo) B, the principal structural component necessary for the synthesis and secretion of triglyceride-rich lipoproteins by the intestine and liver, is highly expressed in the yolk sac visceral endoderm of mammals, although its function in this tissue has been hitherto unclear. Disruption of the apoB gene in mice results in embryonic lethality (approximately 9.5 - 10.5 d). Here we demonstrate that apoB is normally expressed at early time points in embryonic development in yolk sac visceral endodermal cells, and that this expression is associated with the synthesis and secretion of apoB-containing lipoproteins. The lack of apoB in the visceral endoderm resulted in an accumulation of intracellular lipid droplets, an absence of lipoproteins from the secretory pathway, and reduced concentrations of cholesterol and alpha-tocopherol in tissues of apoB-/- embryos. Visceral endoderm of apoB+/- embryos exhibited an intermediate phenotype. Our results suggest that apoB plays an essential role in the transport of lipid nutrients to the developing mouse embryo via the yolk sac-mediated synthesis and secretion of apoB-containing lipoproteins.

We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulated, phospholipase D-dependent PC hydrolysis and subsequent translocation of PKC-alpha and PKC-beta to the plasma membrane. Wortmannin did not inhibit PKC directly in vitro, or the PKC-dependent effects of phorbol esters on glucose transport in intact adipocytes. The PKC inhibitor RO 31-8220 did not inhibit PI 3-kinase directly or its activation in situ by insulin, but inhibited both insulin-stimulated and phorbol ester-stimulated glucose transport. Our findings suggest that insulin acts through PI 3-kinase to activate a PC-specific phospholipase D and causes the translocative activation of PKC-alpha and PKC-beta in plasma membranes of rat adipocytes.

As part of an effort to dissect the genetic factors involved in cholesterol homeostasis in the mouse model, we report the mapping of 12 new candidate genes using linkage analysis. The genes include: cytoplasmic HMG-CoA synthase (Hmgcs 1, Chr 13), mitochondrial synthase (Hmgcs 2, Chr 3), a synthase-related sequence (Hmgcs 1-rs, Chr 12), mevalonate kinase (Mvk, Chr 5), farnesyl diphosphate synthase (Fdps, Chr 3), squalene synthase (Fdft 1, Chr 14), acyl-CoA:cholesterol acyltransferase (Acact, Chr 1), sterol regulatory element binding protein-1 (Srebf1, Chr 8) and -2 (Srebf2, Chr 15), apolipoprotein A-I regulatory protein (Tcfcoup2, Chr 7), low density receptor-related protein-related sequence (Lrp-rs, Chr 10), and Lrp-associated protein (Lrpap 1, Chr 5). In addition, the map positions for several lipoprotein receptor genes were refined. These genes include: low density lipoprotein receptor (Ldlr, Chr 9), very low density lipoprotein receptor (Vldlr, Chr 19), and glycoprotein 330 (Gp330, Chr 2). Some of these candidate genes are located within previously defined chromosomal regions (quantitative trait loci, QTLs) contributing to plasma lipoprotein levels, and Acact maps near a mouse mutation, ald, resulting in depletion of cholesteryl esters in the adrenals. The combined use of QTL and candidate gene mapping provides a powerful means of dissecting complex traits such as cholesterol homeostasis.

Effects of insulin of levels of mRNA encoding protein kinase C (PKC)-alpha, PKC-beta, PKC-epsilon and PKC-theta were examined by ribonuclease protection assay in primary cultures of rat adipocytes in vitro, and in rat adipose tissue and gastrocnemius muscle in vivo. In all cases, insulin increased the levels of PKC-alpha mRNA and PKC-beta mRNA, and, in muscle, insulin also increased the level of PKC-theta mRNA. PKC-epsilon mRNA levels, on the other hand, were not altered significantly. Insulin also stimulated the apparent translocation of PKC-alpha, -beta, -epsilon and -theta, to the membrane fractions of adipocytes, adipose tissue and gastrocnemius muscles, and, in some instances, total PKC levels were diminished, e.g. PKC-alpha and PKC-beta in cultured adipocytes in vitro and/or whole adipose tissue in vivo, and PKC-alpha and PKC-theta in the gastrocnemius muscle. Thus, insulin-induced increases in PKC mRNA may have been partly compensatory in nature to restore PKC levels following translocation and proteolytic losses. However, much more severe depletion of PKC-alpha and PKC-beta by phorbol ester treatment in cultured rat adipocytes in vitro resulted in, if anything, smaller increases in PKC-alpha mRNA and PKC-beta mRNA, and it therefore appears that insulin effects on PKC mRNA levels were not simply due to decreases in respective PKC levels. In addition, effects of insulin, particularly on PKC-beta mRNA, could not be attributed to increased glucose metabolism, which alone decreased PKC-beta mRNA in cultured adipocytes in vitro. We conclude that insulin-induced translocation and degradation of PKC-alpha, PKC-beta and PKC-theta are attended by selective increases in their mRNAs. This mechanism of increasing mRNA may be important in maintaining PKC levels during the continued action of insulin.

Apolipoprotein B is synthesized by the intestine and the liver in mammals, where it serves as the main structural component in the formation of chylomicrons and very low density lipoproteins, respectively. Apolipoprotein B is also expressed in mammalian fetal membranes. To examine the consequences of apolipoprotein B deficiency in mice, we used gene targeting in mouse embryonic stem cells to generate mice containing an insertional disruption of the 5' region of the apolipoprotein B gene. Mice that were heterozygous for the disrupted apolipoprotein B allele had an approximately 20% reduction in plasma cholesterol levels, markedly reduced plasma concentrations of the pre-beta and beta-migrating lipoproteins, and an approximately 70% reduction in plasma apolipoprotein B levels. When fed a diet rich in fat and cholesterol, heterozygous mice were protected from diet-induced hypercholesterolemia; these mice, which constitute an animal model for hypobetalipoproteinemia, should be useful for studying the effects of decreased apolipoprotein B expression on atherogenesis. The breeding of heterozygous mice yielded no viable homozygous apolipoprotein B knockout mice. Most homozygous embryos were resorbed by midgestation (before gestational day 11.5); several embryos that survived until later in gestation exhibited exencephalus. The embryonic lethal phenotype was rescued by complementation with a human apolipoprotein B transgene--i.e., human apolipoprotein B transgenic mice that were homozygous for the murine apolipoprotein B knockout mutation were viable. Our findings indicate that apolipoprotein B plays an essential role in mouse embryonic development.

SummaryLipid droplets (LDs) are the major fat storage organelles in eukaryotic cells, but how their size is regulated is unknown. Using genetic screens in C. elegans for LD morphology defects in intestinal cells, we found that mutations in atlastin, a GTPase required for homotypic fusion of endoplasmic reticulum (ER) membranes, cause not only ER morphology defects, but also a reduction in LD size. Similar results were obtained after depletion of atlastin or expression of a dominant-negative mutant, whereas overexpression of atlastin had the opposite effect. Atlastin depletion in Drosophila fat bodies also reduced LD size and decreased triglycerides in whole animals, sensitizing them to starvation. In mammalian cells, co-overexpression of atlastin-1 and REEP1, a paralog of the ER tubule-shaping protein DP1/REEP5, generates large LDs. The effect of atlastin-1 on LD size correlates with its activity to promote membrane fusion in vitro. Our results indicate that atlastin-mediated fusion of ER membranes is important for LD size regulation.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease primarily affecting motor neurons. Mutations in the gene encoding TDP-43 cause some forms of the disease, and cytoplasmic TDP-43 aggregates accumulate in degenerating neurons of most individuals with ALS. Thus, strategies aimed at targeting the toxicity of cytoplasmic TDP-43 aggregates may be effective. Here, we report results from two genome-wide loss-of-function TDP-43 toxicity suppressor screens in yeast. The strongest suppressor of TDP-43 toxicity was deletion of DBR1, which encodes an RNA lariat debranching enzyme. We show that, in the absence of Dbr1 enzymatic activity, intronic lariats accumulate in the cytoplasm and likely act as decoys to sequester TDP-43, preventing it from interfering with essential cellular RNAs and RNA-binding proteins. Knockdown of Dbr1 in a human neuronal cell line or in primary rat neurons is also sufficient to rescue TDP-43 toxicity. Our findings provide insight into TDP-43-mediated cytotoxicity and suggest that decreasing Dbr1 activity could be a potential therapeutic approach for ALS.

Glucocorticoids are important regulators of lipid homeostasis, and chronically elevated glucocorticoid levels induce hypertriglyceridemia, hepatic steatosis, and visceral obesity. The occupied glucocorticoid receptor (GR) is a transcription factor. However, those genes regulating lipid metabolism under GR control are not fully known. Angiopoietin-like 4 (ANGPTL4, fasting-induced adipose factor), a protein inhibitor of lipoprotein lipase, is synthesized and secreted during fasting, when circulating glucocorticoid levels are physiologically increased. We therefore tested whether the ANGPTL4 gene (Angptl4) is transcriptionally controlled by GR. We show that treatment with the synthetic glucocorticoid dexamethasone increased Angptl4 mRNA levels in primary hepatocytes and adipocytes (2-3-fold) and in the livers and white adipose tissue of mice (approximately 4-fold). We tested the mechanism of this increase in H4IIE hepatoma cells and found that dexamethasone treatment increased the transcriptional rate of Angptl4. Using bioinformatics and chromatin immunoprecipitation, we identified a GR binding site within the rat Angptl4 sequence. A reporter plasmid containing this site was markedly activated by dexamethasone, indicative of a functional glucocorticoid response element. Dexamethasone treatment also increased histone H4 acetylation and DNase I accessibility in genomic regions near this site, further supporting that it is a glucocorticoid response element. Glucocorticoids promote the flux of triglycerides from white adipose tissue to liver. We found that mice lacking ANGPTL4 (Angptl4(-/-)) had reductions in dexamethasone-induced hypertriglyceridemia and hepatic steatosis, suggesting that ANGPTL4 is required for this flux. Overall, we establish that ANGPTL4 is a direct GR target that participates in glucocorticoid-regulated triglyceride metabolism.

Calorie restriction results in leanness, which is linked to metabolic conditions that favor longevity. We show here that deficiency of the triglyceride synthesis enzyme acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), which promotes leanness, also extends longevity without limiting food intake. Female DGAT1-deficient mice were protected from age-related increases in body fat, tissue triglycerides, and inflammation in white adipose tissue. This protection was accompanied by increased mean and maximal life spans of ~25% and ~10%, respectively. Middle-aged Dgat1−/− mice exhibited several features associated with longevity, including decreased levels of circulating insulin growth factor 1 (IGF1) and reduced fecundity. Thus, deletion of DGAT1 in mice provides a model of leanness and extended lifespan that is independent of calorie restriction.

The Goto-Kakizaki (G/K) rat is an animal model of non-insulin-dependent diabetes mellitus, with early hyperglycaemia, hyperinsulinaemia, and insulin resistance. We have studied the effect of insulin on the activation of glycogen synthase in the G/K rat and in the original parent strain, the Wistar rat. After insulin injection, glycogen synthase I activity, glycogen synthase phosphatase activity and glucose 6-phosphate content in skeletal muscle were significantly increased in the Wistar rats. In the G/K rats, insulin injection resulted in a reduced activation of skeletal muscle glycogen synthase, which was not significant when compared with the control rats without insulin, and no increases in glycogen synthase phosphatase and glucose 6-phosphate were seen. In adipose tissue the activation of glycogen synthase by insulin was normal in the G/K rats. Previous investigations have shown that glucose disappearance rates are low in the G/K rat. However, stimulation of glucose transport was reported to be normal in the G/K rat. A defective activation of glucose accumulation into glycogen by skeletal muscle may contribute to explain the hyperglycaemia in the G/K rat.

Pertussis toxin was used to block insulin-stimulated phosphatidylinositol (PI)-glycan hydrolysis, consequent de novo synthesis of phosphatidic acid (PA) and the diacylglycerol (DAG) production that results from these two related processes in BC3H-1 myocytes. In contrast, pertussis toxin pretreatment did not inhibit insulin-stimulated hydrolysis of phosphatidylcholine (PC) which was found to be at least partly due to activation of a phospholipase D. Moreover, pertussis toxin-insensitive PC hydrolysis was accompanied by rapid biphasic increases in DAG and translocative activation of protein kinase C (PKC). Insulin-stimulated glucose transport was also insensitive to pertussis toxin pretreatment. Our findings suggest that insulin-stimulated PC hydrolysis pays an important role in DAG/PKC signalling during insulin action.

Excessive activity of hepatic atypical protein kinase (aPKC) is proposed to play a critical role in mediating lipid and carbohydrate abnormalities in obesity, the metabolic syndrome, and type 2 diabetes mellitus. In previous studies of rodent models of obesity and type 2 diabetes mellitus, adenoviral-mediated expression of kinase-inactive aPKC rapidly reversed or markedly improved most if not all metabolic abnormalities. Here, we examined effects of 2 newly developed small-molecule PKC-ι/λ inhibitors. We used the mouse model of heterozygous muscle-specific knockout of PKC-λ, in which partial deficiency of muscle PKC-λ impairs glucose transport in muscle and thereby causes glucose intolerance and hyperinsulinemia, which, via hepatic aPKC activation, leads to abdominal obesity, hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia. One inhibitor, 1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)], binds to the substrate-binding site of PKC-λ/ι, but not other PKCs. The other inhibitor, aurothiomalate, binds to cysteine residues in the PB1-binding domains of aPKC-λ/ι/ζ and inhibits scaffolding. Treatment with either inhibitor for 7 days inhibited aPKC, but not Akt, in liver and concomitantly improved insulin signaling to Akt and aPKC in muscle and adipocytes. Moreover, both inhibitors diminished excessive expression of hepatic, aPKC-dependent lipogenic, proinflammatory, and gluconeogenic factors; and this was accompanied by reversal or marked improvements in hyperglycemia, hyperinsulinemia, abdominal obesity, hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia. Our findings highlight the pathogenetic importance of insulin signaling to hepatic PKC-ι in obesity, the metabolic syndrome, and type 2 diabetes mellitus and suggest that 1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)] and aurothiomalate or similar agents that selectively inhibit hepatic aPKC may be useful treatments.

Lipid accumulation in organs is strongly associated with insulin resistance. However, the causal relationship is uncertain. For hepatic steatosis, this relationship has given rise to many studies and theories. One hypothesis, suggested as unifying (1), posits simply that accumulation of diacylglycerol induces hepatic insulin resistance by interfering with insulin signaling.

Acetylation is increasingly recognized as an important metabolic regulatory posttranslational protein modification, yet the metabolic consequence of mitochondrial protein hyperacetylation is unknown. We find that high-fat diet (HFD) feeding induces hepatic mitochondrial protein hyperacetylation in mice and downregulation of the major mitochondrial protein deacetylase SIRT3. Mice lacking SIRT3 (SIRT3KO) placed on a HFD show accelerated obesity, insulin resistance, hyperlipidemia, and steatohepatitis compared to wild-type (WT) mice. The lipogenic enzyme stearoyl-CoA desaturase 1 is highly induced in SIRT3KO mice, and its deletion rescues both WT and SIRT3KO mice from HFD-induced hepatic steatosis and insulin resistance. We further identify a single nucleotide polymorphism in the human SIRT3 gene that is suggestive of a genetic association with the metabolic syndrome. This polymorphism encodes a point mutation in the SIRT3 protein, which reduces its overall enzymatic efficiency. Our findings show that loss of SIRT3 and dysregulation of mitochondrial protein acetylation contribute to the metabolic syndrome.

Calorie restriction [CR; ∼65% of ad libitum (AL) intake] improves insulin-stimulated glucose uptake (GU) and Akt phosphorylation in skeletal muscle. We aimed to elucidate the effects of CR on 1) processes that regulate Akt phosphorylation [insulin receptor (IR) tyrosine phosphorylation, IR substrate 1-phosphatidylinositol 3-kinase (IRS-PI3K) activity, and Akt binding to regulatory proteins (heat shock protein 90, Appl1, protein phosphatase 2A)]; 2) Akt substrate of 160-kDa (AS160) phosphorylation on key phosphorylation sites; and 3) atypical PKC (aPKC) activity. Isolated epitrochlearis (fast-twitch) and soleus (slow-twitch) muscles from AL or CR (6 mo duration) 9-mo-old male F344BN rats were incubated with 0, 1.2, or 30 nM insulin and 2-deoxy-[3H]glucose. Some CR effects were independent of insulin dose or muscle type: CR caused activation of Akt (Thr308and Ser473) and GU in both muscles at both insulin doses without CR effects on IRS1-PI3K, Akt-PP2A, or Akt-Appl1. Several muscle- and insulin dose-specific CR effects were revealed. Akt-HSP90 binding was increased in the epitrochlearis; AS160 phosphorylation (Ser588and Thr642) was greater for CR epitrochlearis at 1.2 nM insulin; and IR phosphorylation and aPKC activity were greater for CR in both muscles with 30 nM insulin. On the basis of these data, our working hypothesis for improved insulin-stimulated GU with CR is as follows: 1) elevated Akt phosphorylation is fundamental, regardless of muscle or insulin dose; 2) altered Akt binding to regulatory proteins (HSP90 and unidentified Akt partners) is involved in the effects of CR on Akt phosphorylation; 3) Akt effects on GU depend on muscle- and insulin dose-specific elevation in phosphorylation of Akt substrates, including, but not limited to, AS160; and 4) greater IR phosphorylation and aPKC activity may contribute at higher insulin doses.

The B apolipoproteins, apo-B48 and apo-B100, are key structural proteins in those classes of lipoproteins considered to be atherogenic [e.g., chylomicron remnants, beta-VLDL, LDL, oxidized LDL, and Lp(a)]. Here we describe the development of transgenic mice expressing high levels of human apo-B48 and apo-B100. A 79.5-kb human genomic DNA fragment containing the entire human apo-B gene was isolated from a P1 bacteriophage library and microinjected into fertilized mouse eggs. 16 transgenic founders expressing human apo-B were generated, and the animals with the highest expression had plasma apo-B100 levels nearly as high as those of normolipidemic humans (approximately 50 mg/dl). The human apo-B100 in transgenic mouse plasma was present largely in lipoproteins of the LDL class as shown by agarose gel electrophoresis, chromatography on a Superose 6 column, and density gradient ultracentrifugation. When the human apo-B transgenic founders were crossed with transgenic mice expressing human apo(a), the offspring that expressed both transgenes had high plasma levels of human Lp(a). Both the human apo-B and Lp(a) transgenic mice will be valuable resources for studying apo-B metabolism and the role of apo-B and Lp(a) in atherosclerosis.

Hepatitis C virus (HCV) infection is closely tied to the lipid metabolism of liver cells. Here we identify the triglyceride-synthesizing enzyme diacylglycerol acyltransferase-1 (DGAT1) as a key host factor for HCV infection. DGAT1 interacts with the viral nucleocapsid core and is required for the trafficking of core to lipid droplets. Inhibition of DGAT1 activity or RNAi-mediated knockdown of DGAT1 severely impairs infectious virion production, implicating DGAT1 as a new target for antiviral therapy.

Obesity, the metabolic syndrome, and aging share several pathogenic features in both humans and non-human primates, including insulin resistance and inflammation. Since muscle and liver are considered key integrators of metabolism, we sought to determine in biopsies from lean and obese aging rhesus monkeys the nature of defects in insulin activation and, further, the potential for mitigation of such defects by an in vivo insulin sensitizer, rosiglitazone, and a thiazolidinedione activator of the peroxisome proliferator-activated receptor gamma. The peroxisome proliferator-activated receptor gamma agonist reduced hyperinsulinemia, improved insulin sensitivity, lowered plasma triglycerides and free fatty acids, and increased plasma adiponectin. In muscle of obese monkeys, previously shown to exhibit defective insulin signaling, the insulin sensitizer improved insulin activation of atypical protein kinase C (aPKC), the defective direct activation of aPKC by phosphatidylinositol (PI)-3,4,5-(PO4)3, and 5′-AMP-activated protein kinase and increased carnitine palmitoyltransferase-1 mRNA expression, but it did not improve insulin activation of insulin receptor substrate (IRS)-1-dependent PI 3-kinase (IRS-1/PI3K), protein kinase B, or glycogen synthase. We found that, although insulin signaling was impaired in muscle, insulin activation of IRS-1/PI3K, IRS-2/PI3K, protein kinase B, and aPKC was largely intact in liver and that rosiglitazone improved insulin signaling to aPKC in muscle by improving responsiveness to PI-3,4,5-(PO4)3. Antioxid. Redox Signal. 14, 207–219.