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1.  Akt-Dependent Phosphorylation of Hepatic FoxO1 Is Compartmentalized on a WD40/ProF Scaffold and Is Selectively Inhibited by aPKC in Early Phases of Diet-Induced Obesity 
Diabetes  2014;63(8):2690-2701.
Initiating mechanisms that impair gluconeogenic enzymes and spare lipogenic enzymes in diet-induced obesity (DIO) are obscure. Here, we examined insulin signaling to Akt and atypical protein kinase C (aPKC) in liver and muscle and hepatic enzyme expression in mice consuming a moderate high-fat (HF) diet. In HF diet–fed mice, resting/basal and insulin-stimulated Akt and aPKC activities were diminished in muscle, but in liver, these activities were elevated basally and were increased by insulin to normal levels. Despite elevated hepatic Akt activity, FoxO1 phosphorylation, which diminishes gluconeogenesis, was impaired; in contrast, Akt-dependent phosphorylation of glycogenic GSK3β and lipogenic mTOR was elevated. Diminished Akt-dependent FoxO1 phosphorylation was associated with reduced Akt activity associated with scaffold protein WD40/Propeller/FYVE (WD40/ProF), which reportedly facilitates FoxO1 phosphorylation. In contrast, aPKC activity associated with WD40/ProF was increased. Moreover, inhibition of hepatic aPKC reduced its association with WD40/ProF, restored WD40/ProF-associated Akt activity, restored FoxO1 phosphorylation, and corrected excessive expression of hepatic gluconeogenic and lipogenic enzymes. Additionally, Akt and aPKC activities in muscle improved, as did glucose intolerance, weight gain, hepatosteatosis, and hyperlipidemia. We conclude that Akt-dependent FoxO1 phosphorylation occurs on the WD/Propeller/FYVE scaffold in liver and is selectively inhibited in early DIO by diet-induced increases in activity of cocompartmentalized aPKC.
PMCID: PMC4113067  PMID: 24705403
2.  Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting 
eLife  2014;3:e01607.
Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids, such as triacylglycerol (TG), as reservoirs of metabolic energy and membrane precursors. The Arf1/COPI protein machinery, known for its role in vesicle trafficking, regulates LD morphology, targeting of specific proteins to LDs and lipolysis through unclear mechanisms. Recent evidence shows that Arf1/COPI can bud nano-LDs (∼60 nm diameter) from phospholipid-covered oil/water interfaces in vitro. We show that Arf1/COPI proteins localize to cellular LDs, are sufficient to bud nano-LDs from cellular LDs, and are required for targeting specific TG-synthesis enzymes to LD surfaces. Cells lacking Arf1/COPI function have increased amounts of phospholipids on LDs, resulting in decreased LD surface tension and impairment to form bridges to the ER. Our findings uncover a function for Arf1/COPI proteins at LDs and suggest a model in which Arf1/COPI machinery acts to control ER-LD connections for localization of key enzymes of TG storage and catabolism.
eLife digest
Just like the body contains organs that perform different jobs, the cells within the body contain organelles that carry out different tasks. The endoplasmic reticulum, for example, makes proteins that are sent to other organelles or to destinations outside the cell. Each organelle is typically sealed within a membrane made from a double layer of phospholipids—molecules that have a phosphate ‘head’ group at one end, and two fatty acid ‘tails’ at the other. Proteins are shuttled between the organelles inside membrane-bound packages called vesicles.
There is, however, an exception to this rule. Lipid droplets are organelles that store fats and oils inside a single layer of phospholipids. This layer can include enzymes that break down the contents of the droplet, or make new fat molecules, depending on the needs of the cell and the organism. However, it is not clear how these enzymes get from the endoplasmic reticulum to the lipid droplet.
Previous work had suggested that a protein complex called Arf1/COP—which is also involved in the movement of vesicles around the cell—might recruit the enzymes to the lipid droplets. However, none of the other proteins known to be involved in vesicle transport were needed to transport the enzymes to the droplets, which suggested that the Arf1/COPI complex was using a previously unknown mechanism to move the enzymes.
Now Wilfling, Thiam et al. have shown that Arf1/COPI complexes trigger the establishment of membrane bridges between the endoplasmic reticulum and the droplets, which means that vesicles are not needed to get the enyzmes to the lipid droplets. It was also shown that the Arf1/COPI complexes could pinch off tiny droplets from full-size lipid droplets taken from living cells. Wilfling, Thiam et al. suggest that this ‘budding’ process changes the composition of the phospholipid layer around the larger droplet in a way that allows it to interact directly with the membrane of the endoplasmic reticulum.
By providing new insights into the trafficking of proteins between organelles, the work of Wilfling, Thiam et al. reveals mechanisms that govern the composition of lipid droplets. In the future, these pathways could be manipulated to treat conditions that result from excessive storage of fat, such as obesity or cardiovascular diseases.
PMCID: PMC3913038  PMID: 24497546
lipid droplet; protein targeting; ER-lipid droplet connections; lipolysis; triglyceride synthesis; D. melanogaster; human; mouse
3.  DGAT1 mutation is linked to a congenital diarrheal disorder  
The Journal of Clinical Investigation  2012;122(12):4680-4684.
Congenital diarrheal disorders (CDDs) are a collection of rare, heterogeneous enteropathies with early onset and often severe outcomes. Here, we report a family of Ashkenazi Jewish descent, with 2 out of 3 children affected by CDD. Both affected children presented 3 days after birth with severe, intractable diarrhea. One child died from complications at age 17 months. The second child showed marked improvement, with resolution of most symptoms at 10 to 12 months of age. Using exome sequencing, we identified a rare splice site mutation in the DGAT1 gene and found that both affected children were homozygous carriers. Molecular analysis of the mutant allele indicated a total loss of function, with no detectable DGAT1 protein or activity produced. The precise cause of diarrhea is unknown, but we speculate that it relates to abnormal fat absorption and buildup of DGAT substrates in the intestinal mucosa. Our results identify DGAT1 loss-of-function mutations as a rare cause of CDDs. These findings prompt concern for DGAT1 inhibition in humans, which is being assessed for treating metabolic and other diseases.
PMCID: PMC3533555  PMID: 23114594
4.  Cellular Fatty Acid Metabolism and Cancer 
Cell metabolism  2013;18(2):153-161.
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.
PMCID: PMC3742569  PMID: 23791484
5.  Pharmacological TLR4 Inhibition Protects against Acute and Chronic Fat-Induced Insulin Resistance in Rats 
PLoS ONE  2015;10(7):e0132575.
To evaluate whether pharmacological TLR4 inhibition protects against acute and chronic fat-induced insulin resistance in rats.
Materials and Methods
For the acute experiment, rats received a TLR4 inhibitor [TAK-242 or E5564 (2x5 mg/kg i.v. bolus)] or vehicle, and an 8-h Intralipid (20%, 8.5 mg/kg/min) or saline infusion, followed by a two-step hyperinsulinemic-euglycemic clamp. For the chronic experiment, rats were subcutaneously implanted with a slow-release pellet of TAK-242 (1.5 mg/d) or placebo. Rats then received a high fat diet (HFD) or a low fat control diet (LFD) for 10 weeks, followed by a two-step insulin clamp.
Acute experiment; the lipid-induced reduction (18%) in insulin-stimulated glucose disposal (Rd) was attenuated by TAK-242 and E5564 (the effect of E5564 was more robust), suggesting improved peripheral insulin action. Insulin was able to suppress hepatic glucose production (HGP) in saline- but not lipid-treated rats. TAK-242, but not E5564, partially restored this effect, suggesting improved HGP. Chronic experiment; insulin-stimulated Rd was reduced ~30% by the HFD, but completely restored by TAK-242. Insulin could not suppress HGP in rats fed a HFD and TAK-242 had no effect on HGP.
Pharmacological TLR4 inhibition provides partial protection against acute and chronic fat-induced insulin resistance in vivo.
PMCID: PMC4510579  PMID: 26196892
6.  Meformin action in human hepatocytes. Co-activation of atypical protein kinase C alters 5′-AMP-activated protein kinase effects on lipogenic and gluconeogenic enzyme expression 
Diabetologia  2013;56(11):2507-2516.
Levels and activity of atypical protein kinase C (aPKC) are elevated in hepatocytes of type 2 diabetic (T2DM) humans and cause excessive increases in expression 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 liver, this activation could amplify T2DM aberrations and offset salutary effects of AMPK. Here, we examined whether metformin activates aPKC in human hepatocytes and metabolic its consequences.
We compared protein kinase activities and alterations of lipogenic and gluconeogenic enzyme expression during actions of AMPK activators, metformin and AICAR, relative to those of an aPKC-ι inhibitor, in hepatocytes of non-diabetic and T2DM humans.
Metformin and AICAR activated aPKC at concentrations comparable to those required for AMPK activation. Moreover, both agents increased lipogenic enzyme expression by an aPKC-dependent mechanism. Thus, whereas insulin-dependent and T2DM-dependent increases in lipogenic enzyme expression were reversed by aPKC inhibition, such expression was increased in non-diabetic hepatocytes and remained elevated in T2DM hepatocytes following metformin and AICAR treatment. Also, whereas aPKC inhibition diminished gluconeogenic enzyme expression in the absence and presence of insulin in both non-diabetic and T2DM hepatocytes, metformin and AICAR increased gluconeogenic enzyme expression in non-diabetic hepatocytes, but nevertheless diminished gluconeogenic enzyme expression in insulin-treated T2DM hepatocytes.
Metformin and AICAR activate aPKC along with AMPK in human hepatocytes. aPKC activation increases lipogenic enzyme expression, alters gluconeogenic enzyme expression and therefore appears to offset salutary effects of AMPK.
PMCID: PMC3973184  PMID: 23933835
Metformin; AICAR; AMP-activated Protein Kinase; Protein Kinase C-iota; Type 2 Diabetes; Hepatocytes
7.  In Vivo Metabolic Fingerprinting of Neutral Lipids with Hyperspectral Stimulated Raman Scattering Microscopy 
Metabolic fingerprinting provides valuable information on the physiopathological states of cells and tissues. Traditional imaging mass spectrometry and magnetic resonance imaging are unable to probe the spatial-temporal dynamics of metabolites at the subcellular level due to either lack of spatial resolution or inability to perform live cell imaging. Here we report a complementary metabolic imaging technique that is based on hyperspectral stimulated Raman scattering (hsSRS). We demonstrated the use of hsSRS imaging in quantifying two major neutral lipids: cholesteryl ester and triacylglycerol in cells and tissues. Our imaging results revealed previously unknown changes of lipid composition associated with obesity and steatohepatitis. We further used stable-isotope labeling to trace the metabolic dynamics of fatty acids in live cells and live Caenorhabditis elegans with hsSRS imaging. We found that unsaturated fatty acid has preferential uptake into lipid storage while saturated fatty acid exhibits toxicity in hepatic cells. Simultaneous metabolic fingerprinting of deuterium-labeled saturated and unsaturated fatty acids in living C. elegans revealed that there is a lack of interaction between the two, unlike previously hypothesized. Our findings provide new approaches for metabolic tracing of neutral lipids and their precursors in living cells and organisms, and could potentially serve as a general approach for metabolic fingerprinting of other metabolites.
PMCID: PMC4073829  PMID: 24869754
8.  Impairment of insulin-stimulated glucose transport and ERK activation by adipocyte-specific knockout of PKC-λ produces a phenotype characterized by diminished adiposity and enhanced insulin suppression of hepatic gluconeogenesis 
Adipocyte  2013;3(1):19-29.
Tissue-specific knockout (KO) of atypical protein kinase C-λ (PKC-λ) impairs insulin-stimulated glucose transport in muscle (M) and lipid synthesis in liver (L), thereby producing insulin resistance in MλKO mice and insulin-hypersensitivity in LλKO mice. Here, we generated mice with KO of PKC-λ in adipocytes, i.e., AλKO mice. In isolated adipocytes of AλKO mice, insulin-stimulated aPKC activity and glucose transport were diminished, as were ERK levels and activity. Insulin-stimulated glucose transport and insulin activation of ERK in adipocytes of wild-type mice were similarly inhibited by acute inhibition of PKC-λ with a highly-specific chemical inhibitor. With impairments in glucose transport and ERK activation, AλKO mice had diminished adiposity and serum leptin levels. In addition, AλKO mice had normal glucose tolerance and insulin hypersensitivity owing to enhanced suppression of hepatic glucose output, which apparently reflected increases in Akt activity and FoxO1 phosphorylation, and subsequent decreases in expression of gluconeogenic phosphoenolpyruvate carboxykinase. We conclude that: PKC-λ is required for insulin-stimulated glucose transport and ERK signaling in mouse adipocytes; and diminution of these processes is attended by leanness and therefore hypoleptinemia. How these and perhaps other PKC-λ-dependent processes communicate to liver and improve insulin suppression of hepatic gluconeogenesis remains unclear.
PMCID: PMC3917928  PMID: 24575365
ERK; PKC-λ; adipocyte; diabetes; glucose transport; insulin; liver
9.  The problem of establishing relationships between hepatic steatosis and hepatic insulin resistance 
Cell metabolism  2012;15(5):570-573.
Excessive deposition of fat in the liver (hepatic steatosis) is frequently accompanied by hepatic insulin resistance. Whether this correlation is due to a causal relationship between the conditions has been the subject of considerable debate, and the literature abounds with conflicting data and theories. Here we provide a perspective by defining the problem and its challenges, analyzing the possible causative relationships, and drawing some conclusions.
PMCID: PMC3767424  PMID: 22560209
triglycerides; liver; diabetes
10.  Progranulin Protects against Amyloid β Deposition and Toxicity in Alzheimer’s Disease Mouse Models 
Nature medicine  2014;20(10):1157-1164.
Haploinsufficiency of progranulin (PGRN) gene (GRN) causes familial frontotemporal lobar degeneration (FTLD), and modulates an innate immune response in humans and mouse models. GRN polymorphism may be linked to late-onset Alzheimer’s disease (AD). However, PRGN’s role in AD pathogenesis is unknown. Here, we show PGRN inhibits amyloid β (Aβ) deposition. Selectively reducing microglial PGRN in AD mice impaired phagocytosis and increased plaque load threefold. Lentivirus-mediated PGRN overexpression lowered plaque load in AD mice with aggressive amyloid plaque pathology. Aβ plaque load correlated negatively with levels of hippocampal PGRN, showing PGRN’s dose-dependent inhibitory effects on plaque deposition. PGRN also protected against Aβ toxicity. Reducing microglial PGRN exacerbated cognitive deficits in AD mice. Lentivirus-mediated PGRN overexpression prevented spatial memory deficits and hippocampal neuronal loss in AD mice. PGRN’s protective effects against Aβ deposition and toxicity have important therapeutic implications. We propose enhancing PGRN as a potential treatment for PGRN-deficient FTD and AD.
PMCID: PMC4196723  PMID: 25261995
11.  Inhibition of RNA lariat debranching enzyme suppresses TDP-43 toxicity in ALS disease models 
Nature genetics  2012;44(12):1302-1309.
ALS is a devastating neurodegenerative disease primarily affecting motor neurons. Mutations in TDP-43 cause some forms of the disease, and cytoplasmic TDP-43 aggregates accumulate in degenerating neurons of most ALS patients. 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 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 away from interfering with essential cellular RNAs and RNA-binding proteins. Knockdown of Dbr1 in a human neuronal cell line or in primary rodent neurons is also sufficient to rescue TDP-43 toxicity. Our findings provide insight into TDP-43 cytotoxicity and suggest decreasing Dbr1 activity could be a potential therapeutic approach for ALS.
PMCID: PMC3510335  PMID: 23104007
12.  Early retinal neurodegeneration and impaired Ran-mediated nuclear import of TDP-43 in progranulin-deficient FTLD 
The Journal of Experimental Medicine  2014;211(10):1937-1945.
Ward et al. report retinal thinning in humans with progranulin mutations that precedes dementia onset, and an age-dependent retinal neurodegenerative phenotype in progranulin null mice. Nuclear depletion of TDP-43 precedes retinal neuronal loss and is accompanied by reduced GTPase Ran, with overexpression of Ran restoring nuclear TDP-43 and neuronal survival.
Frontotemporal dementia (FTD) is the most common cause of dementia in people under 60 yr of age and is pathologically associated with mislocalization of TAR DNA/RNA binding protein 43 (TDP-43) in approximately half of cases (FLTD-TDP). Mutations in the gene encoding progranulin (GRN), which lead to reduced progranulin levels, are a significant cause of familial FTLD-TDP. Grn-KO mice were developed as an FTLD model, but lack cortical TDP-43 mislocalization and neurodegeneration. Here, we report retinal thinning as an early disease phenotype in humans with GRN mutations that precedes dementia onset and an age-dependent retinal neurodegenerative phenotype in Grn-KO mice. Retinal neuron loss in Grn-KO mice is preceded by nuclear depletion of TDP-43 and accompanied by reduced expression of the small GTPase Ran, which is a master regulator of nuclear import required for nuclear localization of TDP-43. In addition, TDP-43 regulates Ran expression, likely via binding to its 3′-UTR. Augmented expression of Ran in progranulin-deficient neurons restores nuclear TDP-43 levels and improves their survival. Our findings establish retinal neurodegeneration as a new phenotype in progranulin-deficient FTLD, and suggest a pathological loop involving reciprocal loss of Ran and nuclear TDP-43 as an underlying mechanism.
PMCID: PMC4172214  PMID: 25155018
13.  Atypical Protein Kinase C in Cardiometabolic Abnormalities 
Current opinion in lipidology  2012;23(3):175-181.
Review aberrations of insulin signaling to atypical protein kinase C (aPKC) in muscle and liver that generate cardiovascular risk factors, including, obesity, hypertriglyceridemia, hypercholesterolemia, insulin resistance and glucose intolerance in type 2 diabetes mellitus (T2DM), and obesity-associated metabolic syndrome (MetSyn).
Recent Findings
aPKC and/or Akt mediate insulin effects on glucose transport in muscle, and synthesis of lipids, cytokines and glucose in liver. In T2DM, whereas Akt and aPKC activation are diminished in muscle, and hepatic Akt activation is diminished, hepatic aPKC activation is conserved. Imbalance between muscle and hepatic aPKC activation (and expression of PKC-ι in humans) by insulin results from differential downregulation of insulin receptor substrates that control phosphatidylinositol 3-kinase. Conserved activation of hepatic aPKC in hyperinsulinemic states of T2DM, obesity and MetSyn is problematic as excessive activation of aPKC-dependent lipogenic, gluconeogenic and proinflammatory pathways increases cardiovascular risk factors. Indeed, selective inhibition of hepatic aPKC by adenoviral-mediated expression of kinase-inactive aPKC, or newly-developed small-molecule biochemicals, dramatically improves abdominal obesity, hepatosteatosis, hypertriglyceridemia, hypercholesterolemia, insulin resistance and glucose intolerance in murine models of obesity and T2DM.
Hepatic aPKC is a unifying target for treating multiple clinical abnormalities that increase cardiovascular risk in insulin-resistant states of obesity, MetSyn and T2DM.
PMCID: PMC3519242  PMID: 22449812
Atypical Protein Kinase C; Obesity; Metabolic Syndrome; Type 2 Diabetes Mellitus; Insulin Signaling in Liver and Muscle
14.  Progranulin deficiency promotes neuroinflammation and neuron loss following toxin-induced injury 
The Journal of Clinical Investigation  2012;122(11):3955-3959.
Progranulin (PGRN) is a widely expressed secreted protein that is linked to inflammation. In humans, PGRN haploinsufficiency is a major inherited cause of frontotemporal dementia (FTD), but how PGRN deficiency causes neurodegeneration is unknown. Here we show that loss of PGRN results in increased neuron loss in response to injury in the CNS. When exposed acutely to 1-methyl-4-(2′-methylphenyl)-1,2,3,6-tetrahydrophine (MPTP), mice lacking PGRN (Grn–/–) showed more neuron loss and increased microgliosis compared with wild-type mice. The exacerbated neuron loss was due not to selective vulnerability of Grn–/– neurons to MPTP, but rather to an increased microglial inflammatory response. Consistent with this, conditional mutants lacking PGRN in microglia exhibited MPTP-induced phenotypes similar to Grn–/– mice. Selective depletion of PGRN from microglia in mixed cortical cultures resulted in increased death of wild-type neurons in the absence of injury. Furthermore, Grn–/– microglia treated with LPS/IFN-γ exhibited an amplified inflammatory response, and conditioned media from these microglia promoted death of cultured neurons. Our results indicate that PGRN deficiency leads to dysregulated microglial activation and thereby contributes to increased neuron loss with injury. These findings suggest that PGRN deficiency may cause increased neuron loss in other forms of CNS injury accompanied by neuroinflammation.
PMCID: PMC3484443  PMID: 23041626
15.  Progranulin: At the interface of neurodegenerative and metabolic diseases 
Trends in endocrinology and metabolism: TEM  2013;24(12):10.1016/j.tem.2013.08.003.
Progranulin is a widely expressed, cysteine-rich, secreted glycoprotein originally discovered for its growth factor–like properties. Its subsequent identification as a causative gene for frontotemporal dementia (FTD), a devastating early-onset neurodegenerative disease, has catalyzed a surge of new discoveries about progranulin’s function in the brain. More recently, progranulin was recognized as an adipokine involved in diet-induced obesity and insulin resistance, revealing its metabolic function. Here, we review progranulin biology in both neurodegenerative and metabolic diseases. In particular, we highlight progranulin’s growth factor–like, trophic, and anti-inflammatory properties as potential unifying themes in these seemingly divergent conditions. We also discuss potential therapeutic options for raising progranulin levels to treat progranulin-deficient FTD, as well as the possible consequences of such treatment.
PMCID: PMC3842380  PMID: 24035620
16.  Deficiency of the lipid synthesis enzyme, DGAT1, extends longevity in mice 
Aging (Albany NY)  2012;4(1):13-27.
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.
PMCID: PMC3292902  PMID: 22291164
DGAT1; adipose tissue; longevity; triglycerides; calorie restriction
17.  A Specific Role for Dgat1 in Hepatic Steatosis Due to Exogenous Fatty Acids 
Hepatology (Baltimore, Md.)  2009;50(2):434-442.
Nonalcoholic fatty liver disease, characterized by accumulation of triacylglycerols (TG) and other lipids in the liver, often accompanies obesity and is a risk factor for nonalcoholic steatohepatitis and fibrosis. To treat or prevent fatty liver, a thorough understanding of hepatic fatty acid and TG metabolism is crucial. To investigate the role of acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), a key enzyme of TG synthesis, in fatty liver development, we studied mice with global and liver-specific knockout of Dgat1. DGAT1 was required for hepatic steatosis induced by high-fat diet and prolonged fasting, which are both characterized by delivery of exogenous fatty acids to the liver. Studies in primary hepatocytes showed that DGAT1 deficiency protected against hepatic steatosis by reducing synthesis and increasing the oxidation of fatty acids. In contrast, lipodystrophy (aP2-SREBP-1c436) and liver X receptor activation (T0901317), which increase de novo fatty acid synthesis in liver, caused steatosis independently of DGAT1. Pharmacologic inhibition of Dgat1 with antisense oligonucleotides protected against fatty liver induced by a high-fat diet. In conclusion, our findings identify a specific role for hepatic DGAT1 in esterification of exogenous fatty acids and indicate that DGAT1 contributes to hepatic steatosis induced by this mechanism.
PMCID: PMC3097135  PMID: 19472314
Triglyceride synthesis; obesity; diabetes; DGAT; fatty liver
18.  Hepatic Atypical Protein Kinase C: An Inherited Survival-Longevity Gene that Now Fuels Insulin-Resistant Syndromes of Obesity, the Metabolic Syndrome and Type 2 Diabetes Mellitus 
Journal of Clinical Medicine  2014;3(3):724-740.
This review focuses on how insulin signals to metabolic processes in health, why this signaling is frequently deranged in Western/Westernized societies, how these derangements lead to, or abet development of, insulin-resistant states of obesity, the metabolic syndrome and type 2 diabetes mellitus, and what our options are for restoring insulin signaling, and glucose/lipid homeostasis. A central theme in this review is that excessive hepatic activity of an archetypal protein kinase enzyme, “atypical” protein kinase C (aPKC), plays a critically important role in the development of impaired glucose metabolism, systemic insulin resistance, and excessive hepatic production of glucose, lipids and proinflammatory factors that underlie clinical problems of glucose intolerance, obesity, hepatosteatosis, hyperlipidemia, and, ultimately, type 2 diabetes. The review suggests that normally inherited genes, in particular, the aPKC isoforms, that were important for survival and longevity in times of food scarcity are now liabilities in times of over-nutrition. Fortunately, new knowledge of insulin signaling mechanisms and how an aberration of excessive hepatic aPKC activation is induced by over-nutrition puts us in a position to target this aberration by diet and/or by specific inhibitors of hepatic aPKC.
PMCID: PMC4449650
insulin resistance; obesity; type 2 diabetes mellitus
19.  High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation[S] 
Journal of Lipid Research  2014;55(7):1465-1477.
Accurate protein inventories are essential for understanding an organelle’s functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae. Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism.
PMCID: PMC4076087  PMID: 24868093
lipid droplets; lipid metabolism; lipids; protein targeting; polyprenol synthesis
20.  DGAT1-dependent triacylglycerol storage by macrophages protects mice from diet-induced insulin resistance and inflammation 
Diet-induced obesity (DIO) leads to inflammatory activation of macrophages in white adipose tissue (WAT) and subsequently to insulin resistance. PPARγ agonists are antidiabetic agents known to suppress inflammatory macrophage activation and to induce expression of the triacylglycerol (TG) synthesis enzyme acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) in WAT and in adipocytes. Here, we investigated in mice the relationship between macrophage lipid storage capacity and DIO-associated inflammatory macrophage activation. Mice overexpressing DGAT1 in both macrophages and adipocytes (referred to herein as aP2-Dgat1 mice) were more prone to DIO but were protected against inflammatory macrophage activation, macrophage accumulation in WAT, systemic inflammation, and insulin resistance. To assess the contribution of macrophage DGAT1 expression to this phenotype, we transplanted wild-type mice with aP2-Dgat1 BM. These mice developed DIO similar to that of control mice but retained the protection from WAT inflammation and insulin resistance seen in aP2-Dgat1 mice. In isolated macrophages, Dgat1 mRNA levels correlated directly with TG storage capacity and inversely with inflammatory activation by saturated fatty acids (FAs). Moreover, PPARγ agonists increased macrophage Dgat1 mRNA levels, and the protective effects of these agonists against FA-induced inflammatory macrophage activation were absent in macrophages isolated from Dgat1-null mice. Thus, increasing DGAT1 expression in murine macrophages increases their capacity for TG storage, protects against FA-induced inflammatory activation, and is sufficient to reduce the inflammatory and metabolic consequences of DIO.
PMCID: PMC2827941  PMID: 20124729
21.  Effects of DGAT1 deficiency on energy and glucose metabolism are independent of adiponectin 
Mice lacking acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), an enzyme that catalyzes the terminal step in triacylglycerol synthesis, have enhanced insulin sensitivity and are protected from obesity, a result of increased energy expenditure. In these mice, factors derived from white adipose tissue (WAT) contribute to the systemic changes in metabolism. One such factor, adiponectin, increases fatty acid oxidation and enhances insulin sensitivity. To test the hypothesis that adiponectin is required for the altered energy and glucose metabolism in DGAT1-deficient mice, we generated adiponectin-deficient mice and introduced adiponectin deficiency into DGAT1-deficient mice by genetic crosses. Although adiponectin-deficient mice fed a high-fat diet were heavier, exhibited worse glucose tolerance, and had more hepatic triacylglycerol accumulation than wild-type controls, mice lacking both DGAT1 and adiponectin, like DGAT1-deficient mice, were protected from diet-induced obesity, glucose intolerance, and hepatic steatosis. These findings indicate that adiponectin is required for normal energy, glucose, and lipid metabolism but that the metabolic changes induced by DGAT1-deficient WAT are independent of adiponectin and are likely due to other WAT-derived factors. Our findings also suggest that the pharmacological inhibition of DGAT1 may be useful for treating human obesity and insulin resistance associated with low circulating adiponectin levels.
PMCID: PMC1552042  PMID: 16595853
triglyceride; adipose tissue; gene knockout; acyl-coenzyme A:diacyl-glycerol acyltransferase 1
22.  Muscle-specific knockout of PKC-λ impairs glucose transport and induces metabolic and diabetic syndromes 
Journal of Clinical Investigation  2007;117(8):2289-2301.
Obesity, the metabolic syndrome, and type 2 diabetes mellitus (T2DM) are major global health problems. Insulin resistance is frequently present in these disorders, but the causes and effects of such resistance are unknown. Here, we generated mice with muscle-specific knockout of the major murine atypical PKC (aPKC), PKC-λ, a postulated mediator for insulin-stimulated glucose transport. Glucose transport and translocation of glucose transporter 4 (GLUT4) to the plasma membrane were diminished in muscles of both homozygous and heterozygous PKC-λ knockout mice and were accompanied by systemic insulin resistance; impaired glucose tolerance or diabetes; islet β cell hyperplasia; abdominal adiposity; hepatosteatosis; elevated serum triglycerides, FFAs, and LDL-cholesterol; and diminished HDL-cholesterol. In contrast to the defective activation of muscle aPKC, insulin signaling and actions were intact in muscle, liver, and adipocytes. These findings demonstrate the importance of aPKC in insulin-stimulated glucose transport in muscles of intact mice and show that insulin resistance and resultant hyperinsulinemia owing to a specific defect in muscle aPKC is sufficient to induce abdominal obesity and other lipid abnormalities of the metabolic syndrome and T2DM. These findings are particularly relevant because humans who have obesity, impaired glucose tolerance, and T2DM reportedly have defective activation and/or diminished levels of muscle aPKC.
PMCID: PMC1913489  PMID: 17641777
23.  Progranulin Does Not Bind Tumor Necrosis Factor (TNF) Receptors and Is Not a Direct Regulator of TNF-Dependent Signaling or Bioactivity in Immune or Neuronal Cells 
Progranulin (PGRN) is a secreted glycoprotein expressed in neurons and glia that is implicated in neuronal survival on the basis that mutations in the GRN gene 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 from Grn+/− or Grn−/− 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.
PMCID: PMC3707136  PMID: 23699531
24.  Downregulation of MicroRNA-9 in iPSC-Derived Neurons of FTD/ALS Patients with TDP-43 Mutations 
PLoS ONE  2013;8(10):e76055.
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.
PMCID: PMC3797144  PMID: 24143176
25.  Correction of metabolic abnormalities in a rodent model of obesity, metabolic syndrome, and type 2 diabetes mellitus by inhibitors of hepatic protein kinase C-ι 
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 PBl-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.
PMCID: PMC3786325  PMID: 22225955

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