The Mevalonate Pathway Is Indispensable for Adipocyte Survival.
ABSTRACT: The mevalonate pathway is essential for the synthesis of isoprenoids and cholesterol. Adipose tissue is known as a major site for cholesterol storage; however, the role of the local mevalonate pathway and its synthesized isoprenoids remains unclear. In this study, adipose-specific mevalonate pathway-disrupted (aKO) mice were generated through knockout of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (HMGCR). aKO mice showed serious lipodystrophy accompanied with glucose and lipid metabolic disorders and hepatomegaly. These metabolic variations in aKO mice were dramatically reversed after fat transplantation. In addition, HMGCR-disrupted adipocytes exhibited loss of lipid accumulation and an increase of cell death, which were ameliorated by the supplementation of mevalonate and geranylgeranyl pyrophosphate but not farnesyl pyrophosphate and squalene. Finally, we found that apoptosis may be involved in adipocyte death induced by HMGCR down-regulation. Our findings indicate that the mevalonate pathway is essential for adipocytes and further suggest that this pathway is an important regulator of adipocyte turnover.
Project description:The cholesterol biosynthetic pathway produces not only sterols but also non-sterol mevalonate metabolites involved in isoprenoid synthesis. Mevalonate metabolites affect transcriptional and post-transcriptional events that in turn affect various biological processes including energy metabolism. In the present study, we examine whether mevalonate metabolites activate PPARγ (peroxisome-proliferator-activated receptor γ), a ligand-dependent transcription factor playing a central role in adipocyte differentiation. In the luciferase reporter assay using both GAL4 chimaera and full-length PPARγ systems, a mevalonate metabolite, FPP (farnesyl pyrophosphate), which is the precursor of almost all isoprenoids and is positioned at branch points leading to the synthesis of other longer-chain isoprenoids, activated PPARγ in a dose-dependent manner. FPP induced the in vitro binding of a co-activator, SRC-1 (steroid receptor co-activator-1), to GST (glutathione transferase)-PPARγ. Direct binding of FPP to PPARγ was also indicated by docking simulation studies. Moreover, the addition of FPP up-regulated the mRNA expression levels of PPARγ target genes during adipocyte differentiation induction. In the presence of lovastatin, an HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase inhibitor, both intracellular FPP levels and PPARγ-target gene expressions were decreased. In contrast, the increase in intracellular FPP level after the addition of zaragozic acid, a squalene synthase inhibitor, induced PPARγ-target gene expression. The addition of FPP and zaragozic acid promotes lipid accumulation during adipocyte differentiation. These findings indicated that FPP might function as an endogenous PPARγ agonist and regulate gene expression in adipocytes.
Project description:Insulin resistance is a key feature of obesity and type 2 diabetes. PU.1 is a master transcription factor predominantly expressed in macrophages but after HFD feeding PU.1 expression is also significantly increased in adipocytes. We generated adipocyte specific PU.1 knockout mice using adiponectin cre to investigate the role of PU.1 in adipocyte biology, insulin and glucose homeostasis. In HFD-fed obese mice systemic glucose tolerance and insulin sensitivity were improved in PU.1 AKO mice and clamp studies indicated improvements in both adipose and liver insulin sensitivity. At the level of adipose tissue, macrophage infiltration and inflammation was decreased and glucose uptake was increased in PU.1 AKO mice compared with controls. While PU.1 deletion in adipocytes did not affect the gene expression of PPARg itself, we observed increased expression of PPARg target genes in eWAT from HFD fed PU.1 AKO mice compared with controls. Furthermore, we observed decreased phosphorylation at serine 273 in PU.1 AKO mice compared with fl/fl controls, indicating that PPARg is more active when PU.1 expression is reduced in adipocytes. Therefore, in obesity the increased expression of PU.1 in adipocytes modifies the adipocyte PPARg cistrome resulting in impaired glucose tolerance and insulin sensitivity.
Project description:UbiA prenyltransferase domain-containing protein-1 (UBIAD1) synthesizes the vitamin K subtype menaquinone-4 (MK-4). Previous studies in cultured cells (Schumacher et al., 2015) revealed that UBIAD1 also inhibits endoplasmic reticulum (ER)-associated degradation (ERAD) of ubiquitinated HMG CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway that produces cholesterol and essential nonsterol isoprenoids. Gene knockout studies were previously attempted to explore the function of UBIAD1 in mice; however, homozygous germ-line elimination of the Ubiad1 gene caused embryonic lethality. We now report that homozygous deletion of Ubiad1 is produced in knockin mice expressing ubiquitination/ERAD-resistant HMGCR. Thus, embryonic lethality of Ubiad1 deficiency results from depletion of mevalonate-derived products owing to enhanced ERAD of HMGCR rather than from reduced synthesis of MK-4. These findings provide genetic evidence for the significance of UBIAD1 in regulation of cholesterol synthesis and offer the opportunity in future studies for the discovery of new physiological roles of MK-4.
Project description:The isoprenoid family of compounds is estimated to contain ? 65,000 unique structures including medicines, fragrances, and biofuels. Due to their structural complexity, many isoprenoids can only be obtained by extraction from natural sources, an inherently risky and costly process. Consequently, the biotechnology industry is attempting to genetically engineer microorganisms that can produce isoprenoid-based drugs and fuels on a commercial scale. Isoprenoid backbones are constructed from two, five-carbon building blocks, isopentenyl 5-pyrophosphate and dimethylallyl 5-pyrophosphate, which are end-products of either the mevalonate or non-mevalonate pathways. By linking the HMG-CoA reductase pathway (which produces mevalonate) to the mevalonate pathway, these building block can be synthesized enzymatically from acetate, ATP, NAD(P)H and CoA. Here, the enzymes in these pathways are used to produce pathway intermediates and end-products in single-pot reactions and in remarkably high yield, ? 85%. A strategy for the regio-specific incorporation of isotopes into isoprenoid backbones is developed and used to synthesize a series of isotopomers of diphosphomevalonate, the immediate end-product of the mevalonate pathway. The enzymatic system is shown to be robust and capable of producing quantities of product in aqueous solutions that meet or exceed the highest levels achieved using genetically engineered organisms in high-density fermentation.
Project description:OBJECTIVE:Cold and ?3-adrenergic receptor (AR) agonists activate beige adipocyte biogenesis in white adipose tissue (WAT). The two stimuli also induce expression of inflammatory cytokines in WAT. The low-grade inflammation may further promote WAT browning. However, the mechanisms to reconcile these two biological processes remain to be elucidated. In this study, we aim to investigate the roles of the rate-limiting polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SAT1) in regulating beige adipocyte biogenesis and inflammation. METHODS:Adipose-specific SAT1 knockout mice (SAT1-aKO) were generated by crossing adiponectin-cre to SAT1-lox/lox mice. Metabolic phenotype was investigated. Primary pre-adipocytes were isolated from inguinal WAT (iWAT) and differentiated to adipocytes for studying beige adipocyte biogenesis. RESULT:The expression and enzymatic activity of SAT1 were up-regulated in iWAT upon cold and ?3-AR stimulation. SAT1-aKO mice developed late-onset obesity on a high-fat diet with impaired cold-induced beige adipocyte biogenesis and energy expenditure. RNA-seq analysis of iWAT from cold-challenged SAT1-aKO mice revealed that, in addition to beige adipocyte biogenesis signatures, the immune response markers were highly enriched among reduced genes. In cultured adipocytes, SAT1 overexpression or pharmacological activation with N1, N11-diethylnorspermine (DENSpm) elevated oxygen consumption and increased the expression of beige adipocyte marker UCP1 and PGC-1?. DENSpm treatment of adipocytes also increased the expression of inflammatory genes. SAT1 activation enhanced hydrogen peroxide production in adipocytes. Antioxidant N-acetylcysteine abrogated the elevated UCP1 expression and reversed some inflammatory genes induced by SAT1 activation. CONCLUSIONS:SAT1 activation plays a key role in cold and ?3-AR agonist-induced beige adipocyte biogenesis and low-grade inflammation.
Project description:Isoprenoids make up a remarkably diverse class of more than 25000 biomolecules that include familiar compounds such as cholesterol, chlorophyll, vitamin A, ubiquinone, and natural rubber. The two essential building blocks of all isoprenoids, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), are ubiquitous in the three domains of life. In most eukaryotes and archaea, IPP and DMAPP are generated through the mevalonate pathway. We have identified two novel enzymes, mevalonate-3-kinase and mevalonate-3-phosphate-5-kinase from Thermoplasma acidophilum, which act sequentially in a putative alternate mevalonate pathway. We propose that a yet unidentified ATP-independent decarboxylase acts upon mevalonate 3,5-bisphosphate, yielding isopentenyl phosphate, which is subsequently phosphorylated by the known isopentenyl phosphate kinase from T. acidophilum to generate the universal isoprenoid precursor, IPP.
Project description:The mevalonate pathway is a well-known metabolic route that provides biosynthetic precursors for myriad isoprenoids. An unexpected variety of the pathway has been discovered from recent studies on microorganisms, mainly on archaea. The most recently discovered example, called the "archaeal" mevalonate pathway, is a modified version of the canonical eukaryotic mevalonate pathway and was elucidated in our previous study using the hyperthermophilic archaeon Aeropyrum pernix This pathway comprises four known enzymes that can produce mevalonate 5-phosphate from acetyl coenzyme A, two recently discovered enzymes designated phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase, and two more known enzymes, i.e., isopentenyl phosphate kinase and isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase. To show its wide distribution in archaea and to confirm if its enzyme configuration is identical among species, the putative genes of a lower portion of the pathway-from mevalonate to isopentenyl pyrophosphate-were isolated from the methanogenic archaeon Methanosarcina mazei, which is taxonomically distant from A. pernix, and were introduced into an engineered Escherichia coli strain that produces lycopene, a red carotenoid pigment. Lycopene production, as a measure of isoprenoid productivity, was enhanced when the cells were grown semianaerobically with the supplementation of mevalonolactone, which demonstrates that the archaeal pathway can function in bacterial cells to convert mevalonate into isopentenyl pyrophosphate. Gene deletion and complementation analysis using the carotenogenic E. coli strain suggests that both phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase from M. mazei are required for the enhancement of lycopene production.IMPORTANCE Two enzymes that have recently been identified from the hyperthermophilic archaeon A. pernix as components of the archaeal mevalonate pathway do not require ATP for their reactions. This pathway, therefore, might consume less energy than other mevalonate pathways to produce precursors for isoprenoids. Thus, the pathway might be applicable to metabolic engineering and production of valuable isoprenoids that have application as pharmaceuticals. The archaeal mevalonate pathway was successfully reconstructed in E. coli cells by introducing several genes from the methanogenic or hyperthermophilic archaeon, which demonstrated that the pathway requires the same components even in distantly related archaeal species and can function in bacterial cells.
Project description:Obesity occurs when excess energy accumulates in white adipose tissue (WAT), whereas brown adipose tissue (BAT), which is specialized in dissipating energy through thermogenesis, potently counteracts obesity. White adipocytes can be converted to thermogenic "brown-like" cells (beige cells; WAT browning) under various stimuli, such as cold exposure. AMP-activated protein kinase (AMPK) is a crucial energy sensor that regulates energy metabolism in multiple tissues. However, the role of AMPK in adipose tissue function, especially in the WAT browning process, is not fully understood. To illuminate the effect of adipocyte AMPK on energy metabolism, we generated Adiponectin-Cre-driven adipose tissue-specific AMPK ?1/?2 KO mice (AKO). These AKO mice were cold intolerant and their inguinal WAT displayed impaired mitochondrial integrity and biogenesis, and reduced expression of thermogenic markers upon cold exposure. High-fat-diet (HFD)-fed AKO mice exhibited increased adiposity and exacerbated hepatic steatosis and fibrosis and impaired glucose tolerance and insulin sensitivity. Meanwhile, energy expenditure and oxygen consumption were markedly decreased in the AKO mice both in basal conditions and after stimulation with a ?3-adrenergic receptor agonist, CL 316,243. In contrast, we found that in HFD-fed obese mouse model, chronic AMPK activation by A-769662 protected against obesity and related metabolic dysfunction. A-769662 alleviated HFD-induced glucose intolerance and reduced body weight gain and WAT expansion. Notably, A-769662 increased energy expenditure and cold tolerance in HFD-fed mice. A-769662 treatment also induced the browning process in the inguinal fat depot of HFD-fed mice. Likewise, A-769662 enhanced thermogenesis in differentiated inguinal stromal vascular fraction (SVF) cells via AMPK signaling pathway. In summary, a lack of adipocyte AMPK? induced thermogenic impairment and obesity in response to cold and nutrient-overload, respectively, whereas chronic AMPK activation by A-769662 promoted WAT browning in inguinal WAT and protected against HFD-induced obesity and related metabolic dysfunction. These findings reveal a vital role for adipocyte AMPK in regulating the browning process in inguinal WAT and in maintaining energy homeostasis, which suggests that the targeted activation of adipocyte AMPK may be a promising strategy for anti-obesity therapy.
Project description:Sterol-regulated HMG-CoA reductase (HMGCR) degradation and SREBP-2 cleavage are two major feedback regulatory mechanisms governing cholesterol biosynthesis. Reportedly, lanosterol selectively stimulates HMGCR degradation, and cholesterol is a specific regulator of SREBP-2 cleavage. However, it is unclear whether other endogenously generated sterols regulate these events. Here, we investigated the sterol intermediates from the mevalonate pathway of cholesterol biosynthesis using a CRISPR/Cas9-mediated genetic engineering approach. With a constructed HeLa cell line expressing the mevalonate transporter, we individually deleted genes encoding major enzymes in the mevalonate pathway, used lipidomics to measure sterol intermediates, and examined HMGCR and SREBP-2 statuses. We found that the C4-dimethylated sterol intermediates, including lanosterol, 24,25-dihydrolanosterol, follicular fluid meiosis activating sterol, testis meiosis activating sterol, and dihydro-testis meiosis activating sterol, were significantly upregulated upon mevalonate loading. These intermediates augmented both degradation of HMGCR and inhibition of SREBP-2 cleavage. The accumulated lanosterol induced rapid degradation of HMGCR, but did not inhibit SREBP-2 cleavage. The newly synthesized cholesterol from the mevalonate pathway is dispensable for inhibiting SREBP-2 cleavage. Together, these results suggest that lanosterol is a bona fide endogenous regulator that specifically promotes HMGCR degradation, and that other C4-dimethylated sterol intermediates may regulate both HMGCR degradation and SREBP-2 cleavage.
Project description:Dysregulation of cell metabolism is a hallmark of cancer. The mevalonate pathway in lipid metabolism has been implicated as a potential target of cancer therapy for hepatocellular carcinoma (HCC). The role of the Forkhead Box M1 (FoxM1) transcription factor in HCC development has been well documented, however, its involvement in cancer metabolism of HCC has not been fully determined. Here, we hypothesized that FoxM1 is involved in the mevalonate pathway of cholesterol biosynthesis in HCC. Inhibition of the mevalonate pathway by statins, inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR), resulted in reduced expression of FoxM1 and increased cell death in human hepatoma cells. Re-exposure of mevalonate, a product of HMGCR, restored these effects. Likewise, knockdown of HMGCR reduced FoxM1 expression, indicating that FoxM1 expression was regulated by the mevalonate pathway in HCC. Mechanistically, protein geranylgeranylation was found to be responsible for FoxM1 expression and geranylgeranylated proteins, including RhoA, Rac1 or Cdc42, were shown to be involved in this process. In surgically resected human HCC tissues, the gene expression of FoxM1 had a positive correlation with that of the mevalonate pathway-related genes, such as HMGCR or sterol regulatory element-binding protein 2 (SREBP2). Furthermore, the gene expression of FoxM1 along with that of HMGCR or SREBP2 defined prognosis of HCC patients, suggesting the clinical significance of the mevalonate-FoxM1 pathway in human HCC. Our data indicate that FoxM1 links the mevalonate pathway to oncogenic signals in HCC. Thus, we propose a novel therapeutic approach to inhibit FoxM1 by targeting the mevalonate pathway for HCC.