Shupei Wang

Lipolysis and the integrated physiology of lipid energy metabolism

Fat cell lipolysis, the cleavage of triglycerides and release of fatty acids and glycerol, evolved to enable survival during prolonged food deprivation but is paradoxically increased in obesity, in which a surfeit of all energy metabolites is found. Essential, previously-unsuspected components have been discovered in the lipolytic machinery, at the protective interface of the lipid droplet surface and in the signaling pathways that control lipolysis. At least two adipocyte lipases are important for controlling lipolysis, hormone-sensitive lipase (HSL) and adipocyte triglyceride lipase (ATGL). Perilipin (PLIN) and possibly other proteins of the lipid droplet surface are master regulators of lipolysis, protecting or exposing the triglyceride core of the droplet to lipases. The prototypes for hormonal lipolytic control are beta adrenergic stimulation and suppression by insulin, both of which affect cyclic AMP levels and hence the protein kinase A-mediated phosphorylation of HSL and PLIN. Newly-recognized mediators of lipolysis include atrial natriuretic peptide, cyclic GMP, the ketone body 3-hydroxybutyrate, AMP kinase and mitogen-activated kinases. Lipolysis must be interpreted in its physiological context since similar rates of basal or stimulated lipolysis occur under different conditions and by different mechanisms. Age, sex, anatomical site, genotype and species differences are each important variables. Manipulation of lipolysis has therapeutic potential in several inborn errors and in the metabolic syndrome that frequently complicates obesity.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency: clinical course and description of causal mutations in two patients.

Hereditary deficiency of mitochondrial HMG-CoA synthase (mHS, OMIM 600234) is a poorly defined, treatable, probably underdiagnosed condition that can cause episodes of severe hypoketotic hypoglycemia. We present clinical follow-up and molecular analysis of the two known mHS-deficient patients. The diagnosis of mHS deficiency is challenging because the symptoms and metabolite pattern are not specific. Moreover, enzyme analysis is technically difficult and requires sampling of an expressing organ such as liver. The patients, now aged 16 and 6 y, have normal development and have had no further decompensations since diagnosis. Patient 1 is homozygous for a phenylalanine-to-leucine substitution at codon 174 (F174L). Interestingly, although the F174 residue is conserved in vertebrate mHS and cytoplasmic HS isozymes, a Leu residue is predicted in the corresponding position of HS-like sequences from Caenorhabditis elegans, Arabidopsis thaliana, and Brassica juncea. Bacterial expression of human F174L-mHS produces a low level of mHS polypeptide with no detectable activity. Similarly, in purified cytoplasmic HS, which in contrast to purified human mHS is stable and can be studied in detail, the corresponding F→L substitution causes a 10,000-fold decrease in Vmax and a 5-fold reduction in thermal stability. Patient 2 is a genetic compound of a premature termination mutation, R424X, and an as-yet uncharacterized mutant allele that is distinguishable by intragenic single nucleotide polymorphisms that we describe. Molecular studies of mHS are useful in patients with a suggestive clinical presentation.

3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL): cloning and characterization of a mouse liver HL cDNA and subchromosomal mapping of the human and mouse HL genes

Abstract3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL) is a homodimeric mitochondrial matrix enzyme that catalyzes the last step of ketogenesis. Using a human HL cDNA as a probe, we isolated a 1.4-kb mouse HL cDNA (HLM) from a mouse liver library and extended the sequence in the 5′ direction, using RACE PCR to include the complete coding sequence. The nucleotide sequence of the mouse HL coding region is 85.7% identical to human HL, and 52.6% to Ps. mevalonii HL. Peptide identities of 87.4% and 54.3% respectively were observed. Southern analysis of 29 strains of laboratory mice and of Mus spretus revealed a total of about 25 kb of hybridizing fragments and three polymorphic fragments in both EcoRI and HindIII digestions. The mouse HL locus (Hmgcl) was localized on Chromosome (Chr) 4: Pmv-19-12.6±3.6 cM-Hmgcl-7.3±2.3 cM-Xmv-8-1.5±1.0 cM-Gpd1. The human HL locus (HMGCL) was mapped to distal Chr 1p by analysis of a human-hamster hybrid cell panel and by in situ hybridization.

Hormone-sensitive lipase (Lipe): Sequence analysis of the 129Sv mouse Lipe gene

Hormone-sensitive lipase (Lipe) catalyzes both the release of fatty acids from storage triglycerides in adipocytes and the liberation of cholesterol from cholesterol esters in steroidogenic tissues. Lipe activity is regulated in a tissue-, development- and hormone-specific fashion, the latter in large part by serine phosphorylation. We cloned and sequenced the Lipe gene from the 129Sv strain mouse, including 2.7 kb of the 5′ nontranslated region. The primary transcript of the 129Sv Lipe locus spans 9.6 kb and contains 9 exons. We studied the curious hypervariable region immediately 5′ to the regulatory serine residues by aligning the peptide and nucleic acid sequences of mouse, human, and rat Lipe. We propose that much of the variability is attributable to differences in the copy number of a 12-nucleotide repeat that shifts the intron 7 acceptor splice site. Introns 1 and 7 contain B1 elements, which in intron 7 are immediately adjacent to a tetranucleotide repeat. The mouse Lipe promoter region contains numerous potential binding motifs for factors implicated in adipose tissue expression and hormone responsiveness including adipocyte determination- and differentiation-dependent factor 1 (ADD1/SREBP1).

The stoichiometry of protein phosphorylation in adipocyte lipid droplets: Analysis by N-terminal isotope tagging and enzymatic dephosphorylation

Most phosphoproteomic studies to date have been limited to the identification of phosphoproteins and their phosphorylation sites, and have not assessed the stoichiometry of protein phosphorylation, a critical parameter reflecting the dynamic equilibrium between phosphorylated and non‐phosphorylated pools of proteins. Here, we used a method for measuring phosphorylation stoichiometry through isotope tagging and enzymatic dephosphorylation of tryptic peptides. Using this method, protein digests are divided into two equal aliquots that are modified with either light or heavy isotope tags. One aliquot is dephosphorylated by alkaline phosphatase. Finally, the peptide mixtures are recombined and LC‐MS/MS analysis is performed. With this method, we studied adipocytes of mice stimulated with CL316,243, a β‐3 adrenergic agonist known to induce lipolysis and marked phosphorylation changes in proteins of the lipid droplet surface. In lipid droplet preparations, CL316,243 administration increased phosphorylation of proteins related to regulation of signaling, metabolism and intracellular trafficking in white adipose tissue, including hormone‐sensitive lipase which was 80% phosphorylated at the previously reported site, Ser‐559, and the lipid surface protein perilipin, which was phosphorylated by ∼60 and ∼40% at previously unreported sites, Ser‐410 and Ser‐460.