Obesity is a disorder of energy balance. Hormone-sensitive lipase (HSL) mediates the hydrolysis of triacylglycerol, the major form of stored energy in the body. Perilipin (encoded by the gene Plin), an adipocyte protein, has been postulated to modulate HSL activity. We show here that targeted disruption of Plin results in healthy mice that have constitutively activated fat-cell HSL. Plin -/- mice consume more food than control mice, but have normal body weight. They are much leaner and more muscular than controls, have 62% smaller white adipocytes, show elevated basal lipolysis that is resistant to beta-adrenergic agonist stimulation, and are cold-sensitive except when fed. They are also resistant to diet-induced obesity. Breeding the Plin -/- alleles into Leprdb/db mice reverses the obesity by ncreasing the metabolic rate of the mice. Our results demonstrate a role for perilipin in reining in basal HSL activity and regulating lipolysis and energy balance; thus, agents that inactivate perilipin may prove useful as anti-obesity medications.

Contents 1. Introduction 1315 2. Phosphorus-31 Chemical Shifts 1316 1. Introduction and Basic Principles 1316 2. Theoretical 31P Chemical Shift Calculations 1316 and Empirical Observations 3. Bond Angle Effects 1317 4. Stereoelectronic Effects on 31P Chemical 1318 Shifts 5. Extrinsic and Other Effects on 31P 1318 Chemical Shifts 3. Assignment of 31P Signals of Oligonucleotides 1318 4. 31P Heteronuclear Coupling Constants 1319 5. 31P Melting Curves 1322 6. 31P NMR of Polynucleic Acids 1322 7. 31P NMR of DNA-Drug Complexes 1322 8. Sequence-Specific Variation in 31P Chemical 1323 Shifts and Coupling Constants of Duplex Oligonucleotides 1. 31P Chemical Shifts as a Function of 1324 Sequence and Position 2. Conformation and Dynamics of the 1325 Phosphate Ester Backbone 3. Origin of Sequence-Specific Variation in 1326 the « and f Torsional Angles and P-H3' Coupling Constants; C4'-C4' Interresidue Distances 4. 31P NMR Spectra of a Tandem 1328 GA-Mismatch Duplex, d(CCAAGATTGG)2 5. Comparison of the Phosphate Backbone

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A nonanucleotide in which (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9,10-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (7-hydroxy group and epoxide oxygen are trans) is covalently bonded to the exocyclic N6-amino group of deoxyadenosine through trans addition at C10 of the epoxide (10R adduct) has been synthesized. The modified oligonucleotide d(GGTCA*CGAG) was incorporated into the duplex d(GGTCA*CGAG).d(CTCGGGACC), containing a dG mismatch opposite the modified base (dA*). Proton assignments for the solution structure of the duplex containing the 10R adduct were made using 2D TOCSY and NOESY NMR spectra. The complete hybrid relaxation matrix program, MORASS2.0, was used to generate NOESY distance constraints for iterative refinement using distance-restrained molecular dynamics calculations with AMBER4.0. The iteratively refined structure showed the hydrocarbon intercalated from the major groove immediately below the dC4-dG15 base pair and oriented toward the 5'-end of the modified strand. The modified dA is in an anti configuration, with the dG of the GA mismatch turned out into the major groove. Chemical shifts of the hydrocarbon protons and unusual chemical shifts of sugar protons were accounted for by this orientation of the adduct. The information available currently provides the foundation for the rational explanation of observed benzo[a]pyrene (BaP) structures and predictions for other BaP dG and dA adducts.

A nonanucleotide, d(G1G2T3C4[BaP]A5C6G7A8G9), in which (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (7-hydroxyl group and epoxide oxygen are trans) is covalently bonded to the exocyclic N6-amino group of deoxyadenosine (dA5) through trans addition at C10 of the epoxide (to give a 10S adduct) has been synthesized. The solution structure of the duplex, d(G1G2T3C4[BaP]A5C6G7A8G9).d(C10T11C12G13G14G15A16C17C18+ ++), containing a dG mismatch opposite the modified dA (designated 10S-[BaP]dA.dG 9-mer duplex) has been investigated using a combination of 1D and 2D (including COSY, PECOSY, TOCSY, NOESY, and indirect detection of 1H-31P HETCOR) NMR spectroscopies. The NMR results together with restrained molecular dynamics/energy minimization calculations show that the modified dA5 adopts a syn glycosidic torsion angle whereas all other nucleotide residues adopt anti glycosidic torsion angles. The sugar ring of dA5 is in the C3'-endo conformation, and the sugar rings of the other residues are in the C2'-endo conformation. The hydrocarbon attached at dA5 orients toward the 3' end of the modified strand (i.e., dC6 direction) and intercalates between and parallel to bases of dG13 and dG14 of the complementary strand directly opposite dC6 and dA5, respectively. The edge of the hydrocarbon bearing H11 and H12 is positioned between the imino protons of dG13 and dG14 in the interior of the duplex, whereas H4 and H5 at the opposite edge are positioned near the sugar H1' and H2" protons of dG13 and facing the exterior of the duplex. The mismatched AG base pair is stabilized by dAsyn-dGanti base pairing in which the imino proton and the O6 of dG14 are hydrogen bonded to N7- and the single N6-amino proton, respectively, of the modified dA5. The modified DNA duplex remains in a right-handed helix, which bends at the site of intercalation about 20 to 30 degrees away from the helical axis and toward the direction of the modified strand.