Glucose and other reducing sugars react with proteins by a nonenzymatic, post-translational modification process called nonenzymatic glycosylation or glycation. The sugar-derived carbonyl group adds to a free amine, forming a reversible adduct which over time rearranges to produce a class of products termed advanced-glycation end-products (AGEs). These remain irreversibly bound to macromolecules and can covalently crosslink proximate amino groups. The formation of AGEs on long-lived connective tissue and matrix components accounts largely for the increase in collagen crosslinking that accompanies normal ageing and which occurs at an accelerated rate in diabetes. AGEs can activate cellular receptors and initiate a variety of pathophysiological responses. They modify an appreciable fraction of circulating low-density lipoproteins preventing uptake of these particles by their high-affinity tissue receptors. Advanced glycation has also been implicated in the pathology of Alzheimer's disease. Because AGEs may form by a pathway involving reactive alpha-dicarbonyl intermediates, we investigated a potential pharmacological strategy for selectively cleaving the resultant glucose-derived protein crosslinks. We now describe a prototypic AGE crosslink 'breaker', N-phenacylthiazolium bromide (PTB), which reacts with and cleaves covalent, AGE-derived protein crosslinks. The ability of PTB to break AGE crosslinks in vivo points to the importance of an alpha-dicarbonyl intermediate in the advanced glycation pathway and offers a potential therapeutic approach for the removal of established AGE crosslinks.

Hormone-sensitive lipase activity (HSL), which is found in the supernatant of centrifuged homogenates of lipolytically quiet isolated rat adipocytes, was greatly reduced in or absent from the supernatant of lipolytically stimulated cells. The lipase was purified 100-to 250-fold from the supernatant of lipolytically quiet cells to 10-20% purity by a single passage over phenyl-Sepharose resin with high (>70%) activity yields. Western blotting of adipocyte homogenate fractions with polyclonal antiserum raised against HSL showed that the enzyme shifted quantitatively from the supernatant of control cells to the floating "fat cake" of lipolytically stimulated cells. A similar shift to the fat cake was observed when cells were disrupted by hypotonic lysis and centrifugation rather than by homogenization. We propose that upon lipolytic activation of adipocytes and phosphorylation ofHSL by cAMPdependent protein kinase, the critical event is not an increase in catalytic activity (i.e., turnover number) but a translocation of the lipase to its substrate at the surface of the lipid storage droplet.

Glucose and other reducing sugars react with proteins by a nonenzymatic, posttranslational modification process called nonenzymatic glycation. The formation of advanced glycation end products (AGEs) on connective tissue and matrix components accounts largely for the increase in collagen crosslinking that accompanies normal aging and which occurs at an accelerated rate in diabetes, leading to an increase in arterial stiffness. A new class of AGE crosslink ''breakers'' reacts with and cleaves these covalent, AGEderived protein crosslinks. Treatment of rats with streptozotocin-induced diabetes with the AGE-breaker ALT-711 for 1-3 weeks reversed the diabetes-induced increase of large artery stiffness as measured by systemic arterial compliance, aortic impedance, and carotid artery compliance and distensibility. These findings will have considerable implications for the treatment of patients with diabetes-related complications and aging.

The major cAMP-dependent protein kinase (A-kinase) substrate in adipocytes is perilipin, a protein found exclusively at the surface of the lipid storage droplets. Using anti-perilipin serum, we have isolated two related classes of funl-length coding cDNAs, designated perilipin A and B, from a rat adipocyte cDNA expression library. The two cDNAs derive from two mRNA species that arise by differential splicing. The mRNAs are predicted to encode perilipins A and B, proteins of 517 aa (56,870 Da) and 422 aa (46,420 Da), respectively, which share a common 406-aa N-terminal sequence. The predicted perilipin A contains peptides present in proteolytic digests ofthe purified 62-kDa form ofperilipin from rat adipocytes, as well as the requisite consensus A-kinase phosphorylation sites. Like perilipin A, the B form is expressed in adipocytes and is associated with lipid storage droplets. Modeling of predicted secondary structures fails to reveal an underlying basis for the tenacious association of perilipins with lipid droplets. These proteins exhibit a sicant sequence relationship (=65% similarity through 105 aa) with only one other known protein, the adipocyte differentiation-related protein (ADRP). Like the perilipins, ADRP appears to be adipocyte-specific, which suggests that they interact in a related intracellular pathway. The molecular probes for perilipins A and B described here will permit detailed analyses of their functional role(s) in lipid metabolism.The major reservoir of stored energy in animals is the triacylglycerol pool in lipid storage droplets of adipose cells. As reviewed briefly (1)