Systemic fate of the adipocyte-derived factor adiponectin.

OBJECTIVE: The adipocyte-derived secretory protein adiponectin has been widely studied and shown to have potent insulin-sensitizing, antiapoptotic, and anti-inflammatory properties. While its biosynthesis is well understood, its fate, once in circulation, is less well established. RESEARCH DESIGN AND METHODS: Here, we examine the half-life of adiponectin in circulation by tracking fluorescently labeled recombinant adiponectin in the circulation, following it to its final destination in the hepatocyte. RESULTS: Despite its abundant presence in plasma, adiponectin is cleared rapidly with a half-life of approximately 75 min. A more bioactive version carrying a mutation at cysteine 39 is cleared within minutes. Even though steady-state levels of adiponectin differ between male and female mice, we failed to detect any differences in clearance rates, suggesting that differences in plasma are mostly due to differential production rates. In a metabolically challenged state (high-fat diet exposure or in an ob/ob background), adiponectin levels are reduced in plasma and clearance is significantly prolonged, reflecting a dramatic drop in adiponectin production levels. CONCLUSIONS: Combined, these results show a surprisingly rapid turnover of adiponectin with multiple fat pads contributing to the plasma levels of adiponectin and clearance mediated primarily by the liver. It is surprising that despite high-level production and rapid clearance, plasma levels of adiponectin remain remarkably constant.

PANIC-ATTAC: a mouse model for inducible and reversible beta-cell ablation.

OBJECTIVE: Islet transplantations have been performed clinically, but their practical applications are limited. An extensive effort has been made toward the identification of pancreatic beta-cell stem cells that has yielded many insights to date, yet targeted reconstitution of beta-cell mass remains elusive. Here, we present a mouse model for inducible and reversible ablation of pancreatic beta-cells named the PANIC-ATTAC (pancreatic islet beta-cell apoptosis through targeted activation of caspase 8) mouse. RESEARCH DESIGN AND METHODS: We efficiently induce beta-cell death through apoptosis and concomitant hyperglycemia by administration of a chemical dimerizer to the transgenic mice. In contrast to animals administered streptozotocin, the diabetes phenotype and beta-cell loss are fully reversible in the PANIC-ATTAC mice, and we find significant beta-cell recovery with normalization of glucose levels after 2 months. RESULTS: The rate of recovery can be enhanced by various pharmacological interventions with agents acting on the glucagon-like peptide 1 axis and agonists of peroxisome proliferator-activated receptor-gamma. During recovery, we find an increased population of GLUT2(+)/insulin(-) cells in the islets of PANIC-ATTAC mice, which may represent a novel pool of potential beta-cell precursors. CONCLUSIONS: The PANIC-ATTAC mouse may be used as an animal model of inducible and reversible beta-cell ablation and therefore has applications in many areas of diabetes research that include identification of beta-cell precursors, evaluation of glucotoxicity effects in diabetes, and examination of pharmacological interventions.

Secretion of the adipocyte-specific secretory protein adiponectin critically depends on thiol-mediated protein retention.

Adiponectin is a secretory protein abundantly secreted from adipocytes. It assembles into a number of different higher-order complexes. Adipocytes maintain tight control over circulating plasma levels, suggesting the existence of a complex, highly regulated biosynthetic pathway. However, the critical mediators of adiponectin maturation within the secretory pathway have not been elucidated. Previously, we found that a significant portion of de novo-synthesized adiponectin is not secreted and retained in adipocytes. Here, we show that there is an abundant pool of properly folded adiponectin in the secretory pathway that is retained through thiol-mediated retention, as judged by the release of adiponectin in response to treatment of adipocytes with reducing agents. Adiponectin is covalently bound to the ER chaperone ERp44. An adiponectin mutant lacking cysteine 39 fails to stably interact with ERp44, demonstrating that this residue is the primary site mediating the covalent interaction. Another ER chaperone, Ero1-Lalpha, plays a critical role in the release of adiponectin from ERp44. Levels of both of these proteins are highly regulated in adipocytes and are influenced by the metabolic state of the cell. While less critical for the secretion of trimers, these chaperones play a major role in the assembly of higher-order adiponectin complexes. Our data highlight the importance of posttranslational events controlling adiponectin levels and the release of adiponectin from adipocytes. One mechanism for increasing circulating levels of specific adiponectin complexes by peroxisome proliferator-activated receptor gamma agonists may be selective upregulation of rate-limiting chaperones.

Platelets from Munc18c heterozygous mice exhibit normal stimulus-induced release.

A critical aspect of hemostasis is the release of clot-forming components from the three intra-platelet stores: dense core granules, alpha-granules and lysosomes. Exocytosis from these granules is mediated by soluble (SNAPs and NSF) and integralmembrane proteins (v- and t-SNAREs). Three SM (Sec1/Munc18) proteins are present in mouse platelets (Munc18a, 18b and 18c) and each potentially regulates exocytosis via modulation of their cognate syntaxin binding partner. To define the molecular machinery required for platelet exocytosis, we analyzed platelets from Munc18c heterozygous knockout mice. These platelets show a decrease in Munc18c but no apparent reduction in other secretory machinery components. No differences in the rates of aggregation or of secretion of [(3)H]-5HT (dense core granules), platelet factor 4 (alpha-granules), or hexosaminidase (lysosomes) were detected between platelets from Munc18c heterozygous knockout or wild-type mice. The platelets also show normal morphology. Contrary to a predicted requirement for Munc18c in platelet secretion, data reported here show that reducing Munc18c levels does not substantially alter platelet function. These data show that despite Munc18c's role in platelet secretion, the lack of a secretion defect may be attributed to compensation by other Munc18 isoforms or that one allele is sufficient to maintain secretion under standard conditions.

Granule stores from cellubrevin/VAMP-3 null mouse platelets exhibit normal stimulus-induced release.

It is widely accepted that the platelet release reaction is mediated by heterotrimeric complexes of integral membrane proteins known as SNAREs (SNAP receptors). In an effort to define the precise molecular machinery required for platelet exocytosis, we have analyzed platelets from cellubrevin/VAMP-3 knockout mice. Cellubrevin/VAMP-3 has been proposed to be a critical v-SNARE for human platelet exocytosis; however, data reported here suggest that it is not required for platelet function. Upon stimulation with increasing concentrations of thrombin, collagen, or with thrombin for increasing time there were no differences in secretion of [3H]-5HT (dense core granules), platelet factor IV (alpha granules), or hexosaminidase (lysosomes) between null and wild-type platelets. There were no gross differences in bleeding times nor in agonist-induced aggregation measured in platelet-rich plasma or with washed platelets. Western blotting of wild-type, heterozygous, and null platelets confirmed the lack of cellubrevin/VAMP-3 in nulls and showed that most elements of the secretion machinery are expressed at similar levels. While the secretory machinery in mice was similar to humans, mice did express apparently higher levels of synaptobrevin/VAMP-2. These data show that the v-SNARE, cellubrevin/VAMP-3 is not a requirement for the platelet release reaction in mice.

A role for Sec1/Munc18 proteins in platelet exocytosis.

A critical aspect of haemostasis is the release of clot-forming components from the three intra-platelet stores: dense-core granules, alpha granules and lysosomes. Exocytosis from these granules is mediated by soluble proteins [N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs)] and integral membrane proteins [vesicle and target SNAP receptors (v- and t-SNAREs)]. Three Sec1/Munc18 proteins (SM proteins) are present in platelets (Munc18a, Munc18b and Munc18c) and they bind to and potentially regulate specific syntaxin t-SNAREs. In resting platelets, these SM proteins associate with granules and open canalicular system membranes predominantly but not with the plasma membrane. Munc18a binds to syntaxin 2 alone and does not associate with other members of the core SNARE complex. Munc18b associates with a larger complex that contains synaptosome-associated protein of 23 kDa (SNAP-23) and cellubrevin/vesicle-associated membrane protein 3. Munc18c associates with both syntaxins 2 and 4, with synaptosome-associated protein of 23 kDa (SNAP-23) and with a v-SNARE. On stimulation, most of the platelet SM proteins are still found in membrane fractions. Phosphorylation of each Munc18 increases in thrombin-treated cells and phosphorylated Munc18c remains associated with syntaxins 2 and 4, but its affinity for the SNAREs appears to be reduced. To determine the functional role of the platelet SM proteins, we examined the effects of Munc18-based peptides (Munc18a peptide 3 and Munc18c peptide 3). Addition of the peptides to permeabilized platelets inhibits secretion from all three platelet granules. These peptides also inhibit agonist-induced aggregation in saponin-permeabilized platelets. These studies demonstrate a clear role for SM proteins in platelet exocytosis and aggregation and suggest a dominant role for Munc18c in all three granule-release events.