ABSTRACT
BACKGROUND AND PURPOSE: Adiponectin, an adipokine possessing profound insulin-sensitizing and anti-inflammatory properties, is a potent biotherapeutic agent . The trimeric adiponectin subunit assembles into hexameric and functionally important higher molecular weight (HMW) forms, controlled by the endoplasmic reticulum protein 44 (ERp44). Obesity-induced ER stress decreases the HMW form in serum, contributing to the development of insulin resistance and Type 2 diabetes. In this study, a panel of synthetic peptides, designed to target ERp44-adiponectin interactions, were tested for their effects on circulating levels of HMW adiponectin. EXPERIMENTAL APPROACH: Peptides derived from the ERp44 binding region of adiponectin and immunoglobulin IgM were synthesized with or without a cell-penetrating sequence. Cultures of 3T3-L1 adipocytes were incubated with the peptides for assessing the assembly and secretion of HMW adiponectin. Mice given standard chow or a high-fat diet were treated acutely or chronically, with the peptides to investigate the therapeutic effects on insulin sensitivity and energy metabolism. RESULTS: The designed peptides interfered with ERp44-adiponectin interactions and modulated adiponectin assembly and release from adipocytes. In particular, IgM-derived peptides facilitated the release of endogenous adiponectin (especially the HMW form) from adipose tissue, enhanced its circulating level and the ratio of HMW-to-total-adiponectin in obese mice. Long-term treatment of mice fed with high-fat diet by IgM-derived peptides reduced the circulating lipid levels and improved insulin sensitivity. CONCLUSIONS AND IMPLICATIONS: Targeting ERp44-adiponectin interactions with short peptides represents an effective strategy to treat of obesity-related metabolic disorders, such as insulin resistance and Type 2 diabetes.
Subject(s)
Adiponectin/metabolism , Metabolic Diseases/drug therapy , Obesity/complications , Peptides/pharmacology , 3T3-L1 Cells , Animals , Diabetes Mellitus, Type 2/etiology , Disease Models, Animal , Drug Design , Endoplasmic Reticulum Stress/drug effects , Energy Metabolism/drug effects , Insulin Resistance , Male , Membrane Proteins/metabolism , Metabolic Diseases/etiology , Mice , Mice, Inbred C57BL , Molecular Chaperones/metabolism , Molecular Weight , Peptides/chemical synthesisABSTRACT
Adiponectin, a collagenous hormone secreted abundantly from adipocytes, possesses potent antidiabetic and anti-inflammatory properties. Mediated by the conserved Cys(39) located in the variable region of the N terminus, the trimeric (low molecular weight (LMW)) adiponectin subunit assembles into different higher order complexes, e.g. hexamers (middle molecular weight (MMW)) and 12-18-mers (high molecular weight (HMW)), the latter being mostly responsible for the insulin-sensitizing activity of adiponectin. The endoplasmic reticulum (ER) chaperone ERp44 retains adiponectin in the early secretory compartment and tightly controls the oxidative state of Cys(39) and the oligomerization of adiponectin. Using cellular and in vitro assays, we show that ERp44 specifically recognizes the LMW and MMW forms but not the HMW form. Our binding assays with short peptide mimetics of adiponectin suggest that ERp44 intercepts and converts the pool of fully oxidized LMW and MMW adiponectin, but not the HMW form, into reduced trimeric precursors. These ERp44-bound precursors in the cis-Golgi may be transported back to the ER and released to enhance the population of adiponectin intermediates with appropriate oxidative state for HMW assembly, thereby underpinning the process of ERp44 quality control.
Subject(s)
Adiponectin/metabolism , Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Molecular Chaperones/metabolism , Adiponectin/chemistry , Amino Acid Sequence , Animals , HEK293 Cells , Humans , Mice , Molecular Sequence Data , Protein Binding , Protein Interaction Maps , Protein MultimerizationABSTRACT
The chemical synthesis is described of a polypeptide construct possessing both the variable and the collagen-like domain of adiponectin, which can be used as a model system for probing the influence of the variable domain on multimerization of this important circulating hormone. Using a collagen domain repeat peptide unit derived from native adiponectin or a glutamic acid analogue was ineffective due to noncollagenous conformational properties in both cases. However, employing a collagen model peptide and linking this to the variable domain thioester peptide using native chemical ligation proved effective. The 63 residue peptide was characterized by circular dichroism and mass spectrometry which demonstrated that a collagen-like triple-helical structure was preserved.