Joel P. Berger

COMPLEX DISTRIBUTION, NOT ABSOLUTE AMOUNT OF ADIPONECTIN, CORRELATES WITH THIAZOLIDINEDIONE-MEDIATED IMPROVEMENT IN INSULIN SENSITIVITY

Adiponectin is an adipocyte-specific secretory protein that circulates in serum as a hexamer of relatively low molecular weight (LMW) and a larger multimeric structure of high molecular weight (HMW). Serum levels of the protein correlate with systemic insulin sensitivity. The full-length protein affects hepatic gluconeogenesis through improved insulin sensitivity, and a proteolytic fragment of adiponectin stimulates β oxidation in muscle. Here, we show that the ratio, and not the absolute amounts, between these two oligomeric forms (HMW to LMW) is critical in determining insulin sensitivity. We define a new index, SA, that can be calculated as the ratio of HMW/(HMW + LMW). db/db mice, despite similar total adiponectin levels, display decreased SA values compared with wild type littermates, as do type II diabetic patients compared with insulin-sensitive individuals. Furthermore, SA improves with peroxisome proliferator-activated receptor-γ agonist treatment (thiazolidinedione; TZD) in mice and humans. We demonstrate that changes in SA in a number of type 2 diabetic cohorts serve as a quantitative indicator of improvements in insulin sensitivity obtained during TZD treatment, whereas changes in total serum adiponectin levels do not correlate well at the individual level. Acute alterations in SA (ΔSA) are strongly correlated with improvements in hepatic insulin sensitivity and are less relevant as an indicator of improved muscle insulin sensitivity in response to TZD treatment, further underscoring the conclusions from previous clamp studies that suggested that the liver is the primary site of action for the full-length protein. These observations suggest that the HMW adiponectin complex is the active form of this protein, which we directly demonstrate in vivo by its ability to depress serum glucose levels in a dose-dependent manner.

International union of pharmacology. LXI. Peroxisome proliferator-activated receptors

The three peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily. They share a high degree of structural homology with all members of the superfamily, particularly in the DNA-binding domain and ligand- and cofactor-binding domain. Many cellular and systemic roles have been attributed to these receptors, reaching far beyond the stimulation of peroxisome proliferation in rodents after which they were initially named. PPARs exhibit broad, isotype-specific tissue expression patterns. PPARα is expressed at high levels in organs with significant catabolism of fatty acids. PPARβ/δ has the broadest expression pattern, and the levels of expression in certain tissues depend on the extent of cell proliferation and differentiation. PPARγ is expressed as two isoforms, of which PPARγ2 is found at high levels in the adipose tissues, whereas PPARγ1 has a broader expression pattern. Transcriptional regulation by PPARs requires heterodimerization with the retinoid X receptor (RXR). When activated by a ligand, the dimer modulates transcription via binding to a specific DNA sequence element called a peroxisome proliferator response element (PPRE) in the promoter region of target genes. A wide variety of natural or synthetic compounds was identified as PPAR ligands. Among the synthetic ligands, the lipid-lowering drugs, fibrates, and the insulin sensitizers, thiazolidinediones, are PPARα and PPARγ agonists, respectively, which underscores the important role of PPARs as therapeutic targets. Transcriptional control by PPAR/RXR heterodimers also requires interaction with coregulator complexes. Thus, selective action of PPARs in vivo results from the interplay at a given time point between expression levels of each of the three PPAR and RXR isotypes, affinity for a specific promoter PPRE, and ligand and cofactor availabilities.

Integrated Genomic and Proteomic Analyses of Gene Expression in Mammalian Cells

Using DNA microarrays together with quantitative proteomic techniques (ICAT reagents, two-dimensional DIGE, and MS), we evaluated the correlation of mRNA and protein levels in two hematopoietic cell lines representing distinct stages of myeloid differentiation, as well as in the livers of mice treated for different periods of time with three different peroxisome proliferative activated receptor agonists. We observe that the differential expression of mRNA (up or down) can capture at most 40% of the variation of protein expression. Although the overall pattern of protein expression is similar to that of mRNA expression, the incongruent expression between mRNAs and proteins emphasize the importance of posttranscriptional regulatory mechanisms in cellular development or perturbation that can be unveiled only through integrated analyses of both proteins and mRNAs.

Induction of Adipocyte Complement-Related Protein of 30 Kilodaltons by PPARγ Agonists: A Potential Mechanism of Insulin Sensitization

Adipocyte complement-related protein of 30 kDa (Acrp30, adiponectin, or AdipoQ) is a fat-derived secreted protein that circulates in plasma. Adipose tissue expression of Acrp30 is lower in insulin-resistant states and it is implicated in the regulation of in vivo insulin sensitivity. Here we have characterized the ability of PPARγ agonists to modulate Acrp30 expression. After chronic treatment of obese-diabetic (db/db) mice with PPARγ agonists (11 d), mean plasma Acrp30 protein levels increased (>3×). Similar effects were noted in a nongenetic type 2 diabetes model (fat-fed and low-dose streptozotocin-treated mice). In contrast, treatment of mice (db/db or fat-fed) with metformin or a PPARα agonist did not affect plasma Acrp30 protein levels. In a cohort of normal human subjects, 14-d treatment with rosiglitazone also produced a 130% increase in circulating Acrp30 levels vs. placebo. In addition, circulating Acrp30 levels were suppressed 5-fold in patients with severe insulin resistance in association wit...

NOVEL PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR) GAMMA AND PPARDELTA LIGANDS PRODUCE DISTINCT BIOLOGICAL EFFECTS

The peroxisome proliferator-activated receptors (PPARs) include three receptor subtypes encoded by separate genes: PPARα, PPARδ, and PPARγ. PPARγ has been implicated as a mediator of adipocyte differentiation and the mechanism by which thiazolidinedione drugs exert in vivo insulin sensitization. Here we characterized novel, non-thiazolidinedione agonists for PPARγ and PPARδ that were identified by radioligand binding assays. In transient transactivation assays these ligands were agonists of the receptors to which they bind. Protease protection studies showed that ligand binding produced specific alterations in receptor conformation. Both PPARγ and PPARδ directly interacted with a nuclear receptor co-activator (CREB-binding protein) in an agonist-dependent manner. Only the PPARγ agonists were able to promote differentiation of 3T3-L1 preadipocytes. In diabeticdb/db mice all PPARγ agonists were orally active insulin-sensitizing agents producing reductions of elevated plasma glucose and triglyceride concentrations. In contrast, selectivein vivo activation of PPARδ did not significantly affect these parameters. In vivo PPARα activation with WY-14653 resulted in reductions in elevated triglyceride levels with minimal effect on hyperglycemia. We conclude that: 1) synthetic non-thiazolidinediones can serve as ligands of PPARγ and PPARδ; 2) ligand-dependent activation of PPARδ involves an apparent conformational change and association of the receptor ligand binding domain with CREB-binding protein; 3) PPARγ activation (but not PPARδ or PPARα activation) is sufficient to potentiate preadipocyte differentiation; 4) non-thiazolidinedione PPARγ agonists improve hyperglycemia and hypertriglyceridemia in vivo; 5) although PPARα activation is sufficient to affect triglyceride metabolism, PPARδ activation does not appear to modulate glucose or triglyceride levels.

Activation of PPARδ alters lipid metabolism in db/db mice

Peroxisome proliferator‐activated receptors (PPARs) are nuclear receptors, which heterodimerize with the retinoid X receptor and bind to peroxisome proliferator response elements in the promoters of regulated genes. Despite the wealth of information available on the function of PPARα and PPARγ, relatively little is known about the most widely expressed PPAR subtype, PPARδ. Here we show that treatment of insulin resistant db/db mice with the PPARδ agonist L‐165 041, at doses that had no effect on either glucose or triglycerides, raised total plasma cholesterol concentrations. The increased cholesterol was primarily associated with high density lipoprotein (HDL) particles, as shown by fast protein liquid chromatography analysis. These data were corroborated by the chemical analysis of the lipoproteins isolated by ultracentrifugation, demonstrating that treatment with L‐165 041 produced an increase in circulating HDL without major changes in very low or low density lipoproteins. White adipose tissue lipoprotein lipase activity was reduced following treatment with the PPARδ ligand, but was increased by a PPARγ agonist. These data suggest both that PPARδ is involved in the regulation of cholesterol metabolism in db/db mice and that PPARδ ligands could potentially have therapeutic value.

Retinaldehyde represses adipogenesis and diet-induced obesity.

The metabolism of vitamin A and the diverse effects of its metabolites are tightly controlled by distinct retinoid-generating enzymes, retinoid-binding proteins and retinoid-activated nuclear receptors. Retinoic acid regulates differentiation and metabolism by activating the retinoic acid receptor and retinoid X receptor (RXR), indirectly influencing RXR heterodimeric partners. Retinoic acid is formed solely from retinaldehyde (Rald), which in turn is derived from vitamin A. Rald currently has no defined biologic role outside the eye. Here we show that Rald is present in rodent fat, binds retinol-binding proteins (CRBP1, RBP4), inhibits adipogenesis and suppresses peroxisome proliferator-activated receptor-γ and RXR responses. In vivo, mice lacking the Rald-catabolizing enzyme retinaldehyde dehydrogenase 1 (Raldh1) resisted diet-induced obesity and insulin resistance and showed increased energy dissipation. In ob/ob mice, administrating Rald or a Raldh inhibitor reduced fat and increased insulin sensitivity. These results identify Rald as a distinct transcriptional regulator of the metabolic responses to a high-fat diet.

Insulin- and Mitogen-activated Protein Kinase-mediated Phosphorylation and Activation of Peroxisome Proliferator-activated Receptor γ

Peroxisome proliferator-activated receptor (PPAR) γ plays an important role in adipocyte differentiation and the regulation of adipocyte gene expression. Insulin also serves to promote adipogenesis. We report that insulin and a PPARγ ligand (thiazolidinedione (TZD)) stimulate in a synergistic manner the expression of an adipocyte-specific gene (aP2) in rat adipocytes and 3T3-L1 cells. Potential cross-talk between insulin signaling and PPARγ was studied in Chinese hamster ovary cells expressing insulin receptors (CHO.T), PPARγ, and reporter genes. Both TZD and insulin independently stimulated PPARγ-mediated transactivation of aP2 promoter-luciferase reporter genes; both agents combined resulted in a synergistic effect. Co-transfection of CHO.T cells with dominant-negative mitogen-activated protein (MAP) kinase-kinase (MKK1) abrogated both insulin- and TZD-mediated activation of PPARγ; transactivation was markedly increased in cells co-transfected with constitutively active MKK1. Both insulin and constitutively active MKK1 also stimulated 32P incorporation into PPARγ in vivo. The conclusions are: 1) Insulin synergizes with a PPARγ ligand and can activate the receptor in a ligand-independent fashion. 2) PPARγ is phosphorylated in vivo by insulin stimulation or activation of the MAP kinase pathway. 3) MAP kinase is an important mediator of cross-talk between insulin signal transduction pathways and PPARγ function.

Adipose Fibroblast Growth Factor 21 Is Up-Regulated by Peroxisome Proliferator-Activated Receptor γ and Altered Metabolic States

Adipose tissue is a metabolically responsive endocrine organ that secretes a myriad of adipokines. Antidiabetic drugs such as peroxisome proliferator-activated receptor (PPAR) γ agonists target adipose tissue gene expression and correct hyperglycemia via whole-body insulin sensitization. The mechanism by which altered gene expression in adipose tissue affects liver and muscle insulin sensitivity (and thus glucose homeostasis) is not fully understood. One possible mechanism involves the alteration in adipokine secretion, in particular the up-regulation of secreted factors that increase whole-body insulin sensitivity. Here, we report the use of transcriptional profiling to identify genes encoding for secreted proteins the expression of which is regulated by PPARγ agonists. Of the 379 genes robustly regulated by two structurally distinct PPARγ agonists in the epididymal white adipose tissue (EWAT) of db/db mice, 33 encoded for known secreted proteins, one of which was FGF21. Although FGF21 was recently reported to be up-regulated in cultured adipocytes by PPARγ agonists and in liver by PPARα agonists and induction of ketotic states, we demonstrate that the protein is transcriptionally up-regulated in adipose tissue in vivo by PPARγ agonist treatment and under a variety of physiological conditions, including fasting and high fat diet feeding. In addition, we found that circulating levels of FGF21 protein were increased upon treatment with PPARγ agonists and under ketogenic states. These results suggest a role for FGF21 in mediating the antidiabetic activities of PPARγ agonists.

Activation of Peroxisome Proliferator-Activated Receptor γ Does Not Inhibit IL-6 or TNF-α Responses of Macrophages to Lipopolysaccharide In Vitro or In Vivo

We have investigated the potential use of peroxisome proliferator-activated receptor γ (PPARγ) agonists as anti-inflammatory agents in cell-based assays and in a mouse model of endotoxemia. Human peripheral blood monocytes were treated with LPS or PMA and a variety of PPARγ agonists. Although 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) at micromolar concentrations significantly inhibited the production of TNF-α and IL-6, four other high affinity PPARγ ligands failed to affect cytokine production. Similar results were obtained when the monocytes were allowed to differentiate in culture into macrophages that expressed significantly higher levels of PPARγ or when the murine macrophage cell line RAW 264.7 was used. Furthermore, saturating concentrations of a potent PPARγ ligand not only failed to block cytokine production, but also were unable to block the inhibitory activity of 15d-PGJ2. Thus, activation of PPARγ does not appear to inhibit the production of cytokines by either monocytes or macrophages, and the inhibitory effect observed with 15d-PGJ2 is most likely mediated by a PPARγ-independent mechanism. To examine the anti-inflammatory activity of PPARγ agonists in vivo, db/db mice were treated with a potent thiazolidinedione that lowered their elevated blood glucose and triglyceride levels as expected. When thiazolidinedione-treated mice were challenged with LPS, they displayed no suppression of cytokine production. Rather, their blood levels of TNF-α and IL-6 were elevated beyond the levels observed in control db/db mice challenged with LPS. Comparable results were obtained with the corresponding lean mice. Our data suggest that compounds capable of activating PPARγ in leukocytes will not be useful for the treatment of acute inflammation.