Nils Halberg

Receptor-mediated activation of ceramidase activity initiates the pleiotropic actions of adiponectin

The adipocyte-derived secretory factor adiponectin promotes insulin sensitivity, decreases inflammation and promotes cell survival. No unifying mechanism has yet explained how adiponectin can exert such a variety of beneficial systemic effects. Here, we show that adiponectin potently stimulates a ceramidase activity associated with its two receptors, AdipoR1 and AdipoR2, and enhances ceramide catabolism and formation of its antiapoptotic metabolite—sphingosine-1-phosphate (S1P)—independently of AMP-dependent kinase (AMPK). Using models of inducible apoptosis in pancreatic beta cells and cardiomyocytes, we show that transgenic overproduction of adiponectin decreases caspase-8-mediated death, whereas genetic ablation of adiponectin enhances apoptosis in vivo through a sphingolipid-mediated pathway. Ceramidase activity is impaired in cells lacking both adiponectin receptor isoforms, leading to elevated ceramide levels and enhanced susceptibility to palmitate-induced cell death. Combined, our observations suggest a unifying mechanism of action for the beneficial systemic effects exerted by adiponectin, with sphingolipid metabolism as its core upstream signaling component.The adipocyte-derived secretory factor adiponectin promotes insulin sensitivity, decreases inflammation and promotes cell survival. To date, no unifying mechanism explains how adiponectin can exert such a variety of beneficial systemic effects. Here, we show that adiponectin potently stimulates a ceramidase activity associated with its two receptors, adipoR1 and adipoR2, and enhances ceramide catabolism and formation of its anti-apoptotic metabolite – sphingosine-1-phosphate (S1P), independently of AMPK. Using models of inducible apoptosis in pancreatic β-cells and cardiomyocytes, we show that transgenic overproduction of adiponectin decreases caspase-8 mediated death, while genetic adiponectin ablation enhances apoptosis in vivo through a sphingolipid-mediated pathway. Ceramidase activity is impaired in cells lacking both adiponectin receptor isoforms, leading to elevated ceramide levels and enhanced susceptibility to palmitate-induced cell death. Combined, our observations suggest a novel unifying mechanism of action for the beneficial systemic effects exerted by adiponectin, with sphingolipid metabolism as its core upstream component.

Hypoxia-Inducible Factor 1α Induces Fibrosis and Insulin Resistance in White Adipose Tissue

ABSTRACT Adipose tissue can undergo rapid expansion during times of excess caloric intake. Like a rapidly expanding tumor mass, obese adipose tissue becomes hypoxic due to the inability of the vasculature to keep pace with tissue growth. Consequently, during the early stages of obesity, hypoxic conditions cause an increase in the level of hypoxia-inducible factor 1α (HIF1α) expression. Using a transgenic model of overexpression of a constitutively active form of HIF1α, we determined that HIF1α fails to induce the expected proangiogenic response. In contrast, we observed that HIF1α initiates adipose tissue fibrosis, with an associated increase in local inflammation. “Trichrome- and picrosirius red-positive streaks,” enriched in fibrillar collagens, are a hallmark of adipose tissue suffering from the early stages of hypoxia-induced fibrosis. Lysyl oxidase (LOX) is a transcriptional target of HIF1α and acts by cross-linking collagen I and III to form the fibrillar collagen fibers. Inhibition of LOX activity by β-aminoproprionitrile treatment results in a significant improvement in several metabolic parameters and further reduces local adipose tissue inflammation. Collectively, our observations are consistent with a model in which adipose tissue hypoxia serves as an early upstream initiator for adipose tissue dysfunction by inducing a local state of fibrosis.

A microRNA regulon that mediates endothelial recruitment and metastasis by cancer cells

Metastatic progression of cancer is a complex and clinically daunting process. We previously identified a set of human microRNAs (miRNAs) that robustly suppress breast cancer metastasis to lung and bone and which display expression levels that predict human metastasis. Although these findings revealed miRNAs as suppressors of cell-autonomous metastatic phenotypes, the roles of non-coding RNAs in non-cell-autonomous cancer progression processes remain unknown. Here we reveal that endogenous miR-126, an miRNA silenced in a variety of common human cancers, non-cell-autonomously regulates endothelial cell recruitment to metastatic breast cancer cells, in vitro and in vivo. It suppresses metastatic endothelial recruitment, metastatic angiogenesis and metastatic colonization through coordinate targeting of IGFBP2, PITPNC1 and MERTK—novel pro-angiogenic genes and biomarkers of human metastasis. Insulin-like growth factor binding protein 2 (IGFBP2) secreted by metastatic cells recruits endothelia by modulating IGF1-mediated activation of the IGF type-I receptor on endothelial cells; whereas c-Mer tyrosine kinase (MERTK) receptor cleaved from metastatic cells promotes endothelial recruitment by competitively antagonizing the binding of its ligand GAS6 to endothelial MERTK receptors. Co-injection of endothelial cells with breast cancer cells non-cell-autonomously rescues their miR-126-induced metastatic defect, revealing a novel and important role for endothelial interactions in metastatic initiation. Through loss-of-function and epistasis experiments, we delineate an miRNA regulatory network’s individual components as novel and cell-extrinsic regulators of endothelial recruitment, angiogenesis and metastatic colonization. We also identify the IGFBP2/IGF1/IGF1R and GAS6/MERTK signalling pathways as regulators of cancer-mediated endothelial recruitment. Our work further reveals endothelial recruitment and endothelial interactions in the tumour microenvironment to be critical features of metastatic breast cancer.

The Adipocyte as an Endocrine Cell

Adipose tissue contains many cell types. Among the more abundant are adipocytes, preadipocytes, immune cells, and endothelial cells. During times of excess caloric intake, these cells have to adjust and remodel to accommodate the increased demand for triglyceride storage. Based on a comprehensive analysis of the total adipose tissue secretome, this article focuses on three areas of adipokine biology: (1) How does the adipocyte interact with the extracellular matrix over the course of obesity? (2) Does the adipocyte, per se, play a role in the innate immune response? (3) How is the angiogenic profile of adipose tissue linked to the development of insulin resistance? The authors present a comprehensive overview of all of the currently available secreted adipose tissue products that have been identified at the protein level.

N6-methyladenosine marks primary microRNAs for processing

The first step in the biogenesis of microRNAs is the processing of primary microRNAs (pri-miRNAs) by the microprocessor complex, composed of the RNA-binding protein DGCR8 and the type III RNase DROSHA. This initial event requires recognition of the junction between the stem and the flanking single-stranded RNA of the pri-miRNA hairpin by DGCR8 followed by recruitment of DROSHA, which cleaves the RNA duplex to yield the pre-miRNA product. While the mechanisms underlying pri-miRNA processing have been determined, the mechanism by which DGCR8 recognizes and binds pri-miRNAs, as opposed to other secondary structures present in transcripts, is not understood. Here we find in mammalian cells that methyltransferase-like 3 (METTL3) methylates pri-miRNAs, marking them for recognition and processing by DGCR8. Consistent with this, METTL3 depletion reduced the binding of DGCR8 to pri-miRNAs and resulted in the global reduction of mature miRNAs and concomitant accumulation of unprocessed pri-miRNAs. In vitro processing reactions confirmed the sufficiency of the N6-methyladenosine (m6A) mark in promoting pri-miRNA processing. Finally, gain-of-function experiments revealed that METTL3 is sufficient to enhance miRNA maturation in a global and non-cell-type-specific manner. Our findings reveal that the m6A mark acts as a key post-transcriptional modification that promotes the initiation of miRNA biogenesis.

Plasma Adiponectin Complexes Have Distinct Biochemical Characteristics

Adipocytes release the secretory protein adiponectin in a number of different higher-order complexes. Once synthesized and assembled in the secretory pathway of the adipocyte, these complexes circulate as biochemically distinct and stable entities with little evidence of interchange between the different forms that include a high-molecular-weight (HMW) species, a hexamer (low-molecular-weight form), and a trimeric form of the complexes. Here, we validate a high-resolution gel filtration method that reproducibly separates the three complexes in recombinant adiponectin and adiponectin from human and murine samples. We demonstrate that the HMW form is prominently reduced in male vs. female subjects and in obese, insulin-resistant vs. lean, insulin-sensitive individuals. A direct comparison of human and mouse adiponectin demonstrates that the trimer is generally more abundant in human serum. Furthermore, when the production of adiponectin is reduced, either by obesity or in mice carrying only a single functional allele of the adiponectin locus, then the amount of the HMW form is selectively reduced in circulation. The complex distribution of adiponectin can be regulated in several ways. Both mouse and human HMW adiponectin are very stable under basic conditions but are exquisitely labile under acidic conditions below pH 7. Murine and human adiponectin HMW forms also display differential susceptibility to the presence of calcium in the buffer. A mutant form of adiponectin unable to bind calcium is less susceptible to changes in calcium concentrations. However, the lack of calcium binding results in a destabilization of the structure. Disulfide bond formation (at position C39) is also important for complex formation. A mutant form of adiponectin lacking C39 prominently forms HMW and trimer but not the low-molecular-weight form. Injection of adiponectin with a fluorescent label reveals that over time, the various complexes do not interconvert in vivo. The stability of adiponectin complexes highlights that the production and secretion of these forms from fat cells has a major influence on the circulating levels of each complex.

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 ∼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.

Selective Inhibition of Hypoxia-Inducible Factor 1α Ameliorates Adipose Tissue Dysfunction

ABSTRACT Hypoxia-inducible factor 1α (HIF1α) induction in adipocytes is a critical component of the “fibrotic response,” directly linked to metabolic dysfunction in adipose tissues under hypoxic conditions. We reasoned that inhibition of HIF1α may ameliorate the negative aspects of the obesity-associated fat pad expansion. We used the selective HIF1α inhibitor PX-478, whose effectiveness has previously been established in tumor models. We demonstrate that PX-478 treatment effectively suppresses the high-fat-diet (HFD)-induced HIF1α activation in adipose tissue. HIF1α inhibition causes a reduction of weight gain in mice on an HFD but not on a chow diet. Treatment increases energy expenditure and prompts resistance to HFD-mediated deterioration of metabolic parameters. Moreover, PX-478-treated mice have reduced fibrosis and fewer inflammatory infiltrates in their adipose tissues. We confirm the metabolic effects obtained with PX-478 treatment using an adipose tissue-specific, doxycycline-inducible dominant negative HIF1α mutant (dn-HIF1α). Consistent with the pharmacological results, genetic inhibition of endogenous HIF1α activity prompts similar metabolic improvements in HFD-fed mice. Collectively, our results demonstrate that HIF1α inhibition in the adipocyte leads to significant metabolic improvements, suggesting that selective HIF1α inhibition in adipose tissue may be an effective therapeutic avenue in the context of metabolic dysfunction.

Paradoxical Elevation of High–Molecular Weight Adiponectin in Acquired Extreme Insulin Resistance Due to Insulin Receptor Antibodies

Total plasma adiponectin and high–molecular weight (HMW) polymeric adiponectin are strongly positively correlated with insulin sensitivity. However, we have recently reported paradoxical hyperadiponectinemia in patients with severe insulin resistance due to genetically defective insulin receptors. This implies either that the insulin receptor has a critical physiological role in controlling adiponectin production and/or clearance or that constitutive insulin receptor dysfunction influences adiponectin levels through developmental effects. The aim of the current study was to distinguish between these possibilities using a human model of reversible antibody-mediated insulin receptor dysfunction and to refine the previous observations by determining adiponectin complex distribution. Cross-sectional and longitudinal determination of fasting plasma adiponectin and adiponectin complex distribution was undertaken in patients with extreme insulin resistance due to insulin receptor mutations, anti-insulin receptor antibodies (type B insulin resistance), or an undefined cause. Despite extreme insulin resistance, patients with type B insulin resistance (all women; mean age 42 years [range 12–54]) had dramatically elevated total plasma adiponectin compared with the general population (mean 43.0 mg/l [range 31.3–54.2] vs. 8.9 mg/l [1.5–28.5 for BMI <25 kg/m2]), which was accounted for largely by HMW polymers. Hyperadiponectinemia resolved in parallel with reduction of insulin receptor antibodies and clinical resolution of insulin resistance. Although the well-established inverse relationship between plasma insulin and adiponectin levels may, in part, reflect positive effects of adiponectin on insulin sensitivity, these data suggest that the magnitude of the effect of insulin action on adiponectin levels may have been underestimated.

Chronic Intermittent Hypoxia Induces Atherosclerosis via Activation of Adipose Angiopoietin-like 4

RATIONALE Obstructive sleep apnea is a risk factor for dyslipidemia and atherosclerosis, which have been attributed to chronic intermittent hypoxia (CIH). Intermittent hypoxia inhibits a key enzyme of lipoprotein clearance, lipoprotein lipase, and up-regulates a lipoprotein lipase inhibitor, angiopoietin-like 4 (Angptl4), in adipose tissue. The effects and mechanisms of Angptl4 up-regulation in sleep apnea are unknown. OBJECTIVES To examine whether CIH induces dyslipidemia and atherosclerosis by increasing adipose Angptl4 via hypoxia-inducible factor-1 (HIF-1). METHODS ApoE(-/-) mice were exposed to intermittent hypoxia or air for 4 weeks while being treated with Angptl4-neutralizing antibody or vehicle. MEASUREMENTS AND MAIN RESULTS In vehicle-treated mice, hypoxia increased adipose Angptl4 levels, inhibited adipose lipoprotein lipase, increased fasting levels of plasma triglycerides and very low density lipoprotein cholesterol, and increased the size of atherosclerotic plaques. The effects of CIH were abolished by the antibody. Hypoxia-induced increases in plasma fasting triglycerides and adipose Angptl4 were not observed in mice with germline heterozygosity for a HIF-1α knockout allele. Transgenic overexpression of HIF-1α in adipose tissue led to dyslipidemia and increased levels of adipose Angptl4. In cultured adipocytes, constitutive expression of HIF-1α increased Angptl4 levels, which was abolished by siRNA. Finally, in obese patients undergoing bariatric surgery, the severity of nocturnal hypoxemia predicted Angptl4 levels in subcutaneous adipose tissue. CONCLUSIONS HIF-1-mediated increase in adipose Angptl4 and the ensuing lipoprotein lipase inactivation may contribute to atherosclerosis in patients with sleep apnea.