LRP1 is the cell-surface endocytosis receptor for vaspin in adipocytes.

Vaspin is a serine protease inhibitor that protects against adipose tissue inflammation and insulin resistance, two key drivers of adipocyte dysfunction and metabolic disorders in obesity. Inhibition of target proteases such as KLK7 has been shown to reduce adipose tissue inflammation in obesity, while vaspin binding to cell surface GRP78 has been linked to reduced obesity-induced ER stress and insulin resistance in the liver. However, the molecular mechanisms by which vaspin directly affects cellular processes in adipocytes remain unknown. Using fluorescently labeled vaspin, we found that vaspin is rapidly internalized by mouse and human adipocytes, but less efficiently by endothelial, kidney, liver, and neuronal cells. Internalization occurs by active, clathrin-mediated endocytosis, which is dependent on vaspin binding to the LRP1 receptor, rather than GRP78 as previously thought. This was demonstrated by competition experiments and RNAi-mediated knock-down in adipocytes and by rescuing vaspin internalization in LRP1-deficient Pea13 cells after transfection with a functional LRP1 minireceptor. Vaspin internalization is further increased in mature adipocytes after insulin-stimulated translocation of LRP1. Although vaspin has nanomolar affinity for LRP1 clusters II-IV, binding to cell surface heparan sulfates is required for efficient LRP1-mediated internalization. Native, but not cleaved vaspin, and also vaspin polymers are efficiently endocytosed, and ultimately targeted for lysosomal degradation. Our study provides mechanistic insight into the uptake and degradation of vaspin in adipocytes, thereby broadening our understanding of its functional repertoire. We hypothesize the vaspin-LRP1 axis to be an important mediator of vaspin effects not only in adipose tissue but also in other LRP1-expressing cells.

Differential expression of immunoregulatory cytokines in adipose tissue and liver in response to high fat and high sugar diets in female mice.

A comprehensive understanding of how dietary components impact immunoregulatory gene expression in adipose tissue (AT) and liver, and their respective contributions to metabolic health in mice, remains limited. The current study aimed to investigate the metabolic consequences of a high-sucrose diet (HSD) and a high-fat diet (HFD) in female mice with a focus on differential lipid- and sucrose-induced changes in immunoregulatory gene expression in AT and liver. Female C57BL/6 J mice were fed a purified and macronutrient matched high fat, high sugar, or control diets for 12 weeks. Mice were extensively phenotyped, including glucose and insulin tolerance tests, adipose and liver gene and protein expression analysis by qPCR and Western blot, tissue lipid analyses, as well as histological analyses. Compared to the control diet, HSD- and HFD-fed mice had significantly higher body weights, with pronounced obesity along with glucose intolerance and insulin resistance only in HFD-fed mice. HSD-fed mice exhibited an intermediate phenotype, with mild metabolic deterioration at the end of the study. AT lipid composition was significantly altered by both diets, and inflammatory gene expression was only significantly induced in HFD-fed mice. In the liver however, histological analysis revealed that both HSD- and HFD-fed mice had pronounced ectopic lipid deposition indicating hepatic steatosis, but more pronounced in HSD-fed mice. This was in line with significant induction of pro-inflammatory gene expression specifically in livers of HSD-fed mice. Overall, our findings suggest that HFD consumption in female mice induces more profound inflammation in AT with pronounced deterioration of metabolic health, whereas HSD induced more pronounced hepatic steatosis and inflammation without yet affecting glucose metabolism.

A novel compound heterozygous leptin receptor mutation causes more severe obesity than in Leprdb/db mice.

The leptin receptor (Lepr) pathway is important for food intake regulation, energy expenditure, and body weight. Mutations in leptin and the Lepr have been shown to cause early-onset severe obesity in mice and humans. In studies with C57BL/6NCrl mice, we found a mouse with extreme obesity. To identify a putative spontaneous new form of monogenic obesity, we performed backcross studies with this mouse followed by a quantitative trait locus (QTL) analysis and sequencing of the selected chromosomal QTL region. We thereby identified a novel Lepr mutation (C57BL/6N-LeprL536Hfs*6-1NKB), which is located at chromosome 4, exon 11 within the CRH2-leptin-binding site. Compared with C57BL/6N mice, LeprL536Hfs*6 develop early onset obesity and their body weight exceeds that of Leprdb/db mice at an age of 30 weeks. Similar to Leprdb/db mice, the LeprL536Hfs*6 model is characterized by hyperphagia, obesity, lower energy expenditure and activity, hyperglycemia, and hyperinsulinemia compared with C57BL/6N mice. Crossing Leprdb/wt with LeprL536Hfs*6/wt mice results in compound heterozygous LeprL536Hfs*6/db mice, which develop even higher body weight and fat mass than both homozygous Leprdb/db and LeprL536Hfs*6 mice. Compound heterozygous Lepr deficiency affecting functionally different regions of the Lepr causes more severe obesity than the parental homozygous mutations.

Effects of Whole-Body Adenylyl Cyclase 5 (Adcy5) Deficiency on Systemic Insulin Sensitivity and Adipose Tissue.

Genome-wide association studies have identified adenylyl cyclase type 5 (ADCY5) as candidate gene for diabetes-related quantitative traits and an increased risk of type 2 diabetes. Mice with a whole-body deletion of Adcy5 (Adcy5-/-) do not develop obesity, glucose intolerance and insulin resistance, have improved cardiac function and increased longevity. Here, we investigated Adcy5 knockout mice (Adcy5-/-) to test the hypothesis that changes in adipose tissue (AT) may contribute to the reported healthier phenotype. In contrast to previous reports, we found that deletion of Adcy5 did not confer any physiological or biochemical benefits. However, this unexpected finding allowed us to investigate the effects of Adcy5 depletion on AT independently of lower body weight and a metabolically healthier phenotype. Adcy5-/- mice exhibited an increased number of smaller adipocytes, lower mean adipocyte size and a distinct AT gene expression pattern with midline 1 (Mid1) as the most significantly downregulated gene compared to control mice. Our Adcy5-/- model challenges previously described beneficial effects of Adcy5 deficiency and suggests that targeting Adcy5 does not improve insulin sensitivity and may therefore limit the relevance of ADCY5 as potential drug target.

Does C-C Motif Chemokine Ligand 2 (CCL2) Link Obesity to a Pro-Inflammatory State?

The mechanisms of how obesity contributes to the development of cardio-metabolic diseases are not entirely understood. Obesity is frequently associated with adipose tissue dysfunction, characterized by, e.g., adipocyte hypertrophy, ectopic fat accumulation, immune cell infiltration, and the altered secretion of adipokines. Factors secreted from adipose tissue may induce and/or maintain a local and systemic low-grade activation of the innate immune system. Attraction of macrophages into adipose tissue and altered crosstalk between macrophages, adipocytes, and other cells of adipose tissue are symptoms of metabolic inflammation. Among several secreted factors attracting immune cells to adipose tissue, chemotactic C-C motif chemokine ligand 2 (CCL2) (also described as monocyte chemoattractant protein-1 (MCP-1)) has been shown to play a crucial role in adipose tissue macrophage infiltration. In this review, we aimed to summarize and discuss the current knowledge on CCL2 with a focus on its role in linking obesity to cardio-metabolic diseases.

Role of Kallikrein 7 in Body Weight and Fat Mass Regulation.

Increased plasma and adipose tissue protease activity is observed in patients with type 2 diabetes and obesity. It has been proposed that specific proteases contribute to the link between obesity, adipose tissue inflammation and metabolic diseases. We have recently shown that ablation of the serine protease kallikrein-related peptidase 7 (Klk7) specifically in adipose tissue preserves systemic insulin sensitivity and protects mice from obesity-related AT inflammation. Here, we investigated whether whole body Klk7 knockout (Klk7-/-) mice develop a phenotype distinct from that caused by reduced Klk7 expression in adipose tissue. Compared to littermate controls, Klk7-/- mice gain less body weight and fat mass both under chow and high fat diet (HFD) feeding, are hyper-responsive to exogenous insulin and exhibit preserved adipose tissue function due to adipocyte hyperplasia and lower inflammation. Klk7-/- mice exhibit increased adipose tissue thermogenesis, which is not related to altered thyroid function. These data strengthen our recently proposed role of Klk7 in the regulation of body weight, energy metabolism, and obesity-associated adipose tissue dysfunction. The protective effects of Klk7 deficiency in obesity are likely linked to a significant limitation of adipocyte hypertrophy. In conclusion, our data indicate potential application of specific KLK7 inhibitors to regulate KLK7 activity in the development of obesity and counteract obesity-associated inflammation and metabolic diseases.

The Fabp4-Cre-Model is Insufficient to Study Hoxc9 Function in Adipose Tissue.

Developmental genes are important regulators of fat distribution and adipose tissue (AT) function. In humans, the expression of homeobox c9 (HOXC9) is significantly higher in subcutaneous compared to omental AT and correlates with body fat mass. To gain more mechanistic insights into the role of Hoxc9 in AT, we generated Fabp4-Cre-mediated Hoxc9 knockout mice (ATHoxc9-/-). Male and female ATHoxc9-/- mice were studied together with littermate controls both under chow diet (CD) and high-fat diet (HFD) conditions. Under HFD, only male ATHoxc9-/- mice gained less body weight and exhibited improved glucose tolerance. In both male and female mice, body weight, as well as the parameters of glucose metabolism and AT function were not significantly different between ATHoxc9-/- and littermate control CD fed mice. We found that crossing Hoxc9 floxed mice with Fabp4-Cre mice did not produce a biologically relevant ablation of Hoxc9 in AT. However, we hypothesized that even subtle reductions of the generally low AT Hoxc9 expression may cause the leaner and metabolically healthier phenotype of male HFD-challenged ATHoxc9-/- mice. Different models of in vitro adipogenesis revealed that Hoxc9 expression precedes the expression of Fabp4, suggesting that ablation of Hoxc9 expression in AT needs to be achieved by targeting earlier stages of AT development.

Membrane Phospholipids and Polyphosphates as Cofactors and Binding Molecules of SERPINA12 (vaspin).

Visceral adipose tissue derived serine protease inhibitor (vaspin) is a member of the serpin family and has been shown to have beneficial effects on glucose tolerance, insulin stability as well as adipose tissue inflammation, parameters seriously affected by obesity. Some of these effects require inhibition of target proteases such as kallikrein 7(KLK7) and many studies have demonstrated vaspin-mediated activation of intracellular signaling cascades in various cells and tissues. So far, little is known about the exact mechanism how vaspin may trigger these intracellular signaling events. In this study, we investigated and characterized the interaction of vaspin with membrane lipids and polyphosphates as well as their potential regulatory effects on serpin activity using recombinant vaspin and KLK7 proteins and functional protein variants thereof. Here, we show for the first time that vaspin binds to phospholipids and polyphosphates with varying effects on KLK7 inhibition. Vaspin binds strongly to monophosphorylated phosphatidylinositol phosphates (PtdInsP) with no effect on vaspin activation. Microscale thermophoresis (MST) measurements revealed high-affinity binding to polyphosphate 45 (KD: 466 ± 75 nM) and activation of vaspin in a heparin-like manner. Furthermore, we identified additional residues in the heparin binding site in ß-sheet A by mutating five basic residues resulting in complete loss of high-affinity heparin binding. Finally, using lipid overlay assays, we show that these residues are additionally involved in PtdInsP binding. Phospholipids play a major role in membrane trafficking and signaling whereas polyphosphates are procoagulant and proinflammatory agents. The identification of phospholipids and polyphosphates as binding partners of vaspin will contribute to the understanding of vaspins involvement in membrane trafficking, signaling and beneficial effects associated with obesity.

Repin1 deficiency improves insulin sensitivity and glucose metabolism in db/db mice by reducing adipose tissue mass and inflammation.

Replication initiator 1 (Repin1) is a zinc finger protein playing a role in insulin sensitivity, body fat mass and lipid metabolism by regulating the expression key genes of glucose and lipid metabolism. Here, we tested the hypothesis that introgression of a Repin1 deletion into db/db mice improves glucose metabolism in vivo. We generated a whole body Repin1 deficient db/db double knockout mouse (Rep1(-/-)x db/db) and systematically characterized the consequences of Repin1 deficiency on insulin sensitivity, glucose and lipid metabolism parameters and fat mass. Hyperinsulinemic-euglycemic clamp studies revealed significantly improved insulin sensitivity in Rep1(-/-)x db/db mice, which are also characterized by lower HbA1c, lower body fat mass and reduced adipose tissue (AT) inflammation area. Our study provides evidence that loss of Repin1 in db/db mice improves insulin sensitivity and reduces chronic hyperglycemia most likely by reducing fat mass and AT inflammation.