Ingrid Wernstedt Asterholm

Adipocyte Inflammation Is Essential for Healthy Adipose Tissue Expansion and Remodeling

Chronic inflammation constitutes an important link between obesity and its pathophysiological sequelae. In contrast to the belief that inflammatory signals exert a fundamentally negative impact on metabolism, we show that proinflammatory signaling in the adipocyte is in fact required for proper adipose tissue remodeling and expansion. Three mouse models with an adipose tissue-specific reduction in proinflammatory potential were generated that display a reduced capacity for adipogenesis in vivo, while the differentiation potential is unaltered in vitro. Upon high-fat-diet exposure, the expansion of visceral adipose tissue is prominently affected. This is associated with decreased intestinal barrier function, increased hepatic steatosis, and metabolic dysfunction. An impaired local proinflammatory response in the adipocyte leads to increased ectopic lipid accumulation, glucose intolerance, and systemic inflammation. Adipose tissue inflammation is therefore an adaptive response that enables safe storage of excess nutrients and contributes to a visceral depot barrier that effectively filters gut-derived endotoxin.

Dichotomous effects of VEGF-A on adipose tissue dysfunction

Obese fat pads are frequently undervascularized and hypoxic, leading to increased fibrosis, inflammation, and ultimately insulin resistance. We hypothesized that VEGF-A–induced stimulation of angiogenesis enables sustained and sufficient oxygen and nutrient exchange during fat mass expansion, thereby improving adipose tissue function. Using a doxycycline (Dox)-inducible adipocyte-specific VEGF-A overexpression model, we demonstrate that the local up-regulation of VEGF-A in adipocytes improves vascularization and causes a “browning” of white adipose tissue (AT), with massive up-regulation of UCP1 and PGC1α. This is associated with an increase in energy expenditure and resistance to high fat diet-mediated metabolic insults. Similarly, inhibition of VEGF-A–induced activation of VEGFR2 during the early phase of high fat diet-induced weight gain, causes aggravated systemic insulin resistance. However, the same VEGF-A–VEGFR2 blockade in ob/ob mice leads to a reduced body-weight gain, an improvement in insulin sensitivity, a decrease in inflammatory factors, and increased incidence of adipocyte death. The consequences of modulation of angiogenic activity are therefore context dependent. Proangiogenic activity during adipose tissue expansion is beneficial, associated with potent protective effects on metabolism, whereas antiangiogenic action in the context of preexisting adipose tissue dysfunction leads to improvements in metabolism, an effect likely mediated by the ablation of dysfunctional proinflammatory adipocytes.

Enhanced Metabolic Flexibility Associated with Elevated Adiponectin Levels

Metabolically healthy individuals effectively adapt to changes in nutritional state. Here, we focus on the effects of the adipocyte-derived secretory molecule adiponectin on adipose tissue in mouse models with genetically altered adiponectin levels. We found that higher adiponectin levels increased sensitivity to the lipolytic effects of adrenergic receptor agonists. In parallel, adiponectin-overexpressing mice also display enhanced clearance of circulating fatty acids and increased expansion of subcutaneous adipose tissue with chronic high fat diet (HFD) feeding. These adaptive changes to the HFD were associated with increased mitochondrial density in adipocytes, smaller adipocyte size, and a general transcriptional up-regulation of factors involved in lipid storage through efficient esterification of free fatty acids. The physiological response to adiponectin overexpression resembles in many ways the effects of chronic exposure to beta3-adrenergic agonist treatment, which also results in improvements in insulin sensitivity. In addition, using a novel computed tomography-based method for measurements of hepatic lipids, we resolved the temporal events taking place in the liver in response to acute HFD exposure in both wild-type and adiponectin-overexpressing mice. Increased levels of adiponectin potently protect against HFD-induced hepatic lipid accumulation and preserve insulin sensitivity. Given these profound effects of adiponectin, we propose that adiponectin is a factor that increases the metabolic flexibility of adipose tissue, enhancing its ability to maintain proper function under metabolically challenging conditions.

Altered Mitochondrial Function and Metabolic Inflexibility Associated with Loss of Caveolin-1

Caveolin-1 is a major structural component of raft structures within the plasma membrane and has been implicated as a regulator of cellular signal transduction with prominent expression in adipocytes. Here, we embarked on a comprehensive characterization of the metabolic pathways dysregulated in caveolin-1 null mice. We found that these mice display decreased circulating levels of total and high molecular weight adiponectin and a reduced ability to change substrate use in response to feeding/fasting conditions. Caveolin-1 null mice are extremely lean but retain muscle mass despite lipodystrophy and massive metabolic dysfunction. Hepatic gluconeogenesis is chronically elevated, while hepatic steatosis is reduced. Our data suggest that the complex phenotype of the caveolin-1 null mouse is caused by altered metabolic and mitochondrial function in adipose tissue with a subsequent compensatory response driven mostly by the liver. This mouse model highlights the central contributions of adipose tissue for system-wide preservation of metabolic flexibility.

Targeted Deletion of Adipocytes by Apoptosis Leads to Adipose Tissue Recruitment of Alternatively Activated M2 Macrophages

Obesity is frequently associated with an infiltration of macrophages into adipose tissue. Adipocyte dysfunction causes a phenotypic switch of macrophages from an alternatively activated M2-like phenotype towards a proinflammatory M1 phenotype. The cross talk between adipocytes and infiltrating immune cells, in particular macrophages, is thought to contribute to local and eventually systemic inflammation. Here, we tested the phenotypic impact of a lack of adipocytes on the inflammatory status of macrophages. We took advantage of the fat apoptosis through targeted activation of caspase-8 (FAT-ATTAC) mouse model that allows for the inducible system-wide elimination of adipocytes through a proapoptotic mechanism and followed the degree and type of inflammatory response upon ablation of live adipocytes. Analysis of depots 2 wk after elimination of adipocytes resulted in markedly reduced levels of adipose tissue and a robust down-regulation of circulating adipokines. Quantitative PCR and immunohistochemistry on epididymal and inguinal fat depots revealed an increase of the macrophage markers F4/80 and CD11c. Using polychromatic flow cytometry, we observed an up-regulation of alternatively activated M2 macrophage markers (CD206 and CD301) on the majority of F4/80 positive cells. Apoptosis of adipocytes is sufficient to initiate a large influx of macrophages into the remnant fat pads. However, these macrophages are alternatively activated, antiinflammatory M2 macrophages and not M1 cells. We conclude that adipocyte death is sufficient to initiate macrophage infiltration, and live adipocytes are required to initiate and/or sustain a proinflammatory response within the infiltrating macrophages in adipose tissue.

Joint analysis of individual participants' data from 17 studies on the association of the IL6 variant -174GC with circulating glucose levels, interleukin-6 levels, and body mass index

Background. Several studies have investigated associations between the -174G>C single nucleotide polymorphism (rs1800795) of the IL6 gene and phenotypes related to type 2 diabetes mellitus (T2DM) but presented inconsistent results. Aims. This joint analysis aimed to clarify whether IL6 -174G>C was associated with glucose and circulating interleukin-6 concentrations as well as body mass index (BMI). Methods. Individual-level data from all studies of the IL6-T2DM consortium on Caucasian subjects with available BMI were collected. As study-specific estimates did not show heterogeneity (P>0.1), they were combined by using the inverse-variance fixed-effect model. Results. The main analysis included 9440, 7398, 24,117, or 5659 non-diabetic and manifest T2DM subjects for fasting glucose, 2-hour glucose, BMI, or circulating interleukin-6 levels, respectively. IL6 -174 C-allele carriers had significantly lower fasting glucose (−0.091 mmol/L, P=0.014). There was no evidence for association between IL6 -174G>C and BMI or interleukin-6 levels, except in some subgroups. Conclusions. Our data suggest that C-allele carriers of the IL6 -174G>C polymorphism have lower fasting glucose levels on average, which substantiates previous findings of decreased T2DM risk of these subjects.

Interleukin‐6 mediates exercise‐induced increase in insulin sensitivity in mice

Interleukin‐6 (IL‐6) is released from working skeletal muscle during exercise. We investigated the acute and the long‐term beneficial effects of IL‐6 on exercise‐induced glucose uptake in skeletal muscle and insulin sensitivity. The acute effect on exercise‐induced glucose uptake was measured in IL‐6‐deficient (IL‐6−/−) mice and wild‐type control animals using a tracer technique. There was no difference in serum disappearance of 3[H]2‐deoxyglucose after a single bout of exercise between IL‐6−/− and wild‐type mice (13565 ± 426 versus 14343 ± 1309 d.p.m. min ml−1, P= 0.5). The glucose uptake rate in the extensor digitorum longus muscle was, however, lower in IL‐6−/− compared with wild‐type mice (398 ± 44 versus 657 ± 41 nmol g−1 min−1, P < 0.01). In a long‐term study, we monitored insulin sensitivity, serum retinol‐binding protein‐4 (RBP‐4) levels, running activity, food intake, body weight and body composition in IL‐6−/− and wild‐type mice on a high‐fat diet (HFD), with or without access to running wheels. In sedentary IL‐6−/− and wild‐type mice, the HFD decreased insulin sensitivity (glucose area under the concentration–time curve increased about 20% during an insulin tolerance test, P < 0.05 for both genotypes versus baseline) and led to a 30% increase in serum RBP‐4 levels (P < 0.01 for both genotypes versus baseline). Wild‐type mice with access to running wheels were protected against these effects of the HFD and maintained their baseline insulin sensitivity and serum RBP‐4 levels. In contrast, IL‐6−/− mice did not benefit from running to the same extent as wild‐type animals. The IL‐6−/− mice with access to running wheels had a similar decrease in insulin sensitivity to their sedentary littermates (glucose area under the concentration–time curve during an insulin tolerance test in runners versus sedentary IL‐6−/− HFD mice, 312 ± 14 versus 340 ± 22 mmol min l−1, P= 0.4) and displayed a 14% increase in serum RBP‐4 compared with baseline levels (P < 0.01). Our results indicate that endogenous IL‐6 contributes to the exercise‐induced increase in insulin sensitivity, but plays only a minor role for glucose uptake into skeletal muscle during exercise.

Rgs16 and Rgs8 in embryonic endocrine pancreas and mouse models of diabetes

SUMMARY Diabetes is characterized by the loss, or gradual dysfunction, of insulin-producing pancreatic β-cells. Although β-cells can replicate in younger adults, the available diabetes therapies do not specifically target β-cell regeneration. Novel approaches are needed to discover new therapeutics and to understand the contributions of endocrine progenitors and β-cell regeneration during islet expansion. Here, we show that the regulators of G protein signaling Rgs16 and Rgs8 are expressed in pancreatic progenitor and endocrine cells during development, then extinguished in adults, but reactivated in models of both type 1 and type 2 diabetes. Exendin-4, a glucagon-like peptide 1 (Glp-1)/incretin mimetic that stimulates β-cell expansion, insulin secretion and normalization of blood glucose levels in diabetics, also promoted re-expression of Rgs16::GFP within a few days in pancreatic ductal-associated cells and islet β-cells. These findings show that Rgs16::GFP and Rgs8::GFP are novel and early reporters of G protein-coupled receptor (GPCR)-stimulated β-cell expansion after therapeutic treatment and in diabetes models. Rgs16 and Rgs8 are likely to control aspects of islet progenitor cell activation, differentiation and β-cell expansion in embryos and metabolically stressed adults.

Lack of “immunological fitness” during fasting in metabolically challenged animals

Subclinical inflammation is frequently associated with obesity. Here, we aim to better define the acute inflammatory response during fasting. To do so, we analyzed representatives of immune-related proteins in circulation and in tissues as potential markers for adipose tissue inflammation and modulation of the immune system. Lipopolysaccharide treatment or high-fat diet led to an increase in circulating serum amyloid (SAA) and α1-acid glycoprotein (AGP), whereas adipsin levels were reduced. Mouse models that are protected against diet-induced challenges, such as adiponectin-overexpressing animals or mice treated with PPARγ agonists, displayed lower SAA levels and higher adip-sin levels. An oral lipid gavage, as well as prolonged fasting, increased circulating SAA concurrent with the elevation of free FA levels. Moreover, prolonged fasting was associated with an increased number of Mac2-positive crown-like structures, an increased capillary permeability, and an increase in several M2-type macrophage markers in adipose tissue. This fasting-induced increase in SAA and M2-type macrophage markers was impaired in metabolically challenged animals. These data suggest that metabolic inflexibility is associated with a lack of “immunological fitness.”

Elevated resistin levels induce central leptin resistance and increased atherosclerotic progression in mice

Aims/hypothesisResistin was originally identified as an adipocyte-derived factor upregulated during obesity and as a contributor to obesity-associated insulin resistance. Clinically, resistin has also been implicated in cardiovascular disease in a number of different patient populations. Our aim was to simultaneously address these phenomena.MethodsWe generated mice with modest adipocyte-specific resistin overexpression. These mice were crossed with mice deficient in the LDL receptor (Ldlr−/−) to probe the physiological role of resistin. Both metabolic and atherosclerotic assessments were performed.ResultsResistin overexpression led to increased atherosclerotic progression in Ldlr−/− mice. This was in part related to elevated serum triacylglycerol levels and a reduced ability to clear triacylglycerol upon a challenge. Additional phenotypic changes, such as increased body weight and reduced glucose clearance, independent of the Ldlr−/− background, confirmed increased adiposity associated with a more pronounced insulin resistance. A hallmark of elevated resistin was the disproportionate increase in circulating leptin levels. These mice thus recapitulated both the proposed negative cardiovascular correlation and the insulin resistance. A unifying mechanism for this complex phenotype was a resistin-mediated central leptin resistance, which we demonstrate directly both in vivo and in organotypic brain slices. In line with reduced sympathetic nervous system outflow, we found decreased brown adipose tissue (BAT) activity. The resulting elevated triacylglycerol levels provide a likely explanation for accelerated atherosclerosis.Conclusions/interpretationResistin overexpression leads to a complex metabolic phenotype driven by resistin-mediated central leptin resistance and reduced BAT activity. Hypothalamic leptin resistance thus provides a unifying mechanism for both resistin-mediated insulin resistance and enhanced atherosclerosis.