Summary

Background
In spite of new treatments, the incidence of type 2 diabetes (T2D) and its morbidities continue to rise. The key feature of T2D is resistance of adipose tissue and other organs to insulin. Approaches to overcome insulin resistance are limited due to a poor understanding of the mechanisms and inaccessibility of drugs to relevant intracellular targets. We previously showed in mice and humans that CD248, a pre/adipocyte cell surface glycoprotein, acts as an adipose tissue sensor that mediates the transition from healthy to unhealthy adipose, thus promoting insulin resistance.
Methods
Molecular mechanisms by which CD248 regulates insulin signaling were explored using in vivo insulin clamp studies and biochemical analyses of cells/tissues from CD248 knockout (KO) and wild-type (WT) mice with diet-induced insulin resistance. Findings were validated with human adipose tissue specimens.
Findings
Genetic deletion of CD248 in mice, overcame diet-induced insulin resistance with improvements in glucose uptake and lipolysis in white adipose tissue depots, effects paralleled by increased adipose/adipocyte GLUT4, phosphorylated AKT and GSK3β, and reduced ATGL. The insulin resistance of the WT mice could be attributed to direct interaction of the extracellular domains of CD248 and the insulin receptor (IR), with CD248 acting to block insulin binding to the IR. This resulted in dampened insulin-mediated autophosphorylation of the IR, with reduced downstream signaling/activation of intracellular events necessary for glucose and lipid homeostasis.
Interpretation
Our discovery of a cell-surface CD248-IR complex that is accessible to pharmacologic intervention, opens research avenues toward development of new agents to prevent/reverse insulin resistance.
Funding
Funded by Canadian Institutes of Health Research (CIHR), Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Foundations for Innovation (CFI), the Swedish Diabetes Foundation, Family Ernfors Foundation and Novo Nordisk Foundation.

Introduction

Type 2 diabetes (T2D) is a chronic inflammatory disease that is associated with multiple morbidities, including in particular, a heightened risk of cardio-cerebro-vascular disorders, thrombosis and cancer.1,  ۲,  ۳ More than 450 million people are living with diabetes, a number expected to grow to ∼۶۵۰ million by 2040. A key feature of T2D is resistance of adipose tissue and other organs to insulin, often accompanied by hyperinsulinemia.4

Insulin controls key metabolic activities, including inducing glucose uptake, glycogenesis and lipogenesis, inhibiting lipolysis, stimulating protein synthesis, and enhancing adipogenesis.5,  ۶,  ۷ Its effects are mediated via cell surface-expressed tyrosine kinase receptors, triggering signaling cascades leading to a plethora of cellular responses, dysregulation of which are believed to result in the associated morbidities. Thus, for example, the increased risk of cancer in T2D is likely due in part to sustained insulin-induced signaling that promotes pathologic angiogenesis, dysregulated cell differentiation and proliferation, with alterations in cell metabolism.8 Mechanisms that regulate insulin signaling are incompletely understood, as are those that underly insulin resistance.9 It is known however, that with obesity and white adipose tissue (WAT) expansion, extracellular matrix remodeling occurs.10 This is associated with inadequate angiogenesis11 due to microenvironmental hypoxia, the latter which is accompanied by dampened adipogenesis and dysregulated expression and/or activation of the insulin receptor (IR).12,13 Adipocytes become dysfunctional with multiple changes, including mitochondrial disturbances, reduced adiponectin release, upregulation of pro-apoptotic and pro-fibrotic genes, increased release of cytokines and adipokines, infiltration with pro-inflammatory cells and suppression of preadipocyte differentiation.14 The associated disturbances in glucose metabolism and insulin sensitivity may occur early, even before evidence of inflammation.

CD248 is a multi-domain, transmembrane glycoprotein expressed on the surface of pre/adipocytes, perivascular cells and macrophages. From studies with gene targeted mice, we and others showed that CD248 participates in hypoxic regulation and angiogenesis,15,  ۱۶,  ۱۷,  ۱۸ and promotes inflammation, fibrosis, and tumorigenesis. Mice lacking CD248 (KO) are healthy and protected against tumor growth,16,19,20 atherosclerosis,21 arthritis,16 thrombosis,22 liver and renal fibrosis23,  ۲۴,  ۲۵ and lipid accumulation.26 We also recently reported that mice lacking CD248 either globally or specifically in mature adipocytes, are resistant to high fat diet (HFD)-induced weight gain, and are protected against insulin resistance, glucose intolerance and steatosis.18 Notably, WAT health, insulin sensitivity and glucose tolerance could be restored to normal after onset of HFD-induced diabetes by genetically excising CD248 from the mature adipocyte. The relevance of CD248 in glucometabolic health in humans was confirmed by studies of several independent clinical cohorts, showing that CD248 expression in adipocytes strongly and directly correlates with dysfunctional WAT and insulin resistance.18

Results

We previously showed that global lack of CD248 in mice (KO) confers protection of male mice against 9-weeks of a HFD that induced obesity, insulin resistance, glucose intolerance, steatosis, WAT hypoxia, inflammation and fibrosis.18 For the current studies, we found that even exposure to 2 weeks of the HFD induced differences in glucometabolism between male KO mice and their wild-type (WT) counterparts (Supplemental Figures S1–S4). Thus, the KO mice had significantly lower fasting plasma insulin and insulin growth factor (IGF)-1 levels (Supplemental Figure S1) as compared to sibling control, sex matched HFD-fed WT mice, with significantly better responses in glucose tolerance and insulin tolerance tests (GTT & ITT, respectively) (Supplemental Figure S2). ]Immunohistochemical analyses of epididymal (e)WAT from the HFD-fed KO mice followed by fasting x 5 h, revealed less evidence of fibrosis, less infiltration with F480 inflammatory monocytes, and reduced hypoxia, as compared to fasting HFD-fed wild-type (WT) mice (Supplemental Figures S3 and S4). There was no evidence of steatosis in either group (not shown) following the 2 week HFD. Based on these findings, we used this 2-week HFD model to study the mechanisms by which CD248 regulates glucometabolism and insulin sensitivity.