Laminin-alpha 4 (LAMA4) is an extracellular matrix protein implicated in the regulation of adipocyte differentiation and function. Prior research describes a role for LAMA4 in modulating adipocyte thermogenesis and uncoupling protein-1 (UCP1) expression in white adipose; however, the mechanisms involved are poorly understood. Here, we describe that Lama4 knockout mice (Lama4-/-) exhibit heightened mitochondrial biogenesis and peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) expression in subcutaneous white adipose tissue (sWAT). Furthermore, the acute silencing of LAMA4 with small interfering RNA in primary murine adipocytes was sufficient to upregulate the expression of thermogenic markers UCP1 and PR domain containing 16 (PRDM16). Silencing also resulted in an upregulation of PGC1-alpha and adenosine 5'-monophosphate-activated protein kinase (AMPK)-alpha expression. Subsequently, we show that integrin-linked kinase (ILK) is downregulated in the sWAT of Lama4-/- mice, and its silencing in adipocytes similarly resulted in elevated expression of UCP1 and AMPK alpha. Last, we demonstrate that treatment of human induced pluripotent stem cell-derived thermogenic adipocytes with LAMA4 (LN411) inhibited the expression of thermogenic markers and AMPK alpha. Overall, our results indicate that LAMA4 negatively regulates a thermogenic phenotype and pathways involving mitochondrial biogenesis in adipocytes through the suppression of AMPK alpha.

An accurate in vitro model of human adipose tissue could assist in the study of adipocyte function and allow for better tools for screening new therapeutic compounds. Cell culture models on two-dimensional surfaces fall short of mimicking the three-dimensional in vivo adipose environment, while three-dimensional culture models are often unable to support long-term cell culture due, in part, to insufficient mass transport. Microfluidic systems have been explored for adipose tissue models. However, current systems have primarily focused on 2D cultured adipocytes. In this work, a 3D human adipose microtissue was engineered within a microfluidic system. Human adipose-derived stem cells (ADSCs) were used as the cell source for generating differentiated adipocytes. The ADSCs differentiated within the microfluidic system formed a dense lipid-loaded mass with the expression of adipose tissue genetic markers. Engineered adipose tissue showed a decreased adiponectin secretion and increased free fatty acid secretion with increasing shear stress. Adipogenesis markers were downregulated with increasing shear stress. Overall, this microfluidic system enables the on-chip differentiation and development of a functional 3D human adipose microtissue supported by the interstitial flow. This system could potentially serve as a platform for in vitro drug testing for adipose tissue-related diseases.

Laminins are extracellular matrix proteins that reside in the basement membrane and provide structural support in addition to promoting cellular adhesion and migration. Through interactions with cell surface receptors, laminins stimulate intracellular signaling cascades which direct specific survival and differentiation outcomes. In metabolic tissues such as the pancreas, adipose, muscle, and liver, laminin isoforms are expressed in discrete temporal and spatial patterns suggesting that certain isoforms may support the development and function of particular metabolic cell types. This review focuses on the research to date detailing the expression of laminin isoforms, their potential function, as well as known pathways involved in laminin signaling in metabolic tissues. We will also discuss the current biomedical therapies involving laminins in these tissues in addition to prospective applications, with the goal being to encourage future investigation of laminins in the context of metabolic disease.

Abstract As research into the adipocyte microenvironment has advanced, it is becoming more widely accepted that the extracellular matrix (ECM) contributes to adipocyte dysfunction. The majority of current published work focuses on the role of collagens in metabolic disease while less emphasis has been placed on the contribution of laminins, an important component of the adipocyte basement membrane. Laminins are trimeric ECM proteins composed of α, β, and γ chains. The α chains contain sites which can interact with cell surface receptors and is considered the driver of tissue-specific expression and specialized signaling. Our group has shown that the laminin-α4 (LAMA4) chain, which is highly expressed in mature adipocytes, plays a role in adipocyte function and thermogenesis in mice (1). In this study we investigate the relationship between laminin α chain expression and obesity by assessing gene expression of LAMA1-5 in subcutaneous white adipose tissue (sWAT) from mice fed chow (RCD) and 45% high fat diet (HFD) for 8 weeks. Expression of LAMA2 and LAMA4 was significantly increased in the HFD sWAT compared to chow (6.1 fold, p=0.01 and 4.9 fold, p=0.001 respectively), however LAMA4 displayed a much stronger positive correlation with weight (R2=0.697) than did LAMA2 (R2=0.382). In order to validate the relevance of these findings in human models of obesity, we evaluated gene expression of LAMA2, LAMA4, and LAMA5 in sWAT biopsies from non-diabetic adult females with obesity (class II or higher). sWAT samples from obese subjects exhibited 4.5 fold higher LAMA4 expression (p=0.0089) than samples from non-obese control subjects, suggesting that the LAMA4 chain may play an important role in human obesity. Lastly we examined changes in sWAT LAMA4 expression following a period of weight loss in obese mice and in human subjects after bariatric surgery, and found that LAMA4 expression levels remain largely unchanged in both cases. In this study we demonstrate the relationship between LAMA4 expression and obesity and present findings that can be extended to human models of obesity. Reference: (1) Vaicik et al., Endocrinology. 2018 Jan;159(1):356–67.

Obesity affects nearly one billion globally and can lead to life-threatening sequelae. Consequently, there is an urgent need for novel therapeutics. We have previously shown that laminin, alpha 4 (Lama4) knockout in mice leads to resistance to adipose tissue accumulation; however, the relationship between LAMA4 and obesity in humans has not been established. In this study we measured laminin-α chain and collagen mRNA expression in the subcutaneous white adipose tissue (sWAT) of mice placed on chow (RCD) or 45% high fat diet (HFD) for 8 weeks, and also in HFD mice then placed on a “weight loss” regimen (8 weeks HFD followed by 6 weeks RCD). To assess extracellular matrix (ECM) components in humans with obesity, laminin subunit alpha mRNA and protein expression was measured in sWAT biopsies of female control subjects (BMI<30) or subjects with obesity undergoing bariatric surgery at the University of Chicago Medical Center (BMI>35) both before and three months after surgery. Lama4 was significantly higher in sWAT of HFD compared to RCD mice at both the RNA and protein level (p<0.001, p<0.05 respectively). sWAT from human subjects with obesity also showed significantly higher LAMA4 mRNA (p<0.01) and LAMA4 protein expression (p<0.05) than controls. Interestingly, even though LAMA4 expression was increased in both humans and murine models of obesity, no significant difference in Lama4 or LAMA4 expression was detected following short-term weight loss in either mouse or human samples, respectively. From these results we propose a significant association between obesity and elevated LAMA4 expression in humans, as well as in mouse models of obesity. Further studies should clarify the mechanisms underlying this association to target LAMA4 effectively as a potential therapy for obesity.

Obesity and the metabolic disease epidemic has led to an increase in morbidity and mortality. A rise in adipose thermogenic capacity via activation of brown or beige fat is a potential treatment for metabolic diseases. However, an understanding of how local factors control adipocyte fate is limited. Mice with a null mutation in the laminin α4 (LAMA4) gene (KO) exhibit resistance to obesity and enhanced expression of thermogenic fat markers in white adipose tissue (WAT). In this study, changes in WAT extracellular matrix composition in the absence of LAMA4 were evaluated using liquid chromatography/tandem mass spectrometry. KO-mice showed lower levels of collagen 1A1 and 3A1, and integrins α7 (ITA7) and β1 (ITB1). ITA7-ITB1 and collagen 1A1-3A1 protein levels were lower in brown adipose tissue compared to WAT in wild-type mice. Immunohistochemical staining confirmed lower levels and different spatial distribution of ITA7 in KO-WAT. In culture studies, ITA7 and LAMA4 levels decreased following a 12-day differentiation of adipose-derived stem cells into beige fat, and knock-down of ITA7 during differentiation increased beiging. These results demonstrate that extracellular matrix interactions regulate adipocyte thermogenic capacity and that ITA7 plays a role in beige adipose formation. A better understanding of the mechanisms underlying these interactions can be used to improve systemic energy metabolism and glucose homeostasis.

Obesity is characterized by an increase in adipose mass and is the leading risk factor for type 2 diabetes. Increasing adipose thermogenic capacity, by activation of brown or beige fat, could be a treatment for metabolic diseases. Nevertheless, many approaches fail in maintaining transformed adipose tissue in vivo, in part, due to the limited understanding of how environmental factors control cell fate and maintenance. We have shown that mice with a null mutation in the laminin α4 exhibit resistance to obesity, enhanced expression of thermogenic fat markers (UCP1) in subcutaneous white adipose tissue (sWAT), increased energy expenditure and enhanced insulin sensitivity. The knockout (KO) of Lama4 results in complex changes in overall ECM composition. We used liquid chromatography/tandem mass spectrometry (LC/MS) to evaluate ECM composition in sWAT from wild type (WT) and KO mice. In addition to the expected absence of Lama4, collagen 1A1 (Col1A1) and collagen 3A1 (Col3A1) were significantly lower in KO mice. Also, integrins α7 (ITα7) and β1 (ITβ1) were dramatically reduced. Immunohistochemical staining confirmed lower levels of Itα7 in KO compared to WT mice (P<0.0001). Knocking down Itα7 with siRNA promoted UCP1 expression (P=0.003) in vitro. In addition, culture of ADSC on Lama4, Col3A1 and Col1A1 coated surfaces had lower UCP1 expression (P<0.0001). Finally, mRNA levels of ITα7 and Lama4 decreased following 12 days of beige differentiation in human ADSC (P<0.0001). These results demonstrate that ECM can regulate adipocyte thermogenic capacity. Specifically, Lama4, Itα7, Col1A1 and Col3A1 are involved in the modulation of metabolic function in adipocytes. A better understanding of the mechanisms underlying these interactions allows for the potential to specifically manipulate these cells to improve systemic energy metabolism and glucose homeostasis. Disclosure M.A. Gonzalez Porras: None. K. Stojkova: None. M.K. Vaicik: None. A.A. Goddi: None. R.N. Cohen: None. E. Brey: None.

The Silencing Mediator of Retinoid and Thyroid Hormone Receptors (SMRT) is a nuclear corepressor, regulating the transcriptional activity of many transcription factors critical for metabolic processes. While the importance of the role of SMRT in the adipocyte has been well-established, our comprehensive understanding of its in vivo function in the context of homeostatic maintenance is limited due to contradictory phenotypes yielded by prior generalized knockout mouse models. Multiple such models agree that SMRT deficiency leads to increased adiposity, although the effects of SMRT loss on glucose tolerance and insulin sensitivity have been variable. We therefore generated an adipocyte-specific SMRT knockout (adSMRT-/-) mouse to more clearly define the metabolic contributions of SMRT. In doing so, we found that SMRT deletion in the adipocyte does not cause obesity—even when mice are challenged with a high-fat diet. This suggests that adiposity phenotypes of previously described models were due to effects of SMRT loss beyond the adipocyte. However, an adipocyte-specific SMRT deficiency still led to dramatic effects on systemic glucose tolerance and adipocyte insulin sensitivity, impairing both. This metabolically deleterious outcome was coupled with a surprising immune phenotype, wherein most genes differentially expressed in the adipose tissue of adSMRT-/- mice were upregulated in pro-inflammatory pathways. Flow cytometry and conditioned media experiments demonstrated that secreted factors from knockout adipose tissue strongly informed resident macrophages to develop a pro-inflammatory, MMe (metabolically activated) phenotype. Together, these studies suggest a novel role for SMRT as an integrator of metabolic and inflammatory signals to maintain physiological homeostasis.

Amyloid precursor protein (APP) and its metabolites play key roles in Alzheimer’s disease (AD) pathophysiology. Whereas short amyloid-β (Aβ) peptides derived from APP are pathogenic, the APP holoprotein serves multiple purposes in the nervous system through its cell adhesion and receptor-like properties. Our studies focused on the signaling mediated by the APP cytoplasmic tail. We investigated whether sustained APP signaling during brain development might favor neuronal plasticity and memory process through a direct interaction with the heterotrimeric G-protein subunit GαS (stimulatory G-protein alpha subunit). Our results reveal that APP possesses autonomous regulatory capacity within its intracellular domain that promotes APP cell surface residence, precludes Aβ production, facilitates axodendritic development, and preserves cellular substrates of memory. Altogether, these events contribute to strengthening cognitive functions and are sufficient to modify the course of AD pathology.

Silencing Mediator of Retinoid and Thyroid Hormone Receptors (SMRT) is a nuclear corepressor, which regulates the transcriptional activity of metabolically essential transcription factors. While SMRT has been shown to modulate adipocyte function, in vivo experiments utilizing knock-out (KO) models have led to conflicting results. To more rigorously define the role of SMRT in the adipocyte, we generated adipocyte-specific SMRT KO (adSMRT KO) mice by crossing adiponectin-Cre mice with floxed SMRT mice on a C57/BL6 background. When adSMRT KO mice are challenged with a 45% high-fat diet, we observe 20% increase in glucose intolerance (p=0.006) compared to wild type (WT) counterparts. Additionally, RNA-Seq data of adipose tissue from these mice indicate a dramatic up-regulation of inflammatory gene expression. To further characterize the pro-inflammatory phenotype, we utilized flow cytometry to identify infiltration of specific populations of adipose tissue inflammatory cells. We found that overall macrophage infiltration in the adipose tissue of KO mice increased two-fold (p=0.026), with anti-inflammatory M2 macrophages infiltrating in significantly lower proportions (p=0.017), indicating a higher ratio of M1:M2 cells in the KO mice compared to WT. In contrast, body weight and total fat mass were not altered. These data suggest a role for SMRT in the cross-talk between adipocytes and pro-inflammatory macrophages for the regulation of systemic glucose tolerance, distinct from the development of obesity. SMRT therefore integrates metabolic and inflammatory signals to maintain physiological homeostasis. Disclosure J. Kahn: None. A.A. Goddi: None. R.N. Cohen: None.

Laminin α4 (LAMA4) is located in the extracellular basement membrane that surrounds each individual adipocyte. Here we show that LAMA4 null (Lama4−/−) mice exhibit significantly higher energy expenditure (EE) relative to wild-type (WT) mice at room temperature and when exposed to a cold challenge, despite similar levels of food intake and locomotor activity. The Lama4−/− mice are resistant to age- and diet-induced obesity. Expression of uncoupling protein 1 is higher in subcutaneous white adipose tissue of Lama4−/− mice relative to WT animals on either a chow diet or a high-fat diet. In contrast, uncoupling protein 1 expression was not increased in brown adipose tissue. Lama4−/− mice exhibit significantly improved insulin sensitivity compared with WT mice, suggesting improved metabolic function. Overall, these data provide critical evidence for a role of the basement membrane in EE, weight gain, and systemic insulin sensitivity.

Presenilin 1 (PS1) is an essential γ-secretase component, the enzyme responsible for amyloid precursor protein (APP) intramembraneous cleavage. Mutations in PS1 lead to dominant-inheritance of early-onset familial Alzheimer’s disease (FAD). Although expression of FAD-linked PS1 mutations enhances toxic Aβ production, the importance of other APP metabolites and γ-secretase substrates in the etiology of the disease has not been confirmed. We report that neurons expressing FAD-linked PS1 variants or functionally deficient PS1 exhibit enhanced axodendritic outgrowth due to increased levels of APP intracellular C-terminal fragment (APP-CTF). APP expression is required for exuberant neurite outgrowth and hippocampal axonal sprouting observed in knock-in mice expressing FAD-linked PS1 mutation. APP-CTF accumulation initiates CREB signaling cascade through an association of APP-CTF with Gαs protein. We demonstrate that pathological PS1 loss-of-function impinges on neurite formation through a selective APP gain-of-function that could impact on axodendritic connectivity and contribute to aberrant axonal sprouting observed in AD patients.