Martin Werno

Obesogenic and Diabetogenic Effects of High-Calorie Nutrition Require Adipocyte BK Channels

Diabetes, Sep 7, 2016

Adiponectin release and insulin receptor targeting share trans-Golgi-dependent endosomal trafficking routes

Molecular metabolism, Feb 1, 2018

Objective: Intracellular vesicle trafficking maintains cellular structures and functions. The ass... more Objective: Intracellular vesicle trafficking maintains cellular structures and functions. The assembly of cargo-laden vesicles at the trans-Golgi network is initiated by the ARF family of small GTPases. Here, we demonstrate the role of the trans-Golgi localized monomeric GTPase ARFRP1 in endosomal-mediated vesicle trafficking of mature adipocytes. Methods: Control (Arfrp1 flox/flox) and inducible fat-specific Arfrp1 knockout (Arfrp1 iATÀ/À) mice were metabolically characterized. In vitro experiments on mature 3T3-L1 cells and primary mouse adipocytes were conducted to validate the impact of ARFRP1 on localization of adiponectin and the insulin receptor. Finally, secretion and transferrin-based uptake and recycling assays were performed with HeLa and HeLa M-C1 cells. Results: We identified the ARFRP1-based sorting machinery to be involved in vesicle trafficking relying on the endosomal compartment for cell surface delivery. Secretion of adiponectin from fat depots was selectively reduced in Arfrp1 iATÀ/À mice, and Arfrp1-depleted 3T3-L1 adipocytes revealed an accumulation of adiponectin in Rab11-positive endosomes. Plasma adiponectin deficiency of Arfrp1 iATÀ/À mice resulted in deteriorated hepatic insulin sensitivity, increased gluconeogenesis and elevated fasting blood glucose levels. Additionally, the insulin receptor, undergoing endocytic recycling after ligand binding, was less abundant at the plasma membrane of adipocytes lacking Arfrp1. This had detrimental effects on adipose insulin signaling, followed by insufficient suppression of basal lipolytic activity and impaired adipose tissue expansion. Conclusions: Our findings suggest that adiponectin secretion and insulin receptor surface targeting utilize the same post-Golgi trafficking pathways that are essential for an appropriate systemic insulin sensitivity and glucose homeostasis.

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Dietary carbohydrates impair the protective effect of protein restriction against diabetes in NZO mice used as a model of type 2 diabetes

Diabetologia, Mar 17, 2018

Aims/hypothesis Low-protein diets are well known to improve glucose tolerance and increase energy... more Aims/hypothesis Low-protein diets are well known to improve glucose tolerance and increase energy expenditure. Increases in circulating fibroblast growth factor 21 (FGF21) have been implicated as a potential underlying mechanism. Methods We aimed to test whether low-protein diets in the context of a high-carbohydrate or high-fat regimen would also protect against type 2 diabetes in New Zealand Obese (NZO) mice used as a model of polygenetic obesity and type 2 diabetes. Mice were placed on high-fat diets that provided protein at control (16 kJ%; CON) or low (4 kJ%; low-protein/high-carbohydrate [LP/HC] or low-protein/high-fat [LP/HF]) levels. Results Protein restriction prevented the onset of hyperglycaemia and beta cell loss despite increased food intake and fat mass. The effect was seen only under conditions of a lower carbohydrate/fat ratio (LP/HF). When the carbohydrate/fat ratio was high (LP/HC), mice developed type 2 diabetes despite the robustly elevated hepatic FGF21 secretion and increased energy expenditure. Conclusion/interpretation Prevention of type 2 diabetes through protein restriction, without lowering food intake and body fat mass, is compromised by high dietary carbohydrates. Increased FGF21 levels and elevated energy expenditure do not protect against hyperglycaemia and type 2 diabetes per se.

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The zDHHC family of S-acyltransferases

Biochemical Society Transactions, Apr 1, 2015

The discovery of the zDHHC family of S-acyltransferase enzymes has been one of the major breakthr... more The discovery of the zDHHC family of S-acyltransferase enzymes has been one of the major breakthroughs in the S-acylation field. Now, more than a decade since their discovery, major questions centre on profiling the substrates of individual zDHHC enzymes (there are twenty-four ZDHHC genes and several hundred S-acylated proteins), defining the mechanisms of enzymesubstrate specificity, and unravelling the importance of this enzyme family for cellular physiology and pathology. S-acylation The post-translational modification of cellular proteins by S-acylation, the reversible attachment of fatty acids onto cysteine residues, mediates a variety of regulatory effects on a broad range of proteins [1-4]. These effects include facilitating membrane attachment of otherwise soluble proteins [3], regulating protein sorting to specific intracellular compartments [3], modulating protein folding and stability [2], and affecting the lateral distribution of proteins in membranes [3]. These Sacylation-dependent processes impact key physiological molecules such as G protein-coupled receptors, heterotrimeric G proteins, ion channels and membrane receptors [5]. As a result, Sacylation is essential for normal cellular physiology, and there is growing evidence linking dysfunctional S-acylation to specific disease processes [6]. The enzymes that regulate S-acylation dynamics are therefore receiving increasing interest as potential new drug targets.

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Analysis of protein palmitoylation in adipocytes

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The GTPase ARFRP1 affects lipid droplet protein composition and triglyceride release from intracellular storage of intestinal Caco-2 cells

Biochemical and Biophysical Research Communications, Nov 1, 2018

Intestinal release of dietary triglycerides via chylomicrons is the major contributor to elevated... more Intestinal release of dietary triglycerides via chylomicrons is the major contributor to elevated postprandial triglyceride levels. Dietary lipids can be transiently stored in cytosolic lipid droplets (LDs) located in intestinal enterocytes for later release. ADP ribosylation factor-related protein 1 (ARFRP1) participates in processes of LD growth in adipocytes and in lipidation of lipoproteins in liver and intestine. This study aims to explore the impact of ARFRP1 on LD organization and its interplay with chylomicron-mediated triglyceride release in intestinal-like Caco-2 cells. Suppression of Arfrp1 reduced release of intracellularly derived triglycerides (0.69-fold) and increased the abundance of transitional endoplasmic reticulum ATPase TERA/VCP, fatty acid synthase-associated factor 2 (FAF2) and perilipin 2 (Plin2) at the LD surface. Furthermore, TERA/VCP and FAF2 co-occurred more frequently with ATGL at LDs, suggesting a reduced adipocyte triglyceride lipase (ATGL)-mediated lipolysis. Accordingly, inhibition of lipolysis reduced lipid release from intracellular storage pools by the same magnitude as Arfrp1 depletion. Thus, the lack of Arfrp1 increases the abundance of lipolysis-modulating enzymes TERA/VCP, FAF2 and Plin2 at LDs, which might decrease lipolysis and reduce availability of fatty acids for triglyceride synthesis and their release via chylomicrons.

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Investigating the role of palmitoylation in insulin action

Diabetic Medicine, Mar 1, 2013

Investigating the role of palmitoylation in insulin action

Diabetic Medicine, 2013

Impact of the GTPase ARFRP1 on chylomicron maturation

Diabetologie und Stoffwechsel, 2016

Intracellular trafficking of the insulin receptor requires ARFRP1 function in adipose tissue and fat cells

Diabetologie Und Stoffwechsel, 2017

Analysis of protein palmitoylation in adipocytes

Lemonidis, Kimon and Werno, Martin W. and Greaves, Jennifer and Diez Ardanuy, Cinta and Sanchez Perez, Maria C. and Salaun, Christine and Thomson, David M. and Chamberlain, Luke H. (2015) The zDHHC family

The discovery of the zDHHC family of S-acyltransferase enzymes has been one of the major breakthr... more The discovery of the zDHHC family of S-acyltransferase enzymes has been one of the major breakthroughs in the S-acylation field. Now, more than a decade since their discovery, major questions centre on profiling the substrates of individual zDHHC enzymes (there are twenty-four ZDHHC genes and several hundred S-acylated proteins), defining the mechanisms of enzymesubstrate specificity, and unravelling the importance of this enzyme family for cellular physiology and pathology. S-acylation The post-translational modification of cellular proteins by S-acylation, the reversible attachment of fatty acids onto cysteine residues, mediates a variety of regulatory effects on a broad range of proteins [1-4]. These effects include facilitating membrane attachment of otherwise soluble proteins [3], regulating protein sorting to specific intracellular compartments [3], modulating protein folding and stability [2], and affecting the lateral distribution of proteins in membranes [3]. These Sacylati...

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The role of ARFRP1 on adipocyte secretory capacity

Diabetologie und Stoffwechsel

Identification of trafficking proteins involved in adiponectin secretion

Diabetologie und Stoffwechsel, 2016

Adiponectin release and insulin receptor targeting share trans-Golgi-dependent endosomal trafficking routes

Molecular Metabolism

Objective: Intracellular vesicle trafficking maintains cellular structures and functions. The ass... more Objective: Intracellular vesicle trafficking maintains cellular structures and functions. The assembly of cargo-laden vesicles at the trans-Golgi network is initiated by the ARF family of small GTPases. Here, we demonstrate the role of the trans-Golgi localized monomeric GTPase ARFRP1 in endosomal-mediated vesicle trafficking of mature adipocytes. Methods: Control (Arfrp1 flox/flox) and inducible fat-specific Arfrp1 knockout (Arfrp1 iATÀ/À) mice were metabolically characterized. In vitro experiments on mature 3T3-L1 cells and primary mouse adipocytes were conducted to validate the impact of ARFRP1 on localization of adiponectin and the insulin receptor. Finally, secretion and transferrin-based uptake and recycling assays were performed with HeLa and HeLa M-C1 cells. Results: We identified the ARFRP1-based sorting machinery to be involved in vesicle trafficking relying on the endosomal compartment for cell surface delivery. Secretion of adiponectin from fat depots was selectively reduced in Arfrp1 iATÀ/À mice, and Arfrp1-depleted 3T3-L1 adipocytes revealed an accumulation of adiponectin in Rab11-positive endosomes. Plasma adiponectin deficiency of Arfrp1 iATÀ/À mice resulted in deteriorated hepatic insulin sensitivity, increased gluconeogenesis and elevated fasting blood glucose levels. Additionally, the insulin receptor, undergoing endocytic recycling after ligand binding, was less abundant at the plasma membrane of adipocytes lacking Arfrp1. This had detrimental effects on adipose insulin signaling, followed by insufficient suppression of basal lipolytic activity and impaired adipose tissue expansion. Conclusions: Our findings suggest that adiponectin secretion and insulin receptor surface targeting utilize the same post-Golgi trafficking pathways that are essential for an appropriate systemic insulin sensitivity and glucose homeostasis.

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Dietary carbohydrates impair the protective effect of protein restriction against diabetes in NZO mice used as a model of type 2 diabetes

Diabetologia

Aims/hypothesis Low-protein diets are well known to improve glucose tolerance and increase energy... more Aims/hypothesis Low-protein diets are well known to improve glucose tolerance and increase energy expenditure. Increases in circulating fibroblast growth factor 21 (FGF21) have been implicated as a potential underlying mechanism. Methods We aimed to test whether low-protein diets in the context of a high-carbohydrate or high-fat regimen would also protect against type 2 diabetes in New Zealand Obese (NZO) mice used as a model of polygenetic obesity and type 2 diabetes. Mice were placed on high-fat diets that provided protein at control (16 kJ%; CON) or low (4 kJ%; low-protein/high-carbohydrate [LP/HC] or low-protein/high-fat [LP/HF]) levels. Results Protein restriction prevented the onset of hyperglycaemia and beta cell loss despite increased food intake and fat mass. The effect was seen only under conditions of a lower carbohydrate/fat ratio (LP/HF). When the carbohydrate/fat ratio was high (LP/HC), mice developed type 2 diabetes despite the robustly elevated hepatic FGF21 secretion and increased energy expenditure. Conclusion/interpretation Prevention of type 2 diabetes through protein restriction, without lowering food intake and body fat mass, is compromised by high dietary carbohydrates. Increased FGF21 levels and elevated energy expenditure do not protect against hyperglycaemia and type 2 diabetes per se.

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The GTPase ARFRP1 affects lipid droplet protein composition and triglyceride release from intracellular storage of intestinal Caco-2 cells

Biochemical and Biophysical Research Communications

Obesogenic and Diabetogenic Effects of High-Calorie Nutrition Require Adipocyte BK Channels

Diabetes, 2016

Elevated adipose tissue expression of the Ca 2+-and voltage-activated K + (BK) channel was identi... more Elevated adipose tissue expression of the Ca 2+-and voltage-activated K + (BK) channel was identified in morbidly obese men carrying a BK gene variant supporting the hypothesis that K + channels affect metabolic responses of fat cells to nutrients. To establish the role of endogenous BKs for fat cell maturation, storage of excess dietary fat and bodyweight (BW) gain we studied a gene-targeted mouse model with a global ablation of the BK channel (BK L1/L1) and adipocyte-specific BK-deficient (adipoqBK L1/L2) mice. Global BK deficiency afforded protection from high-fat-diet (HFD) induced BW gain and excessive fat accumulation. Expansion of white adipose tissue-derived epididymal BK L1/L1 pre-adipocytes and their differentiation to lipid-filled mature adipocytes in vitro, however, were improved. Moreover, BW gain and total fat masses of usually super-obese ob/ob mice were significantly attenuated in the absence of BK together supporting a central or peripheral role for BKs in the regulatory system that controls adipose tissue and weight. Accordingly, HFD-fed adipoqBK L1/L2 mutants presented with a reduced total BW and overall body fat mass, smaller adipocytes and reduced leptin levels. Protection from pathologic weight gain in the absence of adipocyte BKs was beneficial for glucose handling and related to an increase in body core temperature due to higher levels of uncoupling protein 1 as well as low abundance of the proinflammatory interleukin-6 as a common risk factor for diabetes and metabolic abnormalities. This suggests that adipocyte BK activity is at least partially responsible for excessive BW gain under high-caloric conditions suggesting BK channels as promising drug targets for pharmacotherapy of metabolic disorders and obesity.

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S-acylation of the Insulin-Responsive Aminopeptidase (IRAP): Quantitative analysis and Identification of Modified Cysteines

Scientific Reports, 2015

The insulin-responsive aminopeptidase (IRAP) was recently identified as an S-acylated protein in ... more The insulin-responsive aminopeptidase (IRAP) was recently identified as an S-acylated protein in adipocytes and other tissues. However, there is currently no information on the extent of S-acylation of this protein, the residues that are modified, or the effects of S-acylation on IRAP localisation. In this study, we employ a semi-quantitative acyl-RAC technique to show that approximately 60% of IRAP is S-acylated in 3T3-L1 adipocytes. In contrast, S-acylation of GLUT4, a glucose transporter that extensively co-localises with IRAP, was approximately five-fold lower. Site-directed mutagenesis was employed to map the sites of S-acylation on IRAP to two cysteine residues, one of which is predicted to lie in the cytoplasmic side of the single transmembrane domain and the other which is just upstream of this transmembrane domain; our results suggest that these cysteines may be modified in a mutually-exclusive manner. Although S-acylation regulates the intracellular trafficking of several transmembrane proteins, we did not detect any effects of mutating the modified cysteines on the plasma membrane localisation of IRAP in HEK293T cells, suggesting that S-acylation is not essential for the movement of IRAP through the secretory pathway. Protein S-acylation (also referred to as palmitoylation) is a post-translational modification (PTM) involving the attachment of palmitate and other fatty acids to cysteine residues of proteins via thioester linkage. Numerous studies have demonstrated that S-acylation can mediate membrane attachment of soluble proteins, and regulate the intracellular trafficking of both soluble and transmembrane proteins and their partitioning into membrane micro-domains such as lipid rafts 1. Furthermore, S-acylation can also affect protein-protein interactions and protein stability 2. S-acylation is mediated by a family of membrane-bound enzymes comprising 24 isoforms in mammals 3,4. These palmitoyl-transferases (PATs) are referred to as zDHHC enzymes due to the presence of a conserved DHHC (Asp-His-His-Cys) amino acid motif present within a zinc-finger cysteine-rich domain 5-7. Unlike other lipid modifications, S-acylation is reversible and dynamic due to the labile nature of the thioester bond 8. Depalmitoylation is catalysed by enzymes including acyl-protein thioesterases (APTs) 9 , and cycles of S-acylation and deacylation play an important role in regulating dynamic changes in protein localisation, often in response to specific signals 10,11. Insulin-stimulated translocation of GLUT4 storage vesicles (GSVs) to the plasma membrane in adipocytes and skeletal muscle cells is essential for blood glucose homeostasis 12 , and defects in this pathway are linked to obesity and diabetes 13,14. The insulin-stimulated translocation of GLUT4 to the plasma membrane is regulated by various PTMs, most notably phosphorylation. In addition, some studies have also highlighted the potential involvement of S-acylation in these pathways. A variety of important proteins including the insulin receptor and the SNARE protein SNAP23 have long been known to be

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The zDHHC family of S-acyltransferases

Biochemical Society transactions, 2015

The discovery of the zDHHC family of S-acyltransferase enzymes has been one of the major breakthr... more The discovery of the zDHHC family of S-acyltransferase enzymes has been one of the major breakthroughs in the S-acylation field. Now, more than a decade since their discovery, major questions centre on profiling the substrates of individual zDHHC enzymes (there are 24 ZDHHC genes and several hundred S-acylated proteins), defining the mechanisms of enzyme-substrate specificity and unravelling the importance of this enzyme family for cellular physiology and pathology.

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