Madeleen Bosma

Perilipin 2 Improves Insulin Sensitivity in Skeletal Muscle Despite Elevated Intramuscular Lipid Levels

Type 2 diabetes is characterized by excessive lipid storage in skeletal muscle. Excessive intramyocellular lipid (IMCL) storage exceeds intracellular needs and induces lipotoxic events, ultimately contributing to the development of insulin resistance. Lipid droplet (LD)–coating proteins may control proper lipid storage in skeletal muscle. Perilipin 2 (PLIN2/adipose differentiation–related protein [ADRP]) is one of the most abundantly expressed LD-coating proteins in skeletal muscle. Here we examined the role of PLIN2 in myocellular lipid handling and insulin sensitivity by investigating the effects of in vitro PLIN2 knockdown and in vitro and in vivo overexpression. PLIN2 knockdown decreased LD formation and triacylglycerol (TAG) storage, marginally increased fatty-acid (FA) oxidation, and increased incorporation of palmitate into diacylglycerols and phospholipids. PLIN2 overexpression in vitro increased intramyocellular TAG storage paralleled with improved insulin sensitivity. In vivo muscle-specific PLIN2 overexpression resulted in increased LD accumulation and blunted the high-fat diet–induced increase in protein content of the subunits of the oxidative phosphorylation (OXPHOS) chain. Diacylglycerol levels were unchanged, whereas ceramide levels were increased. Despite the increased IMCL accumulation, PLIN2 overexpression improved skeletal muscle insulin sensitivity. We conclude that PLIN2 is essential for lipid storage in skeletal muscle by enhancing the partitioning of excess FAs toward TAG storage in LDs, thereby blunting lipotoxicity-associated insulin resistance.

Re-evaluating lipotoxic triggers in skeletal muscle: relating intramyocellular lipid metabolism to insulin sensitivity

Ectopic fat accumulation has been linked to lipotoxic events, including the development of insulin resistance in skeletal muscle. Indeed, intramyocellular lipid storage is strongly associated with the development of type 2 diabetes. Research during the last two decades has provided evidence for a role of lipid intermediates like diacylglycerol and ceramide in the induction of lipid-induced insulin resistance. However, recently novel data has been gathered that suggest that the relation between lipid intermediates and insulin resistance is less straightforward than has been previously suggested, and that there are several routes towards lipid-induced insulin resistance. For example, research in this field has shifted towards imbalances in lipid metabolism and lipid droplet dynamics. Next to imbalances in key lipogenic and lipolytic proteins, lipid droplet coat proteins appear to be essential for proper intramyocellular lipid storage, turnover and protection against lipid-induced insulin resistance. Here, we discuss the current knowledge on lipid-induced insulin resistance in skeletal muscle with a focus on the evidence from human studies. Furthermore, we discuss the available data that provides supporting mechanistic information.

Overexpression of PLIN5 in skeletal muscle promotes oxidative gene expression and intramyocellular lipid content without compromising insulin sensitivity.

AIMS/HYPOTHESIS While lipid deposition in the skeletal muscle is considered to be involved in obesity-associated insulin resistance, neutral intramyocellular lipid (IMCL) accumulation per se does not necessarily induce insulin resistance. We previously demonstrated that overexpression of the lipid droplet coat protein perilipin 2 augments intramyocellular lipid content while improving insulin sensitivity. Another member of the perilipin family, perilipin 5 (PLIN5), is predominantly expressed in oxidative tissues like the skeletal muscle. Here we investigated the effects of PLIN5 overexpression - in comparison with the effects of PLIN2 - on skeletal muscle lipid levels, gene expression profiles and insulin sensitivity. METHODS Gene electroporation was used to overexpress PLIN5 in tibialis anterior muscle of rats fed a high fat diet. Eight days after electroporation, insulin-mediated glucose uptake in the skeletal muscle was measured by means of a hyperinsulinemic euglycemic clamp. Electron microscopy, fluorescence microscopy and lipid extractions were performed to investigate IMCL accumulation. Gene expression profiles were obtained using microarrays. RESULTS TAG storage and lipid droplet size increased upon PLIN5 overexpression. Despite the higher IMCL content, insulin sensitivity was not impaired and DAG and acylcarnitine levels were unaffected. In contrast to the effects of PLIN2 overexpression, microarray data analysis revealed a gene expression profile favoring FA oxidation and improved mitochondrial function. CONCLUSIONS/INTERPRETATION Both PLIN2 and PLIN5 increase neutral IMCL content without impeding insulin-mediated glucose uptake. As opposed to the effects of PLIN2 overexpression, overexpression of PLIN5 in the skeletal muscle promoted expression of a cluster of genes under control of PPARα and PGC1α involved in FA catabolism and mitochondrial oxidation.

Augmenting muscle diacylglycerol and triacylglycerol content by blocking fatty acid oxidation does not impede insulin sensitivity

A low fat oxidative capacity has been linked to muscle diacylglycerol (DAG) accumulation and insulin resistance. Alternatively, a low fat oxidation rate may stimulate glucose oxidation, thereby enhancing glucose disposal. Here, we investigated whether an ethyl-2-[6-(4-chlorophenoxy)hexyl]-oxirane-2-carboxylate (etomoxir)-induced inhibition of fat oxidation leads to muscle fat storage and insulin resistance. An intervention in healthy male subjects was combined with studies in human primary myotubes. Furthermore, muscle DAG and triacylglycerol (TAG), mitochondrial function, and insulin signaling were examined in etomoxir-treated C57bl6 mice. In humans, etomoxir administration increased glucose oxidation at the expense of fat oxidation. This effect was accompanied by an increased abundance of GLUT4 at the sarcolemma and a lowering of plasma glucose levels, indicative of improved glucose homeostasis. In mice, etomoxir injections resulted in accumulation of muscle TAG and DAG, yet improved insulin-stimulated GLUT4 translocation. Also in human myotubes, insulin signaling was improved by etomoxir, in the presence of increased intramyocellular lipid accumulation. These insulin-sensitizing effects in mice and human myotubes were accompanied by increased phosphorylation of AMP-activated protein kinase (AMPK). Our results show that a reduction in fat oxidation leading to accumulation of muscle DAG does not necessarily lead to insulin resistance, as the reduction in fat oxidation may activate AMPK.

Interleukin 34: a new modulator of human and experimental inflammatory bowel disease

IBD (inflammatory bowel disease), where CD (Crohns disease) and UC (ulcerative colitis) represent the two main forms, are chronic inflammatory conditions of the intestine. Macrophages play a central role in IBD pathogenesis and are regulated by major differentiation factors such as CSF-1 (colony-stimulating factor 1) in homoeostasis and inflammation. IL (interleukin)-34 has recently been discovered as a second ligand for CSF-1R (CSF-1 receptor). However, expression and involvement of IL-34 in IBD remain unknown. In the present paper, we investigated the expression of IL34, CSF1 and their shared receptor CSF1R in normal human ileum and colon, in inflamed and non-inflamed tissues of CD and UC patients, and in a mouse model of experimental colitis. We found distinct expression patterns of IL34 and CSF1 in ileum and colon, with higher IL34 in ileum and, in contrast, higher CSF1 in colon. Furthermore, IL34 and CSF1 expression was increased with inflammation in IBD patients and in experimental colitis. In humans, infiltrating cells of the lamina propria and intestinal epithelial cells expressed IL-34, and TNF-α (tumour necrosis factor α) regulated IL-34 expression in intestinal epithelial cells through the NF-κB (nuclear factor κB) pathway. These data demonstrate the expression pattern of IL-34 in ileum and colon and suggest IL-34 as a new modulator of inflammation in IBD.

Chemical imaging of lipid droplets in muscle tissues using hyperspectral coherent Raman microscopy.

The accumulation of lipids in non-adipose tissues is attracting increasing attention due to its correlation with obesity. In muscle tissue, ectopic deposition of specific lipids is further correlated with pathogenic development of insulin resistance and type 2 diabetes. Most intramyocellular lipids are organized into lipid droplets (LDs), which are metabolically active organelles. In order to better understand the putative role of LDs in pathogenesis, insight into both the location of LDs and nearby chemistry of muscle tissue is very useful. Here, we demonstrate the use of label-free coherent anti-Stokes Raman scattering (CARS) microscopy in combination with multivariate, chemometric analysis to visualize intracellular lipid accumulations in ex vivo muscle tissue. Consistent with our previous results, hyperspectral CARS microscopy showed an increase in LDs in tissues where LD proteins were overexpressed, and further chemometric analysis showed additional features morphologically (and chemically) similar to mitochondria that colocalized with LDs. CARS imaging is shown to be a very useful method for label-free stratification of ectopic fat deposition and cellular organelles in fresh tissue sections with virtually no sample preparation.

Effects of Bezafibrate Treatment in a Patient and a Carrier With Mutations in the PNPLA2 Gene, Causing Neutral Lipid Storage Disease With Myopathy

Neutral lipid storage disease with myopathy (NLSDM) is a rare but severe genetic disorder characterized by excessive lipid accumulation in tissues including skin, bone marrow, heart, liver, and muscles. Clinically, NLSDM patients present with severe dilated cardiomyopathy, skeletal muscle myopathy, and insulin resistance.1 NLSDM is caused by a defect in the PNPLA2 gene encoding the enzyme adipose triglyceride lipase (ATGL),2 which catalyzes the breakdown of triglycerides in multiple tissues and is the rate-limiting step of lipolysis. Although heterozygous carriers and homozygous patients both present with similar clinical symptoms, the severity of these symptoms in homozygous patients is more dramatic, leading to premature death attributed to dilated cardiomyopathy in some patients.1,2 To date, the only available treatment is strict dietary guidelines and is focused on treating the comorbidities rather than targeting the primary defect. To investigate cardiac lipotoxicity in NLSDM, ATGL-deficient mice have been investigated. Just like NLSDM patients, ATGL-deficient mice are also characterized by excessive lipid storage in skeletal muscle, liver, and heart, and they develop cardiomyopathy at a young age, resulting in premature death.3 Interestingly, we reported recently that a lack of ATGL resulted in a diminished cardiac peroxisome proliferator-activated receptor (PPAR) activity and mitochondrial function, suggesting that lipolysis-derived fatty acids or fatty acid byproducts serve as transcriptional activators of PPARs.4 Subsequently, pharmacological treatment of ATGL-deficient mice with PPAR agonists completely reversed the mitochondrial defects, restored cardiac function, and prevented premature death. These promising findings inspired us to investigate whether PPAR agonist treatment in patients and carriers of a PNPLA2 gene defect could also have beneficial effects. Although NSLDM is a very rare disease, we had the opportunity to study 2 sisters with PNPLA2 gene mutations. Patient 1 is a 37-year-old woman with a body mass index of 21.4 kg/m2. …

Lipid droplet dynamics in skeletal muscle.

The skeletal muscle is subjected to high mechanical and energetic demands. Lipid droplets are an important source of energy substrates for the working muscle. Muscle cells contain a variety of lipid droplets, which are fundamentally smaller than those found in adipocytes. This translates into a greater lipid droplet surface area serving as the interface for intracellular lipid metabolism. The skeletal muscle has a high plasticity, it is subjected to major remodeling following training and detraining. This coincides with adaptations in lipid droplet characteristics and dynamics. The majority of lipid droplets in skeletal muscle are located in the subsarcolemmal region or in-between the myofibrils, in close vicinity to mitochondria. The vastly organized nature of skeletal muscle fibers limits organelle mobility. The high metabolic rate and substrate turnover in skeletal muscle demands a strict coordination of intramyocellular lipid metabolism and LD dynamics, in which lipid droplet coat proteins play an important role. This review provides insights into the characteristics, diversity and dynamics of skeletal muscle lipid droplets.

Reduced Incorporation of Fatty Acids Into Triacylglycerol in Myotubes From Obese Individuals With Type 2 Diabetes

Altered skeletal muscle lipid metabolism is a hallmark feature of type 2 diabetes (T2D). We investigated muscle lipid turnover in T2D versus BMI-matched control subjects (controls) and examined whether putative in vivo differences would be preserved in the myotubes. Male obese T2D individuals (n = 6) and BMI-matched controls (n = 6) underwent a hyperinsulinemic-euglycemic clamp, VO2max test, dual-energy X-ray absorptiometry scan, underwater weighing, and muscle biopsy of the vastus lateralis. 14C-palmitate and 14C-oleate oxidation rates and incorporation into lipids were measured in muscle tissue as well as in primary myotubes. Palmitate oxidation (controls: 0.99 ± 0.17 nmol/mg protein; T2D: 0.53 ± 0.07 nmol/mg protein; P = 0.03) and incorporation of fatty acids (FAs) into triacylglycerol (TAG) (controls: 0.45 ± 0.13 nmol/mg protein; T2D: 0.11 ± 0.02 nmol/mg protein; P = 0.047) were significantly reduced in muscle homogenates of T2D. These reductions were not retained for palmitate oxidation in primary myotubes (P = 0.38); however, incorporation of FAs into TAG was lower in T2D (P = 0.03 for oleate and P = 0.11 for palmitate), with a strong correlation of TAG incorporation between muscle tissue and primary myotubes (r = 0.848, P = 0.008). The data indicate that the ability to incorporate FAs into TAG is an intrinsic feature of human muscle cells that is reduced in individuals with T2D.

FNDC4 acts as an anti-inflammatory factor on macrophages and improves colitis in mice

FNDC4 is a secreted factor sharing high homology with the exercise-associated myokine irisin (FNDC5). Here we report that Fndc4 is robustly upregulated in several mouse models of inflammation as well as in human inflammatory conditions. Specifically, FNDC4 levels are increased locally at inflamed sites of the intestine of inflammatory bowel disease patients. Interestingly, administration of recombinant FNDC4 in the mouse model of induced colitis markedly reduces disease severity compared with mice injected with a control protein. Conversely, mice lacking Fndc4 develop more severe colitis. Analysis of binding of FNDC4 to different immune cell types reveals strong and specific binding to macrophages and monocytes. FNDC4 treatment of bone marrow-derived macrophages in vitro results in reduced phagocytosis, increased cell survival and reduced proinflammatory chemokine expression. Hence, treatment with FNDC4 results in a state of dampened macrophage activity, while enhancing their survival. Thus, we have characterized FNDC4 as a factor with direct therapeutic potential in inflammatory bowel disease and possibly other inflammatory diseases.