Increased FAT/CD36 Cycling and Lipid Accumulation in Myotubes Derived from Obese Type 2 Diabetic Patients

BACKGROUND: Permanent fatty acid translocase (FAT/)CD36 relocation has previously been shown to be related to abnormal lipid accumulation in the skeletal muscle of type 2 diabetic patients, however mechanisms responsible for the regulation of FAT/CD36 expression and localization are not well characterized in human skeletal muscle. METHODOLOGY/PRINCIPAL FINDINGS: Primary muscle cells derived from obese type 2 diabetic patients (OBT2D) and from healthy subjects (Control) were used to examine the regulation of FAT/CD36. We showed that compared to Control myotubes, FAT/CD36 was continuously cycling between intracellular compartments and the cell surface in OBT2D myotubes, independently of lipid raft association, leading to increased cell surface FAT/CD36 localization and lipid accumulation. Moreover, we showed that FAT/CD36 cycling and lipid accumulation were specific to myotubes and were not observed in reserve cells. However, in Control myotubes, the induction of FAT/CD36 membrane translocation by the activation of (AMP)-activated protein kinase (AMPK) pathway did not increase lipid accumulation. This result can be explained by the fact that pharmacological activation of AMPK leads to increased mitochondrial beta-oxidation in Control cells. CONCLUSION/SIGNIFICANCE: Lipid accumulation in myotubes derived from obese type 2 diabetic patients arises from abnormal FAT/CD36 cycling while lipid accumulation in Control cells results from an equilibrium between lipid uptake and oxidation. As such, inhibiting FAT/CD36 cycling in the skeletal muscle of obese type 2 diabetic patients should be sufficient to diminish lipid accumulation

A roadmap to the efficient and robust characterization of temperate terrestrial planet atmospheres with JWST

Ultra-cool dwarf stars are abundant, long-lived, and uniquely suited to enable the atmospheric study of transiting terrestrial companions with JWST. Amongst them, the most prominent is the M8.5V star TRAPPIST-1 and its seven planets, which have been the favored targets of eight JWST Cycle 1 programs. While Cycle 1 observations have started to yield preliminary insights into the planets, they have also revealed that their atmospheric exploration requires a better understanding of their host star. Here, we propose a roadmap to characterize the TRAPPIST-1 system -- and others like it -- in an efficient and robust manner. We notably recommend that -- although more challenging to schedule -- multi-transit windows be prioritized to constrain stellar heterogeneities and gather up to 2$\times$ more transits per JWST hour spent. We conclude that in such systems planets cannot be studied in isolation by small programs, thus large-scale community-supported programs should be supported to enable the efficient and robust exploration of terrestrial exoplanets in the JWST era

Abnormal metabolism flexibility in response to high palmitate concentrations in myotubes derived from obese type 2 diabetic patients

International audienceInsulin resistance in type 2 diabetes (T2D) is associated with intramuscular lipid (IMCL) accumulation. To determine whether impaired lipid oxidation is involved in IMCL accumulation, we measured expression of genes involved in mitochondrial oxidative metabolism or biogenesis, mitochondrial content and palmitate beta-oxidation before and after palmitate overload (600 μM for 16h), in myotubes derived from healthy subjects and obese T2D patients. Mitochondrial gene expression, content and network were not different between groups. Basal palmitate beta-oxidation was not affected in T2D myotubes, whereas after 16h of palmitate pre-treatment, T2D myotubes in contrast to control myotubes, showed an inability to increase palmitate beta-oxidation (p<0.05). Interestingly, acetyl-CoA carboxylase (ACC) phosphorylation was increased with a tendency for statistical significance after palmitate pre-treatment in control myotubes (p=0.06) but not in T2D myotubes which can explain their inability to increase palmitate beta-oxidation after palmitate overload. To determine whether the activation of the AMP activated protein kinase (AMPK)-ACC pathway was able to decrease lipid content in T2D myotubes, cells were treated with AICAR and metformin. These AMPK activators had no effect on ACC and AMPK phosphorylation in T2D myotubes as well as on lipid content, whereas AICAR, but not metformin, increased AMPK phosphorylation in control myotubes. Interestingly, metformin treatment and mitochondrial inhibition by antimycin induced increased lipid content in control myotubes. We conclude that T2D myotubes display an impaired capacity to respond to metabolic stimuli

OBT2D myotubes are characterized by increased lipid accumulation and plasma membrane FAT/CD36 localization.

<p>A. Quantification of lipid accumulation in Control (n = 4) and OBT2D differentiated satellite cells (n = 5) after palmitate treatment (0.6 mM for 16 h). Data are presented normalized to lipid accumulation in Control myotubes. Each point was assayed in triplicate for each of the 9 independent cell cultures. *, p<0.05, OBT2D versus Control differentiated cells. B. Representative immunofluorescence microscopy of satellite cells established from control subjects (Control) and obese type 2 diabetic patients (OBT2D) after 8 days of differentiation. Living myotubes were incubated for 1 h with an antibody against FAT/CD36 (H300, left panel) and for 1 h with an antibody against FAT/CD36 alexa 488 (CD36- alexa488, right panel). For H300 staining a polyclonal secondary antibody conjugated to alexa 488 (green) was added to the cells for 1 h. After fixation and permeabilization, cells were incubated with an antibody against troponin T (TT) visualized using a secondary monoclonal antibody conjugated to alexa 546 (red). Nuclei in cells were stained by dapi (blue). The 4 Control and the 5 OBT2D showed a staining similar to the representative pictures. Scale bar represents 30 µm. C. Top panels: Western blot analysis of total FAT/CD36 and caveolin 3 expression in differentiated satellite cell lysate established from 4 control subjects numbered 1 to 4 (Control) and 3 obese type 2 diabetic (OBT2D) patients numbered 1 to 3. Bottom panels: Western blot analysis of FAT/CD36 and caveolin 3 expression in post-membrane fractions (P) and plasma membrane fractions (Mb) of differentiated satellite cells established from 4 control subjects (1 to 4) and 3 obese type 2 diabetic (1 to 3) patients.</p

AMPK activation increases FAT/CD36 translocation in Control myotubes.

<p>A. Representative immunofluorescence microscopy of myotubes established from control subjects (Control) after 8 days of differentiation, incubated for 1 h at 37°C with CD36-alexa488 antibody (green) followed by insulin stimulation (100 nM for 10 min) or by AICAR stimulation (500 µM for 1 h) or by metformin stimulation (2 mM for 1 h). Nuclei were stained by dapi (blue) after fixation of the cells. The 4 Control cells showed a staining similar to the representative pictures. Scale bar represents 30 µm. B. Western blot analysis of the expression of AMPK and the phosphorylated form of AMPK (PAMPK) after infection with an adenovirus expressing either GFP (GFP) or GFP and the constitutively activated form of AMPK alpha 2 (alpha 2) in differentiated cells established from control subjects (Control). C. Representative immunofluorescence microscopy of myotubes established from control subjects (Control) after 8 days of differentiation infected with an adenovirus expressing either GFP (GFP) or GFP and the constitutively activated form of AMPK alpha 2 (alpha 2). To monitor cell surface FAT/CD36 localization in a co-staining experiment, CD36-alexa488 antibody (green) could not be used because of the GFP expression, as such, the same antibody against FAT/CD36 was used but with PhytoErythrine (CD36-PE) as a red fluorochrome. Living cells were incubated for 1 h with CD36- PE (red). Nuclei in cells were stained by dapi (blue). Scale bar represents 30 µm.</p

AMPK-mediated FAT/CD36 translocation in Control cells does not modify lipid accumulation.

<p>A. Quantification of lipid accumulation in Control (n = 4) (A) after palmitate treatment (0.6 mM for 16 h) or palmitate treatment plus AICAR stimulation (500 µM for 1 h) or palmitate treatment after SSO addition (250 µg/ml for 30 min) or palmitate treatment with or without AICAR stimulation after SSO addition. Data are means ±SEM. Each point was assayed in triplicate for each of the 4 independent cell cultures. B. Representative fluorescence and light microscopy of myotubes established from control subjects (Control) and from obese type 2 diabetic patients (OBT2D) after 8 days of differentiation infected with an adenovirus expressing either GFP (GFP) or GFP and the constitutively activated form of AMPK alpha 2 (alpha 2) stained by oil red O (ORO) after palmitate treatment (0.6 mM for 16 h). C. Palmitate beta-oxidation in differentiated Control cells (n = 3) before (−) and after (+) AICAR stimulation (500 µM for 1 h) expressed relative to protein content. Experiments were performed in triplicate for each of the 3 independent cell cultures. Data are mean ±SEM. *, P<0.05, AICAR-treated versus untreated Control cells.</p

Increased membrane localization of FAT/CD36 during differentiation is responsible for increased lipid accumulation in OBT2D myotubes.

<p>A. Representative light microscopy of myotubes derived from control subjects (Control) or obese type 2 diabetic patients (OBT2D), after 8 days of differentiation, stained by oil red O after palmitate treatment (0.6 mM for 16 h). The 4 Control and the 5 OBT2D cells showed a staining similar to the representative pictures. Arrows show reserve cells. Scale bar represents 30 µm. B. Merged picture of FAT/CD36 (H300), troponin T (TT) and dapi staining in OBT2D differentiated cells (for 8 days). Living cells were incubated for 1 h with an antibody against FAT/CD36 (H300, left panel) and for 1 h with a polyclonal secondary antibody conjugated to alexa 488 (green). After fixation and permeabilization, cells were incubated with an antibody against troponin T (TT) visualized using a secondary monoclonal antibody conjugated to alexa 546 (red). The 5 OBT2D cells showed a staining similar to the representative pictures. Arrows show reserve cells. Scale bar represents 30 µm. C. Left panel: Western blot analysis of the expression of total FAT/CD36 in proliferative (0), and after 2, 4, 6 and 8 days of differentiation of cells established from two control subjects (Control 1 and 2) and two obese type 2 diabetic patients (OBT2D 1 and 2). Troponin T (TT) and caveolin 3 were used as markers of myotube differentiation and α-tubulin as a loading control. Right panel: quantification by density analysis of the 2 controls and the 2 OBT2D. Data are presented normalized to α-tubulin protein expression where the value of Control cells after 8 days of differentiation has been arbitrary chosen as the reference value equal to 1. D. Representative immunofluorescence microscopy of cells established from obese type 2 diabetic patients (OBT2D) in proliferative (0), and after 2, 4 and 8 days of differentiation, treated by palmitate (0.6 mM for 16 h), incubated for the last hour with CD36-alexa488 antibody (green) and stained by oil red O (red) after fixation. The 5 OBT2D cells showed a staining similar to the representative pictures. Scale bar represents 30 µm. E. Percentage of inhibition of lipid content in Control (n = 4) and in OBT2D differentiated satellite cells (n = 5) after phloretin stimulation (400 µM for 30 min) followed by palmitate treatment (0.6 mM for 16 h). Data are means ±SEM. Each point was assayed in triplicate for each of the 9 independent cell cultures. *, p<0.05, OBT2D versus Control cells. F. Percentage of inhibition of lipid content in Control (n = 4) and OBT2D differentiated satellite cells (n = 4) after SSO stimulation (250 µg/ml for 30 min) followed by three PBS washes and by palmitate treatment (0.6 mM for 16 h). Data are means ±SEM. Each point was assayed in triplicate for each of the 8 independent cell cultures. *, p<0.05, OBT2D versus Control cells.</p

A simple yet stable molybdenum(0) carbonyl complex for upconver-sion and photoredox catalysis

Photoactive complexes with earth-abundant metals have attracted increasing interest in the recent years fueled by the promise of sustainable photochemistry. However, sophisticated ligands with complicated syntheses are oftentimes required to enable photoactivity with non-precious metals. Here, we combine a cheap metal with simple ligands to easily access a photoactive complex. Specifically, we synthesize the molybdenum(0) carbonyl complex Mo(CO)3(tpe) featuring the tripodal ligand tris(pyridyl)ethane (tpe) in two steps with high overall yield. The complex shows intense deep-red phosphorescence with excited state lifetimes of several hundred nanoseconds. Time-resolved infrared spectroscopy and laser flash photolysis reveal a triplet metal-to-ligand charge-transfer (3MLCT) state as lowest excited state. Temperature-dependent luminescence complemented by density functional theory (DFT) calculations suggest thermal deactivation of the 3MLCT state via higher lying metal-centered states in analogy to the well-known photophysics of [Ru(bpy)3]2+. Importantly, we found that the title compound is very photostable due to the lack of labilized Mo–CO bonds (as caused by trans-coordinated CO) in the facial configuration of the ligands. Finally, we show the versatility of the molybdenum(0) complex in two applications: (1) green-to-blue photon upconversion via a triplet-triplet annihilation mechanism and (2) photoredox catalysis for a green-light driv-en dehalogenation reaction. Overall, our results establish tripodal carbonyl complexes as a promising design strategy to ac-cess stable photoactive complexes of non-precious metals avoiding tedious multi-step syntheses