Important mitochondrial proteins in human omental adipose tissue show reduced expression in obesity.

Obesity is associated with impaired mitochondrial function. This study compares mitochondrial protein expression in omental fat in obese and non-obese humans. Omental adipose tissue was obtained by surgical biopsy, adipocytes were purified and mitochondria isolated. Using anion-exchange chromatography, SDS-PAGE and mass-spectrometry, 128 proteins with potentially different abundances in patient groups were identified, 62 of the 128 proteins are mainly localized in the mitochondria. Further quantification of 12 of these 62 proteins by immune dot blot analysis revealed four proteins citrate synthase, HADHA, LETM1 and mitofilin being inversely associated with BMI, and mitofilin being inversely correlated with gender.

Important mitochondrial proteins in human omental adipose tissue show reduced expression in obesity.

Impaired mitochondrial function is important in obesity and the development of insulin resistance and diabetes. The aim of this study was to identify human adipocyte-derived mitochondrial proteins associated with obesity. Mitochondrial proteins from 20 abdominal omental adipose tissue biopsies (13 obese and 7 control subjects) were separated by anion-exchange chromatography coupled to SDS-PAGE. Protein contents were compared and identified by MALDI-TOF-TOF mass spectrometry. Proteins of interest were validated, verified and quantified using immuno dot blot assays in a total of 76 mitochondrial preparations from both obese and non-obese patients. Mass spectrometric comparison of 20 mitochondrial proteomes yielded 62 proteins that were differentially expressed in adipose tissue of obese subjects. The immunological quantification of 12 mitochondrial proteins from 76 omental adipose tissue biopsies revealed four proteins, citrate synthase, HADHA, LETM1 and mitofilin inversely being associated with BMI, and mitofilin being inversely correlated with gender. BIOLOGICAL SIGNIFICANCE: The finding that obese human subjects have reduced levels of important mitochondrial proteins in adipocytes of omental adipose tissue as compared to non-obese controls gives new insights in the impairment of mitochondrial function in this specialized compartment of human adipose tissue in obesity and may eventually lead to the definition of valuable obesity markers.

Morphological and functional remodelling of the neuromuscular junction by skeletal muscle PGC-1α.

The neuromuscular junction (NMJ) exhibits high morphological and functional plasticity. In the mature muscle, the relative levels of physical activity are the major determinants of NMJ function. Classically, motor neuron-mediated activation patterns of skeletal muscle have been thought of as the major drivers of NMJ plasticity and the ensuing fibre-type determination in muscle. Here we use muscle-specific transgenic animals for the peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) as a genetic model for trained mice to elucidate the contribution of skeletal muscle to activity-induced adaptation of the NMJ. We find that muscle-specific expression of PGC-1α promotes a remodelling of the NMJ, even in the absence of increased physical activity. Importantly, these plastic changes are not restricted to post-synaptic structures, but extended to modulation of presynaptic cell morphology and function. Therefore, our data indicate that skeletal muscle significantly contributes to the adaptation of the NMJ subsequent to physical activity.

Obesity affects mitochondrial citrate synthase in human omental adipose tissue.

The activities of some key enzymes in mitochondria from 135 human omental adipose tissue samples of obese and nonobese patients were analyzed for potential association with the patients' state of obesity. The activities of respiratory complexes I and II as well as citrate synthase in isolated mitochondria were measured using spectrophotometric enzyme assays. ATP generation of mitochondria was determined with a bioluminescence assay. Protein levels of citrate synthase were quantified by western blot. The rates of ATP generation and the enzymatic activities of complexes I and II did not display associations with age, gender, obesity, or diabetes. By contrast, the enzymatic activities of citrate synthase and its protein levels were significantly reduced in obesity as compared to controls. In diabetic patients, protein levels but not enzymatic activities of citrate synthase were elevated. Thus, this investigation based on enzymatic assay and determination of protein levels revealed that the development of obesity is associated with a significant impact on citrate synthase in mitochondria of human omental adipose tissue. The state of obesity appears to affect mitochondrial function in human omental adipose tissue by limiting this key enzyme of the tricarboxylic acid cycle rather than by limiting the activities of respiratory chain enzymes.

A functional motor unit in the culture dish: co-culture of spinal cord explants and muscle cells.

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures. Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium. The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context. Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Electric pulse stimulation of cultured murine muscle cells reproduces gene expression changes of trained mouse muscle.

Adequate levels of physical activity are at the center of a healthy lifestyle. However, the molecular mechanisms that mediate the beneficial effects of exercise remain enigmatic. This gap in knowledge is caused by the lack of an amenable experimental model system. Therefore, we optimized electric pulse stimulation of muscle cells to closely recapitulate the plastic changes in gene expression observed in a trained skeletal muscle. The exact experimental conditions were established using the peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) as a marker for an endurance-trained muscle fiber. We subsequently compared the changes in the relative expression of metabolic and myofibrillar genes in the muscle cell system with those observed in mouse muscle in vivo following either an acute or repeated bouts of treadmill exercise. Importantly, in electrically stimulated C2C12 mouse muscle cells, the qualitative transcriptional adaptations were almost identical to those in trained muscle, but differ from the acute effects of exercise on muscle gene expression. In addition, significant alterations in the expression of myofibrillar proteins indicate that this stimulation could be used to modulate the fiber-type of muscle cells in culture. Our data thus describe an experimental cell culture model for the study of at least some of the transcriptional aspects of skeletal muscle adaptation to physical activity. This system will be useful for the study of the molecular mechanisms that regulate exercise adaptation in muscle.

Melanoma targeting with DOTA-alpha-melanocyte-stimulating hormone analogs: structural parameters affecting tumor uptake and kidney uptake.

UNLABELLED: Radiolabeled analogs of alpha-melanocyte-stimulating hormone (alpha-MSH) are potential candidates for the diagnosis and therapy of melanoma metastases. After our recent observation that a linear octapeptide alpha-MSH analog incorporating the metal chelator 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) at the C-terminal lysine, [Nle(4),Asp(5),d-Phe(7),Lys(11)(DOTA)]-alpha-MSH(4-11) (DOTA-NAPamide), showed high accumulation in melanomas in a mouse model, low uptake in normal tissues, and moderate uptake in the kidneys, we attempted to identify the structural parameters influencing tumor uptake versus kidney uptake. METHODS: We designed a series of novel DOTA-alpha-MSH analogs differing from DOTA-NAPamide by small alterations, such as the position of DOTA in the peptide, hydrophobicity, and charge, by modifying the C-terminal Lys(11) residue. They were evaluated both for their melanocortin type 1 receptor (MC1R)-binding potency and for their biodistribution by use of the B16F1 melanoma mouse model. RESULTS: When DOTA was shifted to the N terminus of the peptide, a 3-fold increase in kidney retention was obtained. However, when the epsilon-amino group of the Lys(11) residue was acetylated in addition to the DOTA relocation, kidney uptake returned to the low values obtained with DOTA-NAPamide; this result indicated that neutralization of the epsilon-amino group positive charge of the Lys(11) residue rather than the position of DOTA accounted for the low kidney retention. Unexpectedly, no further reduction in kidney uptake was obtained by the introduction of 1 or 2 negative charges on Lys(11). Melanoma uptake was in accordance with MC1R affinity; the highest values were obtained for peptides bearing carboxy-terminal amidation and positioning of DOTA. CONCLUSION: The kidney uptake of DOTA-alpha-MSH analogs could be considerably reduced, without affecting MC1R affinity, by altering (neutralizing) the charge of the Lys(11) residue. Accordingly, the resulting peptides exhibited a high ratio of tumor uptake to kidney uptake that is favorable for diagnostic and therapeutic applications. These structure-activity data may help to improve the performance of DOTA-alpha-MSH analogs and other radiopeptides.

A gallium-labeled DOTA-alpha-melanocyte- stimulating hormone analog for PET imaging of melanoma metastases.

UNLABELLED: Although (18)F-FDG PET is widely used for metastatic melanoma diagnosis, it is less accurate than desirable, particularly for small foci. Since both melanotic and amelanotic melanomas overexpress receptors for alpha-melanocyte-stimulating hormone (alpha-MSH; receptor name, melanocortin type 1 receptor [MC1R]), radiolabeled alpha-MSH analogs are potential candidates for melanoma diagnosis. The aim of this study was to develop a positron emitter-labeled alpha-MSH analog suitable for PET imaging of melanoma metastases. METHODS: A short linear alpha-MSH analog, [Nle(4),Asp(5),D-Phe(7)]-alpha-MSH(4-11) (NAPamide), was newly designed and conjugated to the metal chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to enable radiometal incorporation. Compared with our previously reported DOTA-alpha-MSH analog, DOTA-MSH(oct) ([DOTA-betaAla(3),Nle(4),Asp(5),D-Phe(7),Lys(10)]-alpha-MSH(3-10)), the major modification lies in the conjugation of DOTA to the C-terminal end of the peptide via the epsilon-amino group of Lys(11), as opposed to the N-terminal alpha-amino group. After labeling with (111)In, (67)Ga, and the short-lived positron emitter (68)Ga, DOTA-NAPamide was characterized in vitro and in vivo using the mouse melanoma B16F1cell line. RESULTS: DOTA-NAPamide exhibited an almost 7-fold higher MC1R binding potency as compared with DOTA-MSH(oct). In B16F1 melanoma-bearing mice, both (111)In-DOTA-NAPamide and (67)Ga-DOTA-NAPamide behaved more favorably than (111)In-DOTA-MSH(oct). Both radiopeptides exhibited higher tumor and lower kidney uptake leading to tumor-to-kidney ratios of the 4- to 48-h area under the curve that were 4.6 times ((111)In) and 7.5 times ((67)Ga) greater than that obtained with (111)In-DOTA-MSH(oct). In addition, the 4-h kidney uptake of (67)Ga-DOTA-NAPamide could be reduced by 64% by coinjection of 15 mg L-lysine, without affecting tumor uptake. Skin primary melanoma as well as lung and liver melanoma metastases could be easily visualized on tissue section autoradiographs after systemic injection of (67)Ga-DOTA-NAPamide. The melanoma selectivity of DOTA-NAPamide was confirmed by PET imaging studies using (68)Ga-DOTA-NAPamide. Tumor uptake was found to be highest when the smallest amount of peptide was administered. CONCLUSION: DOTA-NAPamide labeled with either (111)In or (67)Ga/(68)Ga is in every way superior to (111)In-DOTA-MSH(oct) in murine models of primary and metastatic melanoma, which makes it a promising agent for melanoma targeting. High-contrast images obtained in PET studies with an experimental tumor model 1 h after injection augurs well for its clinical potential as an imaging tool.

DOTA alpha-melanocyte-stimulating hormone analogues for imaging metastatic melanoma lesions.

Scintigraphic imaging of metastatic melanoma lesions requires highly tumor-specific radiopharmaceuticals. Because both melanotic and amelanotic melanomas overexpress melanocortin-1 receptors (MC1R), radiolabeled analogues of alpha-melanocyte-stimulating hormone (alpha-MSH) are potential candidates for melanoma diagnosis. Here, we report the in vivo performance of a newly designed octapeptide analogue, [betaAla(3), Nle(4), Asp(5), D-Phe(7), Lys(10)]-alpha-MSH(3-10) (MSH(OCT)), which was conjugated through its N-terminal amino group to the metal chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to enable incorporation of radiometals (e.g., indium-111) into the peptide. DOTA-MSH(OCT) displayed high in vitro MC1R affinity (IC(50) 9.21 nM). In vivo [(111)In]DOTA-MSH(OCT) exhibited a favorable biodistribution profile after injection in B16-F1 tumorbearing mice. The radiopeptide was rapidly cleared from blood through the kidneys and, most importantly, accumulated preferentially in the melanoma lesions. Lung and liver melanoma metastases could be clearly imaged on tissue section autoradiographs 4 h after injection of [(111)In]DOTA-MSH(OCT). A comparative study of [(111)In]DOTA-MSH(OCT) with [(111)In]DOTA-[Nle(4), D-Phe(7)]-alpha-MSH ([(111)In]-DOTA-NDP-MSH) demonstrated the superiority of the DOTA-MSH(OCT) peptide, particularly for the amount of radioactivity taken up by nonmalignant organs, including bone, the most radiosensitive tissue. These results demonstrate that [(111)In]DOTA-MSH(OCT) is a promising melanoma imaging agent.

A novel DOTA-alpha-melanocyte-stimulating hormone analog for metastatic melanoma diagnosis.

UNLABELLED: Scintigraphic imaging of metastatic melanoma lesions requires highly tumor-specific radiolabeled compounds. Because both melanotic and amelanotic melanomas overexpress receptors for alpha-melanocyte-stimulating hormone (alpha-MSH; receptor name: melanocortin type 1 receptor, or MC1R), radiolabeled alpha-MSH analogs are potential candidates for melanoma diagnosis. The aim of this study was to develop a melanoma-selective radiolabeled alpha-MSH analog suitable for melanoma diagnosis. METHODS: The very potent alpha-MSH analog [Nle(4), D-Phe(7)]-alpha-MSH (NDP-MSH) and a newly designed alpha-MSH octapeptide analog, [betaAla(3), Nle(4), Asp(5), D-Phe(7), Lys(10)]-alpha-MSH(3-10) (MSH(oct)), were conjugated to the metal chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to enable radiometal incorporation. The resulting DOTA conjugates were evaluated in vitro for their MC1R-binding affinity and melanogenic activity in isolated mouse B16F1 cells and in vivo for their biodistribution in mouse models of primary and metastatic melanoma after labeling with (111)In. RESULTS: DOTA-MSH(oct) was shown to bind with high affinity (inhibitory concentration of 50% [IC(50)] = 9.21 nmol/L) to the MC1R, although with lower potency than does DOTA-NDP-MSH (IC(50) = 0.25 nmol/L). In B16F1 melanoma-bearing mice, both (111)In-DOTA-NDP-MSH and (111)In-DOTA-MSH(oct) exhibited high MC1R-mediated uptake by melanoma, which differed by a factor of only 1.5 at 4 h after injection. The main route of excretion for both radioconjugates was the kidneys, whereby (111)In-DOTA-MSH(oct) led to somewhat higher kidney values than did (111)In-DOTA-NDP-MSH. In contrast, the latter was much more poorly cleared from other nonmalignant tissues, including bone, the most radiosensitive organ. Therefore, (111)In-DOTA-MSH(oct) displayed higher uptake ratios of tumor to nontarget tissue (e.g., tumor-to-bone ratio 4 h after injection was 4.9 for (111)In-DOTA-NDP-MSH and 53.9 for (111)In-DOTA-MSH(oct)). Lung and liver melanoma metastases could easily be visualized on tissue section autoradiographs after injection of (111)In-DOTA-MSH(oct). Radio-reversed-phase high-performance liquid chromatography analysis of urine samples revealed that most (111)In-DOTA-MSH(oct) is excreted intact 4 h after injection, indicating good in vivo stability. CONCLUSION: (111)In-DOTA-MSH(oct) exhibits more favorable overall performance than does (111)In-DOTA-NDP-MSH in murine models of primary and metastatic melanoma, making it a promising melanoma imaging agent.

Neuroendocrine tumor targeting: study of novel gallium-labeled somatostatin radiopeptides in a rat pancreatic tumor model.

Somatostatin analogs labeled with radionuclides are of considerable interest in the diagnosis and therapy of SSTR-expressing tumors, such as gastroenteropancreatic, small cell lung, breast and frequently nervous system tumors. In view of the favorable physical characteristics of the Ga isotopes (67)Ga and (68)Ga, enabling conventional tumor scintigraphy, PET and possibly internal radiotherapy, we focused on the development of a Ga-labeled somatostatin analog suitable for targeting SSTR-expressing tumors. For this purpose, 3 somatostatin analogs, OC, TOC and TATE were conjugated to the metal chelator DOTA and labeled with the radiometals (111)In, (90)Y and (67)Ga. They were then evaluated for their performance in the AR4-2J pancreatic tumor model by testing SSTR2-binding affinity, internalization/externalization in isolated cells and biodistribution in tumor-bearing nude mice. Surprisingly, we found that, compared to (111)In or (90)Y, labeling with (67)Ga considerably improved the biologic performance of the tested somatostatin analogs with respect to SSTR2 affinity and tissue distribution. (67)Ga-labeled DOTA-somatostatin analogs were rapidly excreted from nontarget tissues, leading to excellent tumor-to-nontarget tissue uptake ratios. Of interest for radiotherapeutic application, [(67)Ga]DOTATOC was strongly internalized by AR4-2J cells. Furthermore, our results suggest a link between the radioligand charge and its kidney retention. The excellent tumor selectivity of Ga-DOTA somatostatin analogs together with the different applications of Ga in nuclear oncology suggests that Ga-DOTA somatostatin analogs will become an important tool in the management of SSTR-positive tumors.