Electrochemical Lipolysis Induces Adipocyte Death and Fat Necrosis: In Vivo Pilot Study in Pigs.

BACKGROUND: Current minimally invasive fat reduction modalities use equipment that can cost thousands of U.S. dollars. Electrochemical lipolysis (ECLL), using low-cost battery and electrodes (approximately $10), creates acid/base within fat (width, approximately 3 mm), damaging adipocytes. Longitudinal effects of ECLL have not been studied. In this pilot study, the authors hypothesize that in vivo ECLL induces fat necrosis, decreases adipocyte number/viability, and forms lipid droplets. METHODS: Two female Yorkshire pigs (50 to 60 kg) received ECLL. In pig 1, 10 sites received ECLL, and 10 sites were untreated. In pig 2, 12 sites received ECLL and 12 sites were untreated. For ECLL, two electrodes were inserted into dorsal subcutaneous fat and direct current was applied for 5 minutes. Adverse effects of excessive pain, bleeding, infection, and agitation were monitored. Histology, live-dead (calcein, Hoechst, ethidium homodimer-1), and morphology (Bodipy and Hoechst) assays were performed on day 0 and postprocedure days 1, 2, 7, 14 (pig 1 and pig 2), and 28 (pig 2). Average particle area, fluorescence signal areas, and adipocytes and lipid droplet numbers were compared. RESULTS: No adverse effects occurred. Live-dead assays showed adipocyte death on the anode on days 0 to 7 and the cathode on days 1 to 2 (not significant). Bodipy showed significant adipocyte loss at all sites ( P < 0.001) and lipid droplet formation at the cathode site on day 2 ( P = 0.0046). Histology revealed fat necrosis with significant increases in average particle area at the anode and cathode sites by day 14 (+277.3% change compared with untreated, P < 0.0001; +143.4%, P < 0.0001) and day 28 (+498.6%, P < 0.0001; +354.5%, P < 0.0001). CONCLUSIONS: In vivo ECLL induces fat necrosis in pigs. Further studies are needed to evaluate volumetric fat reduction. CLINICAL RELEVANCE STATEMENT: In vivo ECLL induces adipocyte death and fat necrosis. ECLL has the potential to be utilized in body fat contouring.

Exploring feedback-controlled versus open-circuit electrochemical lipolysis in ex vivo and in vivo porcine fat: A feasibility study.

OBJECTIVES: Minimally invasive fat sculpting techniques are becoming more widespread with the development of office-based devices and therapies. Electrochemical lipolysis (ECLL) is a needle-based technology that uses direct current (DC) to electrolyze tissue water creating acid and base in situ. In turn, fat is saponified and adipocyte cell membrane lysis occurs. The electrolysis of water can be accomplished using a simple open-loop circuit (V-ECLL) or by incorporating a feedback control circuit using a potentiostat (P-ECLL). A potentiostat utilizes an operational amplifier with negative feedback to allow users to precisely control voltage at specific electrodes. To date, the variation between the two approaches has not been studied. The aim of this study was to assess current and charge transfer variation and lipolytic effect created by the two approaches in an in vivo porcine model. METHODS: Charge transfer measurements from ex vivo V-ECLL and P-ECLL treated porcine skin and fat were recorded at -1 V P-ECLL, -2 V P-ECLL, -3 V P-ECLL, and -5 V V-ECLL each for 5 min to guide dosimetry parameters for in vivo studies. In follow-up in vivo studies, a sedated female Yorkshire pig was treated with both V-ECLL and P-ECLL across the dorsal surface over a range of dosimetry parameters, including -1.5 V P-ECLL, -2.5 V P-ECLL, -3.5 V P-ECLL, and 5 V V-ECLL each treated for 5 min. Serial biopsies were performed at baseline before treatment, 1, 2, 7, 14, and 28 days after treatment. Tissue was examined using fluorescence microscopy and histology to compare the effects of the two ECLL approaches. RESULTS: Both V-ECLL and P-ECLL treatments induced in-vivo fat necrosis evident by adipocyte membrane lysis, adipocyte denuclearization, and an acute inflammatory response across a 28-day longitudinal study. However, -1.5 V P-ECLL produced a smaller spatial necrotic effect compared to 5 V V-ECLL. In addition, 5 V V-ECLL produced a comparable necrotic effect to that of -2.5 V and -3.5 V P-ECLL. CONCLUSIONS: V-ECLL and P-ECLL at the aforementioned dosimetry parameters both achieved fat necrosis by adipocyte membrane lysis and denuclearization. The -2.5 V and -3.5 V P-ECLL treatments created spatially similar fat necrotic effects when compared to the 5 V V-ECLL treatment. Quantitatively, total charge transfer between dosimetry parameters suggests that -2.5 V P-ECLL and 5 V V-ECLL produce comparable electrochemical reactions. Such findings suggest that a low-voltage closed-loop potentiostat-based system is capable of inducing fat necrosis to a similar extent compared to that of a higher voltage direct current system.

Effects of flanking sequences and cellular context on subcellular behavior and pathology of mutant HTT.

Huntington's disease (HD) is caused by an expansion of a poly glutamine (polyQ) stretch in the huntingtin protein (HTT) that is necessary to cause pathology and formation of HTT aggregates. Here we ask whether expanded polyQ is sufficient to cause pathology and aggregate formation. By addressing the sufficiency question, one can identify cellular processes and structural parameters that influence HD pathology and HTT subcellular behavior (i.e. aggregation state and subcellular location). Using Drosophila, we compare the effects of expressing mutant full-length human HTT (fl-mHTT) to the effects of mutant human HTTexon1 and to two commonly used synthetic fragments, HTT171 and shortstop (HTT118). Expanded polyQ alone is not sufficient to cause inclusion formation since full-length HTT and HTTex1 with expanded polyQ are both toxic although full-length HTT remains diffuse while HTTex1 forms inclusions. Further, inclusions are not sufficient to cause pathology since HTT171-120Q forms inclusions but is benign and co-expression of HTT171-120Q with non-aggregating pathogenic fl-mHTT recruits fl-mHTT to aggregates and rescues its pathogenicity. Additionally, the influence of sequences outside the expanded polyQ domain is revealed by finding that small modifications to the HTT118 or HTT171 fragments can dramatically alter their subcellular behavior and pathogenicity. Finally, mutant HTT subcellular behavior is strongly modified by different cell and tissue environments (e.g. fl-mHTT appears as diffuse nuclear in one tissue and diffuse cytoplasmic in another but toxic in both). These observations underscore the importance of cellular and structural context for the interpretation and comparison of experiments using different fragments and tissues to report the effects of expanded polyQ.

Miles to go (mtgo) encodes FNDC3 proteins that interact with the chaperonin subunit CCT3 and are required for NMJ branching and growth in Drosophila.

Analysis of mutants that affect formation and function of the Drosophila larval neuromuscular junction (NMJ) has provided valuable insight into genes required for neuronal branching and synaptic growth. We report that NMJ development in Drosophila requires both the Drosophila ortholog of FNDC3 genes; CG42389 (herein referred to as miles to go; mtgo), and CCT3, which encodes a chaperonin complex subunit. Loss of mtgo function causes late pupal lethality with most animals unable to escape the pupal case, while rare escapers exhibit an ataxic gait and reduced lifespan. NMJs in mtgo mutant larvae have dramatically reduced branching and growth and fewer synaptic boutons compared with control animals. Mutant larvae show normal locomotion but display an abnormal self-righting response and chemosensory deficits that suggest additional functions of mtgo within the nervous system. The pharate lethality in mtgo mutants can be rescued by both low-level pan- and neuronal-, but not muscle-specific expression of a mtgo transgene, supporting a neuronal-intrinsic requirement for mtgo in NMJ development. Mtgo encodes three similar proteins whose domain structure is most closely related to the vertebrate intracellular cytosolic membrane-anchored fibronectin type-III domain-containing protein 3 (FNDC3) protein family. Mtgo physically and genetically interacts with Drosophila CCT3, which encodes a subunit of the TRiC/CCT chaperonin complex required for maturation of actin, tubulin and other substrates. Drosophila larvae heterozygous for a mutation in CCT3 that reduces binding between CCT3 and MTGO also show abnormal NMJ development similar to that observed in mtgo null mutants. Hence, the intracellular FNDC3-ortholog MTGO and CCT3 can form a macromolecular complex, and are both required for NMJ development in Drosophila.

Nonmammalian Models of Huntington's Disease.

Flies, worms, yeast and more recently zebra fish have all been engineered to express expanded polyglutamine repeat versions of Huntingtin with various resulting pathologies including early death, neurodegeneration, and loss of motor function. Each of these models present particular features that make it useful in studying the mechanisms of polyglutamine pathology. However, one particular unbiased readout of mHTT pathology is functional loss of motor control. Loss of motor control is prominent in patients, but it remains unresolved whether pathogenic symptoms in patients result from overt degeneration and loss of neurons or from malfunctioning of surviving neurons as the pathogenic insult builds up. This is why a functional assay such as motor control can be uniquely powerful in revealing early as well as late neurological deficits and does not rely on assumptions such as that the level of inclusions or the degree of neuronal loss can be equated with the level of pathology. Drosophila is well suited for such assays because it contains a functioning nervous system with many parallels to the human condition. In addition, the ability to readily express mHTT transgenes in different tissues and subsets of neurons allows one the possibility of isolating a particular effect to a subset of neurons where one can correlate subcellular events in response to mHTT challenge with pathology at both the cellular and organismal levels. Here we describe methods to monitor the degree of motor function disruption in Drosophila models of HD and we include a brief summary of other nonmammalian models of HD and discussion of their unique strengths.

The phasor-FLIM fingerprints reveal shifts from OXPHOS to enhanced glycolysis in Huntington Disease.

Huntington disease (HD) is an autosomal neurodegenerative disorder caused by the expansion of Polyglutamine (polyQ) in exon 1 of the Huntingtin protein. Glutamine repeats below 36 are considered normal while repeats above 40 lead to HD. Impairment in energy metabolism is a common trend in Huntington pathogenesis; however, this effect is not fully understood. Here, we used the phasor approach and Fluorescence Lifetime Imaging Microscopy (FLIM) to measure changes between free and bound fractions of NADH as a indirect measure of metabolic alteration in living cells. Using Phasor-FLIM, pixel maps of metabolic alteration in HEK293 cell lines and in transgenic Drosophila expressing expanded and unexpanded polyQ HTT exon1 in the eye disc were developed. We found a significant shift towards increased free NADH, indicating an increased glycolytic state for cells and tissues expressing the expanded polyQ compared to unexpanded control. In the nucleus, a further lifetime shift occurs towards higher free NADH suggesting a possible synergism between metabolic dysfunction and transcriptional regulation. Our results indicate that metabolic dysfunction in HD shifts to increased glycolysis leading to oxidative stress and cell death. This powerful label free method can be used to screen native HD tissue samples and for potential drug screening.

A potent and selective Sirtuin 1 inhibitor alleviates pathology in multiple animal and cell models of Huntington's disease.

Protein acetylation, which is central to transcriptional control as well as other cellular processes, is disrupted in Huntington's disease (HD). Treatments that restore global acetylation levels, such as inhibiting histone deacetylases (HDACs), are effective in suppressing HD pathology in model organisms. However, agents that selectively target the disease-relevant HDACs have not been available. SirT1 (Sir2 in Drosophila melanogaster) deacetylates histones and other proteins including transcription factors. Genetically reducing, but not eliminating, Sir2 has been shown to suppress HD pathology in model organisms. To date, small molecule inhibitors of sirtuins have exhibited low potency and unattractive pharmacological and biopharmaceutical properties. Here, we show that highly selective pharmacological inhibition of Drosophila Sir2 and mammalian SirT1 using the novel inhibitor selisistat (selisistat; 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide) can suppress HD pathology caused by mutant huntingtin exon 1 fragments in Drosophila, mammalian cells and mice. We have validated Sir2 as the in vivo target of selisistat by showing that genetic elimination of Sir2 eradicates the effect of this inhibitor in Drosophila. The specificity of selisistat is shown by its effect on recombinant sirtuins in mammalian cells. Reduction of HD pathology by selisistat in Drosophila, mammalian cells and mouse models of HD suggests that this inhibitor has potential as an effective therapeutic treatment for human disease and may also serve as a tool to better understand the downstream pathways of SirT1/Sir2 that may be critical for HD.

Morphometric analysis of Huntington's disease neurodegeneration in Drosophila.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder. The HD gene encodes the huntingtin protein (HTT) that contains polyglutamine tracts of variable length. Expansions of the CAG repeat near the amino terminus to encode 40 or more glutamines (polyQ) lead to disease. At least eight other expanded polyQ diseases have been described. HD can be faithfully modeled in Drosophila with the key features of the disease such as late onset, slowly progressing degeneration, formation of abnormal protein aggregates and the dependence on polyQ length being evident. Such invertebrate model organisms provide powerful platforms to explore neurodegenerative mechanisms and to productively speed the identification of targets and agents that are likely to be effective at treating diseases in humans. Here we describe an optical pseudopupil method that can be readily quantified to provide a fast and sensitive assay for assessing the degree of HD neurodegeneration in vivo. We discuss detailed crossing schemes as well as factors including different drivers, various constructs, the number of UAS sites, genetic background, and temperature that can influence the result of pseudopupil measurements.

Histone deacetylase (HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate polyglutamine-elicited phenotypes in model systems of Huntington's disease.

We have previously demonstrated amelioration of Huntington's disease (HD)-related phenotypes in R6/2 transgenic mice in response to treatment with the novel histone deacetylase (HDAC) inhibitor 4b. Here we have measured the selectivity profiles of 4b and related compounds against class I and class II HDACs and have tested their ability to restore altered expression of genes related to HD pathology in mice and to rescue disease effects in cell culture and Drosophila models of HD. R6/2 transgenic and wild-type (wt) mice received daily injections of HDAC inhibitors for 3 days followed by real-time PCR analysis to detect expression differences for 13 HD-related genes. We find that HDACi 4b and 136, two compounds showing high potency for inhibiting HDAC3 were most effective in reversing the expression of genes relevant to HD, including Ppp1r1b, which encodes DARPP-32, a marker for medium spiny striatal neurons. In contrast, compounds targeting HDAC1 were less effective at correcting gene expression abnormalities in R6/2 transgenic mice, but did cause significant increases in the expression of selected genes. An additional panel of 4b-related compounds was tested in a Drosophila model of HD and in STHdhQ111 striatal cells to further distinguish HDAC selectivity. Significant improvement in huntingtin-elicited Drosophila eye neurodegeneration in the fly was observed in response to treatment with compounds targeting human HDAC1 and/or HDAC3. In STHdhQ111 striatal cells, the ability of HDAC inhibitors to improve huntingtin-elicited metabolic deficits correlated with the potency at inhibiting HDAC1 and HDAC3, although the IC50 values for HDAC1 inhibition were typically 10-fold higher than for inhibition of HDAC3. Assessment of HDAC protein localization in brain tissue by Western blot analysis revealed accumulation of HDAC1 and HDAC3 in the nucleus of HD transgenic mice compared to wt mice, with a concurrent decrease in cytoplasmic localization, suggesting that these HDACs contribute to a repressive chromatin environment in HD. No differences were detected in the localization of HDAC2, HDAC4 or HDAC7. These results suggest that inhibition of HDACs 1 and 3 can relieve HD-like phenotypes in model systems and that HDAC inhibitors targeting these isotypes might show therapeutic benefit in human HD.

The role of feedback in the formation of morphogen territories.

In this paper, we consider a mathematical model for the formation of spatial morphogen territories of two key morphogens: Wingless (Wg) and Decapentaplegic (DPP), involved in leg development of Drosophila. We define a gene regulatory network (GRN) that utilizes autoactivation and cros-sinhibition (modeled by Hill equations) to establish and maintain stable boundaries of gene expression. By computational analysis we find that in the presence of a general activator, neither autoactivation, nor cross-inhibition alone are sufficient to maintain stable sharp boundaries of morphogen production in the leg disc. The minimal requirements for a self-organizing system are a coupled system of two morphogens in which the autoactivation and cross-inhibition have Hill coefficients strictly greater than one. In addition, the GRN modeled here describes the regenerative responses to genetic manipulations of positional identity in the leg disc.

A unique DNA binding domain converts T-cell factors into strong Wnt effectors.

Wnt regulation of gene expression requires binding of LEF/T-cell factor (LEF/TCF) transcription factors to Wnt response elements (WREs) and recruitment of the activator beta-catenin. There are significant differences in the abilities of LEF/TCF family members to regulate Wnt target genes. For example, alternatively spliced isoforms of TCF-1 and TCF-4 with a C-terminal "E" tail are uniquely potent in their activation of LEF1 and CDX1. Here we report that the mechanism responsible for this unique activity is an auxiliary 30-amino-acid DNA interaction motif referred to here as the "cysteine clamp" (or C-clamp). The C-clamp contains invariant cysteine, aromatic, and basic residues, and surface plasmon resonance (SPR) studies with recombinant C-clamp protein showed that it binds double-stranded DNA but not single-stranded DNA or RNA (equilibrium dissociation constant = 16 nM). CASTing (Cyclic Amplification and Selection of Targets) experiments were used to test whether this motif influences WRE recognition. Full-length LEF-1, TCF-1E, and TCF-1E with a mutated C-clamp all bind nearly identical WREs (TYYCTTTGATSTT), showing that the C-clamp does not alter WRE specificity. However, a GC element downstream of the WRE (RCCG) is enriched in wild-type TCF-1E binding sites but not in mutant TCF-1E binding sites. We conclude that the C-clamp is a sequence-specific DNA binding motif. C-clamp mutations destroy the ability of beta-catenin to regulate the LEF1 promoter, and they severely impair the ability of TCF-1 to regulate growth in colon cancer cells. Thus, E-tail isoforms of TCFs utilize two DNA binding activities to access a subset of Wnt targets important for cell growth.

Wingless directly represses DPP morphogen expression via an armadillo/TCF/Brinker complex.

BACKGROUND: Spatially restricted morphogen expression drives many patterning and regeneration processes, but how is the pattern of morphogen expression established and maintained? Patterning of Drosophila leg imaginal discs requires expression of the DPP morphogen dorsally and the wingless (WG) morphogen ventrally. We have shown that these mutually exclusive patterns of expression are controlled by a self-organizing system of feedback loops that involve WG and DPP, but whether the feedback is direct or indirect is not known. METHODS/FINDINGS: By analyzing expression patterns of regulatory DNA driving reporter genes in different genetic backgrounds, we identify a key component of this system by showing that WG directly represses transcription of the dpp gene in the ventral leg disc. Repression of dpp requires a tri-partite complex of the WG mediators armadillo (ARM) and dTCF, and the co-repressor Brinker, (BRK), wherein ARM.dTCF and BRK bind to independent sites within the dpp locus. CONCLUSIONS/SIGNIFICANCE: Many examples of dTCF repression in the absence of WNT signaling have been described, but few examples of signal-driven repression requiring both ARM and dTCF binding have been reported. Thus, our findings represent a new mode of WG mediated repression and demonstrate that direct regulation between morphogen signaling pathways can contribute to a robust self-organizing system capable of dynamically maintaining territories of morphogen expression.