In this study, we present the novel findings that chronic ethanol consumption induces mitochondrial protein hyperacetylation in the kidney and correlates with significantly increased renal oxidative stress. A major proteomic footprint of alcoholic liver disease (ALD) is an increase in hepatic mitochondrial protein acetylation. Protein hyperacetylation has been shown to alter enzymatic function of numerous proteins and plays a role in regulating metabolic processes. Renal mitochondrial targets of hyperacetylation include numerous metabolic and antioxidant pathways, such as lipid metabolism, oxidative phosphorylation, and amino acid metabolism, as well as glutathione and thioredoxin pathways. Disruption of protein lysine acetylation has the potential to impair renal function through metabolic dysregulation and decreased antioxidant capacity. Due to a significant elevation in ethanol-mediated renal oxidative stress, we highlight the acetylation of superoxide dismutase, peroxiredoxins, glutathione reductase, and glutathione transferase enzymes. Since oxidative stress is a known factor in ethanol-induced nephrotoxicity, we examined biochemical markers of protein hyperacetylation and oxidative stress. Our results demonstrate increased protein acetylation concurrent with depleted glutathione, altered Cys redox potential, and the presence of 4-HNE protein modifications in our 6-week model of early-stage alcoholic nephrotoxicity. These findings support the hypothesis that ethanol metabolism causes an influx of mitochondrial metabolic substrate, resulting in mitochondrial protein hyperacetylation with the potential to impact mitochondrial metabolic and antioxidant processes.

Aldehyde dehydrogenase 1B1 (ALDH1B1) is a mitochondrial enzyme sharing 65% and 72% sequence identity with ALDH1A1 and ALDH2 proteins, respectively. Compared to the latter two ALDH isozymes, little is known about the physiological functions of ALDH1B1. Studies in humans indicate that ALDH1B1 may be associated with alcohol sensitivity and stem cells. Our recent in vitro studies using human ALDH1B1 showed that it metabolizes acetaldehyde and retinaldehyde. To investigate the in vivo role of ALDH1B1, we generated and characterized a global Aldh1b1 knockout mouse line. These knockout (KO) mice are fertile and show overtly good health. However, ethanol pharmacokinetic analysis revealed ∼40% increase in blood acetaldehyde levels in KO mice. Interestingly, the KO mice exhibited higher fasting blood glucose levels. Collectively, we show for the first time the functional in vivo role of ALDH1B1 in acetaldehyde metabolism and in maintaining glucose homeostasis. This mouse model is a valuable tool to investigate the mechanism by which alcohol may promote the development of diabetes.

Hypoxia is an independent prognostic indicator of poor outcome in several malignancies. However, precise mechanism through which hypoxia promotes disease aggressiveness is still unclear. Here, we report that under hypoxia (1% O2), human prostate cancer (PCA) cells, and extracellular vesicles (EVs) released by these cells, are significantly enriched in triglycerides due to the activation of lipogenesis-related enzymes and signaling molecules. This is likely a survival response to hypoxic stress as accumulated lipids could support growth following reoxygenation. Consistent with this, significantly higher proliferation was observed in hypoxic PCA cells following reoxygenation associated with rapid use of accumulated lipids. Importantly, lipid utilization inhibition by CPT1 inhibitor etomoxir and shRNA-mediated CPT1-knockdown significantly compromised hypoxic PCA cell proliferation following reoxygenation. Furthermore, COX2 inhibitor celecoxib strongly reduced growth and invasiveness following hypoxic PCA cells reoxygenation, and inhibited invasiveness induced by hypoxic PCA EVs. This establishes a role for COX2 enzymatic products in the enhanced PCA growth and invasiveness. Importantly, concentration and loading of EVs secreted by PCA cells were significantly compromised under delipidized serum condition and by lipogenesis inhibitors (fatostatin and silibinin). Overall, present study highlights the biological significance of lipid accumulation in hypoxic PCA cells and its therapeutic relevance in PCA.

Spaceflight affects numerous organ systems in the body, leading to metabolic dysfunction that may have long-term consequences. Microgravity-induced alterations in liver metabolism, particularly with respect to lipids, remain largely unexplored. Here we utilize a novel systems biology approach, combining metabolomics and transcriptomics with advanced Raman microscopy, to investigate altered hepatic lipid metabolism in mice following short duration spaceflight. Mice flown aboard Space Transportation System -135, the last Shuttle mission, lose weight but redistribute lipids, particularly to the liver. Intriguingly, spaceflight mice lose retinol from lipid droplets. Both mRNA and metabolite changes suggest the retinol loss is linked to activation of PPARα-mediated pathways and potentially to hepatic stellate cell activation, both of which may be coincident with increased bile acids and early signs of liver injury. Although the 13-day flight duration is too short for frank fibrosis to develop, the retinol loss plus changes in markers of extracellular matrix remodeling raise the concern that longer duration exposure to the space environment may result in progressive liver damage, increasing the risk for nonalcoholic fatty liver disease.

Cytoplasmic lipid droplets (CLD) are organelle-like structures that function in neutral lipid storage, transport and metabolism through the actions of specific surface-associated proteins. Although diet and metabolism influence hepatic CLD levels, how they affect CLD protein composition is largely unknown. We used non-biased, shotgun, proteomics in combination with metabolic analysis, quantitative immunoblotting, electron microscopy and confocal imaging to define the effects of low- and high-fat diets on CLD properties in fasted-refed mice. We found that the hepatic CLD proteome is distinct from that of CLD from other mammalian tissues, containing enzymes from multiple metabolic pathways. The hepatic CLD proteome is also differentially affected by dietary fat content and hepatic metabolic status. High fat feeding markedly increased the CLD surface density of perilipin-2, a critical regulator of hepatic neutral lipid storage, whereas it reduced CLD levels of betaine-homocysteine S-methyltransferase, an enzyme regulator of homocysteine levels linked to fatty liver disease and hepatocellular carcinoma. Collectively our data demonstrate that the hepatic CLD proteome is enriched in metabolic enzymes, and that it is qualitatively and quantitatively regulated by diet and metabolism. These findings implicate CLD in the regulation of hepatic metabolic processes, and suggest that their properties undergo reorganization in response to hepatic metabolic demands.

The role of neo-angiogenesis in prostate cancer (PCA) growth and metastasis is well established, but the development of effective and non-toxic pharmacological inhibitors of angiogenesis remains an unaccomplished goal. In this regard, targeting aberrant angiogenesis through non-toxic phytochemicals could be an attractive angiopreventive strategy against PCA. The rationale of the present study was to compare the anti-angiogenic potential of four pure diastereoisomeric flavonolignans, namely silybin A, silybin B, isosilybin A and isosilybin B, which we established previously as biologically active constituents in Milk Thistle extract. Results showed that oral feeding of these flavonolignans (50 and 100 mg/kg body weight) effectively inhibit the growth of advanced human PCA DU145 xenografts. Immunohistochemical analyses revealed that these flavonolignans inhibit tumor angiogenesis biomarkers (CD31 and nestin) and signaling molecules regulating angiogenesis (VEGF, VEGFR1, VEGFR2, phospho-Akt and HIF-1α) without adversely affecting the vessel-count in normal tissues (liver, lung, and kidney) of tumor bearing mice. These flavonolignans also inhibited the microvessel sprouting from mouse dorsal aortas ex vivo, and the VEGF-induced cell proliferation, capillary-like tube formation and invasiveness of human umbilical vein endothelial cells (HUVEC) in vitro. Further studies in HUVEC showed that these diastereoisomers target cell cycle, apoptosis and VEGF-induced signaling cascade. Three dimensional growth assay as well as co-culture invasion and in vitro angiogenesis studies (with HUVEC and DU145 cells) suggested the differential effectiveness of the diastereoisomers toward PCA and endothelial cells. Overall, these studies elucidated the comparative anti-angiogenic efficacy of pure flavonolignans from Milk Thistle and suggest their usefulness in PCA angioprevention.

Increasingly, evidence suggests that exposure to maternal obesity creates an inflammatory environment in utero, exerting long-lasting postnatal signatures on the juvenile innate immune system and microbiome that may predispose offspring to development of fatty liver disease. We found that exposure to a maternal Western-style diet (WD) accelerated fibrogenesis in the liver of offspring and was associated with early recruitment of proinflammatory macrophages at 8-12 weeks and microbial dysbiosis as early as 3 weeks of age. We further demonstrated that bone marrow-derived macrophages (BMDMs) were polarized toward an inflammatory state at 8 weeks of age and that a potent antioxidant, pyrroloquinoline quinone (PQQ), reversed BMDM metabolic reprogramming from glycolytic toward oxidative metabolism by restoring trichloroacetic acid cycle function at isocitrate dehydrogenase. This resulted in reduced inflammation and inhibited collagen fibril formation in the liver at 20 weeks of age, even when PQQ was withdrawn at 3 weeks of age. Beginning at 3 weeks of age, WD-fed mice developed a decreased abundance of Parabacteroides and Lactobacillus, together with increased Ruminococcus and decreased tight junction gene expression by 20 weeks, whereas microbiota of mice exposed to PQQ retained compositional stability with age, which was associated with improved liver health. Conclusion: Exposure to a maternal WD induces early gut dysbiosis and disrupts intestinal tight junctions, resulting in BMDM polarization and induction of proinflammatory and profibrotic programs in the offspring that persist into adulthood. Disrupted macrophage and microbiota function can be attenuated by short-term maternal treatment with PQQ prior to weaning, suggesting that reshaping the early gut microbiota in combination with reprogramming macrophages during early weaning may alleviate the sustained proinflammatory environment, preventing the rapid progression of nonalcoholic fatty liver disease to nonalcoholic steatohepatitis in offspring of obese mothers. (Hepatology Communications 2018;2:313-328).

Serelaxin, a recombinant form of the naturally occurring peptide hormone relaxin-2, is a pleiotropic vasodilating hormone that has been studied in patients with acute heart failure. In this study, the effects of serelaxin on cardiac and renal function, fibrosis, inflammation and lipid accumulation were studied in DOCA-salt treated rats. Uninephrectomized rats were assigned to two groups: controls provided with normal drinking water and DOCA provided with DOCA pellets and sodium chloride drinking water. After 4 weeks, the DOCA-salt rats were randomly selected and implanted with osmotic minipumps delivering vehicle or serelaxin for another 4 weeks. Treatment with serelaxin prevented cardiac and renal dysfunction in DOCA-salt rats. Serelaxin prevented cardiac and renal fibrosis, as determined by Picrosirius Red staining and Second Harmonic Generation (SHG) Microscopy. Treatment of DOCA-salt rats with serelaxin decreased renal inflammation, including the expression of TGF-β, NFκB, MCP-1, IL-1, IL-6, ICAM-1, VCAM-1 and CD68 macrophages. Serelaxin also decreased lipid accumulation in kidney in part by decreasing SREBP-1c, SREBP-2, ChREBP, FATP1, HMGCoAR, and LDL receptor, and increasing Acox1 and ABCA1. In summary, serelaxin reversed DOCA-salt induced cardiac and renal dysfunction.

Adenosine triphosphate-binding cassette subfamily C member 2 (ABCC2/Abcc2) is critically important to biliary excretion of many endobiotic and xenobiotic compounds, and is a major driving force for bile acid-independent bile flow. Abcc2 expression is reduced at the messenger RNA (mRNA) and protein levels in various forms of experimental cholestasis. In a microRNA (miRNA) screen of mouse liver after biliary obstruction, we found that miRNA let7a-5p was significantly up-regulated approximately 4-fold. Similarly, ABCC2 mRNA was depleted and miRNA let7a-5p was elevated over 4-fold in livers of children with biliary atresia compared with normal livers. In silico analysis predicted that let7a-5p would target the 3' untranslated region (3' UTR) of ABCC2/Abcc2 RNA. The objective of this study was to determine whether let7a-5p contributes to the depletion of ABCC2/Abcc2 in cholestasis. To demonstrate the functional importance of miRNA let7a-5p in regulating the expression of ABCC2, co-transfection of a let7a-5p mimic and an ABCC2-3' UTR luciferase construct into Huh-7 cells led to a marked inhibition of luciferase activity by about 60%-70% compared with controls, which was reversed by a let7a-5p mimic inhibitor. Expression of this mimic led to a significant decrease in endogenous ABCC2 mRNA and protein levels in a Huh-7 liver cell line, which could be blocked by expression of a let7a-5p mimic inhibitor. Injection of a lentivirus let7a-5p inhibitor into normal mouse liver or into mouse liver after common bile duct ligation led to a significant increase in endogenous Abcc2 mRNA and protein levels and a depletion of let7a-5p mRNA levels compared with untreated, saline-injected livers or livers treated with an inactive lentivirus control. Conclusion: These studies demonstrate that miR-let7a-5p is involved in regulating ABCC2/Abcc2 expression, and is aberrantly up-regulated in obstructive cholestasis.

Intake of sugars, especially the fructose component, is strongly associated with the development of obesity and metabolic syndrome, but the relative role of taste versus metabolism in driving preference, intake, and metabolic outcome is not fully understood. We aimed to evaluate the preference for sweet substances and the tendency to develop metabolic syndrome in response to these sugars in mice lacking functional taste signaling [P2X2 (P2X purinoreceptor 2)/P2X3 (P2X purinoreceptor 3) double knockout mice (DKO)] and mice unable to metabolize fructose (fructokinase knockout mice). Of interest, our data indicate that despite their inability to taste sweetness, P2X2/3 DKO mice still prefer caloric sugars (including fructose and glucose) to water in long-term testing, although with diminished preference compared with control mice. Despite reduced intake of caloric sugars by P2X2/3 DKO animals, the DKO mice still show increased levels of the sugar-dependent hormone FGF21 (fibroblast growth factor 21) in plasma and liver. Despite lower sugar intake, taste-blind mice develop severe features of metabolic syndrome due to reduced sensitivity to leptin, reduced ability to mobilize and oxidize fats, and increased hepatic de novo lipogenesis. In contrast to P2X2/3 DKO and wild-type mice, fructokinase knockout mice, which cannot metabolize fructose and are protected against fructose-induced metabolic syndrome, demonstrate reduced preference and intake for all fructose-containing sugars tested but not for glucose or artificial sweeteners. Based on these observations, we conclude that sugar can induce metabolic syndrome in mice independently of its sweet properties. Furthermore, our data demonstrate that the metabolism of fructose is necessary for sugar to drive intake and preference in mice.

Curcumin is a bioactive natural compound with a wide range of pharmacological properties, including antitumor activity; however, its clinical application has been limited because of its low solubility, stability, and bioavailability. In this study, a solid phase approach was proposed for the combinatorial synthesis of a mini library of the mimics of curcumin in good purity and yield. The non-effective findings in pancreatic cancer cells switched to strong growth inhibition and cell death efficacy for PC3 prostate cancer cells, and mimic 9, in which tyrosol (TYR) and homovanillyl alcohol (HVA) units were linked by a phosphodiester bond, was quite effective not only in cell growth inhibition but also in causing strong cell death under the study conditions and treatments that were not effective in PANC1 cells. The results got more exciting when we also consider the findings in SW480 human colorectal carcinoma cell line, where the growth inhibitor effects were more in line with that of the PC3 cells, but the lack of cell death effect was more in line with the PANC1 cells.

Latent varicella zoster virus (VZV) has been detected in human adrenal glands, raising the possibility of virus-induced adrenal damage and dysfunction during primary infection or reactivation. Rare cases of bilateral adrenal hemorrhage and insufficiency associated with VZV reactivation have been reported. Since there is no animal model for VZV infection of adrenal glands, we obtained adrenal glands from two non-human primates (NHPs) that spontaneously developed varicella from primary simian varicella virus (SVV) infection, the NHP VZV homolog. Histological and immunohistochemical analysis revealed SVV antigen and DNA in the adrenal medulla and cortex of both animals. Adrenal glands were observed to have Cowdry A inclusion bodies, cellular necrosis, multiple areas of hemorrhage, and varying amounts of polymorphonuclear cells. No specific association of SVV antigen with βIII-tubulin-positive nerve fibers was found. Overall, we found that SVV can productively infect NHP adrenal glands, and is associated with inflammation, hemorrhage, and cell death. These findings suggest that further studies are warranted to examine the contribution of VZV infection to human adrenal disease. This study also suggests that VZV infection may present itself as acute adrenal dysfunction with "long-hauler" symptoms of fatigue, weakness, myalgias/arthralgias, and hypotension.

Sulfur mustard (SM) is a cytotoxic, vesicating, chemical warfare agent, first used in 1917; corneas are particularly vulnerable to SM exposure. They may develop inflammation, ulceration, neovascularization (NV), impaired vision, and partial/complete blindness depending upon the concentration of SM, exposure duration, and bio-physiological conditions of the eyes. Comprehensive in vivo studies have established ocular structural alterations, opacity, NV, and inflammation upon short durations (<4 min) of SM exposure. In this study, detailed analyses of histopathological alterations in corneal structure, keratocytes, inflammatory cells, blood vessels, and expressions of cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), and cytokines were performed in New Zealand white rabbits, in a time-dependent manner till 28 days, post longer durations (5 and 7 min) of ocular SM exposure to establish quantifiable endpoints of injury and healing. Results indicated that SM exposure led to duration-dependent increases in corneal thickness, opacity, ulceration, epithelial-stromal separation, and epithelial degradation. Significant increases in NV, keratocyte death, blood vessels, and inflammatory markers (COX-2, MMP-9, VEGF, and interleukin-8) were also observed for both exposure durations compared to the controls. Collectively, these findings would benefit in temporal delineation of mechanisms underlying SM-induced corneal toxicity and provide models for testing therapeutic interventions.

Heme metabolism is a key regulator of inflammatory responses. Cobalt protoporphyrin IX (CoPP) is a heme analog and mimic that potently activates the NRF2/heme oxygenase-1 (HO-1) pathway, especially in monocytes and macrophages. We investigated the influence of CoPP on inflammatory responses using a murine model of colitis. Surprisingly, conditional deletion of myeloid HO-1 did not impact the colonic inflammatory response or the protective influence of CoPP in the setting of dextran sodium sulfate-induced colitis. Rather, we reveal that CoPP elicits a contradictory shift in blood myeloid populations relative to the colon during active intestinal inflammation. Major population changes include markedly diminished trafficking of CCR2+Ly6Chi monocytes to the inflamed colon, despite significant mobilization of this population into circulation. This resulted in significantly diminished colonic expansion of monocyte-derived macrophages and inflammatory cytokine expression. These findings were linked with significant induction of systemic CCL2 leading to a disrupted CCL2 chemoattractant gradient toward the colon and concentration-dependent suppression of circulating monocyte CCR2 expression. Administration of CoPP also induced macrophage differentiation toward a MarcohiHmox1hi anti-inflammatory erythrophagocytic phenotype, contributing to an overall decreased inflammatory profile. Such findings redefine protective influences of heme metabolism during inflammation, and highlight previously unreported immunosuppressive mechanisms of endogenous CCL2 induction.

Since the Nobel Prize-winning work of Huggins, androgen ablation has been a mainstay for treatment of recurrent prostate cancer. While initially effective for most patients, prostate cancers inevitably develop the ability to survive, grow, and metastasize further, despite ongoing androgen suppression. Here, we briefly review key preclinical studies over decades and include illustrative examples from our own laboratories that suggest prostate cancer cells titrate androgen signaling to optimize growth. Such laboratory-based studies argue that adaptations that allow growth in a low-androgen environment render prostate cancer sensitive to restoration of androgens, especially at supraphysiologic doses. Based on preclinical data as well as clinical observations, trials employing high-dose testosterone (HDT) therapy have now been conducted. These trials suggest a clinical benefit in cancer response and quality of life in a subset of castration-resistant prostate cancer patients. Laboratory studies also suggest that HDT may yet be optimized further to improve efficacy or durability of response. However, laboratory observations suggest that the cancer will inevitably adapt to HDT, and, as with prior androgen deprivation, disease progression follows. Nonetheless, the adaptations made to render tumors resistant to hormonal manipulations may reveal vulnerabilities that can be exploited to prolong survival and provide other clinical benefits.

The portion of the global population that is over the age of 65 is growing rapidly and this presents a number of clinical complications, as the aged population is at higher risk for various diseases, including infection. For example, advanced age is a risk factor for heightened morbidity and mortality following infection with Streptococcus pneumoniae. This increased vulnerability is due, at least in part, to age-related dysregulation of the immune response, a phenomenon termed immunosenescence. However, our understanding of the mechanisms influencing the immunosenescent state and its effects on the innate immune response to pneumonia remain incomplete. Recently, a role for the gut microbiome in age-specific alterations in immunity has been described. Here, we utilized a murine model of intranasal Streptococcus pneumoniae infection to investigate the effects of age on both the innate immune response and the intestinal microbial populations after infection. In aged mice, compared to their younger counterparts, infection with Streptococcus pneumoniae led to increased mortality, impaired lung function and inadequate bacterial control. This poor response to infection was associated with increased influx of neutrophils into the lungs of aged mice 24 h after infection. The exacerbated pulmonary immune response was not associated with increased pro-inflammatory cytokines in the lung compared to young mice but instead heightened expression of immune cell recruiting chemokines by lung neutrophils. Bacterial 16S-rRNA gene sequencing of the fecal microbiome of aged and young-infected mice revealed expansion of Enterobacteriaceae in the feces of aged, but not young mice, after infection. We also saw elevated levels of gut-derived bacteria in the lung of aged-infected mice, including the potentially pathogenic symbiote Escherichia coli. Taken together, these results reveal that, when compared to young mice, Streptococcus pneumoniae infection in age leads to increased lung neutrophilia along with potentially pathogenic alterations in commensal bacteria and highlight potential mechanistic targets contributing to the increased morbidity and mortality observed in infections in age.

Nonalcoholic steatohepatitis (NASH) is the most common chronic liver disease in the US, partly due to the increasing incidence of metabolic syndrome, obesity, and type 2 diabetes. The roles of bile acids and their receptors, such as the nuclear receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5, on the development of NASH are not fully clear. C57BL/6J male mice fed a Western diet (WD) develop characteristics of NASH, allowing determination of the effects of FXR and TGR5 agonists on this disease. Here we show that the FXR-TGR5 dual agonist INT-767 prevents progression of WD-induced hepatic steatosis, inflammation, and fibrosis, as determined by histological and biochemical assays and novel label-free microscopy imaging techniques, including third harmonic generation, second harmonic generation, and fluorescence lifetime imaging microscopy. Furthermore, we show INT-767 decreases liver fatty acid synthesis and fatty acid and cholesterol uptake, as well as liver inflammation. INT-767 markedly changed bile acid composition in the liver and intestine, leading to notable decreases in the hydrophobicity index of bile acids, known to limit cholesterol and lipid absorption. In addition, INT-767 upregulated expression of liver p-AMPK, SIRT1, PGC-1α, and SIRT3, which are master regulators of mitochondrial function. Finally, we found INT-767 treatment reduced WD-induced dysbiosis of gut microbiota. Interestingly, the effects of INT-767 in attenuating NASH were absent in FXR-null mice, but still present in TGR5-null mice. Our findings support treatment and prevention protocols with the dual FXR-TGR5 agonist INT-767 arrest progression of WD-induced NASH in mice mediated by FXR-dependent, TGR5-independent mechanisms.

Hepatocellular carcinoma (HCC) is a major global health concern, representing one of the leading causes of cancer-related deaths. Despite various treatment options, the prognosis for HCC patients remains poor, emphasizing the need for a deeper understanding of the factors contributing to HCC development. This study investigates the role of poly(ADP-ribosyl)ation in hepatocyte maturation and its impact on hepatobiliary carcinogenesis. A conditional Parg knockout mouse model was employed, utilizing Cre recombinase under the albumin promoter to target Parg depletion specifically in hepatocytes. The disruption of the poly(ADP-ribosyl)ating pathway in hepatocytes affects the early postnatal liver development. The inability of hepatocytes to finish the late maturation step that occurs early after birth causes intensive apoptosis and acute inflammation, resulting in hypertrophic liver tissue with enlarged hepatocytes. Regeneration nodes with proliferative hepatocytes eventually replace the liver tissue and successfully fulfill the liver function. However, early developmental changes predispose these types of liver to develop pathologies, including with a malignant nature, later in life. In a chemically induced liver cancer model, Parg-depleted livers displayed a higher tendency for hepatocellular carcinoma development. This study underscores the critical role of the poly(ADP-ribosyl)ating pathway in hepatocyte maturation and highlights its involvement in liver pathologies and hepatobiliary carcinogenesis. Understanding these processes may provide valuable insights into liver biology and liver-related diseases, including cancer.

Perilipin-1 (Plin1), a prominent cytoplasmic lipid droplet (CLD) binding phosphoprotein and key physiological regulator of triglyceride storage and lipolysis in adipocytes, is thought to regulate the fragmentation and dispersion of CLD that occurs in response to β-adrenergic activation of adenylate cyclase. Here we investigate the dynamics and molecular determinants of these processes using cell lines stably expressing recombinant forms of Plin1 and/or other members of the perilipin family. Plin1 and a C-terminal CLD-binding fragment of Plin1 (Plin1CT) induced formation of single dense CLD clusters near the microtubule organizing center, whereas neither an N-terminal CLD-binding fragment of Plin1, nor Plin2 or Plin3 induced clustering. Clustered CLD coated by Plin1, or Plin1CT, dispersed in response to isoproterenol, or other agents that activate adenylate cyclase, in a process inhibited by the protein kinase A inhibitor, H89, and blocked by microtubule disruption. Isoproterenol-stimulated phosphorylation of CLD-associated Plin1 on serine 492 preceded their dispersion, and live cell imaging showed that cluster dispersion involved initial fragmentation of tight clusters into multiple smaller clusters, which then fragmented into well-dispersed individual CLD. siRNA knockdown of the cortical actin binding protein, moesin, induced disaggregation of tight clusters into multiple smaller clusters, and inhibited the reaggregation of dispersed CLD into tight clusters. Together these data suggest that the clustering and dispersion processes involve a complex orchestration of phosphorylation-dependent, microtubule-dependent and independent, and microfilament dependent steps.

Carbohydrates with high glycaemic index are proposed to promote the development of obesity, insulin resistance and fatty liver, but the mechanism by which this occurs remains unknown. High serum glucose concentrations are known to induce the polyol pathway and increase fructose generation in the liver. Here we show that this hepatic, endogenously produced fructose causes systemic metabolic changes. We demonstrate that mice unable to metabolize fructose are protected from an increase in energy intake and body weight, visceral obesity, fatty liver, elevated insulin levels and hyperleptinaemia after exposure to 10% glucose for 14 weeks. In normal mice, glucose consumption is accompanied by aldose reductase and polyol pathway activation in steatotic areas. In this regard, we show that aldose reductase-deficient mice are protected against glucose-induced fatty liver. We conclude that endogenous fructose generation and metabolism in the liver represents an important mechanism by which glucose promotes the development of metabolic syndrome.