Correction: A clinical and computational study on anti-obesity effects of hydroxycitric acid.

[This corrects the article DOI: 10.1039/C9RA01345H.].

Modulation of de novo purine biosynthesis leads to activation of AMPK and results in improved glucose handling and insulin sensitivity.

BACKGROUND: AMP activated protein kinase (AMPK) regulates key metabolic reactions and plays a major role in glucose homeostasis. Activating the AMPK is considered as one of the potential therapeutic strategies in treating type-2 diabetes. However, targeting AMPK by small molecule mediated approach can be challenging owing to diverse isoforms of the enzyme and their varied combination in different tissues. In the current study we employ a novel strategy of achieving AMPK activation through increasing the levels of cellular AMP (an allosteric activator of AMPK) levels by activating the enzyme involved in AMP biosynthesis namely Adenylosuccinate lyase (ADSL). METHODS: Rat primary hepatocytes were cultured under metabolic overload conditions (500 µM palmitate) to induce insulin resistance. ADSL was overexpressed in these hepatocytes and its effect on hepatic glucose output, and triglyceride accumulation was checked. In addition to this, ADSL was overexpressed in high fat diet induced obese mice by hydrodynamic tail vein injection and its effect on fasting glucose, glucose tolerance and pyruvate tolerance were checked. RESULTS: Rat primary hepatocytes when cultured under metabolic overload conditions developed insulin resistance as measured in terms of failure of insulin to suppress the glucose output. Overexpressing the ADSL in these hepatocytes resulted in increased AMPK phosporylation and improved the insulin sensitivity and also resulted in reduced triglyceride accumulation and inflammatory cytokine levels. In addition to this, when ADSL was overexpressed in high fat diet induced obese mice, it resulted in reduced the fasting hyperglycemia (20% reduction), and increased glucose and pyruvate tolerance. CONCLUSIONS: This study indicates that activating ADSL can be a potential mechanism to achieve the activation of AMPK in the cells. This leads to a novel idea of exploring the purine nucleotide metabolic pathway as a promising therapeutic target for diabetes and metabolic syndrome.

Ceramide transfer protein deficiency compromises organelle function and leads to senescence in primary cells.

Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum (ER) to the Golgi complex. Its deficiency in mouse leads to embryonic death at E11.5. CERT deficient embryos die from cardiac failure due to defective organogenesis, but not due to ceramide induced apoptotic or necrotic cell death. In the current study we examined the effect of CERT deficiency in a primary cell line, namely, mouse embryonic fibroblasts (MEFs). We show that in MEFs, unlike in mutant embryos, lack of CERT does not lead to increased ceramide but causes an accumulation of hexosylceramides. Nevertheless, the defects due to defective sphingolipid metabolism that ensue, when ceramide fails to be trafficked from ER to the Golgi complex, compromise the viability of the cell. Therefore, MEFs display an incipient ER stress. While we observe that ceramide trafficking from ER to the Golgi complex is compromised, the forward transport of VSVG-GFP protein is unhindered from ER to Golgi complex to the plasma membrane. However, retrograde trafficking of the plasma membrane-associated cholera toxin B to the Golgi complex is reduced. The dysregulated sphingolipid metabolism also leads to increased mitochondrial hexosylceramide. The mitochondrial functions are also compromised in mutant MEFs since they have reduced ATP levels, have increased reactive oxygen species, and show increased glutathione reductase activity. Live-cell imaging shows that the mutant mitochondria exhibit reduced fission and fusion events. The mitochondrial dysfunction leads to an increased mitophagy in the CERT mutant MEFs. The compromised organelle function compromise cell viability and results in premature senescence of these MEFs.

CDase is a pan-ceramidase in Drosophila.

Ceramidases catalyze the conversion of ceramide to sphingosine. They are acylaminohydrolases that catalyze the deacylation of the amide-linked saturated fatty acid from ceramide to generate sphingosine. They also catalyze the reverse reaction of ceramide biosynthesis using sphingosine and fatty acid. In mammals, different proteins catalyze these reactions while individually exhibiting optimal activity over a narrow pH range and have been accordingly called acid, neutral, and alkaline ceramidases. Several genes encode for variants of alkaline ceramidase in mammals. Brainwashing (Bwa) is the only putative alkaline ceramidase homologue present in Drosophila. In this study we have demonstrated that BWA does not exhibit ceramidase activity and that bwa null mutants display no loss of ceramidase activity. Instead, the neutral ceramidase gene CDase encodes the protein that is responsible for all measurable ceramidase activity in Drosophila. Our studies show strong genetic interaction of Bwa with CDase and the Drosophila ceramide kinase gene (DCERK). We show that, although BWA is unlikely to be a ceramidase, it is a regulator of sphingolipid flux in Drosophila. Bwa exhibits strong genetic interaction with other genes coding for ceramide-metabolizing enzymes. This interaction might partly explain its original identification as a ceramidase.

Ceramide kinase regulates phospholipase C and phosphatidylinositol 4, 5, bisphosphate in phototransduction.

Phosphoinositide-specific phospholipase C (PLC) is a central effector for many biological responses regulated by G-protein-coupled receptors including Drosophila phototransduction where light sensitive channels are activated downstream of NORPA, a PLCbeta homolog. Here we show that the sphingolipid biosynthetic enzyme, ceramide kinase, is a novel regulator of PLC signaling and photoreceptor homeostasis. A mutation in ceramide kinase specifically leads to proteolysis of NORPA, consequent loss of PLC activity, and failure in light signal transduction. The mutant photoreceptors also undergo activity-dependent degeneration. Furthermore, we show that a significant increase in ceramide, resulting from lack of ceramide kinase, perturbs the membrane microenvironment of phosphatidylinositol 4, 5, bisphosphate (PIP(2)), altering its distribution. Fluorescence image correlation spectroscopic studies on model membranes suggest that an increase in ceramide decreases clustering of PIP(2) and its partitioning into ordered membrane domains. Thus ceramide kinase-mediated maintenance of ceramide level is important for the local regulation of PIP(2) and PLC during phototransduction.

Mitochondrial degeneration and not apoptosis is the primary cause of embryonic lethality in ceramide transfer protein mutant mice.

Ceramide transfer protein (CERT) functions in the transfer of ceramide from the endoplasmic reticulum (ER) to the Golgi. In this study, we show that CERT is an essential gene for mouse development and embryonic survival and, quite strikingly, is critical for mitochondrial integrity. CERT mutant embryos accumulate ceramide in the ER but also mislocalize ceramide to the mitochondria, compromising their function. Cells in mutant embryos show abnormal dilation of the ER and degenerating mitochondria. These subcellular changes manifest as heart defects and cause severely compromised cardiac function and embryonic death around embryonic day 11.5. In spite of ceramide accumulation, CERT mutant mice do not die as a result of enhanced apoptosis. Instead, cell proliferation is impaired, and expression levels of cell cycle-associated proteins are altered. Individual cells survive, perhaps because cell survival mechanisms are activated. Thus, global compromise of ER and mitochondrial integrity caused by ceramide accumulation in CERT mutant mice primarily affects organogenesis rather than causing cell death via apoptotic pathways.

Sphingolipids and membrane biology as determined from genetic models.

The importance of sphingolipids in membrane biology was appreciated early in the twentieth century when several human inborn errors of metabolism were linked to defects in sphingolipid degradation. The past two decades have seen an explosion of information linking sphingolipids with cellular processes. Studies have unraveled mechanistic details of the sphingolipid metabolic pathways, and these findings are being exploited in the development of novel therapies, some now in clinical trials. Pioneering work in yeast has laid the foundation for identifying genes encoding the enzymes of the pathways. The advent of the era of genomics and bioinformatics has led to the identification of homologous genes in other species and the subsequent creation of animal knock-out lines for these genes. Discoveries from these efforts have re-kindled interest in the role of sphingolipids in membrane biology. This review highlights some of the recent advances in understanding sphingolipids' roles in membrane biology as determined from genetic models.

Ceramide transfer protein function is essential for normal oxidative stress response and lifespan.

Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum to the Golgi complex, a process critical in synthesis and maintenance of normal levels of sphingolipids in mammalian cells. However, how its function is integrated into development and physiology of the animal is less clear. Here, we report the in vivo consequences of loss of functional CERT protein. We generated Drosophila melanogaster mutant flies lacking a functional CERT (Dcert) protein using chemical mutagenesis and a Western blot-based genetic screen. The mutant flies die early between days 10 and 30, whereas controls lived between 75 and 90 days. They display >70% decrease in ceramide phosphoethanolamine (the sphingomyelin analog in Drosophila) and ceramide. These changes resulted in increased plasma membrane fluidity that renders them susceptible to reactive oxygen species and results in enhanced oxidative damage to cellular proteins. Consequently, the flies showed reduced thermal tolerance that was exacerbated with aging and metabolic compromise such as decreasing ATP and increasing glucose levels, reminiscent of premature aging. Our studies demonstrate that maintenance of physiological levels of ceramide phosphoethanolamine by CERT in vivo is required to prevent oxidative damages to cellular components that are critical for viability and normal lifespan of the animal.

Smoke treatment triggers the release of a novel DNA damaging factor by lymphocytes.

Organic fuel smoke is a hazardous agent, which pushes the cells towards"prooxidant state'', leading to 4,46,400 strand breaks/cell/day as against 47,000 strand breaks/cell/day produced by constitutive oxygen radicals. This prooxidants scenario switches on a plethora of intercellular events. Here we report a novel DNA damaging factor released by lymphocytes, upon treatment with smoke condensate. Human lymphocytes, when exposed to cow dung cake smoke condensate, were found to release a low molecular weight factor into the media at 20 min of exposure. The conditioned media, displayed a propensity of inducing DNA damage in fresh, normal lymphocytes, which were not exposed to any damaging agent. The above DNA damaging effect of the conditioned media was not due to any residual presence of Polycyclic Aromatic Hydrocarbons, which were present in the smoke. The release of this factor was in correlation with the DNA damaging event, taking place in the cells. This secondary DNA damaging factor had a molecular weight less than 5 kd. The factor had the cell death inducing propensity when allowed to act on lymphocytes.