Monowarul M. Siddique

Caffeine stimulates hepatic lipid metabolism by the autophagy-lysosomal pathway in mice

Caffeine is one of the worlds most consumed drugs. Recently, several studies showed that its consumption is associated with lower risk for nonalcoholic fatty liver disease (NAFLD), an obesity‐related condition that recently has become the major cause of liver disease worldwide. Although caffeine is known to stimulate hepatic fat oxidation, its mechanism of action on lipid metabolism is still not clear. Here, we show that caffeine surprisingly is a potent stimulator of hepatic autophagic flux. Using genetic, pharmacological, and metabolomic approaches, we demonstrate that caffeine reduces intrahepatic lipid content and stimulates β‐oxidation in hepatic cells and liver by an autophagy‐lysosomal pathway. Furthermore, caffeine‐induced autophagy involved down‐regulation of mammalian target of rapamycin signaling and alteration in hepatic amino acids and sphingolipid levels. In mice fed a high‐fat diet, caffeine markedly reduces hepatosteatosis and concomitantly increases autophagy and lipid uptake in lysosomes. Conclusion: These results provide novel insight into caffeines lipolytic actions through autophagy in mammalian liver and its potential beneficial effects in NAFLD. (Hepatology 2014;59:1366‐1380)

Trp53-dependent DNA-repair is affected by the codon 72 polymorphism

Trp53 is arguably the most critical tumour suppressor gene product that inhibits malignant transformation. Besides mutations that inactivate Trp53 functions, genetic polymorphisms have been suggested to be risk factors for cancer. A polymorphic site at codon 72 in exon 4 encodes either an arginine amino acid (Trp5372R) or a proline residue (Trp5372P). Previous studies have shown that the Trp5372R form is more efficient in apoptosis induction, whereas the Trp5372P form was suggested to induce G1 arrest better. Here we report that Trp5372P is more efficient than Trp5372R in specifically activating several Trp53-dependent DNA-repair target genes in several cellular systems. Moreover, using isogenic cell lines and several DNA-repair assays, we show that Trp5372P cells have a significantly higher DNA-repair capacity than the Trp5372R cells. Furthermore, Trp5372P-expressing cells exhibit reduced micronuclei formation compared to Trp5372R-expressing cells, suggesting that genomic instability is reduced in these cells. Together, the data highlight the functional differences between the Trp53 polymorphic variants, and suggest that their expression status may influence cancer risk.

Fenretinide Prevents Lipid-induced Insulin Resistance by Blocking Ceramide Biosynthesis

Background: Fenretinide, an in-trial chemotherapeutic, improves insulin sensitivity in mice and humans. Results: Fenretinide reduces Des1 expression and prevents ceramide accumulation, while protecting against lipid-induced insulin resistance. Conclusion: Fenretinide decreases ceramide biosynthesis, and increases levels of dihydroceramides, thus preserving insulin responsiveness. Significance: These data suggest that Des1 may be a viable therapeutic target for normalizing glucose homeostasis. Fenretinide is a synthetic retinoid that is being tested in clinical trials for the treatment of breast cancer and insulin resistance, but its mechanism of action has been elusive. Recent in vitro data indicate that fenretinide inhibits dihydroceramide desaturase, an enzyme involved in the biosynthesis of lipotoxic ceramides that antagonize insulin action. Because of this finding, we assessed whether fenretinide could improve insulin sensitivity and glucose homeostasis in vitro and in vivo by controlling ceramide production. The effect of fenretinide on insulin action and the cellular lipidome was assessed in a number of lipid-challenged models including cultured myotubes and isolated muscles strips incubated with exogenous fatty acids and mice fed a high-fat diet. Insulin action was evaluated in the various models by measuring glucose uptake or disposal and the activation of Akt/PKB, a serine/threonine kinase that is obligate for insulin-stimulated anabolism. The effects of fenretinide on cellular lipid levels were assessed by LC-MS/MS. Fenretinide negated lipid-induced insulin resistance in each of the model systems assayed. Simultaneously, the drug depleted cells of ceramide, while promoting the accumulation of the precursor dihydroceramide, a substrate for the reaction catalyzed by Des1. These data suggest that fenretinide improves insulin sensitivity, at least in part, by inhibiting Des1 and suggest that therapeutics targeting this enzyme may be a viable therapeutic means for normalizing glucose homeostasis in the overweight and diabetic.

Ceramides and Glucosylceramides Are Independent Antagonists of Insulin Signaling

Background: Both ceramides and glucosylceramides have been implicated in the pathogenesis of insulin resistance. Results: These two classes of sphingolipids modulate insulin action but differ by both tissue specificity and mechanism of action. Conclusion: Ceramides and glucosylceramides are independent and separable antagonists of insulin signaling. Significance: These observations will contribute to our understanding of how sphingolipids contribute to obesity-related metabolic diseases. Inhibitors of sphingolipid synthesis protect mice from diet induced-insulin resistance, and sphingolipids such as ceramides and glucosylated-ceramides (e.g., GM3) are putative nutritional intermediates linking obesity to diabetes risk. Herein we investigated the role of each of these sphingolipids in muscle and adipose tissue and conclude that they are independent and separable antagonists of insulin signaling. Of particular note, ceramides antagonize insulin signaling in both myotubes and adipocytes, whereas glucosyceramides are only efficacious in adipocytes: 1) In myotubes exposed to saturated fats, inhibitors of enzymes required for ceramide synthesis enhance insulin signaling, but those targeting glucosylceramide synthase have no effect. 2) Exogenous ceramides antagonize insulin signaling in myotubes, whereas ganglioside precursors do not. 3) Overexpression of glucosylceramide synthase in myotubes induces glucosylceramide but enhances insulin signaling. In contrast, glucosylated ceramides have profound effects in adipocytes. For example, either ganglioside addition or human glucosylceramide synthase overexpression suppresses insulin signaling in adipocytes. These data have important mechanistic implications for understanding how these sphingolipids contribute to energy sensing and the disruption of anabolism under conditions of nutrient oversupply.

Ablation of Dihydroceramide Desaturase 1, a Therapeutic Target for the Treatment of Metabolic Diseases, Simultaneously Stimulates Anabolic and Catabolic Signaling

ABSTRACT The lipotoxicity hypothesis posits that obesity predisposes individuals to metabolic diseases because the oversupply of lipids to tissues not suited for fat storage leads to the accumulation of fat-derived molecules that impair tissue function. Means of combating this have been to stimulate anabolic processes to promote lipid storage or to promote catabolic ones to drive fat degradation. Herein, we demonstrate that ablating dihydroceramide desaturase 1 (Des1), an enzyme that produces ceramides, leads to the simultaneous activation of both anabolic and catabolic signaling pathways. In cells lacking Des1, the most common sphingolipids were replaced with dihydro forms lacking the double bond inserted by Des1. These cells exhibited a remarkably strong activation of the antiapoptotic and anabolic signaling pathway regulated by Akt/protein kinase B (PKB), were resistant to apoptosis, and were considerably larger than their wild-type counterparts. Paradoxically, Des1−/− cells exhibited high levels of autophagy. Mechanistic studies revealed that this resulted from impaired ATP synthesis due in part to decreased expression and activity of several complexes of the electron transport chain, particularly complex IV, leading to activation of AMP-activated protein kinase and its induction of the autophagosome. Thus, Des1 ablation enhanced starvation responses but dissociated them from the anabolic, prosurvival, and antiautophagic Akt/PKB pathways.

Ablation of Dihydroceramide Desaturase Confers Resistance to Etoposide-Induced Apoptosis In Vitro

Sphingolipid biosynthesis is potently upregulated by factors associated with cellular stress, including numerous chemotherapeutics, inflammatory cytokines, and glucocorticoids. Dihydroceramide desaturase 1 (Des1), the third enzyme in the highly conserved pathway driving sphingolipid biosynthesis, introduces the 4,5-trans-double bond that typifies most higher-order sphingolipids. Surprisingly, recent studies have shown that certain chemotherapeutics and other drugs inhibit Des1, giving rise to a number of sphingolipids that lack the characteristic double bond. In order to assess the effect of an altered sphingolipid profile (via Des1 inhibition) on cell function, we generated isogenic mouse embryonic fibroblasts lacking both Des1 alleles. Lipidomic profiling revealed that these cells contained higher levels of dihydroceramide than wild-type fibroblasts and that complex sphingolipids were comprised predominantly of the saturated backbone (e.g. sphinganine vs. sphingosine, dihydrosphingomyelin vs. sphingomyelin, etc.). Des1 ablation activated pro-survival and anabolic signaling intermediates (e.g. Akt/PKB, mTOR, MAPK, etc.) and provided protection from apoptosis caused by etoposide, a chemotherapeutic that induces sphingolipid synthesis by upregulating several sphingolipid biosynthesizing enzymes. These data reveal that the double bond present in most sphingolipids has a profound impact on cell survival pathways, and that the manipulation of Des1 could have important effects on apoptosis.

Neurochemistry changes associated with mutations in familial Parkinson's disease.

Parkinsons disease (PD), a common neurodegenerative disease, is characterized by the progressive loss of dopamine neurons and the accumulation of Lewy bodies and neurites. The exact role of genetic and environmental factors in the pathogenesis of PD has frequently been debated. The association of MPTP (methyl-4-phenyl-1, 2, 3, 6- tetrahydropyridine) and toxins (such as rotenone) with parkinsonism highlights the potential etiologic role of environmental toxins in disease causation. The recent discoveries of monogenic (such as α-synuclein, Parkin, UCHL1, PINK1, DJ-1, LRRK2) forms of PD have provided considerable insights into its pathophysiology. Parkin, an ubiquitin protein ligase assists in the degradation of toxic substrates via the ubiquitin proteasome system. It can also mediate a nondegradative form of ubiquitination. PINK1 and LRRK2 are possibly involved in the phosphorylation of substrates important for various cellular functions. Some toxins could interact with α-synuclein, an endogenous protein that is implicated in pathology of PD. Increasing in vitro and in vivo studies suggest that deficits in mitochondrial function, oxidative and nitrosative stress, the accumulation of aberrant or misfolded proteins, and ubiquitin-proteasome system dysfunction underpin the pathogenesis of sporadic and familial forms of PD. Elucidation of the functions of the proteins encoded by the diseasecausing genes will provide an opportunity for identification of specific pathways that could be targeted in neurotherapeutics.