A sexually dimorphic hypothalamic response to chronic high-fat diet consumption.

In this review, we discuss the observations that, following chronic high-fat diet (HFD) exposure, male mice have higher levels of saturated fatty acids (FAs) and total sphingolipids, whereas lower amounts of polyunsaturated FAs in the central nervous system (CNS) than females. Furthermore, males, when compared with female mice, have higher levels of inflammatory markers in the hypothalamus following exposure to HFD. The increase in markers of inflammation in male mice is possibly due to the reductions in proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) and estrogen receptor alpha (ERα), which is not recapitulated in female mice. Consistently, hypothalamic inflammation is induced both in male and female ERα total-body knockout mice when exposed to a HFD, thus confirming the key role of ERα in the regulation of HFD-induced hypothalamic inflammation. Finally, the HFD-induced depletion of hypothalamic ERα is associated with dysregulation in metabolic homeostasis, as evidenced by reductions in glucose tolerance and decrements in myocardial function.

Cardiomyocyte death: mechanisms and translational implications.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. Although treatments have improved, development of novel therapies for patients with CVD remains a major research goal. Apoptosis, necrosis, and autophagy occur in cardiac myocytes, and both gradual and acute cell death are hallmarks of cardiac pathology, including heart failure, myocardial infarction, and ischemia/reperfusion. Pharmacological and genetic inhibition of autophagy, apoptosis, or necrosis diminishes infarct size and improves cardiac function in these disorders. Here, we review recent progress in the fields of autophagy, apoptosis, and necrosis. In addition, we highlight the involvement of these mechanisms in cardiac pathology and discuss potential translational implications.

BH3 mimetics activate multiple pro-autophagic pathways.

The BH3 mimetic ABT737 induces autophagy by competitively disrupting the inhibitory interaction between the BH3 domain of Beclin 1 and the anti-apoptotic proteins Bcl-2 and Bcl-X(L), thereby stimulating the Beclin 1-dependent allosteric activation of the pro-autophagic lipid kinase VPS34. Here, we examined whether ABT737 stimulates other pro-autophagic signal-transduction pathways. ABT737 caused the activating phosphorylation of AMP-dependent kinase (AMPK) and of the AMPK substrate acetyl CoA carboxylase, the activating phosphorylation of several subunits of the inhibitor of NF-κB (IκB) kinase (IKK) and the hyperphosphorylation of the IKK substrate IκB, inhibition of the activity of mammalian target of rapamycin (mTOR) and consequent dephosphorylation of the mTOR substrate S6 kinase. In addition, ABT737 treatment dephosphorylates (and hence likewise inhibits) p53, glycogen synthase kinase-3 and Akt. All these effects were shared by ABT737 and another structurally unrelated BH3 mimetic, HA14-1. Functional experiments revealed that pharmacological or genetic inhibition of IKK, Sirtuin and the p53-depleting ubiquitin ligase MDM2 prevented ABT737-induced autophagy. These results point to unexpected and pleiotropic pro-autophagic effects of BH3 mimetics involving the modulation of multiple signalling pathways.

Illicit survival of cancer cells during polyploidization and depolyploidization.

Tetraploidy and the depolyploidization of tetraploid cells may contribute to oncogenesis. Several mechanisms have evolved to avoid the generation, survival, proliferation and depolyploidization of tetraploids. Cells that illicitly survive these checkpoints are prone to chromosomal instability and aneuploidization. Along with their replication, tetraploids constantly undergo chromosomal rearrangements that eventually lead to pseudodiploidy by two non-exclusive mechanisms: (i) multipolar divisions and (ii) illicit bipolar divisions in the presence of improper microtubule-kinetochore attachments. Here, we describe the regulation and the molecular mechanisms that underlie such a 'polyploidization-depolyploidization' cascade, while focusing on the role of oncogenes and tumor suppressor genes in tetraploidy-driven tumorigenesis. We speculate that the identification of signaling/metabolic cascades that are required for the survival of tetraploid or aneuploid (but not diploid) cancer cells may pave the way for the development of novel broad-spectrum anticancer agents.

Parallel activation of Ca(2+)-induced survival and death pathways in cardiomyocytes by sorbitol-induced hyperosmotic stress.

Hyperosmotic stress promotes rapid and pronounced apoptosis in cultured cardiomyocytes. Here, we investigated if Ca(2+) signals contribute to this response. Exposure of cardiomyocytes to sorbitol [600 mosmol (kg water)(-1)] elicited large and oscillatory intracellular Ca(2+) concentration increases. These Ca(2+) signals were inhibited by nifedipine, Cd(2+), U73122, xestospongin C and ryanodine, suggesting contributions from both Ca(2+) influx through voltage dependent L-type Ca(2+) channels plus Ca(2+) release from intracellular stores mediated by IP(3) receptors and ryanodine receptors. Hyperosmotic stress also increased mitochondrial Ca(2+) levels, promoted mitochondrial depolarization, reduced intracellular ATP content, and activated the transcriptional factor cyclic AMP responsive element binding protein (CREB), determined by increased CREB phosphorylation and electrophoretic mobility shift assays. Incubation with 1 mM EGTA to decrease extracellular [Ca(2+)] prevented cardiomyocyte apoptosis induced by hyperosmotic stress, while overexpression of an adenoviral dominant negative form of CREB abolished the cardioprotection provided by 1 mM EGTA. These results suggest that hyperosmotic stress induced by sorbitol, by increasing Ca(2+) influx and raising intracellular Ca(2+) concentration, activates Ca(2+) release from stores and causes cell death through mitochondrial function collapse. In addition, the present results suggest that the Ca(2+) increase induced by hyperosmotic stress promotes cell survival by recruiting CREB-mediated signaling. Thus, the fate of cardiomyocytes under hyperosmotic stress will depend on the balance between Ca(2+)-induced survival and death pathways.

Caloric restriction and resveratrol promote longevity through the Sirtuin-1-dependent induction of autophagy.

Caloric restriction and autophagy-inducing pharmacological agents can prolong lifespan in model organisms including mice, flies, and nematodes. In this study, we show that transgenic expression of Sirtuin-1 induces autophagy in human cells in vitro and in Caenorhabditis elegans in vivo. The knockdown or knockout of Sirtuin-1 prevented the induction of autophagy by resveratrol and by nutrient deprivation in human cells as well as by dietary restriction in C. elegans. Conversely, Sirtuin-1 was not required for the induction of autophagy by rapamycin or p53 inhibition, neither in human cells nor in C. elegans. The knockdown or pharmacological inhibition of Sirtuin-1 enhanced the vulnerability of human cells to metabolic stress, unless they were stimulated to undergo autophagy by treatment with rapamycin or p53 inhibition. Along similar lines, resveratrol and dietary restriction only prolonged the lifespan of autophagy-proficient nematodes, whereas these beneficial effects on longevity were abolished by the knockdown of the essential autophagic modulator Beclin-1. We conclude that autophagy is universally required for the lifespan-prolonging effects of caloric restriction and pharmacological Sirtuin-1 activators.

The inositol 1,4,5-trisphosphate receptor regulates autophagy through its interaction with Beclin 1.

The inositol 1,4,5-trisphosphate receptor (IP(3)R) is a major regulator of apoptotic signaling. Through interactions with members of the Bcl-2 family of proteins, it drives calcium (Ca(2+)) transients from the endoplasmic reticulum (ER) to mitochondria, thereby establishing a functional and physical link between these organelles. Importantly, the IP(3)R also regulates autophagy, and in particular, its inhibition/depletion strongly induces macroautophagy. Here, we show that the IP(3)R antagonist xestospongin B induces autophagy by disrupting a molecular complex formed by the IP(3)R and Beclin 1, an interaction that is increased or inhibited by overexpression or knockdown of Bcl-2, respectively. An effect of Beclin 1 on Ca(2+) homeostasis was discarded as siRNA-mediated knockdown of Beclin 1 did not affect cytosolic or luminal ER Ca(2+) levels. Xestospongin B- or starvation-induced autophagy was inhibited by overexpression of the IP(3)R ligand-binding domain, which coimmunoprecipitated with Beclin 1. These results identify IP(3)R as a new regulator of the Beclin 1 complex that may bridge signals converging on the ER and initial phagophore formation.

Control of autophagy by oncogenes and tumor suppressor genes.

Multiple oncogenes (in particular phosphatidylinositol 3-kinase, PI3K; activated Akt1; antiapoptotic proteins from the Bcl-2 family) inhibit autophagy. Similarly, several tumor suppressor proteins (such as BH3-only proteins; death-associated protein kinase-1, DAPK1; the phosphatase that antagonizes PI3K, PTEN; tuberous sclerosic complex 1 and 2, TSC1 and TSC2; as well as LKB1/STK11) induce autophagy, meaning that their loss reduces autophagy. Beclin-1, which is required for autophagy induction acts as a haploinsufficient tumor suppressor protein, and other essential autophagy mediators (such as Atg4c, UVRAG and Bif-1) are bona fide oncosuppressors. One of the central tumor suppressor proteins, p53 exerts an ambiguous function in the regulation of autophagy. Within the nucleus, p53 can act as an autophagy-inducing transcription factor. Within the cytoplasm, p53 exerts a tonic autophagy-inhibitory function, and its degradation is actually required for the induction of autophagy. The role of autophagy in oncogenesis and anticancer therapy is contradictory. Chronic suppression of autophagy may stimulate oncogenesis. However, once a tumor is formed, autophagy inhibition may be a therapeutic goal for radiosensitization and chemosensitization. Altogether, the current state-of-the art suggests a complex relationship between cancer and deregulated autophagy that must be disentangled by further in-depth investigation.

Reduction of endoplasmic reticulum Ca2+ levels favors plasma membrane surface exposure of calreticulin.

Some chemotherapeutic agents can elicit apoptotic cancer cell death, thereby activating an anticancer immune response that influences therapeutic outcome. We previously reported that anthracyclins are particularly efficient in inducing immunogenic cell death, correlating with the pre-apoptotic exposure of calreticulin (CRT) on the plasma membrane surface of anthracyclin-treated tumor cells. Here, we investigated the role of cellular Ca(2+) homeostasis on CRT exposure. A neuroblastoma cell line (SH-SY5Y) failed to expose CRT in response to anthracyclin treatment. This defect in CRT exposure could be overcome by the overexpression of Reticulon-1C, a manipulation that led to a decrease in the Ca(2+) concentration within the endoplasmic reticulum lumen. The combination of Reticulon-1C expression and anthracyclin treatment yielded more pronounced endoplasmic reticulum Ca(2+) depletion than either of the two manipulations alone. Chelation of intracellular (and endoplasmic reticulum) Ca(2+), targeted expression of the ligand-binding domain of the IP(3) receptor and inhibition of the sarco-endoplasmic reticulum Ca(2+)-ATPase pump reduced endoplasmic reticulum Ca(2+) load and promoted pre-apoptotic CRT exposure on the cell surface, in SH-SY5Y and HeLa cells. These results provide evidence that endoplasmic reticulum Ca(2+) levels control the exposure of CRT.

Regulation of autophagy by the inositol trisphosphate receptor.

The reduction of intracellular 1,4,5-inositol trisphosphate (IP(3)) levels stimulates autophagy, whereas the enhancement of IP(3) levels inhibits autophagy induced by nutrient depletion. Here, we show that knockdown of the IP(3) receptor (IP(3)R) with small interfering RNAs and pharmacological IP(3)R blockade is a strong stimulus for the induction of autophagy. The IP(3)R is known to reside in the membranes of the endoplasmic reticulum (ER) as well as within ER-mitochondrial contact sites, and IP(3)R blockade triggered the autophagy of both ER and mitochondria, as exactly observed in starvation-induced autophagy. ER stressors such as tunicamycin and thapsigargin also induced autophagy of ER and, to less extent, of mitochondria. Autophagy triggered by starvation or IP(3)R blockade was inhibited by Bcl-2 and Bcl-X(L) specifically targeted to ER but not Bcl-2 or Bcl-X(L) proteins targeted to mitochondria. In contrast, ER stress-induced autophagy was not inhibited by Bcl-2 and Bcl-X(L). Autophagy promoted by IP(3)R inhibition could not be attributed to a modulation of steady-state Ca(2+) levels in the ER or in the cytosol, yet involved the obligate contribution of Beclin-1, autophagy-related gene (Atg)5, Atg10, Atg12 and hVps34. Altogether, these results strongly suggest that IP(3)R exerts a major role in the physiological control of autophagy.