Role of STIM1 in the Regulation of Cardiac Energy Substrate Preference.

The heart requires a variety of energy substrates to maintain proper contractile function. Glucose and long-chain fatty acids (FA) are the major cardiac metabolic substrates under physiological conditions. Upon stress, a shift of cardiac substrate preference toward either glucose or FA is associated with cardiac diseases. For example, in pressure-overloaded hypertrophic hearts, there is a long-lasting substrate shift toward glucose, while in hearts with diabetic cardiomyopathy, the fuel is switched toward FA. Stromal interaction molecule 1 (STIM1), a well-established calcium (Ca2+) sensor of endoplasmic reticulum (ER) Ca2+ store, is increasingly recognized as a critical player in mediating both cardiac hypertrophy and diabetic cardiomyopathy. However, the cause-effect relationship between STIM1 and glucose/FA metabolism and the possible mechanisms by which STIM1 is involved in these cardiac metabolic diseases are poorly understood. In this review, we first discussed STIM1-dependent signaling in cardiomyocytes and metabolic changes in cardiac hypertrophy and diabetic cardiomyopathy. Second, we provided examples of the involvement of STIM1 in energy metabolism to discuss the emerging role of STIM1 in the regulation of energy substrate preference in metabolic cardiac diseases and speculated the corresponding underlying molecular mechanisms of the crosstalk between STIM1 and cardiac energy substrate preference. Finally, we briefly discussed and presented future perspectives on the possibility of targeting STIM1 to rescue cardiac metabolic diseases. Taken together, STIM1 emerges as a key player in regulating cardiac energy substrate preference, and revealing the underlying molecular mechanisms by which STIM1 mediates cardiac energy metabolism could be helpful to find novel targets to prevent or treat cardiac metabolic diseases.

Ketone Body Exposure of Cardiomyocytes Impairs Insulin Sensitivity and Contractile Function through Vacuolar-Type H+-ATPase Disassembly-Rescue by Specific Amino Acid Supplementation.

The heart is metabolically flexible. Under physiological conditions, it mainly uses lipids and glucose as energy substrates. In uncontrolled diabetes, the heart switches towards predominant lipid utilization, which over time is detrimental to cardiac function. Additionally, diabetes is accompanied by high plasma ketone levels and increased utilization of energy provision. The administration of exogenous ketones is currently being investigated for the treatment of cardiovascular disease. Yet, it remains unclear whether increased cardiac ketone utilization is beneficial or detrimental to cardiac functioning. The mechanism of lipid-induced cardiac dysfunction includes disassembly of the endosomal proton pump (named vacuolar-type H+-ATPase; v-ATPase) as the main early onset event, followed by endosomal de-acidification/dysfunction. The de-acidified endosomes can no longer serve as a storage compartment for lipid transporter CD36, which then translocates to the sarcolemma to induce lipid accumulation, insulin resistance, and contractile dysfunction. Lipid-induced v-ATPase disassembly is counteracted by the supply of specific amino acids. Here, we tested the effect of ketone bodies on v-ATPase assembly status and regulation of lipid uptake in rodent/human cardiomyocytes. 3-ß-hydroxybutyrate (3HB) exposure induced v-ATPase disassembly and the entire cascade of events leading to contractile dysfunction and insulin resistance, similar to conditions of lipid oversupply. Acetoacetate addition did not induce v-ATPase dysfunction. The negative effects of 3HB could be prevented by addition of specific amino acids. Hence, in sedentary/prediabetic subjects ketone bodies should be used with caution because of possible aggravation of cardiac insulin resistance and further loss of cardiac function. When these latter maladaptive conditions would occur, specific amino acids could potentially be a treatment option.

Specific amino acid supplementation rescues the heart from lipid overload-induced insulin resistance and contractile dysfunction by targeting the endosomal mTOR-v-ATPase axis.

OBJECTIVE: The diabetic heart is characterized by extensive lipid accumulation which often leads to cardiac contractile dysfunction. The underlying mechanism involves a pivotal role for vacuolar-type H+-ATPase (v-ATPase, functioning as endosomal/lysosomal proton pump). Specifically, lipid oversupply to the heart causes disassembly of v-ATPase and endosomal deacidification. Endosomes are storage compartments for lipid transporter CD36. However, upon endosomal deacidification, CD36 is expelled to translocate to the sarcolemma, thereby inducing myocardial lipid accumulation, insulin resistance, and contractile dysfunction. Hence, the v-ATPase assembly may be a suitable target for ameliorating diabetic cardiomyopathy. Another function of v-ATPase involves the binding of anabolic master-regulator mTORC1 to endosomes, a prerequisite for the activation of mTORC1 by amino acids (AAs). We examined whether the relationship between v-ATPase and mTORC1 also operates reciprocally; specifically, whether AA induces v-ATPase reassembly in a mTORC1-dependent manner to prevent excess lipids from entering and damaging the heart. METHODS: Lipid overexposed rodent/human cardiomyocytes and high-fat diet-fed rats were treated with a specific cocktail of AAs (lysine/leucine/arginine). Then, v-ATPase assembly status/activity, cell surface CD36 content, myocellular lipid uptake/accumulation, insulin sensitivity, and contractile function were measured. To elucidate underlying mechanisms, specific gene knockdown was employed, followed by subcellular fractionation, and coimmunoprecipitation. RESULTS: In lipid-overexposed cardiomyocytes, lysine/leucine/arginine reinternalized CD36 to the endosomes, prevented/reversed lipid accumulation, preserved/restored insulin sensitivity, and contractile function. These beneficial AA actions required the mTORC1-v-ATPase axis, adaptor protein Ragulator, and endosomal/lysosomal AA transporter SLC38A9, indicating an endosome-centric inside-out AA sensing mechanism. In high-fat diet-fed rats, lysine/leucine/arginine had similar beneficial actions at the myocellular level as in vitro in lipid-overexposed cardiomyocytes and partially reversed cardiac hypertrophy. CONCLUSION: Specific AAs acting through v-ATPase reassembly reduce cardiac lipid uptake raising the possibility for treatment in situations of lipid overload and associated insulin resistance.

Augmenting Vacuolar H+-ATPase Function Prevents Cardiomyocytes from Lipid-Overload Induced Dysfunction.

The diabetic heart is characterized by a shift in substrate utilization from glucose to lipids, which may ultimately lead to contractile dysfunction. This substrate shift is facilitated by increased translocation of lipid transporter CD36 (SR-B2) from endosomes to the sarcolemma resulting in increased lipid uptake. We previously showed that endosomal retention of CD36 is dependent on the proper functioning of vacuolar H+-ATPase (v-ATPase). Excess lipids trigger CD36 translocation through inhibition of v-ATPase function. Conversely, in yeast, glucose availability is known to enhance v-ATPase function, allowing us to hypothesize that glucose availability, via v-ATPase, may internalize CD36 and restore contractile function in lipid-overloaded cardiomyocytes. Increased glucose availability was achieved through (a) high glucose (25 mM) addition to the culture medium or (b) adenoviral overexpression of protein kinase-D1 (a kinase mediating GLUT4 translocation). In HL-1 cardiomyocytes, adult rat and human cardiomyocytes cultured under high-lipid conditions, each treatment stimulated v-ATPase re-assembly, endosomal acidification, endosomal CD36 retention and prevented myocellular lipid accumulation. Additionally, these treatments preserved insulin-stimulated GLUT4 translocation and glucose uptake as well as contractile force. The present findings reveal v-ATPase functions as a key regulator of cardiomyocyte substrate preference and as a novel potential treatment approach for the diabetic heart.

A new set of reference housekeeping genes for the normalization RT-qPCR data from the intestine of piglets during weaning.

The intestinal mucosal development of piglets (Sus scrofa) during the weaning stage is important to their disease susceptibility and later growth. Quantitative real-time PCR (RT-qPCR) is commonly used to screen for differentially expressed genes and, for accurate results, proper reference housekeeping genes are essential. Here we assessed the mRNA expression of 18 well-known candidate reference genes at different parts of the gastrointestinal tract (GIT) of piglets during the weaning process by RT-qPCR assay. GeNorm analysis revealed that B2M/HMBS/HPRT1 were the three most stable reference genes and GAPDH was the least stable gene in the duodenum, jejunum, ileum, colon, and whole GIT. BestKeeper analysis found that B2M/HMBS/PGK11, HMBS/B2M/HPRT1, B2M/HMBS/HSPCB, B2M/HPRT1/HMBS, and B2M/HMBS/HPRT1 were the most stable genes in the duodenum, jejunum, ileum, colon, and whole GIT, respectively, whereas GAPDH, B-actin, and 18S rRNA were the least stable genes at different parts of the GIT. To confirm the crucial role of appropriate housekeeping genes in obtaining reliable results, we analyzed the expression of ALP using each of the 18 reference genes to normalize the RT-qPCR data. We found that the expression levels of ALP normalized using the most stable reference genes (B2M/HMBS/HPRT1) differed greatly from the expression levels obtained when the data were normalized using the least stable genes (GAPDH, B-actin, and 18S). We concluded that B2M/HMBS/HPRT1 were the optimal reference genes for gene expression analysis by RT-qPCR in the intestinal mucosal development stages of piglets at weaning. Our findings provide a set of porcine housekeeping reference genes for studies of mRNA expression in different parts of the pig intestine.

Acute and Chronic Effects of Protein Kinase-D Signaling on Cardiac Energy Metabolism.

Protein kinase-D (PKD) is increasingly recognized as a key regulatory signaling hub in cardiac glucose uptake and also a major player in the development of hypertrophy. Glucose is one of the predominant energy substrates for the heart to support contraction. However, a cardiac substrate switch toward glucose over-usage is associated with the development of cardiac hypertrophy. Hence, regulation of PKD activity must be strictly coordinated. This review provides mechanistic insights into the acute and chronic regulatory functions of PKD signaling in the healthy and hypertrophied heart. First an overview of the activation pathways of PKD1, the most abundant isoform in the heart, is provided. Then the various regulatory roles of the PKD isoforms in the heart in relation to cardiac glucose and fatty acid metabolism, contraction, morphology, function, and the development of cardiac hypertrophy are described. Finally, these findings are integrated and the possibility of targeting this kinase as a novel strategy to combat cardiac diseases is discussed.

Microbial-Driven Butyrate Regulates Jejunal Homeostasis in Piglets During the Weaning Stage.

Microbe-derived butyrate plays an important role in the gut health of young mammals during the weaning stage. A greater understanding of how butyrate regulates intestinal development is necessary for overcoming post-weaning diarrheal diseases. We aimed to investigate whether jejunal microbial metabolite butyrate modulates the apoptosis/proliferation balance and immune response in piglets during the post-weaning period of the first 3 weeks of life. On the one hand, during the first week post-weaning, the relative abundances of the dominant bacterial families Erysipelotrichaceae (P < 0.01) and Lachnospiraceae (P < 0.01) were increased, which induced decreases in both butyrate production (P < 0.05) and its receptor (G-protein coupled receptor 43) expression (P < 0.01). The resulting intestinal inflammation (inferred from increased TNF-α and IFN-γ expression) contributed to the onset of cell apoptosis and the inhibition of cell-proliferation along the crypt-villus axis, which were followed by impaired jejunal morphology (i.e., increased crypt-depth) (P < 0.05) and intestinal dysfunction (i.e., decreased creatine kinase, and lactate dehydrogenase) (P < 0.05). On the other hand, during the second week post-weaning, the relative abundances of Lactobacillaceae (P < 0.01) and Ruminococcaceae (P < 0.05) were increased. The increases were accompanied by increased butyrate production (P < 0.05) and its receptor expression (P < 0.01), leading to the inhibition of cell apoptosis and the stimulation of cell proliferation via decreased pro-inflammatory cytokines and thereby the improvement of intestinal development and function. Herein, this study demonstrates that microbial-driven butyrate might be a key modulator in the maintenance of intestinal homeostasis after weaning. The findings suggest that strategies to promote butyrate production can maintain the apoptosis/proliferation balance via minimizing intestinal inflammation, and thereby improving post-weaning jejunal adaptation toward gut health.

Molecular mechanisms underlying inhibition of STIM1-Orai1-mediated Ca2+ entry induced by 2-aminoethoxydiphenyl borate.

Store-operated Ca2+ entry (SOCE) mediated by STIM1 and Orai1 is crucial for Ca2+ signaling and homeostasis in most cell types. 2-Aminoethoxydiphenyl borate (2-APB) is a well-described SOCE inhibitor, but its mechanisms of action remain largely elusive. Here, we show that 2-APB does not affect the dimeric state of STIM1, but enhances the intramolecular coupling between the coiled-coil 1 (CC1) and STIM-Orai-activating region (SOAR) of STIM1, with subsequent reduction in the formation of STIM1 puncta in the absence of Orai1 overexpression. 2-APB also inhibits Orai1 channels, directly inhibiting Ca2+ entry through the constitutively active, STIM1-independent Orai1 mutants, Orai1-P245T and Orai1-V102A. When unbound from STIM1, the constitutively active Orai1-V102C mutant is not inhibited by 2-APB. Thus, we used Orai1-V012C as a tool to examine whether 2-APB can also inhibit the coupling between STIM1 and Orai1. We reveal that the functional coupling between STIM1 and Orai1-V102C is inhibited by 2-APB. This inhibition on coupling is indirect, arising from 2-APB's action on STIM1, and it is most likely mediated by functional channel residues in the Orai1 N-terminus. Overall, our findings on this two-site inhibition mediated by 2-APB provide new understanding on Orai1-activation by STIM1, important to future drug design.

Proteomic mapping of ER-PM junctions identifies STIMATE as a regulator of Ca²âº influx.

Specialized junctional sites that connect the plasma membrane (PM) and endoplasmic reticulum (ER) play critical roles in controlling lipid metabolism and Ca(2+) signalling. Store-operated Ca(2+) entry mediated by dynamic STIM1-ORAI1 coupling represents a classical molecular event occurring at ER-PM junctions, but the protein composition and how previously unrecognized protein regulators facilitate this process remain ill-defined. Using a combination of spatially restricted biotin labelling in situ coupled with mass spectrometry and a secondary screen based on bimolecular fluorescence complementation, we mapped the proteome of intact ER-PM junctions in living cells without disrupting their architectural integrity. Our approaches led to the discovery of an ER-resident multi-transmembrane protein that we call STIMATE (STIM-activating enhancer, encoded by TMEM110) as a positive regulator of Ca(2+) influx in vertebrates. STIMATE physically interacts with STIM1 to promote STIM1 conformational switch. Genetic depletion of STIMATE substantially reduces STIM1 puncta formation at ER-PM junctions and suppresses the Ca(2+)-NFAT signalling. Our findings enable further genetic studies to elucidate the function of STIMATE in normal physiology and disease, and set the stage to uncover more uncharted functions of hitherto underexplored ER-PM junctions.

Inside-out Ca(2+) signalling prompted by STIM1 conformational switch.

Store-operated Ca(2+) entry mediated by STIM1 and ORAI1 constitutes one of the major Ca(2+) entry routes in mammalian cells. The molecular choreography of STIM1-ORAI1 coupling is initiated by endoplasmic reticulum (ER) Ca(2+) store depletion with subsequent oligomerization of the STIM1 ER-luminal domain, followed by its redistribution towards the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca(2+) signalling were largely undefined. By taking advantage of a unique gain-of-function mutation within the STIM1 transmembrane domain (STIM1-TM), here we show that local rearrangement, rather than alteration in the oligomeric state of STIM1-TM, prompts conformational changes in the cytosolic juxtamembrane coiled-coil region. Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition. On the basis of these findings, we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.