Major Regulator Of Energy Metabolism Research Articles

Lactate Release By Bone Marrow Neutrophils Promotes Their Inflammatory Mobilization Via Endothelial GPR81 Signaling

Neutrophils are best known as glycolytic cells and an essential first host defense line of the innate immune system during inflammation. Activation of pro-inflammatory signaling by immune cells accelerates metabolic pathways, leading to increased lactate production. While lactate levels are the best clinical marker for sepsis diagnosis, the mechanisms underlying lactate production by bone marrow (BM) neutrophils and its role during inflammation remain poorly understood. Here, we report that LPS exposure in mice (mimicking bacterial inflammation) activated BM neutrophils leading to their mobilization to peripheral blood (PB) and recruitment to the liver. This happened concomitantly with an increase in BM neutrophil metabolism, reflected by high glucose uptake, high generation of reactive oxygen species (ROS) and increase in HIF-1α expression. Importantly, we identified that BM neutrophils highly express lactate dehydrogenase A (LDHA), an enzyme that converts pyruvate to lactate, as well as the lactate efflux transporter MCT4. On the other hand BM neutrophils express low levels of the lactate influx transporter MCT1 and the lactate receptor GPR81. In addition, we found that BM neutrophil depletion resulted in lower levels of BM derived lactate in mice exposed to LPS. Moreover, LPS administration up-regulated the expression of LDHA and MCT4 on BM neutrophils. Taken together, these results reveal that during acute LPS induced inflammation BM neutrophils produce and release high levels of lactate. Unexpectedly, we found that in vivo lactate treatment specifically induced rapid ROShigh neutrophil activation and mobilization from the BM to PB and liver in an NADPH oxidase/ROS dependent manner. Accordingly, reduced activated neutrophil levels in PB and liver were observed following inhibition of ROS production. Furthermore, in a genetic mouse model of Chronic Granulomatous Disease (CGD) due to NADPH oxidase inactivation, with reduced ROS generation, treatment with lactate partially reduced neutrophil mobilization from the BM. Our results demonstrate that NADPH oxidase/ROS signaling play a role in lactate-induced neutrophil mobilization. HIF-1α is a major regulator of energy metabolism and glycolysis pathway in immune cells (Reviewed by Weidemann and Johnson, Cell Death and Diff. 2008, and by O'Neill and Pearce, J. Exp. Med. 2015). Notably, our results show in myeloid specific HIF-1α-deficient mice exposed to LPS a reduction in the expression of LDHA and MCT4 on BM neutrophils compared to wild type mice, suggesting that HIF-1α is upstream of lactate. Accordingly, exogenous lactate administration restored neutrophil mobilization in myeloid HIF-1α-deficient mice implicating HIF-1α in lactate production and release by BM neutrophils and in their mobilization from the BM following acute inflammation.Further deciphering the mechanisms of lactate-induced neutrophil mobilization, we identified that BM endothelial cells (BMEC) highly express the lactate receptor GPR81. This GPCR was downregulated and internalized by lactate, as well as GPR81 agonist or LPS treatment. Interestingly, we found that lactate or GPR81 agonist reduced BMEC VE-Cadherin expression, leading to increased blood-BM vascular permeability as a possible cause for the enhanced neutrophil mobilization from the BM into the circulation. Lactate effects on BM neutrophils required functional GPR81 signaling since lactate injection together with a GPR81 antagonist, or to GPR81-deficient mice interfered with BM vascular permeability and reduced neutrophil mobilization from the BM. Moreover, in chimeras of GPR81-deficient BM reconstituted in wild type mice recipients we found that lactate induced-neutrophil mobilization is dependent on host BMEC GPR81 signaling rather than hematopoietic cell GPR81. This finding eludes to a role of endothelial GPR81 rather than to neutrophil GPR81 in lactate induced-neutrophil mobilization. In summary, our study links, for the first time, the metabolic by-product lactate secreted by BM neutrophils exposed to inflammatory conditions, to a novel GPR81 signaling axis on the BM endothelial cells which controls vascular permeability and thereby neutrophil mobilization. Our study highlights lactate as a potential target for attenuating blood neutrophilia and neutrophil injury associated with sepsis and other infectious conditions. DisclosuresNo relevant conflicts of interest to declare.

Adipose vascular endothelial growth factor B is a major regulator of energy metabolism.

Excessive fat accumulation causes obesity and many diseases. Previous study demonstrates VEGFB universal knockout induces obese phenotypes including expansion of white adipose tissue, whitening of brown adipose tissue, increase of fat accumulation and reduction in energy consumption. However, roles of VEGFB in adipose tissues are not clear. In this study, we have generated a mouse model with adipose-specific VEGFB repression using CRISPR/dCas9 system (Vegfb AdipoDown ) and investigated the roles of VEGFB in adipose development and energy metabolism. VEGFB repression induced significant changes in adipose tissue structure and function. Vegfb AdipoDown mice have larger body sizes, larger volume of white adipose tissues than its wild type littermates. Adipose-specific VEGFB repression induced morphological and functional transformation of adipose tissues toward white adipose for energy storage. Metabolic processes are broadly changed in Vegfb AdipoDown adipose tissues including carbohydrate metabolism, lipid metabolism, nucleotide metabolism and amino acid metabolism. We have demonstrated that adipose VEGFB repression can recapitulate most of the phenotypes of the whole body VEGFB knockout mouse. Intriguingly, approximately 50% VEGFB repression in adipose tissues can almost completely mimic the effects of universal Vegfb deletion, suggesting adipose VEGFB is a major regulator of energy metabolism and may be important in prevention and treatment of obesity.

SHBG141\u2013161 Domain-Peptide Stimulates GPRC6A-Mediated Response in Leydig and \u03b2-Langerhans cell lines

GPRC6A is acknowledged as a major regulator of energy metabolism and male fertility through the action of undercarboxylated osteocalcin (ucOCN), representing a possible therapeutic target. We recently showed that the sex hormone-binding globulin (SHBG) binds to GPRC6A through the likely involvement of the 141–161 domain. To confirm this model, here we investigated the possible binding and agonist activity of SHBG(141–161) domain-peptide (SHBG141–161) on GPRC6A. The binding of SHBG141–161 to GPRC6A and downstream dissociation from Gαi(GDP) protein was computationally modelled. SHBG141–161 was obtained by solid-phase synthesis, characterized by circular dichroism (CD) and the receptor binding was assessed by displacement of ucOCN on HEK-293 cells transfected with GPRC6A gene. Agonist activity of SHBG141–161 was assessed on Leydig MA-10 and Langerhans β-TC6 cell lines through the GPRC6A-mediated release of testosterone (T) and insulin. SHBG141–161 was predicted to bind to GPRC6A and to reduce the affinity for Gαi(GDP) at computational level. Conformational properties and binding to GPRC6A of the synthetic SHBG141–161 were confirmed by CD and displacement experiments. SHBG141–161 stimulated cell secretion of T and insulin, with dose dependency from 10−13 to 10−11M for T release (respectively P = 0,041 10−13M; P = 0,032 10−12M; P = 0,008 10−11M vs basal) and for 10−12 to 10−10M for insulin (respectively P = 0,041 10−12M; P = 0,007 10−11M; P = 0,047 10−10M; P = 0,045 vs basal). Blockade with anti GPRC6A IgG abolished the response to SHBG141-161, suggesting agonist specificity. SHBG141–161 showed stimulating activity on GPRC6A, representing a template peptide with possible therapeutic use for metabolic and endocrine disorders.

Nucleosides block AICAR-stimulated activation of AMPK in skeletal muscle and cancer cells.

AMP-activated kinase (AMPK) is a major regulator of energy metabolism and a promising target for development of new treatments for type 2 diabetes and cancer. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), an adenosine analog, is a standard positive control for AMPK activation in cell-based assays. Some broadly used cell culture media, such as minimal essential medium α (MEMα), contain high concentrations of adenosine and other nucleosides. We determined whether such media alter AICAR action in skeletal muscle and cancer cells. In nucleoside-free media, AICAR stimulated AMPK activation, increased glucose uptake, and suppressed cell proliferation. Conversely, these effects were blunted or completely blocked in MEMα that contains nucleosides. Addition of adenosine or 2'-deoxyadenosine to nucleoside-free media also suppressed AICAR action. MEMα with nucleosides blocked AICAR-stimulated AMPK activation even in the presence of methotrexate, which normally markedly enhances AICAR action by reducing its intracellular clearance. Other common media components, such as vitamin B-12, vitamin C, and α-lipoic acid, had a minor modulatory effect on AICAR action. Our findings show that nucleoside-containing media, commonly used in AMPK research, block action of the most widely used pharmacological AMPK activator AICAR. Results of cell-based assays in which AICAR is used for AMPK activation therefore critically depend on media formulation. Furthermore, our findings highlight a role for extracellular nucleosides and nucleoside transporters in regulation of AMPK activation.

Divergent Role of Estrogen-Related Receptor α in Lipid- and Fasting-Induced Hepatic Steatosis in Mice.

Given the increasing prevalence of obesity and the metabolic syndrome, identification of intrinsic molecular programs responsible for ensuring fuel homeostasis and preventing metabolic disease is needed. We investigated whether the orphan nuclear receptor estrogen-related receptor α (ERRα), a major regulator of energy metabolism, plays a role in lipid homeostasis and the development of nonalcoholic fatty liver disease (NAFLD) in response to chronic high-fat diet (HFD) consumption and long-term fasting. Systemic ablation of ERRα in mice demonstrated clear beneficial effects for loss of ERRα function in protection from HFD-provoked body weight gain manifested not only from a reduction in white adipose tissue stores but also from an impediment in intrahepatic lipid accumulation. The prevention of HFD-induced NAFLD in ERRα-null mice was underscored by transcriptional repression of de novo lipogenesis, which was upregulated in wild-type mice, a known contributing factor to lipid-stimulated hepatic steatosis. Surprisingly, given these findings, ERRα deficiency had no significant impact on the degree of fasting-induced NAFLD, involving the mobilization of adipocyte triglyceride (TG) stores into the liver. However, the presence of ERRα was essential for acute refeeding-mediated reversal of fasting-induced hepatic TG accretion, underpinned by impaired downregulation of adipose TG lipolysis and reduced hepatic mitochondrial oxidative activity. Taken together, the regulation of lipid handling by ERRα depended on the nutritional state, suggesting that negative modulation of ERRα activity could be envisaged to prevent lipid-induced NAFLD, whereas inducing its activity would be useful to treat and reverse the instilled disease.

Apoptotic effects of extract from Cnidium monnieri (L.) Cusson by adenosine monosphosphate-activated protein kinase-independent pathway in HCT116 colon cancer cells.

Colon cancer, a common malignancy, can occur due to poor eating habits and increasing age. Consequently, careful regulation of eating habits may serve as a possible method for preventing the occurrence or progression of colon cancer. Extracts of the fruit of Cnidium monnieri (L.) Cusson are well‑known as an effective herbal medicine for the treatment of pain in female genitalia and carbuncle. However, there have been no studies on the apoptotic effects of Cnidium monnieri (L.) Cusson (CME). Adenosine monophosphate‑activated protein kinase (AMPK), the major regulator of energy metabolism, is activated by metabolic stress, including hypoxia and glucose deprivation. Activation of AMPK inhibits cell proliferation and induces apoptosis through the inhibition of phosphorylated (p)‑Akt and control of B‑cell lymphoma2 (Bcl‑2) family members. The pro‑apoptotic proteins Bcl‑2‑associatedX protein (Bax) and Bcl‑2‑homologous antagonist killer (Bak), are activated by their translocation to mitochondria from the cytosol. Translocation of Bax/Bak induces outer membrane permeabilization and is likely to lead to apoptosis through cytochromeC release and caspase activity. In the present study, the apoptotic effects and influence on mitochondria‑mediated apoptotic proteins of CME in HCT116 cells were assessed. We hypothesized that CME may have an effect on the inhibition of p‑Akt in an AMPK‑independent pathway. The present study demonstrated that CME induced the release of LDH and apoptosis through its inhibition of p‑Akt to control Bcl‑2 and activate Bax and Bak. Co‑treatment with CME and AMPK inhibitors showed that CME‑induced apoptosis does not occurr through a AMPK‑dependent pathway. Therefore, the present study determined, for the first time, that CME induced apoptosis as a result of causing metabolic stresses due to directly regulation of the de‑phosphorylation of Akt, whereas it did not control the AMPK-dependent pathway in HCT116 colon cancer cells.

Characterizing the interplay between multiple levels of organization within bacterial sigma factor regulatory networks

Bacteria contain multiple sigma factors, each targeting diverse, but often overlapping sets of promoters, thereby forming a complex network. The layout and deployment of such a sigma factor network directly impacts global transcriptional regulation and ultimately dictates the phenotype. Here we integrate multi-omic data sets to determine the topology, the operational, and functional states of the sigma factor network in Geobacter sulfurreducens, revealing a unique network topology of interacting sigma factors. Analysis of the operational state of the sigma factor network shows a highly modular structure with σ(N) being the major regulator of energy metabolism. Surprisingly, the functional state of the network during the two most divergent growth conditions is nearly static, with sigma factor binding profiles almost invariant to environmental stimuli. This first comprehensive elucidation of the interplay between different levels of the sigma factor network organization is fundamental to characterize transcriptional regulatory mechanisms in bacteria.

Ginsenoside Rg3 Reduces Lipid Accumulation with AMP-Activated Protein Kinase (AMPK) Activation in HepG2 Cells

Cardiovascular disease (CVD) is one of the main causes of mortality worldwide, and dyslipidemia is a major risk factor for CVD. Ginseng has been widely used in the clinic to treat CVD. Ginsenoside Rg3, one of the major active components of ginseng, has been reported to exhibit antiobesity, antidiabetic, and cardioprotective effects. However, the effect of ginsenoside Rg3 on hepatic lipid metabolism remains unclear. Therefore, we investigated whether ginsenoside Rg3 would regulate hepatic lipid metabolism with AMP-activated protein kinase (AMPK) activation in HepG2 cells. Ginsenoside Rg3 significantly reduced hepatic cholesterol and triglyceride levels. Furthermore, ginsenoside Rg3 inhibited expression of sterol regulatory element binding protein-2 (SREBP-2) and 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGCR). Ginsenoside Rg3 increased activity of AMPK, a major regulator of energy metabolism. These results suggest that ginsenoside Rg3 reduces hepatic lipid accumulation with inhibition of SREBP-2 and HMGCR expression and stimulation of AMPK activity in HepG2 cells. Therefore, ginsenoside Rg3 may be beneficial as a food ingredient to lower the risk of CVD by regulating dyslipidemia.

Activation of Peroxisome Proliferator–Activated Receptor β/δ Induces Lung Cancer Growth via Peroxisome Proliferator–Activated Receptor Coactivator γ-1α

We previously demonstrated that a selective agonist of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta), GW501516, stimulated human non-small cell lung carcinoma (NSCLC) growth, partly through inhibition of phosphatase and tensin homolog deleted on chromosome 10 expression. Here, we show that GW501516 also decreases the phosphorylation of AMP-activated protein kinase alpha (AMPKalpha), a major regulator of energy metabolism. This was mediated through specific activation of PPARbeta/delta, as a PPARbeta/delta small interfering RNA inhibited the effect. However, AMPKalpha did not mediate the growth-promoting effects of GW501516, as silencing of AMPKalpha did not inhibit GW501516-induced cell proliferation. Instead, we found that GW501516 stimulated peroxisome proliferator-activated receptor coactivator gamma (PGC)-1alpha, which activated the phosphatidylinositol 3 kinase (PI3-K)/Akt mitogenic pathway. An inhibitor of PI3-K, LY294002, had no effect on PGC-1alpha, consistent with PGC-1alpha being upstream of PI3-K/Akt. Of note, an activator of AMPKalpha, 5-amino-4-imidazole carboxamide riboside, inhibited the growth-promoting effects of GW501516, suggesting that although AMPKalpha is not responsible for the mitogenic effects of GW501516, its activation can oppose these events. This study unveils a novel mechanism by which GW501516 and activation of PPARbeta/delta stimulate human lung carcinoma cell proliferation, and suggests that activation of AMPKalpha may oppose this effect.

Adenosine 5′-Monophosphate-Activated Protein Kinase Regulates Progesterone Secretion in Rat Granulosa Cells

The AMP-activated protein kinase (AMPK) is a major regulator of energy metabolism involved in fatty acid and cholesterol synthesis. In the ovary, cholesterol plays a key role in steroid production. We report the presence of AMPK in rat ovaries, and we have investigated its role in granulosa cells. We show using RT-PCR and Western blot that the mRNAs for the alpha1/2 and beta1/2 subunits and the proteins are found in the ovaries. Immunohistochemistry localized the alpha1 AMPK subunit in granulosa cells, corpus luteum, and oocyte and less abundantly in theca cells. Treatment with 1 mm 5-amino-imidazole-4-carboxyamide-1-beta-D-ribofuranoside (AICAR), an activator of AMPK, increased dose-dependent and time-dependent phosphorylation of AMPKalpha1 on Thr172 in primary granulosa cells. Simultaneously, phosphorylation of acetyl-coenzyme A carboxylase at Ser79 was also increased. AICAR treatment for 48 h halved progesterone secretion, 3beta-HSD protein and mRNA levels, and phosphorylation of both basal MAPK ERK1/2 and p38 and in response to IGF-I and/or FSH in granulosa cells. AICAR treatment (1 mM) had no detectable effect on basal and FSH- and/or IGF-I-induced estradiol production and on granulosa cell proliferation or viability. Adenovirus-mediated expression of dominant negative AMPK totally abolished the effects of AICAR on progesterone secretion, 3beta-HSD protein production, and MAPK ERK1/2 and p38 phosphorylation. Moreover, we showed using specific in- hibitors of ERK1/2 and p38 MAPK that the MAPK ERK1/2 and not p38 is involved in progesterone secretion and 3beta-HSD expression, strongly suggesting that the activation of AMPK in response to AICAR reduces progesterone production through the MAPK ERK1/2 signaling pathway in rat granulosa cells.

Reversal of behavioral and metabolic abnormalities, and insulin resistance syndrome, by dietary restriction in mice deficient in brain-derived neurotrophic factor.

Dietary restriction (DR) extends life span and improves glucose metabolism in mammals. Recent studies have shown that DR stimulates the production of brain-derived neurotrophic factor (BDNF) in brain cells, which may mediate neuroprotective and neurogenic actions of DR. Other studies have suggested a role for central BDNF signaling in the regulation of glucose metabolism and body weight. BDNF heterozygous knockout (BDNF+/-) mice are obese and exhibit features of insulin resistance. We now report that an intermittent fasting DR regimen reverses several abnormal phenotypes of BDNF(+/-) mice including obesity, hyperphagia, and increased locomotor activity. DR increases BDNF levels in the brains of BDNF(+/-) mice to the level of wild-type mice fed ad libitum. BDNF(+/-) mice exhibit an insulin-resistance syndrome phenotype characterized by elevated levels of circulating glucose, insulin, and leptin; DR reduces levels of each of these three factors. DR normalizes blood glucose responses in glucose tolerance and insulin tolerance tests in the BDNF(+/-) mice. These findings suggest that BDNF is a major regulator of energy metabolism and that beneficial effects of DR on glucose metabolism are mediated, in part, by BDNF signaling. Dietary and pharmacological manipulations of BDNF signaling may prove useful in the prevention and treatment of obesity and insulin resistance syndrome-related diseases.

Thyroid hormone regulates the obesity gene tub.

Thyroid hormone T3/T4 is a major regulator of energy metabolism in vertebrates, and defects in thyroid status are frequently associated with changes in body weight. It is demonstrated here that thyroid hormone regulates in vivo and in vitro the tub gene, which when mutated in tubby mice causes obesity, insulin resistance and sensory deficits. Hypothyroidism in rats altered tub mRNA and protein in discrete brain areas. These changes could be attributed to thyroid hormone deficiency since T3/T4 treatment restored normal tub expression. T3 also upregulated tub mRNA within 4-6 h in neuronal cells in culture, suggesting that T3 is a positive regulator of tub gene expression. Thus, these results establish a novel pathway of T3 action and provide an important molecular link between thyroid status and the tubby-associated syndrome.