Glucose Uptake In Cells Research Articles

Role of G protein-coupled receptor kinases (GRKs) in β2 -adrenoceptor-mediated glucose uptake.

Truncation of the C-terminal tail of the β2 -AR, transfection of βARKct or over-expression of a kinase-dead GRK mutant reduces isoprenaline-stimulated glucose uptake, indicating that GRK is important for this response. We explored whether phosphorylation of the β2 -AR by GRK2 has a role in glucose uptake or if this response is related to the role of GRK2 as a scaffolding protein. CHO-GLUT4myc cells expressing wild-type and mutant β2 -ARs were generated and receptor affinity for [3 H]-CGP12177A and density of binding sites determined together with the affinity of isoprenaline and BRL37344. Following receptor activation by β2 -AR agonists, cAMP accumulation, GLUT4 translocation, [3 H]-2-deoxyglucose uptake, and β2 -AR internalization were measured. Bioluminescence resonance energy transfer was used to investigate interactions between β2 -AR and β-arrestin2 or between β2 -AR and GRK2. Glucose uptake after siRNA knockdown or GRK inhibitors was measured in response to β2 -AR agonists. BRL37344 was a poor partial agonist for cAMP generation but displayed similar potency and efficacy to isoprenaline for glucose uptake and GLUT4 translocation. These responses to β2 -AR agonists occurred in CHO-GLUT4myc cells expressing β2 -ARs lacking GRK or GRK/PKA phosphorylation sites as well as in cells expressing the wild-type β2 -AR. However, β2 -ARs lacking phosphorylation sites failed to recruit β-arrestin2 and did not internalize. GRK2 knock-down or GRK2 inhibitors decreased isoprenaline-stimulated glucose uptake in rat L6 skeletal muscle cells. Thus, GRK phosphorylation of the β2 -AR is not associated with isoprenaline- or BRL37344-stimulated glucose uptake. However, GRKs acting as scaffold proteins are important for glucose uptake as GRK2 knock-down or GRK2 inhibition reduces isoprenaline-stimulated glucose uptake.

NT5DC2 knockdown suppresses progression, glycolysis, and neuropathic pain in triple-negative breast cancer by blocking the EGFR pathway.

Triple-negative breast cancer (TNBC) is an exceptionally aggressive breast cancer subtype associated with neuropathic pain. This study explores the effects of 5'-nucleotidase domain-containing protein 2 (NT5DC2) on the progression of TNBC and neuropathic pain. Microarray analysis was conducted to identify differentially expressed genes in TNBC and the pathways involved. Gain- and loss-of-function assays of NT5DC2 were performed in TNBC cells, followed by detection of the extracellular acidification rate, adenosine triphosphate (ATP) levels, lactic acid production, glucose uptake, proliferation, migration, and invasion in TNBC cells. Macrophages were co-cultured with TNBC cells to examine the release of polarization-related factors and cytokines. A xenograft tumor model was established for in vivo validation. In addition, a mouse model of neuropathic pain was established through subepineural injection of TNBC cells, followed by measurement of the sciatic functional index and behavioral analysis to assess neuropathic pain. NT5DC2 was upregulated in TNBC and was positively correlated with epidermal growth factor receptor (EGFR). NT5DC2 interacted with EGFR to promote downstream signal transduction in TNBC cells. NT5DC2 knockdown diminished proliferation, migration, invasion, the extracellular acidification rate, ATP levels, lactic acid production, and glucose uptake in TNBC cells. Co-culture with NT5DC2-knockdown TNBC cells alleviated the M2 polarization of macrophages. Furthermore, NT5DC2 knockdown reduced tumor growth and neuropathic pain in mice. Importantly, activation of the EGFR pathway counteracted the effects of NT5DC2 knockdown. NT5DC2 knockdown protected against TNBC progression and neuropathic pain by inactivating the EGFR pathway.

Abstract IA005: PET imaging predictors of response to chemoradiotherapy in cervical cancer

Abstract Although HPV transformed tumors are presumed to be radiation sensitive, approximately one third of locally advanced cervical cancer patients fail the current standard of care (concurrent cisplatin and radiation therapy). Work in our laboratory has combined the results of functional imaging with 18F-fluoro-deoxy-glucose positron emission tomography (FDG-PET) imaging together with bioinformatic analyses of associated translational correlates to define new predictors of resistance to DNA damaging therapies. Results of this work demonstrate that cervical tumors with increased FDG uptake on pretreatment FDG-PET imaging are characterized by myeloid predominant inflammatory infiltrates, including immature monocytes and macrophages. Co-culture of cervical tumor cells with macrophages is associated with increased glucose uptake and pro-survival signaling in tumor cells mediated through macrophage derived IL-6 and signaling through the JAK-STAT pathway. We are currently using a combination of single cell sequencing and spatially resolved approaches to further define PET associated and chemoradiation induced changes in cervix tumor cell metabolism and the immune tumor microenvironment with the goal of defining new targets for personalized therapies. Importantly, cervical cancers retain and transcribe portions of the HPV genome. At the end of the talk, we will review recent work in our lab that highlights the impact of HPV associated gene expression, including splice variants, on the response to DNA damaging agents. Citation Format: Julie K. Schwarz. PET imaging predictors of response to chemoradiotherapy in cervical cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA005.

Dual inhibitory potential of ganoderic acid A on GLUT1/3: computational and in vitro insights into targeting glucose metabolism in human lung cancer.

Human glucose transporters (GLUTs) facilitate the uptake of hexoses into cells. In cancer, the increased proliferation necessitates higher expression of GLUTs, with particular emphasis on GLUT1 and GLUT3. Thus, inhibiting GLUTs holds promise as an anticancer therapy by starving these cells of fuel. Ganoderic acid A (GAA), a triterpene found in Ganoderma lucidum, has anticancer and antidiabetic properties. Recent studies show that GAA reduces glucose uptake in cancer cells, which indicates that GAA may affect GLUT1/GLUT3 by inhibiting glucose uptake. Therefore, this study aimed to inspect whether GAA could target GLUT1/GLUT3 and play an inhibitory role in changing their endofacial and exofacial conformations. To this end, AlphaFold2 was employed to model the endofacial and exofacial conformations of GLUT3 and GLUT1, respectively. Molecular docking, molecular dynamics simulation, cell viability, cellular thermal shift assays (CETSA), glucose uptake, qPCR, and western blotting were harnessed. In comparison to the endofacial (cytochalasin B) and exofacial (phloretin) GLUT1/3 inhibitors, the computational findings unveiled GAA's capacity to bind and stabilize GLUT1/3 in their two conformational states, with a preference for binding the endofacial conformation. A low, non-cytotoxic dose of GAA thermally stabilized both transporters and inhibited glucose uptake in human lung cancer cells, similar to cytochalasin B and phloretin. In conclusion, this study has unearthed novel functionalities of GAA, suggesting its potential utility in cancer therapy by targeting glucose metabolism.

BRIX1 promotes ribosome synthesis and enhances glycolysis by selected translation of GLUT1 in colorectal cancer.

Ribosome biogenesis protein BRX1 homolog (BRIX1) is critically required for the synthesis of the 60S ribosome subunit. However, the role and mechanism of BRIX1 in colorectal cancer (CRC) remain unclear. Kyoto Encyclopedia of Gene and Genome pathway and Gene Ontology analyses were used for bioinformatics analysis. The rRNA levels were detected in CRC tissues and cells. Nascent RNA synthesis was detected via cellular immunofluorescence. The correlation was analyzed between patient Positron Emission Tomography-Computed Tomography (PET-CT) values and their BRIX1 expression. The extracellular acidification rate (ECAR) and oxygen consumption rate were determined via live metabolic analyses. Polysome fractions were collected for BRIX1 mRNA used in translation. The orthotopic model and Cell Counting Kit-8 (CCK8) assay were used to assess BRIX1 function in CRC. BRIX1 is a core protein involved in ribosome-related pathway changes in CRC. Gene Ontology analysis showed that BRIX1 was primarily enriched in ribosome assembly and ribosome biogenesis pathways. In fresh CRC tissue, rRNA levels (5S, 5.8S, 18S and 28S) were higher in the BRIX1 high-expression group than in the BRIX1 low-expression group. Similarly, BRIX1 knockdown significantly decreased rRNA levels for 5S, 5.8S, 18S and 28S in CRC cells, whereas overexpression of BRIX1 significantly increased these levels. In addition, BRIX1 knockdown inhibited nascent RNA synthesis in CRC cells. In clinical data analysis, BRIX1 expression was related to the glucose uptake in PET-CT. BRIX1 knockdown significantly decreased the ECAR value, glucose uptake and lactic acid production in CRC cells, whereas BRIX1 overexpression significantly increased these. Furthermore, BRIX1 knockdown significantly decreased the protein expression of GLUT1, whereas BRIX1 overexpression significantly increased this; however, expression of BRIX1 mRNA was unaffected in either case. Blocking glycolysis by si-GLUT1 or galactose reversed BRIX1 promotion of glycolysis and cell proliferation in CRC cells.

Inhibition of α-glucosidase activity and intestinal glucose transport to assess the in vivo anti-hyperglycemic potential of dodecyl-acylated phlorizin and polydatin derivatives.

A diet containing natural active compounds that can inhibit the hydrolytic activity of α-glucosidase on carbohydrates and intestinal glucose absorption is an effective means of controlling postprandial hyperglycemia. Phlorizin and polydatin as phenolic glycosides have a high affinity for the catalytic site of α-glucosidase, but exhibited unsatisfactory competitive inhibitory capacity, with an IC50 of 0.97 and >2 mM, respectively. However, dodecyl-acylated derivatives of phlorizin and polydatin exerted α-glucosidase inhibitory capacity, with an IC50 of 55.10 and 70.95 μM, respectively, which were greatly enhanced and much stronger than that of acarbose with an IC50 of 2.46 mM. The SPR assay suggested the high affinity of dodecyl phlorizin and dodecyl polydatin to α-glucosidase with equilibrium dissociation constant (KD) values of 12.0 and 7.9 μM, respectively. Both dodecyl phlorizin and dodecyl polydatin reduced the catalytic ability of α-glucosidase by reversible noncompetitive and uncompetitive mixed inhibition, which bind noncovalently to the allosteric site 2 through hydrogen bonds and hydrophobic interactions, thereby inducing the secondary structure unfolding and intrinsic fluorescence quenching of α-glucosidase. Confocal microscopy detection visually showed significant inhibitory effects on FITC-labeled glucose uptake in intestinal Caco-2 cells by phlorizin, polydatin, dodecyl phlorizin and dodecyl polydatin. In addition, based on the differentiated Caco-2 cell monolayer model, dodecyl phlorizin and dodecyl polydatin suppressed intestinal glucose transport more effectively than phlorizin and polydatin, suggesting that they were promising in vivo hypoglycemic active compounds.

Glucolipotoxicity: A Novel Different Perspective on the Causes of Cancer

The Warburg effect, characterized by increased glucose uptake and lactate production in cancer cells even in the presence of oxygen, has long been recognized as a hallmark of cancer metabolism. This metabolic alteration provides cancer cells with a growth advantage, facilitating their rapid proliferation. The underlying mechanisms driving the Warburg effect involve dysregulated glucose metabolism, upregulation of glucose transporters, and metabolic reprogramming favoring glycolysis. The resulting accumulation of metabolic intermediates, such as lactate, contributes to the acidic tumor microenvironment, promoting tumor progression. However, a novel perspective proposed by Maher Akl suggests that dysregulated glycolipid metabolism, particularly the accumulation of glycolipids within cells, plays a pivotal role in tumor development. This glucolipotoxicity hypothesis offers a broader understanding of the primary causes of cancer, emphasizing the interference of accumulated glycolipids with cellular processes and the activation of oncogenic pathways. In this abstract, we summarize the mechanisms underlying the Warburg effect and glucolipotoxicity, highlighting their implications for tumor growth. Understanding these paradoxical conditions that activate tumor growth provides insights for the development of innovative therapeutic strategies targeting the primary cause of cancer.

The novel family of Warbicin® compounds inhibits glucose uptake both in yeast and human cells and restrains cancer cell proliferation.

Many cancer cells share with yeast a preference for fermentation over respiration, which is associated with overactive glucose uptake and breakdown, a phenomenon called the Warburg effect in cancer cells. The yeast tps1Δ mutant shows even more pronounced hyperactive glucose uptake and phosphorylation causing glycolysis to stall at GAPDH, initiation of apoptosis through overactivation of Ras and absence of growth on glucose. The goal of the present work was to use the yeast tps1Δ strain to screen for novel compounds that would preferentially inhibit overactive glucose influx into glycolysis, while maintaining basal glucose catabolism. This is based on the assumption that the overactive glucose catabolism of the tps1Δ strain might have a similar molecular cause as the Warburg effect in cancer cells. We have isolated Warbicin ® A as a compound restoring growth on glucose of the yeast tps1Δ mutant, showed that it inhibits the proliferation of cancer cells and isolated structural analogs by screening directly for cancer cell inhibition. The Warbicin ® compounds are the first drugs that inhibit glucose uptake by both yeast Hxt and mammalian GLUT carriers. Specific concentrations did not evoke any major toxicity in mice but increase the amount of adipose tissue likely due to reduced systemic glucose uptake. Surprisingly, Warbicin ® A inhibition of yeast sugar uptake depends on sugar phosphorylation, suggesting transport-associated phosphorylation as a target. In vivo and in vitro evidence confirms physical interaction between yeast Hxt7 and hexokinase. We suggest that reversible transport-associated phosphorylation by hexokinase controls the rate of glucose uptake through hydrolysis of the inhibitory ATP molecule in the cytosolic domain of glucose carriers and that in yeast tps1Δ cells and cancer cells reversibility is compromised, causing constitutively hyperactive glucose uptake and phosphorylation. Based on their chemical structure and properties, we suggest that Warbicin ® compounds replace the inhibitory ATP molecule in the cytosolic domain of the glucose carriers, preventing hexokinase to cause hyperactive glucose uptake and catabolism.

NGF effects promote the maturation of rat pancreatic beta cells by regulating GLUT2 levels and distribution, and glucokinase activity.

The nerve growth factor (NGF) participates in cell survival and glucose-stimulated insulin secretion (GSIS) processes in rat adult beta cells. GSIS is a complex process in which metabolic events and ionic channel activity are finely coupled. GLUT2 and glucokinase (GK) play central roles in GSIS by regulating the rate of the glycolytic pathway. The biphasic release of insulin upon glucose stimulation characterizes mature adult beta cells. On the other hand, beta cells obtained from neonatal, suckling, and weaning rats are considered immature because they secrete low levels of insulin and do not increase insulin secretion in response to high glucose. The weaning of rats (at postnatal day 20 in laboratory conditions) involves a dietary transition from maternal milk to standard chow. It is characterized by increased basal plasma glucose levels and insulin levels, which we consider physiological insulin resistance. On the other hand, we have observed that incubating rat beta cells with NGF increases GSIS by increasing calcium currents in neonatal cells. In this work, we studied the effects of NGF on the regulation of cellular distribution and activity of GLUT2 and GK to explore its potential role in the maturation of GSIS in beta cells from P20 rats. Pancreatic islet cells from both adult and P20 rats were isolated and incubated with 5.6 mM or 15.6 mM glucose with and without NGF for 4 hours. Specific immunofluorescence assays were conducted following the incubation period to detect insulin and GLUT2. Additionally, we measured glucose uptake, glucokinase activity, and insulin secretion assays at 5.6 mM or 15.6 mM glucose concentrations. We observed an age-dependent variation in the distribution of GLUT2 in pancreatic beta cells and found that glucose plays a regulatory role in GLUT2 distribution independently of age. Moreover, NGF increases GLUT2 abundance, glucose uptake, and GSIS in P20 beta cells and GK activity in adult beta cells. Our results suggest that besides increasing calcium currents, NGF regulates metabolic components of the GSIS, thereby contributing to the maturation process of pancreatic beta cells.

IL-37 inhibits glycolysis of lung adenocarcinoma by inhibiting the expression of PFKFB3.

Aerobic glycolysis is one of the hallmarks of cancer. The metabolic phenotype of tumor cells is characterized by preferential dependence on glycolysis under aerobic conditions. Recent researchers have provided a piece of information on the effectiveness of targeting glycolysis. Thus, targeted glucose metabolism therapy is still a research hotspot. Interleukin 37 (IL-37) plays an important role in tumor development. Previous studies have found that IL-37 can inhibit the progression of lung adenocarcinoma in a variety of ways. For example, IL-37 can inhibit the migration and invasion of lung adenocarcinoma by inhibiting the interleukin 6(IL-6)/ Signal transducing activator of transcription 3(STAT3) pathway. IL-37 inhibits tumor growth by regulating RNA methylation at the M6A site of lung adenocarcinoma. It has been found that overexpression of IL-37 in macrophages can reverse the Warburg effect. The mechanism of IL-37 on glucose metabolism of tumor cells has not been studied. In research, glucose uptake and lactic acid production were inhibited in A549 cells with recombinant human IL-37(rhIL-37). Also, rhIL-37 inhibited the expression level of PFKFB3 in A549 cells. To verify whether the two aspects of rhIL-37's effects on A549 cells are related, we applied PFK15, a specific inhibitor of PFKFB3, to prove that rhIL-37 inhibits the glucose uptake and lactate production of A549 cells by inhibiting the expression of PFKFB3, and further inhibits the progression of lung adenocarcinoma.

Developing a new multi-featured chitosan-quinoline Schiff base with potent antibacterial, antioxidant, and antidiabetic activities: design and molecular modeling simulation

A new chitosan Schiff base was developed via the reaction of chitosan (CH) with 2-chloro-3-formyl-7-ethoxy quinoline (Q) derivative. The alteration in the chemical structure and morphology of CHQ derivative was confirmed by 1H NMR, FT-IR spectroscopy and SEM analysis. The antibacterial activity was considerably promoted with increasing quinoline concentration up to 1 M with maximal inhibition reached 96 and 77% against Staphylococcus haemolyticus and Escherichia coli, respectively. Additionally, CHQ derivative afforded higher ABTS·+ radical scavenging activity reached 59% compared to 13% for native chitosan, approving its acceptable antioxidant activity. Moreover, the developed CHQ derivative can stimulate the glucose uptake in HepG-2 and yeast cells, while better inhibition of α-amylase and α-glucosidase was accomplished with maximum values of 99.78 and 92.10%, respectively. Furthermore, the molecular docking simulation clarified the binding mode of CHQ derivative inside the active site of α-amylase and α-glucosidase, suggesting its potential use as diabetes mellitus drug. The DFT calculations indicated an improvement in the electronic properties of CHQ with a lower energy band gap reached 4.05eV compared to 5.94eV for CH. The cytotoxicity assay revealed the safety of CHQ towards normal HSF cells, hypothesizing its possible application as non-toxic antibacterial, antioxidant, and antidiabetic agent for biomedical applications.

MiR-338-3p and miR-34a-5p act as antagonists in phycocyanin-mediated process of ameliorating glycometabolism in T2DM HepG2 cells

Phycocyanin has been demonstrated to regulate glycometabolism. Previously, we discovered phycocyanin could protect against type 2 diabetes mellitus (T2DM), while its mechanism is still unclear. In this work, a miRNA-seq was employed to investigate potential vital miRNAs. The results indicated that miR-338-3p expression was decreased in T2DM, which was rescued by phycocyanin, while the expression of miR-34a-5p showed opposite trend. Knockdown miR-338-3p expression or overexpression of miR-34a-5p both reduced the glucose utilization of cells, while could be significantly reversed by phycocyanin. Dual-luciferase reporter assays suggested that PTPRM and NR3C2 are direct targets of miR-34a-5p and miR-338-3p, respectively. Strikingly, knockdown PTPRM expression impacted the uptake of glucose in normal cells, while interfering NR3C2 expression alleviated the decreased glucose availability caused by insulin resistance (IR). Thus, the present work demonstrated that miR-338-3p and miR-34a-5p act as antagonists in phycocyanin-mediated process of ameliorating glycometabolism, which highlights a critical role of miRNAs in regulating glycometabolism.

Discovery and biological evaluation of novel dual PTP1B and ACP1 inhibitors for the treatment of insulin resistance

In this study, a virtual screening pipeline comprising ligand-based and structure-based approaches was established and applied for the identification of dual PTP1B and ACP1 inhibitors. As a result, a series of benzoic acid derivatives was discovered, and compound H3 and S6 demonstrated PTP1B and ACP1 inhibitory activity, with IC50 values of 3.5 and 8.2 μM for PTP1B, and 2.5 and 5.2 μM for ACP1, respectively. Molecular dynamics simulations illustrated that H3 interacted with critical residues in the active site, such as Cys215 and Arg221 for PTP1B, and Cys17 and Arg18 for ACP1. Enzymatic kinetic research indicated that identified inhibitors competitively inhibited PTP1B and ACP1. Additionally, cellular assays demonstrated that H3 and S6 effectively increased glucose uptake in insulin-resistant HepG2 cells while displaying very limited cytotoxicity at their effective concentrations. In summary, H3 and S6 represent novel dual-target inhibitors for PTP1B and ACP1, warranting further investigation as potential agents for the treatment of diabetes.

Pharmacological Evidence That Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake Partly via an Increase in Intracellular cAMP Content in Mouse 3T3-L1 Cells

Differentiation-inducing factor 1 (DIF-1) isolated from the cellular slime mold Dictyostelium discoideum can inhibit mammalian calmodulin-dependent cAMP/cGMP phosphodiesterase (PDE1) in vitro. DIF-1 also promotes glucose uptake, at least in part, via a mitochondria- and AMPK-dependent pathway in mouse 3T3-L1 fibroblast cells, but the mechanism underlying this effect has not been fully elucidated. In this study, we investigated the effects of DIF-1 on intracellular cAMP and cGMP levels, as well as the effects that DIF-1 and several compounds that increase cAMP and cGMP levels have on glucose uptake in confluent 3T3-L1 cells. DIF-1 at 20 μM (a concentration that promotes glucose uptake) increased the level of intracellular cAMP by about 20% but did not affect the level of intracellular cGMP. Neither the PDE1 inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine at 10–200 μM nor the broad-range PDE inhibitor 3-isobutyl-1-methylxanthine at 40–400 μM had any marked effects on glucose uptake. The membrane-permeable cAMP analog 8-bromo-cAMP at 200–1000 μM significantly promoted glucose uptake (by 20–25%), whereas the membrane-permeable cGMP analog 8-bromo-cGMP at 3–100 μM did not affect glucose uptake. The adenylate cyclase activator forskolin at 1–10 μM promoted glucose uptake by 20–30%. Thus, DIF-1 may promote glucose uptake by 3T3-L1 cells, at least in part, via an increase in intracellular cAMP level.

DDX24 promotes tumor progression by mediating hexokinase-1 induced glycolysis in gastric cancer

Metabolic reprogramming allows tumor cells to meet high demand of biogenesis and increased energy for rapid proliferation. Gastric cancer (GC) ranks among the most prevalent malignancies globally. Exploring the underlying mechanisms of glycolytic reprogramming in GC could provide new therapeutic target for GC treatment. Here, we showed that DEAD-box helicase 24 (DDX24) played a critical role in hexokinase-1 (HK1) induced glycolysis. DDX24 expression was significantly elevated in GC tissues and was closely associated with worse survival in GC patients. In addition, DDX24 promoted glucose uptake and lactate production in GC cells. Mechanistically, DDX24 could bind the HK1 mRNA and positively regulated HK1 level at the transcriptional level. Moreover, DDX24 promoted the proliferation, migration, and invasion ability of GC cells by upregulating HK1. Collectively, these results suggested that DDX24 was a critical player in the regulation of glycolytic reprogramming and also implicated DDX24 as a valuable therapeutic target for GC.

LPS-Induced Modifications in Macrophage Transcript and Secretion Profiles Are Linked to Muscle Wasting and Glucose Intolerance.

Macrophages are versatile immune cells that play crucial roles in tissue repair, immune defense, and the regulation of immune responses. In the context of skeletal muscle, they are vital for maintaining muscle homeostasis but macrophage-induced chronic inflammation can lead to muscle dysfunction, resulting in skeletal muscle atrophy characterized by reduced muscle mass and impaired insulin regulation and glucose uptake. Although the involvement of macrophage-secreted factors in inflammation-induced muscle atrophy is well-established, the precise intracellular signaling pathways and secretion factors affecting skeletal muscle homeostasis require further investigation. This study aimed to explore the regulation of macrophage-secreted factors and their impact on muscle atrophy and glucose metabolism. By employing RNA sequencing (RNA-seq) and proteome array, we uncovered that factors secreted by lipopolysaccharide (LPS)-stimulated macrophages upregulated markers of muscle atrophy and pro-inflammatory cytokines, while concurrently reducing glucose uptake in muscle cells. The RNA-seq analysis identified alterations in gene expression patterns associated with immune system pathways and nutrient metabolism. The utilization of gene ontology (GO) analysis and proteome array with macrophage-conditioned media revealed the involvement of macrophage-secreted cytokines and chemokines associated with muscle atrophy. These findings offer valuable insights into the regulatory mechanisms of macrophage-secreted factors and their contributions to muscle-related diseases.

The Characterization of Metabolic Profile and Substrates Dependencies of Richter's Syndrome Cells Open for Translational Opportunities

Richter's syndrome (RS) is a high-grade aggressive lymphoma transformed from chronic lymphocytic leukemia (CLL), with a generally poor clinical outcome. The presence of sites of abnormally increased fluorodeoxyglucose F18 (18F-FDG) uptake on PET/CT of patients with CLL is predictive of Richter's transformation. The higher uptake of glucose in RS infiltrated lymph nodes compared to non-infiltrated ones together with evidence of metabolic reprogramming as a hallmark of cancer, prompt us to investigate the metabolic profile of RS cells and characterize their substrates dependencies. Exploiting 4 RS-patient-derived xenograft (PDX) models and the RS cell line U-RT1, we aimed to i) provide a complete picture of the metabolic landscape of RS cells, ii) identify the undeniable metabolites for these neoplastic cells, and iii) provide a proof-of-principle that metabolism can be a target for RS cells. Taking advantage of primary RS and CLL publicly available transcriptomic data, a clear shift in RNA expression was highlighted between the two disease phases, with an enrichment in metabolic processes-related terms within the upregulated genes. These results were validated in an internal cohort of primary RS samples and in the 4 RS-PDXs, with a complete overlap of the enriched “metabolic terms”, making these models representative tools to study the metabolic profile of RS cells. Specifically, gene sets enrichment analyses revealed oxidative phosphorylation, reactive oxygen species pathway, glycolysis, and fatty acid metabolism among the most significantly enriched gene sets in RS. This expression profile was supported by the measurement of the enzymatic activity of key enzymes belonging to glucose, glutamine, and fatty acid metabolic pathways. Three out of 4 PDXs and the U-RT1 cell line were characterized by an elevated metabolic rate compared to CLL, in terms of Krebs cycle, oxidative phosphorylation and glutamine metabolism, while the remaining RS-PDX model showed a more pronounced Warburg profile, with a stronger lactate dehydrogenase activity. These pathways are sustained by an increased glucose and glutamine uptake in RS cells leading to an elevated ATP content. Simultaneously, RS cells showed activation of anabolic processes that result in the synthesis of nucleotides and fatty acids. In line with these data, an unbiased approach based on metabolomic analyses identified a limited number of products of reaction, mainly attributable to pathways necessary both to produce energy and to provide building blocks (nucleotides, proteins and lipids) for cell proliferation. The metabolic dependencies of RS cells from glucose and glutamine were formally demonstrated by ex-vivo treatment with highly selective metabolic inhibitors. BPTES, a glutaminase inhibitor, and UK5099, a mitochondrial pyruvate carrier blocker, led to a significant decrease in the oxygen consumption rate and ATP production, while limited effects were observed when treating RS cells with Etomoxir, a fatty acid oxidation inhibitor. These reductions ultimately led to a significant induction of apoptosis in RS-PDXs derived cells. Finally, we showed that selective inhibitors of PI3K (duvelisib) and NF-kB (p65 inhibitor), two major regulators of energy metabolism and biosynthetic pathways, by interfering with the activity of these molecules, impacted on RS cells metabolism with a marked reduction of the oxidative phosphorylation and glycolytic cascade. Overall, these data depict the metabolic features of RS cells and their dependency from glucose and glutamine, at variance with CLL cells that mostly depend on fatty acid oxidation, thus suggesting a metabolic rewiring because of disease evolution. Moreover, they provide a proof-of-principle that metabolism can be envisaged as a candidate target for RS using either direct metabolic inhibitors or drugs that inhibit key metabolic regulators.

Downhill running and caloric restriction attenuate insulin resistance associated skeletal muscle atrophy via the promotion of M2-like macrophages through TRIB3-AKT pathway

BackgroudDownhill running has recently become a promising exercise modality for metabolic syndrome, but the effect and precise mechanism of downhill running training on insulin resistance (IR) induced skeletal muscle atrophy remains unclear. The current study aimed to explore the benefits of downhill running training accompanied by a low-fat diet on skeletal muscle atrophy in IR mice and its possible mechanisms. MethodsFor in vivo study, high fat diet (HFD) -induced IR mice were submitted to the downhill running training or/and caloric restriction for 8 weeks. In vitro study was performed using co-cultured RAW264.7 macrophages and C2C12 myoblasts model. Glucose tolerance test (GTT), insulin tolerance test (ITT), immunofluorescence staining, Western blot analysis, hematoxylin and eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), Cell counting kit-8 (CCK-8) assays and glucose uptake assays were employed to explore the benefits and possible mechanisms of downhill running training accompanied by a low-fat diet on IR mice. ResultsOur data revealed that HFD induces IR, which leading to skeletal muscle atrophy. Downhill running accompanied by caloric restriction mitigated HFD-induced IR and improve skeletal muscle atrophy. Further study suggested that descended TRIB3 mediated the favorable impact of downhill running on IR induced skeletal muscle atrophy by suppressing M1-like macrophages and promoting M2-like macrophages. Macrophages-specific knockdown of TRIB3 exerted similar effects on the macrophage polarization and IR related myogenesis to downhill running training accompanied by caloric restriction. In contrast, macrophages-specific overexpression of TRIB3 descended phosphorylation of AKT, further activated M1-like macrophages and aggravated IR related inhibition of myogenesis. ConclusionsThis finding demonstrated the beneficial effects of downhill running training and caloric restriction on IR related skeletal muscle atrophy by promoting M2-like macrophages through TRIB3-AKT pathway.

HCV affects KATP channels through GnT-IVa-mediated N-glycosylation of GLUT2 on the surface of pancreatic β-cells leading to impaired insulin secretion.

To explore the mechanism of insulin secretion dysfunction in pancreatic beta cells induced by N-glycosylation mediated by an infection from the hepatitis C virus (HCV). Min6 cell models infected with HCV and stimulated with glucose were constructed. Meanwhile, an HCV-infected animal model and a type 2 diabetes mellitus (T2DM) rat model were constructed. Glucose uptake in the Min6 cells was detected, and insulin secretion was detected by ELISA. Flow cytometry, immunofluorescence staining, Western blotting, RT-qPCR, and lectin blotting were used to detect the expression levels of related proteins and mRNA, as well as the level of N-glycosylation. HE staining was used to observe the pathological changes in the pancreatic tissue, and an oral glucose tolerance test (OGTT) was used to evaluate the glucose tolerance of the rats. Compared with the NC group, the expression levels of GnT-IVa, GLUT2, galectin-9, and voltage-dependent calcium channel 1.2 (Cav1.2) were significantly downregulated in the HCV-infected group. The ATP-sensitive potassium channel (KATP) component proteins SUR1 and Kir6.2 were significantly upregulated, while intracellular glucose intake and insulin secretion decreased, N-glycosylation levels and ATP levels significantly decreased, and the overexpression of GnT-IVa reversed the effect of the HCV infection. However, treatment with the glycosylation inhibitor kifunensine (KIF) or the KATP channel activator diazine (Dia) reversed the effects of the overexpression of GnT-IVa. In the animal experiments, HE staining revealed serious pathological injuries in the pancreatic tissue of the HCV-infected rats, with decreased glucose tolerance and glycosylation levels, decreased insulin secretion, downregulated expression of GnT-IVa, GLUT2, and Cav1.2, and upregulated expression of SUR1 and Kir6.2. The overexpression treatment of GnT-IVa or the KATP channel antagonist miglinide reversed the effects of HCV. HCV infection inhibits GLUT2 N-glycosylation on the pancreatic β cell surface by downregulating the expression of GnT-IVa and then activates the KATP pathway, which ultimately leads to disturbances in insulin secretion.

A Propolis-Derived Small Molecule Tectochrysin Ameliorates Type 2 Diabetes in Mice by Activating Insulin Receptor β.

Propolis has been found to decrease glucose levels and increase insulin sensitivity in type 2 diabetes. However, the active ingredient responsible for these effects and its regulating mechanism are not fully understood. To address this, molecular docking screening is used to screen the effective hypoglycemic ingredient in propolis and found that tectochrysin (TEC) has a high affinity to the insulin receptor (IR), highlighting its potential for glycemic control. In vivo tests show that TEC decreases glucose levels and enhances insulin sensitivity in db/db mice. By hyperinsulinemic euglycemic clamp test, this study further finds that TEC promotes glucose uptake in adipose tissue and skeletal muscle, as well as inhibits hepatic gluconeogenesis. Moreover, it finds that TEC promotes glucose uptake and adipocytes differentiation in 3T3-L1 cells like insulin, suggesting that TEC exerts an insulin mimetic effect. Mechanistically, pharmacology inhibition of IRβ abolishes the effects of TEC on glucose uptake and the phosphorylation of IR. The study further demonstrates that TEC binds to and activates IRβ by targeting its E1077 and M1079. Therefore, this study sheds light on the mechanism underlying propolis' potential for ameliorating type 2 diabetes, offering a natural food-derived compound as a promising therapeutic option.