Biology subjects

Thurmond, D. C.

Publications and source records attributed to Thurmond, D. C..

4 recordsLinked to original sources

bioRxiv2025

TAS1R3 is dually required for GTPase signaling in human islet β-cells and skeletal muscle cells

BackgroundTaste receptor type 1 member 3 (TAS1R3) is a class C G protein-coupled receptor (GPCR) traditionally associated with taste perception. While its role in insulin secretion is established, its contribution to skeletal muscle glucose uptake a process responsible for 70-80% of postprandial glucose disposal remains unclear. MethodsTAS1R3 expression was assessed in skeletal muscle biopsies from non-diabetic and type 2 diabetes (T2D) donors using qPCR and immunoblotting. Functional studies in human LHCN-M2 myotubes involved TAS1R3 inhibition with lactisole or siRNA-mediated knockdown, followed by measurement of insulin-stimulated glucose uptake using radiolabeled glucose assays. Rac1 activation and phospho-cofilin were analyzed by G-LISA and Western blotting, and Galphaq/11 involvement was tested using YM-254890. ResultsTAS1R3 mRNA and protein levels were significantly reduced in T2D skeletal muscle. Pharmacological inhibition or knockdown of TAS1R3 impaired insulin-stimulated glucose uptake in myotubes. ConclusionTAS1R3 regulates skeletal muscle glucose uptake through a non-canonical insulin signaling pathway involving Rac1 and phospho-cofilin, independent of IRS1-AKT and Galphaq/11 signaling. These findings identify TAS1R3 as a key determinant of Rac1-mediated glucose uptake and a potential therapeutic target for improving insulin sensitivity in T2D.

physiology↗

bioRxiv2025

STX4 is indispensable for mitochondrial homeostasis in skeletal muscle

BackgroundMitochondrial homeostasis is vital for optimal skeletal muscle integrity. Mitochondrial quality control (MQC) mechanisms that are essential for maintaining proper functions of mitochondria include mitochondrial biogenesis, dynamics and mitophagy. Previously, Syntaxin 4 (STX4) traditionally considered a cell surface protein known for glucose uptake in skeletal muscle, was also identified at the outer mitochondrial membrane. STX4 enrichment was sufficient to reverse Type 2 diabetes-associated mitochondrial damage in skeletal muscle by inactivation of mitochondrial fission. However, whether STX4 could modulate skeletal muscle mitochondrial homeostasis through MQC mechanisms involving mitochondrial biogenesis or mitophagy remains to be determined. MethodsTo determine the requirements of STX4 in mitochondrial structure, function and MQC processes of biogenesis and mitophagy, we implemented our in-house generated inducible skeletal muscle-specific STX4-knockout (skmSTX4-iKO) mice (Stx4fl/fl; Tg(HSA-rtTA/TRE-Cre)/B6) and STX4-depleted immortalized L6.GLUT4myc myotubes via siRNA knockdown (siSTX4). ResultsWe found that non-obese skmSTX4-iKO male mice (>50% reduced STX4 abundance, Soleus and Gastrocnemius ***p<0.001, Tibialis anterior (TA) ****p<0.0001) developed insulin resistance (**p<0.01), together with reduced energy expenditure (AUC *p<0.05), respiratory exchange ratio (AUC **p<0.01), and grip strength (*p<0.05). STX4 ablation in muscle also impaired mitochondrial oxygen consumption rate (****p<0.0001). Mitochondrial morphological damage was heterogenous in STX4 depleted muscle, presenting with small fragmented mitochondria (****p<0.0001) and deceased electron transport chain (ETC) abundance (CI ***p<0.001, CII *p<0.05, CIV **p<0.01) in oxidative soleus muscle, while glycolytic TA fibers display enlarged swollen mitochondria (****p<0.0001) with no change in ETC abundance. Notably, >60% reduction of STX4 in siSTX4 L6.GLUT4myc myotubes (****p<0.0001) also decreased ETC abundance (CI ****p<0.0001, CII ****p<0.0001, CIV *p<0.05) without changes in mitochondrial glucose metabolism, as shown by [U-13C] glucose isotope tracing. For MQC, both skmSTX4-iKO male mice (*p<0.05) and siSTX4 L6.GLUT4myc myotubes (*p<0.05) showed decreased mitochondrial DNA levels alongside reduced mRNA expression of mitochondrial biogenesis genes Ppargc1a (PGC1-, *p<0.05) and Tfam (*p<0.05) in skmSTX4-iKO soleus muscle and PGC1- (mRNA *p<0.05, protein ***p<0.001), NRF1 (mRNA and protein *p<0.05) and Tfam (mRNA *p<0.05) in siSTX4 L6.GLUT4myc myotubes. Furthermore, live cell imaging using mt-Keima mitophagy biosensor in siSTX4 L6.GLUT4myc cells revealed significantly impaired mitochondrial turnover by mitophagy (*p<0.05) and mitochondria-lysosome colocalization (*p<0.05). STX4 depletion also reduced canonical mitophagy markers, PINK1 and PARKIN in both skmSTX4-iKO muscle (PARKIN *p<0.05, PINK1 **p<0.01) and siSTX4 L6.GLUT4myc myotubes (PARKIN ****p<0.0001, PINK1 *p<0.05). ConclusionsOur study demonstrated STX4 as a key mitochondrial regulator required for mitochondrial homeostasis in skeletal muscle.

cell biology↗

bioRxiv2024

DOC2b enrichment mitigates proinflammatory cytokine-induced CXCL10 expression by attenuating IKKβ and STAT-1 signaling in human islets

IntroductionType 1 diabetic human islet {beta}-cells are deficient in double C 2 like domain beta (DOC2b) protein. Further, DOC2b protects against cytokine-induced pancreatic islet {beta}-cell stress and apoptosis. However, the mechanisms underpinning the protective effects of DOC2b remain unknown. MethodsBiochemical studies, qPCR, proteomics, and immuno-confocal microscopy were conducted to determine the underlying protective mechanisms of DOC2b in {beta}-cells. DOC2b- enriched or-depleted primary islets (human and mouse) and {beta}-cell lines challenged with or without proinflammatory cytokines, global DOC2b heterozygous knockout mice subjected to multiple-low-dose-streptozotocin (MLD-STZ), were used for these studies. ResultsA significant elevation of stress-induced CXCL10 mRNA was observed in DOC2b- depleted {beta}-cells and primary mouse islets. Further, DOC2b enrichment markedly attenuated cytokine-induced CXCL10 levels in primary non-diabetic human islets and {beta}-cells. DOC2b enrichment also reduced total-NF-{kappa}B p65 protein levels in human islets challenged with T1D mimicking proinflammatory cytokines. IKK{beta}, NF-{kappa}B p65, and STAT-1 are capable of associating with DOC2b in cytokine-challenged {beta}-cells. DOC2b enrichment in cytokine-stressed human islets and {beta}-cells corresponded with a significant reduction in activated and total IKK{beta} protein levels. Total I{kappa}B{beta} protein was increased in DOC2b-enriched human islets subjected to acute cytokine challenge. Cytokine-induced activated and total STAT-1 protein and mRNA levels were markedly reduced in DOC2b-enriched human islets. Intriguingly, DOC2b also prevents ER-stress-IKK{beta} and STAT-1 crosstalk in the rat INS1-832/13 {beta}-cell line. ConclusionThe mechanisms underpinning the protective effects of DOC2b involve attenuation of IKK{beta}-NF-{kappa}B p65 and STAT-1 signaling, and reduced CXCL10 expression. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/629540v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1b9d0acorg.highwire.dtl.DTLVardef@32db73org.highwire.dtl.DTLVardef@e5645aorg.highwire.dtl.DTLVardef@1f7619_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Insulin-Independent Regulation of Type 1 Diabetes via Brown Adipocyte-Secreted Proteins and the Novel Glucagon Regulator Nidogen-2.

Current treatments for type 1 diabetes (T1D) focus on insulin replacement. We demonstrate the therapeutic potential of a secreted protein fraction from embryonic brown adipose tissue (BAT), independent of insulin. The large molecular weight secreted fraction mediates insulin receptor-dependent recovery of euglycemia in a T1D animal model, nonobese diabetic (NOD) mice, by suppressing glucagon secretion. This fraction also promotes white adipocyte differentiation and browning, maintains healthy BAT, and enhances glucose uptake in adipose tissue, skeletal muscle, and liver. From this fraction, we identify nidogen-2 as a critical BAT-secreted protein that reverses hyperglycemia in NOD mice, inhibits glucagon secretion from pancreatic -cells, and mimics other actions of the entire secreted fraction. These findings confirm that BAT transplants affect physiology and demonstrate that BAT-secreted peptides represent a novel therapeutic approach to diabetes management. Furthermore, our research reveals a novel signaling role for nidogen-2, beyond its traditional classification as an extracellular matrix protein. HIGHLIGHTSO_LIThe large molecular weight brown adipocyte-secreted protein fraction suppresses glucagon secretion and normalizes glycemia in mouse models of type 1 diabetes (T1D), independent of insulin, offering a novel therapeutic strategy for disease management. C_LIO_LINidogen-2, a critical component of this fraction, is identified as an inhibitor of glucagon secretion in pancreatic -cells by regulating intracellular messenger activities. C_LIO_LIThe large-secreted protein fraction prevents T1D-related whitening of brown adipose tissue, promotes adipocyte differentiation, and enhances browning of inguinal white adipose tissue. C_LIO_LIThis fraction enhances glucose uptake in adipose tissue, skeletal muscle, and liver through an insulin receptor-dependent pathway. C_LI

cell biology↗