Biology subjects

Huang, S. M.

Publications and source records attributed to Huang, S. M..

4 recordsLinked to original sources

bioRxiv2026

iG6PSnFR: A genetically encoded fluorescent sensor for observing glucose-6-phosphate dynamics in living preparations

Glucose-6-phosphate (G6P) is a key intermediate in multiple energetic and anabolic pathways, and quantifying its dynamics is essential for understanding cellular physiology. We have previously developed a syndicate of intensity-based, genetically encoded sensors based on the insertion of circularly permuted GFP into a Venus-flytrap-like analyte-binding protein. Here we use the same approach to develop an intensity-based G6P Sensing Fluorescent Reporter (iG6PSnFR). We present two variants: a G6P-activated sensor that increases fluorescence and a G6P-inactivated sensor that decreases fluorescence. We validate performance across progressively more complex preparations, including purified protein in vitro, immortalized and primary neuronal cultures, isolated pancreatic islets, in an intravital liver model, and finally in vivo in C. elegans neurons. In each context, iG6PSnFR reports G6P changes consistent with expected responses to physiological perturbations.

biochemistry↗

bioRxiv2026

Compartmentalized glycolysis powers ATP production in primary cilia and engages mitochondria via the phosphoenolpyruvate cycle

Primary cilia are antenna-like sensory and signaling organelles present on most mammalian cells, including glucose-sensing pancreatic {beta}-cells. Here, we show that the local energetic demands of primary cilia require the ATP-producing enzyme pyruvate kinase, with loss of PKm1, but not PKm2, impairing ciliary glycolytic flux. While the entire glycolytic machinery localizes to cilia, our data indicate that mitochondria are a critical source of phosphoenolpyruvate (PEP), the high-energy glycolytic intermediate that drives the pyruvate kinase reaction. Abolishing PCK2, the mitochondrial enzyme that generates PEP, prevents cilia from sensing not only glucose but also the amino acids glutamine and leucine. Finally, by mislocalizing glycolysis, we demonstrate that primary cilia can utilize ATP generated within the cell body when glucose is limiting. These findings indicate that primary cilia, while possessing the capacity for local ATP generation, leverage a ciliary-mitochondrial signaling axis to meet their bioenergetic needs.

cell biology↗

bioRxiv2025

Amino acid sensing by the α-cell mitochondrial phosphoenolpyruvate cycle regulates intracellular Ca2+ levels without impacting glucagon secretion

Pancreatic islet -cells are increasingly recognized as amino acid sensors for the organism. Building on our prior work in {beta}-cells, we sought to determine whether the mitochondrial phosphoenolpyruvate (PEP) cycle is involved in -cell amino acid sensing. Three different methods were used to probe the PEP cycle, including pyruvate kinase activators (TEPP-46), and mice with -cell specific deletion of pyruvate kinase (PKM1/2-KO) or mitochondrial PEP carboxykinase (PCK2-KO). The mitochondrial fuels glutamine/leucine antagonized alanine/arginine-stimulated Ca2+ influx and glucagon secretion under hypoglycemic conditions. Both PKM1/2 and PCK2 were required for glutamine/leucine to close KATP channels and limit amino acid-stimulated membrane depolarization. The Ca2+ response to amino acids was suppressed by pyruvate kinase activation with TEPP-46, and enhanced by -cell deletion of pyruvate kinase or PCK2 - all without changing glucagon secretion. Finally, using diazoxide/KCl to probe the pathways downstream of membrane depolarization, we identified an essential role of the PEP cycle in homeostatically restoring intracellular Ca2+ levels. In sum, the -cell mitochondrial PEP cycle senses glutamine/leucine and inhibits KATP channels similarly to {beta}-cells, while restricting amino acid-stimulated membrane depolarization and Ca2+ influx. However, defying expectations, none of the amino acids tested, including alanine/arginine, regulate glucagon secretion by modulating membrane depolarization or intracellular Ca2+. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/656009v2_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@18a29f0org.highwire.dtl.DTLVardef@1c77041org.highwire.dtl.DTLVardef@9adc1dorg.highwire.dtl.DTLVardef@e91407_HPS_FORMAT_FIGEXP M_FIG Graphical abstract-cells as amino acids sensors. Arginine, alanine, and glutamine potentiate glucagon secretion, while leucine has a suppressive effect (left). Independently of glucagon secretion, glutamine and leucine suppress alanine and arginine-stimulated Ca2+ influx via the phosphoenolpyruvate (PEP) cycle, which closes KATP channels and suppresses Vm depolarization (right). C_FIG HighlightsO_LIOur studies identify a role for the -cell PEP cycle in sensing amino acids under hypoglycemic conditions. C_LIO_LIPyruvate kinase and PCK2 are required for glutamine/leucine to close -cell KATP channel and limit membrane depolarization and Ca2+ influx. C_LIO_LIGlutamine/leucine oppose alanine/arginine-stimulated Ca2+ influx and glucagon secretion. C_LIO_LIAll of the amino acids tested regulate glucagon secretion, but none do so by modulating membrane depolarization or intracellular Ca2+ levels. C_LI

cell biology↗

bioRxiv2023

Glutamine antagonist DRP-104 suppresses tumor growth and enhances response to checkpoint blockade in KEAP1 mutant lung cancer

Loss-of-function mutations in KEAP1 frequently occur in lung cancer and are associated with resistance to standard of care treatment, highlighting the need for the development of targeted therapies. We have previously shown that KEAP1 mutant tumors have increased glutamine consumption to support the metabolic rewiring associated with NRF2 activation. Here, using patient-derived xenograft models and antigenic orthotopic lung cancer models, we show that the novel glutamine antagonist DRP-104 impairs the growth of KEAP1 mutant tumors. We find that DRP-104 suppresses KEAP1 mutant tumor growth by inhibiting glutamine-dependent nucleotide synthesis and promoting anti-tumor CD4 and CD8 T cell responses. Using multimodal single-cell sequencing and ex vivo functional assays, we discover that DRP-104 reverses T cell exhaustion and enhances the function of CD4 and CD8 T cells culminating in an improved response to anti-PD1 therapy. Our pre-clinical findings provide compelling evidence that DRP-104, currently in phase 1 clinical trials, offers a promising therapeutic approach for treating patients with KEAP1 mutant lung cancer. Furthermore, we demonstrate that by combining DRP-104 with checkpoint inhibition, we can achieve suppression of tumor intrinsic metabolism and augmentation of anti-tumor T cell responses.

cancer biology↗