Biology subjects

Neukam, M.

Publications and source records attributed to Neukam, M..

5 recordsLinked to original sources

bioRxiv2026

pH-dependent trapping of cationic amphiphilic drugs perturbs insulin granule homeostasis

Pancreatic {beta}-cells store insulin in acidic secretory granules (SGs), specialized organelles that also contain monoamine neurotransmitters such as serotonin. Many neuroactive drugs with monoaminergic activity are cationic amphiphilic drugs (CADs) that accumulate in acidic compartments by pH-dependent trapping. Yet, whether insulin SGs represent a site of CAD accumulation and if this affects their properties such as monoamine storage and pH remain unclear. Here, we show that Slc18a1/VMAT1 is required for vesicular monoamine uptake and maintenance of cellular serotonin levels in insulinoma INS-1 cells. In contrast, neuroactive CADs accumulate via pH-dependent trapping at luminal pH values characteristic of insulin SGs. CADs inhibit VMAT-mediated uptake of the fluorescent monoamine probe FFN206 and induce its efflux to the extracellular space without detectable changes in SG luminal pH. Conversely, natural VMAT substrates such as serotonin and dopamine increase SG pH in a VMAT-dependent manner. These findings identify insulin SGs as acidic organelles susceptible to CAD accumulation and uncover distinct mechanisms regulating secretory granule homeostasis.

cell biology↗

bioRxiv2025

Improved conditioning for hematopoietic chimerism induces islet tolerance to cure diabetes

Mixed hematopoietic chimerism after hematopoietic cell transplantation (HCT) can modulate the immune system and induce tolerance to allogeneic tissues. However, bone marrow conditioning-related toxicities preclude wider adoption of HCT for transplant allotolerance. We sought agents that reduced conditioning intensity, while promoting durable mixed chimerism after HCT across complete major histocompatibility complex (MHC) mismatch in diabetic mice, permitting islet allotransplantation and diabetes reversal. We systematically tested baricitinib (JAK1/2 inhibitor), venetoclax (Bcl2 inhibitor), and CD47 antibody, agents in current clinical use, and quantified hematopoietic chimerism after HCT. Combined with CD117 antibody, transient T cell depletion, and just 10 centigray (cGy) total body irradiation (TBI), these agents enabled durable mixed chimerism and matching allo-islet tolerance, to cure diabetes without evidence of GVHD. Thus, we have developed a conditioning regimen to promote allogeneic mixed hematopoietic chimerism and transplanted islet allotolerance that minimizes conditioning radiation and cures diabetes, a significant achievement.

immunology↗

bioRxiv2021

Sequential in vivo labeling of insulin secretory granule pools in INS-SNAP transgenic pigs

{beta}-cells produce, store and secrete insulin upon elevated blood glucose levels. Insulin secretion is a highly regulated process. The probability for insulin secretory granules to undergo fusion with the plasma membrane or being degraded is correlated with their age. However, the molecular features and stimuli connected to this behavior have not yet been fully understood. Furthermore, our understanding of {beta}-cell function is mostly derived from studies of ex vivo isolated islets and/or rodent models. To overcome this translational gap and study insulin secretory granule turnover in vivo, we have generated a transgenic pig model with the SNAP-tag fused to insulin. We demonstrate the correct targeting and processing of the tagged insulin and normal glycemic control of the pig model. Furthermore, we show specific single- and dual-color granular labeling of in vivo labeled pig pancreas. This model may provide unprecedented insights into the in vivo insulin secretory granule behavior in an animal close to humans.

cell biology↗

bioRxiv2020

Purification of age-distinct insulin secretory granules through antigen restriction using the CLIP-tag

Endocrine cells employ regulated exocytosis of secretory granules to secrete hormones and neurotransmitters. The competence of secretory granules for exocytosis depends on spatio-temporal variables such as proximity to the plasma membrane and age, with newly-generated granules being preferentially released. Despite recent advances, we still lack a comprehensive view about the molecular composition of insulin granules and how this may change over the lifetime of the organelles. Here we report a strategy for the purification of insulin secretory granules of distinct age from insulinoma INS-1 cells. Tagging the granule-resident protein phogrin with a cleavable CLIP tag, we obtained intact fractions of age-distinct granules for proteomic and lipidomic analyses. We find that the lipid composition changes over time along with the physical properties of the membrane.

cell biology↗

bioRxiv2017

FLIM-based pH measurements reveal incretin-induced rejuvenation of aged insulin secretory granules.

Insulin is stored in dense-core secretory granules (SGs) and is released from beta cells in two distinct phases upon glucose stimulation. Newly synthesized insulin SGs are secreted preferentially, but the underlying mechanism of this phenomenon remains elusive. The relationship of SG age with their intraluminal pH is of particular interest: proinsulin conversion by prohormone convertases follows the acidification of immature SGs by the vacuolar proton-translocating ATPase (v-ATPase). v-ATPases may also participate in the formation of the fusion pore for SG exocytosis, with intraluminal alkalinization inhibiting membrane fusion. Previous studies examined the luminal pH of SGs on a population level only. Here we measured the pH-dependent lifetime changes of eCFP fused to the ICA512-RESP18 homology domain to assess for the first time the luminal pH of individual age-defined SGs in insulinoma INS-1 cells by fluorescence-lifetime imaging microscopy. We show that 2-4-hour-old young SGs have a pH of ~5.5, while 26-28-hour-old SGs have a pH of ~6.2. Remarkably, the GLP-1 receptor agonist Exendin-4 prompted the re-acidification of old SGs in a glutamate-dependent fashion, while it did not affect the pH of young SGs. This study demonstrates that insulin SGs change their pH over time - a change that is reversible by insulin secretagogues. Hence, it provides novel insight into the mechanisms accounting for aging and exocytosis of SGs and suggests that their rejuvenation may be exploited to enhance insulin secretion in diabetes.

cell biology↗