Biology subjects

Knudsen, J. G.

Publications and source records attributed to Knudsen, J. G..

6 recordsLinked to original sources

bioRxiv2026

Primary cilia coordinate c-KIT signaling induced proliferation in alpha cells

Circulating glucagon levels are elevated in patients with diabetes and obesity and contribute to hyperglycemia. The mechanisms underlying hyperglucagonemia remain poorly understood, but expansion of pancreatic -cell mass is thought to play an important role. Primary cilia are sensory organelles that act as signaling hubs, for pathways controlling cell differentiation, proliferation, and function, yet their contribution to -cell biology remains poorly defined. Here we investigated the role of primary cilia in the regulation of -cell proliferation. To this end we generated the first -cell primary cilia proteome identifying 167 cilia-enriched proteins. Among these, we identified and validated the proto-oncogene receptor tyrosine kinase, c-KIT, as a novel ciliary receptor in -cells. We further show that c-KIT signaling depends on intact primary cilia and that stimulation of isolated mouse islets with its endogenous ligand, stem cell factor (SCF), promotes -cell proliferation. In human pancreatic islets KITLG mRNA expression, but not KIT mRNA expression, correlated positively with donor BMI, suggesting that increased ligand availability drives c-KIT signaling in obesity. Together, our findings identify the primary cilium as a signaling platform c-KIT in cells and reveal a ciliary axis that may drive -cell expansion and hyperglucagonemia in obesity and diabetes.

physiology↗

bioRxiv2026

Distribution of the glucagon receptor in periventricular brain barrier interfaces including motile and primary cilia in rat brain

Glucagon is a key metabolic hormone regulating blood glucose and appetite, yet little is known about its actions within the brain. Here, we investigated its receptor (GCGR) localization in periventricular brain barrier interfaces in young rats using immunohistochemical and immunofluorescence approaches. GCGR was enriched in the proximal region of motile ependymal cilia lining the ventricles, as well as in tanycytic primary cilia and cytoplasmic extensions within the hypothalamus. Additional immunostaining was observed in ciliated cells of the subcommissural organ and, more heterogeneously, in choroid plexus epithelium and associated primary cilia, while other circumventricular organs lacked detectable GCGR. These findings identify brain cilia and tanycytes as previously unrecognized sites of glucagon receptor localization and suggest that glucagon signaling at brain barrier interfaces may contribute to integrating peripheral metabolic cues with central homeostatic circuits.

neuroscience↗

bioRxiv2026

Glucose derived redox equivalents preserve PKA activity and glucagon secretion during hypoglycaemia

The release of glucagon from pancreatic alpha cells is a core component of hypoglycaemic counter regulation. Several mechanisms regulate glucagon release including paracrine control by neighbouring cell types, and changes in extracellular glucose. While the inhibitory effect of glucose on glucagon secretion is well established, the exact way in which glucose metabolism contributes to alpha cell function remains unclear. Here, we use live-cell imaging of the redox potential in alpha cells within intact islets to investigate whether non-mitochondrial glucose metabolism contributes to the potentiation of glucagon secretion at low glucose. Our findings show that increased glucose metabolism through the pentose phosphate pathway elevates the cytosolic redox potential in alpha cells. Using a combination of antioxidant treatment and pre-incubation in 5 mM glucose, we find that the cytosolic redox potential affects PKA activity in alpha cells and that changes in whole body redox state affects the counterregulatory response in mice. These findings indicate that prior glucose-driven redox potential charging is essential for maintaining glucagon secretion at low glucose.

physiology↗

bioRxiv2023

Validation of Tau Antibodies for Use in Western Blotting and Immunohistochemistry

BackgroundThe microtubule-associated protein Tau has attracted diverse and increasing research interest, with Tau being mentioned in the title/abstract of nearly 34,000 PubMed-indexed publications to date. To accelerate studies into Tau biology, the characterisation of its multiple proteoforms, including disease-relevant post-translational modifications (PTMs), and its role in neurodegeneration, a multitude of Tau-targeting antibodies have been developed, with hundreds of distinct antibody clones currently available for purchase. Nonetheless, concerns over antibody specificity and limited understanding of the performance of many of these reagents has hindered research. MethodsWe have employed a range of techniques in combination with samples of murine and human origin to characterise the performance and specificity of 53 commercially-available Tau antibodies by Western blot, and a subset of these, 35 antibodies, in immunohistochemistry. ResultsContinued expression of residual protein was found in presumptive Tau "knockout" human cells and further confirmed through mass-spectrometry proteomics, providing evidence of Tau isoforms generated by exon skipping. Importantly, many total and isoform-specific antibodies failed to detect this residual Tau, as well as Tau expressed at low, endogenous levels, thus highlighting the importance of antibody choice. Our data further reveal that the binding of several "total" Tau antibodies, which are assumed to detect Tau independently of post-translational modifications, was partially inhibited by phosphorylation. Many antibodies also displayed non-specific cross-reactivity, with some total and phospho-Tau antibodies cross-reacting with MAP2 isoforms, while the "oligomer-specific" T22 antibody detected monomeric Tau on Western blot. Regardless of their specificity, with one exception, the phospho-Tau antibodies tested were found to not detect the unphosphorylated protein. ConclusionsWe identify Tau antibodies across all categories (total, PTM-dependent and isoform-specific) that can be employed in Western blot and/or immunohistochemistry applications to reliably detect even low levels of Tau expression with high specificity. This is of particular importance for studying Tau in non-neuronal cells and peripheral tissues, as well as for the confident validation of knockout cells and/or animal models. This work represents an extensive resource that serves as a point of reference for future studies. Our findings may also aid in the re-interpretation of existing data and improve reproducibility of Tau research.

neuroscience↗

bioRxiv2020

Reduced somatostatin signalling leads to hypersecretion of glucagon in mice fed a high fat diet

Elevated plasma glucagon is an early symptom of diabetes, occurring in subjects with impaired glucose regulation. Here we explored alpha-cell function in female mice fed a high fat diet (HFD) - a widely used mouse model of pre-diabetes. In vivo, HFD-fed mice have increased fed plasma glucagon levels that are unaffected by elevation of plasma glucose. To explore the underlying mechanisms, we conducted experiments on isolated islets and in the perfused pancreas. In both experimental models, glucagon secretion under both hypo- and hyperglycaemic conditions was elevated. Because Ca2+ is an important intracellular regulator of glucagon release in alpha-cells, we fed mice expressing the Ca2+ indicator GCaMP3 specifically in alpha-cells the HFD. In mice fed a control (CTL) diet, increasing glucose reduced intracellular Ca2+ ([Ca2+]i) (oscillation frequency and amplitude). This effect was not observed in HFD mice where both the frequency and amplitude of the [Ca2+]i oscillations were higher than in CTL alpha-cells. Given that alpha-cells are under strong paracrine control from neighbouring somatostatin-secreting delta-cells, we hypothesised that this elevation of alpha-cell output was due to a lack of somatostatin (SST) secretion. Indeed, SST secretion in isolated islets from HFD mice was reduced but exogenous SST also failed to suppress glucagon secretion and Ca2+ activity from HFD alpha-cells, in contrast to observations in CTL mice. These findings suggest that reduced delta-cell function, combined with intrinsic changes in alpha-cell sensitivity to somatostatin, accounts for the hyperglucagonaemia in mice fed a HFD.

cell biology↗

bioRxiv2020

AVP-induced counter-regulatory glucagon is diminished in type 1 diabetes

Insulin-induced hypoglycemia is a major barrier to the treatment of type-1 diabetes (T1D). Accordingly, it is important that we understand the mechanisms regulating the circulating levels of glucagon - the bodys principal blood glucose-elevating hormone which is secreted from alpha-cells of the pancreatic islets. Varying glucose over the range of concentrations that occur physiologically between the fed and fuel-deprived states (from 8 to 4 mM) has no significant effect on glucagon secretion in isolated islets (in vitro) and yet associates with dramatic changes in plasma glucagon in vivo. The identity of the systemic factor(s) that stimulates glucagon secretion remains unknown. Here, we show that arginine-vasopressin (AVP), secreted from the posterior pituitary, stimulates glucagon secretion. Glucagon-secreting alpha-cells express high levels of the vasopressin 1b receptor gene (Avpr1b). Activation of AVP neurons in vivo increased circulating copeptin (the C-terminal segment of the AVP precursor peptide, a stable surrogate marker of AVP) and increased blood glucose; effects blocked by pharmacological antagonism of either the glucagon receptor or vasopressin 1b receptor. AVP also mediates the stimulatory effects of hypoglycemia produced by exogenous insulin and 2-deoxy-D-glucose on glucagon secretion. We show that the A1/C1 neurons of the medulla oblongata drive AVP neuron activation in response to insulin-induced hypoglycemia. Exogenous injection of AVP in vivo increased cytoplasmic Ca2+ in alpha-cells (implanted into the anterior chamber of the eye) and glucagon release. Hypoglycemia also increases circulating levels of AVP in humans and this hormone stimulates glucagon secretion from isolated human islets. In patients with T1D, hypoglycemia failed to increase both plasma copeptin and glucagon levels. These findings suggest that AVP is a physiological systemic regulator of glucagon secretion and that this mechanism becomes impaired in T1D.

cell biology↗