Biology subjects

Holst, J. J.

Publications and source records attributed to Holst, J. J..

10 recordsLinked to original sources

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↗

bioRxiv2026

Impaired renal base excretion in secretin receptor knock-out mice during prolonged base-loading

AimSecretin was recently found to play a pivotal role in the renal adaptation to acute base excess. Here, secretin increases pendrin-dependent HCO3- secretion from the beta-intercalated cells in the cortical collecting ducts. Whether secretin and its receptor play a role during prolonged base-loading remains unknown. MethodsUrine and blood acid-base analyses were carried out in secretin receptor (SCTR) KO and WT mice at baseline and after 1 and up to 8 days of base-loading with NaHCO3-enriched drinking water. Changes in pendrin protein abundance and function were assessed by immunoblotting and isolated tubule perfusion experiments. Plasma secretin levels and renal SCTR expression were assessed after 24 hours of acid/base-loading by radioimmunoassay and qPCR, respectively. ResultsSCTR KO mice responded with diminished urine alkalization and a lesser reduction of urinary acid excretion when base-loaded for 48 hours. Concordantly, SCTR KO mice presented with increased blood base retention compared with WTs. Base-loaded SCTR WT and KO mice showed comparable total pendrin protein abundance. Despite this, pendrin function was markedly lower in SCTR KO mice. Base-loaded mice had higher plasma secretin and renal SCTR levels compared with acid-loaded mice. Higher arterial HCO - associated with higher renal SCTR mRNA expression. ConclusionLoss of the SCTR diminishes renal base excretion capacity and exacerbates systemic base accumulation during prolonged base-loading. Further, plasma secretin and renal SCTR mRNA levels are modulated by acid-base intake. These findings further support a central role of secretin and its receptor in the regulation of both acute and prolonged base excess. Key pointsO_LIIn mice, the receptor of the gut hormone secretin is required for the kidney to adequately handle a sustained base load. In mice with secretin receptor knockout, renal base excretion is impaired and metabolic alkalosis is aggravated and slower to resolve. C_LIO_LIBase-loaded knockout mice up-regulate pendrin protein abundance normally, the bicarbonate-secreting Cl-/HCO3-exchanger in collecting-duct {beta}-intercalated cells, but exhibit lower pendrin function. This suggests that the defect lies in the activation of pendrin, rather than in the regulation of its abundance. C_LIO_LIRenal secretin-receptor expression increases with higher arterial bicarbonate and plasma secretin, and renal secretin receptor expression, levels are lower in acid-loaded mice compared to base-loaded mice. This indicates that the secretin/secretin receptor system is itself affected by acid-base status/acid-base intake. C_LI

physiology↗

bioRxiv2025

Physical activity promotes gut adaptation, responses to nutrients, and sensitivity to gut peptides

Physical activity is essential for body weight maintenance after body weight-loss, partly by promoting the coupling between energy intake and expenditure. However, the underlying mechanisms remain largely unknown. Here we demonstrate that running induces small intestine growth independently of GLP-2. In addition, exercise increases L-cell density in the small intestine and glucose-stimulated GLP-1 secretion, and improves the sensitivity both to the gut-derived hormones PYY, CCK and ghrelin, and to treatment with GLP-1 receptor agonist. Moreover, increased physical activity enhances satiation and satiety post-fasting, regulates the gene expression of appetite signals in the intestine, nodose ganglia and brainstem, and induces a greater feeding-response in the activation of hypothalamic and brainstem neurons. This improves overall appetite regulation, and in turn, promotes body weight maintenance. In summary, the present data suggest that increased physical activity improves body weight maintenance by inducing adaptations in the gut and in gut- to-brain communication that control appetite. HIGHLIGHTSO_LIIncreased physical activity promotes intestinal growth C_LIO_LISensitivity to gut-derived hormones PYY, CCK and ghrelin is improved in active mice C_LIO_LIIncreased physical activity enhances glucose-stimulated GLP-1 secretion from the small intestine C_LIO_LIIncreased physical activity regulates effectively satiation and satiety post-fasting C_LI

physiology↗

bioRxiv2024

Selective Agonism of Liver and Gut FXR Prevents Cholestasis and Intestinal Atrophy in Parenterally Fed Neonatal Pigs

BACKGROUND & AIMSWe aimed to investigate the relative efficacy of feeding different bile acids in preventing PNALD in neonatal pigs. METHODSNewborn pigs given total parenteral nutrition (TPN) combined with minimal enteral feeding of chenodeoxycholic acid (CDCA), or increasing doses of obeticholic acid (OCA) for 19 days. RESULTSEnteral OCA (5 and 15 mg/kg), but not CDCA (30 mg/kg) reduced blood cholestasis markers compared to TPN controls and increased bile acids in the gallbladder and intestine. Major bile acids in the liver and distal intestine were CDCA, HCA, HDCA and OCA, and their relative proportions were increased by the type of bile acid (CDCA or OCA) given enterally. High doses of OCA increased the total NR1H4-agonistic bile acid profile in the liver and intestine above 50% total bile acids. Both CDCA and OCA treatments suppressed hepatic cyp7a1 expression, but only OCA increased hepatobiliary transporters, ABCB11, ABCC$ and ABCB1. Plasma phytosterol levels were reduced and biliary levels were increased by CDCA and OCA and hepatic sterol transporters, abcg5/8, expression were increased by OCA. Both CDCA and OCA increased plasma FGF19 and OCA increased intestinal FGF19, FABP6, and SLC51A. Both CDCA and OCA increased intestinal mucosal growth, whereas CDCA increased the plasma GLP-2, GLP-1 and GIP. CONCLUSIONSEnteral OCA prevented cholestasis and phytosterolemia by increased hepatic bile acid and sterol transport via induction of hepatobiliary transporter FXR target genes and not by suppression of bile acid synthesis genes. We also showed an intestinal trophic action of OCA that demonstrates a dual clinical benefit of FXR agonism in the prevention of PNALD in piglets.

physiology↗

bioRxiv2024

Proinflammatory cytokine-induced alpha-cell impairment in human islet microtissues is partially restored by dual incretin receptor agonism

Aims/hypothesisIn type 1 diabetes, the counterregulatory glucagon response to low plasma glucose is impaired. The resulting increased risk of hypoglycaemia necessitates novel strategies to ameliorate alpha-cell impairment. Here, we aimed to establish an in vitro model of alpha-cell impairment in type 1 diabetes using human islet microtissues (MTs) exposed to proinflammatory cytokines. Additionally, we investigated the therapeutic potential of incretin receptor agonists in improving alpha-cell responses to low glucose. MethodsHuman islet MTs were exposed to proinflammatory cytokines (IL-1{beta}, IFN-{gamma}, and TNF-) for 1 day (short-term) and 6 days (long-term). Alpha-cell function was assessed by sequential glucose-dependent secretion assays at 2.8 and 16.7 mmol/l glucose, followed by glucagon measurements. Additional evaluations included ATP content, caspase-3/7 activity, chemokine secretion, and expression of islet transcription factors and hormones. The effects of incretin receptor agonist treatment (glucose-dependent insulinotropic polypeptide (GIP) analogue [D-Ala2]-GIP {+/-} liraglutide) alongside or after cytokine exposure were also investigated, focusing on low glucose-dependent glucagon secretion. ResultsShort-term cytokine exposure increased glucagon secretion at both 2.8 and 16.7 mmol/l glucose. In contrast, long-term cytokine exposure caused dose-dependent suppression of glucagon secretion at 2.8 mmol/l glucose, resembling a type 1 diabetes phenotype. Long-term cytokine exposure also diminished somatostatin secretion, reduced ATP content, increased caspase 3/7 activity, and decreased islet transcription factor and hormone expression. Despite cytokine-induced impairment, alpha cells partially retained secretory capacity to L-arginine stimulation. Treatment with incretin receptor agonists during long-term cytokine exposure did not prevent alpha-cell impairment. However, acute treatment with [D-Ala2]-GIP {+/-} liraglutide or the single-molecule dual agonist tirzepatide partially restored glucagon secretion at low glucose. Conclusions/interpretationLong-term cytokine exposure of human islet MTs impaired glucagon secretion to low glucose, creating a type 1 diabetes alpha-cell phenotype. This cytokine-induced alpha-cell impairment was partially restored by [D-Ala2]-GIP {+/-} liraglutide and tirzepatide, respectively. Research in contextO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIThe counterregulatory alpha-cell response to low glucose is impaired in type 1 diabetes, increasing the risk of hypoglycaemia. C_LIO_LILimited translatability of rodent islet findings highlights the need for human islet models. C_LIO_LIActions of the incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) have mainly been studied in the context of type 2 diabetes and hyperglycaemia but less in type 1 diabetes and hypoglycaemia. C_LI What is the key question?O_LICan alpha-cell impairment in type 1 diabetes be modelled in vitro by exposing human islet microtissues (MTs) to proinflammatory cytokines, and could incretins protect against this? C_LI What are the new findings?O_LILong-term (6-day) exposure to proinflammatory cytokines produces a type 1 diabetes phenotype of alpha-cell impairment to low glucose in islet MTs C_LIO_LIAcute dual treatment with incretin receptor agonists partially restored glucose-dependent glucagon secretion in cytokine-exposed islet MTs -- an effect mainly carried by GIP receptor agonism and not opposed by GLP-1 receptor agonism. C_LI How might this impact on clinical practice in the foreseeable future?O_LIInvestigating incretin receptor agonists in a preclinical in vitro model of alpha-cell impairment may reveal their potential and fast-track their use as safeguards against hypoglycaemia in type 1 diabetes. C_LI

cell biology↗

bioRxiv2023

Inhibition of dipeptidyl peptidase 4 allows accurate measurement of GLP-1 secretion in mice

Dipeptidyl peptidase (DPP)-4 and neprilysin (NEP) within few minutes degrade glucagon-like peptide 1 (GLP-1) in mice, generating small, inactive fragments of the peptide. Commercially available sandwich ELISA kits may not accurately detect intact GLP-1 (that is GLP-1[7-36]NH2) and these moieties, leading to underestimation of secretion and potentially misleading results. Single-site antibody approaches may pick up some fragments, yet require large plasma volumes and the accuracy is uncertain. We aimed to find a way to stabilize GLP-1 in mice allowing reliable measurement with sensitive commercially available ELISA kits. Non-anesthetized male C57Bl/6JRj mice were subjected to an oral glucose tolerance test (OGTT; 2 g/kg glucose via oral gavage). Blood was drawn from the retrobulbar plexus before and repeatedly during the OGTT and total and intact GLP-1 were measured by commercially available sandwich ELISA kits (Mercodia and Alpco, respectively). In blood samples taken 15 minutes after the glucose load, there were no increase in plasma GLP-1 concentration. There was a small insignificant increase in total GLP-1 (1-2 pmol/L) in samples taken at t=5 and t=10 minutes following the OGTT, but no rise in intact GLP-1. We then administered control (saline), or a DPP-4 inhibitor (valine pyrrolidide, 0.1 {micro}mol/g or sitagliptin, 10 mg/kg) with or without a NEP-inhibitor (sacubitril, 0.3 mg/kg) 30 minutes before the OGTT. In the four inhibitor groups, intact GLP-1 increased during the OGTT (levels ranging between 4.04 [SD 2.87] and 15.53 [SD 6.76] pmol/L). The combination of sitagliptin with sacubitril gave the largest increase in intact GLP-1 levels. Finally, after injecting male C57Bl/6JRj mice with a known dose of GLP-1(7-36)NH2, the peak GLP-1 levels were significantly higher during sitagliptin, but not with the combination of sitagliptin/sacubitril. Both inhibitor groups, however, showed prolonged half-life of the GLP-1 plasma disappearance. We conclude that for measurements of GLP-1 secretion in mice with commercially available sandwich ELISA kits, it is necessary to consider both timing of blood sampling and in vivo inhibition of DPP-4. The described approach allows improved estimates of GLP-1 secretion for future studies. It is a limitation that DPP-4 and NEP inhibition may have metabolic effects by stabilizing the intact GLP-1 peptide (e.g. influencing levels of insulin and glucagon).

physiology↗

bioRxiv2021

Evaluation of Commercially Available Glucagon Receptor Antibodies and Glucagon Receptor Expression

Glucagon is a key regulator of numerous metabolic functions including glucose, protein and lipid metabolism, and glucagon-based therapies are explored for diabetes, fatty liver disease and obesity. Insight into tissue and cell specific expression of the glucagon receptor (GCGR) is important to understand the biology of glucagon as well as to differentiate between direct and indirect actions of glucagon. However, it has been challenging to accurately localize the GCGR in tissue due to low expression levels and lack of specific methodologies. Immunohistochemistry has frequently been used for GCGR localization, but G-protein-coupled receptors (GPCRs) targeting antibodies are notoriously unreliable. In this study, we systematically evaluated all commercially available GCGR antibodies. Initially, twelve GCGR antibodies were evaluated using HEK293 cells transfected with mouse or human GCGR cDNA. Of the twelve antibodies tested, eleven showed positive staining of GCGR protein from both species. Human liver tissue was investigated using the same GCGR antibodies. Five antibodies failed to stain human liver biopsies (despite explicit claims to the contrary from the vendors). Immunohistochemical (IHC) staining demonstrated positive staining of liver tissue from glucagon receptor knockout (Gcgr-/-) mice and their wild-type littermates (Gcgr+/+) with only one out of the twelve available GCGR antibodies. Three antibodies were selected for further evaluation by western blotting and bands corresponding to the predicted size of the GCGR (62 kDa) were identified using two of these. Finally, a single antibody (no. 11) was selected for specific GCGR localization studies in various tissues. In mouse tissue the most intense immunostainings were found in lever, kidney, ileum, heart, and pancreas. Western blotting, performed on liver tissue from Gcgr+/+ and Gcgr-/- mice, confirmed the specificity of antibody no. 11, detecting a band at high intensity in material from Gcgr+/+and no bands in liver tissue from Gcgr-/-mice. Staining of human kidney tissue, with antibody no. 11, showed GCGR localization to the distal tubules. Autoradiography was used as an antibody-independent approach to support the antibody-based findings, revealing specific binding in liver, pancreas, and kidney. As a final approach, RNA-sequencing and single-cell RNA (scRNA)-sequencing were implemented. RNA-sequencing confirmed GCGR presence within liver and kidney tissue. The GCGR was specifically found to be expressed in hepatocytes by scRNA-sequencing and potentially also in collecting and distal tubule cells in the kidney. Our results clearly indicate the liver and the kidneys as the primary targets of glucagon action.

physiology↗

bioRxiv2019

Alanine, arginine, and proline but not glutamine are the feed-back regulators in the liver-alpha cell axis in mice

AimTo identify the amino acids that stimulate glucagon secretion in mice and whether the metabolism of these relies on glucagon receptor signaling.\n\nMethodsPancreata of female C57BL/6JRj mice were perfused with 19 individual amino acids (1 mM) and secretion of glucagon was assessed using a specific glucagon radioimmunoassay. Separately, a glucagon receptor antagonist (GRA; 25-2648, 100 mg/kg) or vehicle was administered to female C57BL/6JRj mice three hours prior to an intraperitoneal injection of four different isomolar (in total 7 {micro}mol/g body weight) amino acid mixtures; mixture 1: alanine, arginine, cysteine, and proline; mixture 2: asparatate, glutamate, histidine, and lysine; mixture 3: citrulline, methionine, serine, and threonine; and mixture 4: glutamine, leucine, isoleucine, and valine. Blood glucose, plasma glucagon, amino acid, and insulin concentrations were measured using well characterized methodologies.\n\nResultsAlanine (P=0.03), arginine (P<0.001), and proline (P=0.03) but not glutamine (P=0.2) stimulated glucagon secretion from the perfused mouse pancreas. Cysteine had the numerically largest effect on glucagon secretion but did not reach statistical significance (P=0.08). However, when the four isomolar amino acid mixtures were administered there were no significant difference (P>0.5) in plasma concentrations of glucagon across mixture 1-4. Plasma concentrations of total amino acids were higher after administration of GRA when mixture 1 (P=0.004) or mixture 3 (P=0.04) were injected.\n\nConclusionOur data suggest that alanine, arginine, and proline but not glutamine are involved in the liver-alpha cell axis in mice as they all increased glucagon secretion and their disappearance rate was altered by GRA.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC=\"FIGDIR/small/792119v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (19K):\norg.highwire.dtl.DTLVardef@b78a3corg.highwire.dtl.DTLVardef@1d475d1org.highwire.dtl.DTLVardef@1830a00org.highwire.dtl.DTLVardef@59c51_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2019

Secretin release after Roux-en-Y Gastric Bypass reveals a population of glucose-sensitive S-cells in distal small intestine

ObjectiveGastrointestinal hormones contribute to the beneficial effects of Roux-en-Y gastric bypass surgery (RYGB) on glycemic control. Secretin is secreted from duodenal S-cells in response to low luminal pH, but it is unknown whether its secretion is altered after RYGB and if secretin contributes to the post-operative improvement in glycemic control. We hypothesized that secretin secretion increases after RYGB as a result of the diversion of nutrients to more distal parts of the small intestine, and thereby affects islet hormone release.\n\nMethodsA specific secretin radioimmunoassay was developed, evaluated biochemically, and used to quantify plasma concentrations of secretin in 13 obese individuals before, 1 week after and 3 months after RYGB. Distribution of secretin and its receptor was assessed by RNA-sequencing, mass-spectrometry and in situ hybridization in human and rat tissues. Isolated, perfused rat intestine and pancreas were used to explore the molecular mechanism underlying glucose-induced secretin secretion and to study direct effects of secretin on glucagon, insulin and somatostatin secretion. Secretin was administered alone or in combination with GLP-1 to non-sedated rats to evaluate effects on glucose regulation.\n\nResultsPlasma postprandial secretin was more than doubled in humans after RYGB (P<0.001). The distal small intestine harbored secretin expressing cells in both rats and humans. Glucose increased secretion of secretin in a sodium-glucose co-transporter dependent manner when administered to the distal part but not into the proximal part of the rat small intestine. Secretin stimulated somatostatin secretion (fold change: 1.59, P<0.05) from the perfused rat pancreas but affected neither insulin (P=0.2) nor glucagon (P=0.97) secretion. When administered to rats in vivo, insulin secretion was attenuated and glucagon secretion increased (P=0.04), while blood glucose peak time was delayed (from 15 min to 45 min) and gastric emptying time prolonged (P=0.004).\n\nConclusionGlucose-sensing secretin cells located in the distal part of the small intestine may contribute to increased plasma concentrations observed after RYGB. The metabolic role of the distal S-cells warrants further studies.

physiology↗

bioRxiv2019

Promiscuous receptor activation mediates glucagonostatic effects of GLP-1(9-36) and GLP-1(7-36)

The incretin hormone glucagon-like peptide 1(7-36) (GLP-1(7-36)) stimulates insulin and inhibits glucagon secretion. The mechanisms by which GLP-1 suppresses glucagon release are unclear as glucagon-secreting -cells express GLP-1 receptors (GLP-1Rs) at very low levels. Here, we examine the underlying mechanisms. We find that both GLP-1(7-36) and its degradation product GLP-1(9-36) inhibit glucagon secretion at physiological (pM) concentrations. Whereas the effect of GLP-1(7-36) is sensitive to PKA inhibition, GLP-1(9-36) exerts its effect by a PKA-independent mechanism sensitive to pretreatment with pertussis. The glucagonostatic effects of both GLP-1(7-36) and (9-36) are retained in islets from Glp1r knockout mice but only GLP-1(9-36) remains glucagonostatic in the presence of the DPP-4 (the peptidase catalyzing the formation of GLP-1(9-36)) inhibitor sitagliptin. Glucagon receptor (GCGR) antagonism specifically prevents the inhibitory effects of GLP-1(9-36) whilst not affecting that of GLP-1(7-36). We conclude that GLP-1(7-36) and GLP-1(9-36) regulate glucagon secretion via interaction with GLP-1R and GCGR, respectively. HighlightsO_LIGLP-1(7-36) and GLP-1(9-36) inhibit glucagon secretion from alpha-cells C_LIO_LIGLP-1(7-36) and (9-36) retain glucagonostatic effect in Glp1r-/- islets C_LIO_LIGLP-1(7-36) and (9-36) activate distinct signal transduction mechanisms C_LIO_LIGLP-1(7-36) acts via GLP-1R and GLP-1(9-36) via GCGR C_LI

molecular biology↗