In the kidney, the flow rate of the pro-urine through the renal tubules is highly variable. The tubular epithelial cells sense these variations in pro-urinary flow rate in order to regulate various... Show moreIn the kidney, the flow rate of the pro-urine through the renal tubules is highly variable. The tubular epithelial cells sense these variations in pro-urinary flow rate in order to regulate various physiological processes, including electrolyte reabsorption. One of the mechanosensitive pathways activated by flow is the release of ATP, which can then act as a autocrine or paracrine factor. Increased ATP release is observed in various kidney diseases, among others autosomal dominant polycystic kidney disease (ADPKD). However, the mechanisms underlying flow-induced ATP release in the collecting duct, especially in the inner medullary collecting duct, remain understudied. Using inner medullary collecting duct 3 (IMCD3) cells in a microfluidic setup, we show here that administration of a high flow rate for 1 min results in an increased ATP release compared to a lower flow rate. Although the ATP release channel pannexin-1 contributed to flow-induced ATP release in Pkd1(-/- )IMCD3 cells, it did not in wildtype IMCD3 cells. In addition, flow application increased the expression of the putative ATP release channel connexin-30.3 (CX30.3) in wildtype and Pkd1(-/-) IMCD3 cells. However, CX30.3 knockout IMCD3 cells exhibited a similar flow-induced ATP release as wildtype IMCD3 cells, suggesting that CX30.3 does not drive flow-induced ATP release in wildtype IMDC3 cells. Collectively, our results show differential mechanisms underlying flow-induced ATP release in wildtype and Pkd1(-/- )IMCD3 cells and further strengthen the link between ADPKD and pannexin-1-dependent ATP release. Show less
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts within the kidney due to mutations in PKD1 or PKD2. Although the disease remains... Show moreAutosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts within the kidney due to mutations in PKD1 or PKD2. Although the disease remains incompletely understood, one of the factors associated with ADPKD progression is the release of nucleotides (including ATP), which can initiate autocrine or paracrine purinergic signaling by binding to their receptors. Recently, we and others have shown that increased extracellular vesicle (EVs) release from PKD1 knockout cells can stimulate cyst growth through effects on recipient cells. Given that EVs are an important communicator between different nephron segments, we hypothesize that EVs released from PKD1 knockout distal convoluted tubule (DCT) cells can stimulate cyst growth in the downstream collecting duct (CD). Here, we show that administration of EVs derived from Pkd1(-/-) mouse distal convoluted tubule (mDCT15) cells result in a significant increase in extracellular ATP release from Pkd1(-/-) mouse inner medullary collecting duct (iMCD3) cells. In addition, exposure of Pkd1(-/-) iMCD3 cells to EVs derived from Pkd1(-/-) mDCT15 cells led to an increase in the phosphorylation of the serine/threonine-specific protein Akt, suggesting activation of proliferative pathways. Finally, the exposure of iMCD3 Pkd1(-/-) cells to mDCT15 Pkd1(-/-) EVs increased cyst size in Matrigel. These findings indicate that EVs could be involved in intersegmental communication between the distal convoluted tubule and the collecting duct and potentially stimulate cyst growth. Show less
Panhuis, W.I.H.; Schoenke, M.; Siebeler, R.; Banen, D.; Pronk, A.C.M.; Streefland, T.C.M.; ... ; Kooijman, S. 2023
Circadian disruption (CD) is the consequence of a mismatch between endogenous circadian rhythms and behavior, and frequently occurs in shift workers. CD has often been linked to impairment of... Show moreCircadian disruption (CD) is the consequence of a mismatch between endogenous circadian rhythms and behavior, and frequently occurs in shift workers. CD has often been linked to impairment of glucose and lipid homeostasis. It is, however, unknown if these effects are sex dependent. Here, we subjected male and female C57BL/6J mice to 6-h light phase advancements every 3 days to induce CD and assessed glucose and lipid homeostasis. Within this model, we studied the involvement of gonadal sex hormones by injecting mice with gonadotropin-releasing hormone-antagonist degarelix. We demonstrate that CD has sex-specific effects on glucose homeostasis, as CD elevated fasting insulin levels in male mice while increasing fasting glucose levels in female mice, which appeared to be independent of behavior, food intake, and energy expenditure. Absence of gonadal sex hormones lowered plasma insulin levels in male mice subjected to CD while it delayed glucose clearance in female mice subjected to CD. CD elevated plasma triglyceride (TG) levels and delayed plasma clearance of TG-rich lipoproteins in both sexes, coinciding with reduced TG-derived FA uptake by adipose tissues. Absence of gonadal sex hormones did not notably alter the effects of CD on lipid metabolism. We conclude that CD causes sex-dependent effects on glucose metabolism, as aggravated by male gonadal sex hormones and partly rescued by female gonadal sex hormones. Future studies on CD should consider the inclusion of both sexes, which may eventually contribute to personalized advice for shift workers. Show less
Schonke, M.; Ying, Z.X.; Kovynev, A.; Panhuis, W.I.H.; Binnendijk, A.; Poel, S. van der; ... ; Rensen, P.C.N. 2023
The metabolic and inflammatory processes that are implicated in the development of cardiovascular diseases are under control of the biological clock. While skeletal muscle function exhibits... Show moreThe metabolic and inflammatory processes that are implicated in the development of cardiovascular diseases are under control of the biological clock. While skeletal muscle function exhibits circadian rhythms, it is unclear to what extent the beneficial health effects of exercise are restricted to unique time windows. We aimed to study whether the timing of exercise training differentially modulates the development of atherosclerosis and elucidate underlying mechanisms. We endurance-trained atherosclerosis-prone female APOE*3-Leiden.CETP mice fed a Western-type diet, a well-established human-like model for cardiometabolic diseases, for 1 h five times a week for 4 weeks either in their early or in their late active phase on a treadmill. We monitored metabolic parameters, the development of atherosclerotic lesions in the aortic root and assessed the composition of the gut microbiota. Late, but not early, exercise training reduced fat mass by 19% and the size of early-stage atherosclerotic lesions by as much as 29% compared to sedentary animals. No correlation between cholesterol exposure and lesion size was evident, as no differences in plasma lipid levels were observed, but circulating levels of the pro-inflammatory markers ICAM-1 and VCAM-1 were reduced with late exercise. Strikingly, we observed a time-of-day-dependent effect of exercise training on the composition of the gut microbiota as only late training increased the abundance of gut bacteria producing short-chain fatty acids with proposed anti-inflammatory properties. Together, these findings indicate that timing is a critical factor to the beneficial anti-atherosclerotic effects of exercise with a great potential to further optimize training recommendations for patients. Show less
Schonke, M.; Ying, Z.X.; Kovynev, A.; Panhuis, W.I.H.; Binnendijk, A.; Poel, S. van der; ... ; Rensen, P.C.N. 2023
The metabolic and inflammatory processes that are implicated in the development of cardiovascular diseases are under control of the biological clock. While skeletal muscle function exhibits... Show moreThe metabolic and inflammatory processes that are implicated in the development of cardiovascular diseases are under control of the biological clock. While skeletal muscle function exhibits circadian rhythms, it is unclear to what extent the beneficial health effects of exercise are restricted to unique time windows. We aimed to study whether the timing of exercise training differentially modulates the development of atherosclerosis and elucidate underlying mechanisms. We endurance-trained atherosclerosis-prone female APOE*3-Leiden.CETP mice fed a Western-type diet, a well-established human-like model for cardiometabolic diseases, for 1h five times a week for 4weeks either in their early or in their late active phase on a treadmill. We monitored metabolic parameters, the development of atherosclerotic lesions in the aortic root and assessed the composition of the gut microbiota. Late, but not early, exercise training reduced fat mass by 19% and the size of early-stage atherosclerotic lesions by as much as 29% compared to sedentary animals. No correlation between cholesterol exposure and lesion size was evident, as no differences in plasma lipid levels were observed, but circulating levels of the pro-inflammatory markers ICAM-1 and VCAM-1 were reduced with late exercise. Strikingly, we observed a time-of-day-dependent effect of exercise training on the composition of the gut microbiota as only late training increased the abundance of gut bacteria producing short-chain fatty acids with proposed anti-inflammatory properties. Together, these findings indicate that timing is a critical factor to the beneficial anti-atherosclerotic effects of exercise with a great potential to further optimize training recommendations for patients. Show less
Schönke, M.; Ying, Z.X.; Kovynev, A.; Panhuis, W.I.H.; Binnendijk, A.; Poel, S. van der; ... ; Rensen, P.C.N. 2023
The metabolic and inflammatory processes that are implicated in the development of cardiovascular diseases are under control of the biological clock. While skeletal muscle function exhibits... Show moreThe metabolic and inflammatory processes that are implicated in the development of cardiovascular diseases are under control of the biological clock. While skeletal muscle function exhibits circadian rhythms, it is unclear to what extent the beneficial health effects of exercise are restricted to unique time windows. We aimed to study whether the timing of exercise training differentially modulates the development of atherosclerosis and elucidate underlying mechanisms. We endurance-trained atherosclerosis-prone female APOE*3-Leiden.CETP mice fed a Western-type diet, a well-established human-like model for cardiometabolic diseases, for 1 h five times a week for 4 weeks either in their early or in their late active phase on a treadmill. We monitored metabolic parameters, the development of atherosclerotic lesions in the aortic root and assessed the composition of the gut microbiota. Late, but not early, exercise training reduced fat mass by 19% and the size of early-stage atherosclerotic lesions by as much as 29% compared to sedentary animals. No correlation between cholesterol exposure and lesion size was evident, as no differences in plasma lipid levels were observed, but circulating levels of the pro-inflammatory markers ICAM-1 and VCAM-1 were reduced with late exercise. Strikingly, we observed a time-of-day-dependent effect of exercise training on the composition of the gut microbiota as only late training increased the abundance of gut bacteria producing short-chain fatty acids with proposed anti-inflammatory properties. Together, these findings indicate that timing is a critical factor to the beneficial anti-atherosclerotic effects of exercise with a great potential to further optimize training recommendations for patients. Show less
Bogaards, F.A.; Gehrmann, T.; Beekman, M.; Akker, E. ben van den; Rest, O. van de; Hangelbroek, R.W.J.; ... ; Slagboom, P.E. 2022
The response to lifestyle intervention studies is often heterogeneous, especially in older adults. Subtle responses that may represent a health gain for individuals are not always detected by... Show moreThe response to lifestyle intervention studies is often heterogeneous, especially in older adults. Subtle responses that may represent a health gain for individuals are not always detected by classical health variables, stressing the need for novel biomarkers that detect intermediate changes in metabolic, inflammatory, and immunity-related health. Here, our aim was to develop and validate a molecular multivariate biomarker maximally sensitive to the individual effect of a lifestyle intervention; the Personalized Lifestyle Intervention Status (PLIS). We used H-1-NMR fasting blood metabolite measurements from before and after the 13-week combined physical and nutritional Growing Old TOgether (GOTO) lifestyle intervention study in combination with a fivefold cross-validation and a bootstrapping method to train a separate PLIS score for men and women. The PLIS scores consisted of 14 and four metabolites for females and males, respectively. Performance of the PLIS score in tracking health gain was illustrated by association of the sex-specific PLIS scores with several classical metabolic health markers, such as BMI, trunk fat%, fasting HDL cholesterol, and fasting insulin, the primary outcome of the GOTO study. We also showed that the baseline PLIS score indicated which participants respond positively to the intervention. Finally, we explored PLIS in an independent physical activity lifestyle intervention study, showing similar, albeit remarkably weaker, associations of PLIS with classical metabolic health markers. To conclude, we found that the sex-specific PLIS score was able to track the individual short-term metabolic health gain of the GOTO lifestyle intervention study. The methodology used to train the PLIS score potentially provides a useful instrument to track personal responses and predict the participant's health benefit in lifestyle interventions similar to the GOTO study. Show less
Our daily 24-h rhythm is synchronized to the external light-dark cycle resulting from the Earth's daily rotation. In the mammalian brain, the suprachiasmatic nucleus (SCN) serves as the master... Show moreOur daily 24-h rhythm is synchronized to the external light-dark cycle resulting from the Earth's daily rotation. In the mammalian brain, the suprachiasmatic nucleus (SCN) serves as the master clock and receives light-mediated input via the retinohypothalamic tract. Abrupt changes in the timing of the light-dark cycle (e.g., due to jet lag) cause a phase shift in the circadian rhythms in the SCN. Here, we investigated the effects of a 6-h delay in the light-dark cycle on PERIOD2::LUCIFERASE expression at the single-cell level in mouse SCN organotypic explants. The ensemble pattern in phase shift response obtained from individual neurons in the anterior and central SCN revealed a bimodal distribution; specifically, neurons in the ventrolateral SCN responded with a rapid phase shift, while neurons in the dorsal SCN generally did not respond to the shift in the light-dark cycle. We also stimulated the hypothalamic tract in acute SCN slices to simulate light-mediated input to the SCN; interestingly, we found similarities between the distribution and fraction of rapid shifting neurons (in response to the delay) and neurons that were excited in response to electrical stimulation. These results suggest that a subpopulation of neurons in the ventral SCN that have an excitatory response to light input, shift their clock more readily than dorsal located neurons, and initiate the SCN's entrainment to the new light-dark cycle. Thus, we propose that light-excited neurons in the anterior and central SCN play an important role in the organism's ability to adjust to changes in the external light-dark cycle. Show less
In both diurnal and nocturnal mammals, the timing of activity is regulated by the central circadian clock of the suprachiasmatic nucleus (SCN). The SCN is synchronized to the external light cycle... Show moreIn both diurnal and nocturnal mammals, the timing of activity is regulated by the central circadian clock of the suprachiasmatic nucleus (SCN). The SCN is synchronized to the external light cycle via the retinohypothalamic tract (RHT). To investigate potential differences in light processing between nocturnal mice and the diurnal rodent Rhabdomys pumilio, we mimicked retinal input by stimulation of the RHT ex vivo. Using Ca2+ imaging, we observed excitations as well as inhibitions of SCN neurons in response to electrical RHT stimulation. In mice, the vast majority of responses were excitatory (85%), whereas in Rhabdomys, the proportion of excitatory and inhibitory responses was similar (51% excitatory, 49% inhibitory). Glutamate blockers AP5 and CNQX blocked the excitatory responses to RHT stimulation but did not abolish the inhibitory responses in mice or Rhabdomys, indicating that the inhibitions were monosynaptically transmitted via the RHT. Simultaneous application of glutamate blockers with the GABA(A) antagonist gabazine blocked all inhibitory responses in mice, but not in Rhabdomys. Collectively, our results indicate that in Rhabdomys, considerably more inhibitory responses to light are present and that these responses are driven directly by the RHT. We propose that this increased proportion of inhibitory input could reflect a difference in the entrainment mechanism employed by diurnal rodents. Show less
G protein-coupled receptors (GPCRs) are known to be involved in tumor progression and metastasis. The adenosine A1 receptor (A1 AR) has been detected to be over-expressed in various cancer cell... Show moreG protein-coupled receptors (GPCRs) are known to be involved in tumor progression and metastasis. The adenosine A1 receptor (A1 AR) has been detected to be over-expressed in various cancer cell lines. However, the role of A1 AR in tumor development is not yet well characterized. A series of A1 AR mutations were identified in the Cancer Genome Atlas from cancer patient samples. In this study, we have investigated the pharmacology of mutations located outside of the 7-transmembrane domain by using a "single-GPCR-one-G protein" yeast system. Concentration-growth curves were obtained with the full agonist CPA for 12 mutant receptors and compared to the wild-type hA1 AR. Most mutations located at the extracellular loops (EL) reduced the levels of constitutive activity of the receptor and agonist potency. For mutants at the intracellular loops (ILs) of the receptor, an increased constitutive activity was found for mutant receptor L211R5.69 , while a decreased constitutive activity and agonist response were found for mutant receptor L113F34.51 . Lastly, mutations identified on the C-terminus did not significantly influence the pharmacological function of the receptor. A selection of mutations was also investigated in a mammalian system. Overall, similar effects on receptor activation compared to the yeast system were found with mutations located at the EL, but some contradictory effects were observed for mutations located at the IL. Taken together, this study will enrich the insight of A1 AR structure and function, enlightening the consequences of these mutations in cancer. Ultimately, this may provide potential precision medicine in cancer treatment. Show less
Buurstede, J.C.; Paul, S.N.; Bosscher, K. de; Meijer, O.C.; Kroon, J. 2022
Glucocorticoids exert their pleiotropic effects by activating the glucocorticoid receptor (GR), which is expressed throughout the body. GR-mediated transcription is regulated by a multitude of... Show moreGlucocorticoids exert their pleiotropic effects by activating the glucocorticoid receptor (GR), which is expressed throughout the body. GR-mediated transcription is regulated by a multitude of tissue- and cell type-specific mechanisms, including interactions with other transcription factors such as the androgen receptor (AR). We previously showed that the transcription of canonical glucocorticoid-responsive genes is dependent on active androgen signaling, but the extent of this glucocorticoid-androgen crosstalk warrants further investigation. In this study, we investigated the overall glucocorticoid-androgen crosstalk in the hepatic transcriptome. Male mice were exposed to GR agonist corticosterone and AR antagonist enzalutamide in order to determine the extent of androgen-dependency after acute and chronic exposure. We found that a substantial proportion of the hepatic transcriptome is androgen-dependent after chronic exposure, while after acute exposure the transcriptomic effects of glucocorticoids are largely androgen-independent. We propose that prolonged glucocorticoid exposure triggers a gradual upregulation of AR expression, instating a situation of androgen dependence which is likely not driven by direct AR-GR interactions. This indirect mode of glucocorticoid-androgen interaction is in accordance with the absence of enriched AR DNA-binding near AR-dependent corticosterone-regulated genes after chronic exposure. In conclusion, we demonstrate that glucocorticoid effects and their interaction with androgen signaling are dependent on the duration of exposure and believe that our findings contribute to a better understanding of hepatic glucocorticoid biology in health and disease. Show less
In mammals, behavioral activity is regulated both by the circadian system, orchestrated by the suprachiasmatic nucleus (SCN), and by arousal structures, including the serotonergic system. While the... Show moreIn mammals, behavioral activity is regulated both by the circadian system, orchestrated by the suprachiasmatic nucleus (SCN), and by arousal structures, including the serotonergic system. While the SCN is active at the same astronomical time in diurnal and nocturnal species, little data are available concerning the serotonergic (5HT) system in diurnal mammals. In this study, we investigated the functioning of the 5HT system, which is involved both in regulating the sleep/wake cycle and in synchronizing the SCN, in a diurnal rodent, Arvicanthis ansorgei. Using in situ hybridization, we characterized the anatomical extension of the raphe nuclei and we investigated 24 h mRNA levels of the serotonin rate-limiting enzyme, tryptophan hydroxylase 2 (tph2). Under both 12 h:12 h light/dark (LD) and constant darkness (DD) conditions, tph2 mRNA expression varies significantly over 24 h, displaying a bimodal profile with higher values around the (projected) light transitions. Furthermore, we considered several SCN outputs, namely melatonin, corticosterone, and locomotor activity. In both LD and DD, melatonin profiles display peak levels during the biological night. Corticosterone plasma levels show a bimodal rhythmic profile in both conditions, with higher levels preceding the two peaks of Arvicanthis locomotor activity, occurring at dawn and dusk. These data demonstrate that serotonin synthesis in Arvicanthis is rhythmic and reflects its bimodal behavioral phenotype, but differs from what has been previously described in nocturnal species. Show less
In mammals, behavioral activity is regulated both by the circadian system, orchestrated by the suprachiasmatic nucleus (SCN), and by arousal structures, including the serotonergic system. While... Show moreIn mammals, behavioral activity is regulated both by the circadian system, orchestrated by the suprachiasmatic nucleus (SCN), and by arousal structures, including the serotonergic system. While the SCN is active at the same astronomical time in diurnal and nocturnal species, little data are available concerning the serotonergic (5HT) system in diurnal mammals. In this study, we investigated the functioning of the 5HT system, which is involved both in regulating the sleep/wake cycle and in synchronizing the SCN, in a diurnal rodent, Arvicanthis ansorgei. Using in situ hybridization, we characterized the anatomical extension of the raphe nuclei and we investigated 24 h mRNA levels of the serotonin rate-limiting enzyme, tryptophan hydroxylase 2 (tph2). Under both 12 h:12 h light/dark (LD) and constant darkness (DD) conditions, tph2 mRNA expression varies significantly over 24 h, displaying a bimodal profile with higher values around the (projected) light transitions. Furthermore, we considered several SCN outputs, namely melatonin, corticosterone, and locomotor activity. In both LD and DD, melatonin profiles display peak levels during the biological night. Corticosterone plasma levels show a bimodal rhythmic profile in both conditions, with higher levels preceding the two peaks of Arvicanthis locomotor activity, occurring at dawn and dusk. These data demonstrate that serotonin synthesis in Arvicanthis is rhythmic and reflects its bimodal behavioral phenotype, but differs from what has been previously described in nocturnal species. Show less
Endo, Y.; Baldino, K.; Li, B.; Zhang, Y.T.; Sakthivel, D.; MacArthur, M.; ... ; Sinha, I. 2020
The ability of skeletal muscle to regenerate declines significantly with aging. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT), a critical component of the hypoxia... Show moreThe ability of skeletal muscle to regenerate declines significantly with aging. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT), a critical component of the hypoxia signaling pathway, was less abundant in skeletal muscle of old (23-25 months old) mice. This loss of ARNT was associated with decreased levels of Notch1 intracellular domain (N1ICD) and impaired regenerative response to injury in comparison to young (2-3 months old) mice. Knockdown of ARNT in a primary muscle cell line impaired differentiation in vitro. Skeletal muscle-specific ARNT deletion in young mice resulted in decreased levels of whole muscle N1ICD and limited muscle regeneration. Administration of a systemic hypoxia pathway activator (ML228), which simulates the actions of ARNT, rescued skeletal muscle regeneration in both old and ARNT-deleted mice. These results suggest that the loss of ARNT in skeletal muscle is partially responsible for diminished myogenic potential in aging and activation of hypoxia signaling holds promise for rescuing regenerative activity in old muscle. Show less
