The cAMP‐activated transcription factor CREB stimulates proliferation of myogenic progenitor cells during embryonic development, and genetic activation of CREB stimulates proliferation during muscle ...regeneration. Proliferation of muscle progenitor cells, known as satellite cells, declines during aging. We hypothesized that pharmacologic activation of the cAMP‐CREB pathway would promote satellite cell proliferation after injury and that cAMP‐regulated CREB co‐activators (CRTCs) contribute to CREB‐regulated target gene expression during satellite cell proliferation. To test this, we created ROSA26‐GsDREADD knock‐in mice for tissue‐specific expression of a Gs‐coupled DREADD (Designer Receptor Exclusively Activated by Designer Drugs, “GsD”). After tamoxifen‐induced expression of GsD in muscle satellite cells in young adult mice, activation of the receptor by the GsD ligand CNO (clozapine N‐oxide) increased proliferation after muscle injury. Consistent with our hypothesis, myoblasts over‐expressing constitutively active CRTC2 had increased cell proliferation in vitro, and Crtc2 knockout mice had reduced proliferation after muscle injury. Our results indicate that cAMP signaling enhances satellite cell proliferation in vivo and cAMP regulated CREB coactivator 2 (CRTC2) contributes to the proliferative response after muscle injury. ROSA26‐GsDREADD mice can therefore be used to stimulate cAMP signaling in specific cell populations in vivo to study the effects of pharmacologic activation of cAMP signaling without using ligands that have pleiotropic effects in other cell types.
Support or Funding Information
Research reported in this was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01‐AR059847. DA was supported by a Postdoctoral Fellowship from the American Heart Association (15POST25090134). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Mitochondrial Ca
2+ signals have been proposed to accelerate oxidative metabolism and ATP production to match Ca
2+-activated energy-consuming processes. Efforts to understand the signaling role of ...mitochondrial Ca
2+ have been hampered by the inability to manipulate matrix Ca
2+ without directly altering cytosolic Ca
2+. We were able to selectively buffer mitochondrial Ca
2+ rises by targeting the Ca
2+-binding protein S100G to the matrix. We find that matrix Ca
2+ controls signal-dependent NAD(P)H formation, respiration, and ATP changes in intact cells. Furthermore, we demonstrate that matrix Ca
2+ increases are necessary for the amplification of sustained glucose-dependent insulin secretion in β cells. Through the regulation of NAD(P)H in adrenal glomerulosa cells, matrix Ca
2+ also acts as a positive signal in reductive biosynthesis, which stimulates aldosterone secretion. Our dissection of cytosolic and mitochondrial Ca
2+ signals reveals the physiological importance of matrix Ca
2+ in energy metabolism required for signal-dependent hormone secretion.
► Mitochondrial matrix Ca
2+ activates energy metabolism in situ ► Matrix Ca
2+ is a regulator of hormone secretion distinct from cytosolic Ca
2+ ► Matrix Ca
2+ is an essential signal for the amplification of insulin secretion ► NAD(P)H formation and aldosterone secretion are regulated by matrix Ca
2+
Glucose-induced insulin secretion from pancreatic β-cells depends on mitochondrial activation. In the organelle, glucose-derived pyruvate is metabolised along the oxidative and anaplerotic pathway to ...generate downstream signals leading to insulin granule exocytosis. Entry into the oxidative pathway is catalysed by pyruvate dehydrogenase (PDH) and controlled in part by phosphorylation of the PDH E1α subunit blocking enzyme activity. We find that glucose but not other nutrient secretagogues induce PDH E1α phosphorylation in INS-1E cells and rat islets. INS-1E cells and primary β-cells express pyruvate dehydrogenase kinase (PDK) 1, 2 and 3, which mediate the observed phosphorylation. In INS-1E cells, suppression of the two main isoforms, PDK1 and PDK3, almost completely prevented PDH E1α phosphorylation. Under basal glucose conditions, phosphorylation was barely detectable and therefore the enzyme almost fully active (90% of maximal). During glucose stimulation, PDH is only partially inhibited (to 78% of maximal). Preventing PDH phosphorylation in situ after suppression of PDK1, 2 and 3 neither enhanced pyruvate oxidation nor insulin secretion. In conclusion, although glucose stimulates E1α phosphorylation and therefore inhibits PDH activity, this control mechanism by itself does not alter metabolism-secretion coupling in INS-1E clonal β-cells.
► Glucose induces phosphorylation of PDH in INS-1E cells and islets. ► Glucose-induced PDH E1α phosphorylation is mainly mediated by PDK1 and PDK3. ► Glucose stimulation causes a partial inhibition of pyruvate dehydrogenase activity. ► Preventing PDH E1α phosphorylation does not alter glucose-induced insulin secretion.
Glucose-evoked mitochondrial signals augment ATP synthesis in the pancreatic β cell. This activation of energy metabolism increases the cytosolic ATP/ADP ratio, which stimulates plasma membrane ...electrical activity and insulin granule exocytosis. We have recently demonstrated that matrix pH increases during nutrient stimulation of the pancreatic β cell. Here, we have tested whether mitochondrial matrix pH controls oxidative phosphorylation and metabolism-secretion coupling in the rat β-cell line INS-1E. Acidification of the mitochondrial matrix pH by nigericin blunted nutrient-dependent respiratory and ATP responses (continuously monitored in intact cells). Using electrophysiology and single cell imaging, we find that the associated defects in energy metabolism suppress glucose-stimulated plasma membrane electrical activity and cytosolic calcium transients. The same parameters were unaffected after direct stimulation of electrical activity with tolbutamide, which bypasses mitochondrial function. Furthermore, lowered matrix pH strongly inhibited sustained, but not first-phase, insulin secretion. Our results demonstrate that the matrix pH exerts a control function on oxidative phosphorylation in intact cells and that this mode of regulation is of physiological relevance for the generation of downstream signals leading to insulin granule exocytosis. We propose that matrix pH serves a novel signaling role in sustained cell activation.--Akhmedov, D., Braun, M., Mataki, C., Park, K.-S., Pozzan, T., Schoonjans, K., Rorsman, P., Wollheim, C. B., Wiederkehr, A. Mitochondrial matrix pH controls oxidative phosphorylation and metabolism-secretion coupling in INS-1E clonal β cells.
Diet-induced obesity (DIO) represents the major cause for the current obesity epidemic, but the mechanism underlying DIO is unclear. β-Adrenergic receptors (β-ARs) play a major role in sympathetic ...nervous system-mediated (SNS-mediated) diet-induced energy expenditure (EE). Rbc express abundant β-ARs; however, a potential role for rbc in DIO remains untested. Here, we demonstrated that high-fat, high-caloric diet (HFD) feeding increased both EE and blood O2 content, and the HFD-induced increases in blood O2 level and in body weight gain were negatively correlated. Deficiency of β-ARs in rbc reduced glycolysis and ATP levels, diminished HFD-induced increases in both blood O2 content and EE, and resulted in DIO. Importantly, specific activation of cAMP signaling in rbc promoted HFD-induced EE and reduced HFD-induced tissue hypoxia independent of obesity. Both HFD and pharmacological activation cAMP signaling in rbc led to increased glycolysis and ATP levels. These results identify a previously unknown role for rbc β-ARs in mediating the SNS action on HFD-induced EE by increasing O2 supply, and they demonstrate that HFD-induced EE is limited by blood O2 availability and can be augenmented by increased O2 supply.
Correct endoplasmic reticulum (ER) function is critical for the health of secretory cells, such as the pancreatic β-cell, and ER stress is often a contributory factor to β-cell death in type 2 ...diabetes. We have used an insulin-secreting cell line with inducible expression of dominant negative (DN) HNF1α, a transcription factor vital for correct β-cell development and function, to show that HNF1α is required for Xbp1 transcription and maintenance of the normal ER stress response. DN HNF1α expression sensitizes the β-cell to ER stress by directly down-regulating Xbp1 transcription, whereas Atf6 is unaffected. Furthermore, DN HNF1α alters calcium homeostasis, resulting in elevated cytoplasmic calcium and increased store-operated calcium entry, whereas mitochondrial calcium uptake is normal. Loss of function of XBP1 is toxic to the β-cell and decreases production of the ER chaperone BiP, even in the absence of ER stress. DN HNF1α-induced sensitivity to cyclopiazonic acid can be partially rescued with the chemical chaperone tauroursodeoxycholate. Rat insulin 2 promoter-DN HNF1α mouse islets express lower levels of BiP mRNA, synthesize less insulin, and are sensitized to ER stress relative to matched control mouse islets, suggesting that this mechanism is also operating in vivo.
During cycles of fasting and feeding, liver function is regulated by both transcriptional and post-translational events. Regulated protein degradation has recently emerged as a key mechanism to ...control abundance of specific hepatic proteins under different nutritional conditions. As glucagon signaling through cAMP and PKA is central to glucose output during fasting, we hypothesized that this signaling pathway may also regulate ubiquitin ligases in the fasted state. Here we show that fasting stimuli promote expression of the short isoform of the E3 ubiquitin ligase Nedd4l in primary mouse hepatocytes. Nedd4l-short mRNA and NEDD4L (short isoform) protein accumulate in glucagon-treated primary mouse hepatocytes and in liver tissues during fasting. We identified a functional cAMP response element in the alternate Nedd4l-short promoter; mutation of this element blunts cAMP-induced expression of a Nedd4l reporter construct. CREB occupies the endogenous Nedd4l locus near this element. CREB and its co-activator CRTC2, both activated by fasting stimuli, contribute to glucagon-stimulated Nedd4l-short expression in primary hepatocytes. siRNA-mediated Nedd4l depletion in primary hepatocytes did not affect gluconeogenic gene expression, glucose output or glycogen synthesis. Our findings reveal a new mechanism of Nedd4l transcriptional regulation in liver cells.
To assess the prevalence and clinical features of individuals affected by spinocerebellar ataxia 36 (SCA36) at a large tertiary referral center in the United States.
A total of 577 patients with ...undiagnosed sporadic or familial cerebellar ataxia comprehensively evaluated at a tertiary referral ataxia center were molecularly evaluated for SCA36. Repeat primed PCR and fragment analysis were used to screen for the presence of a repeat expansion in the
gene.
Fragment analysis of triplet repeat primed PCR products identified a GGCCTG hexanucleotide repeat expansion in intron 1 of
in 4 index cases. These 4 SCA36-positive families comprised 2 distinct ethnic groups: white (European) (2) and Asian (Japanese 1 and Vietnamese 1). Individuals affected by SCA36 exhibited typical clinical features with gait ataxia and age at onset ranging between 35 and 50 years. Patients also suffered from ataxic or spastic limbs, altered reflexes, abnormal ocular movement, and cognitive impairment.
In a US population, SCA36 was observed to be a rare disorder, accounting for 0.7% (4/577 index cases) of disease in a large undiagnosed ataxia cohort.
Glucose-evoked mitochondrial signals augment ATP synthesis in the pancreatic b cell. This activation of energy metabolism increases the cytosolic ATP/ADP ratio, which stimulates plasma membrane ...electrical activity and insulin granule exocytosis. We have recently demonstrated that matrix pH increases during nutrient stimulation of the pancreatic b cell. Here, we have tested whether mitochondrial matrix pH controls oxidative phosphorylation and metabolism-secretion coupling in the rat b-cell line INS-1E. Acidification of the mitochondrial matrix pH by nigericin blunted nutrient-dependent respiratory and ATP responses (continuously monitored in intact cells). Using electrophysiology and single cell imaging, we find that the associated defects in energy metabolism suppress glucose-stimulated plasma membrane electrical activity and cytosolic calcium transients. The same parameters were unaffected after direct stimulation of electrical activity with tolbutamide, which bypasses mitochondrial function. Furthermore, lowered matrix pH strongly inhibited sustained, but not first-phase, insulin secretion. Our results demonstrate that the matrix pH exerts a control function on oxidative phosphorylation in intact cells and that this mode of regulation is of physiological relevance for the generation of downstream signals leading to insulin granule exocytosis. We propose that matrix pH serves a novel signaling role in sustained cell activation.-Akhmedov, D., Braun, M., Mataki, C., Park, K.-S., Pozzan, T., Schoonjans, K., Rorsman, P., Wollheim, C. B., Wiederkehr, A. Mitochondrial matrix pH controls oxidative phosphorylation and metabolism-secretion coupling in INS-1E clonal b cells.
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