bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating the effects of hyperbaric oxygen on mitochondrial function of endothelial progenitor cells.
Authors: Aylin‐Burns Nukhet , Krager Kimberly J. , Zhou Daohong , Hauer‐Jensen Martin
Published: November 2023

Hyperbaric oxygen treatment (HBOT) is a FDA approved adjunctive therapy for 14 different conditions, including delayed radiation injury, impaired wound healing, compromised skin grafts and compression sickness. Patients typically receive multiple daily treatments in chambers pressurized up to 3.0 atmospheres with 100% oxygen, depending on the disease state/type. We previously demonstrated that HBOT stimulates vasculogenic stem cell growth and differentiation in vivo, by creating a physiological redox‐active autocrine loop in circulating progenitor cells. Recent studies also supported the role of HBOT in activation of redox‐regulated pathways including hypoxia inducible factor (HIF), mitogen‐activated protein kinase (MAPK), thioredoxin (Trx) system, nuclear factor erythroid 2 (Nrf2) as well as nitric oxide (NO) signaling. In this study we proposed that alterations in mitochondrial function is an early event following HBOT. Mice were treated with HBO for 90 min at 2.8 ATA. Cellular bioenergetics and mitochondrial respiration were assessed immediately following treatment in primary cells using Seahorse Extracellular flux analyzer. Oxygen consumption rate (OCR) was measured after the sequential injections of oligomycin (inhibitor of ATP synthase), carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP; uncoupler of mitochondrial inner membrane), and antimycin A (inhibitor of complex III). HBOT significantly altered the basal respiration as well as spare respiratory capacity. Furthermore, the mitochondrial mass is significantly increased following a single HBOT session. Future studies will determine the effect of multiple HBOT sessions on mitochondrial function and cellular bioenergetics, which can provide novel targets to improve the efficacy of an already safe and approved therapy.

Support or Funding Information

This project is funded by NIGMS, Center of Biomedical Research Excellence Award, P20GM109005
Early Stage Type 1 Diabetes Augments the Mechanoreflex in Rats
Authors: Garcia Elizabeth A. , Grotle Ann‐Katrin , Stone Audrey J
Published: November 2023

Studies have shown that type 1 diabetic (T1DM) rats develop mechanical allodynia as early as four days post streptozotocin (STZ) injection. This is thought to occur through mechanisms affecting the same thin fiber afferents that evoke the mechanoreflex. In this study we attempted to determine the effects of T1DM on the mechanoreflex at two different time points of the disease as well as determine the role of Piezo channels in evoking the mechanoreflex in T1DM rats. We injected (i.p.) 50 mg/kg of Streptozotocin (STZ) or the vehicle (CTL) in either sex and waited 1wk (STZ: BW= 257±16 g, glucose=472±29 mg/dL, HbA1C=5.9±0.3%; CTL: BW=294±42 g, glucose=172±9 mg/dL, HbA1C=4.26±0.1%) or 3wks (STZ: BW=285±9 g, glucose=485±33 mg/dL, HbA1C=10.13±0.3%; CTL: BW=340±30 g, glucose=220±44 mg/dL, HbA1C=4.5±0.1%). In unanaesthetized decerebrate rats, we stretched the Achilles tendon for 30s and measured the pressor and cardioaccelerator responses. We then compared the pressor and cardioaccelerator responses to tendon stretch before and after injecting GsMTx‐4 (10mg/100ml), a selective mechano‐gated Piezo channel inhibitor, into the arterial supply of the hindlimb muscles in rats 1wk after STZ injection. We found that the pressor (STZ: ΔMAP=49.17±10 mmHg, n=6; CTL: ΔMAP=22.13±6 mmHg, n=8; p=0.01) as well as the cardioaccelerator (STZ: ΔHR=16.33±4 bpm, n=6; CTL: ΔHR=9.25±2 bpm, n=8; p=0.05) responses to tendon stretch were exaggerated 1wk after injecting STZ. Conversely, the pressor (STZ: ΔMAP=30.8±6 mmHg, n=10; CTL: ΔMAP=32.67±7 mmHg, n=6; p=0.42) as well as the cardioaccelerator (STZ: ΔHR=13.7±4 bpm, n=10; CTL: ΔHR=11.83±4 bpm, n=6; p=0.76) responses to tendon stretch were not different 3wks after injecting STZ compared to CTL. Furthermore, GsMTx‐4 appears to attenuate the pressor (before GsMTx‐4: ΔMAP=70±3 mmHg; after GsMTx‐4: ΔMAP=50±2 mmHg, n=2; p=0.07) and cardioaccelerator (before GsMTx‐4: ΔHR=12.5±0.5 bpm; after GsMTx‐4: ΔHR=20±2 bpm, n=2; p=0.06) responses to tendon stretch in rats 1wk after STZ injection. The developed tensions from tendon stretch were similar within each comparison. We conclude that the mechanoreflex is augmented in the early stage of T1DM and that Piezo channels likely play a role in this response.
Exaggerated sympathetic and pressor responses to exercise pressor reflex activation are attenuated by intracerebroventricular insulin administration in type 2 diabetic rats
Authors: Mizuno Masaki , Mitchell Jere , Smith Scott A
Published: November 2023

The cardiovascular response to exercise is abnormally exaggerated in patients with type 2 diabetes mellitus (T2D). We have previously demonstrated that stimulation of the exercise pressor reflex (EPR) during muscle contraction elicits exaggerated increases in renal sympathetic nerve activity (RSNA) contributing significantly to this heightened circulatory responsiveness. However, the mechanisms underlying this EPR dysfunction remain to be elucidated. Evidence suggests that the availability of brain insulin is reduced in T2D. Given that sympathetic activity is primarily regulated within the brain, we hypothesized that a decrease in brain insulin contributes to EPR‐induced sympathetic overactivity in this disease. To test this hypothesis, we measured changes in RSNA and mean arterial pressure (MAP) during stimulation of the EPR in decerebrated Sprague‐Dawley rats given either a normal diet (control) or a high fat diet (in combination with a one‐time low dose of streptozotocin to produce T2D) for 14–16 weeks. Compared to normal diet‐fed rats, animals receiving the high fat diet had significantly increased plasma insulin (1.6±0.2 vs. 4.1±0.4 ng/mL, P<0.05) as well as fasting blood glucose (94±5 vs. 120±5 mg/dL, P<0.05). Compared to control animals, stimulation of the EPR by electrically‐induced muscle contraction evoked significantly greater increases in RSNA (Δ = 36±10 vs. 122±20 %, P<0.05) and MAP (Δ = 15±1 vs. 49±5 mmHg, P<0.05) in T2D rats. Intracerebroventricular insulin administration significantly attenuated the exaggerated sympathetic (Δ = 111±20 vs. 50±17 %, P=0.05) and pressor responses (Δ = 49±5 vs. 12±4 mmHg, P<0.05) to EPR activation in T2D rats but did not have a significant effect in control animals (RSNA: Δ = 43±2 vs. 33±13 %; MAP: Δ = 15±1 vs. 7±4 mmHg). These findings suggest that the heightened cardiovascular response to exercise in T2D may be mediated by EPR‐induced sympathetic dysfunction resulting as a consequence of decreased insulin availability within the brain.

Support or Funding Information

Supported by the Lawson & Rogers Lacy Research Fund in Cardiovascular Disease.
Expression and Distribution of Pro‐Inflammatory Cytokines within Dorsal Root Ganglion of Rats with Femoral Artery Occlusion
Authors: Li Jianhua , Lu Jian , Xing Jihong
Published: November 2023

Using a rat model of peripheral arterial disease (PAD), prior studies have demonstrated that augmented sympathetic nerve and blood pressure responses evoked by activation of the exercise pressor reflex are attenuated after blocking activities of pro‐inflammatory cytokines (PICs) such as IL‐6 and TNF‐α. The present study was to examine the protein expression levels of PICs in dorsal root ganglion (DRG) of control rats and rats with 72 hours of femoral artery occlusion. Our results show that femoral occlusion significantly enhanced the levels of IL‐1β, IL‐6 and TNF‐α as compared with controls (P<0.05, n=6 in each group). In addition, peripherin and NF200 were used to determine DRG neurons supplied by A‐fiber containing group III fiber and C‐fiber/group IV thin fiber, respectively. We observed that IL‐1β and IL‐6 were present in peripherin‐ and NF200‐positive DRG neurons, whereas TNF‐α was found for the most part in only peripherin‐positive DRG neurons. Expression levels of PIC receptors including IL‐6R and TNFR1 were also found to be upregulated in DRG of rats with femoral artery occlusion (P<0.05 vs. control rats). Overall, our data suggest that PIC mechanisms contribute to augmented sympathetic responsiveness likely via the mechanical and metabolic component of the exercise pressor reflex when arterial blood supply to the hindlimb is deficient under ischemic conditions as observed in PAD.

Support or Funding Information

NIH P01 HL096570 and AHA Established Investigator Award 0840130N
Sex Differences in Sympathetic Vasoconstrictor Responsiveness and Sympatholysis
Authors: Just Timothy P , DeLorey Darren S
Published: November 2023

Sympathetic nervous system control of blood pressure and skeletal muscle vascular resistance appears to differ between males and females. The mechanism(s) responsible for sex differences in neurovascular control have not been fully elucidated. Nitric Oxide (NO) has been shown to inhibit sympathetic vasoconstriction in resting and contracting skeletal muscle and estrogen appears to modulate NO synthase (NOS) expression and NO bioavailability. The relative abundance of estrogen in females compared to males suggests that NO‐mediated inhibition of sympathetic vasoconstriction may be augmented in females relative to males. Therefore, the purpose of the present study was to investigate the hypothesis that compared to males, females would have blunted sympathetic vasoconstrictor responsiveness and augmented NO‐mediated inhibition of sympathetic vasoconstriction at rest and during exercise. Male (n=8) and Female (n=10) Sprague‐Dawley rats were anesthetized and surgically instrumented for measurement of arterial blood pressure and femoral artery vascular conductance (FVC) and stimulation of the lumbar sympathetic chain. The percentage change of FVC (%FVC) in response to sympathetic chain stimulation delivered at 2 and 5 Hz was determined at rest and during triceps surae muscle contraction before (control) and after NOS‐blockade (L‐NAME; 10 mg·kg−1 IV). At rest, vasoconstrictor responsiveness to sympathetic stimulation delivered at 2Hz was augmented (p<0.05) in females (−33 ± 8%) compared to males (−26 ± 6%), whereas vasoconstrictor responsiveness to sympathetic stimulation delivered at 5Hz was not different (p>0.05) between females (−55 ± 7%) and males (−47 ± 7%). Muscle contraction blunted (p<0.05) sympathetic vasoconstrictor responsiveness in both female and male rats; however the magnitude of sympatholysis was greater (p<0.05) in female (2Hz: 22 ± 8%; 5Hz: 30 ± 7%) compared to male (2Hz: 12 ± 6%; 5Hz: 13 ± 8%) rats. NOS blockade did not alter (p>0.05) sympatholysis of responses evoked at 2Hz in females or males; however L‐NAME reduced (p<0.05) sympatholysis of responses evoked at 5Hz in female rats. These data demonstrate that resting sympathetic vasoconstrictor responsiveness was augmented in female rats compared to male rats in a stimulation frequency‐dependent manner. Furthermore, sympatholysis was enhanced in female compared to male rats and this was partly attributable to augmented NO‐mediated inhibition of sympathetic vasoconstriction.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada (NSERC), Canadian Foundation for Innovation, and Alberta Advanced Education and Technology.
Role of Alpha‐Adrenergic Vasoconstriction in Regulating Skeletal Muscle Blood Flow during Single Leg Knee Extension Exercise with Advancing Age
Authors: Ratchford Stephen M , Clifton Heather L , Barret‐O'Keefe Zachary M , Broxterman Ryan M , Gifford Jayson R , Hydren Jay R , Rossman Matthew J , Ives Stephen J , Trinity Joel D , Witman Melissa AH , Garten Ryan S , Morgan David E , Nelson Ashley D , Richardson Russell S , Wray David Walter
Published: November 2023

Background

Aging is associated with augmented sympathetic nervous system activity, which promotes vasoconstriction in the peripheral circulation through the alpha‐adrenergic pathway. However, whether this age‐related sympathoexcitation restrains exercising skeletal muscle blood flow and vascular conductance remains poorly understood.

Methods

In nine young (4F/5M, 25 ± 5 yr, 178 ± 8 cm, 73 ± 12 kg) and seven old (2F/5M, 63 ± 11 yr, 175 ± 9 cm, 78 ± 15 kg) healthy volunteers, we evaluated changes in leg blood flow (LBF, Doppler ultrasound), mean arterial pressure (MAP) and leg vascular conductance (LVC) before and after femoral intra‐arterial infusion of phentolamine (a nonselective alpha‐adrenergic antagonist) at rest and during knee‐extensor exercise (0, 5, and 10W). Results are presented as the change from control to adrenergic blockade conditions.

Results

At rest, similar increases in LBF (269 ± 45 vs. 250 ± 60 ml/min, young vs. older) and LVC (3.4 ± 0.6 vs. 2.9 ± 0.6 ml/min/mmHg, young vs. old) were observed between groups following the adrenergic blockade. During exercise, the adrenergic blockade increased LBF to a similar degree in both young (0W: 330 ± 116, 5W: 178 ± 130, and 10W: 239 ± 127ml/min) and old (0W: 209 ± 57, 5W: 251 ± 22, and 10W: 165 ± 75 ml/min), with no difference between work rates in either group. Similarly, LVC increased as a consequence of the adrenergic blockade in both young (0W: 4.6 ± 1.2, 5W: 3.0 ± 1.2, and 10W: 3.1 ± 1.2 ml/min/mmHg) and old (0W: 3.6 ± 1.1, 5W: 4.2 ± 0.9, and 10W: 3.8 ± 0.9 ml/min/mmHg), with no difference between work rates. Both at rest and during exercise, MAP did not differ between groups or as a consequence of drug administration.

Conclusions

Using a pharmacologic approach to locally block alpha adrenergic vasoconstriction, we observed a similar drug‐induced change in leg hemodynamics in both the young and old groups. These data suggest that sympathetic restraint of limb blood flow persists during small muscle mass exercise, and that this adrenergic response is not impacted by advancing age.

Support or Funding Information

Funded in part by the National Institutes of Health (HL118313) and the U.S. Department of Veterans Affairs (RX001697, RX001418, E6910R).
Revisiting “Resetting” of Baroreflexes During Canine Exercise
Authors: Wurster Robert D.
Published: November 2023

At rest and during exercise, systemic arterial blood pressure(SAP) is regulated by classic arterial baroreceptor reflexes mediated by receptors located largely in the carotid sinus(CS) and the ascending aorta. Many investigators have concluded that exercise causes these controls to be “reset” to higher pressures.

Presently, changes of SAP and heart rate(HR) during treadmill exercise (up a 10% grade) were investigated using 5 exercise‐trained dogs that were surgically instrumented with a bilateral, vascularly‐isolated CS preparation and aortic nerve denervations. This allows systematic, stepwise changes of CS pressures(CSP) (25 mm Hg steps between 50 and 200 mm Hg) independent of SAP changes and at various exercise levels (5 min. steps from 0 to almost 10 Km/hr) without involvement of the aortic baroreceptors.

At all levels of exercise, SAP responses to CSP's below 100 mm Hg were nearly identical. At low exercise levels (3.2 Km/hr), SAP changes at all CSP's (50 to 200 mm Hg) remained unaltered compared to at rest. However, with higher exercise levels at higher CSP's (from 100 to 200 mm Hg), changes in SAP became greatly attenuated.

On the other hand, HR changes occurred with every change in exercise level, regardless of the CSP. At rest, changes in HR occurred with every rise or fall of CSP. However, during exercise, HR responses to CSP changes above 100 mm Hg were markedly attenuated, especially at higher CSP's.

These data suggest that baroreflex control of SAP is not significantly altered by the exercise intensity, suggesting little role of central command and mechanoreflex resetting. However, metaboreflexes limit minimal levels of SAP, resulting in elevation of SAP at higher exercise levels. Baroreflex control of HR is influenced by higher levels of exercise, suggesting a “resetting” role of central command and/or mechanoreflexes. Similar to SAP control, the minimal HR responses are limited by activation of metaboreflexes resulting in higher HR responses at all CS pressures. In conclussion, CS reflex control of SAP and HR during exercise are different but have in common the influences of metaboreflexes.

Support or Funding Information

Public Health Service Grant HL 08682
AdipoRon, pan‐adiponectin receptor agonist, activates mitochondrial metabolism via AMPK‐PGC1α pathway in the skeletal muscle
Authors: Balasubramanian Priya , Howell Porsha , Martin Stephen , Anderson Rozalyn
Published: November 2023

Adiponectin is an adipose tissue‐derived peptide multimer that impinges on skeletal muscle cellular metabolism to activate lipid utilization and mitochondrial oxidative pathways. The actions of adiponectin resemble exercise in that adiponectin signaling activates AMPK and PGC1α downstream of AdipoR1 and R2 receptor in the skeletal muscle. It is known that interventions that increase PGC1a in the skeletal muscle enhance endurance and protect against muscle aging. AdipoRon is a recently identified pan‐adiponectin receptor agonist. We hypothesize that activation of adiponectin receptor using AdipoRon will activate PGC1α and mitochondrial metabolism in the skeletal muscle. Our results show that AdipoRon increases phosphorylation of AMPK (Thr172) and its downstream target acetyl‐coA carboxylase (ACC) within 10 minutes of treatment both in cells (NIH 3T3 fibroblasts and primate peripheral blood mononuclear cells (PBMCs)) and in skeletal muscle of treated mice. AdipoRon treatment also caused an increase in the expression of gene targets of PGC‐1a involved in lipid metabolism namely Cpt1a, Acadm and Acox1 both in cells and in skeletal muscle of AdipoRon treated mice. Interestingly, the effects of AdipoRon were fiber‐type specific as the transcriptional changes were only observed in gastrocnemius muscle with predominantly type II glycolytic fibers that are more vulnerable for aging and not in soleus enriched for type I oxidative fibers. Chronic AdipoRon treatment also resulted in the up regulation of genes involved in mitochondrial fusion process, Mfn1 and Mfn2, suggesting its involvement in modulating mitochondrial dynamics to facilitate oxidative metabolism. Further investigations are needed to determine the precise metabolic consequence of adiponectin receptor activation by AdipoRon and to determine the therapeutic potential of this promising new drug to modulate the processes implicated in skeletal muscle aging.

Support or Funding Information

Supported by NIHAG047358
Satellite Cell‐Mediated Myonuclear Accretion Is Required For Mechanical Overload‐Induced Hypertrophy In Young Growing Mice
Authors: Murach Kevin , White Sarah , Wen Yuan , Ho Angel , Kosmac Kate , McCarthy John , Peterson Charlotte
Published: November 2023

When skeletal muscle hypertrophy is induced by mechanical or genetic means, numerous investigations report that satellite cell‐mediated myonuclear accretion is not required to promote muscle growth in adult mice (≥16 weeks old). However, recent evidence suggests that the satellite cell requirement during hypertrophy is influenced by maturational age. That is, if a hypertrophic stimulus is imposed on growing muscle during adolescence, satellite cell‐mediated myonuclear accretion may become necessary to support the additional growth. The purpose of this investigation was to rigorously test whether maturational age alters the requirement for satellite cell‐mediated myonuclear accretion during mechanically‐induced skeletal muscle hypertrophy. Using the Pax7CreR‐R26RDTA mouse, we conditionally depleted satellite cells in adolescent mice (8 weeks of age) via five consecutive daily tamoxifen injections, then allowed a two‐week washout (SC−). Age‐matched vehicle‐treated Pax7CreR‐R26RDTA mice were used as controls (SC+). Tamoxifen‐ and vehicle‐treated mice were then sham surgerized or synergist ablated for 10 days via a modified technique that overloads the plantaris muscle but minimizes muscle regeneration (n=6–8 per group). Satellite cell density (Pax7+ cells/fiber), embryonic myosin heavy chain expression (eMyHC), and muscle fiber cross sectional area were evaluated via immunohistochemistry. Myonuclear counts (myonuclei/100 millimeters) were performed on isolated single muscle fibers. Only tamoxifen‐treated mice with ≥90% satellite cell depletion were included in this analysis. Following 10 days of mechanical overload of the plantaris, SC+ mice experienced a 60% increase in satellite cell density (P<0.05). Muscle fiber cross sectional area and myonuclear number increased by 20% in SC+ (P<0.05), but did not change in SC− mice (P>0.05). Expression of eMyHC across all mice was <2%, indicating that overload surgery did not induce a regenerative response. Resident myonuclei of young growing mice appear inherently different than those of adult mice in their ability to compensate during hypertrophy without satellite cells.

Support or Funding Information

NIH RO1 AR060701 07
Fn14 Expression is Lower in Female Compared to Male Skeletal Muscle, Independent of Adult Age
Authors: Blum Jamie , Roman Heather , Thalacker‐Mercer Anna
Published: November 2023

Background

Tweak, a pro‐inflammatory cytokine, and its receptor, Fn14, are recognized as important inflammatory mediators. In skeletal muscle (SkM), elevated TWEAK signaling is associated with reduced insulin sensitivity, reduced mitochondrial biogenesis and SkM atrophy. Further, TWEAK signaling is dysregulated with aging, underlying SkM deterioration and pathological remodeling. Several studies suggest a relationship between estrogen and TWEAK signaling, such that estrogen increases TWEAK expression while suppressing Fn14 expression. Sex‐specific differences in SkM TWEAK and Fn14 have not be identified, but are likely relevant for SkM health in aging.

Objectives

We sought to evaluate whether SkM of young females has lower Fn14 and higher TWEAK expression compared to young males, and whether Fn14 and TWEAK changes with age in both sexes.

Methods

SkM biopsies were collected from the vastus lateralis of young (n=5) and old females (n=6) and young (n=6) and old males (n=4). RNA was extracted from biopsy tissue using TRIzol reagent. Gene expression was measured using qPCR. In a follow‐up experiment human myogenic progenitor cells (MPCs) were isolated from young females (YF‐MPC, n=5) and young males (YM‐MPC, n=6). Cells were cultured in Ham's F12 supplemented with 20% FBS and 5ng/ml bFGF (GM). Once cells reached 75% confluence, all cultures were switched to differentiation media [(DM), Ham's F12 + 2% HI equine serum]. MPCs were cultured in DM for 72 hours and then continued in DM for an additional 48 hours with or without 10ng/ml TNFα. RNA was isolated and gene expression measured using qPCR.

Results

At the whole tissue level, females had a 2.86 fold lower Fn14 expression (p<0.05) and 1.34 fold higher TWEAK expression (p<0.05) compared males, independent of age. In response to TNFα treatment, differentiated YF‐MPC experienced a 1.7 fold increase in Fn14 expression (p<0.05) while no change in Fn14 expression was observed in differentiated YM‐MPC. TNFα treatment did not affect TWEAK expression in either differentiated YF‐ or YM‐MPC (p>0.05).

Conclusion

As hypothesized, females have lower expression of Fn14 and higher expression of TWEAK in skeletal muscle, likely driven, in part, through estrogen‐related mechanisms. However, differences in Fn14 and TWEAK were not differential with age in the female SkM. The young female SkM was collected when the participants had low levels of estrogen, which could partially explain why we observed no difference with age in the females. Intriguingly, TNFα induced Fn14 in differentiated YF‐MPCs, but had no effect in differentiated YM‐MPCs. The significance of this difference for pathological remodeling of skeletal muscle, especially during regeneration, requires further elucidation.

Support or Funding Information

Cornell University
Age‐Related Alterations in Cutaneous Vascular and Sweating Responses Depend on Sex and Types of Receptor Agonist
Authors: Fujii Naoto , McNeely Brendan D , Halili Lyra , Zhang Sarah Y , Danquah Mercy O , Kenny Glen P
Published: November 2023

In humans, peripheral cutaneous vascular and sweating function may be altered by aging. For example, cholinergic sweating (Stapleton et al. Physiol Rep, 2(7), pii: e12078, 2014) and cutaneous vasodilation (Bruning et al. J Appl Physiol, 112 (12), 2019, 2012) are markedly lower in older relative to young adults. However, it remains unclear if aging also modulates cutaneous vasodilation and sweating in response to non‐cholinergic receptor activation, and if so, to what extent. Our recent studies show that aging does not affect cutaneous vasodilation elicited by prostacyclin in males (Fujii et al. J Physiol 594 (21), 6419, 2016) whereas in older females we observed an augmented cutaneous vasodilation (unpublished data). Therefore, the objective of this study was to determine how aging might differentially modulate cutaneous vascular and sweating responses to cholinergic and non‐cholinergic receptor activations between males and females. In this preliminary study, 8 young males (23±3 years), 10 young females (23±5 years), 12 older males (58±8 years), and 9 older females (61±9 years) were recruited. In each group, cutaneous vascular conductance (CVC, measured via laser Doppler flowmetry) and sweat rate (measured via sweat capsule) was evaluated at four intradermal forearm skin sites that perfused with the following different receptor agonists: 1) methacholine (cholinergic receptor agonist, 5 doses: 0.0125, 0.25, 5, 100, 2000 mM), 2) ATP (purinergic receptor agonist, 5 doses: 0.03, 0.3, 3, 30, 300 mM), 3) nicotine (nicotinic receptor agonist, 5 doses: 1.2, 3.6, 11, 33, 100 mM), and 4) isoproterenol (β‐adrenergic receptor agonist, 3 doses: 1, 10, 100 μM). An age‐related reduction for cholinergic sweating was observed in females (P≤0.05) but not males (P>0.05). Moreover, purinergic cutaneous vasodilation was attenuated in older relative to young adults for males only (P≤0.05). β‐adrenergic cutaneous vasodilation was greater in both older males and females as compared to their younger counterparts (P≤0.05). Furthermore, there were no age‐related differences in cholinergic cutaneous vasodilation, nicotinic cutaneous vasodilation and sweating, as well as β‐adrenergic sweating, irrespective of sex (P>0.05). Purinergic receptor activation did not elicit an increase in sweating among all groups (all P>0.05). Our results show that aging modulates cutaneous vascular and sweating responses, however this modulation is dependent on sex and types of receptor agonist.

Support or Funding Information

This study was supported by the Natural Sciences and Engineering Research Council of Canada (Discovery grant, RGPIN‐06313‐2014; Discovery Grants Program ‐ Accelerator Supplement, RGPAS‐462252‐2014; funds held by Dr. Glen P. Kenny).
Raw Garlic Aqueous Extract (RGAE) Augments MafA Expression in the Liver of STZ‐Induced Type‐1 Diabetic Rats
Authors: Al‐Qattan Khaled K , Al‐Otaibi Anood , Al‐Adsani Amani , Al‐Bader Maie , Thomson Martha , Divya SJ , Miranda Lavina , Al‐Bustan Suzanne
Published: November 2023

Background

Treatment with raw garlic aqueous extract (RGAE) increases plasma insulin concentration in the rat model of streptozotocin (STZ)‐induced type‐1 diabetes mellitus (DM). The regulatory gene MafA plays an important role in insulin gene expression and production.

Objective

This study investigated the effect of RGAE on MafA expression in the liver of STZ‐type‐1 DM rats.

Method

A group of normal rats (n=8, NR‐NS) and a group of DM rats (n=8, DR‐NS) were treated with normal saline (NS). Additionally, a group of normal rats (n=8, NR‐RGAE) and a group of diabetic rats (n=8, DR‐RGAE) were treated with RGAE. All treatments were carried out for 8 weeks after which all rats' liver and pancreas were analyzed for MafA expression at week 1 (n=4, each group) and week 8 (n=4, each group). Total mRNA was extracted from both the liver and pancreatic tissues and then converted to cDNA for gene expression profiling using real‐time PCR with Taqman assay.

Results

At week 8 and compared to week 1, MafA expression levels in the liver did not reveal any significant changes in response to either NS or RGAE in normal rats. Alternatively, MafA expression showed differential expressions in response to RGAE treatment in the diabetic rats.

Conclusion

The results indicate that RGAE may augment the expression of MafA and therefore could contribute towards the generation of insulin producing cells from the liver.

Support or Funding Information

This study was supported by The Research Sector – Kuwait University (YS04/13)
Proinsulin C‐Peptide Effects in Retinal Pigment Epithelium Health and VEGF Release: Implications for Diabetic Retinopathy
Authors: Grote Stephen M. , Kolar Grant R. , Yosten Gina L. C.
Published: November 2023

Connecting peptide (C‐peptide) is derived from the proinsulin molecule and is released with insulin in equimolar amounts by beta cells of the pancreas. For many years, C‐peptide was known only as a marker of insulin secretion, but recent work has demonstrated that C‐peptide has important bioactivity, particularly in the microvasculature including the microvessels of the eye. In diabetic retinopathy, a leading cause of blindness in the developed world, the retinal pigment epithelium (RPE) becomes dysfunctional. The RPE normally plays vital metabolic and secretory roles in maintaining the retina through interactions with the choriocapillaris and the photoreceptor layer. However, exposure to high glucose, as in uncontrolled diabetes, may encourage transdifferentiation of RPE cells into fibroblast‐like cells that disrupt the normal functioning of this essential cell layer. Furthermore, high glucose can induce pathological hypersecretion of VEGF from the RPE, which contributes to retinopathy by stimulating retinal neovascularization and by increasing permeability of the RPE via an autocrine action. Since RPE cells express the putative C‐peptide receptor, GPR146, we hypothesized that C‐peptide in physiological concentrations might normalize RPE‐specific gene expression and inhibit VEGF release. To test this hypothesis, we first performed transdifferentiation assays using the spontaneously transformed human RPE cell line, ARPE‐19, in which cells were incubated in either high or normal glucose containing media, with or without C‐peptide for 30 days. We measured changes in gene expression of RPE functional genes using qPCR. C‐peptide enhanced expression of RPE‐specific genes, and the changes in gene expression appeared to be dependent on glucose conditions. In addition, we treated ARPE‐19 cells with C‐peptide in normal and high glucose conditions and measured VEGF secretion by ELISA. C‐peptide significantly reduced VEGF secretion; however, VEGF secretion was enhanced by treating cells with the Na+/K+ ATPase inhibitor, ouabain, suggesting that C‐peptide inhibits VEGF release by increasing Na+/K+ ATPase activity. We conclude that C‐peptide prevents transdifferentiation of RPE cells to disruptive fibroblast‐like cells by enhancing the expression of RPE‐specific genes, and that C‐peptide diminishes release of VEGF, possibly by increasing Na+/K+ ATPase activity. Future experiments exploring the mechanisms underlying these effects are essential next steps towards the development of C‐peptide‐based therapeutics for the treatment of diabetes‐associated eye disease.

Support or Funding Information

HL121456 National Institutes of Health
KNOCKOUT OF MIR‐223 INDUCES BETA‐CELL DYSFUNCTION THROUGH ACTIVATION OF THE FOXO1 SIGNALING PATHWAY
Authors: Li Yutian , Peng Jiangtong , Deng Shan , Wang Xiaohong , Kuhel David , Cash James , Essandoh Kobina , Mu Xingjiang , Salem Esam , Hui David , Fan Guo‐chang
Published: November 2023

Background

MicroRNAs (miRNAs) are small, endogenous non‐coding RNAs, well‐known to regulate wide ranges of biological processes and tissue metabolism homeostasis. Recently, miR‐223 has been reported to coordinate systemic cholesterol under basal conditions and ameliorate adipose tissue‐associated insulin resistance in mice fed with high‐fat diet. However, the functional role of miR‐223 in the diabetic pancreas remains less studied.

Method and Results

Diabetes was induced in C57BL6 mice (5‐week old, male) by either daily intraperitoneal injection of streptozotocin (STZ, 50μg/g body weight) for 5 days (type 1 diabetes, or T1D), or 60% high‐fat diet (HFD) feeding for 24 weeks (type 2 diabetes, or T2D). Subsequently, pancreases were collected from such diabetic mice to measure miR‐223 levels using RT‐PCR. We observed that the levels of miR‐223 were increased by 8‐fold in the T1D pancreas, and by 2‐fold in T2D pancreas, compared to respective controls. Similarly, in vitro studies using Min6 β‐cell line revealed a 3‐fold increase of miR‐223 levels upon high glucose treatment. These results indicate that miR‐223 may involve in the development of diabetes. Of important, global miR‐223 knockout (KO) mice displayed insulin resistance, as evidenced by higher levels of plasma glucose and lower levels of insulin than wild‐type (WT) mice under both basal and diabetic conditions (n=5–11, p<0.05)). Further investigation showed that the diabetic phenotype of miR‐223‐KO mice may be attributed to diminished β‐cell proliferation and increased β‐cell apoptosis. Using luciferase reporter assay, we mechanistically identified that miR‐223 could directly target the 3′‐UTR of Foxo1, a critical transcriptional factor that regulates β‐cell function and survival. Using Western‐blotting, we validated that the protein levels of Foxo1 were increased by 3‐fold in the pancreas of miR‐223‐KO mice, compared to WTs, leading to subsequent decreased levels of Pdx1 and Glut2, two key regulators of β‐cell proliferation and function.

Conclusion

Our study reveals that loss of miR‐223 triggers β‐cell dysfunction by post‐transcriptional activation of the Foxo1 signaling pathway, resulting in the development of diabetes.

Support or Funding Information

NIH R01 GM‐112930
Cytokines Activate Src Kinase and Inhibit EGFR‐Mediated Repair Mechanisms via Mig6 in Pancreatic Beta Cells
Authors: Fong Kimberley M. , Rezaeizadeh Alireza , Chen Yi‐Chun , Fueger Patrick T.
Published: November 2023

Type 1 diabetes (T1D) is caused by autoimmune‐mediated destruction of insulin‐producing beta cells. Following beta cell injury, the pancreas attempts to launch a cellular repair and regenerative program, yet it fails to completely restore functional beta cell mass. One component of the regenerative program is epidermal growth factor receptor (EGFR) signaling. Upon irreparable beta cell damage, EGFR signaling is dampened, disrupting attempts to restore functional beta cell mass and maintain normoglycemia. We previously demonstrated that the negative feedback inhibitor of EGFR, Mitogen‐inducible gene 6 (Mig6), is induced by the pro‐inflammatory cytokines central to the beta cell destruction in T1D. We also established that pro‐inflammatory cytokines suppress EGFR activation, and siRNA‐mediated suppression of Mig6 restores EGFR signaling. Recently, Mig6 was reported to be phosphorylated and activated by Src kinase. Interestingly, Src increases cell proliferation by activating receptor tyrosine kinases, such as EGFR, yet it is also implicated in the production of reactive oxygen species, including nitric oxide (NO). We hypothesized that pro‐inflammatory cytokines activate Src kinase, and that along with NO, disrupt EGFR repair mechanisms through the induction and activation of Mig6. To test this hypothesis, we treated 832/13 cells independently with cytokines and high glucose and identified that these conditions increase the activation of Src. We confirmed these results ex vivo in mouse islets. Additionally, NO induces Mig6, which attenuates EGFR signaling, and NO synthase inhibition blocks the cytokine‐mediated induction of Mig6, thereby restoring cytokine‐impaired EGFR signaling. Interestingly, Src knockout mice appear to have a higher beta cell mass, and mice lacking pancreatic Mig6 have preserved beta cell mass following treatment with streptozotocin, which also induces NO. Our work suggests that Src kinase may be involved in the progression to T1D by activating Mig6, and may be a component of a feedback mechanism that can be targeted to preserve beta cell mass in T1D.
Transcriptional regulation and therapeutic targeting of P2X7 receptors in diabetic nephropathy
Authors: Menzies Robert I , Booth John W , Mullins John J , Bailey Matthew A , Tam Frederick W , Norman Jill T , Unwin Robert J
Published: November 2023

Purine P2X7 receptors are essential components of pro‐inflammatory signalling, functionally expressed on multiple immune and non‐immune cells. Renal biopsies from patients with diabetic nephropathy show widespread, glomerular mesangial expansion, immune cell infiltration and fibrosis. Here we show that in diabetic patients, renal P2X7 expression is associated with severe mesangial expansion, impaired glomerular filtration (≥40ml/min), and increased fibrosis (50%+) compared with non‐diabetic controls. P2rx7‐deficient mice developed STZ‐induced hyperglycaemia similar to controls but had reduced glomerular macrophage attraction and collagen IV deposition. Activation of P2X7 was investigated in a rat model of diabetic nephropathy. Wister rats underwent uninephrectomy and STZ‐induction. Whole kidney P2rx7 mRNA expression was increased (2.2‐fold; P<0.05) compared with sham controls by 8 weeks of induction. Glomerular and interstitial macrophage accrual increased, reflecting the pathology seen in patient biopsies. Rats were then randomly selected to receive drug vehicle or selective P2X7R antagonist (AZ11657312; 50mg/kg IP) twice daily for the following 4‐weeks. P2X7R antagonism supressed transcription of pro‐inflammatory signalling molecules compared with the vehicle group, and reduced interstitial macrophage accrual. These data suggest that P2rx7 contributes to renal inflammation in diabetics and P2X7R antagonists may have therapeutic potential.

Support or Funding Information

RIM is a BHF Immediate Postdoctoral Basic Science Research Fellow (Award number FS/15/60/31510). JWRB received a MRC/Kidney Research UK Joint Clinical Training Fellowship (Award number G0901956). We would like to thank GSK for gifting the P2X7k expressing gene‐targeted mice. FWKT is supported by the Diamond Fund from Imperial College Healthcare Charity. Part of this work is supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Imperial College Healthcare NHS Trust and Imperial College London.
Suppression of Albumin‐Induced Matrix Metalloproteinase‐9 by Rosiglitazone in Renal Tubular Epithelial Cells
Authors: Cobbs Alyssa , Chen Xiaoming , Khamdy Anjulyna , Chima Ashmeer , George Jasmine , Zhao Xueying
Published: November 2023

Matrix metalloproteinase‐9 (MMP‐9) activation is closely linked to albuminuria in chronic kidney disease. This study was designed to explore the role of albumin on MMP‐9 expression and activity in renal tubular epithelial cells (TECs). The effect of rosiglitazone, a PPARγ agonist, on MMP‐9 activation was further examined in the diabetic kidneys and in vitro primary cultured TECs. In vivo, in situ zymography and immunostaining revealed an increase in gelatinase activity and MMP‐9 protein in the damaged tubules of Zucker diabetic rats. Increased tubular MMP‐9 staining was mainly observed in the tubules expressing kidney injury molecule‐1 (KIM‐1) in the diabetic kidneys. Additionally, urinary excretion of MMP‐9 was associated with an increase in urinary albumin in untreated diabetic rats. Chronic administration of rosiglitazone led to a downregulation of KIM‐1 and osteopontin genes in the diabetic kidneys. Moreover, urinary albumin and MMP‐9 protein were significantly decreased in rosiglitazone‐treated diabetic rats. To evaluate the effect of albumin on expression and activity of MMP‐9, primary cultured rat TECs were incubated with rat serum albumin (RSA, 0.1 – 0.5 mg/ml) for 24 – 48 hrs. RSA administration resulted in a dose‐dependent increase in MMP‐9 protein and activity in culture supernatants of TECs. Rosiglitazone pretreatment significantly attenuated albumin‐induced MMP‐9 expression and secretion from TECs. Taken together, these results suggest that albuminuria may accelerate kidney disease progression by activating tubular MMP‐9. The renoprotective effects exerted by rosiglitazone may be attributed partially to its suppression of albumin‐induced MMP‐9 activity in diabetic kidney disease.

Support or Funding Information

This work is supported by the NIH SDK096441, 1SC1DK112151, 8G12MD007602 and 8U54MD007588.
Modeling the Effects of Functional Adaptation to Reduction in Renal Mass
Authors: Layton Anita , Edwards Aurelie , Vallon Volker
Published: November 2023

When kidney tissue is removed or damaged, the remaining nephrons undergo compensatory growth and adaptive functional changes. The objective of this study is to assess the impact of such structural and functional adaptation. To do so, we developed computational models of solute transport along different nephron populations of a uninephrectomized and a 5/6‐nephrectomized rat kidney. The model represents detailed epithelial and paracellular transport processes along both the superficial and juxtamedullary nephrons. The loop of Henle of each model nephron extends to differing depths of the inner medulla. Each nephron segment is represented as a rigid tubule lined by a layer of epithelial cells, with apical and basolateral transporters that vary according to cell type. To simulate a uninephrectomized or 5/6‐nephrectomized kidney, we reduced the nephron population, increased single‐nephron glomerular filtration rate, and increased nephron size based on data in the literature. Model simulations were conducted to determine what tubular transport changes are necessary to preserve baseline salt excretion. Model results also revealed the differing degrees to which salt transport and oxygen consumption are changed along the different nephron segments.

Support or Funding Information

This research was supported in part by NIH grant DK‐106102.
Neuronal nitric oxide synthase inhibits store‐operated Ca2+ entry in cardiomyocytes via S‐nitrosylation of stromal interaction molecule‐1
Authors: Feng Qingping , Gui Le , Zhu Jinhui , Lu Xiangru , Sims Stephen M. , Lu Wei‐Yang , Stathopulos Peter B.
Published: November 2023

Store‐operated Ca2+ entry (SOCE) mediated by stromal interacting molecule‐1 (STIM1) and Orai1 represents a major route of Ca2+ entry in mammalian cells. The luminal domain of STIM1 is critical to Ca2+ sensing, STIM1 oligomerization and SOCE activation. Notably, the luminal domain contains two cysteine sites (Cys49 and Cys56). It is not known if neuronal nitric oxide synthase (nNOS), which is associated to the sarcoplasmic reticulum (SR) membrane, regulates STIM1 oligomerization and SOCE activity in cardiomyocytes via S‐nitrosylation of these cysteine sites. Here, we show that nNOS deficiency or treatment with a nNOS inhibitor L‐VNIO significantly increased SOCE in cardiomyocytes. Further, the Ca2+ release‐activated Ca2+ channel current (ICRAC) was significantly enhanced in cardiomyocytes treated with L‐VNIO and in nNOS−/− cardiomyocytes. Consistently, STIM1 S‐nitrosylation was significantly decreased in nNOS−/− hearts. Treatment of HEK293 cells co‐expressing YFP‐Orai1 and mCherry‐STIM1 with the NO donor S‐nitrosoglutathione (GSNO) inhibited STIM1 puncta formation and ICRAC. Remarkably, no functional inhibition was observed in cells expressing the Cys49Ser/Cys56Ser STIM1 double mutant. Mechanistically, we found that NO donors caused Cys49 and Cys56‐specific structural changes associated with reduced protein backbone mobility, significantly increased the thermal stability and suppressed Ca2+‐depletion‐dependent oligomerization of the luminal Ca2+‐sensing region of STIM1. Collectively, our data reveal that nNOS inhibits SOCE in cardiomyocytes through NO‐mediated STIM1 S‐nitrosylation which suppresses oligomerization via enhanced luminal domain stability and rigidity.

Support or Funding Information

Canadian Institutes of Health Research
Hydrogen peroxide inhibits neurons in the paraventricular nucleus of the hypothalamus
Authors: Kline David D. , Matott Michael P.
Published: November 2023

The paraventricular nucleus (PVN) of the hypothalamus is an important center for neuroendocrine, respiratory and autonomic regulation. The PVN has reciprocal innervation with the nucleus tractus solitarii (nTS) and both are activated by exposure to low oxygen (hypoxia). Hypoxia induces increased reactive oxygen species (ROS) production. Previously we've shown in the nTS that the ROS hydrogen peroxide (H2O2) hyperpolarized membrane potential and reduced action potential discharge via an increase in potassium conductance that operates at the resting potential. The goal of this study was to examine the electrophysiological effect of H2O2 on PVN parvocellular neurons. Anesthetized Sprague‐Dawley rats were rapidly decapitated and the cortex removed and blocked to make 300 μm acute PVN slices. PVN neurons were recorded at 32 °C in whole cell patch clamp mode and exposed to 150 μM H2O2 (5 min) in artificial cerebrospinal fluid (aCSF). H2O2 induced a significant positive (outward) current of 71 ± 50.5 pA when neurons were held at −60 mV. Peroxide also significantly decreased spontaneous excitatory post‐synaptic current (sEPSC) frequency by 3 ± 1 Hz but did not change sEPSC amplitude. In some neurons inhibitory post‐synaptic currents (IPSCs) became evident after H2O2. Peroxide significantly decreased outward potassium currents (IK) across voltage steps between +10 and +40 mV. Under current clamp, peroxide hyperpolarized membrane potential by −21 ± 6 mV and decreased action potential discharge (APD), evoked by a depolarizing ramp, from 13 ± 3 APs to 2 ± 1 APs. All of these electrophysiological properties except for APD and IK were reversed by wash with aCSF. These data indicate that H2O2 alters pre‐ and postsynaptic properties of PVN neurons to likely alter cardiorespiratory reflexes.

Support or Funding Information

HL098602