bluuebunny/crossref_metadata_2025 huggingface dataset:
Litter Size Reduction Alters the Lipid Metabolism of Adult Male Rat Offspring of Dams Fed a Diet with Caloric Restriction
Authors: da Silva Sartori Denise Rangel , Morceli Lilian , Stefaniszen Amanda Garcia , Barroso Bruna Artico , de Almeida Giovanna Passaroni Marques , Maeda Isabel Cabral , Gonçalves Natalia Volpi , Sartori Maria Marcia Pereira
Published: November 2023

Recent studies have showed that nutritional changes during critical periods of development can lead to permanent alterations in the metabolism of the offspring which can become more susceptible to chronic diseases in adult age. The aim of this study was to evaluate the effect of litter size on the body weight and the lipid metabolism of adult male offspring rats born to caloric restricted dams. Dams were fed with either ad libitum standard diet (C) or a 30% caloric restricted diet (R) throughout gestation. After birth, litters were reduced to eight pups or four pups in both C and R dams. All dams were fed ad libitum during lactation. After weaning (3 wk), the male rat offspring born to control dams (C8 and C4) and male rat offspring born to restricted dams (R8 and R4) were maintained in individual cages until they were 20 weeks old. An interaction between the diet of control or restricted dams and the litter size was observed for the hepatic lipid % (P=0.015). Reduction of litter size decreased the hepatic lipid % in control rats but increased it in rats to restricted dams. The caloric restriction in the dams decreased the hepatic lipids only in the large litter offspring, but no effect on the small litter was observed. There was no interaction between litter size reduction and the diet of the dams (P=0.689). The reduction of litter size induced an increase (P=0.035) of carcass weight, regardless the diet of the dams. No effects of caloric restriction during gestation and of the litter size on body weight (P=0.187) and on blood cholesterol (P=0.171) were found in C or R adult male rat offspring. It is concluded that the effect of reduction of litter size on hepatic lipid metabolism depends on the diet of the dams during gestation. The increase of carcass weight resulting from litter size reduction is not affected by the diet of the dams.

Support or Funding Information

Research supported by Institute of Biosciences
Diminazene Aceturate Decreases Fat Mass with No Effect on Food Intake in Young Diet‐Induced Obese Rats
Authors: Bruce Erin , Sakarya Yasemin , Kirichenko Nataliya , Toklu Hale , Morgan Drake , Tumer Nihal , Carter Christy S. , Scarpace Philip J.
Published: November 2023

Obesity is characterized as having an excessive amount of adipose tissue. Current evidence suggests that Angiotensin‐(1–7) activation of the Mas receptor may decrease body weight. We hypothesize that activation of ACE2, via Diminazene Aceturate (DIZE), will significantly reduce body weight of young obese rats.

Male Fisher 344 x Brown Norway rats, age 3 months were fed either Standard Chow or 60% high fat (HF) diet for six weeks, whereupon, animals were further divided, and given either 7.5mg/kg/day DIZE s.c. (Chow+DIZE, n=8; HF+DIZE, n=8) or vehicle (Chow Control, n=8; HF Control, n=8) for another six weeks. Body weight and food intake were measured throughout the experiment. Time Domain‐Nuclear Magnetic Resonance (TD‐NMR) was used to assess body composition at weeks 0, 3, 6, 9, and 12. Retroperitoneal, perirenal, and epididymal white adipose tissue (WAT) depots, as well as the interscapular brown adipose tissue (BAT) depot, and the tibialis anterior (TA) muscle, were excised at sacrifice and weighed.

After six weeks, animals fed 60% HF diet had a significant increase in bodyweight compared to Chow controls. During the six week treatment period, DIZE prevented further weight gain in both the Chow and HF fed groups. However, TD‐NMR analyses revealed a differential effect of DIZE on body composition in Chow and HF groups. There was a significant difference in absolute fat mass between HF control and HF+DIZE treated animals, but no significant difference between the Chow control and Chow+DIZE groups. Furthermore, there was no change in lean mass in either group. Tissue weights at the time of sacrifice confirm that all DIZE treated animals had significantly lower weights of WAT and BAT, with no significant difference in TA muscle weights. DIZE treatment had a greater effect on lowering perirenal fat mass and BAT mass specifically in HF fed animals. Our results indicate that DIZE may be a potential anti‐obesity agent, specifically targeting the accumulation of adipose tissue with no effect on food intake or lean mass.

Support or Funding Information

R01 DK091710‐01A1, P30 AG028740, T32 HL083810‐01 Postdoctoral Fellow
Analysis of Iron Dysregulation in High‐Fat and Iron‐Deficient Rat Models
Authors: Klabacka Randy , Crandall Amy , Anderson Jacob , Wright David , Hancock Chad
Published: November 2023

Iron (Fe) dysregulation has been identified as a contributor to insulin resistance (IR). Fe chelators and phlebotomy have been shown to improve insulin sensitivity. Due to the central role of the liver in Fe regulation, the objective of our study was to examine hepatic total Fe and critical Fe regulatory proteins in both high‐fat (HF) fed model of IR as well as a model of Fe deficiency (FeDEF).

METHODS

Rats in the HF group were fed a diet with 50% of calories from fat (lard and corn oil) or standard chow for a four‐week period. For the FeDEF model, rats were either fed an FeDEF diet (2–6 mg/kg Fe) or a control diet (50–58 mg/kg Fe). Western blots quantified levels for ferroportin (FPN), ferritin heavy chain (FHC), and ferritin light chain (FLC). Colorimetric analysis was used to quantify total hepatic Fe.

RESULTS

FPN and ferritin levels were reduced in the FeDEF group (FPN: −49%, p<0.01; ferritin: −44.7%, p < 0.01). While FHC did not show a significant difference in the HF group, FLC levels were elevated (42.1%, p<0.05) and FPN levels were reduced (−56.2%, p<0.05). Total hepatic Fe was found to be elevated in the HF group compared to control (64.1%, p < 0.05).

CONCLUSIONS

As expected, in the FeDEF group we observed reductions in both Fe storage proteins (ferritin) and the Fe export protein (FPN) compared to control. Interestingly, with the HF group we observed an increase in both Fe storage proteins and total hepatic Fe while the Fe exporting protein FPN was reduced. Our findings suggest the development of IR due to HF feeding is associated with the dysregulation of hepatic Fe.

Support or Funding Information

Funding was provided by Brigham Young University
Detecting Metabolism of Gluconeogenic Precursors Lactate and Pyruvate in Kidney & Liver with Hyperpolarized 13C MRI
Authors: von Morze Cornelius , Chang Gene‐Yuan , Larson Peder E , Shang Hong , Allu Prasanna KR , Bok Robert A , Crane Jason C , Olson Marram P , Tan Chou T , Marco‐Rius Irene , Nelson Sarah J , Kurhanewicz John , Pearce David , Vigneron Daniel B
Published: November 2023

Objective

The objective of this study was to apply new methods in hyperpolarized (HP) 13C magnetic resonance imaging (MRI) to characterize tissue‐specific alterations in the metabolism of hyperpolarized HP gluconeogenic precursors 13C‐lactate and 13C‐pyruvate by rat liver and kidneys in vivo in both fasting and insulin deprivation in diabetes.

Methods

Solutions of 13C‐lactate and 13C‐pyruvate were magnetically hyperpolarized using the method of dissolution dynamic nuclear polarization (DNP), for use in spectroscopic MRI imaging of anesthetized rats. Seven normal rats were scanned by HP 13C MRI of both 13C‐lactate and 13C‐pyruvate in both normal fed and 24h fasting states, and seven additional rats were scanned after induction of diabetes by streptozotocin (STZ) with subsequent withdrawal of insulin (24–36h). Separate images of the injected substrates and their individual metabolic products (lactate, pyruvate, alanine, and bicarbonate) were acquired. Following the imaging experiments, phosphoenolpyruvate carboxykinase (PEPCK) expression levels were also measured in liver and kidney tissues of the STZ‐treated rats, and compared with seven vehicle‐treated controls.

Results

Multiple sets of significant metabolic modulations were detected, with graded intensity in general from fasting to diabetes (Figure 1). An approximate two‐fold reduction in the ratio of 13C‐bicarbonate to total 13C signal was observed in both organs in fasting. The ratio of HP lactate‐to‐alanine was markedly altered, ranging from a liver‐specific 54% increase in fasting, to increases of 69% and 92% in liver and kidney, respectively, in diabetes. Diabetes resulted in a 40% increase in renal lactate signal. STZ resulted in 5.86‐fold and 2.73‐fold increases in PEPCK expression in liver and kidney, respectively.

Conclusion

The intermediary metabolism of gluconeogenic precursors 13C‐lactate and 13C‐pyruvate can be monitored non‐invasively in vivo using HP 13C MRI. Localized changes in the HP metabolic spectra of these probes in the liver and kidneys were detected in states of increasing gluconeogenic activity, with generally graded intensity of response from fasting to diabetes. These HP 13C methods could be extremely useful for non‐invasive monitoring of hepatic and renal components of gluconeogenesis in future preclinical and ultimately clinical studies.

Support or Funding Information

We gratefully acknowledge grant support from NIH K01DK099451, P41EB013598, and R01DK56695. We also acknowledge support from the Manning Foundation, a Ben J. Lipps

Research Fellowship Award from the American Society of Nephrology, as well as T32DK007219.
Janus‐faced Sestrin2 controls ROS and mTOR signalling through two separate functional domains
Authors: Ro Seung‐Hyun , Kim Hanseong , An Sojin , Cho Uhn‐Soo , Lee Jun Hee
Published: November 2023

Sestrins are stress‐inducible metabolic regulators with two seemingly unrelated but physiologically important functions: reduction of reactive oxygen species (ROS) and inhibition of the mechanistic target of rapamycin complex 1 (mTORC1). How Sestrins fulfil this dual role has remained elusive so far. Here we report the crystal structure of human Sestrin2 (hSesn2), and show that hSesn2 is twofold pseudo‐symmetric with two globular subdomains, which are structurally similar but functionally distinct from each other. While the N‐terminal domain (Sesn‐A) reduces alkylhydroperoxide radicals through its helix‐turn‐helix oxidoreductase motif, the C‐terminal domain (Sesn‐C) modified this motif to accommodate physical interaction with GATOR2 and subsequent inhibition of mTORC1. These findings clarify the molecular mechanism of how Sestrins can attenuate degenerative processes such as aging and diabetes by acting as a simultaneous inhibitor of ROS accumulation and mTORC1 activation.

Support or Funding Information

This research was supported mainly by a Pilot and Feasibility Grant from the Michigan Diabetes Research and Training Center (NIH Grant 2P30‐DK020572 to U.‐S.C. and J.H.L.) and partially by grants from Ellison Medical Foundation (AG‐NS‐0932‐12 to J.H.L.) and NIH (1R21AG050903 to U.‐S.C. and J.H.L., 1R01DK102850 to J.H.L.)
Histamine receptor‐mediated sustained post‐exercise vasodilation in young trained and untrained adults
Authors: Luttrell Meredith J , Abdala Pedro M , Ely Matthew R , Sieck Dylan C , Mangum Joshua E , Halliwill John R
Published: November 2023

Histamine‐mediated sustained post‐exercise vasodilation in response to one hour of dynamic knee extension has been established in young, recreationally active adults. Endurance training leads to an increased muscle blood flow from vascular remodeling and enhanced vasodilation during exercise in young adults. However, it is unknown whether endurance exercise training modifies histamine mediated post‐exercise arterial vasodilation in young adults. The purpose of this study was to test the hypothesis that sedentary young adults have similar histamine receptor mediated sustained post‐exercise vasodilator responses compared to endurance trained young adults. A randomized, double‐blind placebo‐controlled crossover study including 6 endurance trained (3F, 3M; ages 18–26) and 5 sedentary (3F, 2M; ages 22–29) adults was completed. Subjects orally consumed either a combined histamine H1 and H2 receptor blockade with 540 mg fexofenadine (Allegra, H1 receptor blocker) and 300mg ranitidine (Zantac, H2 receptor blocker), or placebo pills containing only the inactive ingredients of fexofenadine and ranitidine. Femoral vascular conductance was calculated by dividing femoral artery blood flow obtained using duplex Doppler ultrasound by brachial artery blood pressure measurements. Pre‐exercise measurements were made every 30 minutes for 90 minutes, while post‐exercise measurements were made every 30 minutes for 2 hours. After 90 minutes of supine rest, subjects completed 1 hour of dynamic knee extension exercise at 60% of maximal work rate. Subject demographics and baseline variables were analyzed using independent t‐tests. Post‐exercise differences between groups in the blockade and placebo conditions were analyzed using a mixed effect model ANOVA with repeated measures. All values are reported as mean ± SE. The endurance trained group had higher self‐reported weekly physical activity compared to the sedentary group (73.03 ± 10.5 vs. 27.4 ± 9.5 METhr/wk, p=0.012). Pre‐exercise mean arterial pressure was not different between the two groups in either placebo or blockade conditions (trained placebo: 83±2, trained blockade: 84±2, sedentary placebo: 84±1, sedentary blockade: 86±3 mmHg, p=0.745). Baseline femoral vascular conductance was not different between groups in either placebo or blockade condition (p=0.905). At 60 minutes post exercise, the change in femoral vascular conductance from baseline was not different between the groups for either placebo or blockade condition, although there was a trend towards drug effect of histamine receptor blockade (p=0.192). These preliminary results suggest that there is no difference in post‐exercise femoral vascular conductance between endurance trained and sedentary young adults.

Support or Funding Information

Support provided by NIH grant HL115027
Flow‐Induced Dilation of Skeletal Muscle Feed Arteries: Relevance to Exercise Hyperemia
Authors: Moore Brittni N. , Jasperse Jeffrey L.
Published: November 2023

Flow‐induced dilation is the dilatory response seen in arteries as a result of an increase in shear stress along the endothelium. It is often thought that flow‐induced dilation contributes to arterial dilation during exercise as blood flow is increased to working muscle. Previous research compared in vivo shear stress values during rest and exercise to in vitro shear stress values and concluded that flow‐induced dilation of rat soleus feed arteries (Jasperse & Laughlin) is unlikely to contribute to exercise hyperemia. However, the predominantly slow‐twitch soleus is unique in that it contributes to postural maintenance and receives higher amounts of blood flow during stance compared to other hindlimb muscles. Therefore, the aim of the present study was to determine if feed arteries from the predominantly fast‐twitch rat gastrocnemius muscle (GFA) demonstrated flow‐induced dilation and to further assess the potential role of flow‐induced dilation in GFA during exercise. GFA (n=12) from male Sprague‐Dawley rats were isolated and cannulated on two glass micropipettes and intraluminal diameter was measured using videomicroscopy. Pressure was maintained at 90 cmH2O using two independent fluid reservoirs. Changing the height of the fluid reservoirs created a pressure gradient that induced flow through the arteries. Pressure gradients of 2, 4, 6, 8, 10, 15, 20, 30, and 40 cmH2O were used. GFA dilated to increasing flow and shear stress values ranged up to 8.0 ± 0.8 dyn/cm2. The in vitro shear stress response range was compared to calculated in vivo shear stress values from previously published studies. Estimated GFA blood flow values in standing rats are 431 μl/min, in walking rats 816 μl/min, and in running rats 1510 μl/min. Corresponding shear stress values were 95 dyn/cm2 (standing), 180 dyn/cm2 (walking), and 332 dyn/cm2 (running). These in vivo shear stress values far exceed the shear stress values to which GFA dilated in vitro. This comparison suggests that maximal flow‐induced dilation occurs at shear stress values far below those present in GFA in non‐exercising rats and that flow‐induced dilation of GFA is an unlikely contributor to the increase in blow flow seen during exercise.

Support or Funding Information

Seaver Research Council of Pepperdine University
Sympatholytic effect of calcitonin gene‐related peptide in rat soleus and gastrocnemius feed arteries
Authors: Brown Megan E. , Humphries M. Harrison , Moore Brittni N. , Jasperse Jeffrey L.
Published: November 2023

Exercise has been shown to increase sympathetic nerve activity and augment the release of the neurotransmitter norepinephrine at the arterial wall and into the blood. Norepinephrine binds to arterial adrenergic receptors causing vasoconstriction, yet arteries and arterioles constrict less to sympathetic stimulation in contracting skeletal muscle in comparison to resting skeletal muscle. This phenomenon is defined as sympatholysis. Calcitonin gene‐related peptide (CGRP) is a potent vasodilator initially studied in the cerebral circulation but with effects on vascular beds throughout the peripheral circulation. In skeletal muscle, CGRP is released from both sensory and motor neurons. Therefore, we hypothesized that increased levels of CGRP could contribute to sympatholysis. Dose response curves to CGRP (10−12 M to 10−6 M) in soleus feed arteries (SFA) and gastrocnemius feed arteries (GFA) isolated from male Sprague‐Dawley rats were used to compare the vasodilation response to CGRP in muscles of different fiber type. SFA (n = 12) and GFA (n = 8) were isolated and cannulated on two glass micropipettes for in vitro videomicroscopy. Both SFA and GFA dilated significantly to CGRP (SFA: 93.6 ± 2.5% and GFA: 87.8 ± 4.5%) and there was no difference between SFA and GFA in the maximal dilation or sensitivity to CGRP. The constriction response to the α‐1 agonist phenylephrine (PE; 10−9 M to 10−4 M, 0.5 log increments) was measured under control conditions and in the presence of two levels of CGRP (Low: 0.5 nM and Medium: 50 nM) in SFA (n = 6) and GFA (n = 5). Maximal constriction to PE was reduced by CGRP (Control: 82.2 ± 4.4%, Low: 68.4 ± 4.3%, Medium: 70.4 ± 5.1%). Sensitivity to PE (EC50) was also reduced by CGRP. In GFA, neither maximal dilation nor sensitivity to PE was reduced by CGRP. We conclude that CGRP reduces α‐1 mediated constriction in feed arteries from the predominantly slow‐twitch soleus muscle, but not in feed arteries from the predominantly fast‐twitch gastrocnemius muscle.

Support or Funding Information

Seaver Research Council of Pepperdine University
Molecular mechanism for cereblon‐dependent inhibition of AMP‐activated protein kinase
Authors: Yang Seung‐joo , Lee Kwang Min , Jeon Seung‐je , Park Chul‐Seung
Published: November 2023

Cereblon (CRBN), initially identified as a target protein of human mental retardation, is a substrate receptor of cullin‐ring E3 ubiquitin ligase (CRL) and a primary target of thalidomide‐induced teratogenicity. We previously reported that CRBN can negatively regulate the functional activity of AMP‐activated protein kinase (AMPK) by binding to the a subunit of the AMPK complex. We also noticed that the exogenous expression of CRBN reduced the content of g subunit in AMPK complex. However, the molecular mechanism for the CRBN‐dependent reduction of g subunit from AMPK complex was not clear. Thus, we investigated the molecular mechanism of CRBN‐dependent inhibition of AMPK. We noticed that the amount of g subunit in AMPK complex was increased in Crbn‐knockout (KO) MEFs and the g subunit was degraded more rapidly in wild‐type MEFs than in Crbn‐KO MEFs. After treating MG132, a proteasome inhibitor, the g subunit is accumulated more rapidly in wild‐type MEFs compared to Crbn‐KO MEFs. Moreover, ectopic expression of CRBN promoted the degradation of g subunit in Crbn‐knockout MEFs only in the presence of a subunit. These results strongly suggest that CRBN negatively regulates the AMPK activity by recruiting AMPK complex to CRL and degrading its g subunit.
Novel Method for Measuring Hepatic Collagen Turnover Rate in Nonalcoholic Fatty Liver Disease (NAFLD): Comparison of Blood and Liver Biopsy Biomarkers of Fibrogenesis
Authors: Hellerstein Marc
Published: November 2023

Background and Objectives

Excess collagen synthesis in the liver plays a causal role in progression from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). Markers that identify a patient's risk for progression, characterize disease activity and provide early assessment of treatment efficacy are needed. In particular, the availability of a non‐invasive metric of the rates of liver collagen synthesis (fibrogenesis) and turnover (fibrolysis) would be extremely valuable. We evaluated novel clinical biomarkers that directly quantify hepatic fibrogenesis rate in well‐characterized patients with biopsy‐proven NAFLD.

Methods

Patients with suspected NAFLD and a clinical indication for liver biopsy received the stable, non‐radioactive tracer heavy water (2H2O, 50 ml, 2–3 times daily) by mouth for 20–35 days prior to biopsy. A plasma sample was collected weekly. Collagen fractional synthesis rate (FSR) from liver biopsy tissue and FSR of lumican (a proteoglycan involved in collagen fibril assembly that exhibits increased synthesis when collagen synthesis is increased) from plasma were measured by 2H incorporation, using tandem mass spectrometry (LC‐MS/MS). Magnetic resonance elastography (MRE) measurements of liver stiffness were obtained within 2 weeks of biopsy.

Results

FSR of hepatic type I collagen (Col1a1) was significantly elevated in NASH relative to NAFL (0.97% vs 0.43 %/day, p=0.03). Liver Col1a1 FSR also significantly correlated with fibrosis severity by histological score (r= 0.63), with liver stiffness by MRE (r=0.65), and with HbA1c levels (r=0.47). FSRs of fibrillar hepatic collagen subtypes (I and III) were highly correlated (r= 0.94). FSR of circulating plasma lumican correlated strongly with liver collagen FSR (r=0.69) and with liver stiffness (r=0.71).

Conclusion

We conclude that hepatic collagen synthesis rates increase with fibrosis severity in NAFLD patients, indicating not only markedly increased absolute rates of collagen synthesis with disease progression but also suggesting that hepatic scar is dynamic even in advanced disease and may therefore be potentially reversible. Importantly, plasma lumican FSR correlates closely with liver collagen FSR and with liver stiffness. Moreover, established clinical risk factors for disease progression (NASH diagnosis, histologic fibrosis score, diabetes) were associated with higher collagen FSR. Non‐invasive kinetic measurements of fibrogenesis, such as plasma lumican FSR, may be helpful as markers of risk for disease progression and as early read outs of treatment response in anti‐fibrotic trials in NASH. Prospective studies are required to establish the predictive power of fibrogenesis rates for disease progression or reversal.

Support or Funding Information

KineMed, Inc.
Experimental Validation of Evolutionarily Conserved Genes Regulating Hypoxia Adaption in Drosophila and Humans
Authors: Zhou Dan , Jha Aashish R , Xue Jin , White Kevin P , Haddad Gabriel G
Published: November 2023

Hypoxia tolerance is regulated by polygenic mechanisms in metazoan organisms. The following study was performed to determine whether such polygenic mechanisms are shared evolutionarily between organisms like Drosophila and humans. Using a generalized linear mixed model we identified significant allele frequency differences between three independently evolved hypoxia‐tolerant populations and normoxic control populations of Drosophila. Around 50% of the ~3,800 SNP variants are clustered in 66 distinct genomic regions that contain genes that are differentially expressed when hypoxia‐tolerant and normoxic populations were compared. In addition, using genome‐wide SNP data from four high‐altitude human populations (i.e., Sherpas, Tibetans, Ethiopians, and Andeans), we found that several human orthologs of the genes under selection in flies are also likely under positive selection in all four high‐altitude human populations, suggesting that these conserved genes and pathways may regulate hypoxia tolerance in both flies and humans. Currently we have experimentally evaluated the role of these candidate genes in hypoxia tolerance in flies using RNAi‐mediated knockdown. We found that down‐regulation of several candidate genes’ expression, such as pyd, significantly enhanced survival in severe hypoxia in flies. Our results demonstrated that fundamental genetic mechanisms regulating hypoxia tolerance have indeed remained conserved throughout evolution. Such mechanisms may help us to develop novel therapeutic strategies to treat hypoxia‐related diseases in humans.

Support or Funding Information

This work was supported by the following grants from National Institute of Health: Training in Oxygen in Health and Disease Grant T32HL094282 to ARJ; 5P01HD32573 to GGH; and P50 GM081892 to KPW.
Nucleoprotein‐enriched diet prevents disuse‐induced muscle atrophy in soleus muscle of rats
Authors: Nakanishi Ryosuke , Hirayama Yusuke , Ueno Mizuki , Yoshikawa Madoka , Maeshige Noriaki , Fujino Hidemi
Published: November 2023

Disuse leads to a decrease of mass, fiber cross‐sectional area, protein synthesis, and satellite cell activity in skeletal muscle. It is known that the depletion of amino acids reduces muscle protein synthesis. In addition, nucleotides affect cell proliferation and differentiation. Thus, amino acids and nucleotides are essential nutrition for protein synthesis, cell proliferation, and differentiation in skeletal muscle. Therefore, the present study investigated the effect of nucleoprotein, which has combined amino acids and nucleotides, enriched diet during unloading on protein synthesis, activity of satellite cell, and muscle atrophy. Female Wistar rats were assigned randomly into 4 groups: control group (CON), control with nucleoprotein group (CON+NP), hindlimb unloading (HU), and hindlimb unloading with nucleoprotein group (HU+NP). Fiber cross‐sectional area (FCSA) was significantly higher in HU+NP group than HU group although unloading resulted in a decrease of CSA. In addition, the levels of phosphorylated‐ribosomal protein S6 (rpS6) and extracellular signal‐regulated kinase 1/2 (ERK1/2) were significantly higher in HU+NP group than HU group, whereas the levels of phosphorylated‐p70S6 kinase (p70S6K) were equivalent among these groups. Furthermore, the numbers of MyoD and myogenin‐positive nuclei cells were significantly higher in HU+NP group than HU group. These results suggest that nucleoprotein exerts preventive effects on muscle atrophy via activations of rpS6 and satellite cells, which are regulated with ERK signaling independently from p70S6K pathway.

Support or Funding Information

Supported by Grants‐in‐Aid for Science Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and the internal funds of Nissan Chemical Industries, Ltd.
Action by Shakuyaku‐kanzo‐to (SKT) and one of its Chemical Components, Glycyrrhetic acid (GA), on Calcium Influx through L‐type Calcium Channel
Authors: Noble Denis , Sam Cynthia , Tasaki Kazuyo Maria
Published: November 2023

Introduction

Our previous work has shown that SKT, a standardized 1:1 combination of root extracts from paeony and licorice, rapidly relaxes smooth muscle within 1–2 mins, and slowly relaxes skeletal muscle within about 30 minutes (Sam, Terrar, Tasaki & Noble, 2015a). We have also shown that increasing extracellular potassium from 5.4 to 10.8 mM is a powerful facilitator of the relaxing action of SKT in skeletal muscle (Sam, Terrar, Noble, Tasaki & Noble, 2015b).

Methods

To investigate possible molecular mechanisms we recorded calcium influx through the alpha1 pore‐forming subunit of the L‐Type calcium channel expressed in HEK293 cells. Influx was estimated by measuring the difference in Fura‐2 fluorescence before and after activating the channels with high (100 mM) potassium. Patch clamp recordings were done at −80 mV and depolarising pulses were applied from −80 to +40 mV. Pulse duration was between 50 and 200 ms. In some experiments ramps were applied from −100 to +100 mV.

Results

SKT produced up to 50% inhibition of calcium influx through the pore‐forming subunit of the L‐type calcium channel in HEK cells (Figure 1). The results also show considerable variability that may be consistent with differing actions of some of the individual chemical components.

One of the pure chemical components, GA, however produces a consistent and statistically significant inhibition of calcium influx, which is up to 80% (Figure 2).

Discussion

In muscle cells, such inhibitions of calcium influx would be expected to deplete cell calcium and so reduce the phasic contractile response and relax tonic resting tension. Since ICaL plays an immediate role in smooth muscle but only a long term role in skeletal muscle, this would be consistent with our previous observation of different time courses between SKT's actions on smooth and skeletal muscles.

There was, however, no statistically significant inhibition of calcium current using controlled voltage patch clamp recording. The action on inhibiting influx could therefore be indirect.

Support or Funding Information

Acknowledgement: This research is conducted by the University of Oxford Innovative Systems Biology Project Sponsored by Tsumura. We thank TSUMURA & CO for their support.
Hypothalamic APPL2 Acts as A Key Mediator in Maintaining Energy Homeostasis by Controlling Thermogenesis in Subcutaneous White Adipose Tissue
Authors: Wang Baile , Cheng Kenneth King Yip , Li Xiaomu , Wu Kelvin Ka Lok , Jin Leigang , Jiang Xue , Liu Zhuohao , Zhou Yawen , Xu Aimin
Published: November 2023

Objective

To investigate the role of hypothalamic APPL2, a close homolog of APPL1 (which has been reported to have regulatory effects on glucose metabolism), in regulation of energy metabolism.

Introduction

Induction of browning in white adipose tissue (WAT), defined by transformation from white adipocytes to brown adipocytes, not only promotes adaptive thermogenesis but acts as a defensive mechanism to protect against obesity and its related metabolic disorders. Browning is tightly controlled by the cross‐talk between the energy controlling center hypothalamus and WAT, yet the underlying mechanism is unclear. In this study, we investigated whether the adaptor protein APPL2, a key player in adiponectin and insulin signaling, controls browning by mediating the cross‐talk between hypothalamus and WAT.

Method

APPL2flox/flox mice were crossed with rat insulin promoter Cre transgenic mice to generate hypothalamus and pancreatic β cell specific APPL2 knockout (RIP‐APPL2‐KO) mice, or locally injected with adenovirus‐associated virus (AAV) encoding Cre into ventromedial hypothalamus to generate APPL2 hypothalamus specific knockout mice. Basic metabolic parameters were monitored for 24 weeks. Cold challenge was employed to induce browning in the above mice.

Results

RIP‐APPL2‐KO mice displayed increased adiposity accompanied by a dramatic reduction of oxygen consumption. Such impairments were exacerbated when these mice were fed with a high‐fat diet. Upon 6‐hour cold exposure, RIP‐APPL2‐KO mice were cold sensitive due to the defective lipolytic and thermogenic responses in WAT but not BAT. During chronic cold acclimation, browning program was almost absent in subcutaneous WAT (sWAT) of RIP‐APPL2‐KO mice, whereas no obvious change appeared in their BAT. The impaired browning in RIP‐APPL2‐KO mice was due to decreased neuronal activation in raphe pallidus and subsequent diminished sympathetic outflow to sWAT. Peripheral treatment of CL316243 (β3 adrenergic receptor agonist), which bypassed SNS activation, could reverse the impaired browning phenotype in sWAT of RIP‐APPL2‐KO mice, indicating that the defect does not lie in sWAT but in hypothalamus. Likewise, adeno‐associated virus (AAV)‐mediated specific knockout of APPL2 in ventromedial hypothalamus recapitulated the defective browning phenotypes of RIP‐APPL2‐KO mice.

Conclusion

Our study uncovers a novel role of ventromedial hypothalamic RIP neurons in maintaining energy homeostasis by controlling browning in sWAT with the involvement of the adaptor protein APPL2.

Support or Funding Information

This work was supported by Health and Medical Research Fund (HMRF 01231606 to KKY Cheng).
A Comparison of Device‐Guided and Self‐Paced Breathing on Forearm Venous Occlusion Plethysmography
Authors: Poteracki James Michael , Kelly Kevin , Lin Jeff , Adams Katelynn , Kelly Catherine , Wegner Brian , Newhouse Lauren , Moser Jason , Wehrwein Erica A
Published: November 2023

Slow, deep breathing (SDB) is thought to lead to reductions in blood pressure by decreasing vascular‐sympathetic coupling. Venous Occlusion Plethysmography (VOP) is used to assess forearm blood flow (FBF) (ml/min/100 dl tissue). We used this technique to determine the vasodilatory effect of device‐guided (DG) vs self‐paced (SP) slow, deep breathing. We hypothesized that SP would lead to a larger vasodilation response than DG due to a more profound reduction in vascular‐sympathetic coupling, and that this would be correlated with psychological evaluations. Young, healthy male subjects (Mean age: 20 yr; DG: n=10; SP: n=11) rested supine for 20 minutes (baseline), followed by 20 minutes of SP or DG slow, deep breathing, then completed 20 minutes of endogenous breathing (recovery). At baseline, there was no difference in breathing rate between SP and DG (14.36±1.03 vs 15.39±1.29 breaths per minute, p=0.27) and respiratory rate was reduced to 8.36±0.74 breaths per minute (BPM) in the SP group (p<0.01 compared to baseline) and to 6.37±0.10 BPM in the DG group (p<0.01 compared to baseline). Respiratory rates between the two groups differed significantly during SDB (p<0.01 between groups). There was a significant increase in SP FBF compared to baseline at the end of the 20 minute intervention (p<0.05),while the DG group saw no significant increases. In DG, there was a strong negative correlation with the cognitive interference scores to VOP responsiveness (−0.77; R2=0.591), as well as between the cognitive failure score and VOP responsiveness (−0.61; R2=0.3693). In contrast, the SP groups had positive correlations when comparing VOP responsiveness to cognitive interference (0.1; R2=0.093) or cognitive failure (0.30; R2=0.011). For subjects in either breathing group, those who had an increase in FBF in response to SDB also had reductions in STAI anxiety score after the intervention whereas those with a decrease in FBF to SDB had an increase in anxiety score during the slow breathing (p=0.04). Overall, male subjects have a variable vasodilator responses to two types of SDB practices related to underlying psychological metrics. Our data underscores the importance of psychological metrics in physiological outcomes related to SDB practices.
Cardiac Autonomic Function is Impaired in Adolescents with Mild Traumatic Brain Injury
Authors: Abbott Kolten C. , Badrov Mark B. , Elfassy Jonah , Moir M. Erin , Fischer Sara A. , Fischer Lisa K. , Fraser Douglas D. , Shoemaker J. Kevin
Published: November 2023

Mild traumatic brain injury (mTBI), of which concussion is a subset, are a major pediatric public health concern. Children and adolescent populations not only report the highest incidence of concussions, but are also more likely to experience symptoms which persist beyond the 2‐week period of spontaneous healing. In order to reduce the transition of acute symptoms into long‐term negative consequences, objective diagnostic and management tools are essential. However despite increased awareness, clinicians remain reliant on subjective symptom analyses and neuropsychological evaluations to diagnose and manage concussive injuries. Furthering our understanding of the pathophysiological response of an adolescent brain following a concussive injury may provide the foundation for the development of objective biomarkers. Therefore, the purpose of this investigation was to evaluate cardiac autonomic function in adolescents acutely diagnosed with a concussion. More specifically, autonomic function was evaluated while in the supine position, via heart rate variability (HRV; time‐and frequency‐domain) and cardiovagal baroreflex sensitivity (BRS; sequence method) in 16 adolescents diagnosed with a concussion (mTBI; 11 females; 16 ± 2 yrs, 173 ± 9 cm, 69 ± 15 kg) and 16 age‐matched controls with no previous history of concussion (CTRL; 7 females; 14 ± 2 yrs, 168 ± 8 cm, 66 ± 15 kg). mTBI patients were 9 ± 4 days post‐injury and presented with a SCAT3 symptom score of 14 ± 7. Compared to CTRL, mTBI patients had significantly lower standard deviation of normal RR intervals (SDNN; 56 ± 20 vs. 88 ± 40 ms; P < 0.01, Cohen's d = 1.06) and root mean square of successive differences (RMSSD; 59 ± 27 vs. 98 ± 57 ms; P < 0.05, d = 0.94). Furthermore, in the frequency domain, both total power (3491 ± 2482 vs. 8954 ± 7932 ms2; P < 0.05, d = 1.05) and high frequency power (1555 ± 1223 vs. 3644 ± 3334 ms2; P < 0.05, d = 0.92) were reduced in mTBI patients compared to CTRL, while a trend for reduced low frequency power in mTBI patients was observed (989 ± 726 vs. 2986 ± 4028 ms2; P = 0.07, d = 0.84). Finally, compared to CTRL, cardiovagal BRS was lower in mTBI patients (32 ± 14 vs. 52 ± 36 ms/mmHg; P < 0.05; d = 0.81). The results of this study suggest that adolescent concussions are characterized by cardiac autonomic dysregulation. The utility of HRV and BRS to assist clinicians in the early diagnosis of adolescent concussion warrants further investigation.

Support or Funding Information

This work was supported by the Children's Health Research Institute (London, Canada).
Cervical neurons control locomotion
Authors: Karadimas Spyridon , Satkunendrarajah Kajana , Sivakumaran Sanghavy , Gosgnach Simon , Fehlings Michael G.
Published: November 2023

The locomotor neural network consisting of genetically defined interneuronal populations is primarily located in the ventromedial region of the lumbar enlargement. Long direct reticulospinal projections have shown to be critical for the activation of this lumbar locomotor network. While, much less is known about the functional connectivity of the cervical propriospinal input during overground locomotion. Here, we first prove that the cervical neurons constitute a major excitatory pathway to locomotor areas of the lumbar spinal cord. Further, we found that cervical spondylotic myelopathy (CSM), the most common form of spinal cord injury selectively disrupts the cervical propriospinal input, and not the reticulospinal, to the lumbar locomotor network. This finding was associated with specific loss of glutamatergic neurons in the rhythmogenic segments of lumbar cord and subsequent decrease in cadence, speed and mobility during overground locomotion. The gradual loss of speed and cadence over time with intact reticulospinal tracts and early disruption of the cervical excitatory monosynaptic input onto excitatory lumbar neurons early in CSM indicate an important role of specific cervico–lumbar pathway in locomotion. To further prove this, we initially ablated the lumbar projecting cervical neurons in naïve mice by employing a dual‐injection experiment. Gait analysis during overground locomotion demonstrated that ablation of these neurons resulted in progressively decreased speed and cadence compared to controls. These neurobehavioural results closely mirrored the locomotion deficits seen in severe human and mice CSM. In addition and similar to the neuroanatomical changes noticed in the severe CSM, stereological analysis demonstrated a decreased number of neurons in the lumbar enlargement of the AAV‐DTA treated animals compared to controls. To further confirm the role of lumbar projecting cervical neurons in initiating and maintaining locomotion, we next selectively silenced the lumbar‐projecting cervical neurons using TeLC. Overtime, TeLC treated animals demonstrated decreased locomotor speed, cadence and overall mobility compared to controls. This phenotype was characterized by a significant increase in the inactive time and decrease in the mobile time and mobile counts compared to control mice indicative of a perturbed ability to initiate and maintain mobility. TeLC‐mediated silencing closely reproduced the locomotor phenotype of CSM and DTA treated animals.
Central Angiotensin‐II Increases Renal Sympathetic Nerve Activity Via Rho Kinase Activation in Conscious Rabbits
Authors: Pellegrino Peter Ricci , Schiller Alicia Marie , Haack Karla K. , Zucker Irving H.
Published: November 2023

Background

A growing body of evidence implicates elevated renal sympathetic tone and activation of the renin‐angiotensin system in the pathogenesis and progression of cardiovascular disease. The RhoA/Rho kinase pathway is an important mediator of the effects of Angiotensin‐II (AngII) in the periphery, but the functional role of this pathway in AngII‐induced renal sympatho‐excitation is not well‐characterized.

Hypothesis

We hypothesized that central inhibition of the RhoA/Rho kinase pathway prevents AngII‐mediated renal sympatho‐excitation.

Methods

Sixteen male New Zealand White rabbits were chronically instrumented with renal sympathetic nerve electrodes, arterial pressure telemetries, and lateral ventricle cannulae. Each rabbit received an intracerebroventricular infusion of AngII, the Rho kinase inhibitor Fasudil (Fas), AngII and Fas, or a vehicle infusion. Seven days after renal nerve electrode implantation, baseline renal sympathetic nerve activity was recorded and quantified by normalization to the nasopharyngeal reflex. Following the study, Rho kinase activity was assessed by performing quantitative immunofluorescence for a phosphorylated form of the Rho kinase target MYPT on midsagittal brain slices using a LiCor Odyssey scanner.

Results

Central AngII infusion significantly raised baseline renal sympathetic nerve activity, but coadministration of the Fas blocked this renal sympatho‐excitation. Importantly, Fas alone had no effect on baseline renal sympathetic nerve activity, resulting in a statistically significant interaction between AngII and Fas. Central AngII infusion increased phospho‐MYPT fluorescence, both diffusely and in the subfornical organ, an important autonomic center, while Fasudil coadministration blocked this Rho kinase activation.

Conclusion

Activation of the RhoA/Rho kinase pathway is necessary for central AngII‐mediated renal sympatho‐excitation.

Support or Funding Information

Supported by NIH F30 HL118974 and NIH P01 HL062222.
Assesing the role of B7‐1 in Diabetic Nephropathy
Authors: Herrera Marcela , Soderberg Magnus , Molne Joan , Santamaria Beatriz , Balverde Angela , Heasman Stephanie , Jermutus Lutz , Baker David , Moreno Carol
Published: November 2023

Diabetic Nephropathy (DN) remains an unmet medical challenge as its prevalence is projected to continue to increase and specific medicines for treatment remain undeveloped. Activation of the immune system, in particular T‐cells, is emerging as a possible mechanism underlying DN disease progression in humans and animal models. We hypothesized that inhibition of T‐cell activation will ameliorate DN. Interaction of B7‐1 (CD80) on the surface of antigen presenting cells with its binding partners, CTLA4 (CD152) and CD28 on T‐cells, is essential for T‐cell activation. In this study we used a soluble CTLA4‐Fc fusion protein to block cell surface B7‐1, preventing the cellular interaction and inhibiting T‐cell activation.

The CTLA4‐Fc Abatacept was dosed i.p. in the Streptozotocin‐induced Diabetic Nephropathy mouse model in both prevention and intervention modes. Renal damage was assessed by albuminuria and renal B7.1 positive cells and lymphocytes were measured by immunohistochemistry. Gene expression interrogated by real‐time PCR and protein expression by Western blotting. Differentiated human immortalized podocytes were used for in vitro studies.

When CTLA4‐Fc was dosed in an animal model of DN, it reduced albuminuria in both prevention and intervention modes. The number of T‐cells infiltrating the kidneys of DN animals correlated with the degree of albuminuria and treatment with CTLA4‐Fc reduced the number of renal T‐cells. As B7‐1 induction has been recently proposed to underlie podocyte damage in DN, CLTA4‐Fc could be efficacious in DN by protecting podocytes. However, this does not appear to be the case as B7‐1 was not expressed in: 1) kidneys of DN animals; 2) stimulated human podocytes in culture; or 3) glomeruli of DN patients.

We conclude that CTLA4‐Fc ameliorates DN by blocking T‐cell activation and not by protecting podocytes.
Role of Myosins IIA and VI in Mediating the in vivo Hormonal Regulation of NHE3 Activity in the Rat Renal Proximal Tubule
Authors: Crajoinas Renato Oliveira , Castelo‐Branco Regiane Cardoso , Malnic Gerhard , Mello‐Aires Margarida , Girardi Adriana Castello Costa
Published: November 2023

Rapid regulation of NHE3 activity may occur through subcellular redistribution of the transporter between the microdomains of the proximal tubule (PT) microvilli (MV). Myosin motor proteins that move along actin filaments on the MV may mediate NHE3 translocation in response to a variety of stimuli. The present study was undertaken to test the following hypothesis: (i) that myosin IIA may prevent NHE3 stimulation by angiotensin II (Ang II), which is known to increase NHE3 activity at least in part by translocating NHE3 from the base to the body of the PT MV; (ii) that myosin VI may prevent NHE3 inhibition by parathyroid hormone (PTH), which is known to reduce NHE3 activity at least in part by translocating NHE3 from the body to the base of the PT MV. To this end, NHE3 activity of Wistar rats was evaluated by net bicarbonate reabsorption (JHCO3−) of in vivo renal proximal tubules using H+ ion‐sensitive microelectrodes in the presence or absence of 10−10 M Ang II, 10−6 M PTH, 10−5 M blebbistatin (a specific myosin IIA inhibitor) or 4×10−6 M 2,4,6‐triiodophenol (TIP) (a specific myosin VI inhibitor). As expected, Ang II increased [4.36 ± 0.18 nmol/cm2.s (N=25)] whereas PTH reduced [1.18 ± 0.10 nmol/cm2.s (N=17)] NHE3‐mediated bicarbonate reabsorption compared to CTRL [2.74 ± 0.15 nmol/cm2.s (N=9)]. Simultaneous tubular perfusion of Ang II with blebbistatin completely prevent Ang II‐induced NHE3 stimulation [2.92 ± 0.14 (N=21) vs. 4.36 ± 0.18 nmol/cm2.s (N=25), P < 0.001]. Likewise, simultaneous tubular perfusion of PTH with TIP was capable of blocking the inhibitory effect of PTH [2.91 ± 0.15 (N=22) vs. 1.18 ± 0.10 nmol/cm2.s (N=17), P < 0.001] on NHE3 activity. Neither blebbistatin [2.51 ± 0.11 (N=15) vs. 2.74 ± 0.15 nmol/cm2.s, (N=9)] nor TIP [2.49 ± 0.18 (N=14) vs. 2.74 ± 0.15 nmol/cm2.s (N=9)] alone affected baseline NHE3 activity. These results suggest that myosin IIA and VI may play a role in mediating hormonal regulation of NHE3 in the rat kidney proximal tubule.

Support or Funding Information

FAPESP