bluuebunny/crossref_metadata_2025 huggingface dataset:
The SGLT2 Inhibitor, Empagliflozin, Attenuates Some Markers of Renal Fibrosis without Improving Albuminuria in Diabetic Db/Db Mice
Authors: Gallo Linda A , Ward Micheal S , Fotheringham Amelia K , Zhuang Aowen , Koepsell Hermann , Vallon Volker , Pollock Carol , Panchapakesan Usha , Forbes Josephine M
Published: November 2023

Sodium‐dependent‐glucose‐transporter 2 (SGLT2) inhibitors are a novel therapy for type 2 diabetes. They block glucose reabsorption by the kidney which promotes urinary glucose excretion and lowers blood glucose levels. We aimed to determine whether SGLT2 inhibition is renoprotective in a mouse model of type 2 diabetes and nephropathy. Male db/db and db/m (C57BLKS/J) mice were administered with once‐daily oral treatment of the SGLT2 inhibitor, empagliflozin (10mg/kg/day), or vehicle from 10–20 weeks of age (n=11/group). These were compared with db/db mice which received metformin (250mg/kg/day) or empagliflozin + metformin co‐therapy (dose as per single). At baseline, all db/db mice were obese with hyperglycemia (P<0.05). Towards the study end, these mice had glomerular hyperfiltration, albuminuria, increased levels of urinary kidney injury molecule‐1 (KIM‐1), increased kidney weight, tubulointerstitial total collagen accumulation, glomerulosclerosis, and increased renal expression of pro‐fibrotic genes (P<0.05). Treatment with empagliflozin, either as a mono‐ or co‐therapy, improved glycemic control compared to vehicle (glycated hemoglobin 6.6% and 5.7%, respectively, vs 9.6%, P<0.05) but did not reduce albuminuria, urinary KIM‐1, kidney weight, or glomerulosclerosis. Empagliflozin in diabetic mice reduced total collagen accumulation and renal expression of Fn1 and Tgfb1 and, when co‐administered with metformin, also attenuated the increased renal cortical expression of ColIV and Ctgf, and glomerular hyperfiltration (P<0.05). Empagliflozin, alone or in combination with metformin, increased intra‐renal renin activity compared to vehicle‐treated diabetic mice (P<0.05) but did not increase cortical angiotensin II content. In this mouse model of diabetic nephropathy, blood glucose lowering with SGLT2 inhibition did not improve albuminuria but attenuated some histological and molecular markers of renal fibrosis. Hyperglycemia and albuminuria‐onset prior to commencement of treatment may have been sufficient for disease development and rendered our study an interventional approach. Thus, early, sufficient, and stable blood glucose lowering with SGLT2 inhibition, possibly in combination with RAS blockade, may be required to achieve maximal renoprotection in diabetes. Further, the potential for SGLT2 inhibition to slow progression to more advanced stages of kidney disease remains to be assessed.

Support or Funding Information

National Health and Medical Council of Australia and Heart Foundation (Australia). Boehringer‐Ingelheim (Germany) provided the empagliflozin.
Dysregulation of Insulin receptor expression affects proximal tubule gluconeogenesis
Authors: Pandey Gaurav , Mandhani Anil , Srivastava Anssesh , Ecelbarger Carolyn , Agnihotri Kripa S , Gaikwad Anil , Tiwari Swasti
Published: November 2023

Reduced insulin receptor protein levels have been reported in the kidney cortex from diabetic human and animals. We have recently reported that targeted deletion of insulin receptor (IR) from proximal tubules (PT) resulted in hyperglycemia in non‐obese mice. To understand the mechanism, we studied human proximal tubule cells (hPTC) and C57bl/c mice fed with high fat diet (HFD, 60% fat for 20 weeks). Immunoblotting revealed a significantly lower expression of IR and blunted rise in p‐AKT levels in the kidney cortex of HFD mice in response to acute insulin (2 unit/kg body weight, i.p) relative to normal chow diet (NCD) n=8/group, p<0.04). Moreover, we found significantly higher transcript levels of phosphoenolpyruvate carboxykinase (PEPCK, rate‐limiting gluconeogenic enzyme) in the kidney cortex from HFD, relative to mice on NCD, (n=8, p<0.05). The higher level of PEPCK in HFD was confirmed by immnoblotting. However, no significant difference was observed in G6Pase and FBPase enzyme transcript levels in the renal cortex of HFD relative to NCD. Furthermore, an in vitro study using primary human PT cells (hPTC) showed insulin's inhibitory effect on cAMP/DEXA –induced gluconeogenesis in these cells. In addition, transcript levels of gluconeogenic enzymes G6Pase and PEPCK were significantly increased in cAMP/DEXA‐stimulated hPTC cells (n=5, p=0.05). However, these effects of insulin on cAMP/DEXA–induced gluconeogenesis and gluconeogenic enzyme induction were blunted in IR silenced hPTC (n=3, p<0.05). Taken together the above data indicate a direct role of insulin receptor in PT‐gluconeogenesis regulation. Thus reduced insulin sensitivity of the proximal tubule could, itself, contribute to hyperglycemia via elevated gluconeogenesis. (BT/HRD/35/02/17/2008)

Support or Funding Information

The work was supported by the Department of Biotechnology, government of India, Ramalingaswami grant (BT/HRD/35/02/17/2008 to S.T). G.P. was supported by Research Fellowship from University Grant Commission.
Engineered FGF21 variant, LY2405319, can protect nonalcoholic fatty liver disease through enhancing hepatic mitochondrial function
Authors: CHUNG HYO KYUN , Kang Yea Eun , Hwang Jin Hee , Kim Koon Soon , Shong Minho
Published: November 2023

Objective

Nonalcoholic fatty liver disease (NAFLD) is one of the causes of fatty liver, linked to insulin resistance, metabolic syndrome, liver fibrosis and cirrhosis. Traditionally, NAFLD has been recognized as a result of excess nutrient intake. However, the molecular target of NAFLD has not been fully addressed. The increased ROS/RNS and mitochondrial dysfunction of hepatocyte can play critical roles in the development of NAFLD and nonalcoholic fatty liver (NASH) through chronic inflammation and insulin resistance. We here show that the beneficial effects of LY2405319, an Engineered FGF21 Variant (lilly, USA), on NAFND using ob/ob mice model.

Methods

The 6‐week‐old male ob/+ control and ob/ob C57BL/6J Lep(−/−) mice were divided into three groups, including ob/+ control, vehicle‐treated (ob/ob‐vehicle) and LY2405319‐treated group (ob/ob‐LY). The vehicle and LY2405319 (5mg/kg) were administered oral gavage for 3 weeks. Body weight was recorded every 2 days. Intraperitoneal glucose tolerance test and energy expenditure were performed after LY2405319 treatment for 3 weeks. The effects of LY2405319 on NAFLD progression were evaluated using biochemical, hitological and molecular markers. Oxygen consumption rate (OCR) and mitochondrial gene expressions were measured to determine the effects on mitochondria in ob/ob mouse and primary hepatocyte.

Results

LY2405319 treated ob/ob mice revealed decreased body weight and serum glucose levels and markedly decrease of serum AST, serum ALT and hepatic triglycerides compared to control group. And also, hepatic steatosis and lipid peroxidation significantly improved compared to control group. Interestingly, LY2405319 treatment mice group showed increased energy expenditures and significantly increased in mitochondrial transcriptional‐, and translational gene expression than control group. Furthermore, LY2405319 increased OCR by dose dependent manner in primary hepatocytes.

Support or Funding Information

This work was supported from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning, Korea (NRF‐2015R1A2A1A13000951). H.K.C. and J.H.H were supported by NTF grant, the Ministry of Education (MOE), Korea (NRF‐2013R1A1A2058889).
The Role of P2X7R in Priming the NLRP3 Inflammasome after Mechanical Strain
Authors: Albalawi Farraj , Lu Wennan , Lim Jason , Mitchell Claire H
Published: November 2023

The NLRP3 inflammasome is a cytosolic multiprotein complex whose activation results in the maturation and release of cytokine IL‐1β. There are two steps in inflammasome involvement; first the relevant proteins must be primed and upregulated on a transcriptional and translational level. Second, the proteins must be activated to assemble and process the maturation of caspase 1 and IL‐1β. The P2X7 receptor (P2X7R) is known to activate the NLRP3 inflammasome. However, it is currently unclear whether the P2X7R also plays a role in priming the inflammasome components. Rat optic nerve head astrocytes were swollen by 30% hypotonic solution for 4 hours then RNA was extracted. For in vivo experiments, we elevated intraocular pressure (IOP) moderately to 50–60 mmHg for 4 hours, with RNA extracted 24 hrs later. qPCR analysis was used to measure the expression of inflammasome related genes. Rat optic nerve head astrocytes release ATP when swollen. Swelling these cells for 4 hours upregulated mRNA for NLRP3 and IL‐1β but not caspase 1. This upregulation was inhibited by 4 μM Bay11‐7082 (NFκB inhibitor), implicating NFκB. Inhibiting the P2X7R with 10 μM Brilliant blue G (BBG), 50 nM A839977, or 5 μM A740003 prevented the swelling‐induced upregulation of IL‐1β but not NLRP3. In vivo, the expression of IL‐1β, NLRP3 and caspase 1 were all increased at the mRNA level in optic nerve head astrocytes of eyes subjected to moderate elevation of IOP. Intraocular injection of BBG prevented the rise in IL‐1β but not NLRP3. In conclusion, mechanical strain upregulates NLRP3 and IL‐1β mRNA via NFκB; but only the upregulation of IL‐1β required P2X7R stimulation.

Support or Funding Information

NIH Grants EY013434, EY015537
Training Enhances the Recovery of Monocyte Subset Response to a Bout of Resistance Exercise
Authors: Jajtner Adam R , Hoffman Jay R , Townsend Jeremy R , Beyer Kyle S , Varanoske Alyssa N , Church David D , Riffe Josh J , Muddle Tyler WD , Radom‐Aizik Shlomit , Fukuda David H , Stoute Jeffrey R
Published: November 2023

Monocytes play a key role in inflammatory processes. Little is known on monocyte response to resistance exercise. The purpose of this study was to examine the acute changes in monocyte subsets following an acute muscle damaging bout of resistance exercise before (PRE) and after (POST) six weeks of resistance training. Fifteen recreationally active men (22.1 ± 2.2 y) completed an acute, muscle damaging protocol PRE and POST that consisted of the squat (6 sets), leg press (4 sets) and leg extension (4 sets) exercises. Ten repetitions at 70% of 1‐RM were completed for each set. Blood was drawn at baseline (BL), immediately (IP), one (1H), five (5H), 24 (24H) and 48 hours (48H) post‐exercise. Flow cytometry was used to identify monocyte subpopulations: classical (CLAS; CD14++/CD16−) and intermediate (INT; CD14++/CD16+). Repeated measures ANOVA was applied. A significant training x time interaction was observed for CLAS (p = 0.008) and INT (p = 0.036). CLAS significantly increased at 1H (BL, 89.3±5.5% vs.1H, 95.10 ± 4.19%; p < 0.001) and 5H (p = 0.012; 91.79 ± 4.14%) in PRE, while at POST, only a trend toward a significant increase was noted at 1H (BL, 90.66 ± 4.62% vs. 1H, 92.50 ± 3.75%, p = 0.067). Additionally, CLAS was significantly higher PRE than POST at 1H (95.10 ± 4.19% vs. 92.50± 3.75%; p = 0.048) and 5H (91.79 ± 4.14% vs. 88.42 ± 5.46%; p = 0.005). During PRE, the proportion of INT appeared to increase (p < 0.001) from BL (4.23 ± 2.19%) at IP (5.62 ± 2.77%), but decreased at both 1H (p < 0.001; 2.01 ± 1.56%) and 5H (p = 0.001; 3.16 ± 1.82%). However, increases from BL were noted at 24H (p = 0.010; 5.83 ± 3.35%) and 48H (p = 0.049; 6.15 ± 3.25%). At POST, changes in INT were similar to PRE, except proportion of INT returned to BL levels at 5H and remained there throughout the 48H recovery period. In conclusion, resistance training appears to enhance the recovery of monocyte subsets to resting proportions following an acute bout of resistance exercise.
Contribution of Endogenous Hydrogen Sulfide to Hypoxic Inhibition of Sodium Transport by Lung Epithelial Cells
Authors: Althaus Mike , Krause Nicole Catherine , Pott Jennifer , Münch Lena Katharina , Olson Kenneth R
Published: November 2023

In lung epithelial cells, hypoxia decreases the expression and activity of sodium transporting molecules, thereby reducing the rate of transepithelial sodium absorption. The mechanisms underlying the sensing of hypoxia and subsequent coupling to sodium transporting molecules remain unclear. Hydrogen sulfide (H2S) has recently been recognized as a cellular signaling molecule whose intracellular concentrations critically depend on oxygen levels. Therefore it was questioned whether endogenously produced H2S contributes to hypoxic inhibition of sodium transport. H2S is generated by multiple pathways involving cystathionine‐γ‐lyase (CSE), cystathionine‐β‐synthase (CBS) and 3‐mercaptopyruvate sulfurtransferase (3‐MST). RT‐PCR and immunoblotting experiments revealed the presence of all of these enzymes in human H441 lung epithelial cells. Using an amperometric H2S‐sensitive electrode, H2S‐production by H441 lysates was detected. H2S‐production inversely correlated with oxygen concentrations. In electrophysiological Ussing chamber experiments, exogenous application of H2S by the sulfur salt Na2S inhibited amiloride‐sensitive sodium transport by cultured H441 monolayers. Furthermore, acute hypoxia which was established by decreasing oxygen concentrations from 10 % to 0.1 % for 30 min decreased amiloride‐sensitive sodium transport by these cells. Pretreatment with the CSE‐inhibitor D/L‐propargylglycine (PAG) decreased hypoxic inhibition of sodium transport, whereas inhibition of CBS (with aminooxy‐acetic acid; AOAA) or 3‐MST (with aspartate) had no effect. Inhibition of all H2S‐generating enzymes with a combination of AOAA, PAG and aspartate abrogated the hypoxic inhibition of sodium transport. By contrast, increasing the concentrations of L‐cysteine, pyridoxalphosphate and α‐ketoglutarate, precursors of H2S production, did not enhance the effect of hypoxia. These data indicate that H441 lung epithelial cells endogenously produce H2S during hypoxia and this might contribute to hypoxic inhibition of sodium absorption.

Support or Funding Information

This work was funded by the German Research Foundation (AL‐1453/1‐2).
Effect of Age on Sexually Dimorphic Expression of X‐linked Genes in the Developing Mouse Cortex and Hippocampus
Authors: Moreira Debbie , Armoskus Chris , Rath Faviolanny , Reyes Rosa , Tsai Houng‐Wei
Published: November 2023

Among different brain regions, the cortex and hippocampus are responsible for regulating cognitive and social behaviors, and considerable evidence suggests that these functions are sexually dimorphic. However, little is known about the molecular and genetic mechanisms underlying sexual dimorphism in these two brain regions. An earlier study observed higher mRNA expression of six X‐linked genes, Ddx3x, Eif2s3x, Kdm5c, Kdm6a, Uba1, and Usp9x, in the female mouse brain than the male during early development and adulthood, suggesting that sex chromosomes may act directly in brain to cause sex differences in development and function. Recently, we have identified eight candidate genes located on X chromosome differentially expressed in the neonatal mouse cortex/hippocampus between the sexes, including Ebp, Eif2s3x, Ftsj1, Kdm6a, Psmd10, and Xist as well as two pseudoautosomal genes, Erdr1 and Mid1. We hypothesized that similar to those observed in neonates, dimorphic expression of these genes along with Ddx3x, Kdm5c, Uba1, and Usp9x, might be persistently present in the mouse cortex/hippocampus during early development, responsible for establishing distinct structures and circuits underlying sex‐biased cortical and hippocampal function. To test this hypothesis, we employed RT‐qPCR to measure mRNA levels of Ddx3x, Ebp, Eif2s3x, Erdr1, Kdm5c, Kdm6a, Mid1, Psmd10, Uba1, Usp9x, and Xist in the male and female cortex/hippocampus of C57BL/6J mice collected on the day of birth (PN0), and 7 (PN7), 14 (PN14), and 21(PN21) days after birth (n=7–9 per group). We found that among the 11 genes tested, female mice expressed higher mRNA levels of Eif2s3x, Erdr1, Kdm5c, Kdm6a, and Xist than males. Interestingly, the female‐biased expression Erdr1 and Kdm6a was only observed at PN21 and PN0 & PN21, respectively. As compared to PN0, Eif2s3x and Xist showed a decrease in expression at PN7 and PN14 while reduced Kdm5c levels were found at PN7 only. Based on our data, we concluded that during early development, sexually dimorphic expression of selected X‐linked genes in the mouse cortex/hippocampus is dynamically altered with age, suggesting that these genes might participate in brain feminization at different developmental stages.

Support or Funding Information

This research project was supported by the National Institute of General Medical Sciences Grant SC3‐GM102051.
Regulation of the Sexually Dimorphic Gene, Rora, in the Developing Mouse Cerebellum Is Independent of Sex Steroids
Authors: Mota Thomas , Tsai Houng‐Wei
Published: November 2023

The cerebellum is important for motor coordination and non‐motor functions, such as cognition, language, and social function. Furthermore, there is mounting evidence of cerebellar involvement in gender‐biased neurological disorders and mental illness. For example, decreased cerebellar expression of retinoic acid receptor‐related orphan receptor α (Rora) is observed in autism, a male‐biased neurodevelopmental disorder characterized by deficits in social behavior and interpersonal communication. In addition, Rora expression is up‐regulated and downregulated in vitro by estrogen receptor α (ERα) and androgen receptor (AR), respectively. Since a perinatal rise in testosterone secreted by the developing testes acts directly on AR or indirectly on ERα via estradiol synthesized locally by aromatase to masculinize brain structures and behaviors, we hypothesized that Rora expression in the mouse cerebellum during early development might be sexually dimorphic, which is regulated by the activation of AR and/or ERα. To test our hypothesis, we measured mRNA levels of Rora in male and female mouse cerebella collected on the day of birth (PN0), 7 (PN7), 14 (PN14), and 21 days (PN21) after birth by quantitative RT‐PCR. While Rora levels significantly increased with age (p<0.001), they were male‐biased on PN0 (p=0.006), but female‐biased at PN14 (p=0.02). We next determined if the male‐biased expression of Rora in the neonatal cerebellum was regulated by AR and/or ERα by treating pregnant female mice daily with vehicle, testosterone propionate (TP), or estradiol benzoate (EB) starting on embryonic day 16 until PN0. The cerebellum was collected from vehicle‐, TP‐, and EB‐treated, male and female neonatal pups. As compared to vehicle‐treated controls, there was no effect of TP (p=0.812) or EB (p=0.624) on Rora expression, suggesting that sex steroids might not be required for the control of Rora expression. In summary, sexual dimorphism in Rora expression in the neonatal mouse cerebellum is transient, followed by a robust change with age during early development. Although the Rora gene in the neonatal cerebellum does not seem to be regulated by sex steroids and their receptors, sex‐ and age‐specific expression of the Rora gene might still be involved in the control of cognitive behaviors between the sexes and the etiology of sex‐biased neurological disorders and mental illnesses.

Support or Funding Information

This research was supported by the National Institute of General Medical Sciences Grant SC3‐GM102051.
Loss of Cdk5 Function May Mediate Age‐Related Decline in Voluntary Physical Activity
Authors: Ruegsegger Gregory , Toedebusch Ryan , Booth Frank
Published: November 2023

Increases in age are associated with declining levels of physical activity in multiple species, including humans, which in turn promotes the development of numerous chronic diseases. However, the neuro‐molecular mechanisms that regulate physical activity motivation at various ages are poorly understood. Our lab has previously observed that, in female Wistar rats, lifetime voluntary wheel running behavior is greatest at 8 wks of age.

Objective

We aimed to assess differences in the nucleus accumbens (NAc), a specific brain nucleus postulated to influence rewarding behavior, of wheel running and sedentary female Wistar rats at 8 and 14 weeks (wks) of age (n= 5–8).

Methods

Wheel running rats were introduced to wheels at 5 wks of age. RNA‐seq, qRT‐PCR and Western blotting were used to assess molecular changes between 8‐ and 14‐wk‐old wheel running and sedentary rats.

Results

Voluntary wheel running was greatest at 8 wks (17.7± 1.0 km/d) and had significantly decreased by 14 wks (11.1± 1.2 km/d). From 619 differentially expressed genes, bioinformatics suggested that cAMP‐mediated signaling, DARPP‐32 feedback, PKA signaling, and synaptic plasticity were increased at 8‐ compared to 14‐wk in wheel running rats. While analyzing transcripts specific to these functions, we observed significant (~20–30%; p< 0.05) decreases in Cadm4, Cdk5, and p39 (Cdk5 activator) mRNAs and proteins from 8 to 14 wks of age in voluntary running rats. Further, Cadm4 (r= 0.84), Cdk5 (r= 0.57), and p39 (r= 0.71) mRNAs were significantly correlated with voluntary running distance during the week of sacrifice. Follow‐up analysis of sedentary 8‐ and 14‐wk‐old rats (n= 5–8) by qRT‐PCR found similar decreases in Cadm4 and p39 mRNAs and proteins from 8 to 14 wks of age (p< 0.05), suggesting an age‐dependent reduction in synaptic function and Cdk5 activity may mediate decreases in wheel running. In light of these transcriptomic and protein changes, we assessed wheel‐running distance following NAc injection of the Cdk5 inhibitor roscovitine (ROS) (80 nmol/0.5μg) (n= 7) or vehicle (n= 5). Compared to vehicle, ROS significantly decreased wheel running distance 0–30 and 30–60 min post‐injection (p< 0.001), and 60–90 min post‐injection (p< 0.01). These effects were present after both 1 day and 5 days of daily injections.

Conclusion

Collectively, these experiments suggest that an age‐dependent loss in synaptic function and Cdk5/p39 activity in the NAc may mediate the age‐related decline in voluntary running behavior.

Significance

Given the epidemic levels of physical inactivity and its associated consequences, these data may foster the development of therapies targeted to reduce age‐dependent increases sedentary behavior.

Support or Funding Information

Research was supported by an MU Life Sciences pre‐doctoral fellowship (GNR) and MU development funds (FWB).
Angiotensin‐(1‐7) Delays Onset of Dilated Cardiomyopathy in Mice with Muscular Dystrophy
Authors: Sabharwal Rasna , Yang Liping
Published: November 2023

We have demonstrated that angiotensin II‐induced autonomic and skeletal muscle dysfunction precedes and predicts development of dilated cardiomyopathy (DCM) and premature mortality in sarcoglycan‐delta deficient mice, i.e. murine model of muscular dystrophy [Exp Physiol 2015]. In addition, we have shown that ang‐(1‐7) administered for 8 weeks markedly improves autonomic function and locomotor activity in young (>15 weeks of age) Sgcd−/− mice by attenuating levels of circulating and skeletal muscle Ang‐II [Clin Sci 2014]. In this study, our goals were – (i) to determine whether Ang‐(1‐7) infusion delays onset of DCM in older mice (>50 weeks of age); and (ii) to evaluate the performance of the recently released SMP‐300 iPRECIO Infusion Pumps (Primetech Corporation®, Japan) in delivering 500 ng of Ang‐(1‐7) at a rate 0.1 ml/hour for 49 days. Blood pressure (BP), heart rate (HR) and sympathovagal tone were measured by telemetry (PA‐C10, DSI®) in older control C57BL6 (untreated n=3) and Sgcd−/− (untreated n=4; treated n=4) mice. Compared to untreated control mice, untreated Sgcd−/− mice exhibit increased sympathetic tone (HR response to propranolol), reduced HR variability (parasympathetic tone), increased cardiac fibrosis (Masson's staining), and cardiac dysfunction (echocardiography) (all variables P<0.05). Ang‐(1‐7) effectively restored autonomic function, reduced cardiac fibrosis by 44%, increased ejection fraction by 21%, and reduced end‐diastolic volume/mass by 57% (P<0.05 vs. untreated Sgcd−/− mice). We conclude: 1) Ang‐(1‐7) treatment restores sympathovagal balance, reduces fibrosis and delays onset of DCM in older aged mice with muscular dystrophy. 2) iPRECIO infusion pumps are refillable, reprogrammable, and delivers drugs easily and successfully. 3) Albeit the combined weights of iPRECIO pump (3.3 g) and DSI transmitter (1.4 g) do not interfere with data collection, it is recommended to use mice not smaller than 25 g of body weight.

Support or Funding Information

Tarix Pharmaceuticals, Primetech, DSI
Developing manganese‐enhanced MRI as a non‐invasive measure of in vivo neuronal activity in the rostral ventrolateral medulla (RVLM) of sedentary and physically active rats
Authors: Huereca Daniel J , Holt Avril G , Berkowitz Bruce A , Mueller Patrick J
Published: November 2023

Elevated sympathetic nerve activity (SNA) is implicated as a major risk factor for cardiovascular disease (CVD). We have previously demonstrated greater SNA in sedentary versus physically active rats following direct and indirect activation of the RVLM, the primary regulator of SNA. We have also reported changes in structure among spinally projecting neurons in the RVLM, including greater dendritic branching in sedentary versus physically active rats. These studies indicate that sedentary conditions promote structural and functional changes within the RVLM linked with increased SNA. The aim of the present study was to develop a new method, manganese‐enhanced magnetic resonance imaging (MEMRI), to compare changes in RVLM activity over time in sedentary versus physically active rats. MEMRI utilizes both the paramagnetic properties of manganese (Mn2+) as well as its uptake by active neurons. Mn2+ is a T1 MRI contrast agent that enters active neurons via calcium channels, thus, greater manganese uptake indicates greater neuronal activity. We hypothesized that the enhanced SNA in sedentary versus physically active rats is due to greater activity of bulbospinal RVLM neurons. To evaluate the influence of sedentary and physically active conditions on in vivo neuronal activity of the RVLM longitudinally, five week old male Sprague Dawley rats (n=4) were anesthetized (2% isoflurane) and RVLMs were imaged on a 7T small animal MRI (Bruker ClinScan). One week later, changes in T1 were evaluated 24 hours after an i.p. injection of manganese (66mg/kg MnCl2). Two rats were then each assigned randomly to a sedentary or physically active group (spontaneous wheel running). After five weeks (24 hours after a 2nd injection of MnCl2), Mn2+ uptake was greater in the RVLM of sedentary versus physically active rats, regardless of the method used for collection and analysis of the MRI data; T1 weighted (+16.3% vs +6.8% signal increase) or T1 mapping (1/T1=0.81 vs 0.69). Furthermore, physically active rats had similar or decreased Mn2+ uptake after versus before wheel running (6.8 vs 11.0% change), while sedentary rats appeared to have enhanced Mn2+ uptake after versus before five weeks of sedentary conditions (16.3 vs 7.4% change). These initial findings support our hypothesis that sedentary conditions promote exaggerated neuronal activity in the RVLM, as indicated by greater Mn2+ accumulation in sedentary versus physically active rats. Enhanced RVLM activity due to sedentary conditions may contribute to greater SNA and contribute to the mechanisms by which a sedentary lifestyle increases the risk of developing CVD in humans.

Support or Funding Information

DJH AHA 15PRE25700308; PJM NIH HL096787‐05A1, AHA 25810010; AGH USDVA 1I01RX001095‐011Rx; BAB NEI EY021619
Rostrocaudal changes in the protein expression of GABAA receptor subunits in the rostral ventrolateral medulla (RVLM) of sedentary and physically active rats
Authors: Mueller Patrick J , Dombrowski Maryetta D , Fyk‐Kolodziej Bozena E
Published: November 2023

It has been well established that GABA is the major inhibitory neurotransmitter in the RVLM, acting primarily through GABAA receptors to regulate tonic activity of pre‐sympathetic RVLM neurons. We have reported that GABAergic modulation of sympathoexcitation in the RVLM is enhanced in sedentary versus physically active rats, despite an overall increase in sympathoexcitation in sedentary animals. The purpose of the present study was to test the hypothesis that sedentary conditions enhance protein expression of GABAA receptor subunits within the RVLM. Physically active or sedentary, male Sprague‐Dawley rats (n=3 ea.) were sacrificed after 10 weeks of wheel running or sedentary conditions, respectively. The brainstem was cryosectioned serially (80 μm) and bilateral micropunches were collected for Western blotting. Punched sections were stained with cresyl violet to verify the rostrocaudal boundary of RVLM and orient pooled punches relative to the caudal pole of the facial nucleus (FN). GABAAα1 and GABAAα2 subunit expressions were normalized to GAPDH and examined in two caudal (FN −480 μm, FN −240 μm) and two rostral (FN +240 μm, FN +480 μm) regions. GABAAα1 expression was significantly lower in sedentary vs. physically active rats overall (p<0.05, main effect), but was higher in rostral vs. caudal regions in both groups (p<0.05, main effect, no interaction). In contrast, GABAAα2 expression did not exhibit a significant rostrocaudal distribution, but was still lower in sedentary vs. physically active (p<0.05, main effect, no interaction). These results suggest that GABAAα1 and GABAAα2 subunit expressions are not responsible for enhanced GABAergic modulation of sympathoexcitation in the RVLM of sedentary rats. Despite results contrary to our original hypothesis, our studies further demonstrate activity‐dependent neuroplasticity in the region of the RVLM involved in control of sympathetic outflow. Further studies including examination of other receptor subunits and the time course by which activity‐dependent neuroplasticity occurs in the RVLM will be important in elucidating and better understanding the mechanisms by which a sedentary lifestyle promotes cardiovascular disease via influences on the sympathetic nervous system.

Support or Funding Information

Supported by HL096787‐05; HL096787‐05S1; AHA25810010.
Acute Effects of Intermittent Hypoxia on Leptin‐Mediated Increases in Adiponectin Expression
Authors: Becari Christiane , Somers Kiran R , Polonis Katarzyna , Pfeifer Michaela A. , Prachi Singh
Published: November 2023

Background

Obstructive sleep apnea (OSA) patients have elevated levels of plasma leptin and are exposed to repeated nocturnal intermittent hypoxia (IH). Leptin induces increased adiponectin expression, suggesting that adiponectin should be increased in OSA. However, levels of plasma adiponectin, an anti‐atherogenic and insulin‐sensitizing adipokine, are inconsistent in OSA, and adiponectin levels are often low. Both increases in leptin and decreases in adiponectin may contribute to increased cardiometabolic risk in OSA. In this study we sought to examine the interactions between IH, leptin, and adiponectin. Using an in‐vitro approach in differentiated human white preadipocytes (dHWP), we tested the hypothesis that adiponectin expression in response to acute IH‐mediated increases in leptin may be modulated in the presence of hypoxia.

Methods

dHWP were exposed to 24 cycles of IH (30 min 0.1% O2 followed by 30 min exposure to 21% O2), continuous hypoxia (0.1% O2) or continuous normoxia (21% O2, control). Secretion of leptin and adiponectin in the 24h conditioned media was determined by ELISA and intracellular changes in adiponectin were determined by Western blot analysis.

Results

We show that acute 24 hour exposure to IH or sustained hypoxia increased leptin secretion (p = 0.013) but did not alter adiponectin secretion (p = 0.48). In fact, the intracellular expression of adiponectin actually decreased during the acute exposure to both IH and to continuous hypoxia (p = 0.02). Also, while leptin increased intracellular adiponectin expression in normoxic conditions (p = 0.03), it was unable to do so in the presence of IH and hypoxia (p = 0.36).

Conclusions

Acute IH and continuous hypoxia mediate increases in leptin, but not in adiponectin secretion. Furthermore, although leptin increases adiponectin in normoxic conditions, IH and continuous hypoxia cause decreases in intra‐cellular adiponectin expression and also attenuate leptin‐mediated increases in adiponectin expression. These hypoxia‐effects on adiponectin expression may help explain the low adiponectin levels in OSA, which may contribute to increased cardiovascular and diabetes risk.

Support or Funding Information

Christiane Becari was supported by a grant from CNPq Brazil (# 203802/2014‐4); Michaela Pfeifer was supported by the APS STRIDE summer undergraduate research fellowship (NHLBI 1R25HL115473‐01); Prachi Singh was supported by AHA scientist development grant 11SDG7260046.
Geographic distribution and functionality of cuticular hydrocarbons in Drosophila athabasca
Authors: Harvey Taylor , Yukilevich Roman
Published: November 2023

Various races of Drosophila athabasca exhibit differences in cuticular hydrocarbon profiles. Races Eastern A and West Northern exemplify these morphologies and thus were used for experimentation. Despite previous work postulating a connection between these traits and sexual selection, we have found that they play little to no role in female choice. Rather, we believe that cuticular hydrocarbons are instead used to aid in desiccation resistance. These waxy molecules provide a layer of protection for the flies against water loss as a result of environmental conditions. Flies were placed into vials containing silica gel in order to induce desiccation and show which lines were able to survive the for the longest period of time. We will construct mortality curves of D. athabasca from various geographic areas in which each race is found in order to determine which pheromone profile is best equipped to resist desiccation. Further, we aim to explore differences within each race based on geographic range of the populations collected. We hypothesize that lines of D. athabasca that inhabit warmer and drier climates will have a pheromone profile that is more resistant to desiccation.

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Union College Undergraduate Research Committee
High glucose‐mediated oxidative stress impairs vasodilation of small resistance skeletal muscle arteries from mourning doves (Zenaida macroura)
Authors: Ahmed Zoha S , Jarrett Catherine L , Faust James J , Sweazea Karen L
Published: November 2023

Plasma glucose concentrations are naturally 1.5–2 times higher in birds than mammals of similar body mass. In mammals, hyperglycemia is associated with superoxide‐mediated endothelial dysfunction resulting in hypertension. In contrast to mammals with hyperglycemia, oxidative stress is typically low in birds. This apparent avian resistance to oxidative stress may be attributed to higher antioxidant status. Therefore, we hypothesized that endothelium‐dependent vasodilation of isolated arteries from mourning doves (Zenaida macroura) would not change after acute exposure to high glucose. Mourning doves were captured in Tempe, Arizona using walk‐in style funnel traps. Following euthanasia with sodium pentobarbital (200 mg/kg body mass), small resistance cranial tibial arteries were isolated, cannulated and pressurized in a vessel chamber. Isolated arteries were then exposed to either physiological (20 mM/L) or high (30 mM/L) glucose concentrations for 1 hour at 41°C (normal dove body temperature). Vessels were then pre‐constricted to 50% of resting inner diameter with phenylephrine in the superfusate. Next, vessels were exposed to increasing concentrations of the endothelium‐dependent vasodilator acetylcholine (ACh, 10‐9 to 10‐3 M, 5 mins each step) in the presence or absence of the superoxide dismutase mimetic, tiron (10 mM/L). Following the concentration response curve, vessels were exposed to a calcium‐free solution containing the chelator EDTA to obtain passive inner diameter from which percent vasodilation was calculated. A separate set of arteries from each bird was exposed to the superoxide indicator dihydroethidium (DHE, 10mM) in the presence or absence of tiron (10mM/L), embedded in optimal cutting compound, frozen in isopentane cooled by dry ice, and 12μm cryosections were collected onto glass microscope slides. Fluorescence intensity was measured by confocal microscopy. Contrary to our hypothesis, exposure to high glucose increased vascular superoxide concentrations by 168.9±42% (p=0.0072) and impaired endothelium‐mediated vasodilation (p<0.05). Moreover, vasodilation of vessels pre‐treated with tiron (10 mM/L) was not different from controls. These results demonstrate that although doves have naturally higher blood glucose concentrations, isolated arteries are not endogenously protected from glucose‐mediated increases in oxidative stress and impaired vasodilation.

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Funding was provided by the Graduate and Professional Student Association and School of Life Sciences Undergraduate Researcher program, ASU.
Endothelial Dysfunction Predicts Systolic Blood Pressure Slope During Whole Body Maximal Exercise In Patients With Chronic Kidney Disease
Authors: Downey Ryan M. , Liao Peizhou , Millson Erin C. , Quyyumi Arshed A. , Park Jeanie
Published: November 2023

Chronic kidney disease (CKD) patients have exercise intolerance and poor physical capacity that increases the risk of adverse cardiovascular events during and after physical activity. Previous studies in our lab demonstrate that CKD patients have exaggerated increases in blood pressure (BP) and sympathetic nervous system activation during handgrip exercises. However, whether CKD patients also have exaggerated increases in BP during whole‐body exercise or high intensity exercise is unknown. Furthermore, CKD patients often have endothelial dysfunction which could potentially lead to an impaired ability to vasodilate during exercise. Whether this might contribute to the augmented pressor response in CKD is also unknown. We hypothesized that CKD patients have exaggerated increases in BP during maximal whole body exercise and that endothelial dysfunction predicts a greater pressor response during exercise in these patients. 56 participants (35 CKD, 21 age‐matched controls) underwent maximal treadmill exercise using the modified Balke protocol. BP, heart rate (HR), and rate of perceived exertion were continuously monitored. Levels of mixed expired oxygen, carbon dioxide, and ventilation were recorded at rest and every 30 seconds during exercise. The peak oxygen uptake (VO2 max) was defined as the highest VO2 rate observed during maximal exercise. Endothelial function was assessed via brachial artery flow‐mediated dilation (FMD) just before and repeated 1 hour after exercise. CKD patients had significantly lower VO2 max compared to controls (21.8±1.1 ml/min/kg vs. 30.3±1.7, p<0.001). There was no significant difference in maximal systolic BP (SBP), diastolic BP, or mean arterial pressure achieved during exercise between groups. However, CKD patients had a greater slope of rise in SBP (10.3 mmHg/stage vs. 7.7, p<0.001). Maximum achieved HR was significantly lower in CKD versus controls (134.9±4.4 bpm vs. 150.0±3.6, p=0.011). Similar to BP response, the slope of rise in HR was significantly steeper in CKD patients (11.9 bpm/stage vs. 10.7, p=0.032). Before exercise, FMD (2.76±0.42% vs. 5.84±0.97, p=0.007), and FMD controlled for peak shear rate (FMDshear) (4.01±0.62% vs. 6.85±1.11, p=0.033) were significantly lower in CKD patients. A lower pre‐exercise FMD (p=0.039) and FMDshear (p<0.001) were significantly associated with a greater slope of rise in SBP during exercise in CKD, but not in controls. There was no significant change in FMD before and after exercise in either group. CKD patients exhibit an accelerated rise in SBP and HR during whole body maximal exercise compared to controls. Further, impaired FMD predicts an accelerated SBP and HR response during exercise in CKD patients, but not in controls. These data suggest that endothelial dysfunction is associated with the exaggerated pressor response during exercise in CKD patients.

Support or Funding Information

Supported by NIH HL‐098744.
Sex‐differences in the Cardio‐metabolic and Hormonal Responses to Long‐duration Spaceflight
Authors: Hughson Richard L , Shoemaker Kevin , Rush James , Arbeille Philippe
Published: November 2023

The recent NASA review on Sex and Gender effects on cardiovascular adaptations to spaceflight acknowledged the differences between men and women in orthostatic tolerance but suggested more research is required to assess impact on cardiovascular health. The study Vascular had the opportunity to compare responses of 4 men and 4 women who lived on the International Space Station (ISS) for approximately 6‐months. Carotid artery stiffness indicators were obtained pre‐ and immediately post‐flight, and measurements of overnight fasting blood samples were collected twice pre‐flight and twice in the final 30‐days of flight to assess metabolic and hormonal control. Carotid artery distensibility coefficient was reduced ~20% after spaceflight with no sex differences, but carotid artery beta‐stiffness index increased more in women. Sex differences were also seen in glucose regulation with increases in fasting glucose and greater increase in the homeostatic model assessment of insulin resistance in men. No sex differences were seen in glycated albumin, but the change in glucose was correlated with the change in glycated albumin (r2=0.52). Hormones associated with fluid regulation had significant spaceflight by sex interaction terms for renin and aldosterone, with greater increases in women than in men. Atrial natriuretic peptide decreased in men and women. Plasma superoxide dismutase tended to be increased more in women than men. Sex‐differences were found in blood biomarkers that might be related to arterial stiffness; however, the direction of change was not consistent between men and women. Greater glucose and insulin resistance found in men, or the elevation in aldosterone in women might be associated with increased stiffness, while greater superoxide dismutase would be expected to be associated with reduced stiffness. Carotid artery stiffness in spaceflight is probably associated with elevated local arterial pressures in the absence of gravity. Future studies need to consider potential sex‐differences in metabolic and hormonal biomarkers during investigations of cardiovascular adaptations to spaceflight.

Support or Funding Information

Supported by Canadian Space Agency
HDAC1 increases functional β‐cell mass
Authors: Draney Carrie , Hobson Amanda , TESSEM JEFFERY
Published: November 2023

β‐cell mass is lost in both major forms of diabetes. Mature β‐cells have restricted proliferative capacity. Studies aimed at increasing β‐cell mass frequently have the unwanted side effects of decreased glucose stimulated insulin secretion (GSIS) or increased apoptotic rates. Understanding the molecular pathways that enhance β‐cell mass while maintaining or improving GSIS and the response to apoptotic stimuli could be used as a treatment for diabetes. Overexpression of the β‐cell transcription factor Nkx6.1 induces β‐cell proliferation, enhances GSIS and protects against apoptosis. Here we present data demonstrating that HDAC1 is induced by Nkx6.1, in sufficient to induce β‐cell proliferation, and is necessary for Nkx6.1 mediated proliferation. HDAC1 overexpression maintains GSIS and increases β‐cell survival when exposed to apoptotic stimuli. Overexpression of HDAC1 results in increased expression of cell cycle controlling genes, and downregulation of the cell cycle inhibitor p15INK4b. We present a model by which HDAC1 is necessary for Nkx6.1 mediated β‐cell proliferation by downregulating p15 INK4b expression.
A Novel Role for the PKG‐encoding foraging Gene in the Fly ‘Pancreas’ of Drosophila melanogaster
Authors: Hughson Bryon N. , Vesterberg Anders , Sokolowski Marla B.
Published: November 2023

The Drosophila melanogaster corpora cardiaca (CC) is functionally homologous to the mammalian pancreatic alpha cells and anterior pituitary gland. The CC is the site of biosynthesis and secretion of the fly glucagon homolog, adipokinetic hormone (AKH). The foraging gene (for) in D. melanogaster encodes a cGMP‐dependent protein kinase (PKG) and is evolutionarily conserved across taxa. for has a well‐established role in the regulation of feeding behavior and metabolism. Here we identify a CC‐specific role for for in the regulation of adult feeding behavior, lipid metabolism, and starvation stress response. The expression of transgenic for‐RNAi constructs specifically in the CC significantly decreases adult whole body lipid content and starvation resistance, and increases adult feeding behavior. Immunohistochemistry data suggest that the transgenic manipulation of for in the CC alters the secretion of AKH. By restricting the CC‐specific transgenic manipulation of for to precise developmental periods, we demonstrate that the effects on adult metabolism and feeding behavior have their origin in juvenile life. These data demonstrate a novel role for foraging and PKG in the CC, and begin to elucidate a putative mechanism whereby juvenile experience can influence adult metabolism and health outcomes.