Biology subjects

Fueger, P. T.

Publications and source records attributed to Fueger, P. T..

11 recordsLinked to original sources

bioRxiv2025

STX4 is indispensable for mitochondrial homeostasis in skeletal muscle

BackgroundMitochondrial homeostasis is vital for optimal skeletal muscle integrity. Mitochondrial quality control (MQC) mechanisms that are essential for maintaining proper functions of mitochondria include mitochondrial biogenesis, dynamics and mitophagy. Previously, Syntaxin 4 (STX4) traditionally considered a cell surface protein known for glucose uptake in skeletal muscle, was also identified at the outer mitochondrial membrane. STX4 enrichment was sufficient to reverse Type 2 diabetes-associated mitochondrial damage in skeletal muscle by inactivation of mitochondrial fission. However, whether STX4 could modulate skeletal muscle mitochondrial homeostasis through MQC mechanisms involving mitochondrial biogenesis or mitophagy remains to be determined. MethodsTo determine the requirements of STX4 in mitochondrial structure, function and MQC processes of biogenesis and mitophagy, we implemented our in-house generated inducible skeletal muscle-specific STX4-knockout (skmSTX4-iKO) mice (Stx4fl/fl; Tg(HSA-rtTA/TRE-Cre)/B6) and STX4-depleted immortalized L6.GLUT4myc myotubes via siRNA knockdown (siSTX4). ResultsWe found that non-obese skmSTX4-iKO male mice (>50% reduced STX4 abundance, Soleus and Gastrocnemius ***p<0.001, Tibialis anterior (TA) ****p<0.0001) developed insulin resistance (**p<0.01), together with reduced energy expenditure (AUC *p<0.05), respiratory exchange ratio (AUC **p<0.01), and grip strength (*p<0.05). STX4 ablation in muscle also impaired mitochondrial oxygen consumption rate (****p<0.0001). Mitochondrial morphological damage was heterogenous in STX4 depleted muscle, presenting with small fragmented mitochondria (****p<0.0001) and deceased electron transport chain (ETC) abundance (CI ***p<0.001, CII *p<0.05, CIV **p<0.01) in oxidative soleus muscle, while glycolytic TA fibers display enlarged swollen mitochondria (****p<0.0001) with no change in ETC abundance. Notably, >60% reduction of STX4 in siSTX4 L6.GLUT4myc myotubes (****p<0.0001) also decreased ETC abundance (CI ****p<0.0001, CII ****p<0.0001, CIV *p<0.05) without changes in mitochondrial glucose metabolism, as shown by [U-13C] glucose isotope tracing. For MQC, both skmSTX4-iKO male mice (*p<0.05) and siSTX4 L6.GLUT4myc myotubes (*p<0.05) showed decreased mitochondrial DNA levels alongside reduced mRNA expression of mitochondrial biogenesis genes Ppargc1a (PGC1-, *p<0.05) and Tfam (*p<0.05) in skmSTX4-iKO soleus muscle and PGC1- (mRNA *p<0.05, protein ***p<0.001), NRF1 (mRNA and protein *p<0.05) and Tfam (mRNA *p<0.05) in siSTX4 L6.GLUT4myc myotubes. Furthermore, live cell imaging using mt-Keima mitophagy biosensor in siSTX4 L6.GLUT4myc cells revealed significantly impaired mitochondrial turnover by mitophagy (*p<0.05) and mitochondria-lysosome colocalization (*p<0.05). STX4 depletion also reduced canonical mitophagy markers, PINK1 and PARKIN in both skmSTX4-iKO muscle (PARKIN *p<0.05, PINK1 **p<0.01) and siSTX4 L6.GLUT4myc myotubes (PARKIN ****p<0.0001, PINK1 *p<0.05). ConclusionsOur study demonstrated STX4 as a key mitochondrial regulator required for mitochondrial homeostasis in skeletal muscle.

cell biology↗

bioRxiv2025

Metabolic Cage Analysis of Surgically Catheterized C57Bl/6J Mice (Mus musculus) Treated with Carprofen and Sustained-release Buprenorphine

Federal regulations require that appropriate analgesia be provided for laboratory animals for pain control. Carprofen and buprenorphine are two common analgesics used for laboratory mice (Mus musculus). However, given the potential gastrointestinal side effects that these analgesics have in various species, the impact of these analgesics on mice used in metabolic studies could be concerning. To investigate the impact of carprofen and sustained-release buprenorphine on food consumption, activity level, and whole-body metabolism, we administered carprofen alone or in combination with sustained-release buprenorphine to mice that underwent jugular vein and carotid artery catheterization, or a sham surgery. The mice were individually housed in instrumented metabolic cages to continuously quantify food consumption, activity levels, and energy expenditure by indirect calorimetry. We hypothesized that catheterized mice receiving both carprofen and sustained-release buprenorphine would have decreased food consumption and increased activity level compared to mice that received sham surgery and carprofen, and catheterized mice treated with carprofen only would have similar food consumption and activity level as sham mice that received carprofen. Our results demonstrate that during the initial 12h after surgery, catheterized mice that received both carprofen and sustained-release buprenorphine were more active than sham mice that received carprofen, and were more active and consumed more food than catheterized mice that received carprofen only. Our study demonstrated how analgesia regimen can affect metabolic parameters. Therefore, researchers should carefully consider the effects that analgesic drugs can have on mice when designing metabolic or behavioral experiments.

animal behavior and cognition↗

bioRxiv2023

Pharmacokinetics of Extended-release Buprenorphine (Ethiqa) in Female Yorkshire Swine (Sus scrofa domestica)

Despite the use of swine as a large animal translational surgical model, precise dosing regimens for commonly used analgesics such as buprenorphine, are currently lacking in this species. A newly available extended-release formulation of buprenorphine (XRB, Ethiqa) is FDA-indexed and approved for use in mice and rats; however, no studies have examined the efficacy and pharmacokinetic parameters of XRB in swine. The goal of this study was to determine the pharmacokinetics of the newly available XRB in swine. We hypothesized that after a single subcutaneous administration of XRB in adult swine, buprenorphine plasma concentrations would be at or above the therapeutic threshold of 0.1 ng/mL and that local injection side effects would be minimal. XRB was administered once, subcutaneously to two separate cohorts of adult female Yorkshire swine at low and high doses (0.2 and 0.4 mg/kg, respectively; n = 3 and 2). Blood was collected from an indwelling jugular catheter prior to and after XRB administration (13 total time points). Individual animal data indicated all animals reached therapeutic buprenorphine plasma concentrations by 8 h post administration. Average plasma buprenorphine levels for both the low- and high-dose cohorts reached therapeutic concentrations starting at 1.5 h after XRB administration and were maintained above therapeutic concentrations throughout the 96-h study period. In the low-dose cohort, the average half-life was 212.6 {+/-} 107.1 h, whereas the half-lives in the high-dose cohort was 63.8 and 48.9 h. As expected, histology of XRB subcutaneous sites revealed mild injection site reactions characterized by granulomatous inflammation with intralesional cholesterol cleft formation. These results support our hypothesis and indicate that all animals maintained therapeutic plasma buprenorphine levels beginning at 8 h and maintaining past 96 h. Thus, XRB at either dose provide therapeutic levels of plasma buprenorphine and therefore its use should be further explored in swine.

pharmacology and toxicology↗

bioRxiv2023

Longitudinal analysis of a dietary mouse model of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH)

Non-alcoholic fatty liver disease (NAFLD), and resultant non-alcoholic steatohepatitis (NASH), incidence and prevalence are rising globally due to increasing rates of obesity and diabetes. Currently, there are no approved pharmacological treatments for NAFLD, highlighting a need for additional mechanistic studies to develop prevention and/or therapeutic strategies. Diet-induced preclinical models of NAFLD can be used to examine the dynamic changes that occur during NAFLD development and progression throughout the lifespan. To date, most studies utilizing such models have focused exclusively on terminal time points and have likely missed critical early and late changes that are important for NAFLD progression (i.e, worsening). We performed a longitudinal analysis of histopathological, biochemical, transcriptomic, and microbiome changes that occurred in adult male mice fed either a control diet or a NASH-promoting diet (high in fat, fructose, and cholesterol) for up to 30 weeks. We observed progressive development of NAFLD in mice fed the NASH diet compared to the control diet. Differential expression of immune-related genes was observed at an early stage of diet-induced NAFLD development (10 weeks) and persisted into the later stages of the disease (20 and 30 weeks). Differential expression of xenobiotic metabolism related genes was observed at the late stage of diet-induced NAFLD development (30 weeks). Microbiome analysis revealed an increased abundance of Bacteroides at an early stage (10 weeks) that persisted into the later stages of the disease (20 and 30 weeks). These data provide insight into the progressive changes that occur during NAFLD/NASH development and progression in the context of a typical Western diet. Furthermore, these data are consistent with what has been reported in patients with NAFLD/NASH, supporting the preclinical use of this diet-induced model for development of strategies to prevent or treat the disease.

physiology↗

bioRxiv2023

Pancreatic loss of Mig6 alters murine endocrine cell fate and protects functional beta cell mass in an STZ-induced model of diabetes

BackgroundMaintaining functional beta cell mass (BCM) to meet glycemic demands is essential to preventing or reversing the progression of diabetes. Yet the mechanisms that establish and regulate endocrine cell fate are incompletely understood. We sought to determine the impact of deletion of mitogen-inducible gene 6 (Mig6), a negative feedback inhibitor of epidermal growth factor receptor (EGFR) signaling, on mouse endocrine cell fate. The extent to which loss of Mig6 might protect against loss of functional BCM in a multiple very low dose (MVLD) STZ-induced model of diabetes was also determined. MethodsTen-week-old male mice with whole pancreas (Pdx1:Cre, PKO) and beta cell-specific (Ins1:Cre, BKO) knockout of Mig6 were used alongside control (CON) littermates. Mice were given MVLD STZ (35 mg/kg for five days) to damage beta cells and induce hyperglycemia. In vivo fasting blood glucose and glucose tolerance were used to assess beta cell function. Histological analyses of isolated pancreata were utilized to assess islet morphology and beta cell mass. We also identified histological markers of beta cell replication, dedifferentiation, and death. Isolated islets were used to reveal mRNA and protein markers of beta cell fate and function. ResultsPKO mice had significantly increased alpha cell mass with no detectable changes to beta or delta cells. The increase in alpha cells alone did not impact glucose tolerance, BCM, or beta cell function. Following STZ treatment, PKO mice had 18{+/-}8% higher BCM than CON littermates and improved glucose tolerance. Interestingly, beta cell-specific loss of Mig6 was insufficient for protection, and BKO mice had no discernable differences compared to CON mice. The increase in BCM in PKO mice was the result of decreased beta cell loss and increased beta cell replication. Finally, STZ-treated PKO mice had more Ins+/Gcg+ bi-hormonal cells compared to controls suggesting alpha to beta cell trans differentiation. ConclusionsMig6 exerted differential effects on alpha and beta cell fate. Pancreatic loss of Mig6 reduced beta cell loss and promoted beta cell growth following STZ. Thus, suppression of Mig6 may provide relief of diabetes.

physiology↗

bioRxiv2023

Dose-dependent progression of multiple low dose streptozotocin-induced diabetes in mice

This study investigated the effects of different multiple low doses of streptozotocin (STZ), namely 35 and 55 mg/kg, on the onset and progression of diabetes in mice. Both doses are commonly used in research, and while both induced a loss of beta cell mass, they had distinct effects on whole glucose tolerance, beta cell function and gene transcription. Mice treated with 55 mg/kg became rapidly glucose intolerant, whereas those treated with 35 mg/kg had a slower onset and remained glucose tolerant for up to a week before becoming equally glucose intolerant as the 55 mg/kg group. Beta cell mass loss was similar between the two groups, but the 35 mg/kg-treated mice had improved glucose-stimulated insulin secretion in gold-standard hyperglycemic clamp studies. Transcriptomic analysis revealed that the 55 mg/kg dose caused disruptions in nearly five times as many genes as the 35 mg/kg dose in isolated pancreatic islets. Pathways that were downregulated in both doses were more downregulated in the 55 mg/kg-treated mice, while pathways that were upregulated in both doses were more upregulated in the 35 mg/kg treated mice. Moreover, we observed a differential downregulation in the 55 mg/kg-treated islets of beta cell characteristic pathways, such as exocytosis or hormone secretion. On the other hand, apoptosis was differentially upregulated in 35 mg/kg-treated islets, suggesting different transcriptional mechanisms in the onset of STZ-induced damage in the islets. This study demonstrates that the two STZ doses induce distinctly mechanistic progressions for the loss of functional beta cell mass.

physiology↗

bioRxiv2023

Pancreatic Deletion of Mitogen-inducible Gene 6 Promotes Beta Cell Proliferation Following Destruction

Type 1 Diabetes (T1D) is caused by autoimmune-mediated beta cell destruction. Following beta cell injury, the pancreas attempts to launch a cellular repair and regenerative program, yet it fails to completely restore functional beta cell mass. One component of this regenerative program is epidermal growth factor receptor (EGFR) signaling. However, upon irreparable beta cell damage, EGFR signaling is dampened, disrupting attempts to restore functional beta cell mass and maintain normoglycemia. We previously demonstrated that the negative feedback inhibitor of EGFR, Mitogen-inducible gene 6 (Mig6), is induced by the pro-inflammatory cytokines central to the autoimmune-mediated beta cell destruction. We also established that pro-inflammatory cytokines suppress EGFR activation, and siRNA-mediated suppression of Mig6 restores EGFR signaling. Thus, we hypothesized that pro-inflammatory cytokines induce nitric oxide production and that in turn induced Mig6, disrupting EGFR repair mechanisms. We determined that NO induces Mig6, attenuating EGFR signaling, and NO synthase inhibition blocks the cytokine-mediated induction of Mig6, thereby restoring cytokine-impaired EGFR signaling. To that end, we treated mice lacking pancreatic Mig6 and control mice with a streptozotocin (STZ) to induce beta cell death and diabetes in a way that mimics the onset and progression of T1D. Whereas STZ-treated control mice became hyperglycemic and had reduced beta cell mass, STZ-treated Mig6 pancreas-specific knock out (PKO) mice remained euglycemic and glucose tolerant due to preserved beta cell mass. The restoration of beta cell mass in PKO mice was accompanied by enhanced beta cell proliferation. Thus, our work suggests that Mig6 is a promising target to preserve beta cell mass before overt T1D.

cell biology↗

bioRxiv2022

Pharmacokinetics of Sustained-Release Buprenorphine and Extended-Release Buprenorphine in Mice with Surgical Catheterization

The Guide for the Care and Use of Laboratory Animals strongly encourages the use of pharmaceutical grade chemicals and analgesics. The extra-label use of sustained-release buprenorphine (SRB) is commonly administered to rodents to mitigate moderate-to-severe pain. An FDA-indexed buprenorphine formulation, known as extended-release buprenorphine (XRB), has recently become available and is currently the only pharmaceutical grade slow-release buprenorphine approved for use in mice and rats. However, no studies have directly compared the pharmacokinetic (PK) parameters and therapeutic efficacy of SRB and XRB in surgically catheterized mice. Thus, we compared the plasma buprenorphine concentrations and PK parameters of SRB and XRB in mice after surgical catheterization. We hypothesized that mice treated with SRB or XRB would have circulating buprenorphine concentrations exceeding the therapeutic threshold for up to 72-hours post-operatively. Male and female C57Bl/6J mice were anesthetized, treated with either SRB (1 mg/kg, SC, once) or XRB (3.25 mg/kg, SC, once) and underwent surgical catheterization. At 6, 24, 48, and 72 h after SRB or XRB administration, arterial blood samples were collected. Post-operative weight loss was similar between groups with a decline of 11.7 {+/-} 1.6 and 12.3 {+/-} 0.7% in males and 7.6 {+/-} 2.2 and 8.1 {+/-} 1.1% (mean {+/-} SEM) in females treated with SRB and XRB, respectively. Both SRB and XRB maintained circulating buprenorphine concentrations above the therapeutic level of 1.0 ng/mL for 72 h after administration. XRB buprenorphine concentrations were significantly greater (3-4-fold) than SRB concentrations at 6, 24, and 48 h, commensurate with the increase dose concentration of XRB to SRB. These results support the use of either SRB or XRB for the alleviation of postoperative pain in mice. The new availability of FDA-indexed XRB increases the options for safe and effective pharmaceutical grade analgesia in rodents.

pharmacology and toxicology↗

bioRxiv2020

Glucolipotoxicity-induced Mig6 desensitizes EGFR signaling and promotes pancreatic beta cell death

A loss of functional beta cell mass is a final etiological event in the development of frank type 2 diabetes (T2D). To preserve or expand beta cells and therefore treat/prevent T2D, growth factors have been considered therapeutically but have largely failed to achieve robust clinical success. The molecular mechanisms preventing the activation of mitogenic signaling pathways from maintaining functional beta cell mass during the development of T2D remain unknown. We speculated that endogenous negative effectors of mitogenic signaling cascades impede beta cell survival/expansion. Thus, we tested the hypothesis that a stress-inducible epidermal growth factor receptor (EGFR) inhibitor, Mitogen-inducible gene 6 (Mig6), regulates beta cell fate in a T2D milieu. To this end, we determined that: 1) glucolipotoxicity (GLT) induces Mig6, thereby blunting EGFR signaling cascades, and 2) Mig6 mediates molecular events regulating beta cell survival/death. We discovered that GLT impairs EGFR activation, and Mig6 is elevated in human islets from T2D donors as well as GLT-treated rodent islets and 832/13 INS-1 beta cells. Mig6 is essential for GLT-induced EGFR desensitization, as Mig6 suppression rescued the GLT-impaired EGFR and ERK1/2 activation. Further, Mig6 mediated EGFR but not insulin-like growth factor-1 receptor nor hepatocyte growth factor receptor activity in beta cells. Finally, we identified that elevated Mig6 augmented beta cell apoptosis, as Mig6 suppression reduced apoptosis during GLT. In conclusion, we established that T2D and GLT induce Mig6 in pancreatic beta cells. The elevated Mig6 desensitizes EGFR signaling and induces beta cell death. Our findings suggest that Mig6 could be a novel therapeutic target for T2D, as blocking Mig6 could possibly enhance mitogenic signaling cascades in a diabetic milieu to promote beta cell survival and prevent beta cell death.

cell biology↗

bioRxiv2020

Mig6 decreases hepatic EGFR activation and survival during saturated fatty acid-induced endoplasmic reticulum stress

Hyperlipidemia associated with obesity and type 2 diabetes (T2D) promotes excess hepatic lipid storage (steatosis) and endoplasmic reticulum (ER) stress, thereby reducing hepatic cell proliferation and survival. An important receptor tyrosine kinase controlling liver proliferation and survival is the epidermal growth factor receptor (EGFR). EGFR expression and activation are decreased during steatosis in humans and several animal models of obesity. Therefore, restoring EGFR activation in obesity-induced ER stress and diabetes could restore the livers capacity for survival and regeneration. As an inducible feedback inhibitor of EGFR activity, mitogen-inducible gene 6 (Mig6) is a novel target for enhancing EGFR signaling during diet-induced obesity (DIO) and T2D. Thus, we hypothesized hepatic ER stress induces Mig6 expression and decreases EGFR activation during DIO and diabetes. We identified that Mig6 expression was increased during obesity-induced insulin resistance in C57Bl/6J mice fed a high fat diet. We also discovered that both pharmacological- and fatty acid-driven ER stress increased Mig6 expression and decreased EGF-mediated EGFR activation in primary rat hepatocytes and cell lines. Furthermore, siRNA-mediated Mig6 knockdown restored EGFR signaling and reduced caspase 3/7 activation during ER stress. Therefore, we conclude Mig6 is increased during ER stress in DIO, thereby reducing EGFR activation and enhancing cell death. The implications are the induction of Mig6 during DIO and diabetes may decrease hepatocyte survival, thus hindering cellular repair and regenerative mechanisms.

physiology↗

bioRxiv2020

The adaptor protein NumbL is involved in the control of glucolipotoxicity-induced pancreatic beta cell apoptosis

Avoiding loss of functional beta cell mass is critical for preventing or treating diabetes. Currently, the molecular mechanisms underlying beta cell death are partially understood, and there is a need to identify new targets for developing novel therapeutics to treat diabetes. Previously, our group established that Mig6, an inhibitor of EGF signaling, mediates beta cell death under diabetogenic conditions. The objective of this study was to clarify the mechanisms linking diabetogenic stimuli to beta cell death by investigating Mig6-interacting proteins. Using co-immunoprecipitation and mass spectrometry, we evaluated the binding partners of Mig6 under both normal glucose (NG) and glucolipotoxic (GLT) conditions in beta cells. We identified that Mig6 interacts dynamically with NumbL; whereas Mig6 associates with NumbL under NG, this interaction is disrupted under GLT conditions. Further, we demonstrate that siRNA-mediated suppression of NumbL expression in beta cells prevented apoptosis under GLT conditions by blocking activation of NF-{kappa}B signaling. Using co-immunoprecipitation experiments we observed that NumbLs interactions with TRAF6, a key component of NF{kappa}B signaling, are increased under GLT conditions. The interactions among Mig6, NumbL, and TRAF6 are dynamic and context-dependent. We propose a model wherein these interactions activate pro-apoptotic NF-{kappa}B signaling while blocking pro-survival EGF signaling under diabetogenic conditions, leading to beta cell apoptosis. These findings indicate that NumbL should be further investigated as a candidate anti-diabetic therapeutic target.

biochemistry↗