BACKGROUND:Diabetic nephropathy (DN) is a leading cause of chronic kidney failure. We hypothesized that mitochondrial Akt1 dysfunction in renal proximal tubules plays a pathogenic role in DN development and that its activation may reverse DN progression. METHODS:To study this signaling pathway, we generated a transgenic mouse model harboring a renal tubule-specific, Tamoxifen-inducible, mitochondria-targeted constitutively active Akt1 (KMCAKT). Type 2 diabetes was induced by a high-fat, high fructose diet (HFFD) for 40 weeks. Renal histology and function were evaluated, and glucose metabolism was assessed using dynamic glucose testing. RESULTS:HFFD feeding resulted in development of DN in control non-induced KMCAKT mice, whereas KMCAKT mice with constitutively active mitochondrial Akt1, induced by Tamoxifen-injection (TAM), exhibited significant improvement of kidney dysfunction and histology. Urinary albumin, fasting plasma BUN levels, fibrosis and Jablonski scores were all markedly improved in HFFD-TAM-KMCAKT mice compared with controls, while levels of α-smooth muscle actin (αSMA) and transforming growth factor-β1 (TGFβ1) were significantly reduced. HFFD-TAM-KMCAKT mice exhibited lower fasting blood glucose and improved oral glucose tolerance, while basal and stimulated insulin levels were higher, along with increased beta cell mass and insulin secretion (HOMA-β) compared to controls. Hyperglycemic clamp studies confirmed increased insulin secretion in HFFD-TAM-KMCAKT mice. CONCLUSION:Tubular mitochondrial Akt1 plays a key role in DN progression. Restoring tubular mitochondrial Akt1 signaling may represent a novel approach to reverse development of chronic kidney disease. We also identified previously unrecognized metabolic crosstalk between renal tubular mitochondrial Akt1 and pancreatic insulin secretion that may modulate systemic glucose homeostasis.

Federal regulations require that appropriate analgesia be provided to laboratory animals for pain control. Carprofen and buprenorphine are 2 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 with mice which received sham surgery and carprofen, and that catheterized mice treated with carprofen only would have similar food consumption and activity level as sham mice which received carprofen. Our results demonstrate that during the initial 12 hours after surgery, catheterized mice that received both carprofen and sustained-release buprenorphine were more active than sham mice which received carprofen, and they were more active and consumed more food than catheterized mice which received carprofen only. Our study demonstrated that analgesia regimen can affect metabolic parameters, thus, researchers should carefully consider the effects that analgesic drugs can have on mice when designing metabolic or behavioral experiments.

Starting at middle age, adults often suffer from visceral adiposity and associated adverse metabolic disorders. Lineage tracing in mice revealed that adipose progenitor cells (APCs) in visceral fat undergo extensive adipogenesis during middle age. Thus, despite the low turnover rate of adipocytes in young adults, adipogenesis is unlocked during middle age. Transplantations quantitatively showed that APCs in middle-aged mice exhibited high adipogenic capacity cell-autonomously. Single-cell RNA sequencing identified a distinct APC population, the committed preadipocyte, age-enriched (CP-A), emerging at this age. CP-As demonstrated elevated proliferation and adipogenesis activity. Pharmacological and genetic manipulations indicated that leukemia inhibitory factor receptor signaling was indispensable for CP-A adipogenesis and visceral fat expansion. These findings uncover a fundamental mechanism of age-dependent adipose remodeling, offering critical insights into age-related metabolic diseases.

Introduction and Objective: Diabetic nephropathy (DN) is a major cause of end-stage renal disease. We have recently shown that activation of mitochondrial Akt1 in proximal tubules played a renoprotective role in murine acute ischemia-reperfusion injury. However, whether activation of mitochondrial Akt1 in renal proximal tubules can protect against the development of DN remains to be elucidated. Methods: We have generated a transgenic mouse model harboring a renal proximal tubule-specific tamoxifen-inducible mitochondria-targeting constitutively active Akt1 (KMCAKT). Results: High fat+fructose diet (HFFD) induced DN, but KMCAKT mice attenuated renal dysfunction induced by HFFD. Urinary albumin/creatinine ratio, fasting plasma creatinine, glomerular mesangial expansion and fibrosis were all improved compared to controls. To dissect the underlying mechanism, we have determined the contents of renal αSMA and TGFβ1 and both were lower in KMCAKT mice than in controls (p<0.001). This indicated that activation of mitochondrial Akt1 in renal tubule protected against the development of DN induced by HFFD. Interestingly, HFFD-fed KMCAKT mice showed significant reduction in fasting plasma glucose and urinary glucose excretion with an improvement of oral glucose tolerance compared to controls. Fasting plasma insulin levels were higher in HFFD-fed KMCAKT mice as compared to controls, whereas insulin resistance (HOMA-IR) were unchanged from controls, suggesting that renal mitochondrial Akt1 positively modulated systemic glucose metabolism, at least partially, by enhancing beta cell function. Conclusion: These data demonstrated novel roles of renal tubular mitochondrial Akt1 in the regulation of whole-body glucose metabolism and insulin secretion beyond its local effect on kidney function in DN, which suggests renal function and glucose homeostasis could be mediated through a common pathway in renal tubular mitochondria. E. Salem: None. A. Ta: None. P.T. Fueger: None. P.H. Wang: None.

ABSTRACT Background Mitochondrial 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. Methods To 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). Results We 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 decreased electron transport chain (ETC) abundance (CI ***p < 0.001, CII *p < 0.05, CIV **p < 0.01) in oxidative soleus muscle, whereas glycolytic‐rich TA fibres displayed 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 < 0.01, CII ***p < 0.001, CIV **p < 0.01) 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‐α, *p < 0.05) and Tfam (*p < 0.05) in skmSTX4‐iKO soleus muscle and PGC1‐α (mRNA **p < 0.01, protein *p < 0.05), NRF1 (mRNA **p < 0.01 and protein *p < 0.05) and Tfam (mRNA *p < 0.05) in siSTX4 L6.GLUT4myc myotubes. Furthermore, live cell imaging using the 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 < 0.01, PINK1 *p < 0.05). Conclusions Our study demonstrated STX4 as a key mitochondrial regulator required for mitochondrial homeostasis in skeletal muscle.

Objective:To determine the pharmacokinetics of extended-release buprenorphine (XRB) in adult swine. We hypothesized that after a single SC administration of XRB in swine, buprenorphine plasma concentrations would be at or above the therapeutic threshold of 0.1 ng/mL and would not result in major injection site reactions. Methods:Extended-release buprenorphine was administered once SC to 2 cohorts of adult female Yorkshire swine at low (0.2 mg/kg) and high doses (0.4 mg/kg). Blood was collected from an indwelling jugular catheter prior to and after XRB administration for 13 total time points. Buprenorphine plasma concentrations were analyzed by HPLC-MS, and pharmacokinetics were performed using a noncompartmental analysis. Results:Extended-release buprenorphine was present in plasma at the therapeutic concentration of 0.1 ng/mL or above beginning at 8 hours and maintaining throughout 96 hours for all animals in both cohorts. Average plasma buprenorphine levels for both cohorts reached therapeutic concentrations starting at 1.5 hours and were maintained above therapeutic concentrations throughout 96 hours. The low-dose cohort's (n = 3) average half-life was 212.6 ± 107.1 hours, and the high-dose cohort's (n = 2) was 63.8 and 48.9 hours, respectively. The histology of SC sites revealed mild injection site reactions characterized by granulomatous inflammation with intralesional cholesterol cleft formation. Conclusions:All animals reached and maintained therapeutic buprenorphine plasma concentrations of 0.1 ng/mL by 8 hours and maintained it to the end of the study at 96 hours. Clinical Relevance:Extended-release buprenorphine at either dose provides therapeutic levels of plasma buprenorphine, and therefore its use should be further investigated in swine.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a spectrum of chronic liver disease that can progress from benign steatosis to metabolic dysfunction-associated steatohepatitis (MASH) - both of which, if left untreated, can progress to cirrhosis and hepatocellular carcinoma. The strongest risk factor for progression of MASLD to MASH is insulin resistance. Previous work in our lab identified that the adaptor protein mitogen-inducible gene-6 (Mig6) is increased in obese mice and that deletion of Mig6 in the liver (LKO) improves glucose tolerance and insulin action during hyperinsulinemic-euglycemic clamps in diet-induced obese mice. Mig6 is an endogenous feedback inhibitor of epidermal growth factor receptor activation (EGFR) and regulates cellular repair and survival. Thus, we sought to elucidate the extent to which loss of Mig6 modulates the progression of MASLD to MASH. We hypothesized that liver-specific loss of Mig6 during early MASLD improves whole-body glucose homeostasis and protects against MASLD-mediated liver damage. LKO mice and matched, control littermates (CON) were fed a MASH diet (40% fat, 40% carbohydrate, 2% cholesterol; CON n=13-19, LKO n=16-22) or a low-fat-matched diet (LFD; 10% fat, 70% carbohydrate; CON n=8-16, LKO n=16-21) for up to 40 weeks. Body weights were recorded weekly, and body composition assessment, glucose and insulin tolerance tests, and hepatic histological analysis were performed after 20-, 30- and 40-weeks on each diet. Compared to the MASH-fed CON mice, MASH-fed LKO mice had lower body weights starting at 10-weeks and persisting throughout the study. Body composition analysis established these differences in body weight were due to decreases in fat, but not lean, mass. Interestingly, whereas intraperitoneal glucose tolerance tests did not identify significant differences in glucose handling between the LFD and MASH fed mice, oral glucose tolerance was significantly improved in 10- and 20-week MASH-fed LKO mice, suggesting alterations in the incretin response in this MASH model. Lastly, histological analysis revealed MASH-fed LKO mice had a preservation of hepatic lipid zonation and blunted levels of circulating alanine aminotransferase (ALT) compared to MASH-fed CON, indicating less hepatic damage in LKO mice. These results suggest that liver-specific loss of Mig6 improves whole-body glucose handling, which in turn ameliorates hepatic damage during MASLD progression. In conclusion, Mig6 prevents, or at least delays, the progression of MASLD, which may reveal a new therapeutic area for the prevention and treatment of MASH. MS was supported by a National Cancer Institute Cancer Metabolism Training Program Postdoctoral Fellowship (T32CA221709). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

ABSTRACT Exocrine-to-endocrine crosstalk in the pancreas is crucial to maintain beta cell function. However, the molecular mechanisms underlying this crosstalk are largely undefined. Trefoil factor 2 (Tff2) is a secreted factor known to promote the proliferation of beta cells in vitro, but its physiological role in vivo in the pancreas is unknown. Also, it remains unclear which pancreatic cell type expresses Tff2 protein. We therefore created a mouse model with a conditional knockout of Tff2 in the murine pancreas. We find that the Tff2 protein is preferentially expressed in acinar but not ductal or endocrine cells. Tff2 deficiency in the pancreas reduces beta cell mass on embryonic day 16.5. However, homozygous mutant mice are born without a reduction of beta cells and with acinar Tff3 compensation by day 7. When mice are aged to 1 year, both male and female homozygous and male heterozygous mutants develop impaired glucose tolerance without affected insulin sensitivity. Perifusion analysis reveals that the second phase of glucose-stimulated insulin secretion from islets is reduced in aged homozygous mutant compared to controls. Collectively, these results demonstrate a previously unknown role of Tff2 as an exocrine acinar cell-derived protein required for maintaining functional endocrine beta cells in mice. Article Highlights: · Exocrine-to-endocrine crosstalk is important in maintaining pancreatic cell homeostasis, but the molecular mechanisms remain largely undefined. · In the pancreas, the physiological role of the secreted factor Tff2 and the cell type that expresses Tff2 has been unclear. · Pancreatic acinar cells are the major cell type expressing Tff2 protein, and specific loss of Tff2 in the pancreas reduces beta cells during development and attenuates glucose-stimulated insulin secretion during aging in conditional Tff2 knockout mice. · Tff2 is a positive exocrine-produced factor required for the development and function of endocrine beta cells, which has implication in diabetes disease progression and therapy.

Activating PI3K/Akt pathway triggers translocation of activated AKT into mitochondria, and granulosa cells are enriched with mitochondria. The goals of this study were to determine the mechanistic roles of mitochondrial AKT in granulosa cells during folliculogenesis and progression of menstruation cycles, and to explore its role in the regulation of whole body metabolism. To simulate ovarian insulin resistance in mitochondria, we have generated a novel transgenic mice model with Cre-LoxP system to disrupt mitochondrial AKT signaling in granulosa cells. The transgenic mice express a dominant negative mitochondria-targeting AKT in the granulosa cells (ovdnAKT). We used histological analysis, biochemical assays, and scRNA-seq to investigate folliculogenesis. Body composition was determined by EchoMRI and glucose homeostasis by glucose tolerance test. Insulin stimulates AKT phosphorylation and translocation to the mitochondria in granulosa cells in vivo. When mitochondrial AKT was inhibited in granulosa cells in 8-week old ovdnAKT mice, menstrual cycle became irregular and prolonged (ovdnAKT, 11.9 ± 3.7 days; controls, 4.5 ± 0.5 days, p<0.001). Vaginal lavage showed predominant diestrus phase in ovdnAKT mice. The ovary weight was increased (ovdnAKT, 10.19 ± 0.89 mg; controls, 6.28 ± 2.40 mg, p=0.049). Histology analysis revealed increased total number of follicles (ovdnAKT, 533.00 ± 14.00; controls, 337.67 ± 53.38, p=0.001) and increased accumulation of preantral follicles in ovdnAKT mice (ovdnAKT, 438.67 ± 22.59; controls, 284.33 ± 41.50, p=0.002). But morphologically mature antral follicles were nearly absent in the ovdnAKT mice. Ovary scRNA-seq cluster annotation confirmed an increased pool of preantral granulosa cells in the ovdnAKT ovary, decreased number of mature granulosa cells and atretic granulosa cells, and increased number of luteinizing granulosa cells. The population of mesenchymal cells, immune cells, epithelial cells, theca cells, and endothelial cells were not changed. Transcriptional analysis in the granulosa cells at preantral phase showed altered folliculogenesis-promoting and steroidogenesis genes in the ovdnAKT mice. Mitochondrial AKT also modulated whole body metabolism, body weight increased by 20%, fat mass/weight ratio increased by 83%, and lean mass/weight ratio decreased by 11% in the ovdnAKT mice after 16 weeks, indicating development of obesity and metabolic syndrome. Insulin stimulated translocation of active AKT to mitochondria in granulosa cells. Mitochondrial AKT played a critical role in the recruitment and maturation of antral follicles through transcriptional modulation. Disrupting this pathway in granulosa cells led to obesity and metabolic syndrome, which suggests ovary-adipose metabolic crosstalk.

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 beta cells; the elevated Mig6 desensitizes EGFR signaling and induces beta cell death, suggesting Mig6 could be a novel therapeutic target for T2D.

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 although 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, whereas 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.

Avoiding the 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 here 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 interacted dynamically with NumbL, whereas Mig6 associated with NumbL under NG, and this interaction was disrupted under GLT conditions. Further, we demonstrated that the siRNA-mediated suppression of NumbL expression in beta cells prevented apoptosis under GLT conditions by blocking the activation of NF-κB signaling. Using co-immunoprecipitation experiments, we observed that NumbL's interactions with TRAF6, a key component of NFκB signaling, were increased under GLT conditions. The interactions among Mig6, NumbL, and TRAF6 were dynamic and context-dependent. We proposed a model wherein these interactions activated pro-apoptotic NF-κB signaling while blocking pro-survival EGF signaling under diabetogenic conditions, leading to beta cell apoptosis. These findings indicated that NumbL should be further investigated as a candidate anti-diabetic therapeutic target.

ABSTRACT 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.

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.

Background:Maintaining 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. Methods:Ten-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. Results:PKO 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 transdifferentiation. Conclusions:Mig6 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.

Pancreatic islets are three-dimensional cell aggregates consisting of unique cellular composition, cell-to-cell contacts, and interactions with blood vessels. Cell aggregation is essential for islet endocrine function; however, it remains unclear how developing islets establish aggregation. By combining genetic animal models, imaging tools, and gene expression profiling, we demonstrate that islet aggregation is regulated by extracellular matrix signaling and cell-cell adhesion. Islet endocrine cell-specific inactivation of extracellular matrix receptor integrin β1 disrupted blood vessel interactions but promoted cell-cell adhesion and the formation of larger islets. In contrast, ablation of cell-cell adhesion molecule α-catenin promoted blood vessel interactions yet compromised islet clustering. Simultaneous removal of integrin β1 and α-catenin disrupts islet aggregation and the endocrine cell maturation process, demonstrating that establishment of islet aggregates is essential for functional maturation. Our study provides new insights into understanding the fundamental self-organizing mechanism for islet aggregation, architecture, and functional maturation.

Introduction: Acute myeloid leukemia (AML) is a lethal hematopoietic malignancy, characterized by the accumulation of clonal myeloid progenitor cells arrested in development. AML patients have dismal survival odds with a 5-year overall survival (2011 - 2017) of 29.5% (SEER). The main cause of AML treatment failure is the persistence of leukemia stem cells (LSCs). Despite recent approval of new drugs, LSCs are not specifically targeted by AML therapies. MicroRNA (miR)-126 is a non-coding regulatory RNA that is necessary for the maintenance of LSCs in the bone marrow (BM) niche, where vascular endothelial cells (ECs) supply LSCs with miR-126 and promote LSC quiescence. Several lines of evidence validate miR-126 as a target in AML. Preclinical development of CpG-anti-miR126 oligonucleotide, termed miRisten, an inhibitor of miR-126, as a drug to treat AML is described. Hypothesis: Systemic administration of miRisten will result in pharmacological depletion of miR-126 levels in the ECs, AML blast cells and LSCs, leading to the eradication of LSCs. Objective: To investigate the pharmacological properties of miRisten to establish a dosing strategy that would result in prolonged knockdown of miR-126. Experimental Plan: Pharmacokinetic/pharmacodynamic (PK/PD) studies were carried out in healthy wild type mice. miRisten was administered as a bolus or 30 min continuous intravenous infusion (CIVI) at 50 mg/kg (mpk) b.i.d. and the resulting PK parameters compared in plasma. miR126 expression was evaluated in BM mononuclear cells (MNC), lung, liver, kidney, heart and stomach/intestines prior to the fourth dose of miRisten (-30 min; start of infusion = 0 min) and at 30, 60, 90, 120, and 160 min after initiation of infusion. A 21-day preliminary toxicology study was carried out in mice using the same dosing regimen. Results: Plasma miRisten levels revealed a Cmax = 4098 nM (bolus) and 556.1 nM (CIVI), t½ = 0.16h (bolus) and 0.4h (CIVI) and area under the curve (AUC) of 465.0 nMxhr (bolus) and 508.4 nMxhr (CIVI). Lung, which contains a high concentration of ECs, had the highest relative levels of miR-126 normalized by Sno234 "housekeeping gene", followed by the heart and kidney. BMMNC had the lowest relative levels. In all cases, the nadir of miR-126 levels occurred approximately 1 hour after start of infusion. In BMMNC, the nadir of miR-126 was lower than 0.1% of basal level when miRisten was given CIVI while as a bolus, the nadir was ~33% of basal levels. In all tissues except lung and stomach, the level of miR-126 prior to the 4th dose was lower than at basal levels prior to miRisten exposure. The recovery of miR-126 levels varied by organ but was more pronounced in the lung and stomach, possibly reflecting a more dynamic regulation of miR-126 in these tissues and shorter intracellular t½ of the miR-126. No significant toxicities were observed by cage-side observation of behavior, monitoring of food/water intake and weight or by histopathological examination of tissues following 21 days of treatment. Conclusion: These data are consistent with widespread tissue distribution of miRisten and supports administering as CIVI at 50 mpk b.i.d.as miRisten effectively sustains knockdown of miR-126 with this regimen. The AUC was comparable for bolus vs CIVI but the Cmax was ~7.5 fold lower with the CIVI which therefore might be safer. When given for 21 day,s miRisten showed no overt or histologically evident toxicity.

We investigated the role of microRNA (miR-379) in the pathogenesis of obesity, adipose tissue dysfunction, and insulin resistance (IR). We used miR-379 knockout (miR-379KO) mice to test whether loss of miR-379 affects high-fat diet (HFD)-induced obesity and IR via dysregulation of key miR-379 targets in adipose tissue. Increases in body weight, hyperinsulinemia, and IR in wild-type (WT)-HFD mice were significantly attenuated in miR-379KO-HFD mice with some sex differences. Relative to control chow-fed mice, in WT-HFD mice, expression of miR-379 and C/EBP homologous protein (Chop) (pro-endoplasmic reticulum [ER] stress) and inflammation in perigonadal white adipose tissue (gWAT) were increased, whereas adipogenic genes and miR-379 target genes (Vegfb and Edem3) were decreased. These changes, as well as key parameters of brown adipose tissue dysfunction (including mitochondrial defects), were significantly attenuated in miR-379KO-HFD mice. WAT from obese human subjects with and without type 2 diabetes showed increased miR-379 and decreased miR-379 target genes. In cultured 3T3L1 pre-adipocytes, miR-379 inhibitors increased miR-379 targets and adipogenic genes. These data suggest that miR-379 plays an important role in HFD-induced obesity through increased adipose inflammation, mitochondrial dysfunction, and ER stress as well as impaired adipogenesis and angiogenesis. miR-379 inhibitors may be developed as novel therapies for obesity and associated complications.

The Guide for the Care and Use of Laboratory Animals strongly encourages the use of pharmaceutical-grade chemicals and analgesics. Sustained-release buprenorphine (SRB) is administered extralabel to rodents to mitigate moderate to severe pain. An FDA-indexed buprenorphine formulation—extended-release buprenorphine (XRB)—has recently become available and is currently the only pharmaceutical-grade slow-release buprenorphine formulation approved for use in mice and rats. However, no studies have directly compared the pharmacokinetic parameters of SRB and XRB in surgically catheterized mice. To this end, we compared the plasma buprenorphine concentrations and pharmacokinetic parameters of SRB and XRB in mice after surgical catheterization. We hypothesized that mice treated before surgery with SRB or XRB would have circulating buprenorphine concentrations that exceeded the therapeutic threshold for as long as 72 h after surgery. Male and female C57Bl/6J mice were anesthetized, treated with a single dose of either SRB (1 mg/kg SC) or XRB (3.25 mg/kg SC), and underwent surgical catheterization. Arterial blood samples were collected at 6, 24, 48, and 72 h after administration. Weight loss after surgery (mean ± SEM) was similar between groups (SRB: males, 12% ± 2%; females, 8% ± 2%; XRB: males, 12% ± 1%; females, 8% ± 1%). Both SRB and XRB maintained circulating buprenorphine concentrations above the therapeutic level of 1.0 ng/mL for 72 h after administration. Plasma buprenorphine concentrations at 6, 24, and 48 h were significantly greater (3- to 4-fold) with XRB than SRB, commensurate with XRB's higher dose. These results support the use of either SRB or XRB for the alleviation of postoperative pain in mice. The availability of FDA-indexed XRB increases options for safe and effective pharmaceutical-grade analgesia in rodents.

BACKGROUND:Collagen production by activated hepatic stellate cells (HSCs) to encapsulate injury is part of the natural wound-healing response in injured liver. However, persistent activation of HSCs can lead to pathological fibrogenesis. Such persistent HSC activation could be mediated by norepinephrine (NE), a reaction product of dopamine beta-hydroxylase (DBH).OBJECTIVE:To investigate the potential paracrine role of NE in hepatotoxin thioacetamide (TAA)-induced liver fibrosis.METHODS:In TAA-treated mice, fibrotic liver tissue showed significant increases in the mRNA expression of DBH up to 14-fold and collagen up to 7-fold. Immunohistochemical staining showed increased DBH protein expression in fibrotic liver tissue. Parenchymal hepatocyte cell line HepG2 expressed DBH and secreted NE, and the conditioned medium of HepG2 cells promoted collagenesis in nonparenchymal HSC cell line LX-2. TAA treatment increased DBH expression by 170% in HepG2 cells, as well as increased NE by 120% in the conditioned medium of HepG2 cells. The conditioned medium of TAA-treated HepG2 cells was used to culture LX-2 cells, and was found to increase collagen expression by 80% in LX-2 cells. Collagen expression was reduced by pre-treating HepG2 cells with siRNA targeting DBH or by adding NE antagonists to the conditioned medium.RESULTS:Finally, TAA-induced oxidative stress in HepG2 cells was associated with induction of DBH expression. Collectively, our results suggest a potential role for DBH/NE-mediated crosstalk between hepatocytes and HSCs in fibrogenesis.CONCLUSION:From a therapeutic standpoint, antagonism of DBH/NE induction in hepatocytes might be a useful strategy to suppress pathological fibrogenesis.