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 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.

Heterozygous midnolin knockout attenuates severity of nonalcoholic fatty liver disease in mice fed a Western-style diet high in fat, cholesterol, and fructose.

Although midnolin has been studied for over 20 years, its biological roles in vivo remain largely unknown, especially due to the lack of a functional animal model. Indeed, given our recent discovery that the knockdown of midnolin suppresses liver cancer cell tumorigenicity and that this antitumorigenic effect is associated with modulation of lipid metabolism, we hypothesized that knockout of midnolin in vivo could potentially protect from nonalcoholic fatty liver disease (NAFLD) which has become the most common cause of chronic liver disease in the Western world. Accordingly, in the present study, we have developed and now report on the first functional global midnolin knockout mouse model. Although the overwhelming majority of global homozygous midnolin knockout mice demonstrated embryonic lethality, heterozygous knockout mice were observed to be similar to wild-type mice in their viability and were used to determine the effect of reduced midnolin expression on NAFLD. We found that global heterozygous midnolin knockout attenuated the severity of NAFLD in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism. Collectively, our results support a role for midnolin in regulating cholesterol/lipid metabolism in the liver. Thus, midnolin may represent a novel therapeutic target for NAFLD. Finally, our observation that midnolin was essential for survival underscores the broad importance of this gene beyond its role in liver biology.NEW & NOTEWORTHY We have developed and now report on the first functional global midnolin knockout mouse model. We found that global heterozygous midnolin knockout attenuated the severity of nonalcoholic fatty liver disease (NAFLD) in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism.

The Adaptor Protein NumbL Is Involved in the Control of Glucolipotoxicity-Induced Pancreatic Beta Cell Apoptosis.

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.

Suppression of Endothelial AGO1 Promotes Adipose Tissue Browning and Improves Metabolic Dysfunction.

BACKGROUND: Metabolic disorders such as obesity and diabetes mellitus can cause dysfunction of endothelial cells (ECs) and vascular rarefaction in adipose tissues. However, the modulatory role of ECs in adipose tissue function is not fully understood. Other than vascular endothelial growth factor-vascular endothelial growth factor receptor-mediated angiogenic signaling, little is known about the EC-derived signals in adipose tissue regulation. We previously identified Argonaute 1 (AGO1; a key component of microRNA-induced silencing complex) as a crucial regulator in hypoxia-induced angiogenesis. In this study, we intend to determine the AGO1-mediated EC transcriptome, the functional importance of AGO1-regulated endothelial function in vivo, and the relevance to adipose tissue function and obesity. METHODS: We generated and subjected mice with EC-AGO1 deletion (EC-AGO1-knockout [KO]) and their wild-type littermates to a fast food-mimicking, high-fat high-sucrose diet and profiled the metabolic phenotypes. We used crosslinking immunoprecipitation- and RNA-sequencing to identify the AGO1-mediated mechanisms underlying the observed metabolic phenotype of EC-AGO1-KO. We further leveraged cell cultures and mouse models to validate the functional importance of the identified molecular pathway, for which the translational relevance was explored using human endothelium isolated from healthy donors and donors with obesity/type 2 diabetes mellitus. RESULTS: We identified an antiobesity phenotype of EC-AGO1-KO, evident by lower body weight and body fat, improved insulin sensitivity, and enhanced energy expenditure. At the organ level, we observed the most significant phenotype in the subcutaneous and brown adipose tissues of KO mice, with greater vascularity and enhanced browning and thermogenesis. Mechanistically, EC-AGO1 suppression results in inhibition of thrombospondin-1 (THBS1/TSP1), an antiangiogenic and proinflammatory cytokine that promotes insulin resistance. In EC-AGO1-KO mice, overexpression of TSP1 substantially attenuated the beneficial phenotype. In human endothelium isolated from donors with obesity or type 2 diabetes mellitus, AGO1 and THBS1 are expressed at higher levels than the healthy controls, supporting a pathological role of this pathway. CONCLUSIONS: Our study suggests a novel mechanism by which ECs, through the AGO1-TSP1 pathway, control vascularization and function of adipose tissues, insulin sensitivity, and whole-body metabolic state.

HDAC1 overexpression enhances ß-cell proliferation by down-regulating Cdkn1b/p27.

The homeobox transcription factor Nkx6.1 is sufficient to increase functional ß-cell mass, where functional ß-cell mass refers to the combination of ß-cell proliferation, glucose-stimulated insulin secretion (GSIS) and ß-cell survival. Here, we demonstrate that the histone deacetylase 1 (HDAC1), which is an early target of Nkx6.1, is sufficient to increase functional ß-cell mass. We show that HDAC activity is necessary for Nkx6.1-mediated proliferation, and that HDAC1 is sufficient to increase ß-cell proliferation in primary rat islets and the INS-1 832/13 ß-cell line. The increase in HDAC1-mediated proliferation occurs while maintaining GSIS and increasing ß-cell survival in response to apoptotic stimuli. We demonstrate that HDAC1 overexpression results in decreased expression of the cell cycle inhibitor Cdkn1b/p27 which is essential for inhibiting the G1 to S phase transition of the cell cycle. This corresponds with increased expression of key cell cycle activators, such as Cyclin A2, Cyclin B1 and E2F1, which are activated by activation of the Cdk4/Cdk6/Cyclin D holoenzymes due to down-regulation of Cdkn1b/p27. Finally, we demonstrate that overexpression of Cdkn1b/p27 inhibits HDAC1-mediated ß-cell proliferation. Our data suggest that HDAC1 is critical for the Nkx6.1-mediated pathway that enhances functional ß-cell mass.

Mig6 haploinsufficiency protects mice against streptozotocin-induced diabetes.

AIMS/HYPOTHESIS: EGF and gastrin co-administration reverses type 1 diabetes in rodent models. However, the failure of this to translate into a clinical treatment suggests that EGF-mediated tissue repair is a complicated process and warrants further investigation. Thus, we aimed to determine whether EGF receptor (EGFR) feedback inhibition by mitogen-inducible gene 6 protein (MIG6) limits the effectiveness of EGF therapy and promotes type 1 diabetes development. METHODS: We treated Mig6 (also known as Errfi1) haploinsufficient mice (Mig6 (+/-)) and their wild-type littermates (Mig6 (+/+)) with multiple low doses of streptozotocin (STZ), and monitored diabetes development via glucose homeostasis tests and histological analyses. We also investigated MIG6-mediated cytokine-induced desensitisation of EGFR signalling and the DNA damage repair response in 832/13 INS-1 beta cells. RESULTS: Whereas STZ-treated Mig6 (+/+) mice became diabetic, STZ-treated Mig6 (+/-) mice remained glucose tolerant. In addition, STZ-treated Mig6 (+/-) mice exhibited preserved circulating insulin levels following a glucose challenge. As insulin sensitivity was similar between Mig6 (+/-) and Mig6 (+/+) mice, the preserved glucose tolerance in STZ-treated Mig6 (+/-) mice probably results from preserved beta cell function. This is supported by elevated Pdx1 and Irs2 mRNA levels in islets isolated from STZ-treated Mig6 (+/-) mice. Conversely, MIG6 overexpression in isolated islets compromises glucose-stimulated insulin secretion. Studies in 832/13 cells suggested that cytokine-induced MIG6 hinders EGFR activation and inhibits DNA damage repair. STZ-treated Mig6 (+/-) mice also have increased beta cell mass recovery. CONCLUSIONS/INTERPRETATION: Reducing Mig6 expression promotes beta cell repair and abates the development of experimental diabetes, suggesting that MIG6 may be a novel therapeutic target for preserving beta cells.

Trefoil factor 2 expressed by the murine pancreatic acinar cells is required for the development of islets and for beta cell function during aging.

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.

Upregulation of p21 activates the intrinsic apoptotic pathway in ß-cells.

Diabetes manifests from a loss in functional ß-cell mass, which is regulated by a dynamic balance of various cellular processes, including ß-cell growth, proliferation, and death as well as secretory function. The cell cycle machinery comprised of cyclins, kinases, and inhibitors regulates proliferation. However, their involvement during ß-cell stress during the development of diabetes is not well understood. Interestingly, in a screen of multiple cell cycle inhibitors, p21 was dramatically upregulated in INS-1-derived 832/13 cells and rodent islets by two pharmacological inducers of ß-cell stress, dexamethasone and thapsigargin. We hypothesized that ß-cell stress upregulates p21 to activate the apoptotic pathway and suppress cell survival signaling. To this end, p21 was adenovirally overexpressed in pancreatic rat islets and 832/13 cells. As expected, p21 overexpression resulted in decreased [(3)H]thymidine incorporation. Flow cytometry analysis in p21-transduced 832/13 cells verified lower replication, as indicated by a decreased cell population in the S phase and a block in G2/M transition. The sub-G0 cell population was higher with p21 overexpression and was attributable to apoptosis, as demonstrated by increased annexin-positive stained cells and cleaved caspase-3 protein. p21-mediated caspase-3 cleavage was inhibited by either overexpression of the antiapoptotic mitochondrial protein Bcl-2 or siRNA-mediated suppression of the proapoptotic proteins Bax and Bak. Therefore, an intact intrinsic apoptotic pathway is central for p21-mediated cell death. In summary, our findings indicate that ß-cell apoptosis can be triggered by p21 during stress and is thus a potential target to inhibit for protection of functional ß-cell mass.

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

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.

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

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.

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.

miR-379 mediates insulin resistance and obesity through impaired angiogenesis and adipogenesis regulated by ER stress.

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.

Pharmacokinetics of Sustained-release and Extended-release Buprenorphine in Mice after Surgical Catheterization.

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.

Thioacetamide-Induced Norepinephrine Production by Hepatocytes is Associated with Hepatic Stellate Cell Activation and Liver Fibrosis.

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.

Enrichment of the exocytosis protein STX4 in skeletal muscle remediates peripheral insulin resistance and alters mitochondrial dynamics via Drp1.

Mitochondrial dysfunction is implicated in skeletal muscle insulin resistance. Syntaxin 4 (STX4) levels are reduced in human diabetic skeletal muscle, and global transgenic enrichment of STX4 expression improves insulin sensitivity in mice. Here, we show that transgenic skeletal muscle-specific STX4 enrichment (skmSTX4tg) in mice reverses established insulin resistance and improves mitochondrial function in the context of diabetogenic stress. Specifically, skmSTX4tg reversed insulin resistance caused by high-fat diet (HFD) without altering body weight or food consumption. Electron microscopy of wild-type mouse muscle revealed STX4 localisation at or proximal to the mitochondrial membrane. STX4 enrichment prevented HFD-induced mitochondrial fragmentation and dysfunction through a mechanism involving STX4-Drp1 interaction and elevated AMPK-mediated phosphorylation at Drp1 S637, which favors fusion. Our findings challenge the dogma that STX4 acts solely at the plasma membrane, revealing that STX4 localises at/proximal to and regulates the function of mitochondria in muscle. These results establish skeletal muscle STX4 enrichment as a candidate therapeutic strategy to reverse peripheral insulin resistance.

Rap1 promotes multiple pancreatic islet cell functions and signals through mammalian target of rapamycin complex 1 to enhance proliferation.

Recent studies have implicated Epac2, a guanine-nucleotide exchange factor for the Rap subfamily of monomeric G proteins, as an important regulator of insulin secretion from pancreatic beta-cells. Although the Epac proteins were originally identified as cAMP-responsive activators of Rap1 GTPases, the role of Rap1 in beta-cell biology has not yet been defined. In this study, we examined the direct effects of Rap1 signaling on beta-cell biology. Using the Ins-1 rat insulinoma line, we demonstrate that activated Rap1A, but not related monomeric G proteins, promotes ribosomal protein S6 phosphorylation. Using isolated rat islets, we show that this signaling event is rapamycin-sensitive, indicating that it is mediated by the mammalian target of rapamycin complex 1-p70 S6 kinase pathway, a known growth regulatory pathway. This newly defined beta-cell signaling pathway acts downstream of cAMP, in parallel with the stimulation of cAMP-dependent protein kinase, to drive ribosomal protein S6 phosphorylation. Activated Rap1A promotes glucose-stimulated insulin secretion, islet cell hypertrophy, and islet cell proliferation, the latter exclusively through mammalian target of rapamycin complex 1, suggesting that Rap1 is an important regulator of beta-cell function. This newly defined signaling pathway may yield unique targets for the treatment of beta-cell dysfunction in diabetes.

The physiological regulation of glucose flux into muscle in vivo.

Skeletal muscle glucose uptake increases dramatically in response to physical exercise. Moreover, skeletal muscle comprises the vast majority of insulin-sensitive tissue and is a site of dysregulation in the insulin-resistant state. The biochemical and histological composition of the muscle is well defined in a variety of species. However, the functional consequences of muscle biochemical and histological adaptations to physiological and pathophysiological conditions are not well understood. The physiological regulation of muscle glucose uptake is complex. Sites involved in the regulation of muscle glucose uptake are defined by a three-step process consisting of: (1) delivery of glucose to muscle, (2) transport of glucose into the muscle by GLUT4 and (3) phosphorylation of glucose within the muscle by a hexokinase (HK). Muscle blood flow, capillary recruitment and extracellular matrix characteristics determine glucose movement from the blood to the interstitium. Plasma membrane GLUT4 content determines glucose transport into the cell. Muscle HK activity, cellular HK compartmentalization and the concentration of the HK inhibitor glucose 6-phosphate determine the capacity to phosphorylate glucose. Phosphorylation of glucose is irreversible in muscle; therefore, with this reaction, glucose is trapped and the uptake process is complete. Emphasis has been placed on the role of the glucose transport step for glucose influx into muscle with the past assertion that membrane transport is rate limiting. More recent research definitively shows that the distributed control paradigm more accurately defines the regulation of muscle glucose uptake as each of the three steps that define this process are important sites of flux control.

miR-379 deletion ameliorates features of diabetic kidney disease by enhancing adaptive mitophagy via FIS1.

Diabetic kidney disease (DKD) is a major complication of diabetes. Expression of members of the microRNA (miRNA) miR-379 cluster is increased in DKD. miR-379, the most upstream 5'-miRNA in the cluster, functions in endoplasmic reticulum (ER) stress by targeting EDEM3. However, the in vivo functions of miR-379 remain unclear. We created miR-379 knockout (KO) mice using CRISPR-Cas9 nickase and dual guide RNA technique and characterized their phenotype in diabetes. We screened for miR-379 targets in renal mesangial cells from WT vs. miR-379KO mice using AGO2-immunopreciptation and CLASH (cross-linking, ligation, sequencing hybrids) and identified the redox protein thioredoxin and mitochondrial fission-1 protein. miR-379KO mice were protected from features of DKD as well as body weight loss associated with mitochondrial dysfunction, ER- and oxidative stress. These results reveal a role for miR-379 in DKD and metabolic processes via reducing adaptive mitophagy. Strategies targeting miR-379 could offer therapeutic options for DKD.

Differential Kat3 Usage Orchestrates the Integration of Cellular Metabolism with Differentiation.

The integration of cellular status with metabolism is critically important and the coupling of energy production and cellular function is highly evolutionarily conserved. This has been demonstrated in stem cell biology, organismal, cellular and tissue differentiation and in immune cell biology. However, a molecular mechanism delineating how cells coordinate and couple metabolism with transcription as they navigate quiescence, growth, proliferation, differentiation and migration remains in its infancy. The extreme N-termini of the Kat3 coactivator family members, CBP and p300, by far the least homologous regions with only 66% identity, interact with members of the nuclear receptor family, interferon activated Stat1 and transcriptionally competent ß-catenin, a critical component of the Wnt signaling pathway. We now wish to report based on multiomic and functional investigations, utilizing p300 knockdown, N-terminal p300 edited and p300 S89A edited cell lines and p300 S89A knockin mice, that the N-termini of the Kat3 coactivators provide a highly evolutionarily conserved hub to integrate multiple signaling cascades to coordinate cellular metabolism with the regulation of cellular status and function.