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BACKGROUND Digital health programs that incorporate frequent blood pressure (BP) self-monitoring and comprehensive support for behavior change offer a scalable solution for hypertension (HTN) management. OBJECTIVE We examined the impact of a digital HTN self-management and lifestyle change support program on BP over 12 months. METHODS Data was analyzed from a retrospective observational cohort of commercially insured members (n=1,117) that started the Omada for HTN program between 1/1/19-9/30/21. Paired t-tests and linear regression were used to measure change in SBP over 12 months, by SBP control at baseline (≥130 vs. RESULTS Members were on average 50.9 years old, 50.8% were female, 60.5% white and 70.5% had uncontrolled SBP at baseline (≥130). At 12 months, members with uncontrolled SBP experienced significant reductions in both unadjusted (mean (95%CI): -8.1mmHg (-9.0, -7.1)) and adjusted SBP (-8.0mmHg (-9.0, -7.1)). Members with uncontrolled SBP also had significant reductions in diastolic BP (-4.7mmHg; -5.3, -4.1), weight (-6.5lbs (-7.7, -5.3); 2.7% weight loss) and body mass index (-1.1kg/m2) (-1.3, -0.9)). Those with controlled SBP maintained within BP goal range. Additionally, 48% of members with uncontrolled BP experienced enough change in BP to improve their BP category. CONCLUSIONS This study provides real-world evidence that a comprehensive digital health program involving HTN education, at-home BP monitoring, and behavior change coaching support was effective for self-managing HTN over 12 months.

BACKGROUND Modest weight loss is recommended for individuals with type 2 diabetes (T2D) to help improve glycemic control and reduce cardiovascular disease risk. Continuous glucose monitoring (CGM) is recommended as a standard of care for individuals with T2D who use insulin, but more research is needed to inform recommendations for CGM use in all T2D populations. Digital diabetes self-management programs can help individuals with T2D lose weight and improve glycemic control. Less is known about the impact of CGM on program engagement within digital programs for diabetes self-management and the relationship between CGM use and weight loss for those with T2D. OBJECTIVE The primary objective of this study was to examine the difference in percent weight loss from baseline to 6 months between CGM users and non-users among commercially insured members with T2D in a digital program for diabetes self-management (Omada for Diabetes). METHODS A non-randomized retrospective observational cohort study was performed using data from 2,612 Omada for Diabetes program members (mean age=52.3 years, 56.4% female, 63.5% white) who started the program between January 1, 2021 and September 30, 2021. Data analyses examined the difference in percent weight loss over 6 months between CGM users and non-users overall as well as stratified by body mass index, program engagement, and CGM-adherence, and associations between program engagement and CGM group. RESULTS A higher percentage of CGM users (57.6%) than non-users (48.4%) were classified as ‘highly engaged’ with the Omada for Diabetes program (p CONCLUSIONS Members participating in the Omada for Diabetes program had a significant change in mean percent body weight from program start to 6 months regardless of CGM status. A higher percentage of CGM users were more engaged with the program than non-users, and higher engagement across both groups was associated with greater percent weight loss. Further understanding the impact that CGM has in digital diabetes self-management programs for T2D could enhance program effectiveness, encourage sustained engagement, and inform standard of care recommendations. CLINICALTRIAL N/A

Factor inhibiting HIF-1alpha (FIH) is an asparaginyl hydroxylase. Hydroxylation of HIF-alpha proteins by FIH blocks association of HIFs with the transcriptional coactivators CBP/p300, thus inhibiting transcriptional activation. We have created mice with a null mutation in the FIH gene and found that it has little or no discernable role in mice in altering classical aspects of HIF function, e.g., angiogenesis, erythropoiesis, or development. Rather, it is an essential regulator of metabolism: mice lacking FIH exhibit reduced body weight, elevated metabolic rate, hyperventilation, and improved glucose and lipid homeostasis and are resistant to high-fat-diet-induced weight gain and hepatic steatosis. Neuron-specific loss of FIH phenocopied some of the major metabolic phenotypes of the global null animals: those mice have reduced body weight, increased metabolic rate, and enhanced insulin sensitivity and are also protected against high-fat-diet-induced weight gain. These results demonstrate that FIH acts to a significant degree through the nervous system to regulate metabolism.

The hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha are closely related, key transcriptional regulators of the hypoxic response, countering a low oxygen situation with the up-regulation of target genes associated with numerous processes, including vascularization and glycolysis. This involves a dual mechanism of control through both stabilization and transactivation, regulated via prolyl and asparaginyl hydroxylation. Despite high similarity with respect to protein sequence and activation pathway, a growing number of physiological and mechanistic differences between HIF-1alpha and HIF-2alpha are being reported. To further characterize this nonredundancy, the stabilization of endogenous proteins and regulation of the transactivation domains were compared in a graded oxygen environment across a series of cell lines. Although generally similar results were found, interesting and specific differences between the HIF-alpha proteins were observed within certain cell lines, such as rat adrenal PC12s, emphasizing the cell-specific nature of HIF-alpha regulation. We characterize a conserved amino acid substitution between HIF-1alpha and HIF-2alpha that contributes to the intrinsically higher FIH-1-mediated asparaginyl hydroxylation of HIF-1alpha and, hence, lower HIF-1alpha activity. In addition, our data demonstrate that the different cell lines can be classified into two distinct groups: those in which stabilization and transactivation proceed in conjunction (HeLa, 293T, and COS-1) and those cells in which HIF-alpha is stabilized prior to transactivation (PC12, HepG2, and CACO2). Interestingly, the initial stabilization of HIF-alpha prior to transactivation up-regulation predicted from in vitro derived hydroxylation data is only true for a subset of cells.

The hypoxia inducible transcription factors (HIFs) are regulated at the level of protein stability and transcriptional activity in an oxygen-dependent manner by prolyl and asparaginyl hydroxylation, respectively. Factor inhibiting HIF (FIH-1) is the only known HIF asparaginyl hydroxylase, and targets a conserved asparaginyl residue within the C-terminal activation domain (CAD) of HIF-alpha. This represses HIF-mediated transcription by inhibiting the recruitment of p300/CBP coactivators. Recent studies have demonstrated that the function of FIH-1 relative to the HIF prolyl hydroxylases (PHDs) is not redundant, and indicate that FIH-1 is a direct oxygen sensor. This paper will address recent published and unpublished work characterising the role of asparaginyl hydroxylation in the cellular response to hypoxia. The relative oxygen affinities and hypoxic activities of FIH-1 and the PHDs will be discussed. Furthermore, in vitro and cell-based assays demonstrating some novel characteristics regarding the substrate specificity of FIH-1, and their potential biological and therapeutic relevance will be presented.

Cells adapt to hypoxia by a cellular response, where hypoxia-inducible factor 1α (HIF-1α) becomes stabilized and directly activates transcription of downstream genes. In addition to this “canonical” response, certain aspects of the pathway require integration with Notch signaling, i.e., HIF-1α can interact with the Notch intracellular domain (ICD) to augment the Notch downstream response. In this work, we demonstrate an additional level of complexity in this cross-talk: factor-inhibiting HIF-1 (FIH-1) regulates not only HIF activity, but also the Notch signaling output and, in addition, plays a role in how Notch signaling modulates the hypoxic response. We show that FIH-1 hydroxylates Notch ICD at two residues (N 1945 and N 2012 ) that are critical for the function of Notch ICD as a transactivator within cells and during neurogenesis and myogenesis in vivo . FIH-1 negatively regulates Notch activity and accelerates myogenic differentiation. In its modulation of the hypoxic response, Notch ICD enhances recruitment of HIF-1α to its target promoters and derepresses HIF-1α function. Addition of FIH-1, which has a higher affinity for Notch ICD than for HIF-1α, abrogates the derepression, suggesting that Notch ICD sequesters FIH-1 away from HIF-1α. In conclusion, the data reveal posttranslational modification of the activated form of the Notch receptor and an intricate mode of cross-coupling between the Notch and hypoxia signaling pathways.

The hypoxia-inducible transcription factors (HIFs) are essential mediators of the genomic response to oxygen deficiency (hypoxia) in multicellular organisms. The HIFs are regulated by four oxygen-sensitive hydroxylases-three prolyl hydroxylases and one asparaginyl hydroxylase. These hydroxylases are all members of the 2-oxoglutarate (2OG)-dependent dioxygenase superfamily and convey changes in cellular oxygen concentration to the HIF-alpha (alpha) subunit, leading to potent accumulation and activity in hypoxia versus degradation and repression in normoxia. HIF-alpha asparaginyl hydroxylation is catalyzed by factor-inhibiting HIF-1 (FIH-1) and directly regulates the transcription activity of the HIF-alpha proteins. Recent work has demonstrated that, in addition to hydroxylating HIF-alpha, FIH-1 can also hydroxylate the ankyrin domains of a wide range of proteins. This paper presents in vitro and cell-based techniques for the preliminary characterization of ankyrin domain-containing proteins as FIH-1 substrates and interacting proteins. Strategies are presented for the expression and purification of FIH-1 from mammalian or bacterial cells. Similar to the HIF-alpha proteins, the ankyrin-containing substrates are examined as purified proteins expressed in bacteria and overexpressed in mammalian cells or in the form of synthetic peptides. Specific conditions for the efficient expression of ankyrin-containing proteins compared with the HIF-alpha substrates in Escherichia coli are detailed. Hydroxylation is rapidly inferred, utilizing the described in vitro CO(2) capture assay. Finally, substrate and non-substrate interactions are examined using in vitro affinity pull-down assays and mammalian cell-based co-immunoprecipitation assays. Together, these methods are rapid and well suited to the preliminary characterization of potential substrates of the therapeutically relevant oxygen-sensing enzyme FIH-1.

The hypoxia‐inducible transcription factors (HIFs) are essential mediators of the genomic response to oxygen deficiency (hypoxia) in multicellular organisms. The HIFs are regulated by four oxygen‐sensitive hydroxylases—three prolyl hydroxylases and one asparaginyl hydroxylase. These hydroxylases are all members of the 2‐oxoglutarate (2OG)–dependent dioxygenase superfamily and convey changes in cellular oxygen concentration to the HIF‐alpha (α) subunit, leading to potent accumulation and activity in hypoxia versus degradation and repression in normoxia. HIF‐α asparaginyl hydroxylation is catalyzed by factor‐inhibiting HIF‐1 (FIH‐1) and directly regulates the transcription activity of the HIF‐α proteins. Recent work has demonstrated that, in addition to hydroxylating HIF‐α, FIH‐1 can also hydroxylate the ankyrin domains of a wide range of proteins. This paper presents in vitro and cell‐based techniques for the preliminary characterization of ankyrin domain–containing proteins as FIH‐1 substrates and interacting proteins. Strategies are presented for the expression and purification of FIH‐1 from mammalian or bacterial cells. Similar to the HIF‐α proteins, the ankyrin‐containing substrates are examined as purified proteins expressed in bacteria and overexpressed in mammalian cells or in the form of synthetic peptides. Specific conditions for the efficient expression of ankyrin‐containing proteins compared with the HIF‐α substrates in Escherichia coli are detailed. Hydroxylation is rapidly inferred, utilizing the described in vitro CO2 capture assay. Finally, substrate and non‐substrate interactions are examined using in vitro affinity pull‐down assays and mammalian cell‐based co‐immunoprecipitation assays. Together, these methods are rapid and well suited to the preliminary characterization of potential substrates of the therapeutically relevant oxygen‐sensing enzyme FIH‐1.

The hypoxia inducible transcription factors (HIFs) are regulated at the level of protein stability and transcriptional activity in an oxygen-dependent manner by prolyl and asparaginyl hydroxylation, respectively. Factor inhibiting HIF (FIH-1) is the only known HIF asparaginyl hydroxylase, and targets a conserved asparaginyl residue within the C-terminal activation domain (CAD) of HIF-alpha. This represses HIF-mediated transcription by inhibiting the recruitment of p300/CBP coactivators. Recent studies have demonstrated that the function of FIH-1 relative to the HIF prolyl hydroxylases (PHDs) is not redundant, and indicate that FIH-1 is a direct oxygen sensor. This paper will address recent published and unpublished work characterising the role of asparaginyl hydroxylation in the cellular response to hypoxia. The relative oxygen affinities and hypoxic activities of FIH-1 and the PHDs will be discussed. Furthermore, in vitro and cell-based assays demonstrating some novel characteristics regarding the substrate specificity of FIH-1, and their potential biological and therapeutic relevance will be presented.

The hypoxia-inducible factor alpha subunits 1 and 2 (HIF-1alpha and HIF-2alpha) are subjected to oxygen-dependent asparaginyl hydroxylation, a modification that represses the carboxyl-terminal transactivation domain (CAD) at normoxia by preventing recruitment of the p300/cAMP-response element-binding protein coactivators. This hydroxylation is performed by the novel asparaginyl hydroxylase, factor-inhibiting HIF-1' (FIH-1), of which HIF-1alpha and HIF-2alpha are the only reported substrates. Here we investigated the substrate requirements of FIH-1 by characterizing its subcellular localization and by examining amino acids within the HIF-1alpha substrate for their importance in recognition and catalysis by FIH-1. Using immunohistochemistry, we showed that both endogenous and transfected FIH-1 are primarily confined to the cytoplasm and remain there under normoxia and following treatment with the hypoxia mimetic, dipyridyl. Individual alanine mutations of seven conserved amino acids flanking the hydroxylated asparagine in HIF-1alpha revealed the importance of the valine (Val-802) adjacent to the targeted asparagine. The HIF-1alpha CAD V802A mutant exhibited a 4-fold lower V(max) in enzyme assays, whereas all other mutants were hydroxylated as efficiently as the wild type HIF-1alpha CAD. Furthermore, in cell-based assays the transcriptional activity of V802A was constitutive, suggesting negligible normoxic hydroxylation in HEK293T cells, whereas the wild type and other mutants were repressed under normoxia. Molecular modeling of the HIF-1alpha CAD V802A in complex with FIH-1 predicted an alteration in asparagine positioning compared with the wild type HIF-1alpha CAD, providing an explanation for the impaired catalysis observed and confirming the importance of Val-802 in asparaginyl hydroxylation by FIH-1.