Cadaveric islet and stem cell-derived transplantation hold promise as treatments for type 1 diabetes (T1D). To tackle the issue of immunocompatibility, numerous cellular macroencapsulation techniques that utilize diffusion to transport insulin across an immunoisolating barrier have been developed. However, despite several devices progressing to human clinical trials, none have successfully attained physiologic glucose control or insulin independence. Based on empirical evidence, macroencapsulation methods with multilayered, high islet surface density are incompatible with on-demand insulin delivery and physiologic glucose regulation, when solely reliant on diffusion. An additional driving force is essential to overcome the distance limit of diffusion. In this study, we present both theoretical evidence and experimental validation that applying pressure, at levels comparable to physiological diastolic blood pressure, significantly enhances insulin flux across immunoisolation membranes—increasing it by nearly three orders of magnitude. This significant enhancement in transport rate allows for precise, sub-minute regulation of both bolus and basal insulin delivery. By incorporating this technique with a pumpbased extravascular system, we demonstrate the ability to rapidly reduce glucose levels in diabetic rodent models, replicating the timescale and therapeutic effect of subcutaneous insulin injection or infusion. This advance provides a potential path towards achieving insulin independence with islet macroencapsulation.

Mitochondrial bioenergetics are essential for cellular function, specifically the intricacies of the electron transport chain (ETC), with Complex IV playing a crucial role in unraveling the mechanisms governing energy production. Mathematical models offer a valuable approach to simulate these complex processes, providing insights into normal mitochondrial function and aberrations associated with various diseases, including neurodegenerative disorders. Our research focuses on introducing and refining a mathematical model, emphasizing Complex IV in the ETC, with objectives including incorporating mitochondrial activity modulation using inhibiting and uncoupling reagents, akin to oxygen consumption experiments. Rigorous validation, calibrating against Oroboros Oxygraph-2k data from C57BL/6 mouse mitochondria, ensures accurate reproduction of dynamic bioenergetic activities. The developed graphical user interface (GUI) complements objectives, providing an in silico platform for seamless hypothesis testing (in MATLAB). Employing an innovative kinetic methodology, our research integrates inhibiting reagents (oligomycin, rotenone, antimycin A, FCCP) into the developed computational model to simulate bioenergetic responses across varied physiological conditions. Optimization of the Mean Square Error (MSE) objective function using multiple optimizing algorithms, including the genetic algorithm, and calibration against Oroboros Oxygraph-2k data using freshly isolated mitochondria from C57BL/6 mice ensures rigorous validation of the model’s precision under both unperturbed and perturbed scenarios. These outcomes unequivocally affirm the model’s efficacy in accurately simulating the intricate contributions of Complex IV to bioenergetics. The outcomes highlight the model’s efficacy in reproducing bioenergetic activities, mirroring experimental outcomes. The GUI facilitates user-friendly in silico simulations, offering a valuable complement to traditional experiments. Beyond bioenergetics, the model proves beneficial in studying mitochondrial dysfunction, presenting insights into neurodegenerative diseases. The model’s potential for early detection and therapeutic intervention contributes to advancements in understanding and treating neurological disorders. Our refined mathematical model successfully simulates mitochondrial bioenergetics, emphasizing Complex IV dynamics. Validated against experimental data, the model accurately reproduces bioenergetic activities and demonstrates the potential for studying mitochondrial dysfunction and neurodegenerative diseases. The integration of inhibiting and uncoupling reagents, along with the user-friendly GUI, enhances accessibility and usability. Our research contributes to advancing the medical understanding, emphasizing the role of computational models in unraveling mitochondrial complexities in neurological disorders.

This study explores the potential of utilizing β-cells, exemplified with R7T1 β-cell pseudoislets, as a transplantable cell factory for on-demand recombinant protein therapeutic delivery. While mammalian cell lines are widely used for in vitro protein production, the commonly utilized constitutive secretion pathway poses challenges to in vivo cell therapy, especially for delivering proteins requiring precise exposure kinetics. The proposed approach capitalizes on unique aspects of β -cells, including substantial vesicular protein storage capacity and electrochemically-regulated protein release, to facilitate timely and titratable in vivo therapeutic delivery. Examining a variety of strategies to acheive β-cell glucagon or glucagon-like peptide 1 (GLP-1) storage and secretion, we devised a flexible β-cell-based expression platform for efficient cellular peptide production and on-demand release. This platform utilizes the preproinsulin coding sequence as a template, wherein therapeutic peptides of interest (glucagon or GLP-1) are substituted for C-peptide while the A- and B-peptide insulin chains are mutated to prevent bio-active insulin production. This approach overcomes the challenge of efficient bio-active peptide expression by leveraging the endogenous β -cell peptide expression, translation, processing, storage and secretion machinery. Furthermore, β-cells provide a mechanism for scalable electyrochemnically-triggered peptide delivery. This transformative strategy, which may be extended to other proteins and peptide expression cassettes, holds significant promise for targeted and temporally controlled in vivo production and release of recombinant protein therapeutics. The study suggests potential applications in addressing challenges in metabolic disorders, blood disorders, and oncology. Future refinements may focus on optimizing vector design, peptide production, and in vivo adaptation. ### Competing Interest Statement E. A. T., R. A. L.,, J. P. A., and A.S.Y.P. are co-inventors of a patent covering the work described in the manuscript filed by Stanford.

Cadaveric islet and stem cell-derived transplantations hold promise as treatments for type 1 diabetes. To tackle the issue of immunocompatibility, numerous cellular macroencapsulation techniques have been developed that utilize diffusion to transport insulin across an immunoisolating barrier. However, despite several devices progressing to human clinical trials, none have successfully managed to attain physiologic glucose control or insulin independence. Based on empirical evidence, macroencapsulation methods with multilayered, high islet surface density are incompatible with homeostatic, on-demand insulin delivery and physiologic glucose regulation, when reliant solely on diffusion. An additional driving force is essential to overcome the distance limit of diffusion. In this study, we present both theoretical proof and experimental validation that applying pressure at levels comparable to physiological diastolic blood pressure significantly enhances insulin flux across immunoisolation membranes-increasing it by nearly three orders of magnitude. This significant enhancement in transport rate allows for precise, sub-minute regulation of both bolus and basal insulin delivery. By incorporating this technique with a pump-based extravascular system, we demonstrate the ability to rapidly reduce glucose levels in diabetic rodent models, effectively replicating the timescale and therapeutic effect of subcutaneous insulin injection or infusion. This advance provides a potential path towards achieving insulin independence with islet macroencapsulation. One Sentence Summary:Towards improved glucose control, applying sub-minute pressure at physiological levels enhances therapeutic insulin transport from macroencapsulated islets.

Pathogenic INS gene mutations are causative for mutant INS-gene-induced diabetes of youth (MIDY). We characterize a novel de novo heterozygous INS gene mutation (c.289A>C, p.T97P) that presented in an autoantibody-negative 5-month-old male infant with severe diabetic ketoacidosis. In silico pathogenicity prediction tools provided contradictory interpretations, while structural modeling indicated a deleterious effect on proinsulin folding. Transfection of wildtype and INS p.T97P expression and luciferase reporter constructs demonstrated elevated intracellular mutant proinsulin levels and dramatically impaired proinsulin/insulin and luciferase secretion. Notably, proteasome inhibition partially and selectively rescued INS p.T97P-derived luciferase secretion. Additionally, expression of INS p.T97P caused increased intracellular proinsulin aggregate formation and XBP-1s protein levels, consistent with induction of endoplasmic reticulum stress. We conclude that INS p.T97P is a newly identified pathogenic A-chain variant that is causative for MIDY via disruption of proinsulin folding and processing with induction of the endoplasmic reticulum stress response.

A wireless dual-channel potentiostat is fabricated in TSMC 40-nm CMOS and integrated in a mouthguard format for continuous salivary biosensing of glucose and lactate. Continuous glucose monitoring is required for closed-loop insulin dosing in Type 1 Diabetes. Continuous lactate sensing could improve closed-loop control, by serving as a biomarker of exercise, to help prevent life threatening hypoglycemia. Saliva is an ideal sensing fluid as it is readily and non-invasively available. Glucose sensing range is < 0.6 mM with sensitivity of 2.4 µM and lactate sensing range is < 2 mM with sensitivity of 1.6 µM. Data from both channels is packetized for simultaneous transmission over the 902–928 MHz ISM band. Nominal power is 100 µW, which would allow for operation on a size-10 hearing aid battery for > 1 month.

Cold stimuli and the subsequent activation of β-adrenergic receptor (β-AR) potently stimulate adipose tissue thermogenesis and increase whole-body energy expenditure. However, systemic activation of the β3-AR pathway inevitably increases blood pressure, a significant risk factor for cardiovascular disease, and, thus, limits its application for the treatment of obesity. To activate fat thermogenesis under tight spatiotemporal control without external stimuli, here, we report an implantable wireless optogenetic device that bypasses the β-AR pathway and triggers Ca 2+ cycling selectively in adipocytes. The wireless optogenetics stimulation in the subcutaneous adipose tissue potently activates Ca 2+ cycling fat thermogenesis and increases whole-body energy expenditure without cold stimuli. Significantly, the light-induced fat thermogenesis was sufficient to protect mice from diet-induced body-weight gain. The present study provides the first proof-of-concept that fat-specific cold mimetics via activating non-canonical thermogenesis protect against obesity.

The creatine (Cr) energy system has been implicated in Alzheimer’s disease (AD), including reductions in brain phosphoCr and Cr kinase, yet no studies have examined the neurobehavioral effects of Cr supplementation in AD, including the 3xTg mouse model. This studied investigated the effects of Cr supplementation on spatial cognition, plasticity- and disease-related protein levels, and mitochondrial function in the 3xTg hippocampus. Here, 3xTg mice were fed a control or Cr-supplemented (3% Cr (w/w)) diet for 8–9 weeks and tested in the Morris water maze. Mitochondrial oxygen consumption (Seahorse) and protein levels (Western blots) were measured in the hippocampus in subsets of mice. Overall, 3xTg females exhibited impaired memory as compared to males. In females, Cr supplementation decreased escape latency and was associated with increased spatial search strategy use. In males, Cr supplementation decreased the use of spatial search strategies. Pilot data indicated mitochondrial enhancements with Cr supplementation in both sexes. In females, Cr supplementation increased CREB phosphorylation and levels of IκB (NF-κB suppressor), CaMKII, PSD-95, and high-molecular-weight amyloid β (Aβ) species, whereas Aβ trimers were reduced. These data suggest a beneficial preventative effect of Cr supplementation in females and warrant caution against Cr supplementation in males in the AD-like brain.

Aims: Abnormalities in mitochondrial function under diabetic conditions can lead to deficits in function of cortical neurons and their support cells exhibiting a pivotal role in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. We aimed to assess mitochondrial respiration rates and membrane potential or H2O2 generation simultaneously and expression of proteins involved in mitochondrial dynamics, ROS scavenging and AMPK/SIRT/PGC-1 alpha pathway activity in cortex under diabetic conditions. Methods: Cortical mitochondria from streptozotocin (STZ)-induced type 1 diabetic rats or mice, and aged-matched controls were used for simultaneous measurements of mitochondrial respiration rates and mitochondrial membrane potential (mtMP) or H2O2 using OROBOROS oxygraph. Measurements of enzymatic activities of respiratory complexes were performed using spectophotometry. Protein levels in cortical mitochondria and homogenates were determined by Western blotting. Results: Mitochondrial coupled respiration rates and FCCP-induced uncoupled respiration rates were significantly decreased in mitochondria of cortex of STZ-diabetic rats compared to controls. The mtMP in the presence of ADP was significantly depolarized and succinate-dependent respiration rates and H2O2 were significantly diminished in cortical mitochondria of diabetic animals compared to controls, accompanied with reduced expression of CuZn- and Mn-superoxide dismutase. The enzymatic activities of Complex I, II, and IV and protein levels of certain components of Complex I and II, mitofusin 2 (Mfn2), dynamin-related protein 1 (DRP1), P-AMPK, SIRT2 and PGC-1 alpha were significantly diminished in diabetic cortex. Conclusion. Deficits in mitochondrial function, dynamics, and antioxidant capabilities putatively mediated through sub-optimal AMPK/SIRT/PGC-1 alpha signaling, are involved in the development of early sub-clinical neurodegeneration in the cortex under diabetic conditions.

The brain has a high demand for energy, of which creatine (Cr) is an important regulator. Studies document neurocognitive benefits of oral Cr in mammals, yet little is known regarding their physiological basis. This study investigated the effects of Cr supplementation (3%, w/w) on hippocampal function in male C57BL/6 mice, including spatial learning and memory in the Morris water maze and oxygen consumption rates from isolated mitochondria in real time. Levels of transcription factors and related proteins (CREB, Egr1, and IκB to indicate NF-κB activity), proteins implicated in cognition (CaMKII, PSD-95, and Egr2), and mitochondrial proteins (electron transport chain Complex I, mitochondrial fission protein Drp1) were probed with Western blotting. Dietary Cr decreased escape latency/time to locate the platform (P < 0.05) and increased the time spent in the target quadrant (P < 0.01) in the Morris water maze. This was accompanied by increased coupled respiration (P < 0.05) in isolated hippocampal mitochondria. Protein levels of CaMKII, PSD-95, and Complex 1 were increased in Cr-fed mice, whereas IκB was decreased. These data demonstrate that dietary supplementation with Cr can improve learning, memory, and mitochondrial function and have important implications for the treatment of diseases affecting memory and energy homeostasis.

Alzheimer's disease (AD) is the most common late onset neurodegenerative disorder with indications that women are disproportionately affected. Mitochondrial dysfunction has been one of the most discussed hypotheses associated with the early onset and progression of AD, and it has been attributed to intraneuronal accumulation of amyloid β (Aβ). It was suggested that one of the possible mediators for Aβ-impaired mitochondrial function is the nuclear factor kappa B (NF-κB) signaling pathway. NF-κB plays important roles in brain inflammation and antioxidant defense, as well as in the regulation of mitochondrial function, and studies have confirmed altered NF-κB signaling in AD brain. In this study, we looked for sex-based differences in impaired bioenergetic processes and NF-κB signaling in the AD-like brain using transgenic (Tg) CRND8 mice that express excessive brain Aβ, but without tau pathology. Our results show that mitochondrial dysfunction is not uniform in affected brain regions. We observed increased basal and coupled respiration in the hippocampus of TgCRND8 females only, along with a decreased Complex II-dependent respiratory activity. Cortical mitochondria from TgCRND8 mice have reduced uncoupled respiration capacity, regardless of sex. The pattern of changes in NF-κB signaling was the same in both brain structures, but was sex specific. Whereas in females there was an increase in all three subunits of NF-κB, in males we observed increase in p65 and p105, but no changes in p50 levels. These results demonstrate that mitochondrial function and inflammatory signaling in the AD-like brain is region- and sex-specific, which is an important consideration for therapeutic strategies.