Accelerated absorption of regular insulin administered via a vascularizing permeable microchamber implanted subcutaneously in diabetic Rattus norvegicus.

In Type 1 diabetes patients, even ultra-rapid acting insulins injected subcutaneously reach peak concentrations in 45 minutes or longer. The lag time between dosing and peak concentration, as well as intra- and inter-subject variability, render prandial glucose control and dose consistency difficult. We postulated that insulin absorption from subcutaneously implantable vascularizing microchambers would be significantly faster than conventional subcutaneous injection. Male athymic nude R. norvegicus rendered diabetic with streptozotocin were implanted with vascularizing microchambers (single chamber; 1.5 cm2 surface area per side; nominal volume, 22.5 µl). Plasma insulin was assayed after a single dose (1.5 U/kg) of diluted insulin human (Humulin®R U-100), injected subcutaneously or via microchamber. Microchambers were also implanted in additional animals and retrieved at intervals for histologic assessment of vascularity. Following conventional subcutaneous injection, the mean peak insulin concentration was 22.7 (SD 14.2) minutes. By contrast, when identical doses of insulin were injected via subcutaneous microchamber 28 days after implantation, the mean peak insulin time was shortened to 7.50 (SD 4.52) minutes. Peak insulin concentrations were similar by either route; however, inter-subject variability was reduced when insulin was administered via microchamber. Histologic examination of tissue surrounding microchambers showed mature vascularization on days 21 and 40 post-implantation. Implantable vascularizing microchambers of similar design may prove clinically useful for insulin dosing, either intermittently by needle, or continuously by pump including in "closed loop" systems, such as the artificial pancreas.

Enteropathogenic Escherichia coli regulates host-cell mitochondrial morphology.

The diarrheagenic pathogen enteropathogenic Escherichia coli is responsible for significant childhood mortality and morbidity. EPEC and related attaching-and-effacing (A/E) pathogens use a type III secretion system to hierarchically deliver effector proteins into host cells and manipulate epithelial structure and function. Subversion of host mitochondrial biology is a key aspect of A/E pathogen virulence strategy, but the mechanisms remain poorly defined. We demonstrate that the early-secreted effector EspZ and the late-secreted effector EspH have contrasting effects on host mitochondrial structure and function. EspZ interacts with FIS1, a protein that induces mitochondrial fragmentation and mitophagy. Infection of epithelial cells with either wildtype EPEC or an isogenic espZ deletion mutant (ΔespZ) robustly upregulated FIS1 abundance, but a marked increase in mitochondrial fragmentation and mitophagy was seen only in ΔespZ-infected cells. FIS1-depleted cells were protected against ΔespZ-induced fission, and EspZ-expressing transfected epithelial cells were protected against pharmacologically induced mitochondrial fission and membrane potential disruption. Thus, EspZ interacts with FIS1 and blocks mitochondrial fragmentation and mitophagy. In contrast to WT EPEC, ΔespH-infected epithelial cells had minimal FIS1 upregulation and exhibited hyperfused mitochondria. Consistent with the contrasting impacts on organelle shape, mitochondrial membrane potential was preserved in ΔespH-infected cells, but profoundly disrupted in ΔespZ-infected cells. Collectively, our studies reveal hitherto unappreciated roles for two essential EPEC virulence factors in the temporal and dynamic regulation of host mitochondrial biology.

Bacterial pathogens strategically manipulate host cell structures and functions during the process of colonization and expansion, and this eventually contributes to disease symptoms. The diarrhea-causing pathogen enteropathogenic Escherichia coli (EPEC) secretes proteins into host cells to alter their behavior. Two secreted proteins, EspZ and EspH, were previously shown to be essential for causing disease in animal models. In this study, we demonstrate that interplay between EspZ/EspH and host factors modulates the structure and function of host cell mitochondria. Among their various roles, mitochondria generate energy, produce important biomolecules, and protect cells from damage. EPEC infection of epithelial cells results in increased abundance of a key mitochondrial outer-membrane protein, FIS1. FIS1 plays a housekeeping role by breaking down unhealthy mitochondria and targeting them for elimination from cells. In the early stages of infection, EspZ interacts with FIS1 and blocks its action, thereby protecting the host mitochondrial network and consequently, enhancing host cell viability. Our studies are consistent with a model wherein EspZ-dependent preservation of mitochondrial integrity early in infection allows for bacterial colonization. Later in infection, however, EspH-dependent increase in FIS1 results in significant mitochondrial fragmentation and host cell death; this likely facilitates pathogen dispersal. Taken together, EspZ and EspH dynamically impact host biology, and consequently, infection outcomes. Overall, an appreciation of the mechanisms by which EspZ and EspH manipulate host cells could eventually lead to host-directed interventions for EPEC diarrhea, which is currently not vaccine-preventable.