Postnatal Loss of the Insulin Receptor in Osteoprogenitor Cells Does Not Impart a Metabolic Phenotype

The relationship between osteoblast-specific insulin signaling, osteocalcin activation and gluco-metabolic homeostasis has proven to be complex and potentially inconsistent across animal-model systems and in humans. Moreover, the impact of postnatally acquired, osteoblast-specific insulin deficiency on the pancreas-to-skeleton-to-pancreas circuit has not been studied. To explore this relationship, we created a model of postnatal elimination of insulin signaling in osteoprogenitors. Osteoprogenitor-selective ablation of the insulin receptor was induced after ~10 weeks of age in IRl°x/lox/Osx-Cre+/− genotypic male and female mice (designated postnatal-OIRKO). At ~21 weeks of age, mice were then phenotypically and metabolically characterized. Postnatal-OIRKO mice demonstrated a significant reduction in circulating concentrations of undercarboxylated osteocalcin (ucOC), in both males and females compared with control littermates. However, no differences were observed between postnatal-OIRKO and control mice in: body composition (lean or fat mass); fasting serum insulin; HbA1c; glucose dynamics during glucose tolerance testing; or in pancreatic islet area or islet morphology, demonstrating that while ucOC is impacted by insulin signaling in osteoprogenitors, there appears to be little to no relationship between osteocalcin, or its derivative (ucOC), and glucose homeostasis in this model

Large numbers of random point and cluster mutations within the adenovirus VA I gene allow characterization of sequences required for efficient transcription

We have isolated clones with well over 100 randomly dispersed point mutations distributed throughout the 5' half of chemically synthesized adenovirus type 2 VA I genes. In addition, we have isolated clusters of mutations targeted to the regions corresponding to the A and B block consensus sequences of eukaryotic tRNA and adenovirus VA genes

Preserving and Restoring Bone with Continuous Insulin Infusion Therapy in a Mouse Model of Type 1 Diabetes

Those with type 1 diabetes (T1D) are more likely to suffer a fracture than age- and sex-matched individuals without diabetes, despite daily insulin therapy. In rodent studies examining the effect of bone- or glucose-targeting therapies on preventing the T1D-related decrease in bone strength, insulin co-therapy is often not included, despite the known importance of insulin signaling to bone mass accrual. Therefore, working toward a relevant pre-clinical model of diabetic bone disease, we assessed the effect of continuous subcutaneous insulin infusion (CSII) therapy at escalating doses on preserving bone and the effect of delayed CSII on rescuing the T1D-related bone deterioration in an established murine model of T1D. Osmotic minipumps were implanted in male DBA/2 J mice 2 weeks (prevention study) and 6 weeks (rescue study) after the first injection of streptozotocin (STZ) to deliver insulin at 0, 0.0625, 0.125, or 0.25 IU/day (prevention study; n = 4–5 per dose) and 0 or 0.25 IU/day (rescue study; n = 10 per group). CSII lasted 4 weeks in both studies, which also included age-matched, non-diabetic DBA/2 J mice (n = 8–12 per study). As the insulin dose increased, blood glucose decreased, body weight increased, a serum maker of bone resorption decreased, and a serum marker of bone formation increased such that each end-point characteristic was linearly correlated with dose. There were insulin dose-dependent relationships (femur diaphysis) with cross-sectional area of cortical bone and cortical thickness (micro-computed tomography) as well as structural strength (peak force endured by the mid-shaft during three-point bending). Likewise, trabecular bone volume fraction (BV/TV), thickness, and number (distal femur metaphysis) increased as the insulin dose increased. Delayed CSII improved glycated hemoglobin (HbA1c), but blood glucose levels remained relatively high (well above non-diabetic levels). Interestingly, it returned the resorption and formation markers to similar levels as those seen in non-T1D control mice. This apparent return after 4 weeks of CSII translated to a partial rescue of the structural strength of the femur mid-shaft. Delayed CSII also increased Tb.Th to levels seen in non-T1D controls but did not fully restore BV/TV. The use of exogenous insulin should be considered in pre-clinical studies investigating the effect of T1D on bone as insulin therapy maintains bone structure without necessarily lowering glucose below diabetic levels

Palmitate and insulin synergistically induce IL-6 expression in human monocytes

<p>Abstract</p> <p>Background</p> <p>Insulin resistance is associated with a proinflammatory state that promotes the development of complications such as type 2 diabetes mellitus (T2DM) and atherosclerosis. The metabolic stimuli that initiate and propagate proinflammatory cytokine production and the cellular origin of proinflammatory cytokines in insulin resistance have not been fully elucidated. Circulating proinflammatory monocytes show signs of enhanced inflammation in obese, insulin resistant subjects and are thus a potential source of proinflammatory cytokine production. The specific, circulating metabolic factors that might stimulate monocyte inflammation in insulin resistant subjects are poorly characterized. We have examined whether saturated nonesterified fatty acids (NEFA) and insulin, which increase in concentration with developing insulin resistance, can trigger the production of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in human monocytes.</p> <p>Methods</p> <p>Messenger RNA and protein levels of the proinflammatory cytokines IL-6 and TNF-α were measured by quantitative real-time PCR (qRT-PCR) and Luminex bioassays. Student's <it>t</it>-test was used with a significance level of <it>p </it>< 0.05 to determine significance between treatment groups.</p> <p>Results</p> <p>Esterification of palmitate with coenzyme A (CoA) was necessary, while β-oxidation and ceramide biosynthesis were not required, for the induction of IL-6 and TNF-α in THP-1 monocytes. Monocytes incubated with insulin and palmitate together produced more IL-6 mRNA and protein, and more TNF-α protein, compared to monocytes incubated with palmitate alone. Incubation of monocytes with insulin alone did not affect the production of IL-6 or TNF-α. Both PI3K-Akt and MEK/ERK signalling pathways are important for cytokine induction by palmitate. MEK/ERK signalling is necessary for synergistic induction of IL-6 by palmitate and insulin.</p> <p>Conclusions</p> <p>High levels of saturated NEFA, such as palmitate, when combined with hyperinsulinemia, may activate human monocytes to produce proinflammatory cytokines and support the development and propagation of the subacute, chronic inflammatory state that is characteristic of insulin resistance. Results with inhibitors of β-oxidation and ceramide biosynthesis pathways suggest that increased fatty acid flux through the glycerolipid biosynthesis pathway may be involved in promoting proinflammatory cytokine production in monocytes.</p

Mice with Infectious Colitis Exhibit Linear Growth Failure and Subsequent Catch-Up Growth Related to Systemic Inflammation and IGF-1

In developing communities, intestinal infection is associated with poor weight gain and linear-growth failure. Prior translational animal models have focused on weight gain investigations into key contributors to linear growth failure have been lacking. We hypothesized that murine intestinal infection with Citrobacter-rodentium would induce linear-growth failure associated with systemic inflammation and suppressed serum levels of insulin-like growth factor-1 (IGF-1). We evaluated 4 groups of mice infected or sham-infected on day-of-life 28: uninfected-controls, wild-type C.-rodentium-infected, partially-attenuated C. rodentium-infected (with deletion of 3 serine protease genes involved in colonization), and pair-fed (given the amount of daily food consumed by the wild-type C.-rodentium group). Relative to the uninfected group, mice infected with wild-type C.-rodentium exhibited temporal associations of lower food intake, weight loss, linear-growth failure, higher IL-6 and TNF-α and lower IGF-1. However, relative to the pair-fed group, the C.-rodentium-infected group only differed significantly by linear growth and systemic inflammatory cytokines. Between post-infection days 15–20, the infected group exhibited resolution of systemic inflammation. Between days 16–20, both wild-type C.-rodentium and pair-fed groups exhibited rapid linear-growth velocities exceeding the uninfected and mutant C.-rodentium groups; during this time levels of IGF-1 increased to match the uninfected group. We submit this as a model providing important opportunities to study mechanisms of catch-up growth related to intestinal inflammation. We conclude that in addition to known effects of weight loss, infection with C.-rodentium induces linear-growth failure potentially related to systemic inflammation and low levels of IGF-1, with catch-up of linear growth following resolution of inflammation

Loss of Insulin Receptor in Osteoprogenitor Cells Impairs Structural Strength of Bone

Type 1 diabetes mellitus (T1D) is associated with decreased bone mineral density, a deficit in bone structure, and subsequently an increased risk of fragility fracture. These clinical observations, paralleled by animal models of T1D, suggest that the insulinopenia of T1D has a deleterious effect on bone. To further examine the action of insulin signaling on bone development, we generated mice with an osteoprogenitor-selective (osterix-Cre) ablation of the insulin receptor (IR), designated OIRKO. OIRKO mice exhibited an 80% decrease in IR in osteoblasts. Prenatal elimination of IR did not affect fetal survival or gross morphology. However, loss of IR in mouse osteoblasts resulted in a postnatal growth-constricted phenotype. By 10–12 weeks of age, femurs of OIRKO mice were more slender, with a thinner diaphyseal cortex and, consequently, a decrease in whole bone strength when subjected to bending. In male mice alone, decreased metaphyseal trabecular bone, with thinner and more rodlike trabeculae, was also observed. OIRKO mice did not, however, exhibit abnormal glucose tolerance. The skeletal phenotype of the OIRKO mouse appeared more severe than that of previously reported bone-specific IR knockdown models, and confirms that insulin receptor expression in osteoblasts is critically important for proper bone development and maintenance of structural integrity

Characterization of a Reverse-Phase Perfluorocarbon Emulsion for the Pulmonary Delivery of Tobramycin

Background: Aerosolized delivery of antibiotics is hindered by poor penetration within distal and plugged airways. Antibacterial perfluorocarbon ventilation (APV) is a proposed solution in which the lungs are partially or totally filled with perfluorocarbon (PFC) containing emulsified antibiotics. The purpose of this study was to evaluate emulsion stability and rheological, antibacterial, and pharmacokinetic characteristics. Methods: This study examined emulsion aqueous droplet diameter and number density over 24?hr and emulsion and neat PFC viscosity and surface tension. Additionally, Pseudomonas aeruginosa biofilm growth was measured after 2-hr exposure to emulsion with variable aqueous volume percentages (0.25, 1, and 2.5%) and aqueous tobramycin concentrations (Ca=0.4, 4, and 40?mg/mL). Lastly, the time course of serum and pulmonary tobramycin concentrations was evaluated following APV and conventional aerosolized delivery of tobramycin in rats. Results: The initial aqueous droplet diameter averaged 1.9±0.2??m with little change over time. Initial aqueous droplet number density averaged 3.5±1.7?109 droplets/mL with a significant (p<0.01) decrease over time. Emulsion and PFC viscosity were not significantly different, averaging 1.22±0.03?10?3 Pa·sec. The surface tensions of PFC and emulsion were 15.0±0.1?10?3 and 14.6±0.6?10?3 N/m, respectively, and the aqueous interfacial tensions were 46.7±0.3?10?3 and 26.9±11.0?10?3 N/m (p<0.01), respectively. Biofilm growth decreased markedly with increasing Ca and, to a lesser extent, aqueous volume percentage. Tobramycin delivered via APV yielded 2.5 and 10 times larger pulmonary concentrations at 1 and 4?hr post delivery, respectively, and significantly (p<0.05) lower serum concentrations compared with aerosolized delivery. Conclusions: The emulsion is bactericidal, retains the rheology necessary for pulmonary delivery, is sufficiently stable for this application, and results in increased pulmonary retention of the antibiotic.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140105/1/jamp.2013.1058.pd

Dysregulation of the Intrarenal Vitamin D Endocytic Pathway in a Nephropathy-Prone Mouse Model of Type 1 Diabetes

Microalbuminuria in humans with Type 1 diabetes (T1D) is associated with increased urinary excretion of megalin, as well as many megalin ligands, including vitamin-D-binding protein (VDBP). We examined the DBA/2J diabetic mouse, nephropathy prone model, to determine if megalin and VDBP excretion coincide with the development of diabetic nephropathy. Megalin, VDBP, and 25-hydroxy-vitamin D (25-OHD) were measured in urine, and genes involved in vitamin D metabolism were assessed in renal tissues from diabetic and control mice at 10, 15, and 18 weeks following the onset of diabetes. Megalin, VDBP, and 25-OHD were increased in the urine of diabetic mice. 1-α hydroxylase (CYP27B1) mRNA in the kidney was persistently increased in diabetic mice, as were several vitamin D-target genes. These studies show that intrarenal vitamin D handling is altered in the diabetic kidney, and they suggest that in T1D, urinary losses of VDBP may portend risk for intrarenal and extrarenal vitamin D deficiencies

The Impact of SGLT2 Inhibitors, Compared with Insulin, on Diabetic Bone Disease in a Mouse Model of Type 1 Diabetes

Skeletal co-morbidities in type 1 diabetes include an increased risk for fracture and delayed fracture healing, which are intertwined with disease duration and the presence of other diabetic complications. As such, chronic hyperglycemia is undoubtedly a major contributor to these outcomes, despite standard insulin-replacement therapy. Therefore, using the streptozotocin (STZ)-induced model of hypoinsulinemic hyperglycemia in DBA/2J male mice, we compared the effects of two glucose lowering therapies on the fracture resistance of bone and markers of bone turnover. Twelve week-old diabetic (DM) mice were treated for 9 weeks with: 1) oral canagliflozin (CANA, dose range ~10-16 mg/kg/day), an inhibitor of the renal sodium-dependent glucose co-transporter type 2 (SGLT2); 2) subcutaneous insulin, via minipump (INS, 0.125 units/day); 3) co-therapy (CANA + INS); or 4) no treatment (STZ, without therapy). These groups were also compared to non-diabetic control groups. Untreated diabetic mice experienced increased bone resorption and significant deficits in cortical and trabecular bone that contributed to structural weakness of the femur mid-shaft and the lumbar vertebra, as determined by three-point bending and compression tests, respectively. Treatment with either canagliflozin or insulin alone only partially rectified hyperglycemia and the diabetic bone phenotype. However, when used in combination, normalization of glycemic control was achieved, and a prevention of the DM-related deterioration in bone microarchitecture and bone strength occurred, due to additive effects of canagliflozin and insulin. Nevertheless, CANA-treated mice, whether diabetic or non-diabetic, demonstrated an increase in urinary calcium loss; FGF23 was also increased in CANA-treated DM mice. These findings could herald ongoing bone mineral losses following CANA exposure, suggesting that certain CANA-induced skeletal consequences might detract from therapeutic improvements in glycemic control, as they relate to diabetic bone disease