R. Clay Bunn

Insulin-like growth factor binding protein proteolysis

High-affinity interactions between insulin-like growth factors (IGF-I and IGF-II) and insulin-like growth factor-binding proteins (IGFBP-1, -2, -3, -4, -5 and -6) antagonize the binding of IGF to the type 1 IGF receptor. Proteases found in a variety of biological fluids can degrade IGFBP 1-6 into fragments that have a greatly reduced affinity for IGF-I and IGF-II, increasing the concentration of free IGFs at the cell surface and allowing IGFs to bind to and activate the IGF receptor. Therefore, IGFBP proteolysis directly modulates the first step in IGF receptor signaling and thereby indirectly modulates cell survival, mitogenesis and differentiation. Our understanding of IGFBP proteolysis has grown exponentially over the past five years, with the identification of several new IGFBP proteases, a growing appreciation of the potential for IGF-independent actions of IGFBP fragments and the realization that perturbations of IGFBP proteolysis are seen in, and might contribute to, several pathological conditions.

Matrix metalloproteinases: their potential role in the pathogenesis of diabetic nephropathy

Matrix metalloproteinases (MMPs), a family of proteinases including collagenases, gelatinases, stromelysins, matrilysins, and membrane-type MMPs, affect the breakdown and turnover of extracellular matrix (ECM). Moreover, they are major physiologic determinants of ECM degradation and turnover in the glomerulus. Renal hypertrophy and abnormal ECM deposition are hallmarks of diabetic nephropathy (DN), suggesting that altered MMP expression or activation contributes to renal injury in DN. Herein, we review and summarize recent information supporting a role for MMPs in the pathogenesis of DN. Specifically, studies describing dysregulated activity of MMPs and/or their tissue inhibitors in various experimental models of diabetes, including animal models of type 1 or type 2 diabetes, clinical investigations of human type 1 or type 2 diabetes, and kidney cell culture studies are reviewed.

Increasing duration of type 1 diabetes perturbs the strength-structure relationship and increases brittleness of bone.

Type 1 diabetes (T1DM) increases the likelihood of a fracture. Despite serious complications in the healing of fractures among those with diabetes, the underlying causes are not delineated for the effect of diabetes on the fracture resistance of bone. Therefore, in a mouse model of T1DM, we have investigated the possibility that a prolonged state of diabetes perturbs the relationship between bone strength and structure (i.e., affects tissue properties). At 10, 15, and 18 weeks following injection of streptozotocin to induce diabetes, diabetic male mice and age-matched controls were examined for measures of skeletal integrity. We assessed 1) the moment of inertia (I(MIN)) of the cortical bone within diaphysis, trabecular bone architecture of the metaphysis, and mineralization density of the tissue (TMD) for each compartment of the femur by micro-computed tomography and 2) biomechanical properties by three-point bending test (femur) and nanoindentation (tibia). In the metaphysis, a significant decrease in trabecular bone volume fraction and trabecular TMD was apparent after 10 weeks of diabetes. For cortical bone, type 1 diabetes was associated with decreased cortical TMD, I(MIN), rigidity, and peak moment as well as a lack of normal age-related increases in the biomechanical properties. However, there were only modest differences in material properties between diabetic and normal mice at both whole bone and tissue-levels. As the duration of diabetes increased, bone toughness decreased relative to control. If the sole effect of diabetes on bone strength was due to a reduction in bone size, then I(MIN) would be the only significant variable explaining the variance in the maximum moment. However, general linear modeling found that the relationship between peak moment and I(MIN) depended on whether the bone was from a diabetic mouse and the duration of diabetes. Thus, these findings suggest that the elevated fracture risk among diabetics is impacted by complex changes in tissue properties that ultimately reduce the fracture resistance of bone.

Early Developmental Changes in IGF-I, IGF-II, IGF Binding Protein-1, and IGF Binding Protein-3 Concentration in the Cerebrospinal Fluid of Children

IGF-I and IGF-II are ubiquitously expressed growth factors that have profound effects on the growth and differentiation of many cell types and tissues, including cells of the CNS. In biologic fluids, most IGFs are bound to one of six IGF binding proteins (IGFBPs 1–6). Increasing evidence strongly supports a role for IGF-I in CNS development, as it promotes neuronal proliferation and survival. However, little is known about IGF-I and its homolog IGF-II and their carrier proteins, IGFBPs, during the neonatal period in which brain size increases dramatically, myelination takes place, and neurons show limited capacity to proliferate. Herein, we have determined the concentrations of IGF-I, IGF-II, IGFBP-1, and IGFBP-3 in cerebral spinal fluid (CSF) samples that were collected from children who were 1 wk to 18 y of age. The concentrations of IGF-I, IGFBP-1, and IGFBP-3 in CSF from children <6 mo of age were significantly higher than in older children, whereas IGF-II was higher in the older group. This is in contrast to what is observed in the peripheral circulation, where IGF-I and IGFBP-3 are low at birth and rise rapidly during the first year, reaching peak levels during puberty. Higher concentrations of IGF-I, IGFBP-1, and IGFBP-3 in the CSF of very young children suggest that these proteins might participate in the active processes of myelination and synapse formation in the developing nervous system. We propose that IGF-I and certain IGFBPs are likely necessary for normal CNS development during critical stages of neonatal brain growth and development.

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.

Physiological matrix metalloproteinase (MMP) concentrations: comparison of serum and plasma specimens

The authors appreciate the critical comments of Professor K. Jung, and agree with his demonstration, here and elsewhere (1, 2), that differences between serum and plasma samples exist for measurement of MMP-9. We also agree, and have cautioned in our original manuscript, that plasma is the preferred clinical specimen for measurement of both MMP-8 and MMP-9 (3). Serum levels of these MMPs are very likely influenced by release of MMPs following degranulation of leukocytes and platelets during the ex vivo blood clotting process in the specimen collection tube (4). Finally, we agree that clinical research studies reporting on circulating MMP concentrations should clearly specify the sample collection methodology. Serum samples utilized in our analyses were prepared using “gold top” BD Vacutainers® (with clot activators and silicon coating, Becton Dickinson and Company, Franklin Lakes, USA) #367382; by centrifugation at 3400 rpm (2000×g) for 5 min. We would challenge, however, the inference that serum is never “appropriate” for the physiological ascertainment of MMP concentrations or that conclusions relative to MMP-9 can be generalized to all MMP measurements. For disease states characterized by cellular inflammatory responses, such as multiple sclerosis (5, 6) and coronary artery disease (7–9), or malignant proliferation of inflammatory cell types such as multiple myeloma (10, 11) and chronic lymphocytic leukemia (12, 13), differences in serum measurements of MMPs have been detected and may provide a marker of pertinent intracellular protease activity. Alternatively, elevated concentrations of MMPs in serum might emanate from a circulating cell-type associated with a pathological condition. Moreover, recognizing that serum concentrations of MMPs have been assayed in disease states to identify clinically relevant or prognostic biomarkers of disease activation (10), we would suggest that both age-appropriate and specimen-appropriate reference ranges (or control data) be used in the interpretation of MMP concentrations in biological fluids. Finally, in our laboratory, Fluorokine® MultiAnalyte Profiling (F-MAP) methods have been used effectively for the simultaneous measurement of multiple MMPs in various biological fluids, including plasma, serum and urine (data not shown) and these measurements and their interrelationships may provide useful information. To demonstrate that differences in plasma vs. serum MMP-9 concentrations cannot necessarily be generalized to all MMPs, we offer some additional clinical data. We have recently assayed paired plasma (potassium EDTA BD Vacutainers®) and serum (“redtop” BD Vacutainers® with clot activator and silicon coated) specimens for MMP-1, -2, -3, -8 and -9 using F-MAP kits (3). Specimens were collected during morning hours in the fasting state from 29 healthy volunteers. The experimental protocol governing collection of these specimens was approved by the Institutional Review Board of the University of Arkansas for Medical Sciences, and informed written consent was obtained from all participants. Samples were stored at −20°C until batch analysis was performed. The relationship between paired plasma and serum values was examined using Pearson linear correlation and linear regression analysis. The computation of a confidence interval for Pearsons correlation coefficient (r) was conducted using Fishers z transformation since the sampling distribution of r was not normally distributed. Students t-test was used to compare differences between group means and such data are presented as the mean±SEM. Subjects in this cohort ranged in age from 14 to 41 years, with a mean of 21.7±6.4 years. The study population included 59% males and 90% Caucasian individuals. As noted in Table 1, plasma values were significantly related to serum values for MMP-2, MMP-3, MMP-8 and MMP-9. The correlation was strongest for MMP-2 and MMP-3. In addition, these relationships were strongest when the analysis was restricted to subjects ≥18 years of age, consistent with our original report that MMP-2 and MMP-3 values are age-dependent (3) (Table 1). Only for MMP-1 were plasma and serum values not related. For MMP-2, there was also no statistically significant difference between the group mean values for MMP-2 in plasma and MMP-2 in serum (140,100.2±21,721.8 vs. 159,974.4±14,772.5 pg/mL, respectively; p=0.45). For MMP-3, -8, and -9, mean serum values were consistently between two- and four-fold higher than plasma values. Mean values of MMP-1 in serum were approximately 14-fold higher than plasma MMP-1 concentrations. These data suggest that with the relatively high correlation for plasma and serum MMP-2 and MMP-3 (using a scaling factor for MMP-3), analysis of serum samples may provide similarly pertinent clinical information to the analysis of plasma samples. Table 1 Correlation of plasma and serum values for MMPs.

Effects of Type 1 Diabetes on Osteoblasts, Osteocytes, and Osteoclasts

Purpose of ReviewTo describe the effects of type 1 diabetes on bone cells.Recent findingsType 1 diabetes (T1D) is associated with low bone mineral density, increased risk of fractures, and poor fracture healing. Its effects on the skeleton were primarily attributed to impaired bone formation, but recent data suggests that bone remodeling and resorption are also compromised. The hyperglycemic and inflammatory environment associated with T1D impacts osteoblasts, osteocytes, and osteoclasts. The mechanisms involved are complex; insulinopenia, pro-inflammatory cytokine production, and alterations in gene expression are a few of the contributing factors leading to poor osteoblast activity and survival and, therefore, poor bone formation. In addition, the observed sclerostin level increase accompanied by decreased osteocyte number and enhanced osteoclast activity in T1D results in uncoupling of bone remodeling.SummaryT1D negatively impacts osteoblasts and osteocytes, whereas its effects on osteoclasts are not well characterized, although the limited studies available indicate increased osteoclast activity, favoring bone resorption.

SGLT2 inhibitor therapy improves blood glucose but does not prevent diabetic bone disease in diabetic DBA/2J male mice

Persons with type 1 and type 2 diabetes have increased fracture risk, attributed to deficits in the microarchitecture and strength of diabetic bone, thought to be mediated, in part, by the consequences of chronic hyperglycemia. Therefore, to examine the effects of a glucose-lowering SGLT2 inhibitor on blood glucose (BG) and bone homeostasis in a model of diabetic bone disease, male DBA/2J mice with or without streptozotocin (STZ)-induced hyperglycemia were fed chow containing the SGLT2 inhibitor, canagliflozin (CANA), or chow without drug, for 10weeks of therapy. Thereafter, serum bone biomarkers were measured, fracture resistance of cortical bone was assessed by μCT analysis and a three-point bending test of the femur, and vertebral bone strength was determined by compression testing. In the femur metaphysis and L6 vertebra, long-term diabetes (DM) induced deficits in trabecular bone microarchitecture. In the femur diaphysis, a decrease in cortical bone area, cortical thickness and minimal moment of inertia occurred in DM (p<0.0001, for all) while cortical porosity was increased (p<0.0001). These DM changes were associated with reduced fracture resistance (decreased material strength and toughness; decreased structural strength and rigidity; p<0.001 for all). Significant increases in PTH (p<0.0001), RatLAPs (p=0.0002), and urine calcium concentration (p<0.0001) were also seen in DM. Canagliflozin treatment improved BG in DM mice by ~35%, but did not improve microarchitectural parameters. Instead, in canagliflozin-treated diabetic mice, a further increase in RatLAPs was evident, possibly suggesting a drug-related intensification of bone resorption. Additionally, detrimental metaphyseal changes were noted in canagliflozin-treated control mice. Hence, diabetic bone disease was not favorably affected by canagliflozin treatment, perhaps due to insufficient glycemic improvement. Instead, in control mice, long-term exposure to SGLT2 inhibition was associated with adverse effects on the trabecular compartment of bone.

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.

Osteo-promoting effects of insulin-like growth factor I (IGF-I) in a mouse model of type 1 diabetes☆

OBJECTIVE Using a streptozotocin (STZ)-induced mouse model of type 1 diabetes (T1D), we have previously demonstrated that long-term diabetes inhibits regenerative bone formation during tibial distraction osteogenesis (DO) and perturbs skeletal integrity by decreasing cortical thickness, bone mineral density and bones resistance to fracture. Because long-standing T1D is also associated with a deficiency of insulin-like growth factor I (IGF-I), we examined the effects of systemic IGF-I treatment on skeletal microarchitecture and strength, as well as on bone formation in diabetic mice. RESEARCH DESIGN AND METHODS Streptozotocin-induced diabetic or control mice were treated with recombinant human IGF-I (rhIGF-I, 1.5mg/kg/day as subcutaneous infusion) or vehicle throughout a 14day DO procedure. Thereafter, trunk blood was assayed for glucose, insulin, rhIGF-I, mouse IGF-I and leptin. Bone formation in distracted tibiae was quantified. Effects on cortical bone strength and trabecular bone architecture were assessed by μCT analysis and three-point bend testing of contralateral femurs. RESULTS New bone formation during DO was reduced in diabetic mice but significantly improved with rhIGF-I treatment. The contralateral femurs of diabetic mice demonstrated significant reductions in trabecular thickness, yield strength and peak force of cortical bone, which were improved with rhIGF-I treatment. rhIGF-I also reduced intracortical porosity in control mice. However, treatment with rhIGF-I did not normalize serum glucose, or correct concurrent deficiencies of insulin or leptin seen in diabetes. CONCLUSIONS These findings demonstrate that despite persistent hyperglycemia, rhIGF-I promoted new bone formation and improved biomechanical properties of bone in a model of T1D, suggesting that it may be useful as a fracture preventative in this disease.