Hypohalous Acids Contribute to Renal Extracellular Matrix Damage in Experimental Diabetes

Authors

Kyle L. Brown, Carl Darris, Kristie Lindsey Rose, Otto A. Sanchez, Hartman Madu, Josh Avance, Nickolas Brooks, Ming-Zhi Zhang, Agnes Fogo, Raymond Harris, Billy G. Hudson and Paul Voziyan

Abstract

In diabetes, toxic oxidative pathways are triggered by persistent hyperglycemia and contribute to diabetic complications. A major proposed pathogenic mechanism is accumulation of protein modifications called advanced glycation end products (AGEs). However, other non-enzymatic post-translational modifications may also contribute to pathogenic protein damage in diabetes. We demonstrate that hypohalous acid-derived modifications of renal tissues and extracellular matrix (ECM) proteins are significantly elevated in experimental diabetic nephropathy. Moreover, diabetic renal ECM shows diminished binding of α1β1 integrin consistent with modification of collagen IV by hypochlorous (HOCl) and hypobromous (HOBr) acids. NC1 hexamers, key connection modules of collagen IV networks, are modified via oxidation and chlorination of tryptophan and bromination of tyrosine residues. Chlorotryptophan, a relatively minor modification, has not been previously found in proteins. In the NC1 hexamers isolated from diabetic kidneys, levels of HOCl-derived oxidized and chlorinated tryptophan residues W28 and W192 are significantly elevated compared to non-diabetic controls. Molecular dynamics simulations predicted more relaxed NC1 hexamer tertiary structure and diminished assembly competence in diabetes; this was confirmed using limited proteolysis and denaturation/refolding. Our results suggest that hypohalous acid-derived modifications of renal ECM and specifically collagen IV networks contribute to functional protein damage in diabetes.

Link To Article

http://dx.doi.org/10.2337/db14-1001

An insulin-sensitizing thiazolidinedione, which minimally activates PPARγ, does not cause bone loss

Authors

Tomohiro Fukunaga, Wei Zou, Nidhi Rohatgi, Jerry R. Colca and Steven L. Teitelbaum

Abstract

Rosiglitazone is an insulin-sensitizing thiazolidinedione (TZD) which activates the transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ). While rosiglitazone effectively treats type II diabetes mellitus (T2DM), it carries substantial complications including increased fracture risk. This predisposition to fracture is consistent with the fact that PPARγ preferentially promotes formation of adipocytes at the cost of osteoblasts. Rosiglitazone-activated PPARγ, however, also stimulates osteoclast formation. A new TZD analog with low affinity for binding and activation of PPARγ but whose insulin-sensitizing properties mirror those of rosiglitazone, has been recently developed. Because of its therapeutic implications, we investigated the effects of this new TZD analog (MSDC-0602) on skeletal homeostasis, in vitro and in vivo. Confirming it activates the nuclear receptor in osteoclasts, rosiglitazone enhances expression of the PPARγ target gene, CD36. MSDC-0602, in contrast, minimally activates PPARγ and does not alter CD36 expression in the bone resorptive cells. Consistent with this finding, rosiglitazone increases RANKL-induced osteoclast differentiation and number whereas MSDC-0602 fails to do. To determine if this new TZD analog is bone sparing, in vivo, we fed adult male C57BL/6 mice MSDC-0602 or rosiglitazone. 6-months of a rosiglitazone diet results in a 35% decrease in bone mass with increased number of osteoclasts whereas that of MSDC-0602 fed mice is indistinguishable from control. Thus PPARγ-sparing eliminates the skeletal side effects of TZDs while maintaining their insulin-sensitizing properties.

Link To Article

http://dx.doi.org/10.1002/jbmr.2364

Raloxifene Prevents Skeletal Fragility in Adult Female Zucker Diabetic Sprague-Dawley Rats

Authors

Kathleen M. Hill Gallant, Maxime A. Gallant, Drew M. Brown, Amy Y. Sato, Justin N. Williams, David B. Burr

Abstract

Fracture risk in type 2 diabetes is increased despite normal or high bone mineral density, implicating poor bone quality as a risk factor. Raloxifene improves bone material and mechanical properties independent of bone mineral density. This study aimed to determine if raloxifene prevents the negative effects of diabetes on skeletal fragility in diabetes-prone rats. Adult Zucker Diabetic Sprague-Dawley (ZDSD) female rats (20-week-old, n = 24) were fed a diabetogenic high-fat diet and were randomized to receive daily subcutaneous injections of raloxifene or vehicle for 12 weeks. Blood glucose was measured weekly and glycated hemoglobin was measured at baseline and 12 weeks. At sacrifice, femora and lumbar vertebrae were harvested for imaging and mechanical testing. Raloxifene-treated rats had a lower incidence of type 2 diabetes compared with vehicle-treated rats. In addition, raloxifene-treated rats had blood glucose levels significantly lower than both diabetic vehicle-treated rats as well as vehicle-treated rats that did not become diabetic. Femoral toughness was greater in raloxifene-treated rats compared with both diabetic and non-diabetic vehicle-treated ZDSD rats, due to greater energy absorption in the post-yield region of the stress-strain curve. Similar differences between groups were observed for the structural (extrinsic) mechanical properties of energy-to-failure, post-yield energy-to-failure, and post-yield displacement. These results show that raloxifene is beneficial in preventing the onset of diabetes and improving bone material properties in the diabetes-prone ZDSD rat. This presents unique therapeutic potential for raloxifene in preserving bone quality in diabetes as well as in diabetes prevention, if these results can be supported by future experimental and clinical studies.

Link To Article

http://dx.doi.org/10.1371/journal.pone.0108262

Effect of isolated hyperglycemia on native mechanical and biologic shoulder joint properties in a rat model

Authors

Stephen J. Thomas, Joseph J. Sarver, Sarah M. Yannascoli, Jennica J. Tucker, John D. Kelly IV, Rexford S. Ahima, Mary F. Barbe and Louis J. Soslowsky

Abstract

Recently, diabetes has been linked to rotator cuff disease and adhesive capsulitis, conditions with increased stiffness and inflammation. Unfortunately, limited research exists examining how hyperglycemia affects the native shoulder (tendon and capsule) properties. Therefore, the objectives of this study were to compare shoulder joint mechanics, tendon properties (mechanics and immunohistochemistry), and capsule of healthy control and hyperglycemic rats 8 weeks following induction of hyperglycemia with a submaximal dose of streptozotocin (STZ). Eighteen rats were injected with STZ to induce hyperglycemia or citrate buffer (control) and underwent normal cage activity for 8 weeks. Passive joint mechanics demonstrated significantly less external rotation in the hyperglycemic group compared to controls, with no other group differences. Tendon mechanical properties (stiffness and modulus) were not significantly different between groups at both the insertion site and mid-substance. Immunohistochemistry staining of the tendon and capsule demonstrated significantly increased interleukin 1-beta (IL1-β) and advanced glycated end-products (AGE) staining localized to the insertion and mid-substance of the tendon but not the capsule. In addition, tumor necrosis factor alpha (TNF-α) staining was significantly increased in the superior capsule but not the supraspinatus tendon. This study demonstrates that isolated hypergylcemia does not diminish shoulder mechanical properties but does induce a chronic inflammatory response.

Link To Article

http://dx.doi.org/10.1002/jor.22695

Palmitic Acid and DGAT1 Deficiency Enhance Osteoclastogenesis while Oleic Acid-Induced Triglyceride Formation Prevents it

Authors

Zoi Drosatos-Tampakaki, Konstantinos Drosatos, Yasemin Siegelin, Shan Gong, Salmiyeh Khan, Thomas Van Dyke, Ira J. Goldberg, P. Christian Schulze, Ulrike Schulze-Späte

Abstract

Both obesity and diabetes mellitus are associated with alterations in lipid metabolism as well as a change in bone homeostasis and osteoclastogenesis. We hypothesized that increased fatty acid levels affect bone health by altering precursor cell differentiation and osteoclast activation. Here we show that palmitic acid (PA, 16:0) enhances RANKL-stimulated osteoclastogenesis and is sufficient to induce osteoclast differentiation even in the absence of RANKL. TNFα expression is crucial for PA-induced osteoclastogenesis as shown by increased TNFα mRNA levels in PA-treated cells and abrogation of PA-stimulated osteoclastogenesis by TNFα neutralizing antibodies. In contrast, oleic acid (OA, 18:1) does not enhance osteoclast differentiation, leads to increased intracellular triglyceride accumulation and inhibits PA-induced osteoclastogenesis. Adenovirus-mediated expression of diacylglycerol acyl transferase (DGAT) 1, a gene involved in triglyceride synthesis, also inhibits PA-induced osteoclastogenesis, suggesting a protective role of DGAT1 for bone health. Accordingly, Dgat1 knockout mice have larger bone marrow-derived osteoclasts and decreased bone mass indices. In line with these findings, mice on a high fat PA-enriched diet have a greater reduction in bone mass and structure than mice on a high fat OA-enriched diet. Thus, we propose that TNFα mediates saturated fatty acid-induced osteoclastogenesis that can be prevented by DGAT activation or supplementation with OA.

Link To Article

http://dx.doi.org/10.1002/jbmr.2150

Streptozotocin-induced diabetes in rats diminishes the size of the osteoprogenitor pool in bone marrow

Authors

E. Weinberg, T. Maymon, O. Moses, M. Weinre

Abstract

Aims

Bone formation is reduced in animals and humans with type 1 diabetes, leading to lower bone mass and inferior osseous healing. Since bone formation greatly depends on the recruitment of osteoblasts from their bone marrow precursors, we tested whether experimental type 1 diabetes in rats diminishes the number of bone marrow osteoprogenitors.

Methods

Diabetes was induced by 65 mg/kg streptozotocin and after 4 weeks, femoral bone marrow cells were extracted and cultured. Tibia and femur were frozen for further analysis.

Results

The size of the osteoprogenitor pool in bone marrow of diabetic rats was significantly reduced, as evidenced by 1) lower (∼35%) fraction of adherent stromal cells (at 24 h of culture); 2) lower (20-25%) alkaline phosphatase activity at 10 days of culture; and 3) lower (∼40%) mineralized nodule formation at 21 days of culture. Administration of insulin to hyperglycemic rats normalized glycemia and abrogated most of the decline in ex-vivo mineralized nodule formation. Apoptotic cells in tibial bone marrow were more numerous in hyperglycemic rats. Also, the levels of malondialdehyde (indicator of oxidative stress) were significantly elevated in bone marrow of diabetic animals.

Conclusions

Experimental type 1 diabetes diminishes the osteoprogenitor population in bone marrow, possibly due to increased apoptosis via Oxidative Stress. Reduced number of osteoprogenitors is likely to impair osteoblastogenesis, bone formation, and bone healing in diabetic animals.

Link to Article

http://dx.doi.org/10.1016/j.diabres.2013.11.015