Impact of an SGLT2-loss of function mutation on renal architecture, histology, and glucose homeostasis.

Inhibitors of sodium/glucose co-transporter 2 (SGLT2) are currently in clinical use for type 2 diabetes (T2D) treatment due to their anti-hyperglycemic effect exerted by the inhibition of glucose reabsorption in the kidney. Inhibition of SGLT2 is associated with improvement of renal outcomes in chronic kidney disease associated with T2D. Our study aimed to describe the renal-specific phenotypic consequences of the SGLT2-loss of function "Jimbee" mutation within the Slc5a2 mouse gene in a non-diabetic/non-obese background. The Jimbee mice displayed reduced body weight, glucosuria, polyuria, polydipsia, and hyperphagia but were normoglycemic, with no signs of baseline insulin resistance or renal dysfunction. Histomorphological analysis of the kidneys revealed a normal architecture and morphology of the renal cortex, but shrinkage of the glomerular and tubular apparatus, including Bowman's space, glomerular tuft, mesangial matrix fraction, and proximal convoluted tubule (PCT). Immunofluorescent analysis of renal sections showed that SGLT2 was absent from the apical membrane of PCT of the Jimbee mice but remnant positive vesicles were detected within the cytosol or at the perinuclear interface. Renal localization and abundance of GLUT1, GLUT2, and SGLT1 were unchanged in the Jimbee genotype. Intriguingly, the mutation did not induce hepatic gluconeogenic gene expression in overnight fasted mice despite a high glucose excretion rate. The Jimbee phenotype is remarkably similar to humans with SLC5A2 mutations and provides a useful model for the study of SGLT2-loss of function effects on renal architecture and physiology, as well as for identifying possible novel roles for the kidneys in glucose homeostasis and metabolic reprogramming.

Diabetes pharmacotherapy and effects on the musculoskeletal system.

Persons with type 1 or type 2 diabetes have a significantly higher fracture risk than age-matched persons without diabetes, attributed to disease-specific deficits in the microarchitecture and material properties of bone tissue. Therefore, independent effects of diabetes drugs on skeletal integrity are vitally important. Studies of incretin-based therapies have shown divergent effects of different agents on fracture risk, including detrimental, beneficial, and neutral effects. The sulfonylurea class of drugs, owing to its hypoglycemic potential, is thought to amplify the risk of fall-related fractures, particularly in the elderly. Other agents such as the biguanides may, in fact, be osteo-anabolic. In contrast, despite similarly expected anabolic properties of insulin, data suggests that insulin pharmacotherapy itself, particularly in type 2 diabetes, may be a risk factor for fracture, negatively associated with determinants of bone quality and bone strength. Finally, sodium-dependent glucose co-transporter 2 inhibitors have been associated with an increased risk of atypical fractures in select populations, and possibly with an increase in lower extremity amputation with specific SGLT2I drugs. The role of skeletal muscle, as a potential mediator and determinant of bone quality, is also a relevant area of exploration. Currently, data regarding the impact of glucose lowering medications on diabetes-related muscle atrophy is more limited, although preclinical studies suggest that various hypoglycemic agents may have either aggravating (sulfonylureas, glinides) or repairing (thiazolidinediones, biguanides, incretins) effects on skeletal muscle atrophy, thereby influencing bone quality. Hence, the therapeutic efficacy of each hypoglycemic agent must also be evaluated in light of its impact, alone or in combination, on musculoskeletal health, when determining an individualized treatment approach. Moreover, the effect of newer medications (potentially seeking expanded clinical indication into the pediatric age range) on the growing skeleton is largely unknown. Herein, we review the available literature regarding effects of diabetes pharmacotherapy, by drug class and/or by clinical indication, on the musculoskeletal health of persons with diabetes.

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

PURPOSE OF REVIEW: To describe the effects of type 1 diabetes on bone cells. RECENT FINDINGS: Type 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. T1D 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.

The role of vitamin D in the metabolic homeostasis of diabetic bone.

Most studies across a variety of geographic locations suggest that vitamin D insufficiency is more common in individuals with type 1 diabetes (T1D) compared to the general population. In type 2 diabetes (T2D), while obesity is commonplace and lower vitamin D levels are present in obese adolescents and adults, the association between vitamin D insufficiency and T2D is less clear. Studies suggest that the relationship between T2D and vitamin D may be concurrently influenced by ethnicity, geography, BMI and age. None-the-less, diabetic osteopathy is a significant co-morbidity of both forms of diabetes, and is characterized by micro-architectural changes that decrease bone quality leading to an increased risk for bone fracture in both disorders. The question remains, however, to what degree vitamin D homeostasis contributes to or exacerbates skeletal pathology in diabetes. Proposed mechanisms for vitamin D deficiency in diabetes include: 1) genetic predisposition (T1D); 2) increased BMI (T2D); 3) concurrent albuminuria (T1D or T2D); or 4) exaggerated renal excretion of vitamin D metabolites or vitamin D binding protein (T1D, T2D, animal models). The specific effects of vitamin D treatment on diabetic osteoporosis have been examined in rodents, and demonstrate skeletal improvements even in the face of untreated diabetes. However, human clinical trial data examining whether vitamin D status can be directly related to or is predictive of bone quality and fracture risk in those with diabetes is still needed. Herein, we provide a review of the literature linking vitamin D, diabetes and skeletal health.

Biomechanical Stimulation of Muscle Constructs Influences Phenotype of Bone Constructs by Modulating Myokine Secretion.

Diabetes is a chronic metabolic disorder that can lead to diabetic myopathy and bone diseases. The etiology of musculoskeletal complications in such metabolic disorders and the interplay between the muscular and osseous systems are not well understood. Exercise training promises to prevent diabetic myopathy and bone disease and offer protection. Although the muscle-bone interaction is largely biomechanical, the muscle secretome has significant implications for bone biology. Uncoupling effects of biophysical and biochemical stimuli on the adaptive response of bone during exercise training may offer therapeutic targets for diabetic bone disease. Here, we have developed an in vitro model to elucidate the effects of mechanical strain on myokine secretion and its impact on bone metabolism decoupled from physical stimuli. We developed bone constructs using cross-linked gelatin, which facilitated osteogenic differentiation of osteoprogenitor cells. Then muscle constructs were made from fibrin, which enabled myoblast differentiation and myotube formation. We investigated the myokine expression by muscle constructs under strain regimens replicating endurance (END) and high-intensity interval training (HIIT) in hyperglycemic conditions. In monocultures, both regimens induced higher expression of Il15 and Igf1, whereas END supported more myoblast differentiation and myotube maturation than HIIT. When co-cultured with bone constructs, HIIT regimen increased Glut4 expression in muscle constructs more than END, supporting higher glucose uptake. Likewise, the muscle constructs under the HIIT regimen promoted a healthier and more matured bone phenotype than END. Under static conditions, myostatin (Mstn) expression was significantly downregulated in muscle constructs co-cultured with bone constructs compared with monocultures. Together, our in vitro co-culture system allowed orthogonal manipulation of mechanical strain on muscle constructs while facilitating bone-muscle biochemical cross-talk. Such systems can provide an individualized microenvironment that allows decoupled biomechanical manipulation, help identify molecular targets, and develop engineered therapies for metabolic bone disease. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Pharmacologic Inhibition of Myostatin With a Myostatin Antibody Improves the Skeletal Muscle and Bone Phenotype of Male Insulin-Deficient Diabetic Mice.

Type 1 diabetes (T1D) is associated with low bone and muscle mass, increased fracture risk, and impaired skeletal muscle function. Myostatin, a myokine that is systemically elevated in humans with T1D, negatively regulates muscle mass and bone formation. We investigated whether pharmacologic myostatin inhibition in a mouse model of insulin-deficient, streptozotocin (STZ)-induced diabetes is protective for bone and skeletal muscle. DBA/2J male mice were injected with low-dose STZ (diabetic) or vehicle (non-diabetic). Subsequently, insulin or palmitate Linbits were implanted and myostatin (REGN647-MyoAb) or control (REGN1945-ConAb) antibody was administered for 8 weeks. Body composition and contractile muscle function were assessed in vivo. Systemic myostatin, P1NP, CTX-I, and glycated hemoglobin (HbA1c) were quantified, and gastrocnemii were weighed and analyzed for muscle fiber composition and gene expression of selected genes. Cortical and trabecular parameters were analyzed (micro-computed tomography evaluations of femur) and cortical bone strength was assessed (three-point bending test of femur diaphysis). In diabetic mice, the combination of insulin/MyoAb treatment resulted in significantly higher lean mass and gastrocnemius weight compared with MyoAb or insulin treatment alone. Similarly, higher raw torque was observed in skeletal muscle of insulin/MyoAb-treated diabetic mice compared with MyoAb or insulin treatment. Additionally, muscle fiber cross-sectional area (CSA) was lower with diabetes and the combination treatment with insulin/MyoAb significantly improved CSA in type II fibers. Insulin, MyoAb, or insulin/MyoAb treatment improved several parameters of trabecular architecture (eg, bone volume fraction [BV/TV], trabecular connectivity density [Conn.D]) and cortical structure (eg, cortical bone area [Ct. Ar.], minimum moment of inertia [Imin]) in diabetic mice. Lastly, cortical bone biomechanical properties (stiffness and yield force) were also improved with insulin or MyoAb treatment. In conclusion, pharmacologic myostatin inhibition is beneficial for muscle mass, muscle function, and bone properties in this mouse model of T1D and its effects are both independent and additive to the positive effects of insulin. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Mouse models of type 1 diabetes and their use in skeletal research.

PURPOSE OF REVIEW: In this review, we describe the three primary mouse models of insulin-deficiency diabetes that have been used to study the effects of type 1 diabetes (T1D) on skeletal outcomes. These models include streptozotocin (chemically)-induced diabetes, autoimmune-mediated diabetes (the nonobese diabetes mouse), and a mutation in the insulin gene (the Akita mouse). We then describe the skeletal findings and/or skeletal phenotypes that have been delineated using these models. RECENT FINDINGS: Humans with T1D have decreased bone mineral density and an increased risk for fragility fracture. Mouse models of insulin-deficiency diabetes (hereafter denoted as T1D) in many ways recapitulate these skeletal deficits. Utilizing techniques of microcomputed tomography, bone histomorphometry, biomechanical testing and fracture modeling, bone biomarker analysis, and Raman spectroscopy, mouse models of T1D have demonstrated abnormalities in bone mineralization, bone microarchitecture, osteoblast function, abnormal bone turnover, and diminished biomechanical properties of bone. SUMMARY: Mouse models have provided significant insights into the underlying mechanisms involved in the abnormalities of bone observed in T1D in humans. These translational models have provided targets and pathways that may be modifiable to prevent skeletal complications of T1D.

Emerging therapies for the treatment of rare pediatric bone disorders.

In recent years, new therapies for the treatment of rare pediatric bone disorders have emerged, guided by an increasing understanding of the genetic and molecular etiology of these diseases. Herein, we review three such disorders, impacted by debilitating deficits in bone mineralization or cartilage ossification, as well as the novel disease-modifying drugs that are now available to treat these conditions. Specifically, we discuss asfotase alfa, burosumab-twza, and vosoritide, for the treatment of hypophosphatasia, X-linked hypophosphatemia and achondroplasia, respectively. For each skeletal disorder, an overview of the clinical phenotype and natural history of disease is provided, along with a discussion of the clinical pharmacology, mechanism of action and FDA indication for the relevant medication. In each case, a brief review of clinical trial data supporting drug development for each medication is provided. Additionally, guidance as to drug dosing and long-term monitoring of adverse events and pediatric efficacy is presented, to aid the clinician seeking to utilize these novel therapies in their practice, or to become familiar with the healthcare expectations for children receiving these medications through specialized multidisciplinary clinics. The availability of these targeted therapies now significantly augments treatment options for conditions in which past therapy has relied upon less specific, symptomatic medical and orthopedic care.

Heterogeneity and altered ß-cell identity in the TallyHo model of early-onset type 2 diabetes.

A primary underlying defect makes ß-cells "susceptible" to no longer compensate for the peripheral insulin resistance and to trigger the onset of type 2 diabetes (T2D). New evidence suggests that in T2D, ß-cells are not destroyed but experience a loss of identity, reverting to a progenitor-like state and largely losing the ability to sense glucose and produce insulin. We assessed (using fluorescence microscopy and histomorphometry correlated with the glycaemic status) the main ß-cell identity modifications as diabetes progresses in the TallyHo/JngJ (TH) male mice, a polygenic model of spontaneous T2D, akin to the human phenotype. We found that: 1) conversion to overt diabetes is paralleled by a progressive reduction of insulin-expressing cells and expansion of a glucagon-positive population, together with alteration of islet size and shape; 2) the ß-cell population is highly heterogeneous in terms of insulin content and specific transcription factors like PDX1 and NKX6.1, that are gradually lost during diabetes progression; 3) GLUT2 expression is altered early and strongly reduced at late stages of diabetes; 4) an endocrine developmental program dependent on NGN3-expressing progenitors is revived when hyperglycaemia becomes severe; and 5) the re-expression of the EMT-associated factor vimentin occurs as diabetes worsens, representing a possible regenerative response to ß-cell loss. Based on these results, we formulated additional hypotheses for the ß-cell identity alteration in the TH model, together with several limitations of the study, that constitute future research directions.

RASopathies: The musculoskeletal consequences and their etiology and pathogenesis.

The RASopathies comprise an ever-growing number of clinical syndromes resulting from germline mutations in components of the RAS/MAPK signaling pathway. While multiple organs and tissues may be affected by these mutations, this review will focus on how these mutations specifically impact the musculoskeletal system. Herein, we review the genetics and musculoskeletal phenotypes of these syndromes in humans. We discuss how mutations in the RASopathy syndromes have been studied in translational mouse models. Finally, we discuss how signaling molecules within the RAS/MAPK pathway are involved in normal and abnormal bone biology in the context of osteoblasts, osteoclasts and chondrocytes.

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

BACKGROUND: 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. METHODS: 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 t-test was used with a significance level of p < 0.05 to determine significance between treatment groups. RESULTS: Esterification of palmitate with coenzyme A (CoA) was necessary, while ß-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. CONCLUSIONS: 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 ß-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.

Canagliflozin, an SGLT2 inhibitor, corrects glycemic dysregulation in TallyHO model of T2D but only partially prevents bone deficits.

Higher fracture risk in type 2 diabetes (T2D) is attributed to disease-specific deficits in micro-structural and material properties of bone, although the primary cause is not yet established. The TallyHO (TH) mouse is a polygenic model of early-onset T2D and obesity analogous to adolescent-onset T2D in humans. Due to incomplete penetrance of the phenotype, ~25% of male TH mice never develop hyperglycemia, providing a strain-matched, non-diabetic control. Utilizing this model of T2D, we examined the impact of glucose-lowering therapy with canagliflozin (CANA) on diabetic bone. Male TH mice with or without hyperglycemia (High BG, Low BG) were monitored from ~8 to 20 weeks of age, and compared to age-matched, male, TH mice treated with CANA from ~8 to 20 weeks of age. At 20 weeks, untreated TH mice with high BG [High BG: 687 ± 106 mg/dL] exhibited lower body mass, decrements in cortical bone of the femur (decreased cross-sectional area and thickness; increased porosity) and in trabecular bone of the femur metaphysis and L6 vertebra (decreased bone volume fraction, thickness, and tissue mineral density), as well as decrements in cortical and vertebral bone strength (decreased yield force and ultimate force) when compared to untreated TH mice with low BG [Low BG: 290 ± 98 mg/dL; p < 0.0001]. CANA treatment was metabolically advantageous, normalizing body mass, BG and HbA1c to values comparable to the Low BG group. With drug-induced glycemic improvement, cortical area and thickness were significantly higher in the CANA than in the High BG group, but deficits in strength persisted with lower yield force and yield stress (partially independent of bone geometry) in the CANA group. Additionally, CANA only partially prevented the T2D-related loss in trabecular bone volume fraction. Taken together, these findings suggest that the ability of CANA to lower glucose and normalized glycemic control ameliorates diabetic bone disease but not fully.

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.

Constitutive activation of MEK1 in osteoprogenitors increases strength of bone despite impairing mineralization.

Recent clinical studies have revealed that a somatic mutation in MAP2K1, causing constitutive activation of MEK1 in osteogenic cells, occurs in melorheostotic bone disease in humans. We have generated a mouse model which expresses an activated form of MEK1 (MEK1DD) specifically in osteoprogenitors postnatally. The skeletal phenotype of these mice recapitulates many features of melorheostosis observed in humans, including extra-cortical bone formation, abundant osteoid formation, decreased mineral density, and increased porosity. Paradoxically, in both humans and mice, MEK1 activation in osteoprogenitors results in bone that is not structurally compromised, but is hardened and stronger, which would not be predicted based on tissue and matrix properties. Thus, a specific activating mutation in MEK1, expressed only by osteoprogenitors postnatally, can have a significant impact on bone strength through complex alterations in whole bone geometry, bone micro-structure, and bone matrix.

Primary Care Cluster RCT to Increase Diabetes Prevention Program Referrals.

INTRODUCTION: The Diabetes Prevention Program, an intensive lifestyle change program, effectively reduces the risk of progression from prediabetes to type 2 diabetes but is underutilized. An implementation study using formative research was undertaken to increase Diabetes Prevention Program referrals at a primary care clinic. STUDY DESIGN: A pragmatic, cluster randomized, mixed-methods study. SETTING/PARTICPANTS: Clusters were teams of primary care clinicians from 2 primary care clinics. The 3 intervention clusters had 8-11 clinicians, and the 3 control clusters had 7-20 clinicians. INTERVENTION: Implementation activities occurred from December 2017 to February 2019. The activities included targeted clinician education, a prediabetes clinician champion, and a custom electronic health record report identifying patients with prediabetes. MAIN OUTCOME MEASURES: The primary outcome was referral of patients with prediabetes to the institutional Diabetes Prevention Program. Study data, including patient demographic and clinical variables, came from electronic health record. Interviews with clinicians evaluated the implementation strategies. Generalized estimating equation analyses that accounted for multiple levels of correlation and interview content analysis occurred in 2019. RESULTS: Study clinicians cared for 2,992 patients with a prediabetes diagnosis or HbA1c indicative of prediabetes (5.7%-6.4%). Clinicians in the intervention clusters referred 6.9% (87 of 1,262) of patients with prediabetes to the Diabetes Prevention Program and those in the control clusters referred 1.5% (26 of 1,730). When adjusted for patient age, sex, race, HbA1c value, HbA1c test location, and insurance type, intervention clinicians had 3.85 (95% CI=0.40, 36.78) greater odds of referring a patient with prediabetes to the Diabetes Prevention Program. The 11 interviewed intervention clinicians had mixed opinions about the utility of the interventions, reporting the prediabetes clinic champion (n=7, 64%) and educational presentations (n=6, 55%) as most helpful. CONCLUSIONS: Intervention clinicians were more likely to make Diabetes Prevention Program referrals; however, the study lacked power to achieve statistical significance. Clinician interviews suggested that intervention components that triggered Diabetes Prevention Program referrals varied among clinicians.

Genetic ablation of SGLT2 function in mice impairs tissue mineral density but does not affect fracture resistance of bone.

Selective sodium-dependent glucose co-transporter 2 inhibitors (SGLT2Is) are oral hypoglycemic medications utilized increasingly in the medical management of hyperglycemia among persons with type 2 diabetes (T2D). Despite favorable effects on cardiovascular events, specific SGLT2Is have been associated with an increased risk for atypical fracture and amputation in subgroups of the T2D population, a population that already has a higher risk for typical fragility fractures than the general population. To better understand the effect of SGLT2 blockade on skeletal integrity, independent of diabetes and its co-morbidities, we utilized the "Jimbee" mouse model of slc5a2 gene mutation to investigate the impact of lifelong SGLT2 loss-of-function on metabolic and skeletal phenotype. Jimbee mice maintained normal glucose homeostasis, but exhibited chronic polyuria, glucosuria and hypercalciuria. The Jimbee mutation negatively impacted appendicular growth of the femur and resulted in lower tissue mineral density of both cortical and trabecular bone of the femur mid-shaft and distal femur metaphysis, respectively. Several components of the Jimbee phenotype were characteristic only of male mice compared with female mice, including reductions: in body weight; in cortical area of the mid-shaft; and in trabecular thickness within the metaphysis. Despite these decrements, the strength of femur diaphysis in bending (cortical bone), which increased with age, and the strength of L6 vertebra in compression (primarily trabecular bone), which decreased with age, were not affected by the mutation. Moreover, the age-related decline in bone toughness was less for Jimbee mice, compared with control mice, such that by 49-50 weeks of age, Jimbee mice had significantly tougher femurs in bending than C57BL/6J mice. These results suggest that chronic blockade of SGLT2 in this model reduces the mineralization of bone but does not reduce its fracture resistance.

Runt-related transcription factor 2 (RUNX2) and RUNX2-related osteogenic genes are down-regulated throughout osteogenesis in type 1 diabetes mellitus.

Type 1 diabetes mellitus is associated with a number of disorders of skeletal health, conditions that rely, in part, on dynamic bone formation. A mouse model of distraction osteogenesis was used to study the consequences of streptozotocin-induced diabetes and insulin treatment on bone formation and osteoblastogenesis. In diabetic mice compared with control mice, new bone formation was decreased, and adipogenesis was increased in and around, respectively, the distraction gaps. Although insulin treatment restored bone formation to levels observed in nondiabetic control mice, it failed to significantly decrease adipogenesis. Molecular events altered during de novo bone formation in untreated type 1 diabetes mellitus, yet restored with insulin treatment were examined so as to clarify specific osteogenic genes that may contribute to diabetic bone disease. RNA from distraction gaps was analyzed by gene microarray and quantitative RT-PCR for osteogenic genes of interest. Runt-related transcription factor 2 (RUNX2), and several RUNX2 target genes, including matrix metalloproteinase-9, Akp2, integrin binding sialoprotein, Dmp1, Col1a2, Phex, Vdr, osteocalcin, and osterix, were all significantly down-regulated in the insulin-deficient, hyperglycemic diabetic animals; however, insulin treatment of diabetic animals significantly restored their expression. Expression of bone morphogenic protein-2, transcriptional coactivator with PDZ-binding motif, and TWIST2, all important regulators of RUNX2, were not impacted by the diabetic condition, suggesting that the defect in osteogenesis resides at the level of RUNX2 expression and its activity. Together, these data demonstrate that insulin and/or glycemic status can regulate osteogenesis in vivo, and systemic insulin therapy can, in large part, rescue the diabetic bone phenotype at the tissue and molecular level.

Advances in micro- and nanotechnologies for the GLP-1-based therapy and imaging of pancreatic beta-cells.

Therapies to prevent diabetes in particular the progressive loss of ß-cell mass and function and/or to improve the dysregulated metabolism associated with diabetes are highly sought. The incretin-based therapy comprising GLP-1R agonists and DPP-4 inhibitors have represented a major focus of pharmaceutical R&D over the last decade. The incretin hormone GLP-1 has powerful antihyperglycemic effect through direct stimulation of insulin biosynthesis and secretion within the ß-cells; it normalizes ß-cell sensitivity to glucose, has an antiapoptotic role, stimulates ß-cell proliferation and differentiation, and inhibits glucagon secretion. However, native GLP-1 therapy is inappropriate due to the rapid post-secretory inactivation by DPP-4. Therefore, incretin mimetics developed on the backbone of the GLP-1 or exendin-4 molecule have been developed to behave as GLP-1R agonists but to display improved stability and clinical efficacy. New formulations of incretins and their analogs based on micro- and nanomaterials (i.e., PEG, PLGA, chitosan, liposomes and silica) and innovative encapsulation strategies have emerged to achieve a better stability of the incretin, to improve its pharmacokinetic profile, to lower the administration frequency or to allow another administration route and to display fewer adverse effects. An important advantage of these formulations is that they can also be used at the targeted non-invasive imaging of the beta-cell mass. This review therefore focuses on the current state of these efforts as the next step in the therapeutic evolution of this class of antidiabetic drugs.

Exploring the interface between metallo-proteinase activity and growth factor and cytokine bioavailability.

Metallo-proteinases are implicated in many processes involved in tissue remodeling, cell motility, morphogenesis, and cell and organ growth and differentiation. Recent data suggest that several members of the metzincin family including the matrix metallo-proteinases (MMPs), adamalysin-related proteinases, and the newly described pappalysins, are intimately involved in the activation and/or release of cytokines and growth factors. We review how metzincins, working through unique mechanisms, influence the extracellular milieu of several important cytokines and growth factors including transforming growth factor-beta (TGF-beta), TNF-alpha, IGFs and HB-epidermal growth factor (EGF). Because metzincins can modulate the bioavailability of these peptides, they may serve as unique target molecules to control cytokine and growth factor action in the extracellular environment.

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.