Lipocalin-2 Deficiency Impairs Thermogenesis and Potentiates Diet-Induced Insulin Resistance in Mice

OBJECTIVE

Lipocalin (LCN) 2 belongs to the lipocalin subfamily of low–molecular mass–secreted proteins that bind small hydrophobic molecules. LCN2 has been recently characterized as an adipose-derived cytokine, and its expression is upregulated in adipose tissue in genetically obese rodents. The objective of this study was to investigate the role of LCN2 in diet-induced insulin resistance and metabolic homeostasis in vivo.

RESEARCH DESIGN AND METHODS

Systemic insulin sensitivity, adaptive thermogenesis, and serum metabolic and lipid profile were assessed in LCN2-deficient mice fed a high-fat diet (HFD) or regular chow diet.

RESULTS

The molecular disruption of LCN2 in mice resulted in significantly potentiated diet-induced obesity, dyslipidemia, fatty liver disease, and insulin resistance. LCN2^−/− mice exhibit impaired adaptive thermogenesis and cold intolerance. Gene expression patterns in white and brown adipose tissue, liver, and muscle indicate that LCN2^−/− mice have increased hepatic gluconeogenesis, decreased mitochondrial oxidative capacity, impaired lipid metabolism, and increased inflammatory state under the HFD condition.

CONCLUSIONS

LCN2 has a novel role in adaptive thermoregulation and diet-induced insulin resistance.Obesity is a major risk for developing insulin resistance, a hallmark of type 2 diabetes and other metabolic complications such as fatty liver, dyslipidemia, and atherosclerosis. Adipose tissue plays a central role in body weight homeostasis, inflammation, and insulin resistance via regulating lipid metabolism/storage and releasing a range of adipokines/cytokines (1–4). Adipose tissue in a variety of insulin-resistant states has been characterized by dysregulated lipid metabolism and altered production of adipokines/cytokines that, in sum, are important contributors to systemic inflammation and related metabolic disorders.Lipocalin (LCN) 2 (also known as neutrophil gelatinase–associated lipocalin [NGAL]), a lipocalin subfamily member, has been recently identified by our group and others (5,6) as an adipose-derived cytokine. LCN2 is a 25-kDa secreted protein initially identified from human neutrophils (7,8) and other immune cells and tissues that are exposed to microorganisms in the respiratory and gastrointestinal tract and is present abundantly in the circulation (9). Interestingly, lipocalins have structural similarity with fatty acid binding proteins (FABPs), and both are members of the multigene family of up and down β-barrel proteins (10). Both the intracellular FABPs and the extracellular lipocalins have a clearly defined β-barrel motif that forms either an interior cavity (FABP) or a deep pit (lipocalins) that constitutes the lipid binding domain (10). The extracellular lipocalins such as LCN2, retinol binding protein (RBP) 4, and α₂-microglobulin use a series of β-strands to form a globular domain with a deep depression resembling the calyx of a flower. Because of the unique structure, the lipocalins function as efficient transporters for a number of different hydrophobic ligands in extracellular milieus, including a variety of retinoids, fatty acids, biliverdin, pheromones, porphyrins, odorants, steroids, and iron. RBP4, one of the extracellular lipocalins, affects glucose metabolism and insulin sensitivity (11).Previous studies have demonstrated that LCN2 gene expression is upregulated in adipose tissue and liver of genetically obese animals (6). Rosiglitazone administration significantly reduces LCN2 expression in adipose tissue in obese animals (6), suggesting that the protein may function as a proinflammatory factor. Unexpectedly, the addition of LCN2 protein to the culture media of adipocytes and macrophages leads to the suppression of tumor necrosis factor (TNF)α- and lipopolysaccharide-induced cytokine/chemokine production, indicating an anti-inflammatory function (6). Most strikingly, LCN2 appears to protect against TNFα-induced insulin resistance in adipocytes. Unlike RBP4, increased production of LCN2 in obesity may be a protective mechanism against inflammation and insulin resistance.To evaluate this hypothesis, we assessed the metabolic and regulatory consequences of LCN2 deficiency. Herein, we show that the ablation of LCN2 profoundly impairs adaptive thermogenesis and exacerbates high-fat diet (HFD)- or age-induced insulin resistance and glucose homeostasis. LCN2-deficient mice have increased hepatic gluconeogenesis and inflammatory state and exhibit a cold sensitive phenotype.     

Sustained Inflammasome Activity in Macrophages Impairs Wound Healing in Type 2 Diabetic Humans and Mice

The hypothesis of this study was that sustained activity of the Nod-like receptor protein (NLRP)-3 inflammasome in wounds of diabetic humans and mice contributes to the persistent inflammatory response and impaired healing characteristic of these wounds. Macrophages (Mp) isolated from wounds on diabetic humans and db/db mice exhibited sustained inflammasome activity associated with low level of expression of endogenous inflammasome inhibitors. Soluble factors in the biochemical milieu of these wounds are sufficient to activate the inflammasome, as wound-conditioned medium activates caspase-1 and induces release of interleukin (IL)-1β and IL-18 in cultured Mp via a reactive oxygen species–mediated pathway. Importantly, inhibiting inflammasome activity in wounds of db/db mice using topical application of pharmacological inhibitors improved healing of these wounds, induced a switch from proinflammatory to healing-associated Mp phenotypes, and increased levels of prohealing growth factors. Furthermore, data generated from bone marrow–transfer experiments from NLRP-3 or caspase-1 knockout to db/db mice indicated that blocking inflammasome activity in bone marrow cells is sufficient to improve healing. Our findings indicate that sustained inflammasome activity in wound Mp contributes to impaired early healing responses of diabetic wounds and that the inflammasome may represent a new therapeutic target for improving healing in diabetic individuals.     

Endostatin Inhibits Callus Remodeling during Fracture Healing in Mice

Information on the impact of endogenous anti‐angiogenic factors on bone repair is limited. The hypothesis of the present study was endostatin, an endogenous inhibitor of angiogenesis, disturbs fracture healing. We evaluated this hypothesis in a closed femoral fracture model studying two groups of mice, one that was treated by a daily injection of 10 µg recombinant endostatin subcutaneously (n = 38) and a second one that received the vehicle for control (n = 37). Histomorphometric analysis showed a significantly increased callus formation in endostatin‐treated animals at 2 and 5 weeks post‐fracture. This was associated with a significantly higher callus tissue fraction of cartilage and fibrous tissue at 2 weeks and a significantly higher fraction of bone at 5 weeks post‐fracture. Biomechanical testing revealed a significantly higher torsional stiffness in the endostatin group at 2 weeks. For both groups, we could demonstrate the expression of the endostatin receptor unit integrin alpha5 in endothelial cells, osteoblasts, osteoclasts, and chondrocytes at 2 weeks. Immunohistochemical fluorescence staining of CD31 showed a lower number of blood vessels in endostatin‐treated animals compared to controls. The results of the present study indicate endostatin promotes soft callus formation but inhibits callus remodeling during fracture healing most probably by an inhibition of angiogenesis. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1579–1584, 2013.     

Delayed Fracture Healing in Aged Senescence-Accelerated P6 Mice

ABSTRACT

Background: Osteoporosis is characterized by poor bone quality. However, it is still controversially discussed whether osteoporosis compromises fracture healing. Herein, we studied whether the course of healing of a femur fracture is affected by osteoporosis or age. Methods: Using the senescence-accelerated osteoporotic mouse, strain P6 (SAMP6), and a closed femur fracture model, we studied the process of fracture healing in 5- and 10-month-old animals, including biomechanical, histomorphometric, and protein biochemical analysis. Results: In five-month-old osteoporotic SAMP6 mice, bending stiffness, callus size, and callus tissue distribution as well as the concentrations of the bone formation marker osteocalcin and the bone resorption markers tartrate-resistant acid phosphatase form 5b (TRAP) and deoxypyridinoline (DPD) did not differ from that of non-osteoporotic, senescence-resistant, strain 1 (SAMR1) controls. In contrast, femur fractures in 10-month-old SAMP6 mice showed a significantly reduced bending stiffness and an increased callus size compared to fractures in age-matched SAMR1 controls. This indicates a delayed fracture healing in advanced age SAMP6 mice. The delay of fracture healing was associated with higher concentrations of TRAP and DPD. Significant differences in osteocalcin concentrations were not found between SAMP6 animals and SAMR1 controls. Conclusion: In conclusion, the present study indicates that fracture healing in osteoporotic SAMP6 mice is not affected in five-month-old animals, but delayed in animals with an age of 10 months. This is most probably due to the increased osteoclast activity in advanced age SAMP6 animals.     

Hyperhomocysteinemia Is Associated with Impaired Fracture Healing in Mice

Hyperhomocysteinemia (HHCY) has been shown to disturb bone metabolism and to increase the incidence of osteoporosis and osteoporotic fractures. However, there is a complete lack of information on whether these metabolic alterations affect bone repair. The aim of this study was to analyze the impact of HHCY on fracture healing. One group of mice was fed a homocystine-supplemented diet (n = 12), whereas another group received the accordant standard diet for control (n = 13). Four weeks after the stable fixation of a closed femoral fracture, animals were killed to prepare bones for histomorphometric and biomechanical analyses. In addition, blood samples were obtained to evaluate serum concentration of homocysteine (HCY). Quantitative analysis of blood samples revealed severe HHCY as indicated by significantly increased serum concentrations of HCY in animals fed the homocystine-supplemented diet (102.2 ± 64.5 μmol/l) compared to controls (2.8 ± 1.5 μmol/l). Biomechanical evaluation of bone repair revealed significantly decreased bending stiffness of the femora of homocystine-fed animals (45.5 ± 18.2 N/mm) compared with controls (64.6 ± 15.8 N/mm). Histomorphometric analysis demonstrated a slightly smaller callus diameter in HHCY animals but no significant differences in the tissue composition of the callus. In conclusion, the homocystine-supplemented diet leads to severe HHCY, which is associated with an impaired biomechanical quality of the healing bone. J. Schmalenbach and M. Herrmann contributed equally to this work.     

Fracture Healing in Mice Deficient in Plasminogen Activator Inhibitor-1

To evaluate the role of plasminogen activator inhibitor (PAI)-1, a key negative regulator of the plasmin system of extracellular matrix proteases in developmental bone growth and fracture repair, the bone phenotype of male adult PAI-1-deficient mice was determined and femoral fracture healing was compared with that of age- and sex-matched wild-type C57BL/6J control mice. Regarding bone phenotype, the length and size (but not cortical thickness) of the femur of male PAI-1-deficient mice were smaller than those of wild-type controls. Although the total bone mineral content of PAI-1-deficient mice was not significantly different from that of wild-type mice, the total bone area in PAI-1-deficient mice was smaller, leading to an increase in total bone mineral density. With respect to fracture healing, PAI-1-deficient mice developed fracture calluses that were larger and more mineralized than those of wild-type mice but only at 14 days postfracture. These changes were even greater given the smaller size of the normal femur in PAI-1-deficient mice. Surprisingly, the larger fracture callus remodeled rapidly to normal size and mineral content by 21 days postfracture. Examination of fracture histology revealed that these changes were associated with a dramatic increase followed by a rapid remodeling of the fracture callus cartilage. The remodeling of fracture callus cartilage in PAI-1-deficient mice also displayed an abnormal pattern. These findings demonstrate for the first time that PAI-1 (and potentially the plasminogen extracellular matrix protease system) is an important regulator of bone size during developmental growth and plays a regulatory role in the determination of fracture callus size, cartilage formation, and resorption during bone fracture repair.     

Reduced COX‐2 Expression in Aged Mice Is Associated With Impaired Fracture Healing

The cellular and molecular events responsible for reduced fracture healing with aging are unknown. Cyclooxygenase 2 (COX‐2), the inducible regulator of prostaglandin E₂ (PGE₂) synthesis, is critical for normal bone repair. A femoral fracture repair model was used in mice at either 7–9 or 52–56 wk of age, and healing was evaluated by imaging, histology, and gene expression studies. Aging was associated with a decreased rate of chondrogenesis, decreased bone formation, reduced callus vascularization, delayed remodeling, and altered expression of genes involved in repair and remodeling. COX‐2 expression in young mice peaked at 5 days, coinciding with the transition of mesenchymal progenitors to cartilage and the onset of expression of early cartilage markers. In situ hybridization and immunohistochemistry showed that COX‐2 is expressed primarily in early cartilage precursors that co‐express col‐2. COX‐2 expression was reduced by 75% and 65% in fractures from aged mice compared with young mice on days 5 and 7, respectively. Local administration of an EP4 agonist to the fracture repair site in aged mice enhanced the rate of chondrogenesis and bone formation to levels observed in young mice, suggesting that the expression of COX‐2 during the early inflammatory phase of repair regulates critical subsequent events including chondrogenesis, bone formation, and remodeling. The findings suggest that COX‐2/EP4 agonists may compensate for deficient molecular signals that result in the reduced fracture healing associated with aging.     

Pantoprazole, a Proton Pump Inhibitor, Delays Fracture Healing in Mice

Proton pump inhibitors (PPIs), which are widely used in the treatment of dyspeptic problems, have been shown to reduce osteoclast activity. There is no information, however, on whether PPIs affect fracture healing. We therefore studied the effect of the PPI pantoprazole on callus formation and biomechanics during fracture repair. Bone healing was analyzed in a murine fracture model using radiological, biomechanical, histomorphometric, and protein biochemical analyses at 2 and 5 weeks after fracture. Twenty-one mice received 100 mg/kg body weight pantoprazole i.p. daily. Controls (n = 21) received equivalent amounts of vehicle. In pantoprazole-treated animals biomechanical analysis revealed a significantly reduced bending stiffness at 5 weeks after fracture compared to controls. This was associated with a significantly lower amount of bony tissue within the callus and higher amounts of cartilaginous and fibrous tissue. Western blot analysis showed reduced expression of the bone formation markers bone morphogenetic protein (BMP)-2, BMP-4, and cysteine-rich protein (CYR61). In addition, significantly lower expression of proliferating cell nuclear antigen indicated reduced cell proliferation after pantoprazole treatment. Of interest, the reduced expression of bone formation markers was associated with a significantly diminished expression of RANKL, indicating osteoclast inhibition. Pantoprazole delays fracture healing by affecting both bone formation and bone remodeling.     

Effects of Interleukin‐6 Ablation on Fracture Healing in Mice

This study examined the impact of an interleukin‐6 (IL‐6) knockout on fracture healing in terms of histological and biomechanical responses. Following IACUC approval, tibial fractures were produced in 4‐ to 6‐week‐old IL‐6 knockouts (n = 35) and wild‐type mice (n = 36) and harvested along with contralateral limbs at 2 and 6 weeks postsurgery. Histology quantified stage of healing, lymphocyte infiltration, TRAP+ cells, and osteocalcin deposition. Bend testing established maximum load and stiffness. Based on normality assessments, Mann–Whitney U or independent t‐tests were used for data analysis using a p‐value threshold of 0.05. Stage of healing, lymphocyte infiltration, and osteocalcin deposition were similar for all time points (p ≥ 0.243). TRAP+ cell counts were reduced approximately 10‐fold in the knockout at 2 weeks (p = 0.015) but were similar at 6 weeks (p = 0.689). Force‐to‐failure in knockouts was approximately 40% that of wild‐type mice at 2 weeks (p = 0.040) but similar at 6 weeks (p = 0.735). Knockout bone was about 25% less stiff at 2 weeks but approximately 60% stiffer at 6 weeks (p ≥ 0.110). The absence of IL‐6 during early fracture healing significantly reduced osteoclastogenesis and impaired callus strength. By 6 weeks, most histological and biomechanical parameters were similar to fractures in wild‐type bone. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1437–1442, 2011     

Molecular and Cellular Mechanisms of Delayed Fracture Healing in Mmp10 (Stromelysin 2) Knockout Mice

The remodeling of the extracellular matrix is a central function in endochondral ossification and bone homeostasis. During secondary fracture healing, vascular invasion and bone growth requires the removal of the cartilage intermediate and the coordinate action of the collagenase matrix metalloproteinase (MMP)-13, produced by hypertrophic chondrocytes, and the gelatinase MMP-9, produced by cells of hematopoietic lineage. Interfering with these MMP activities results in impaired fracture healing characterized by cartilage accumulation and delayed vascularization. MMP-10, Stromelysin 2, a matrix metalloproteinase with high homology to MMP-3 (Stromelysin 1), presents a wide range of putative substrates identified in vitro, but its targets and functions in vivo and especially during fracture healing and bone homeostasis are not well defined. Here, we investigated the role of MMP-10 through bone regeneration in C57BL/6 mice. During secondary fracture healing, MMP-10 is expressed by hematopoietic cells and its maximum expression peak is associated with cartilage resorption at 14 days post fracture (dpf). In accordance with this expression pattern, when Mmp10 is globally silenced, we observed an impaired fracture-healing phenotype at 14 dpf, characterized by delayed cartilage resorption and TRAP-positive cell accumulation. This phenotype can be rescued by a non-competitive transplant of wild-type bone marrow, indicating that MMP-10 functions are required only in cells of hematopoietic linage. In addition, we found that this phenotype is a consequence of reduced gelatinase activity and the lack of proMMP-9 processing in macrophages. Our data provide evidence of the in vivo function of MMP-10 during endochondral ossification and defines the macrophages as the lead cell population in cartilage removal and vascular invasion. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Impaired Fracture Healing in Sarco-Osteoporotic Mice Can Be Rescued by Vibration Treatment Through Myostatin Suppression

Sarcopenia is highly prevalent in fragility fracture patients and is associated with delayed healing. In this study, we investigated the effect of low-magnitude high-frequency vibration (LMHFV) on osteoporotic fracture with sarcopenia and the potential role of myostatin. Osteoporotic fractures created in sarcopenic SAMP8, non-sarcopenic SAMR1 were randomized to control or LMHFV (SAMP8, SAMR1, SAMP8-V, or SAMR1-V) groups. Healing and myostatin expression were evaluated at 2, 4, and 6 weeks post-fracture. In vitro, conditioned-media were collected from myofibers isolated from aged and young SAMP8 or C2C12 myoblasts with or without LMHFV. Osteoblastic MC3T3-E1 under osteogenic differentiation were treated with plain or conditioned-medium (±myostatin propeptide). LMHFV significantly enhanced callus formation was in non-sarcopenic SAMR1 mice; but the enhancement effect was not significant in SAMP8 mice at week 2. Myostatin expressions in callus and biceps femoris of SAMP8 group were significantly higher all groups with significant negative correlation with callus size (R² = 0.7256; p = 0.0004). Mechanical properties (week 4) and callus remodeling (week 6) were inferior in SAMP8 versus SAMR1 and were significantly enhanced by LMHFV. Alkaline Phosphatase (ALP) and Runx2 expression of MC3T3-E1 was lower in aged myofiber compared with young, but upregulated by LMHFV or myostatin inhibition; also confirmed with C2C12. LMHFV enhanced early callus formation, microarchitecture, callus remodeling and mechanical properties of fracture healing in both SAMP8 and SAMR1; however, more effective in non-sarcopenic SAMR1 mice. Impaired fracture healing in sarcopenic SAMP8 mice is attributed by elevated myostatin expression in callus and muscle, which correlated negatively with callus formation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:277-287, 2020     

Calcium-Sensing Receptors in Chondrocytes and Osteoblasts Are Required for Callus Maturation and Fracture Healing in Mice

Calcium and its putative receptor (CaSR) control skeletal development by pacing chondrocyte differentiation and mediating osteoblast (OB) function during endochondral bone formation—an essential process recapitulated during fracture repair. Here, we delineated the role of the CaSR in mediating transition of callus chondrocytes into the OB lineage and subsequent bone formation at fracture sites and explored targeting CaSRs pharmacologically to enhance fracture repair. In chondrocytes cultured from soft calluses at a closed, unfixed fracture site, extracellular [Ca²⁺] and the allosteric CaSR agonist (NPS-R568) promoted terminal differentiation of resident cells and the attainment of an osteoblastic phenotype. Knockout (KO) of the Casr gene in chondrocytes lengthened the chondrogenic phase of fracture repair by increasing cell proliferation in soft calluses but retarded subsequent osteogenic activity in hard calluses. Tracing growth plate (GP) and callus chondrocytes that express Rosa26-tdTomato showed reduced chondrocyte transition into OBs (by >80%) in the spongiosa of the metaphysis and in hard calluses. In addition, KO of the Casr gene specifically in mature OBs suppressed osteogenic activity and mineralizing function in bony calluses. Importantly, in experiments using PTH (1-34) to enhance fracture healing, co-injection of NPS-R568 not only normalized the hypercalcemic side effects of intermittent PTH (1-34) treatment in mice but also produced synergistic osteoanabolic effects in calluses. These data indicate a functional role of CaSR in mediating chondrogenesis and osteogenesis in the fracture callus and the potential of CaSR agonism to facilitate fracture repair. © 2019 American Society for Bone and Mineral Research.     

外源性雌激素对卵巢切除小鼠骨折愈合影响的组织学观察

目的　探讨外源性雌激素对骨折愈合的影响。方法　在卵巢切除后 3个月已经出现明显骨丢失的balb/c小鼠用折骨器造成右股骨中段闭合性骨折 ,应用不同剂量的雌激素治疗 ,然后分批处理取出骨痂标本进行组织学检查。结果　发现卵巢切除后未用雌激素治疗小鼠骨痂形成较小 ,而应用高剂量雌激素治疗小鼠骨痂发育明显推迟。结论　实验表明 ,补充超生理剂量雌激素对卵巢切除小鼠骨折愈合 (早期 )不利。     

Caloric Restriction Chronically Impairs Metabolic Programming in Mice

Although obesity rates are rapidly rising, caloric restriction remains one of the few safe therapies. Here we tested the hypothesis that obesity-associated disorders are caused by increased adipose tissue as opposed to excess dietary lipids. Fat mass (FM) of lean C57B6 mice fed a high-fat diet (HFD; FMC mice) was “clamped” to match the FM of mice maintained on a low-fat diet (standard diet [SD] mice). FMC mice displayed improved glucose and insulin tolerance as compared with ad libitum HFD mice (P < 0.001) or SD mice (P < 0.05). These improvements were associated with fewer signs of inflammation, consistent with the less-impaired metabolism. In follow-up studies, diet-induced obese mice were food restricted for 5 weeks to achieve FM levels identical with those of age-matched SD mice. Previously, obese mice exhibited improved glucose and insulin tolerance but showed markedly increased fasting-induced hyperphagia (P < 0.001). When mice were given ad libitum access to the HFD, the hyperphagia of these mice led to accelerated body weight gain as compared with otherwise matched controls without a history of obesity. These results suggest that although caloric restriction on a HFD provides metabolic benefits, maintaining those benefits may require lifelong continuation, at least in individuals with a history of obesity.Caloric restriction (CR) has become a popular recommendation to decrease body weight (BW) (1), achieve metabolic benefits, and potentially increase life expectancy (2–4). Although the benefits of weight loss are undeniable, the optimal method to achieve a healthy weight is the subject of continuing debate. Obesity is accompanied by many comorbidities (5), and it is widely accepted as a causal factor. A current hypothesis suggests that obesity provokes inflammation in adipose tissue (6), leading to the release of proinflammatory mediators into systemic circulation. Rodent studies suggest that consumption of a high-fat diet (HFD) leads to infiltration by T cells (7) and macrophages (8), which likely contributes to inflammation in adipose tissue and the development of obesity-associated diseases such as type 2 diabetes (9).Alternative explanations for the obesity-related comorbidities are based on specific harmful effects of HFDs per se. High-fat foods induce stress in the intestinal epithelium (10) and promote inflammation by absorption of antigenic material from the gut (11,12). Furthermore, HFD feeding increases reactive oxygen species production (13) that preceded obesity (14), suggesting that like sucrose and fructose, dietary lipids may contribute to obesity-associated diseases. In the present experiments we tested the hypothesis that obesity-associated disorders are caused by increased adipose tissue, as opposed to the obesogenic diet. To dissect these possibilities, we isolated body fat mass (FM) from fat intake using a “FM clamp” mouse model (Fig. 1A). In related studies, we asked whether a history of obesity affects future energy metabolism. Published reports suggest that counter-regulatory mechanisms that develop during CR lead to enhanced metabolic efficiency and rapid weight regain (15,16). We thus aimed to distinguish if this metabolic reprogramming is related to prior obesity, dietary fat intake, or having been calorically restricted (Fig. 1A).Open in a separate windowFIG. 1.A: Study design and FM clamping. B: FM was measured by nuclear magnetic resonance technology and did not differ between SD and FMC mice. C: BW of SD, HFD fed, and FMC mice was measured daily. Mice from the SD and HFD group were fed with SD or HFD ad libitum. Mice from the FMC group were fed defined amounts of HFD to match FM to the SD group. D: Absolute food intake was lowest in FMC mice (P < 0.001). E: Caloric intake was similar in FMC and SD mice and highest in mice of the HFD group. All mice are male (n = 16 per group). All data are represented as mean ± SEM. ITT, insulin tolerance test; FI, food intake.     

Fracture Healing in Rabbits after Osteotomy Using the CO2 Laser

Fracture healing was studied in 28 rabbits after their femurs had been osteotomized using the carbon dioxide laser. Twenty rabbits whose femurs were osteotomized by means of a Gigli saw served as the control group. Fracture healing was initially delayed in the laser cut femurs, yet after 60 days no significant difference between the groups could be detected. The initial delay was caused by thermal damage to the laser cut bone edges. Further fragmentation of the damaged bone occurred during the immediate postoperative period.