Transthyretin Antisense Oligonucleotides Lower Circulating RBP4 Levels and Improve Insulin Sensitivity in Obese Mice

Circulating transthyretin (TTR) is a critical determinant of plasma retinol-binding protein 4 (RBP4) levels. Elevated RBP4 levels cause insulin resistance, and the lowering of RBP4 levels improves glucose homeostasis. Since lowering TTR levels increases renal clearance of RBP4, we determined whether decreasing TTR levels with antisense oligonucleotides (ASOs) improves glucose metabolism and insulin sensitivity in obesity. TTR-ASO treatment of mice with genetic or diet-induced obesity resulted in an 80–95% decrease in circulating levels of TTR and RBP4. Treatment with TTR-ASOs, but not control ASOs, decreased insulin levels by 30–60% and improved insulin sensitivity in ob/ob mice and high-fat diet–fed mice as early as after 2 weeks of treatment. The reduced insulin levels were sustained for up to 9 weeks of treatment and were associated with reduced adipose tissue inflammation. Body weight was not changed. TTR-ASO treatment decreased LDL cholesterol in high-fat diet–fed mice. The glucose infusion rate during a hyperinsulinemic-euglycemic clamp was increased by 50% in high-fat diet–fed mice treated with TTR-ASOs, demonstrating improved insulin sensitivity. This was also demonstrated by 20% greater inhibition of hepatic glucose production, a 45–60% increase of glucose uptake into skeletal and cardiac muscle, and a twofold increase in insulin signaling in muscle. These data show that decreasing circulating TTR levels or altering TTR-RBP4 binding could be a potential therapeutic approach for the treatment of type 2 diabetes.     

In vivo release and turnover of secreted platelet antiheparin proteins in rhesus monkey (Macaca mulatta)

Human and rhesus monkey platelets secrete at least two antiheparin proteins: platelet factor 4 (PF4) and low affinity platelet factor 4 (LA-PF4). Neither of these proteins showed species-related antigenic differences. As determined by radioimmunoassay, the levels of PF4 and LA-PF4 antigen per 10(9) monkey platelets amounted to 10.7 and 20.3 microgram, respectively. One milliliter of monkey plasma prepared from blood collected into an anticoagulant composed of EDTA, prostaglandin E1, and theophylline solution contained 22.4 ng LA-PF4 and 8.0 ng PF4. Concentrations of these two platelet-specific proteins in monkeys closely resembled levels found in human platelets and plasma. Infusion of prostacyclin (PGI2) (100 or 300 ng/kg/min) into monkeys for 15 min resulted in a significant decrease of plasma levels of LA-PF4 antigen and of PF4 by 40%--60% (p < 0.0001). This decrease was related to the inhibitory effect of PGI2 on the secretion of platelets stimulated by a catheter or by venipuncture. Longer infusion of PGI2 did not produce further significant change. The supernate obtained after aggregation of human platelets stimulated by thrombin was injected into monkeys receiving PGI2 infusion. The disappearance of LA-PF4 antigen in monkey plasma followed a biphasic exponential curve with half-lives for the fast and slow components of 8.4 and 63 min. PF4 disappeared faster but followed the same pattern (half-lives for the fast and slow component of 2.1 and 70 min). Analysis of the experimental data suggests that the low levels of secreted platelet proteins in monkey plasma are related to their minimal in vivo release and to their rapid clearance.     

A novel small molecule that directly sensitizes the insulin receptor in vitro and in vivo

Insulin resistance, an important feature of type 2 diabetes, is manifested as attenuated insulin receptor (IR) signaling in response to insulin binding. A drug that promotes the initiation of IR signaling by enhancing IR autophosphorylation should, therefore, be useful for treating type 2 diabetes. This report describes the effect of a small molecule IR sensitizer, TLK16998, on IR signaling. This compound activated the tyrosine kinase domain of the IR beta-subunit at concentrations of 1 micromol/l or less but had no effect on insulin binding to the IR alpha-subunit even at much higher concentrations. TLK16998 alone had no effect on IR signaling in mouse 3T3-L1 adipocytes but, at concentrations as low as 3.2 micromol/l, enhanced the effects of insulin on the phosphorylation of the IR beta-subunit and IR substrate 1, and on the amount of phosphatidylinositol 3-kinase that coimmunoprecipitated with IRS-1. Phosphopeptide mapping revealed that the effect of TLK16998 on the IR was associated with increased tyrosine phosphorylation of the activation loop of the beta-subunit tyrosine kinase domain. TLK16998 also increased the potency of insulin in stimulating 2-deoxy-D-glucose uptake in 3T3-L1 adipocytes, with a detectable effect at 8 micromol/l and a 10-fold increase at 40 micromol/l. In contrast, only small effects were observed on IGF-1-stimulated 2-deoxy-D-glucose uptake. In diabetic mice, TLK16998, at a dose of 10 mg/kg, lowered blood glucose levels for up to 6 h. These results suggest, therefore, that small nonpeptide molecules that directly sensitize the IR may be useful for treating type 2 diabetes.     

Correction of Lower Limb Deformities Using Ilizarov's Technique

Background

India accounts for approximately 10 million orthopaedically handicapped children and adults with limb deformity. Ilizarov ring fixator could treat most of these deformities.

Methods

Twenty cases of deformities of lower limb managed with Ilizarov technique during period between March 2001 and February 2003 were studied.

Results

55% were in the age group of 11-30 years. Out of the 20 cases studied, 6 were congenital talipes equino varus, 8 were fixed flexion deformity of knee, 4 were equines deformity of the ankle and 2 were malunited fracture shaft of tibia.4 patients who had recurrence were operated for fixed flexion deformity of the knee. The main complication encountered was pin tract infection, which was seen in 15(75%) cases. In 16(80%) cases, the results were excellent with no recurrence of deformity and patients were able to walk independently. In 4 (20%) cases, recurrence was mild to moderate (10 to 20) but all of them were able to ambulate idependently and carry out their routine activities.

Conclusion

Ilizarov ring fixator is a superior compared to conventional methods for correction of deformities of lower limb.Key Words: Ilizarov method, Ligamentotaxis, Distraction     

Efficacy and Toxicity Profile of Methotrexate Chloroquine Combination in Treatment of Active Rheumatoid Arthritis

Background

The present study was conducted to study the efficacy and toxicity profile of methotrexate chloroquine combination in treatment of active rheumatoid arthritis.

Methods

24 patients of rheumatoid arthritis confirming to revised American Rheumatism Association (ARA) criteria were studied prospectively for twenty months. Clinical evaluation was made every 3 months. Clinical disease variables measured at each visit were number of joints with swelling, number of joints with tenderness and pain, duration of morning stiffness and physician and patient assessment of disease activity. Blood counts, liver function tests and other adverse effects due to drugs were monitored every 2 months.

Results

10 patients demonstrated more than 50% improvement. 4 patients withdrew from study, 2 because of excessive nausea and vomiting and 2 because of noncompliance. Other side effects noted were hyperpigmentation, photosensitivity, skin rashes, raised transaminases and stomatitis.

Conclusion

Methotrexate chloroquine combination has good efficacy and toxicity profile. Gastrointestinal side effects are most common and usually responsible for the discontinuation of the drugs.Key Words: Rheumatoid arthritis, Methotrexate, Chloroquine, Efficacy, Toxicity     

Reduction of hepatic and adipose tissue glucocorticoid receptor expression with antisense oligonucleotides improves hyperglycemia and hyperlipidemia in diabetic rodents without causing systemic glucocorticoid antagonism

Glucocorticoids (GCs) increase hepatic gluconeogenesis and play an important role in the regulation of hepatic glucose output. Whereas systemic GC inhibition can alleviate hyperglycemia in rodents and humans, it results in adrenal insufficiency and stimulation of the hypothalamic-pituitary-adrenal axis. In the present study, we used optimized antisense oligonucleotides (ASOs) to cause selective reduction of the glucocorticoid receptor (GCCR) in liver and white adipose tissue (WAT) and evaluated the resultant changes in glucose and lipid metabolism in several rodent models of diabetes. Treatment of ob/ob mice with GCCR ASOs for 4 weeks resulted in approximately 75 and approximately 40% reduction in GCCR mRNA expression in liver and WAT, respectively. This was accompanied by approximately 65% decrease in fed and approximately 30% decrease in fasted glucose levels, a 60% decrease in plasma insulin concentration, and approximately 20 and 35% decrease in plasma resistin and tumor necrosis factor-alpha levels, respectively. Furthermore, GCCR ASO reduced hepatic glucose production and inhibited hepatic gluconeogenesis in liver slices from basal and dexamethasone-treated animals. In db/db mice, a similar reduction in GCCR expression caused approximately 40% decrease in fed and fasted glucose levels and approximately 50% reduction in plasma triglycerides. In ZDF and high-fat diet-fed streptozotocin-treated (HFD-STZ) rats, GCCR ASO treatment caused approximately 60% reduction in GCCR expression in the liver and WAT, which was accompanied by a 40-70% decrease in fasted glucose levels and a robust reduction in plasma triglyceride, cholesterol, and free fatty acids. No change in circulating corticosterone levels was seen in any model after GCCR ASO treatment. To further demonstrate that GCCR ASO does not cause systemic GC antagonism, normal Sprague-Dawley rats were challenged with dexamethasone after treating with GCCR ASO. Dexamethasone increased the expression of GC-responsive genes such as PEPCK in the liver and decreased circulating lymphocytes. GCCR ASO treatment completely inhibited the increase in dexamethasone-induced PEPCK expression in the liver without causing any change in the dexamethasone-induced lymphopenia. These studies demonstrate that tissue-selective GCCR antagonism with ASOs may be a viable therapeutic strategy for the treatment of the metabolic syndrome.     

Pneumothorax preceding pulmonary blastoma in a child

Serial plain radiographic, ultrasound and CT findings of an unusual case of pulmonary blastoma are described with a review of the literature.