Regulation of adiponectin receptor gene expression in diabetic mice

Adiponectin is an adipocyte-derived factor that plays pivotal roles in lipid and glucose metabolism in muscle and liver. The following two adiponectin receptor types were recently identified: AdipoR1 is abundantly expressed in muscle, whereas AdipoR2 is predominantly expressed in the liver. To clarify the regulation of adiponectin receptor gene expression in diabetic states, we examined mRNA levels of AdipoR1 in the muscles of diabetic animals by Northern blotting. The level of AdipoR1 mRNA was increased approximately 2.5-fold in muscle of streptozotocin (STZ) diabetic mice, but the normal level was restored by insulin administration, indicating that insulin has an inhibitory effect on AdipoR1 expression. To confirm this inhibitory effect of insulin, we performed in vitro experiments using C2C12 skeletal muscle cells. Insulin treatment for 24 h decreased AdipoR1 expression by approximately 60% in C2C12 cells. In addition, this effect was mediated by the phosphatidylinositol 3-kinase-dependent pathway rather than the mitogen-activated protein kinase pathway. AdipoR1 expression in insulin-resistant diabetic mice was also investigated. AdipoR1 expression was decreased by 36% in type 2 diabetic obese db/db mice compared with lean mice. In contrast, hepatic AdipoR2 expression was not significantly changed in either STZ mice or genetically obese mice. Our results indicate that regulation of AdipoR1, but not that of AdipoR2, may be involved in glucose and lipid metabolism in diabetic states.     

Effect of adiponectin on bovine granulosa cell steroidogenesis,oocyte maturation and embryo development

Background  

Adiponectin is an adipokine, mainly produced by adipose tissue. It regulates several reproductive processes. The protein expression of the adiponectin system (adiponectin, its receptors, AdipoR1 and AdipoR2 and the APPL1 adaptor) in bovine ovary and its role on ovarian cells and embryo, remain however to be determined.     

High glucose induced rat aorta vascular smooth muscle cell oxidative injury: involvement of protein tyrosine nitration

The alteration and further damage of vascular smooth muscle function have been implicated in the development of vascular complications and diabetes. Little is known about protein tyrosine nitration in vascular smooth muscle cell injury induced by high glucose. In this article, vascular smooth muscle cell was exposed to 30 and 40 mM high glucose for 72 h, and then the cell injury in vascular smooth muscle cell induced by high glucose was studied. It was found that high glucose stimulated vascular smooth muscle cell injury in a dose-dependent manner, including decreasing intracellular and extracellular glutathione contents, increasing malondialdehyde and intracellular reactive oxygen species content, increasing the production of nitric oxide (increased nitrite content in cell and medium), as well as increasing protein tyrosine nitration. By comparing protein tyrosine nitration induced by high glucose conditions and extrinsic factors (hemin–nitrite–glucose oxidase system and 3-morpholinosydnonimine), it may be speculated that protein is nitrated selectively, and specific protein tyrosine nitration is involved in diabetic vascular complications.     

Adiponectin Receptor-1 C-Terminal Fragment (CTF) in Plasma: Putative Biomarker for Diabetes

Introduction

Polypeptide fragments from cell surface receptors when found in plasma may be indicators of receptor regulation in disease conditions. It is known that subjects with diabetes have significantly lower plasma concentrations of adiponectin, a hormone released by adipose tissue, compared with nondiabetic controls. This hormone interacts with cell surface receptors in muscle (AdipoR1) and liver (AdipoR2).

Methods

We analyzed the relative distribution of specific fragments of AdipoR1 in healthy and diabetic individuals using an immunoaffinity mass spectrometry approach. We used antibodies raised against AdipoR1 immobilized on pre-activated protein chip surfaces to determine the molecular weights of bound polypeptide fragments using immunomass spectrometry (immuno-MS).

Results

Initially, immuno-MS analyses using a polyclonal antibody revealed two peaks (m/z 3,902 and 7,812) in plasma from normal, healthy individuals (n?=?5) that were not present in the plasma of diabetics (n?=?5). To confirm the detection of these fragments, a monoclonal antibody was developed against the last 25 amino acids of the AdipoR1 C-terminal fragment (CTF). Using the immuno-MS method, the monoclonal antibody detected the AdipoR1 CTF (m/z 3475) in all healthy controls (n?=?10), but did not detect these fragments in the diabetic patients (n?=?10).

Discussion

These preliminary observations suggest that the plasma levels of this receptor fragment may serve as an indicator of diabetic condition.     

Differential effects of treadmill exercise in early and chronic diabetic stages on parvalbumin immunoreactivity in the hippocampus of a rat model of type 2 diabetes

In the present study, we investigated the effects of treadmill exercise in early and chronic diabetic stages on parvalbumin (PV) immunoreactivity in the subgranular zone of the dentate gyrus of Zucker diabetic fatty (ZDF) and its lean control rats (ZLC). To investigate the effects, ZLC and ZDF rats at 6 or 23 weeks of age were put on a treadmill with or without running for 1 h/day/5 consecutive days at 16–22 m/min for 5 weeks or 12–16 m/min for 7 weeks, respectively. Physical exercise in pre-diabetic rats prevented onset of diabetes, while exercise in rats at chronic diabetic stage significantly reduced blood glucose levels. In addition, physical exercise in the pre-diabetic rats significantly increased PV immunoreactive fibers in the strata oriens and radiatum of the CA1-3 region and in the polymorphic and molecular layers of the dentate gyrus compared to that in sedentary controls. However, in rats at chronic stages, PV immunoreactivity was slightly increased in the CA1-3 region as well as in the dentate gyrus compared to that in the sedentary controls. These results suggest that physical exercise has differential effects on blood glucose levels and PV immunoreactivity according to diabetic stages. Early exercise improves diabetic phenotype and PV immunoreactive fibers in the rat hippocampus.     

Expression of adiponectin receptors in pancreatic beta cells

Pancreatic beta cell dysfunction is an early and crucial pathogenic factor in the development of type 2 diabetes. Free fatty acids (FFA) and adipokines released from adipose tissues lead to both the development of insulin resistance and beta cell dysfunction. Adiponectin is a novel adipokine with antidiabetic properties. Its circulating concentrations are reduced in subjects with increased visceral adiposity, insulin resistance, or type 2 diabetes. Very recently, the cloning of two adiponectin receptors AdipoR1 and AdipoR2 was reported. AdipoR1 is abundantly expressed in muscle, while AdipoR2 is predominantly expressed in liver. Here we report the marked expression of mRNAs for the adiponectin receptors AdipoR1 and AdipoR2 in human and rat pancreatic beta cells, at levels similar to liver and greater than muscle. Adiponectin receptor expression is increased by beta cell exposure to the unsaturated FFA oleate, and treatment of insulin-producing cells with globular adiponectin induces lipoprotein lipase expression. Regulated adiponectin receptor expression on pancreatic beta cells might be a novel mechanism modulating the effects of circulating adiponectin.     

Effect of Long-Term Intraperitoneal Zinc Administration on Liver Glycogen Levels in Diabetic Rats Subjected to Acute Forced Swimming

This study aims to examine the effect of zinc administration on liver glycogen levels of rats in which diabetes was induced with streptozotocin and which were subjected to acute swimming exercise. The study was conducted on 80 adult Sprague–Dawley male rats, which were equally allocated to eight groups: group 1, general control; group 2, zinc-administrated control; group 3, zinc-administrated diabetic control; group 4, swimming control; group 5, zinc-administrated swimming; group 6, zinc-administrated diabetic swimming; group 7, diabetic swimming; group 8, diabetic control group. In order to induce diabetes, animals were injected with 40 mg/kg intraperitoneal (ip) streptozotocin. The injections were repeated in the same dose after 24 h. Animals which had blood glucose at or above 300 mg/dl 6 days after the last injections were accepted as diabetic. Zinc was administrated ip for 4 weeks as 6 mg/kg/day per rat. Hepatic tissue samples taken from the animals at the end of the study were fixed in 95% ethyl alcohol. Cross sections of 5 μm thickness, taken by the help of a microtome from the tissue samples buried in paraffin, were placed on a microscope slide and stained with periodic acid–Schiff and evaluated by light microscope. All microscopic images were transferred to a PC and assessed with the help of Clemex PE3.5 image analysis software. The lowest liver glycogen levels in the study were obtained in groups 3, 4, 6, 7, and 8. Liver glycogen levels in group 5 were higher than groups 3, 4, 6, 7, and 8, but lower than groups 1 and 2 (p < 0.05). Groups 1 and 2 had the highest liver glycogen levels. The results obtained from the study indicate that liver glycogen levels which dropped in acute swimming exercise were restored by zinc administration and that diabetes induced in rats prevented the protective effect of zinc.     

Different effects of rat interferon alpha, beta and gamma on rat hepatic stellate cell proliferation and activation

Background  

Liver fibrosis is the common sequel of chronic liver diseases. Recent studies have identified hepatic stellate cells as the primary cell type mediating hepatic fibrogenesis. It has been demonstrated that hepatic stellate cells undergo a process of activation during the development of liver fibrosis. During the activation process, hepatic stellate cells acquire myofibroblast-like phenotype featuring the expression of smooth muscle alpha actin. Interferons have been employed for the treatment of viral hepatitis. However, it is unclear what is the effect of interferons on the prevention and treatment of liver fibrosis. Moreover, it is not clear whether there are any differences among interferon alpha, interferon beta, and interferon gamma in the treatment of liver fibrosis. Therefore, our objective in current study is to investigate the effects of rat interferon-α, interferon-β, and interferon-γ on the proliferation and activation of rat hepatic stellate cells.     

Oxidation of an oral [13C]glucose load at rest and prolonged exercise in trained and sedentary subjects

The purpose of this study was to compare the oxidation of[¹³C]glucose (100 g)ingested at rest or during exercise in six trained (TS) and sixsedentary (SS) male subjects. The oxidation of plasma glucose was alsocomputed from the volume of¹³CO₂and¹³C/¹²Cin plasma glucose to compute the oxidation rate of glucose released from the liver and from glycogen stores in periphery (mainly muscle glycogen stores during exercise). At rest, oxidative disposal of bothexogenous (8.3 ± 0.3 vs. 6.6 ± 0.8 g/h) and liver glucose (4.4 ± 0.5 vs. 2.6 ± 0.4 g/h) was higher in TS than in SS.This could contribute to the better glucose tolerance observed at rest in TS. During exercise, for the same absolute workload [140 ± 5 W: TS = 47 ± 2.5; SS = 68 ± 3 %maximal oxygen uptake(O_{2 max})], [¹³C]glucose oxidationwas higher in TS than in SS (39.0 ± 2.6 vs. 33.6 ± 1.2 g/h),whereas both liver glucose (16.8 ± 2.4 vs. 24.0 ± 1.8 g/h) and muscle glycogen oxidation (36.0 ± 3.0 vs. 51.0 ± 5.4 g/h) were lower. For the same relative workload (68 ± 3% O_{2 max}:TS = 3.13 ± 0.96; SS = 2.34 ± 0.60 lO₂/min), exogenous glucose(44.4 ± 1.8 vs. 33.6 ± 1.2 g/h) and muscle glycogen oxidation (73.8 ± 7.2 vs. 51.0 ± 5.4 g/h) were higher in TS. However,despite a higher energy expenditure in TS, liver glucose oxidation was similar in both groups (22.2 ± 3.0 vs. 24.0 ± 1.8 g/h). Thus exogenous glucose oxidation was selectively favored in TSduring exercise, reducing both liver glucose and muscle glycogen oxidation.

     

The transduction properties of intercostal muscle mechanoreceptors

Background  

Intercostal muscles are richly innervated by mechanoreceptors. In vivo studies of cat intercostal muscle have shown that there are 3 populations of intercostal muscle mechanoreceptors: primary muscle spindles (1°), secondary muscle spindles (2°) and Golgi tendon organs (GTO). The purpose of this study was to determine the mechanical transduction properties of intercostal muscle mechanoreceptors in response to controlled length and velocity displacements of the intercostal space. Mechanoreceptors, recorded from dorsal root fibers, were localized within an isolated intercostal muscle space (ICS). Changes in ICS displacement and the velocity of ICS displacement were independently controlled with an electromagnetic motor. ICS velocity (0.5 – 100 μm/msec to a displacement of 2,000 μm) and displacement (50–2,000 μm at a constant velocity of 10 μm/msec) parameters encompassed the full range of rib motion.     

Expression profiles of adiponectin receptors in mouse embryos

Adiponectin is a protein secreted from adipocytes and it plays an important endocrine role in glucose and lipid homeostasis. A reverse correlation between plasma adiponectin concentrations and insulin resistance has been established in both animals and humans. Adiponectin exerts its function by interacting with membrane receptors, including AdipoR1 and AdipoR2. We investigated the expression pattern of these two adiponectin receptors in mouse embryos. At stages E12.5 and E15.5, both AdipoR1 and AdipoR2 are highly expressed in the nervous system including the trigeminal ganglion, glossopharyngeal ganglion and dorsal root ganglia. AdipoR1 is highly expressed in many tissues derived from primitive gut, including the lung, liver, pancreas and small intestines. Generally, the expression level of AdipoR2 is weaker and more restricted than AdipoR1 in most of the tissues. In addition, AdipoR1 expression can be found in heart, vertebrate, developing bones and cartilage, and many other tissues. This study reveals that AdipoR1 and AdipoR2 have differential but overlapping expression profiles during mouse development.     

Optimization of glucose feeding approaches for enhanced glucosamine and N-acetylglucosamine production by an engineered Escherichia coli

In this work, a recombinant Escherichia coli was constructed by overexpressing glucosamine (GlcN) synthase and GlcN-6-P N-acetyltransferase for highly efficient production of GlcN and N-acetylglucosamine (GlcNAc). For further enhancement of GlcN and GlcNAc production, the effects of different glucose feeding strategies including constant-rate feeding, interval feeding, and exponential feeding on GlcN and GlcNAc production were investigated. The results indicated that exponential feeding resulted in relatively high cell growth rate and low acetate formation rate, while constant feeding contributed to the highest specific GlcN and GlcNAc production rate. Based on this, a multistage glucose supply approach was proposed to enhance GlcN and GlcNAc production. In the first stage (0–2 h), batch culture with initial glucose concentration of 27 g/l was conducted, whereas the second culture stage (2–10 h) was performed with exponential feeding at μ _set = 0.20 h⁻¹, followed by feeding concentrated glucose (300 g/l) at constant rate of 32 ml/h in the third stage (10–16 h). With this time-variant glucose feeding strategy, the total GlcN and GlcNAc yield reached 69.66 g/l, which was enhanced by 1.59-fold in comparison with that of batch culture with the same total glucose concentration. The time-dependent glucose feeding approach developed here may be useful for production of other fine chemicals by recombinant E. coli.     

Transdermal Delivery of Insulin by Amidated Pectin Hydrogel Matrix Patch in Streptozotocin-Induced Diabetic Rats: Effects on Some Selected Metabolic Parameters

Purpose

Studies in our laboratory are concerned with developing optional insulin delivery routes based on amidated pectin hydrogel matrix gel. We therefore investigated whether the application of pectin insulin (PI)-containing dermal patches of different insulin concentrations sustain controlled release of insulin into the bloodstream of streptozotocin (STZ)-induced diabetic rats with concomitant alleviation of diabetic symptoms in target tissues, most importantly, muscle and liver.

Methods

Oral glucose test (OGT) responses to PI dermal matrix patches (2.47, 3.99, 9.57, 16.80 µg/kg) prepared by dissolving pectin/insulin in deionised water and solidified with CaCl₂ were monitored in diabetic rats given a glucose load after an 18-h fast. Short-term (5 weeks) metabolic effects were assessed in animals treated thrice daily with PI patches 8 hours apart. Animals treated with drug-free pectin and insulin (175 µg/kg, sc) acted as untreated and treated positive controls, respectively. Blood, muscle and liver samples were collected for measurements of selected biochemical parameters.

Results

After 5 weeks, untreated diabetic rats exhibited hyperglycaemia and depleted hepatic and muscle glycogen concentrations. Compared to untreated STZ-induced diabetic animals, OGT responses of diabetic rats transdermally applied PI patches exhibited lower blood glucose levels whilst short-term treatments restored hepatic and muscle glycogen concentrations. Plasma insulin concentrations of untreated diabetic rats were low compared with control non-diabetic rats. All PI treatments elevated plasma insulin concentrations of diabetic rats although the levels induced by high doses (9.57 and 16.80 µg/kg) were greater than those caused by low doses (2.47 and 3.99 µg/kg) but comparable to those in sc insulin treated animals.

Conclusions

The data suggest that the PI hydrogel matrix patch can deliver physiologically relevant amounts of pharmacologically active insulin.

Novelty of the Work

A new method to administer insulin into the bloodstream via a skin patch which could have potential future applications in diabetes management is reported.     

Activation of α1A-adrenergic receptor promotes differentiation of rat-1 fibroblasts to a smooth muscle-like phenotype

Background  

Fibroblasts, as connective tissue cells, are able to transform into another cell type including smooth muscle cells. α_1A-adrenergic receptor (α_1A-AR) stimulation in rat-1 fibroblasts is coupled to cAMP production. However, the significance of an increase in cAMP produced by α_1A-AR stimulation on proliferation, hypertrophy and differentiation in these cells is not known.     

Demographic history of Canary Islands male gene-pool: replacement of native lineages by European

Background  

The origin and prevalence of the prehispanic settlers of the Canary Islands has attracted great multidisciplinary interest. However, direct ancient DNA genetic studies on indigenous and historical 17^th–18^th century remains, using mitochondrial DNA as a female marker, have only recently been possible. In the present work, the analysis of Y-chromosome polymorphisms in the same samples, has shed light on the way the European colonization affected male and female Canary Island indigenous genetic pools, from the conquest to present-day times.     

Differential regulation of adiponectin receptor gene expression by adiponectin and leptin in myotubes derived from obese and diabetic individuals

Objective: This study aimed to investigate the regulation of adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2) gene expression in primary skeletal muscle myotubes, derived from human donors, after exposure to globular adiponectin (gAd) and leptin. Research Methods and Procedures: Four distinct primary cell culture groups were established [Lean, Obese, Diabetic, Weight Loss (Wt Loss); n = 7 in each] from rectus abdominus muscle biopsies obtained from surgical patients. Differentiated myotube cultures were exposed to gAd (0.1 μg/mL) or leptin (2.5 μg/mL) for 6 hours. AdipoR1 and AdipoR2 gene expression was measured by real‐time polymerase chain reaction analysis. Results: AdipoR1 mRNA expression in skeletal muscle myotubes derived from Lean subjects (p < 0.05) was stimulated 1.8‐fold and 2.5‐fold with gAd and leptin, respectively. No increase in AdipoR1 gene expression was measured in myotubes derived from Obese, Diabetic, or Wt Loss subjects. AdipoR2 mRNA expression was unaltered after gAd and leptin exposure in all myotube groups. Discussion: Adiponectin and leptin are rapid and potent stimulators of AdipoR1 in myotubes derived from lean healthy individuals. This effect was abolished in myotubes derived from obese, obese diabetic subjects, and obese‐prone individuals who had lost significant weight after bariatric surgery. The incapacity of skeletal muscle of obese and diabetic individuals to respond to exogenous adiponectin and leptin may be further suppressed as a result of impaired regulation of the AdipoR1 gene.     

Wortmannin inhibits both insulin- and contraction-stimulated glucose uptake and transport in rat skeletal muscle

Wojtaszewski, Jørgen F. P., Bo F. Hansen, BirgitteUrsø, and Erik A. Richter. Wortmannin inhibits both insulin-and contraction-stimulated glucose uptake and transport in rat skeletal muscle. J. Appl. Physiol. 81(4):1501-1509, 1996.The role of phosphatidylinositol (PI) 3-kinasefor insulin- and contraction-stimulated muscle glucose transport wasinvestigated in rat skeletal muscle perfused with a cell-freeperfusate. The insulin receptor substrate-1-associated PI 3-kinaseactivity was increased sixfold upon insulin stimulation but wasunaffected by contractions. In addition, the insulin-stimulated PI3-kinase activity and muscle glucose uptake and transport in individualmuscles were dose-dependently inhibited by wortmannin with one-halfmaximal inhibition values of ~10 nM and total inhibition at 1 µM.This concentration of wortmannin also decreased thecontraction-stimulated glucose transport and uptake by ~30-70%without confounding effects on contractility or on muscle ATP andphosphocreatine concentrations. At higher concentrations(3 and 10 µM), wortmannin completely blocked thecontraction-stimulated glucose uptake but also decreased thecontractility. In conclusion, inhibition of PI 3-kinase with wortmanninin skeletal muscle coincides with inhibition of insulin-stimulated glucose uptake and transport. Furthermore, in contrast to recent findings in incubated muscle, wortmannin also inhibitedcontraction-stimulated glucose uptake and transport. The inhibitoryeffect of wortmannin on contraction-stimulated glucose uptake may beindependent of PI 3-kinase activity or due to inhibition of asubfraction of PI 3-kinase with low sensitivity to wortmannin.

     

Damaging effects of ischemia/reperfusion on intestinal muscle

Periods of ischemia followed by restoration of blood flow cause ischemia/reperfusion (I/R) injury. In the intestine, I/R damage to the mucosa and neurons is prominent. Functionally, abnormalities occur in motility, most conspicuously a slowing of transit, possibly as a consequence of damage to neurons and/or muscle. Here, we describe degenerative and regenerative changes that have not been previously reported in intestinal muscle. The mouse small intestine was made ischemic for 1 h, followed by re-perfusion for 1 h to 7 days. The tissues were examined histologically, after hematoxylin/eosin and Masson’s trichrome staining, and by myeloperoxidase histochemistry to detect inflammatory reactions to I/R. Histological analysis revealed changes in the mucosa, muscle, and neurons. The mucosa was severely but transiently damaged. The mucosal surface was sloughed off at 1–3 h, but re-epithelialization occurred by 12 h, and the epithelium appeared healthy by 1–2 days. Longitudinal muscle degeneration was followed by regeneration, but little effect on the circular muscle was noted. The first signs of muscle change were apparent at 3–12 h, and by 1 and 2 days, extensive degeneration within the muscle was observed, which included clear cytoplasm, pyknotic nuclei, and apoptotic bodies. The muscle recovered quickly and appeared normal at 7 days. Histological evidence of neuronal damage was apparent at 1–7 days. Neutrophils were not present in the muscle layers and were infrequent in the mucosa. However, they were often seen in the longitudinal muscle at 1–3 days and were also present in the circular muscle. Neutrophil numbers increased in the mucosa in both I/R and sham-operated animals and remained elevated from 1 h to 7 days. We conclude that I/R causes severe longitudinal muscle damage, which might contribute to the long-term motility deficits observed after I/R injury to the intestine.