Biomedical subjects

S Azhar

Publications and source records attributed to S Azhar.

At least 55 records · Page 3Linked to original sources

PubMed1996

Isoproterenol decreases LDL receptor expression in rat adipose cells: activation of cyclic AMP-dependent proteolysis.

The low density lipoprotein (LDL) receptor is part of a family of proteins that mediate the uptake of lipoproteins into cells. In this paper we have demonstrated the over-expression in E. coli of a rat LDL receptor fusion protein that contains the region of the receptor sharing homology with the EGF precursor. The fusion protein was utilized to immunize rabbits and successfully generate antibodies that recognize the intact LDL receptor. These anti-LDL receptor/fusion protein antibodies were used to examine the effects of cyclic AMP on the expression of LDL receptors in isolated rat adipocytes. Incubation of adipocytes with isoproterenol caused a dose-dependent diminution in intact LDL receptors in the plasma membrane with the concomitant appearance of smaller immunoreactive proteins. Pulse-chase experiments demonstrated that isoproterenol rapidly shortened the initial half-life of intact, immunoprecipitable LDL receptors in the plasma membrane. The effects of isoproterenol on LDL receptor expression were mimicked by forskolin, by an analog of cyclic AMP, and by ACTH. In contrast, incubation with propranolol blocked the effects of isoproterenol on LDL receptor expression. While antioxidants and several different protease inhibitors had no effects, N-acetyl-leucine-leucine-methionine (ALLM) was able to prevent the isoproterenol-induced effects on LDL receptors. Thus, it appears that agents acting via cyclic AMP cause a rapid decrease in LDL receptors in the plasma membranes of isolated adipose cells due to the apparent stimulation of an ALLM-sensitive protease that degrades the LDL receptor. These results suggest a novel mechanism for the posttranscriptional regulation of LDL receptor expression in adipocytes.

Adipocytes↗

PubMed1995

Generation of antibodies against a human lipoprotein lipase fusion protein.

Antibodies generated against specific proteins are useful tools for studying the physiology and cell biology of the protein of interest. Although antibodies have been successfully generated against lipoprotein lipase (LPL) and used to elucidate many aspects of its biology, there have been problems with the specificity, affinity and availability of these antibodies. To circumvent these problems, we have expressed a portion of human LPL as a bacterial fusion protein. The human LPL bacterial fusion protein was utilized to generate polyclonal antibodies in rabbits that recognize intact human, rat and bovine LPL. Using these antibodies, it was possible to demonstrate a direct correlation between LPL mass and LPL activity from different samples of human post-heparin plasma. In addition, these antibodies were used to develop an ELISA for the measurement of LPL in tissue or plasma. This is a useful means for obtaining polyclonal antibodies to LPL in sufficient quantity and without contaminating mammalian proteins.

Animals↗

PubMed1995

Expression of multiple isoenzymes of protein kinase C in airway smooth muscle.

Protein kinase C (PKC) has been implicated in the control of airway smooth muscle (ASM) tone, and abnormalities in PKC-dependent signaling may be associated with asthma. PKC exists in different isoforms, but the pattern of their expression in ASM has not been previously reported. Accordingly, the purpose of the present study was to identify which isoforms of PKC are expressed in ASM. Tissue samples of canine ASM were homogenized and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by immunoblotting with a range of antipeptide antibodies to PKC-alpha, -beta I, -beta II, -gamma, -delta, -epsilon, -eta, -theta, and -zeta. Positive controls were run in parallel with ASM. Immunoblots revealed a mixture of both calcium-dependent and calcium-independent isozymes: PKC-beta I and PKC-beta II were the only conventional isoforms detected; PKC-delta, PKC-epsilon, and the new muscle-specific isoform, PKC-theta, were all expressed in ASM, but the lung-specific isoform, PKC-eta, was not detected. The calcium- and phospholipid-independent isoform, PKC-zeta, was also present. Thus, expression of a wide variety of both calcium-dependent and calcium-independent isoforms suggests a complex, multifunctional role of PKC in ASM.

Animals↗

PubMed1995

Alteration of the adrenal antioxidant defense system during aging in rats.

The goal of this study was to determine to what extent aging affects the antioxidant defense system of the rat adrenal and to evaluate the impact of any change in this system on the recognized age-related decline in steroidogenic capacity of adrenocortical cells. The studies were conducted on young (2-5 mo) and aging (12-27 mo) Sprague-Dawley rats and involved procedures measuring steroidogenesis; oxidative damage to tissue; non enzymatic antioxidants such as vitamin C, E, and glutathione; and tissue antioxidant enzyme (Mn and CuZn superoxide dismutases, catalase, and glutathione peroxidase) activity and expression (mRNA, protein mass, and location). Some measurements were made also on rats maintained on vitamin E-deficient diets. The data show that adrenals from young animals are especially well protected against oxidative events; i.e., these adrenals show the least endogenous lipid peroxidation and the highest level of resistance to prooxidant-induced damage (of various tissues measured) and show exceedingly high levels of tissue antioxidants. Aging, on the other hand, results in oxidative changes in adrenal tissue that are generally linked in time to a reduction in efficiency of the normally protective antioxidant defense system and to the decline in corticosterone production. We speculate that these events are causally related, i.e., that the age-related reduction in oxidative mechanisms in adrenal tissues leads to oxidative damage of membrane or cytosolic factors important to cholesterol transport, and, as a consequence of this damage, cholesterol cannot reach appropriate mitochondrial cholesterol side chain cleavage sites, and corticosterone production fails.

Adrenal Cortex↗

PubMed1995

Cholesterol uptake by the 'selective' pathway of ovarian granulosa cells: early intracellular events.

Although the 'selective' pathway is a major cholesteryl ester (CE) uptake pathway used by steroidogenic cells, essentially nothing is known about the itinerary of the CE once it is extracted from lipoproteins at the cell surface. In the current report we have begun to trace 'selective' pathway internalized-CE using both native and reconstituted human (h) high density lipoproteins (hHDL3) with variously labeled and tagged CEs to provide information from either a biochemical or morphological perspective. It appears that the amount of hHDL3-CE that is internalized and processed through the 'selective' pathway is directly related to the amount of cholesterol used for steroidogenesis at any given time point. There is a time-related correlation between the level of the Bt2cAMP-stimulated cell steroidogenic response, the level of conversion of freshly obtained hHDL3-CE into progestins, increases in hHDL3-derived CE internalization, hHDL3-CE hydrolysis, re-esterification and/or storage. None of this processing takes place in non-stimulated (non-Bt2cAMP treated) cells which do not secrete progestins despite the availability of hHDL3 as a cholesterol source. The data suggest that the 'selective' pathway has a special role in steroidogenic cells-one of providing sufficient cholesterol to fuel the required production of steroid hormones.

Animals↗

PubMed1995

Changes in insulin receptor tyrosine kinase activity associated with metformin treatment of type 2 diabetes.

This study was performed to define the effect of metformin on glycaemic control and erythrocyte insulin receptor tyrosine kinase activity in patients with non-insulin-dependent (Type 2) diabetes mellitus. A case-control study of the effect of metformin treatment in hyperglycaemic patients with Type 2 diabetes was conducted in outpatients of the Diabetes Clinical Center. The study population consisted of 14 patients with Type 2 diabetes (5 males, 9 females) whose hyperglycaemia was uncontrolled by diet. Patients were treated with metformin 850 mg twice daily for 2 1/2 months. Fasting plasma glucose concentrations decreased from 8.9 to 6.4 mmol/L after 10 weeks of metformin treatment (p < 0.001), in association with significantly lower (p < 0.001) plasma glucose and insulin concentrations in response to an oral glucose load. In addition, both fasting plasma triglyceride and cholesterol concentrations were significantly (p < 0.001) lower after metformin treatment. There was no change in erythrocyte insulin receptor binding associated with metformin treatment, but both basal and insulin-stimulated insulin receptor tyrosine kinase activities of solubilized erythrocyte insulin receptors were significantly higher after 10 weeks of metformin treatment. It is concluded that the increase in insulin-stimulated tyrosine kinase activity contributed to the improvement in glucose insulin and lipoprotein metabolism associated with metformin treatment of Type 2 diabetes.

Adult↗

PubMed1994

Effect of age on cholesterol uptake and utilization by rat adrenals: I. Internalization of lipoprotein-derived cholesteryl esters.

Previous studies from this laboratory have documented a progressive age-related decline in trophic hormone (or second messenger cAMP) stimulated corticosterone production in isolated adrenocortical cells. In the current study, we examined the possibility that the aging process exerts this effect by interfering with an early step in the delivery of lipoprotein-derived cholesteryl esters to the cell. As such, we monitored the ability of two different rat adrenocortical cell model systems (intact perfused adrenal glands and primary cultures of adrenocortical cells from 5- and 18- to 20-month-old rats) to internalize lipoprotein cholesteryl esters, and to convert the newly internalized cholesteryl esters to corticosterone production. The results indicate that lipoprotein (hHDL3 and rHDL) cholesteryl ester internalization (by both the endocytic and 'selective' pathways) is comparable in adrenocortical cells of the young and old rats. However, despite this, both the mass of corticosterone produced and the ratio of newly internalized (radiolabeled) cholesteryl ester incorporated into corticosterone is dramatically reduced in cells of the older animals. Thus, the lipoprotein uptake pathway appears to be intact in adrenals of older rats, but the intracellular processing of internalized cholesteryl ester is defective.

Adenine↗

PubMed1994

Effect of age on cholesterol uptake and utilization by rat adrenals: II. Lipoproteins from young and old rats.

The current study examines whether age-related changes in high density lipoproteins (HDL) influences how these particles are handled by adrenal cells. It appears that HDL from 18- to 20-month-old Sprague-Dawley rats show a seven- to eightfold increase in content of apolipoprotein E compared to HDL from 2- to 5-month-old rats. The 'aged' particles show increased binding to susceptible hepatic membranes, and show a doubling in whole particle endocytosis by cortical cells of the perfused adrenal gland and by isolated adrenal cells from all rats regardless of age. Despite this twofold increase in particle uptake, the increase in total cholesteryl ester uptake by either the perfused adrenal or incubated adrenal cells is minor, amounting to less than 10% of the total cholesteryl ester internalized. This discrepancy occurs since the high apo E content of the 'aged' HDL only affects cholesteryl ester uptake by the 'endocytic' pathway; uptake via the 'selective' pathway (where cholesteryl ester is separated from the rest of the particle at the cell surface and directly internalized) is not altered.

Adrenal Cortex↗

PubMed1994

Enhanced expression of granulosa cell low density lipoprotein receptor activity in response to in vitro culture conditions.

Previous studies have shown that the B/E (low density lipoprotein [LDL]) receptor pathway plays a minor role in cholesterol uptake in the intact rat ovary, but when granulosa cells are isolated and maintained in culture, the cells develop a fully functional B/E receptor system. In the current study we examined the development of the B/E receptor over time (96 h) in culture and compared its physiological function, expression of mRNA and protein levels, and morphological events to the upregulation induced in 24 h by hormone (human chorionic gonadotropin [hCG] or Bt2cAMP). With both protocols, increased progestin production occurs and is associated with elevated binding, uptake, and degradation of LDL in the medium although the impact of Bt2cAMP stimulation on all these measurements is several times that observed with time alone. Only the hormone-stimulated LDL receptor response was associated with an increase in receptor protein (Western blot) or mRNA levels (RNase protection assay). We conclude that unstimulated granulosa cells show posttranslational increases in B/E receptor activity with time in culture, but transcriptional changes in B/E receptor follow stimulation with trophic hormone or its second messenger, cAMP.

Animals↗

PubMed1994

Glucose transporter (GLUT 4) content and insulin receptor kinase activity in muscles of the LA/N-cp rat.

In this study we compared insulin binding activity, insulin receptor tyrosine kinase activity, and GLUT 4 protein content in six muscles from LA/N-cp rats and their lean controls. LA/N-cp rats had an approximate 20-fold increase in insulin concentration (837 +/- 113 vs 40 +/- 1), associated with significant (p < 0.01) decreases in both insulin binding activity per mg muscle and in muscle GLUT 4 content. Maximum insulin tyrosine kinase activity was also lower in muscle from LA/N-cp rats, but no difference was noted when tyrosine kinase activity was expressed per receptor. These data indicate that there are at least two defects in the insulin action cascade in muscle from LA/N-cp rats that contribute to the insulin resistance in these animals.

Animals↗

PubMed1994

Insulin-receptor tyrosine kinase activity is decreased in erythrocytes from non-obese patients with NIDDM.

We have examined insulin binding, and insulin receptor associated tyrosine kinase activity in detergent solubilized and Ricin II-agarose purified receptor preparations from erythrocytes of obese and non-obese subjects with normal glucose tolerance and non-obese patients with NIDDM. Insulin receptor activity, as assessed by [125I Tyr A14] insulin binding, was significantly lower in erythrocyte preparations from the obese group when compared with similar preparations from non-obese subjects, with either normal glucose tolerance or NIDDM. The affinity of the receptor for insulin, however, was reduced in both obese subjects and patients with NIDDM as compared to non-obese subjects with normal glucose tolerance. Insulin receptor tyrosine kinase activity, measured in the absence (basal) and presence of insulin (0.3-3000 nM), was decreased in obese and NIDDM subjects with normal glucose tolerance and in patients with NIDDM. Insulin sensitivity, measured as the dose of insulin required for half-maximal activation of kinase activity, however, was comparable among three groups. In contrast, insulin-stimulated tyrosine kinase activity, when normalized to insulin binding activity, was unchanged in both non-obese and obese subjects with normal glucose tolerance, but was reduced approximately 60% in the NIDDM group. These findings indicate that the functional behavior of insulin receptor-kinase signaling system is markedly impaired in non-obese patients with NIDDM. Furthermore, the insulin receptor-tyrosine kinase defect (i.e. decrease in activity) observed in patients with NIDDM is probably related to a reduction in coupling efficiency between insulin binding and the activation of the receptor tyrosine kinase activity.

Adult↗

PubMed1994

Expression of the major isoenzyme of protein kinase-C in skeletal muscle, nPKC theta, varies with muscle type and in response to fructose-induced insulin resistance.

The ability of insulin to stimulate glucose uptake by skeletal muscle varies as a function of muscle type, but the biochemical mechanisms that regulate insulin-mediated glucose transport under normal conditions and in pathological states of insulin resistance are poorly understood. We evaluated differences in the expression of nPKC theta (the major isoform of protein kinase-C in skeletal muscle) in hind limb muscles of different fiber type composition from normal Sprague-Dawley rats and rats made insulin resistant by feeding a fructose-enriched diet. In total muscle homogenates from normal rats, the amount of nPKC theta per unit total protein quantified by immunoblotting using a specific antipeptide antibody was 2.5 times higher in pure white muscle (tensor fascia latae) compared with red muscle (soleus), with two mixed muscles showing intermediate expression. The development of insulin resistance after a fructose-enriched diet was associated with significant increases in diacylglycerol and nPKC theta mass in the membrane fraction of tensor fascia latae, but fructose feeding had no effect on conventional PKC enzyme activity and immunoreactive protein. Thus, expression of nPKC theta varies as a function of muscle type, and fructose-induced insulin resistance appears to be associated with diacylglycerol-mediated isoenzyme-specific changes in nPKC theta in white muscle.

Animals↗

PubMed1994

Potability of water obtained through boring in Karachi.

This study was carried out to detect the faecal contamination in water obtained from indigenously designed boring facilities. The presence of escherichia coli (major indicator of faecal contamination) was detected after performing the coliform test along with biochemical studies in 32 boring water samples out of 60 samples collected from various localities of Karachi city. Its presence in underground water resources definitely indicates the possible presence of water-borne pathogens.

Escherichia coli↗

PubMed1994

Effect of okadaic acid on utilization of lipoprotein-derived cholesteryl esters by rat steroidogenic cells.

This study examines various functional, biochemical, and structural changes in rat adrenocortical and ovarian granulosa cells that could account for the decline in lipoprotein-supported hormone production after cell treatment with the protein phosphatase inhibitor, okadaic acid. Although the steroidogenic pathway enzymes in these cells are not in themselves affected by okadaic acid, the intracellular transport of cholesterol to important cellular processing sites is defective. That is, okadaic acid does not interfere with the internalization of lipoprotein-derived cholesteryl esters, but the mitochondrial utilization of cholesterol obtained from intracellular cholesterol storage sites is 50% reduced as compared to control cells. Two-dimensional electrophoresis gels from okadaic acid-treated cells demonstrate a number of hyperphosphorylated proteins. Morphological examination of the affected cells reveal completely disrupted Golgi complexes with attendant structures, but otherwise the cells appear unchanged. The results suggest that some necessary sterol transport protein (or cofactor or associated membrane) is adversely phosphorylated by okadaic acid, and is rendered dysfunctional.

Animals↗

PubMed1993

The mechanism of equilibrium binding of microtubule-associated protein 2 to microtubules. Binding is a multi-phasic process and exhibits positive cooperativity.

The mechanism of binding of microtubule-associated protein 2 (MAP2) to taxol-stabilized microtubules (MTs) was examined through Scatchard analysis of equilibrium binding and by immunoelectron microscopy. We demonstrate the following. 1) Binding is a cooperative process as indicated by sigmoidal binding curves, prominent humps in Scatchard plots, and an all-or-none response in binding during ligand titrations. At high tubulin/MAP2 ratios, the Kd for noncontiguous binding (5-25 microM) is estimated to be 100-1500 times greater than that predicted for contiguous binding, suggesting a high degree of cooperativity. 2) Cooperativity is indicated independently by a highly clustered or patchy distribution of MAP2 on MTs as revealed by immunoelectron microscopy. 3) The binding of truncated constructs of mouse MAP2 protein suggests that a domain of MAP2 conferring cooperativity is located in or near the MT binding site near the carboxyl terminus. We speculate that in the cell, cooperativity may generate MTs with uniform biochemical properties and contribute to the segregation of MAPs in neuronal cell processes.

Animals↗

PubMed1993

Pathogenesis of cholesteryl lipidosis of adrenocortical and ovarian interstitial cells in F344 rats caused by tricresyl phosphate and butylated triphenyl phosphate.

Triaryl phosphates including tricresyl phosphate (TCP) and butylated triphenyl phosphate (BTP) are organophosphates used in the commercial manufacture of plastics, lubricants, and hydraulic fluids. Rat steroidogenic tissues such as adrenocortical (AC), ovarian interstitial (OI), and Leydig cells use an intracellular pathway to store cholesterol (substrate for biosynthesis of steroid hormones) as cholesteryl ester (CE). This pathway and the pathway for uptake of serum cholesterol are less used in Leydig cells of the adult male rat, resulting in a lower CE pool. BTP and TCP caused cholesteryl lipidosis in steroid hormone-synthesizing AC and OI, but not Leydig cells in the adult rat. The objectives of this study were to determine if the administration of triaryl phosphate fluids caused a defect in the cholesterol storage pathway of AC and OI cells and to determine the mechanism of action. Female rats received daily oral doses of 0 or 0.4 g/kg TCP in sesame oil vehicle or 1.7 g/kg neat BTP for 40 days. Adrenal glands from both treatment groups and ovaries from TCP-treated rats were heavier than controls. Microscopic and biochemical studies revealed cholesteryl lipidosis composed of CE in the adrenal glands and ovaries in BTP- and TCP-treated rats with the latter group affected most severely. The activity of neutral CE hydrolase (nCEH), an enzyme that converts CE to cholesterol in the uptake and storage pathways, also was inhibited most in the TCP-treated group (97% inhibition compared to that of control). The activity of acyl coenzyme A:cholesterol acyl transferase, an enzyme that esterifies cholesterol to make CE, was depressed 27% compared to that of control adrenal glands of the TCP group, resulting in elevated intracellular cholesterol levels in AC cells. An inhibition of nCEH in the storage and uptake pathways by triaryl phosphates most likely resulted in the striking accumulation of CE in cytoplasmic lipid droplets of AC and OI cells in F344 rats.

Adrenal Cortex Diseases↗

PubMed1993

Age-related decline in the steroidogenic capacity of isolated rat Leydig cells: a defect in cholesterol mobilization and processing.

This study was designed to evaluate the effects of aging on steroidogenesis and intracellular cholesterol processing in rat Leydig cells. Maximum gonadotropin-induced testosterone secretion was significantly reduced in Leydig cells from 18 to 27-month-old rats compared to 2 to 5-month-old rats. The decreased production of testosterone in older groups persisted after incubation with cAMP analogs or other non-specific stimulatory agents. This age-related loss in testosterone response was not due to changes in gonadotropin receptor concentration, cAMP concentration, protein kinase A activation or the activity of key steroidogenic enzymes. The content of cellular cholesteryl esters doubled as rats aged from 5 to 18 months, and this high cholesteryl esters level remained constant through 27 months. The ability of hCG to mobilize (hydrolyze) stored cholesteryl ester for testosterone production was significantly reduced (65-75%) in cells from the older rats. This change could be accounted for by the decline in activity of neutral cholesteryl esterase in Leydig cells from 18-month-old rats. In contrast, the activity of a non-specific lysosomal acidic cholesteryl esterase did not change with age. The activity of HMG CoA reductase, the rate limiting enzyme in cholesterol biosynthesis decreased about 70% between 5 and 18 months and fell slightly further as the rats aged to 27 months. Also, [14C]acetate or [3H]H2O incorporation into cellular sterols showed a similar decline. Cyanoketone plus hCG stimulated pregnenolone production was reduced about 70-80% in old as compared to young cells. Leydig cells from young rats responded to hCG with increased accumulation of mitochondrial cholesterol in the presence and absence of steroidogenic inhibitors. On the other hand, old cells responded poorly to hCG and mitochondrial cholesterol levels were little affected by hCG plus cycloheximide or aminoglutethimide. Together, these data indicate that alterations in the intracellular processing and metabolism of cholesteryl esters occur in Leydig cells of aging rats, and we suggest they may be responsible for the observed age-related changes in testosterone production.

Aging↗

PubMed1993

Why does insulin resistance develop during maturation?

We compared skeletal muscle glucose uptake between young and mature rats. Hindlimb perfusions at insulin concentrations of 0, 100, 250, or 10,000 microU/mL were performed on male Sprague-Dawley rats at 5 weeks or 4 months of age. Basal glucose uptake, and glucose uptake at all insulin concentrations were significantly lower in the 4-month-old mature rats (p < .05). This difference was most pronounced at maximally stimulating insulin concentrations. Skeletal muscle insulin receptor binding, autophosphorylation, and tyrosine kinase activity did not differ between young and mature rats. Surprisingly, GLUT-4 glucose transporter content was significantly higher in several muscles of the mature rats (p < .05). Therefore, the decline in insulin-stimulated glucose uptake in hindlimbs of mature rats cannot be explained by decreased activity of these steps in the glucose transport system.

Aging↗