A Glimpse into Milestones of Insulin Resistance and an Updated Review of Its Management.

Insulin is the main metabolic regulator of fuel molecules in the diet, such as carbohydrates, lipids, and proteins. It does so by facilitating glucose influx from the circulation into the liver, adipose tissue, and skeletal myocytes. The outcome of which is subjected to glycogenesis in skeletal muscle and lipogenesis in adipose tissue, as well as in the liver. Therefore, insulin has an anabolic action while, on the contrary, hypoinsulinemia promotes the reverse process. Protein breakdown in myocytes is also encountered during the late stages of diabetes mellitus. The balance of the blood glucose level in physiological conditions is maintained by virtue of the interactive functions of insulin and glucagon. In insulin resistance (IR), the balance is disturbed because glucose transporters (GLUTs) of cell membranes fail to respond to this peptide hormone, meaning that glucose molecules cannot be internalized into the cells, the consequence of which is hyperglycemia. To develop the full state of diabetes mellitus, IR should be associated with the impairment of insulin release from beta-cells of the pancreas. Periodic screening of individuals of high risk, such as those with obesity, hypercholesterolemia, and pregnant nulliparous women in antenatal control, is vital, as these are important checkpoints to detect cases of insulin resistance. This is pivotal as IR can be reversed, provided it is detected in its early stages, through healthy dietary habits, regular exercise, and the use of hypoglycemic agents. In this review, we discuss the pathophysiology, etiology, diagnosis, preventive methods, and management of IR in brief.

Neuroleptic malignant syndrome: an easily overlooked neurologic emergency.

Neuroleptic malignant syndrome is an unpredictable iatrogenic neurologic emergency condition, mainly arising as an idiosyncratic reaction to antipsychotic agent use. It is characterized by distinctive clinical features including a change in mental status, generalized rigidity, hyperpyrexia, and dysautonomia. It can be lethal if not diagnosed and treated properly. Mortality and morbidity attributed to this syndrome have recently declined markedly due to greater awareness, earlier diagnosis, and intensive care intervention. In most cases, the syndrome occurs as a result of a rapid increase in a dose of neuroleptic, especially one of the long-acting ones. Pathophysiology behind this syndrome is attributed to a dopamine receptor blockade inside the neurons rendered by the offending drug and excessive calcium release from the sarcoplasmic reticulum of skeletal myocytes. Laboratory tests, although not diagnostic, may assist in assessing the severity of the syndrome and also the consequent complications. The syndrome has been described in all age groups and occurs more in males than in females. Genetics appears to be central regarding the etiology of the syndrome. Stopping the use of the offending agent, cold intravenous fluids, and removal of the causative agent and its possible active metabolites is the cornerstone of treatment. Periodic observation of psychotic patients recently started on antipsychotic medications, especially those being treated with depot preparations, may aid to an early diagnosis of the syndrome and lead to early treatment.

Lithium: a review of pharmacology, clinical uses, and toxicity.

A radical drug treatment for bipolar affective disorder (BD) is currently unavailable. This is attributed to the fact that the precise pathophysiology of this ailment is unclear though a genetic factor is an essential element in etiology. Dissimilar to other serious psychiatric categories such as psychoses and major depression the forecast of this disease is unpredictable. There is a high suicidal risk among BD affected individuals. In this review we will consider lithium, the drug of choice in treatment of this disorder with special emphasis on pharmacology and toxicity. We have also elucidated the alternatives to lithium, since it has a wide spectrum of side-effects. Lithium is known to interact with many types of drugs used to treat different ailments in humans. This could cause either augmentation or minimization of the therapeutic action, causing secondary undesired effects of the agent. This necessitates a search for other alternatives and/or different combinations to lithium in order to decrease the range of unwanted effects for which it has received discredit. These alternatives should be potent mood stabilizers as monotherapy so as to avoid polypharmacy. If not, one should find the best combination of drugs (synergistic agents) such that the lithium dose can be minimized, thereby securing a more potent drug therapy. This study also focuses on the provision of instruction to psychiatric care givers, such as junior doctors in residency, nurses in psychiatric units, psychiatric emergency personnel and, additionally, medical and pharmacy students.

CUG binding protein 1 binds to a specific region within the human albumin 3' untranslated region.

3' Untranslated regions (3'UTRs) of messenger RNAs have important roles in post-transcriptional regulation of gene expression and this is partly achieved through binding of specific proteins to sequences or structures within these regions. Previously, replacement of a native luciferase 3'UTR with the human albumin 3'UTR has been found to lead to a 10-fold increase in luciferase reporter activity. In this work we investigated protein binding to the human albumin 3'UTR. Electrophoretic mobility shift and UV cross-linking assays indicate that a ∼50kDa protein from Chinese Hamster Ovary (CHO) cells binds to the albumin 3'UTR, and affinity experiments followed by proteomics identified this protein as CUG binding protein 1 (CUG-BP1, also known as CELF1). Deletion analysis of the albumin 3'UTR showed that nucleotides 1-50 and nucleotides 101-150 are not required for binding but that removal of nucleotides 51-100 caused a loss in binding. The results suggest that CUG-BP1 binds to nucleotides 51-100 of the human albumin 3'UTR. In human cells CUG-BP1 binding may thus play a role in regulation of albumin expression and, additionally, it may have a function in post-transcriptional control in CHO cells.

Albumin 3'untranslated region facilitates increased recombinant protein production from Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are used for recombinant protein production in the pharmaceutical industry but there is a need to improve expression levels. In the present work experiments were carried out to test the effectiveness of different 3'untranslated regions (3'UTRs) in promoting production of a naturally secreted luciferase. Seamless cloning was used to produce expression vectors in which Gaussia princeps luciferase coding sequences were linked to the human albumin, immunoglobulin or chymotrypsinogen 3'UTR. Stably transfected CHO cells expressing these constructs were selected. Luciferase activity in the culture medium was increased 2-3-fold by replacing the endogenous 3'UTR with the albumin 3'UTR and decreased by replacement with immunoglobulin or chymotrypsinogen 3'UTR. Replacement of the native 3'UTR with the albumin 3'UTR led to a 10-fold increase in luciferase mRNA levels. Deletion analysis of the albumin 3'UTR showed that loss of nucleotides 1-50, which removed an AU-rich complex stem loop region, caused significant reductions in both luciferase protein expression and luciferase mRNA levels. The results suggest that recombinant protein expression and yield could be improved by the careful selection of appropriate 3'UTR sequences.

An intercalation mechanism as a mode of action exerted by psychotropic drugs: results of altered phospholipid substrate availabilities in membranes?

Patients respond differently to psychotropic drugs, and this is currently a controversial theme among psychiatrists. The effects of 16 psychotropics on cell membrane parameters have been reported. These drugs belong to three major groups used in therapeutic psychiatry: antipsychotics, antidepressants, and anxiolytic/hypnotics. Human platelets, lacking dopamine (D(2)) receptors (proposed targets of most psychotropics), have been used as a cell model. Here we discuss the effects of these drugs on three metabolic phenomena and also results from Langmuir experiments. Diazepam, in contrast to the remaining drugs, had negligible effects on metabolic phenomena and had no effects in Langmuir experiments. Psychotropic drugs may work through intercalation in membrane phospholipids. It is possible that the fluidity of membranes, rich in essential fatty acids, the content being influenced by diet, could be a contributing factor to the action of psychotropics. This might in turn explain the observed major differences in therapeutic response among patients.

In thrombin stimulated human platelets Citalopram, Promethazine, Risperidone, and Ziprasidone, but not Diazepam, may exert their pharmacological effects also through intercalation in membrane phospholipids in a receptor-independent manner.

Intercalation of drugs in the platelet membrane affects phospholipid-requiring enzymatic processes according to the drugs' intercalation capability. We investigated effects of Promethazine, Citalopram, Ziprasidone, Risperidone, and Diazepam on phospholipase A(2) (PLA(2)) and polyphosphoinositide (PPI) metabolism in thrombin-stimulated human platelets. We also examined effects of the drugs on monolayers of glycerophospholipids using the Langmuir technique. Diazepam did not influence PLA( 2 ) activity, had no effects on PPI cycle, and caused no change in mean molecular area of phospholipid monolayers. The remaining psychotropic drugs affected these parameters in different ways and levels of potency suggesting that they act by being intercalated between the molecules of adjacent membrane phospholipids, thus causing changes in substrate availability for phospholipid-hydrolyzing enzymes (PLA(2) and Phospholipase C). We show that several psychotropic drugs can also have other cellular effects than receptor antagonism. These effects may be implicated in the psychotropic effects of the drugs and/or their side effects.

Effects of psychotropic drugs on the thrombin-induced liberation of arachidonate in human platelets.

OBJECTIVE: To compare the effects of chlorpromazine (CPZ), prochlorperazine (PCP), trifluoperazine (TFP), clozapine (CLO), haloperidol (HPD), quetiapine (QTP), pimozide (PMZ), and olanzapine (OLP) as well as the tricyclic antidepressants amitriptyline AMI, imipramine IMI, and nortriptyline NTP on thrombin-induced liberation of arachidonic acid AA in platelets. METHODS: This work was carried out at the Department of Biomedicine, University of Bergen, Norway in 2006-2007. Human platelets pre labelled with [3H] arachidonate were incubated with thrombin in the absence and presence of the drugs, and the amount of free [3H] arachidonate liberated was determined. Myosin light chain (MLC) phosphorylation was determined in [32P] phosphate-labelled platelets after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The effects of the drugs on the molecular area and surface pressure of phospholipid monolayers were determined in the Langmuir apparatus. RESULTS: All drugs reduced arachidonate liberation with the ranking order of increasing potency: OLP

Psychotropic drugs interfere with the tight coupling of polyphosphoinositide cycle metabolites in human platelets: a result of receptor-independent drug intercalation in the plasma membrane?

Incubation of platelets with increasing concentrations of thrombin produced large amounts of phosphatidic acid (PA) and distinct changes in phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2), prominent metabolites in the polyphosphoinositide (PPI) cycle. The relation between normalized PA and PIP or PIP2 levels in such thrombin-treated platelets from 22 normal donors gave a very similar pattern, suggesting tight control of the metabolites in the polyphosphoinositide (PPI) cycle. Prochlorperazine (PCP), trifluoperazine (TFP), haloperidol (HPD), quetiapine (QTP), pimozide (PMZ) and clozapine (CLO) interfered with this tight coupling produced by treating platelets with increasing thrombin concentrations. All drugs decreased the formation of PA at a given thrombin concentration, a decrease that varied greatly among platelets from different donors. This made it difficult to treat the PIP/PA and PIP2/PA relationships with ordinary, descriptive statistics. The data were therefore subjected to regression analysis using polynomials of second or first degree and gave the interference ranking order: PCP>TFP>>PMZ = HPD>CLO>QTP. All six drugs increased the mean molecular area of monolayers of dipalmitoyl phosphatidylserine on pure water at 37 degrees C by 20-50%, while they had little effect on monolayers of dipalmitoyl phosphatidylcholine. These results suggest that the drugs are membrane-active and may intercalate in biomembranes containing negatively charged phospholipids. Since human platelets do not contain D2 receptors, the interference with the tight coupling of PPI cycle metabolites was not receptor-mediated. We suggest that the drugs are intercalated in the plasma membrane and alter the relative, spatial positioning of phospholipid-consuming enzymes and thereby alter the velocities of the enzyme-catalyzed reactions. Such intercalation could be part of the side effects of the drugs and may explain their psychotropic action(s).

The level of synthesis and secretion of Gaussia princeps luciferase in transfected CHO cells is heavily dependent on the choice of signal peptide.

There is a great demand for the improvement of mammalian cell production systems such that they can compete economically with their prokaryotic counterparts. Of a number of parameters that need to be explored to accomplish this we have tested the effects of different signal peptides on the synthesis and secretion of Gaussia princeps luciferase in mammalian cells. A series of plasmids were transfected into CHO cells where the coding region for the marine luciferase was fused to the signal peptide coding regions derived from different sources. Both cell extracts and medium samples were analysed for luciferase activity. When the native Gaussia luciferase signal sequence in the vector was substituted by that from human interleukin-2 or albumin then the amount of active recombinant protein produced was substantially reduced, both in transiently and stably transfected cells. Western blotting showed that enzyme activity and protein levels mirrored one another. The major decrease in luciferase activity was shown not to be a result of decreased mRNA levels, indicating the involvement of a post-transcriptional event. When the coding region of human endostatin was fused to that of the Gaussia luciferase signal peptide then an elevated level of secreted endostatin was observed compared to when that of the albumin signal peptide was used. Stable transfection of HepG2 cells with the different signal peptide constructs gave essentially the same results as seen in CHO cells. The overall results indicate that the choice of signal peptide can be imperative to ensure an optimal synthesis and secretion of a recombinant protein in a mammalian cell culture system.

Vectors encoding seven oikosin signal peptides transfected into CHO cells differ greatly in mediating Gaussia luciferase and human endostatin production although mRNA levels are largely unaffected.

The signal peptide of the luciferase secreted by the marine copepod Gaussia princeps has been shown to promote high-level protein synthesis/secretion of recombinant proteins, being far superior to mammalian counterparts. The main aim of the present study was to investigate the effects of seven selected signal peptides derived from oikosins, house proteins of the marine organism Oikopleura dioica, on synthesis/secretion of recombinant proteins. Vector constructs were made in which the coding regions of two naturally secreted proteins, Gaussia luciferase and human endostatin (hEndostatin), were "seamlessly" fused to the signal peptide coding sequences of interest. CHO cells were transfected with the plasmids and populations of stably transfected cells established. The amounts of reporter proteins in cell extract and medium samples were determined and the results compared to those obtained from cells stably transfected with a reference vector construct. In addition, the amounts of luciferase or hEndostatin encoding mRNAs in the cells were determined and related to the protein levels obtained. The levels of reporter protein produced varied greatly among the seven oikosin signal peptides tested. Whereas the oikosin 1 signal peptide resulted in about 40% production of Gaussia luciferase compared to the reference construct, oikosins 2-7 were extremely ineffective (<1%). mRNA levels were not dramatically affected such that inadequate availability of transcript for translation was not the underlying reason for the observations. The oikosin 1 signal peptide was also the most effective regarding synthesis/secretion of hEndostatin. No secreted product was observed using the oikosin 3 signal peptide. Interestingly, the molecular weight of hEndostatin in cell extracts prepared from cells transfected with oikosin 2 and 3 constructs was higher than that using the oikosin 1 signal peptide. The overall findings indicate that the signal peptide affects the efficiency of protein synthesis and secretion through a mechanism operating at the post-transcriptional level. The results described here provide substantial support to our previous observations which suggested that the choice of the signal peptide is imperative when aiming to achieve optimal synthesis and secretion of a recombinant protein using transfected mammalian cells.

A mistletoe lectin (ML-1)-containing diet reduces the viability of a murine non-Hodgkin lymphoma tumor.

In this study we show that the characteristics of non-Hodgkin lymphoma (NHL) tumors in female Naval Medical Research Institute (NMRI, USA) mice fed mistletoe lectin (ML)-containing diets were different from those in mice fed control diet. The non-Hodgkin lymphoma tumor was originally established from a spontaneous tumor which developed in the inguinal region of a male mouse. Mice (five animals per group) were fed a lactalbumin (LA)-based control diet or a diet which provided up to 10 mg lectin per day. At the highest daily intake (10 mg lectin) the degree of mitotic activity in tumors was reduced by 75% and the nuclear area had diminished by 21%. The overall level of lymphocyte infiltration (CD3 positive cells) in tumors from mistletoe lectin fed mice was increased by a factor of two. Other morphological studies showed a high incidence of apoptotic bodies in non-Hodgkin lymphoma tumors obtained from mice fed mistletoe lectin diets. The feeding of such diets thus produced several identifiable changes in the morphology of non-Hodgkin lymphoma tumors. These were consistent with the observed reduction in tumor mass. In 4/15 mice fed a mistletoe lectin diet for 11 days there was no longer evidence of viable tumor. The results show that this lectin exerts powerful anti-tumor effects when provided by the oral route.

In vivo studies on possible health consequences of genetically modified food and feed--with particular regard to ingredients consisting of genetically modified plant materials.

This synopsis reviews published in vivo studies on possible health consequences of genetically modified food and feed where the ingredients in question have consisted of genetically modified plant materials. The following, however, have not been taken into consideration:--ingredients consisting of genetically modified microorganisms or parts of animals/fish--ingredients produced by/from genetically modified organisms but without any DNA present--studies on consequences for the environment or biodiversity--in vitro studies or computer simulations. According to a Norwegian report "Gen-mat" (NOU 2000:29), and a more recent search in Medline and Citations Index, to our knowledge a total of ten studies have been published on the health effects of GM-foods and feeds. In this minireview the data made available in these published studies is discussed.

Sodium butyrate stimulates the synthesis of firefly luciferase in transfected CHO cells but levels of BiP chaperone are unaffected.

A stably transfected CHO cell line (LUCLEAD) was used where the coding region of native Firefly luciferase was linked to the 3'-UTR of the bovine growth hormone, and the 5'-nucleotides coding for the albumin signal peptide were linked to the N-terminal end of the luciferase coding region. Incubation of cells with 1 or 2 mM sodium butyrate (SB) for 72 h had no effect on cell growth since cultures reached confluency at the same time as control cells. Although cell cultures incubated with SB at a concentration of 4 mM were only about 60% confluent the luciferase content was about 5-fold higher than that in control cells. Cells incubated with either 1 or 2 mM SB showed intermediate levels of luciferase content. The amount of the chaperone BiP in the cells was not affected by incubation with SB. The results indicate that SB can be used to effectively promote synthesis of recombinant luciferase.