inositol-1-4-5-trisphosphate and Diabetes-Mellitus--Type-1

The present study was conducted to determine whether and how store-operated Ca(2+) entry (SOCE) in glomerular mesangial cells (MCs) was altered by high glucose (HG) and diabetes. Human MCs were treated with either normal glucose or HG for different time periods. Cyclopiazonic acid-induced SOCE was significantly greater in the MCs with 7-day HG treatment and the response was completely abolished by GSK-7975A, a selective inhibitor of store-operated Ca(2+) channels. Similarly, the inositol 1,4,5-trisphosphate-induced store-operated Ca(2+) currents were significantly enhanced in the MCs treated with HG for 7 days, and the enhanced response was abolished by both GSK-7975A and La(3+). In contrast, receptor-operated Ca(2+) entry in MCs was significantly reduced by HG treatment. Western blotting showed that HG increased the expression levels of STIM1 and Orai1 in cultured MCs. A significant HG effect occurred at a concentration as low as 10 mM, but required a minimum of 7 days. The HG effect in cultured MCs was recapitulated in renal glomeruli/cortex of both type I and II diabetic rats. Furthermore, quantitative real-time RT-PCR revealed that a 6-day HG treatment significantly increased the mRNA expression level of STIM1. However, the expressions of STIM2 and Orai1 transcripts were not affected by HG. Taken together, these results suggest that HG/diabetes enhanced SOCE in MCs by increasing STIM1/Orai1 protein expressions. HG upregulates STIM1 by promoting its transcription but increases Orai1 protein through a posttranscriptional mechanism.

Topics: Animals; Calcium Channel Agonists; Calcium Channel Blockers; Calcium Channels; Calcium Signaling; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Glucose; Humans; Inositol 1,4,5-Trisphosphate; Ion Channel Gating; Male; Membrane Glycoproteins; Membrane Proteins; Mesangial Cells; Neoplasm Proteins; ORAI1 Protein; Rats; Rats, Sprague-Dawley; RNA Processing, Post-Transcriptional; RNA, Messenger; Stromal Interaction Molecule 1; Time Factors; Transcriptional Activation; Up-Regulation

Diabetes is associated with a pro-inflammatory status characterized by an increased production of inflammatory molecules. Reactive oxygen species (ROS) and cAMP elevating agents represent two molecular systems, normally generated during inflammation. These molecules could be responsible for the alteration of signaling pathways. In the present paper we have studied the correlation between ROS generation and inositolpolyphosphates (InsP(1), InsP(2) InsP(3) and InsP(4)) released by granulocytes from Type 1 diabetic patients (DM1) in the presence or in the absence of cyclic AMP-elevating agents.. The effect of cAMP on ROS production was quantified in a chemoluminescence assay luminol-dependent (RLU/min). InsP(1), InsP(2) InsP(3) and InsP(4) were quantified by inositol-H(3) in a Beta-counter and the results were expressed as count per minute (CPM).. The elevation of intracellular level of cAMP inhibited both InsP(3) and ROS production in granulocytes from healthy subjects and activated in the cells from Type 1 diabetic patients. InsP(1), InsP(2) and InsP(4) did not show significant alteration in both studied cells. There was a significant correlation between InsP(3) and ROS in the presence of elevated content of cAMP. This correlation was observed in a 15 minutes reaction for healthy subjects and in 120 minutes for DM1.. The importance of both InsP(3) release and ROS production in an inflammatory process and tissue pathophysiology in Type 1 diabetic patients is still under debate because hyperglycemia accelerates generation of oxidative stress and may play an important role in the development of complications in diabetes. Thus, our results demonstrated alteration in metabolic response in granulocytes from Type 1 diabetic patients and it may be important for the development of therapeutic processes and drugs that interfere with signaling of ROS generation and may contribute to the improvement of the severe complications of diabetes.

Topics: Adult; Case-Control Studies; Cyclic AMP; Cyclic CMP; Diabetes Mellitus, Type 1; Granulocytes; Humans; Inositol 1,4,5-Trisphosphate; Kinetics; Middle Aged; Reactive Oxygen Species

The pathophysiological mechanisms responsible for diabetic gastroparesis remain unclear. Diabetes mellitus occurs spontaneously in 90% of a partially inbred colony of BB/W rats. This animal model resembles human insulin-dependent diabetes and is suitable for investigating the mechanism of diabetic gastroparesis. Diabetic BB/W rats were killed 6 mo after the onset of diabetes. Muscle contraction experiments and [3H]acetylcholine release studies were performed with muscle strips of the gastric body. Biochemical measurements of inositol trisphosphate (IP3) and protein kinase C (PKC) in gastric muscle were performed to characterize abnormalities of the intracellular signal transduction system in gastric myocytes. Circular muscle contractions in response to direct myogenic stimulants, carbachol (10(-7) - 10 (-3)M) or substance P (10(-7) - 10(-5)M), were significantly impaired in diabetic BB/W rats compared with controls. Similarly, muscle contractions in response to NaF (10 mM), a direct stimulant of G proteins, were also impaired in diabetic BB/W rats. In contrast, muscle contractions in response to KCl (25-75 mM) were similar between control and diabetic BB/W rats, indicating normal voltage-dependent Ca2+ entry in muscle strips obtained from diabetics BB/W rats. [3H]acetylcholine release from gastric myenteric plexus in response to electrical transmural stimulation remained intact in diabetic BB/W rats. In separate studies, we demonstrated that carbachol (10(-6) - 10(-4)M) -induced IP3 responses were significantly reduced in diabetic rats compared with control. In addition, there was also impairment of translocation of PKC in diabetic BB/W rats. These observations indicate that myogenic impairment occurred in diabetic BB/W rats. This resulted from altered intracellular signal transduction involving abnormal IP3 production and PKC translocation.

Topics: Acetylcholine; Animals; Calcium; Carbachol; Cell Membrane; Cells, Cultured; Cytosol; Diabetes Mellitus, Type 1; Electric Stimulation; Humans; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Male; Muscle Contraction; Muscle, Smooth; Potassium Chloride; Protein Kinase C; Rats; Rats, Inbred BB; Reference Values; Signal Transduction; Sodium Fluoride; Stomach; Substance P; Tetradecanoylphorbol Acetate