Are there any tissues that are insulin independent

The alpha chains are entirely extracellular and house insulin binding domains, while the linked beta chains penetrate through the plasma membrane.

The insulin receptor is a tyrosine kinase. In other words, it functions as an enzyme that transfers phosphate groups from ATP to tyrosine residues on intracellular target proteins. Binding of insulin to the alpha subunits causes the beta subunits to phosphorylate themselves autophosphorylation , thus activating the catalytic activity of the receptor.

The activated receptor then phosphorylates a number of intracellular proteins, which in turn alters their activity, thereby generating a biological response.

Several intracellular proteins have been identified as phosphorylation substrates for the insulin receptor, the best-studied of which is insulin receptor substrate 1 or IRS When IRS-1 is activated by phosphorylation, a lot of things happen. Among other things, IRS-1 serves as a type of docking center for recruitment and activation of other enzymes that ultimately mediate insulin's effects. A more detailed look at these processes is presented in the section on Insulin Signal Transduction.

Glucose is liberated from dietary carbohydrate such as starch or sucrose by hydrolysis within the small intestine , and is then absorbed into the blood. Elevated concentrations of glucose in blood stimulate release of insulin, and insulin acts on cells thoughout the body to stimulate uptake, utilization and storage of glucose.

The effects of insulin on glucose metabolism vary depending on the target tissue. Two important effects are:. Insulin facilitates entry of glucose into muscle, adipose and several other tissues.

The only mechanism by which cells can take up glucose is by facilitated diffusion through a family of hexose transporters. In many tissues - muscle being a prime example - the major transporter used for uptake of glucose called GLUT4 is made available in the plasma membrane through the action of insulin.

When insulin concentrations are low, GLUT4 glucose transporters are present in cytoplasmic vesicles, where they are useless for transporting glucose. Binding of insulin to receptors on such cells leads rapidly to fusion of those vesicles with the plasma membrane and insertion of the glucose transporters, thereby giving the cell an ability to efficiently take up glucose. When blood levels of insulin decrease and insulin receptors are no longer occupied, the glucose transporters are recycled back into the cytoplasm.

It should be noted here that there are some tissues that do not require insulin for efficient uptake of glucose: important examples are brain and the liver. This is because these cells don't use GLUT4 for importing glucose, but rather, another transporter that is not insulin-dependent. Insulin stimulates the liver to store glucose in the form of glycogen.

A large fraction of glucose absorbed from the small intestine is immediately taken up by hepatocytes, which convert it into the storage polymer glycogen. Insulin has several effects in liver which stimulate glycogen synthesis.

First, it activates the enzyme hexokinase, which phosphorylates glucose, trapping it within the cell. Coincidently, insulin acts to inhibit the activity of glucosephosphatase. Image processing was performed with the included ZEN software.

Cell quantifications were performed with Neurolucida. Fluorescence intensity was measured with the ImageJ 1. Student t -test was used for two-group comparisons.

Since the insulin-dependent transporter GLUT4 was previously assessed in hypertension models and in diabetes as a model of small vessel disease, we focused on this specific transporter for the gene product studies in relation to the insulin-independent GLUT2.

Figure 1. These data together suggest that the glucose uptake receptors are impaired in these arterial cells. Figure 2. The samples were visualized using the Odyssey CLx Imager. The immunoblot is representative of one experiment. Validation of glucose uptake was carried out using 2-NDBG, a fluorescent indicator for monitoring glucose uptake into living cells, which could be assessed by flow cytometry. These analyses indicated that the reduced gene expression was in line with an overall functional effect.

Figure 3. The 2-NBDG uptake was measured by flow cytometry. To further confirm the above findings, we assessed the extent to which insulin could rescue the deficit in glucose uptake in the cellular model of CADASIL. We also found that carnitine palmitoyltransferase I Cpt1 , specifically the Cpt1c isoform, highly expressed in brain Schreurs et al. This indicated that the reduced glucose uptake in VSMCs was not associated with changes in beta-oxidation of fatty acids and insulin sensitivity in the VSMCs.

Preliminary observations indicated that there was also a correlation between vessel diameter and GLUT4 expression data not shown , but further investigation is needed to confirm this finding. Figure 4. A strong GLUT4-immunostaining in control within the cortex was observed.

ArgCys mutation, C p. ArgCys, and D p. ArgRCys mutation is shown. In accord with our previous work Craggs et al. Figure 5. ArgCys mutation. Figures 4E , 5C. It is difficult to set specific thresholds on what degree of reduction in GLUTs would alter physiological conditions. However, we were careful to compare like VSMCs from controls and patients that were isolated, maintained and cultured in parallel in a similar manner.

GLUT4 has also previously been shown to exhibit an altered expression level in arterial myocytes in different disease models Marcus et al. Thus, if the vascular entry routes for glucose are impaired or that arterial muscle cells exhibit reduced GLUTs arteriosclerosis may ensue to affect perfusion and flow affecting the WM. Among the known GLUTs, GLUT4 is the main insulin-dependent transporter, which makes it a suitable candidate for developing a potential medical intervention.

Indeed, our functional glucose uptake experiment indicated that a significant proportion of glucose uptake was through non-insulin dependent transporters. Since CADASIL is largely characterized as a cerebral small vessel disease with consequent brain pathology and dysfunction we focused on the brain. Furthermore, the brain microvasculature is more of interest because it is endowed with the blood-brain barrier unlike systemic the vessels.

Further research is needed to clarify the disruption of glucose metabolism in VSMCs derived from other organs and ascertain whether this phenomenon is only seen in VSMCs derived from brain vessels. Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy causes degeneration in the walls of small vessels of the brain leading to lacunar infarcts and leukoencephalopathy.

Like in other diseases, vascular risk factors such as hypertension and diabetes, and lifestyle modifications including obesity and smoking can also exacerbate disease progression. The same factors might also affect expression of GLUT2, as evident in the current study. However, given that in this study we used CADASIL brain tissue collected post-mortem, it is impossible for us to definitively control for such variables.

We gathered tissue sections from different Brain Bank sources and some of these tissues are from different individuals that have the same mutation on the NOTCH3 gene.

Another limitation in this study is that the in vitro data was based on a few available cerebral VSMCs cell lines models. It was too small to address the effect of various mutations known to cause this disease. Animal models would be of interest in following up the findings in this study.

Since all of our experimental material is obtained from human subjects with varying genetic backgrounds, it would be most interesting to study the same phenotype using animals with the same genetic background. It would also be noteworthy to study glucose metabolism in VSMCs in animals that have both copies of NOTCH3 mutated, considering that in our experiments the mutations are heterozygous.

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