There are an estimated 20 million people in the United States who have some form of peripheral neuropathy. Peripheral neuropathy also is the most prevalent complication of both type 1 and type 2 diabetes and affects approximately 60 to 70 percent of patients with diabetes. Clinically, diabetic neuropathy exhibits a pronounced similarity to the neuropathy of patients with hereditary sensory and autonomic neuropathy type 1 (HSAN1). HSAN1 patients exhibit elevated plasma levels of a newly identified sphingolipid class, deoxysphingolipids (DSL) and DSL were shown to have pronounced neurotoxic effects on cultured sensory neurons. We have shown that plasma levels of deoxy-C24-ceramide, one species of DSL, are elevated in neuropathy patients with type 1 diabetes. Furthermore, we now provide compelling new evidence that both VLDL with DSL content supple- mented in vitro, and VLDL from a diabetic patient with neuropathy with DSL levels increased in vivo, each can modify the DSL composition of membranes from human Schwann cells and alter matrix metalloproteinase (MMP) metabolism in the cell. It is not known if the increases in plasma DSL concentration we observed in patients with neuropathy precede the onset of neuropathy. It also is not known if the increase in DSL level in plasma from patients with neuropathy is localized to a specific lipoprotein class, nor how DSL in lipoproteins might alter neural cell metabolism and contribute to neuropathy. Our overarching hypothesis is that plasma sphingolipid levels are altered in patients with complications that commonly associate with diabetes mellitus compared to sphingolipid concentrations in patients without complications. More specific to this application, we hypothesize that the increased plasma levels of DSL which we observed in neuropathy patients with type 1 diabetes precede the onset of neuropathy. We further hypothesize that the increase in plasma DSL level a) is localized primarily to VLDL, and b) alters matrix metalloproteinase metabolism in human Schwann cells. To test these hypotheses we will:
Aim 1 - Determine if plasma DSL concentrations measured at the end of the DCCT (1993) in patients with type 1 diabetes can predict the development of incident peripheral neuropathy cases through EDIC year 13/14 (2007). We will determine the concentrations of DSL in previously collected, banked plasma samples collected at the end of the DCCT from 1,375 patients with type 1 diabetes and determine their ability to predict the development of incident peripheral neuropathy up to 14 years later (EDIC year 13/14). Additionally, we will conduct a cross-sectional analysis of these data to determine whether plasma DSL levels determined at DCCT closeout are elevated in patients with prevalent neuropathy compared to DSL levels in patients without neuropathy when neuropathy is defined using the more stringent DCCT definition compared to the disease criteria employed in our original investigations.
Aim 2 - Determine if the elevated plasma DSL levels observed in patients with type 2 diabetes with neuropathy are secondary to increased DSL levels in the VLDL, LDL or HDL lipoprotein fractions. We will compare the concentrations of DSL in plasma and in VLDL, LDL, and HDL prepared from 25 Veterans with type 2 diabetes and neuropathy to DSL levels determined in samples from 25 age-, race-, and gender-matched patients without neuropathy to determine if VLDL is the primary lipoprotein fraction which transports DSL in plasma.
Aim 3 - Determine whether changes in the DSL composition of plasma membranes isolated from human Schwann cells after incubation with lipoproteins isolated from patients with type 2 diabetes and neuropathy will elicit changes in cell matrix metalloproteinase metabolism. Using the lipoproteins prepared from the same Veterans recruited in Aim 2, we will incubate each lipoprotein fraction with human Schwann cells, and compare the DSL composition of cell plasma membranes that results after the incubation with lipoproteins from patients with, compared to those without neuropathy and relate the changes in Schwann cell membrane DSL composition to alterations in Schwann cell matrix metalloproteinase (MMP) and tissue inhibitor of matrix metalloproteinase (TIMP) metabolism.
Sphingolipids are complex lipid molecules that control a variety of cellular functions. Knowledge of the transport of these lipids in blood and their contribution to the development of neuropathy is limited. We will measure the amounts of these lipids in blood from Veterans with type 1 diabetes without neuropathy and determine if differences in the blood levels of specific sphingolipids can predict the future development of neuropathy. We also will investigate potential mechanisms whereby sphingolipids in lipoproteins may contribute to diabetic neuropathy by incubating human Schwann cells in culture with lipoproteins prepared from Veterans with neuropathy and determine the changes in cell metabolism which result. These are the first studies to analyze the sphingolipid composition in blood from Veterans with the neuropathy associated with diabetes and most importantly, to determine the potential for plasma sphingolipid concentrations to predict the development of neuropathy in Veterans with diabetes.