The anti-diabetogenic effect of essential fatty acid deficiency in multiple low-dose streptozotocin-treated mice persists if essential fatty acid repletion occurs outside of a brief window of susceptibility

Abstract

We have previously shown that essential fatty acid deficiency prevents diabetes mellitus and ameliorates insulitis in multiple low-dose streptozotocin-treated male CD-1 mice and that repletion with 99% pure methyl linoleate 3 days after the last injection causes diabetes. In the present study, we examined whether repletion of low-dose streptozotocin-treated deficient mice will cause diabetes whenever repletion occurs. Essential fatty acid deficiency was induced by dietary manipulation and was confirmed biochemically. Groups of deficient mice were repleted 6 h, 1 week, 2 weeks, 4 weeks and 8 weeks after the last low-dose streptozotocin treatment; the incidence of diabetes (i.e., non-fasting plasma glucose levels greater than 11.2 mmol/l) and group mean plasma glucose levels were 93% (19.4 mmol/l), 37% (14.8 mmol/l), 40% (13.7 mmol/l), 20% (9.0 mmol/l), and 8% (7.8 mmol/l) respectively. The incidence and mean glucose levels for low-dose streptozotocin-induced control chow-fed and non-repleted essential fatty acid deficient CD-1 mice were 100% (30.2 mmol/l) and 0% (4.2 mmol/l). The incidence and severity of insulitis also decreased with increasing repletion intervals. These results demonstrate a brief window of susceptibility of less than 8 weeks duration during which repletion will initiate an autoimmune response directed at low-dose streptozotocin-induced Beta-cell neoantigens in low-dose streptozotocin-treated essential fatty acid deficient mice.

Immunophenotyping of insulitis in control and essential fatty acid deficient mice treated with multiple low-dose streptozotocin

“…EFA deficiency prevented diabetes in LDS treated mice (data not shown) as in all previous studies [10,11,12]. Frozen sections of pancreata from all mice contained multiple islets, usually between four and 12.…”

“…Weanling male CD-1 mice (Charles River, Montreal, Quebec, Canada) were placed on EFA-deficient (Purina 5803C low essential fatty acid purified diet; Purina Test Diets, Richmond, Ind., USA) or control (Agway Prolab 3000, Syracuse, N.Y., USA) chow for 2 months as in previous studies [10,11,13,14]. The EFAD and control mice received LDS (Sigma, St.Louis, Mo., USA) treatment (40 rag.…”

“…Insulitis was graded on the H&E stained slides according to previously published methods [11]. Briefly, all pancreata were assigned a grade using the following scheme: (0) normal (i. e. no inflammation), (1)peri-ductal lymphocytic infiltrates (i.e.…”

“…We have previously shown that essential fatty acid (EFA) deficiency ameliorates insulitis and prevents diabetes in LDS-treated highly susceptible CD-1 mice [11,12] as well as in partially susceptible strains, with or without selective immunosuppression directed at suppressor T cells [10]. The following study tests the hypothesis that EFA deficiency prevents or diminishes the influx of macrol:/hages (single cell insulitis) into islets after LDS treatment in male CD-1 mice.…”

“…We have previously hypothesized that LDS insulitis and diabetes require five distinct steps [11]: (1) LDS-induced beta-cell "damage" causing neoantigen expression; (2) influx of activated macrophages ("single-cell insulitis") into the damaged islet amplifying the damage by free radical and cytokine production; (3) processing and presentation of beta-cell fragments by activated macrophages; (4)activation of T-helper cells; and (5)lymphocytic infiltration. We believe that susceptible strains like CD-1 pass through all five steps, but that the partially susceptible strains most likely only progress through the first three steps, resulting in less severe and sometimes transient diabetes.…”