Jean Thuma

Slow restoration of LH pulsatility by refeeding in energetically disrupted women

In other energy-restricted mammals, a single large meal restores luteinizing hormone (LH) pulsatility within a few hours. To determine whether this is so in women, we measured LH pulsatility during the 5th day of low energy availability [dietary energy intake - exercise energy expenditure = 10 kcal ⋅ kg lean body mass (LBM)-1 ⋅ day-1] and during a 6th day of aggressive refeeding (90 kcal ⋅ kg LBM-1 ⋅ day-1) in 15 meals providing 4,100 kcal for an energy availability of 75 kcal ⋅ kg LBM-1 ⋅ day-1. Low energy availability raised β-hydroxybutyrate 1,000% ( P < 0.001) and reduced plasma glucose 15% ( P < 0.01), insulin 63% ( P < 0.001), and triiodothyronine 22% ( P < 0.005). In five of eight subjects, low energy availability also unambiguously suppressed LH pulse frequency 57% to 8.2 ± 1.5 pulses/24 h ( P < 10-4) and raised LH pulse amplitude 94% to 3.1 ± 0.3 IU/l ( P < 10-4), levels below the 5th and above the 95th percentile, respectively, in energy-balanced women. Aggressive refeeding restored β-hydroxybutyrate, glucose, and insulin, but not triiodothyronine. In the five women with unambiguously disrupted LH pulsatility, aggressive refeeding had no effect on LH pulse amplitude ( P > 0.9) and raised LH pulse frequency only slightly (2.4 ± 0.6 pulses/24 h, P = 0.04) and not above the fifth percentile. This striking contrast between women and other mammals may be another clue to the unidentified mechanism mediating the effect of energy availability on LH pulsatility.In other energy-restricted mammals, a single large meal restores luteinizing hormone (LH) pulsatility within a few hours. To determine whether this is so in women, we measured LH pulsatility during the 5th day of low energy availability [dietary energy intake - exercise energy expenditure = 10 kcal . kg lean body mass (LBM)-1 . day-1] and during a 6th day of aggressive refeeding (90 kcal . kg LBM-1 . day-1) in 15 meals providing 4,100 kcal for an energy availability of 75 kcal . kg LBM-1 . day-1. Low energy availability raised beta-hydroxybutyrate 1,000% (P < 0.001) and reduced plasma glucose 15% (P < 0.01), insulin 63% (P < 0.001), and triiodothyronine 22% (P < 0.005). In five of eight subjects, low energy availability also unambiguously suppressed LH pulse frequency 57% to 8.2 +/- 1.5 pulses/24 h (P < 10(-4)) and raised LH pulse amplitude 94% to 3.1 +/- 0.3 IU/l (P < 10(-4)), levels below the 5th and above the 95th percentile, respectively, in energy-balanced women. Aggressive refeeding restored beta-hydroxybutyrate, glucose, and insulin, but not triiodothyronine. In the five women with unambiguously disrupted LH pulsatility, aggressive refeeding had no effect on LH pulse amplitude (P > 0.9) and raised LH pulse frequency only slightly (2.4 +/- 0.6 pulses/24 h, P = 0.04) and not above the fifth percentile. This striking contrast between women and other mammals may be another clue to the unidentified mechanism mediating the effect of energy availability on LH pulsatility.

Toll-Like Receptor 3 Is Critical for Coxsackievirus B4-Induced Type 1 Diabetes in Female NOD Mice

Group B coxsackieviruses (CVBs) are involved in triggering some cases of type 1 diabetes mellitus (T1DM). However, the molecular mechanism(s) responsible for this remain elusive. Toll-like receptor 3 (TLR3), a receptor that recognizes viral double-stranded RNA, is hypothesized to play a role in virus-induced T1DM, although this hypothesis is yet to be substantiated. The objective of this study was to directly investigate the role of TLR3 in CVB-triggered T1DM in nonobese diabetic (NOD) mice, a mouse model of human T1DM that is widely used to study both spontaneous autoimmune and viral-induced T1DM. As such, we infected female wild-type (TLR3(+/+)) and TLR3 knockout (TLR3(-/-)) NOD mice with CVB4 and compared the incidence of diabetes in CVB4-infected mice with that of uninfected counterparts. We also evaluated the islets of uninfected and CVB4-infected wild-type and TLR3 knockout NOD mice by immunohistochemistry and insulitis scoring. TLR3 knockout mice were markedly protected from CVB4-induced diabetes compared with CVB4-infected wild-type mice. CVB4-induced T-lymphocyte-mediated insulitis was also significantly less severe in TLR3 knockout mice compared with wild-type mice. No differences in insulitis were observed between uninfected animals, either wild-type or TLR3 knockout mice. These data demonstrate for the first time that TLR3 is 1) critical for CVB4-induced T1DM, and 2) modulates CVB4-induced insulitis in genetically prone NOD mice.

Phenylmethimazole Suppresses dsRNA-Induced Cytotoxicity and Inflammatory Cytokines in Murine Pancreatic Beta Cells and Blocks Viral Acceleration of Type 1 Diabetes in NOD Mice

Accumulating evidence supports a role for viruses in the pathogenesis of type 1 diabetes mellitus (T1DM). Activation of dsRNA-sensing pathways by viral dsRNA induces the production of inflammatory cytokines and chemokines that trigger beta cell apoptosis, insulitis, and autoimmune-mediated beta cell destruction. This study was designed to evaluate and describe potential protective effects of phenylmethimazole (C10), a small molecule which blocks dsRNA-mediated signaling, on preventing dsRNA activation of beta cell apoptosis and the inflammatory pathways important in the pathogenesis of T1DM. We first investigated the biological effects of C10, on dsRNA-treated pancreatic beta cells in culture. Cell viability assays, quantitative real-time PCR, and ELISAs were utilized to evaluate the effects of C10 on dsRNA-induced beta cell cytotoxicity and cytokine/chemokine production in murine pancreatic beta cells in culture. We found that C10 significantly impairs dsRNA-induced beta cell cytotoxicity and up-regulation of cytokines and chemokines involved in the pathogenesis of T1DM, which prompted us to evaluate C10 effects on viral acceleration of T1DM in NOD mice. C10 significantly inhibited viral acceleration of T1DM in NOD mice. These findings demonstrate that C10 (1) possesses novel beta cell protective activity which may have potential clinical relevance in T1DM and (2) may be a useful tool in achieving a better understanding of the role that dsRNA-mediated responses play in the pathogenesis of T1DM.

A novel small molecule 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose mimics the antiplatelet actions of insulin.

Background We have shown that 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose (α-PGG), an orally effective hypoglycemic small molecule, binds to insulin receptors and activates insulin-mediated glucose transport. Insulin has been shown to bind to its receptors on platelets and inhibit platelet activation. In this study we tested our hypothesis that if insulin possesses anti-platelet properties then insulin mimetic small molecules should mimic antiplatelet actions of insulin. Principal Findings Incubation of human platelets with insulin or α-PGG induced phosphorylation of insulin receptors and IRS-1 and blocked ADP or collagen induced aggregation. Pre-treatment of platelets with α-PGG inhibited thrombin-induced release of P-selectin, secretion of ATP and aggregation. Addition of ADP or thrombin to platelets significantly decreased the basal cyclic AMP levels. Pre-incubation of platelets with α-PGG blocked ADP or thrombin induced decrease in platelet cyclic AMP levels but did not alter the basal or PGE1 induced increase in cAMP levels. Addition of α-PGG to platelets blocked agonist induced rise in platelet cytosolic calcium and phosphorylation of Akt. Administration of α-PGG (20 mg kg−1) to wild type mice blocked ex vivo platelet aggregation induced by ADP or collagen. Conclusions These data suggest that α-PGG inhibits platelet activation, at least in part, by inducing phosphorylation of insulin receptors leading to inhibition of agonist induced: (a) decrease in cyclic AMP; (b) rise in cytosolic calcium; and (c) phosphorylation of Akt. These findings taken together with our earlier reports that α-PGG mimics insulin signaling suggest that inhibition of platelet activation by α-PGG mimics antiplatelet actions of insulin.

L-Arginine Supplementation in Type II Diabetic Rats Preserves Renal Function and Improves Insulin Sensitivity by Altering the Nitric Oxide Pathway.

Rat studies demonstrated that type II diabetes mellitus (T2DM) decreases both the production and bioavailability of nitric oxide (NO). L-arginine (LA) provides the precursor for the production of NO. We hypothesized that LA dietary supplementation will preserve NO production via endothelial nitric oxide synthase (eNOS) causing renal microvascular vasodilation and increased glomerular blood flow and thus increasing glomerular filtration rate (GFR). This would impede the formation of reactive oxygen species which contributes to cell damage and death. LA supplementation preserved GFR in the treated diabetic rats compared to untreated diabetic rats. We provide evidence that this effect may be due to increased levels of eNOS and urinary cyclic guanosine monophosphate, which leads to renal microvascular vasodilation. Plasma nitrotyrosine was decreased in the LA treated rats; however, plasma nitrite levels remained unaffected as expected. Marked improvements in glucose tolerance were also observed in the LA treated diabetic rats. These results demonstrate that LA supplementation preserves NO activity and may delay the onset of insulin resistance and renal dysfunction during hyperglycemic stress. These results suggest the importance of the NO pathway in consequent renal dysfunction and in the development of insulin resistance in diabetic rats.

Diet Is Critical for Prolonged Glycemic Control after Short-Term Insulin Treatment in High-Fat Diet-Induced Type 2 Diabetic Male Mice

Background Clinical studies suggest that short-term insulin treatment in new-onset type 2 diabetes (T2DM) can promote prolonged glycemic control. The purpose of this study was to establish an animal model to examine such a “legacy” effect of early insulin therapy (EIT) in long-term glycemic control in new-onset T2DM. The objective of the study was to investigate the role of diet following onset of diabetes in the favorable outcomes of EIT. Methodology As such, C57BL6/J male mice were fed a high-fat diet (HFD) for 21 weeks to induce diabetes and then received 4 weeks of daily insulin glargine or sham subcutaneous injections. Subsequently, mice were either kept on the HFD or switched to a low-fat diet (LFD) for 4 additional weeks. Principal Findings Mice fed a HFD gained significant fat mass and displayed increased leptin levels, increasing insulin resistance (poor HOMA-IR) and worse glucose tolerance test (GTT) performance in comparison to mice fed a LFD, as expected. Insulin-treated diabetic mice but maintained on the HFD demonstrated even greater weight gain and insulin resistance compared to sham-treated mice. However, insulin-treated mice switched to the LFD exhibited a better HOMA-IR compared to those mice left on a HFD. Further, between the insulin-treated and sham control mice, in spite of similar HOMA-IR values, the insulin-treated mice switched to a LFD following insulin therapy did demonstrate significantly better HOMA-B% values than sham control and insulin-treated HFD mice. Conclusion/Interpretation Early insulin treatment in HFD-induced T2DM in C57BL6/J mice was only beneficial in animals that were switched to a LFD after insulin treatment which may explain why a similar legacy effect in humans is achieved clinically in only a portion of cases studied, emphasizing a vital role for diet adherence in diabetes control.

Early antioxidant supplementation in the prevention of diabetic nephropathy: alterations in NOS expression precede renal functional decline

Rat and human studies have shown that good glycemic control alone is not sufficient to prevent the complications associated with diabetes, such as nephropathy. A proposed culprit is excessive oxidative stress in the cells and vasculature of the kidney resulting in endothelial cell dysfunction, which is one of the earliest and most pivotal consequences seen in early diabetes. We hypothesized that an antioxidant (AO) diet would alleviate the oxidative stress and prevent and/or delay the progression of diabetic nephropathy, even with poor glycemic control. Early nephropathy in diabetes is associated with increased intra renal nitric oxide (NO) production. Nitric oxide synthases (NOS) are a family of enzymes that catalyze the production of NO. We report significantly lower endothelial nitric oxide synthase (eNOS) with AO supplementation compared to the other 2 groups, which means the AO diet led to the decrease in eNOS in the early phase of the onset of diabetic nephropathy. Also, we noted a significant decrease in neuronal nitric oxide synthase (nNOS) with AO supplementation, which is consistent with renal protection in the later phase of diabetic nephropathy. Our results reveal evidence of molecular damage to the endothelium and specialized structures of the kidney that precedes the onset of diabetes. These results suggest early AO supplementation safeguards the kidneys from some of the forces that induce hyperfiltration in early-stage diabetic nephropathy.