Caloric restriction and intermittent fasting alter hepatic lipid droplet proteome and diacylglycerol species and prevent diabetes in NZO mice

AbstractCaloric restriction and intermittent fasting are known to improve glucose homeostasis and insulin resistance in several species including humans. The aim of this study was to unravel potential mechanisms by which these interventions improve insulin sensitivity and protect from type 2 diabetes. Diabetes-susceptible New Zealand Obese mice were either 10% calorie restricted (CR) or fasted every other day (IF), and compared to ad libitum (AL) fed control mice. AL mice showed a diabetes prevalence of 43%, whereas mice under CR and IF were completely protected against hyperglycemia. Proteomic analysis of hepatic lipid droplets revealed significantly higher levels of PSMD9 (co-activator Bridge-1), MIF (macrophage migration inhibitor factor), TCEB2 (transcription elongation factor B (SIII), polypeptide 2), ACY1 (aminoacylase 1) and FABP5 (fatty acid binding protein 5), and a marked reduction of GSTA3 (glutathione S-transferase alpha 3) in samples of CR and IF mice. In addition, accumulation of diacylglycerols (DAGs) was significantly reduced in livers of IF mice (P=0.045) while CR mice showed a similar tendency (P=0.062). In particular, 9 DAG species were significantly reduced in response to IF, of which DAG-40:4 and DAG-40:7 also showed significant effects after CR. This was associated with a decreased PKCε activation and might explain the improved insulin sensitivity. In conclusion, our data indicate that protection against diabetes upon caloric restriction and intermittent fasting associates with a modulation of lipid droplet protein composition and reduction of intracellular DAG species

Endocrine Determinants of Changes in Insulin Sensitivity and Insulin Secretion during a Weight Cycle in Healthy Men

<div><p>Objective</p><p>Changes in insulin sensitivity (IS) and insulin secretion occur with perturbations in energy balance and glycemic load (GL) of the diet that may precede the development of insulin resistance and hyperinsulinemia. Determinants of changes in IS and insulin secretion with weight cycling in non-obese healthy subjects remain unclear.</p><p>Methods</p><p>In a 6wk controlled 2-stage randomized dietary intervention 32 healthy men (26±4y, BMI: 24±2kg/m²) followed 1wk of overfeeding (OF), 3wks of caloric restriction (CR) containing either 50% or 65% carbohydrate (CHO) and 2wks of refeeding (RF) with the same amount of CHO but either low or high glycaemic index at ±50% energy requirement. Measures of IS (basal: HOMA-index, postprandial: Matsuda-ISI), insulin secretion (early: Stumvoll-index, total: tAUC-insulin/tAUC-glucose) and potential endocrine determinants (ghrelin, leptin, adiponectin, thyroid hormone levels, 24h-urinary catecholamine excretion) were assessed.</p><p>Results</p><p>IS improved and insulin secretion decreased due to CR and normalized upon RF. Weight loss-induced improvements in basal and postprandial IS were associated with decreases in leptin and increases in ghrelin levels, respectively (r = 0.36 and r = 0.62, p<0.05). Weight regain-induced decrease in postprandial IS correlated with increases in adiponectin, fT3, TSH, GL of the diet and a decrease in ghrelin levels (r-values between -0.40 and 0.83, p<0.05) whereas increases in early and total insulin secretion were associated with a decrease in leptin/adiponectin-ratio (r = -0.52 and r = -0.46, p<0.05) and a decrease in fT4 (r = -0.38, p<0.05 for total insulin secretion only). After controlling for GL associations between RF-induced decrease in postprandial IS and increases in fT3 and TSH levels were no longer significant.</p><p>Conclusion</p><p>Weight cycling induced changes in IS and insulin secretion were associated with changes in all measured hormones, except for catecholamine excretion. While leptin, adiponectin and ghrelin seem to be the major endocrine determinants of IS, leptin/adiponectin-ratio and fT4 levels may impact changes in insulin secretion with weight cycling.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01737034" target="_blank">NCT01737034</a></p></div

Subject characteristics at baseline of the whole study population, and changes in body weight and fat mass during intervention periods, and changes of body weight and fat mass during CR and RF by intervention group.

<p>Values are means ±SD, n = 32.</p><p>^†††p<0.001 significantly different from baseline</p><p>***p<0.001 significantly different from previous period; Repeated measures ANOVA with Bonferroni adjustments</p><p>OF, overfeeding; CR, caloric restriction; RF, refeeding; FM, fat mass; CHO, carbohydrate; GI, glycemic index</p><p>At baseline, fasting insulin levels ranged from 1.3 to 22.4 mU/l and mean values of IS were in the normal range according to cut-off values by Radikova et al. (HOMA-IR <2.3 and Matsuda >5.0) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117865#pone.0117865.ref033" target="_blank">33</a>], while three study participants had HOMA-IR values >2.3 and Matsuda indices <5.0 together with an increased FM (>23% FM). <i>M</i>-values were significantly associated with HOMA-index and Matsuda-ISI [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117865#pone.0117865.ref022" target="_blank">22</a>](r = -0.43 and r = 0.42, both p<0.05) at baseline as well as after RF with Matsuda-ISI (r = 0.55, p<0.05), while association with HOMA-IR tended to be associated (r = -0.34, p = 0.06).</p><p>Subject characteristics at baseline of the whole study population, and changes in body weight and fat mass during intervention periods, and changes of body weight and fat mass during CR and RF by intervention group.</p

Changes in endocrine parameters due to overfeeding (OF) caloric restriction (CR) and refeeding (RF) (values are means ±SD).

<p>^†p<0.05</p><p>^††p<0.01</p><p>^†††p<0.001 significantly different from baseline</p><p>*p<0.05</p><p>**p<0.01</p><p>***p<0.001 significantly different from previous period; Repeated measures ANOVA with Bonferroni adjustments</p><p><i>OGTT</i>, oral glucose tolerance test; iAUC, incremental area under the curve</p><p>Changes in endocrine parameters due to overfeeding (OF) caloric restriction (CR) and refeeding (RF) (values are means ±SD).</p

Schematic overview of the study protocol.

<p>OGTT, oral glucose tolerance test, CHO, carbohydrate; LGI, low glycaemic index; HGI, high glycaemic index</p

Comparison between fasting insulin level, IS (HOMA-index and Matsuda-ISI) as well as insulin secretion (insulin-iAUC, insulin-tAUC/glucose-tAUC, Stumvoll-index) at baseline (T0), after caloric restriction (CR, T2) and refeeding (RF, T3).

<p>*p<0.05, **p<0.01, ***p<0.001; Repeated measures ANOVA with Bonferroni adjustments. HOMA, homeostasis model assessment; ISI, insulin sensitivity index, iAUC, incremental area under the curve; tAUC, total area under the curve</p

Changes in insulin secretion and IS due to overfeeding (OF) caloric restriction (CR) and refeeding (RF) (values are means ±SD).

<p>^†p<0.05</p><p>^††p<0.01</p><p>^†††p<0.001 significantly different from baseline</p><p>*p<0.05</p><p>**p<0.01</p><p>***p<0.001 significantly different from previous period; Repeated measures ANOVA with Bonferroni adjustments</p><p>IS, insulin sensitivity; HOMA-index, homeostasis model assessment of insulin resistance; OGTT, oral glucose tolerance test; iAUC, incremental area under the curve; tAUC, total area under the curve; Matsuda-ISI, Mastsuda insulin sensitivity index</p><p>Changes in insulin secretion and IS due to overfeeding (OF) caloric restriction (CR) and refeeding (RF) (values are means ±SD).</p

CONSORT flow chart, showing the passage of participants through the different stages of the present trial: enrollment, first allocation after OF to the 65%CHO and 50%CHO intervention and second allocation after CR to 65%CHO-HGI, 65%CHO-LGI, 50%CHO-HGI and 50%CHO-LGI intervention, follow-up, and analysis.

<p>OF, overfeeding; CR, caloric restriction; CHO: carbohydrate, HGI, high glyceamic index; LGI, low glyceamic index</p