Mechanisms of Fasting-Induced Insulin Resistance

DELIVER is a single-center, randomized crossover study investigating the molecular and physiological mechanisms of insulin resistance induced by prolonged fasting. The study will enroll 18 healthy, sedentary men and women aged 18-40 years with a body mass index of 20-25 kg/m².

Each participant will undergo two experimental conditions in randomized order, separated by a washout period of at least four weeks: a 60-hour fasting condition and a control condition following a standard 12-hour overnight fast.

The primary objective is to determine whether fasting-induced skeletal muscle insulin resistance is associated with the accumulation of specific diacylglycerol (DAG) stereoisomers in distinct intracellular compartments, activation of protein kinase C (PKC) isoforms, and alterations in insulin signalling. Alternative mechanisms involving ceramides and PKCζ will also be investigated.

Secondary objectives include evaluating changes in adipose tissue metabolism and extracellular vesicle lipid cargo, assessing the effects of adipose tissue-derived extracellular vesicles on insulin sensitivity in cultured human skeletal muscle cells, and characterizing hepatic energy metabolism and insulin sensitivity during fasting. Potential sex differences will be examined as an exploratory objective.

Insulin sensitivity will be assessed using a two-step hyperinsulinemic-euglycemic clamp with stable glucose isotope tracers, allowing assessment of hepatic, adipose tissue, and peripheral insulin sensitivity. Skeletal muscle and subcutaneous abdominal adipose tissue biopsies will be obtained for lipidomic, molecular, mitochondrial, and extracellular vesicle analyses. Magnetic resonance spectroscopy will be used to assess skeletal muscle and liver lipid content and energy metabolism. Indirect calorimetry, blood sampling, 24-hour urine collection, continuous glucose monitoring, and physical activity monitoring will provide additional information on metabolic adaptations.

The crossover design allows each participant to serve as their own control. By integrating tissue-specific molecular analyses with measurements of whole-body metabolism, DELIVER aims to determine whether fasting-induced insulin resistance involves mechanisms similar to those implicated in obesity-associated insulin resistance or represents a distinct physiological adaptation.

Participants undergo 60 hours of complete fasting under medical supervision. At the end of the fasting period, insulin sensitivity and metabolic responses are assessed using a two-step hyperinsulinemic-euglycemic clamp, stable isotope tracers, magnetic resonance spectroscopy, indirect calorimetry, blood and urine sampling, and skeletal muscle and subcutaneous adipose tissue biopsies.

Other: 60-hour Fasting

Participants undergo a standard 12-hour overnight fast before metabolic assessments, without prolonged fasting. The same principal metabolic and tissue assessments are performed as in the prolonged fasting condition, allowing within-participant comparison of insulin sensitivity, tissue lipid metabolism, and molecular responses.

Other: 12-hour Overnight Fasting (control)

Participants undergo a 60-hour fasting period under medical supervision, during which water and broth are permitted according to the study protocol. The fasting period is followed by standardized metabolic assessments, including a two-step hyperinsulinemic-euglycemic clamp.

Standard overnight fasting for approximately 12 hours before metabolic assessments, serving as the control condition for comparison with prolonged fasting.

Plazo: At the end of each study condition in both crossover periods (washout of at least 4 weeks): Day 2 of the control condition, after a 12-hour overnight fast, and Day 4 of the fasting condition, after 60 hours of fasting.

Difference in plasma membrane-associated sn-1,2-diacylglycerol (DAG) stereoisomer content in skeletal muscle between the 60-hour fasting condition and the control condition, assessed in basal vastus lateralis muscle biopsies.

Plazo: At the end of each study condition in both crossover periods (washout of at least 4 weeks): Day 2 of the control condition, after a 12-hour overnight fast, and Day 4 of the fasting condition, after 60 hours of fasting.

Difference in sn-1,2-diacylglycerol content in mitochondrial/endoplasmic reticulum and cytosolic fractions of basal skeletal muscle biopsies between fasting and control conditions

Plazo: At the end of each study condition in both crossover periods (washout of at least 4 weeks): Day 2 of the control condition, after a 12-hour overnight fast, and Day 4 of the fasting condition, after 60 hours of fasting.

Difference in PKCθ, PKCε and PKCζ translocation, assessed as membrane-to-cytosol ratios in basal skeletal muscle biopsies between conditions

Plazo: At the end of each study condition in both crossover periods (washout of at least 4 weeks): Day 2 of the control condition, after a 12-hour overnight fast, and Day 4 of the fasting condition, after 60 hours of fasting.

Difference in basal and insulin-stimulated phosphorylation of insulin receptor (Tyr1162/1163), IRS-1 (Tyr612, Ser307, Ser1101), Akt (Ser473, Thr308), TBC1D4 (Thr642), GSK3β (Ser9) and S6K1 (Thr389) between conditions

Plazo: At the end of each study condition in both crossover periods (washout of at least 4 weeks): Day 2 of the control condition, after a 12-hour overnight fast, and Day 4 of the fasting condition, after 60 hours of fasting.

Difference in skeletal muscle ceramide content and ceramide ratios between fasting and control conditions

Plazo: At the end of each study condition in both crossover periods (washout of at least 4 weeks): Day 2 of the control condition, after a 12-hour overnight fast, and Day 4 of the fasting condition, after 60 hours of fasting.

Difference in whole-body insulin sensitivity, expressed as M/I (glucose infusion rate corrected for changes in the glucose pool, divided by steady-state plasma insulin), during the last 30 minutes of the high-dose insulin step of the two-step hyperinsulinemic-euglycemic clamp, between fasting and control conditions.

Plazo: At the end of each study condition in both crossover periods (washout of at least 4 weeks): Day 2 of the control condition, after a 12-hour overnight fast, and Day 4 of the fasting condition, after 60 hours of fasting.

Difference in percentage suppression of EGP during the low-dose insulin step of the clamp, relative to basal EGP, between fasting and control conditions.

Plazo: At the end of each study condition in both crossover periods (washout of at least 4 weeks): Day 2 of the control condition, after a 12-hour overnight fast, and Day 4 of the fasting condition, after 60 hours of fasting.

Difference in percentage suppression of plasma non-esterified fatty acids (NEFA) during the low-dose insulin step of the clamp, relative to basal NEFA, between fasting and control conditions.

Plazo: At the end of each study condition in both crossover periods (washout of at least 4 weeks): Day 2 of the control condition, after a 12-hour overnight fast, and Day 4 of the fasting condition, after 60 hours of fasting.

Difference in insulin clearance, calculated as insulin infusion rate divided by the steady-state increment in plasma insulin above baseline during the clamp, between fasting and control conditions.

Plazo: On the day before the clamp in each study condition, in both crossover periods: Day 1 of the control condition, and Day 3 of the fasting condition, after approximately 42 hours of fasting.

Difference in intramyocellular lipid (IMCL) content in the tibialis anterior muscle, assessed by ¹H-magnetic resonance spectroscopy, between fasting and control conditions.

Plazo: On the clamp day of each study condition, before the start of insulin infusion, in both crossover periods: Day 2 of the control condition, after a 12-hour overnight fast, and Day 4 of the fasting condition, after 60 hours of fasting.

Difference in absolute hepatic γ-ATP concentration, assessed by ³¹P-magnetic resonance spectroscopy, between fasting and control conditions.

Plazo: At the end of each study condition in both crossover periods (washout of at least 4 weeks): Day 2 of the control condition, after a 12-hour overnight fast, and Day 4 of the fasting condition, after 60 hours of fasting.

Differences in adipose tissue gene expression profiles, assessed by transcriptomic analysis of subcutaneous abdominal adipose tissue biopsies, between fasting and control conditions.

Plazo: At the end of each study condition in both crossover periods (washout of at least 4 weeks): Day 2 of the control condition, after a 12-hour overnight fast, and Day 4 of the fasting condition, after 60 hours of fasting.

Difference in diacylglycerol and ceramide content of extracellular vesicles isolated from subcutaneous abdominal adipose tissue biopsies, assessed by lipidomic profiling, between fasting and control conditions.

Plazo: At the end of each study condition in both crossover periods (washout of at least 4 weeks): Day 2 of the control condition, after a 12-hour overnight fast, and Day 4 of the fasting condition, after 60 hours of fasting.

Exploratory comparison of the fasting-induced change in plasma membrane sn-1,2-DAG content between men and women (sex-by-condition interaction), reported with effect sizes and confidence intervals.

Plazo: At the end of each study condition in both crossover periods (washout of at least 4 weeks): Day 2 of the control condition, after a 12-hour overnight fast, and Day 4 of the fasting condition, after 60 hours of fasting.

Exploratory comparison of the fasting-induced change in M/I between men and women (sex-by-condition interaction), reported with effect sizes and confidence intervals.

Plazo: At the end of each study condition in both crossover periods (washout of at least 4 weeks): Day 2 of the control condition, after a 12-hour overnight fast, and Day 4 of the fasting condition, after 60 hours of fasting.

Exploratory comparison of the fasting-induced change in percentage EGP suppression during the low-dose insulin step between men and women (sex-by-condition interaction), reported with effect sizes and confidence intervals.

Diacylglycerol, Extracellular Vesicles, and Liver Energy Metabolism in Fasting-Induced Insulin Resistance