according to a previously published protocol The short-term treatment of resistin did not impair or improve endothelium-dependent or -independent vasorelaxation Figures 4C,D , suggesting that short-term resistin administration may not affect vascular function.

Similarly, Lepr db mice showed increased circulating leptin due to leptin receptor deficiency. Circulating leptin was further enhanced by ADF, suggesting that an increase in circulating leptin itself was unlikely to prevent the vascular benefits of ADF.

Thus, our study provided some mechanistic insights into the contribution of adipokines to ADF-mediated vascular effects in type 2 diabetes. Figure 4. The effects of adipokines on endothelium-dependent vasorelaxation of SMA.

A Adenovirus-mediated adiponectin supplementation improved ACh-induced endothelium-dependent vasorelaxation of SMA in Lepr db mice, without affecting SNP-induced endothelium-independent vasorelaxation B. C,D Treatment with recombinant resistin did not affect endothelium-dependent or endothelium-independent vasorelaxation of SMA.

Nitrotyrosine protein levels were elevated in both SMA and MAT of Lepr db diabetic mice compared with m Lepr db control mice. ADF reduced SMA nitrotyrosine protein levels in Lepr db diabetic mice without affecting that in the m Lepr db control mice Figure 5A.

ADF, however, did not significantly decrease MAT nitrotyrosine protein levels Figure 5B. Figure 5. ADF reduced nitrotyrosine protein levels in SMA, but not MAT, of Lepr db mice. A Nitrotyrosine protein levels were higher in SMA of Lepr db mice. ADF reduced nitrotyrosince protein in SMA of Lepr db mice.

B Nitrotyrosine protein levels were higher in MAT of Lepr db mice. ADF did not alter nitrotyrosince protein in MAT of Lepr db mice. Studies demonstrate that intermittent fasting improves cardiometabolic risk factors such as blood pressure, levels of low-density lipoprotein cholesterol and triglycerides, insulin resistance, and HbA1c 5.

A better understanding of how intermittent fasting affects cardiovascular function and the underlying mechanisms will facilitate its clinical application in obesity and diabetes-associated cardiovascular complications.

Our study revealed the profound benefits of ADF in rescuing endothelial dysfunction. The benefits are at least partly mediated through enhanced adiponectin, while resistin and leptin were unlikely to be involved.

Adiponectin thus provides a mechanistic link between the role of ADF in regulating adipokine profile and endothelial function in type 2 diabetes. ADF reduced the marker of oxidative stress in resistance arteries but not adipose tissue, suggesting tissue-specific regulatory roles by ADF.

ADF may also exert metabolic and vascular benefits in non-obese control mice. Overall, our data support that ADF presents as promising lifestyle intervention for treating diabetes-associated endothelial dysfunction.

Intermittent fasting is emerging as a popular alternative dietary intervention strategy. Despite limited numbers of clinical trials directly comparing the long-term effects of intermittent fasting and daily calorie restriction, current evidence supports equivalent or superior metabolic benefits of intermittent fasting 5.

Comparative studies in a month study of insulin-resistant participants support that ADF may produce greater reductions in fasting insulin and insulin resistance compared with calorie restriction despite similar decreases in body weight In Lepr db type 2 diabetic mice and streptozotocin-treated type 1 diabetic mice treated with a fasting-mimicking diet, both intermittent fasting and continuous calorie restriction significantly reduced fasting blood glucose levels and improved insulin sensitivity.

Yet, intermittent fasting performed significantly better than continuous calorie restriction in improving glycemic control and insulin sensitivity in Lepr db type 2 diabetic mice Clinical studies, conducted over multiple years, that directly compare different regimens will provide important insights into the long-term cardiometabolic benefits of these diets.

There are currently no clinical studies determining the vascular benefits of long-term ADF in patients with diabetes. Clinical trials of short-term ADF, e. Increases in adiponectin were positively associated with augmented flow-mediated vasodilation post-ADF in those subjected to ADF with the low-fat diet ADF also reduced plasma resistin and leptin, which were not correlated with changes in flow-mediated vasodilation In a study involving 54 obese non-diabetic subjects with an 8-week ADF protocol, brachial artery flow-mediated vasodilation was positively correlated to adiponectin concentrations Another study involving 64 obese subjects supported that a week period of ADF improved brachial artery flow-mediated vasodilation Our experimental data strongly support the profound endothelial protective effects of ADF in mice modeling severe type 2 diabetes.

To our knowledge, this is the first experimental study determining the role of ADF in diabetes-associated vascular dysfunction. The above clinical studies in obese subjects and our experimental study in type 2 diabetic mice provide premises to further explore the clinical benefits of long-term ADF in diabetes-associated cardiovascular complications.

Our study has shed light on the mechanisms of the endothelial protective effects of ADF partly through enhanced circulating adiponectin. Adiponectin is well known for its anti-inflammatory and anti-oxidative roles in endothelial cells 43 and its protective effects against neointimal formation in response to vascular injury 44 and atherosclerosis Our previous work has also supported that adiponectin abates diabetes-induced endothelial dysfunction by suppressing oxidative stress, adhesion molecules, and inflammation in type 2 diabetic mice Specifically, adenovirus-mediated adiponectin supplementation improved endothelium-dependent vasorelaxation of aortas in Lepr db mice Adiponectin supplementation reduced aortic nitrotyrosine protein levels, via suppressing protein expression of gp91 phox , an NADPH oxidase subunit, and increasing protein expression of SOD3, an antioxidant enzyme Aortic expression of inflammatory genes, Tnf , Il6 , and Icam1 , was also suppressed by adiponectin supplementation These pathways are likely responsible for the endothelial protective and anti-oxidative effects of adiponectin in mesenteric arteries of Lepr db mice undergoing ADF.

The adiponectin-independent endothelial protective and anti-oxidative effects of ADF remain to be further dissected, and we speculate that the metabolic benefits of ADF may play important roles. Alternate day fasting exerts profound metabolic benefits in both control and diabetic mice with remarkably improved glycemic control and insulin sensitivity.

The effects of ADF on weight loss and visceral adiposity were, however, modest. Consistent with our observation, an independent study also suggested that a week period of intermittent fasting, using a fasting mimicking diet protocol, improved glucose homeostasis in Lepr db mice without causing weight loss Thus, the metabolic benefits of ADF in Lepr db diabetic mice are likely not entirely dependent on weight loss effects.

Since the Lepr db mice resemble severe type 2 diabetes, whether ADF may also exert limited benefits in weight management in patients with type 2 diabetes, despite profound metabolic effects, should be studied clinically. Further, the benefits of ADF in non-obese, healthy humans thus may also warrant further investigation.

There are many questions that remain to be explored. Future studies may further elucidate if the knockout of adiponectin abolishes the vascular protective effects of ADF, the involvement of other adipokines, and the molecular mechanisms by which ADF modulates adipokine expression and secretion.

Comparative studies are required to tackle how different intermittent fasting regimens affect metabolic, vascular, and hormonal parameters. Findings generated from such studies could inform whether one regimen is superior to the others and elucidate the mechanisms that underlie the cardiometabolic benefits.

The discovery of pharmacological agents mimicking fasting can potentially provide novel therapeutic strategies. A potential limitation of the present studies is that they were performed only in male mice and mesenteric resistance arteries.

In summary, our study examined the role and mechanisms of ADF in diabetes-associated endothelial dysfunction using murine models of type 2 diabetes. We have revealed that ADF in type 2 diabetic mice exerts profound endothelial protective effects, partly through modulating the adipose-derived hormone, adiponectin.

Thus, this study improves our understanding of how ADF affords significant protection against endothelial dysfunction partly by regulating adipose-derived hormones.

Our work also elaborated on the metabolic benefits and potential cardiovascular protective actions of ADF in the management of type 2 diabetes. The manuscript is in memory of Dr. Cuihua Zhang, who was deceased on October 1, The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by the Animal Care Committee at the University of Missouri Columbia, MO, United States. JC, HZ, and CZ conceived the study. JC, SL, and HZ performed the experiments. JC and HZ analyzed the data.

JC, YL, and HZ interpreted results of experiments and drafted the manuscript. JC, YS, and HZ prepared the tables and figures. JC, SL, YS, MH, YL, and HZ edited and revised the manuscript.

All authors contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

ACh, acetylcholine; ADF, alternate-day fasting; MAT, mesenteric adipose tissue; NO, nitric oxide; PE, phenylephrine; SMA, small mesenteric artery; SNP, sodium nitroprusside. Powell-Wiley TM, Poirier P, Burke LE, Després JP, Gordon-Larsen P, Lavie CJ, et al.

Obesity and cardiovascular disease: a scientific statement from the american heart association. doi: PubMed Abstract CrossRef Full Text Google Scholar. Varady KA, Cienfuegos S, Ezpeleta M, Gabel K. Cardiometabolic benefits of intermittent fasting.

Annu Rev Nutr. Flanagan EW, Most J, Mey JT, Redman LM. Calorie restriction and aging in humans. Santos HO, Genario R, Tinsley GM, Ribeiro P, Carteri RB, Coelho-Ravagnani CF, et al.

A scoping review of intermittent fasting, chronobiology, and metabolism. Am J Clin Nutr. Clinical application of intermittent fasting for weight loss: progress and future directions. Nat Rev Endocrinol. Vasim I, Majeed CN, DeBoer MD. Intermittent fasting and metabolic health. Zang B-Y, He L-X, Xue L.

Intermittent fasting: potential bridge of obesity and diabetes to health? Martens CR, Seals DR. Practical alternatives to chronic caloric restriction for optimizing vascular function with ageing. J Physiol. Carter S, Clifton PM, Keogh JB. Effect of intermittent compared with continuous energy restricted diet on glycemic control in patients with type 2 diabetes: a randomized noninferiority trial.

JAMA Netw Open. Chen H, Charlat O, Tartaglia LA, Woolf EA, Weng X, Ellis SJ, et al. CrossRef Full Text Google Scholar. Chua SC Jr. Phenotypes of mouse diabetes and rat fatty due to mutations in the Ob Leptin receptor.

Zhou J, Jiang Z, Lin Y, Li C, Liu J, Tian M, et al. The daily caloric restriction and alternate-day fasting ameliorated lipid dysregulation in type 2 diabetic mice by downregulating hepatic pescadillo 1. Eur J Nutr. Zhang H, Zhang W, Yun D, Li L, Zhao W, Li Y, et al. Alternate-day fasting alleviates diabetes-induced glycolipid metabolism disorders: roles of Fgf21 and bile acids.

J Nutr Biochem. Kim KE, Jung Y, Min S, Nam M, Heo RW, Jeon BT, et al. Sci Rep. Beli E, Yan Y, Moldovan L, Vieira CP, Gao R, Duan Y, et al. Liu Z, Dai X, Zhang H, Shi R, Hui Y, Jin X, et al. Gut microbiota mediates intermittent-fasting alleviation of diabetes-induced cognitive impairment.

Nat Commun. Zhang H, Zhang C. Obesity Silver Spring. Regulation of microvascular function by adipose tissue in obesity and type 2 diabetes: evidence of an adipose-vascular loop. Am J Biomed Sci. Zhang H, Cui J, Zhang C.

Emerging role of adipokines as mediators in atherosclerosis. World J Cardiol. Lee S, Zhang H, Chen J, Dellsperger KC, Hill MA, Zhang C. Adiponectin abates diabetes-induced endothelial dysfunction by suppressing oxidative stress, adhesion molecules, and inflammation in type 2 diabetic mice.

Am J Physiol Heart Circ Physiol. Zhang H, Park Y, Zhang C. Coronary and aortic endothelial function affected by feedback between adiponectin and tumor necrosis factor α in type 2 diabetic mice.

Arterioscler Thromb Vasc Biol. Lee S, Park Y, Dellsperger KC, Zhang C. Exercise training improves endothelial function via adiponectin-dependent and independent pathways in type 2 diabetic mice.

Reinehr T, Roth C, Menke T, Andler W. Adiponectin before and after weight loss in obese children. J Clin Endocrinol Metab. Zorena K, Jachimowicz-Duda O, Ślęzak D, Robakowska M, Mrugacz M.

Adipokines and obesity. Potential link to metabolic disorders and chronic complications. Int J Mol Sci. Varady KA, Tussing L, Bhutani S, Braunschweig CL. Degree of weight loss required to improve adipokine concentrations and decrease fat cell size in severely obese women. Steppan CM, Bailey ST, Bhat S, Brown EJ, Banerjee RR, Wright CM, et al.

The hormone resistin links obesity to diabetes. Zhang H, Wang Y, Zhang J, Potter BJ, Sowers JR, Zhang C. Bariatric surgery reduces visceral adipose inflammation and improves endothelial function in type 2 diabetic mice.

Chen X, Zhang H, McAfee S, Zhang C. The reciprocal relationship between adiponectin and lox-1 in the regulation of endothelial dysfunction in apoe knockout mice. Ste Marie L, Palmiter RD. Norepinephrine and epinephrine-deficient mice are hyperinsulinemic and have lower blood glucose. Matsuda M, Shimomura I, Sata M, Arita Y, Nishida M, Maeda N, et al.

Role of adiponectin in preventing vascular stenosis. the missing link of adipo-vascular axis. J Biol Chem.

Su J, Lucchesi PA, Gonzalez-Villalobos RA, Palen DI, Rezk BM, Suzuki Y, et al. Role of advanced glycation end products with oxidative stress in resistance artery dysfunction in type 2 diabetic mice. Zhang H, Potter BJ, Cao JM, Zhang C. Interferon-gamma induced adipose tissue inflammation is linked to endothelial dysfunction in type 2 diabetic mice.

Basic Res Cardiol. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, et al. Primer3—new capabilities and interfaces. Nucleic Acids Res.

Zhang H, Zhang J, Ungvari Z, Zhang C. Resveratrol improves endothelial function: role of tnf{alpha} and vascular oxidative stress. Chen X, Zhang H, Hill MA, Zhang C, Park Y. Regulation of coronary endothelial function by interactions between Tnf-A, Lox-1 and adiponectin in apolipoprotein e knockout mice.

J Vasc Res. Zhao S, Kusminski CM, Scherer PE. Adiponectin, leptin and cardiovascular disorders. Circ Res. Gabel K, Kroeger CM, Trepanowski JF, Hoddy KK, Cienfuegos S, Kalam F, et al. Differential effects of alternate-day fasting versus daily calorie restriction on insulin resistance.

Varady KA, Allister CA, Roohk DJ, Hellerstein MK. Improvements in body fat distribution and circulating adiponectin by alternate-day fasting versus calorie restriction. Wei S, Zhao J, Bai M, Li C, Zhang L, Chen Y.

Comparison of glycemic improvement between intermittent calorie restriction and continuous calorie restriction in diabetic mice. Nutr Metab Lond. Klempel MC, Kroeger CM, Norkeviciute E, Goslawski M, Phillips SA, Varady KA.

Benefit of a low-fat over high-fat diet on vascular health during alternate day fasting. Nutr Diabet. Hoddy KK, Bhutani S, Phillips SA, Varady KA. Effects of different degrees of insulin resistance on endothelial function in obese adults undergoing alternate day fasting.

Nutr Healthy Aging. Bhutani S, Klempel MC, Kroeger CM, Trepanowski JF, Phillips SA, Norkeviciute E, et al. Alternate day fasting with or without exercise: effects on endothelial function and adipokines in obese humans.

Protein content in m. vastus lateralis was examined using SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis and Western blotting techniques see Supplementary Material.

Analysis of mitochondrial function was performed in permeabilized skeletal muscle fibers. Mitochondrial respiratory capacity was determined as previously described Dohlmann et al. See Supplementary Material for the detailed protocols.

Citrate synthase CS activity was measured as previously described Larsen et al. Image analysis was performed with Fiji software Schindelin et al. Insulin sensitivity was estimated from glucose infusion rates during the final 30 min of the clamp. Glucose clearance rates were calculated as glucose infusion rates divided by the prevailing plasma glucose concentration.

Statistical analyses of parameters measured across the intervention phases e. insulin secretion, insulin sensitivity, blood lipids, body weight were performed with mixed model analyses GraphPad Prism 8.

In the absence of missing values, this method gives the same p values and multiple comparisons tests as repeated measures ANOVA. In the presence of missing values missing completely at random , the results can be interpreted like repeated measures ANOVA. Based on data from previous studies Halberg et al.

Maximal oxygen uptake was similar in the patients with type 2 diabetes T2DM and obesity OB 2. Apart from the volume of visceral fat measured by MRI, the two groups were well matched concerning anthropometrics at baseline Table 1.

However, the decrease was primarily due to the last part of the intervention protocol, where a weight loss was allowed. Plasma concentrations of insulin and C-peptide were as expected elevated on the double diet day Table 2.

β-hydroxybutyrate representing ketone bodies were increased after 30 h Fasting, and higher in T2DM vs. Total cholesterol, LDL, and HDL cholesterol were not different between the groups and did not change with the intervention Table 2.

Plasma glycerol concentrations displayed a similar pattern, however, with no group differences, and only OB displayed a significant increase with fasting Table 2.

Plasma hsCRP values were generally very low, and no changes were seen Table 2. The interventions did not change the insulin response in the control group.

FIGURE 2. In column graphs are shown area under the curve AUC for insulin E and glucose F. Data are shown as mean ± SD. T2DM patients displayed insulin resistance glucose infusion rate compared with OB Table 1. the insulin sensitivity increased as a main effect and not with ADF alone.

Glucose rate of appearance Ra after an overnight fast in the baseline test was increased in T2DM compared with OB 3. Glucose Ra was unchanged in the fasting state following 3 weeks of ADF T2DM: 3. Muscle glycogen content Table 2 was also measured in the evening after a double diet day and in the morning after a fasting day after 30 h fasting see protocol, Figure 1.

No significant difference was seen with the analysis of the OB and T2DM group separately. The purpose was to monitor adherence to the fasting, double diet and ad libitum diet days and all subjects followed the intervention. Glucose levels were higher in T2DM compared with OB at all times Figure 3.

During ADF, CGM data from weeks one to three illustrates that eating on double diet days increased after the first week, probably also due to encouragement to the patients based upon the daily home-based weighing.

No hypoglycemic events were recorded. FIGURE 3. Continuous glucose monitoring CGM traces in patients with obesity OB and type 2 diabetes T2DM. Data are shown as mean values.

FIGURE 4. Intrahepatic triglyceride content measured by 1 H-magnetic resonance spectroscopy in eleven subjects with obesity OB and eleven patients with type 2 diabetes T2DM. Experiments were performed at baseline, after 3 weeks of alternate-day fasting with double diet on non-fasting days ADF , and after 3 weeks of alternate-day fasting with ad libitum diet, i.

Triglyceride content in m. psoas major by 1 H-MRS was not different between the groups and did not change with the intervention baseline: 5. Muscle lipid droplet density, size of droplets, or the fractional area of the droplets did not differ between T2DM and OB, and no change with ADF was seen Figure 5A, B, C, F.

These data were confirmed by analysis of intramuscular triglyceride IMTG content Figure 5D. In addition, IMTG content was also measured in Tru-Cut biopsies obtained in the evening after a double diet day and in the morgen after 30 h fasting Figure 5E.

after an overnight fast , but this could not be detected in OB Figure 5E. No difference in IMTG between double diet day and 30 h fasting could be seen in either group Figure 5E.

FIGURE 5. Lipid droplets LD density per area of muscle fibre A. LD size B. Intramuscular triglyceride IMTG content in muscle at baseline clamp and at the clamp after ADF D. IMTG content in muscle after one double diet day and after 30 h fasting E. Representative images of Bodipy staining F.

OB, Obese subjects, T2DM, patients with type 2 diabetes. In figure f, scale bars represent µm top and 25 µm bottom.

The expression of proteins relevant for insulin-mediated glucose metabolism is shown in Supplementary Figure S1A-I. Proteins involved in glucose transport GLUT4 and glycolysis hexokinase and pyruvate kinase and glycogen storage glycogen synthase and phosphorylase were similar between the two groups and did not change with ADF.

Proteins relevant for GLUT4 vesicle formation showed minor changes. Thus, Akt was significantly higher in OB compared with T2DM, and AMPKα1 increased significantly with ADF in OB, but not in T2DM.

AS and PKCθ remained unchanged and similar in the two groups. The expression of proteins relevant to lipid metabolism is shown in Supplementary Figure S1J-S.

Proteins involved in fatty acids transport into muscle cells were studied by analysis of proteins located in the plasma membrane. This was carried out to determine if alternate day fasting had any effect on fatty acid transport in healthy or diabetic skeletal muscle tissue.

Fatty acid translocase FAT or CD36 binds long-chain fatty acids and is a key player in fatty acid transport across the plasma membrane. Fatty acid transport protein 4 FATP4 esterifies long-chain fatty acids and has a role in fatty acid transport across the plasma membrane.

Plasma membrane fatty acid binding protein FABPpm is also involved in myocellular uptake of long-chain fatty acids. No change was observed in the T2DM group or between the two groups for all fatty acid transporters. Citrate synthase CS activity was used as an index for mitochondrial mass Larsen et al.

Oxygen consumption ex vivo was measured with a sequential substrate protocol, with state 2 respiration complex I; malate and glutamate followed by state 3 respiration with increasing concentrations of ADP complex I and dual electron input to complex I and II glutamate, malate, succinate, and ADP , ending with uncoupled respiration FCCP as protonophore Supplementary Figure S2A.

First of all, there were no differences in respiration between T2DM and OB, and no significant effect of ADF was observed Supplementary Figure S2A. Reactive oxygen species ROS displayed no difference between the groups and no effect of ADF was observed Supplementary Figure S2B.

ADP sensitivity and maximal oxygen flux Vmax was calculated from the oxygen flux during increasing ADP concentrations, and no differences between groups or effects of ADF were seen Supplementary Figures S2C, D.

The present study represents a comprehensive characterization of the effects of alternate-day fasting regimens on the human metabolism, studied in obese patients with and without type 2 diabetes.

The energy balance is essential in every attempt to lose bodyweight, and if weight loss is the primary focus it is fundamental to achieve a negative energy balance, no matter how this is brought about.

This was not the primary focus here. With the present study protocol, we aimed to study the metabolic effects of oscillations in energy intake and thus energy balance. An improvement of the insulin secretory capacity in patients with type 2 diabetes is a therapeutic goal that is difficult to achieve non-pharmacologically, and only a few studies in patients with type 2 diabetes have reported improvements in β-cell secretion following physical training Krotkiewski et al.

The purpose of the present study was to mimic the oscillations that occur in energy stores with frequent exercise training, but at the same time avoid the physiological impact on metabolism that takes place with exercise training i. muscle contractions.

ADF would largely accomplish this, but from previous studies, it is known that weight loss often follows ADF. Therefore, we divided the study into two 3-week periods of ADF, where weight loss was allowed in the latter period, as would be the every day practice.

The first-phase insulin response in the patients with type 2 diabetes was, however, not restored albeit the insulin response curve displayed a more marked first phase profile compared with baseline Figure 2C. An indication that time-restricted feeding i. not the same protocol as used in the present study without weight loss may increase ß-cell responsiveness in pre-diabetic people has been published Sutton et al.

The mechanism for improvements in insulin secretion has been attributed to a decrease in intrapancreatic triacylglycerol Lim et al. This is also a likely explanation in the present study, where we observed large decreases in visceral fat Table 1 and intrahepatic triglyceride content Figure 4 where the latter correlated significantly with the improvement in insulin secretion.

The elevated plasma concentrations of FFA, glycerol, and β-hydroxybutyrate during fasting Table 2 testified to an increased lipolytic rate during fasting, contributing to the marked decrease of adipose tissue during the interventions Table 1.

An additional mechanism for the improvement in insulin secretion could also be due to an overall reduced glycemic load on the β-cells i. reduced glucotoxicity. Apart from documenting the adherence to the protocol, the continuous glucose monitoring Figure 3 revealed a lessened glycemic burden, which in itself reduces the stress on the β-cells.

It is important to note that the duration of type 2 diabetes, or at least the time since diagnosis, was short among the included patients 2.

This means that the patients had a relatively well-preserved β-cell function, but of course, diminished compared with the obese subjects without type 2 diabetes Figure 2. We have previously shown that patients with a high pre-operative β-cell function experience a superior outcome to gastric bypass surgery compared with those patients with the lowest pre-operative β-cell function Lund et al.

Most likely, patients with severely reduced insulin secretory capacity which can be easily estimated by a 6 min glucagon test Dela et al. Many studies have shown positive effects of training on insulin-mediated glucose uptake in skeletal muscle in patients with type 2 diabetes Dela et al.

For muscle glycogen Table 2 this aim was achieved, but the oscillations did not translate into an improvement of insulin-mediated glucose clearance with ADF alone, which is in contrast to earlier findings in young, healthy subjects Halberg et al.

However, the data are in line with findings in obese people, using a calculated index for insulin sensitivity S I from an intravenous glucose tolerance test Catenacci et al. Insulin action at the hepatic level, i. inhibition of endogenous glucose Ra, did not change with ADF.

This finding is in line with the lack of effect of ADF on peripheral insulin action. It may require an extended period of starvation 3—4 days before a reduction of insulin-induced suppression of hepatic glucose output is seen Fery et al.

This indicates a general improvement in hepatic function elicited by dietary regimen. The lack of increases in insulin sensitivity with ADF is in line with the general lack of increases in proteins relevant for skeletal muscle insulin action, e.

GLUT4, hexokinase, glycogen synthase Supplementary Figure S1. In rodents, a similar lack of change in hexokinase after intermittent fasting has been reported Real-Hohn et al.

The amount of intramyocellular lipids is inversely correlated with insulin sensitivity Pan et al. psoas major fit well with the lack of changes in insulin sensitivity. A similar amount of lipid content in the muscle in T2DM and OB has been shown before Hansen et al.

The latter is in line with earlier findings that demonstrated that it requires prolonged fasting e. Lipolysis increases at the beginning of a fasting period here evidenced by increased FFA and glycerol after 30 h fasting; Table 2. The more so, because the anti-lipolytic effect of insulin diminishes with fasting Jensen et al.

Support for an ADF-induced triglyceride-lowering effect is found in low-calorie refeeding studies that demonstrated increased triglyceride turnover and removal efficiency Streja et al.

During the fasting days in the present ADF protocol, it would be reasonable to assume that a large part of the substrates for energy production comes from lipids. If not from intramuscular stores, of which a decrease could not be detected, then from extramyocellular stores, i.

adipose tissue. The amount of adipose tissue decreased Table 1 during the 6-week intervention, which in turn give rise to the increased availability of fatty acids Table 2 that facilitates an increased fatty acid transport across the sarcolemma.

To this end, we measured fatty acid translocase CD36 , fatty acids transport protein 4 FATP4 , and plasma membrane fatty acid binding protein FABPpm which are important players in the transport of fatty acids across the plasma membrane.

A mixed result was seen, with CD36 increasing significantly in the OB group, FATP4 decreasing in T2DM, and decreasing in FABPpm main effect Supplementary Figure S1. The changes were small, and the data cannot support the notion that fatty acid transport was increased. Most likely, the oscillation of carbohydrate and lipid substrates every other day blurred a potential marked increase in these proteins.

Once inside the muscle cell, fatty acids can be stored as triglycerides and the final step in the synthesis is catalyzed by diglyceride acyltransferase 1 DGAT1.

DGAT1 protein expression did not change with the intervention Supplementary Figure S2M , but even though we did not detect a difference in lipid content between the two groups, DGAT1 was significantly higher expressed in T2DM compared with OB.

To our knowledge, DGAT1 protein expression in skeletal muscle of patients with type 2 diabetes has only been measured in one other study, in which no change was found compared with obese people and athletes Bergman et al.

Our data suggest that T2DM have the capacity to synthesize greater amounts of intramuscular lipids. We measured two proteins involved in lipolysis adipose triglyceride lipase ATGL , monoacylglyceride lipase MGLL and in lipid storage Perilipin 2 adipophilin , perilipin 3 TIP47 , and perilipin 5 OXPAT and in line with the unchanged lipid content in the muscles Figure 5 we found no effect of the intervention on these proteins Supplementary Figure S1.

An increase in medium-chain acyl-CoA dehydrogenase MCAD might have been seen because MCAD is involved in medium-chain fatty acid beta-oxidation, which would be expected to increase with increased fatty acid availability but not with increased lipid storage.

However, no change was detected Supplementary Figure S1. The expression of proteins involved in lipid transport, synthesis and storage presented here, are in line with data on gene expression mRNA of many of these proteins in a study on females undergoing an intermittent fasting regimen with the muscle biopsies obtained in the same condition i.

after an overnight h fast Liu et al. However, in that study Liu et al. Others have also found that CD36 mRNA remains unchanged with a zero-calorie ADF regimen Heilbronn et al.

Compared with minor caloric restriction, ADF over 6 months does not bring about superior health benefits in terms of body weight, body composition, or cardiovascular risk factors in patients with obesity Trepanowski et al.

This difference in design as well as differences in study cohorts between the two studies makes a direct comparison difficult. The second part of the present study, where ad libitum diet was allowed on feast days demonstrated that the study participants did not inadvertently compensate the overall caloric deficit, because body weight decreased faster in the latter part of the study.

If weight loss is the purpose of ADF, zero-calorie intake must therefore be recommended on fast days because it will not be compensated on feast days. The length of the fasting may also play a role. In the study by Trepanowski et al. Trepanowski et al. Varady, personal communication , but since lunch was allowed between and on fasting days, the fasting period was, in fact, two periods of 12 and 10 h.

These relatively short periods of fasting every other day may therefore be the reason that this intervention was not superior in reducing body weight compared to ordinary everyday caloric restriction.

In the present study, each zero-calorie fasting period was 30 h, which is of sufficient length to markedly draw from endogenous energy sources, introducing loss of body weight and also mimicking oscillations in energy stores induced by exercise Dela et al.

In line with previous studies for review see Dela et al. Dela and Helge, and newer studies Lund et al. In the present study, we tested ADP sensitivity of the skeletal muscle mitochondria Supplementary Figure S1 but found no difference between the groups or an effect of ADF. Previously, in patients with type 2 diabetes, we have demonstrated increased sensitivity for complex I glutamate and complex II succinate substrates Larsen et al.

This study has some limitations. We did not randomize patients to a non-intervention control group, because it is a well-known risk that patients assigned to passive control groups may exhibit behavioural changes, especially in studies with a focus on dietary behaviour.

Instead, we performed two baseline experiments that were carried out two to 3 weeks apart Figure 1 to account for any variation in methodology and to avoid a time effect of enrollment into a dietary study per se.

We did not include a group that performed conventional caloric restriction, thus we cannot make a direct comparison between ADF and conventional caloric restriction, and this was not the purpose here.

The intervention was well tolerated by all patients. The strict zero-calorie regimen is a quite demanding approach, but the reports from the participants were that the most difficult task was to eat the double diet on non-fasting days during the first 3 weeks.

However, a double diet every other day was only used for mechanistic reasons, and it is not the recommended approach for the general use of ADF. It should also be noted that oral medication, except antihypertensive drugs but including glucose lowering drugs, was discontinued during the entire intervention.

Yet, the patients with type 2 diabetes experienced an improvement in fasting glucose and even HbA1c. This suggests that shorter term 6 weeks ADF is a feasible approach in patients in treatment with oral glucose-lowering therapy that will bring about loss of weight and improved glycemic control.

Longer-term more than 6 weeks effects i. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The studies involving human participants were reviewed and approved by The Regional Ethics Committee H Conceptualization, FD, and AI; Methodology, FD, SL, CP.

CH, and AI; Investigation, AI, FD, HH, MC, CJ, EN, SL, JH, EC, and CP; Writing—Original Draft, AI, and FD; Writing—Review and Editing, AI, FD, JH, SL, and CP; Funding Acquisition, FD.

The Danish Council for Independent Research grant no: B , Nordea Foundation grant to the Center for Healthy Aging. We thank the participants, and the technical assistance provided by R. Kraunsøe, J. Bach, and T. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Al-Mrabeh A. Hepatic lipoprotein export and remission of human type 2 diabetes after weight loss. Cell Metab. PubMed Abstract CrossRef Full Text Google Scholar. Arnason T.

Effects of intermittent fasting on health markers in those with type 2 diabetes: A pilot study. World J. Diabetes 8 4 , — Barnosky A. Intermittent fasting vs daily calorie restriction for type 2 diabetes prevention: A review of human findings.

Bergman B. Intramuscular triglyceride synthesis: Importance in muscle lipid partitioning in humans. Bille D. Liver fat content investigated by magnetic resonance spectroscopy in obese children and youths included in multidisciplinary treatment.

Carbajo M. Weight loss and improvement of lipid profiles in morbidly obese patients after laparoscopic one-anastomosis gastric bypass: 2-year follow-up. Carter S. Effect of intermittent compared with continuous energy restricted diet on glycemic control in patients with type 2 diabetes: A randomized noninferiority trial.

JAMA Netw. Open 1 3 , e Catenacci V. A randomized pilot study comparing zero-calorie alternate-day fasting to daily caloric restriction in adults with obesity.

Silver Spring 24 9 , — Chabanova E. Dela F. Insulin resistance and mitochondrial function in skeletal muscle. Cell Biol. Effects of one-legged High-intensity Interval Training on insulin-mediated skeletal muscle glucose homeostasis in patients with type 2 diabetes. Acta Physiol. Insulin-stimulated muscle glucose clearance in patients with NIDDM.

Effects of one-legged physical training. Diabetes 44, — Physical training increases muscle GLUT-4 protein and mRNA in patients with NIDDM. Diabetes 43, — Effect of physical training on insulin secretion and action in skeletal muscle and adipose tissue of first-degree relatives of type 2 diabetic patients.

Physical training may enhance beta-cell function in type 2 diabetes. Dohlmann T.

The 80 study participants were divided into four groups: an alternate-day fasting group, an aerobic exercise group, a combined group and a control group who made no changes to their behaviors. The researchers also found that compared to other participants, the group who combined exercise and fasting experienced reduced body weight, fat mass and waist circumference.

The study, published in the journal Cell Metabolism, was funded by the National Institute of Diabetes and Digestive and Kidney Diseases. Co-authors include Mark Ezpeleta, KN assistant professor Kelsey Gabel , Sofia Cienfuegos, Faiza Kalam, Shuhao Lin, Vasiliki Pavlou, KN professor Zhenyuan Song , Jacob M.

Haus, Sean Koppe, Shaina J. Alexandria and KN associate professor Lisa Tussing-Humphreys. The fasting day began at midnight a. and continued for 30 h until a. the following day. During the fasting day only water, coffee and tea were allowed. During the first 3 weeks the participants aimed at maintaining their body weight by doubling their diet intake on the non-fasting days.

No specific diet was prescribed, and the effect on body weight was monitored by daily weighing and reports to the investigators. After the 10th fasting day, a habitual eu-caloric diet for 1 day was followed by a repetition of the experimental procedures performed at baseline.

In the fourth, fifth, and sixth weeks of ADF, the participants followed an ad libitum diet on eating days, i. a weight loss was allowed. Then the baseline tests were repeated after 1 day of the habitual diet.

This design aimed to focus on two different interventions, ADF without weight loss, i. as the everyday practice of the method. Primary endpoints were changes in insulin sensitivity by the hyperinsulinaemic euglycemic clamp, glucose clearance rate per fat-free mass and beta-cell function by IVGTT, insulin area under the curve with ADF.

Changes in energy store levels were secondary outcome measures. Plasma concentrations of substrates and metabolites glucose, free fatty acids FFA , glycerol, ß-hydroxybutyrate, cholesterol, triglyceride, high-sensitive C-reactive peptide hsCRP , alanine aminotranferase ALAT , asparatate aminotransferase ASAT were measured by spectrophotometry Cobas c , Roche, Glostrup, Denmark.

HbA1c was analyzed on a DCA Vantage Analyser Siemens Healthcare Diagnostics Inc. Tarrytown NY, United States. Applied methods for resting energy expenditure, maximal oxygen uptake tests, whole-body dual-energy X-ray absorptiometry DXA scan Hansen et al.

Methods for the measurement of hepatic triglyceride content Chabanova et al. Measurements were T2 corrected, and T2 was measured for each individual at each visit. Muscle intra- and extra myocellular content were not separated in the analysis, thus m.

psoas major results are describing the sum of these. Protein content in m. vastus lateralis was examined using SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis and Western blotting techniques see Supplementary Material.

Analysis of mitochondrial function was performed in permeabilized skeletal muscle fibers. Mitochondrial respiratory capacity was determined as previously described Dohlmann et al.

See Supplementary Material for the detailed protocols. Citrate synthase CS activity was measured as previously described Larsen et al. Image analysis was performed with Fiji software Schindelin et al. Insulin sensitivity was estimated from glucose infusion rates during the final 30 min of the clamp.

Glucose clearance rates were calculated as glucose infusion rates divided by the prevailing plasma glucose concentration. Statistical analyses of parameters measured across the intervention phases e.

insulin secretion, insulin sensitivity, blood lipids, body weight were performed with mixed model analyses GraphPad Prism 8. In the absence of missing values, this method gives the same p values and multiple comparisons tests as repeated measures ANOVA. In the presence of missing values missing completely at random , the results can be interpreted like repeated measures ANOVA.

Based on data from previous studies Halberg et al. Maximal oxygen uptake was similar in the patients with type 2 diabetes T2DM and obesity OB 2.

Apart from the volume of visceral fat measured by MRI, the two groups were well matched concerning anthropometrics at baseline Table 1. However, the decrease was primarily due to the last part of the intervention protocol, where a weight loss was allowed.

Plasma concentrations of insulin and C-peptide were as expected elevated on the double diet day Table 2. β-hydroxybutyrate representing ketone bodies were increased after 30 h Fasting, and higher in T2DM vs.

Total cholesterol, LDL, and HDL cholesterol were not different between the groups and did not change with the intervention Table 2. Plasma glycerol concentrations displayed a similar pattern, however, with no group differences, and only OB displayed a significant increase with fasting Table 2.

Plasma hsCRP values were generally very low, and no changes were seen Table 2. The interventions did not change the insulin response in the control group. FIGURE 2. In column graphs are shown area under the curve AUC for insulin E and glucose F.

Data are shown as mean ± SD. T2DM patients displayed insulin resistance glucose infusion rate compared with OB Table 1. the insulin sensitivity increased as a main effect and not with ADF alone. Glucose rate of appearance Ra after an overnight fast in the baseline test was increased in T2DM compared with OB 3.

Glucose Ra was unchanged in the fasting state following 3 weeks of ADF T2DM: 3. Muscle glycogen content Table 2 was also measured in the evening after a double diet day and in the morning after a fasting day after 30 h fasting see protocol, Figure 1.

No significant difference was seen with the analysis of the OB and T2DM group separately. The purpose was to monitor adherence to the fasting, double diet and ad libitum diet days and all subjects followed the intervention.

Glucose levels were higher in T2DM compared with OB at all times Figure 3. During ADF, CGM data from weeks one to three illustrates that eating on double diet days increased after the first week, probably also due to encouragement to the patients based upon the daily home-based weighing.

No hypoglycemic events were recorded. FIGURE 3. Continuous glucose monitoring CGM traces in patients with obesity OB and type 2 diabetes T2DM. Data are shown as mean values. FIGURE 4. Intrahepatic triglyceride content measured by 1 H-magnetic resonance spectroscopy in eleven subjects with obesity OB and eleven patients with type 2 diabetes T2DM.

Experiments were performed at baseline, after 3 weeks of alternate-day fasting with double diet on non-fasting days ADF , and after 3 weeks of alternate-day fasting with ad libitum diet, i. Triglyceride content in m. psoas major by 1 H-MRS was not different between the groups and did not change with the intervention baseline: 5.

Muscle lipid droplet density, size of droplets, or the fractional area of the droplets did not differ between T2DM and OB, and no change with ADF was seen Figure 5A, B, C, F. These data were confirmed by analysis of intramuscular triglyceride IMTG content Figure 5D.

In addition, IMTG content was also measured in Tru-Cut biopsies obtained in the evening after a double diet day and in the morgen after 30 h fasting Figure 5E.

after an overnight fast , but this could not be detected in OB Figure 5E. No difference in IMTG between double diet day and 30 h fasting could be seen in either group Figure 5E.

FIGURE 5. Lipid droplets LD density per area of muscle fibre A. LD size B. Intramuscular triglyceride IMTG content in muscle at baseline clamp and at the clamp after ADF D. IMTG content in muscle after one double diet day and after 30 h fasting E. Representative images of Bodipy staining F.

OB, Obese subjects, T2DM, patients with type 2 diabetes. In figure f, scale bars represent µm top and 25 µm bottom. The expression of proteins relevant for insulin-mediated glucose metabolism is shown in Supplementary Figure S1A-I.

Proteins involved in glucose transport GLUT4 and glycolysis hexokinase and pyruvate kinase and glycogen storage glycogen synthase and phosphorylase were similar between the two groups and did not change with ADF.

Proteins relevant for GLUT4 vesicle formation showed minor changes. Thus, Akt was significantly higher in OB compared with T2DM, and AMPKα1 increased significantly with ADF in OB, but not in T2DM. AS and PKCθ remained unchanged and similar in the two groups.

The expression of proteins relevant to lipid metabolism is shown in Supplementary Figure S1J-S. Proteins involved in fatty acids transport into muscle cells were studied by analysis of proteins located in the plasma membrane.

This was carried out to determine if alternate day fasting had any effect on fatty acid transport in healthy or diabetic skeletal muscle tissue.

Fatty acid translocase FAT or CD36 binds long-chain fatty acids and is a key player in fatty acid transport across the plasma membrane.

Fatty acid transport protein 4 FATP4 esterifies long-chain fatty acids and has a role in fatty acid transport across the plasma membrane. Plasma membrane fatty acid binding protein FABPpm is also involved in myocellular uptake of long-chain fatty acids. No change was observed in the T2DM group or between the two groups for all fatty acid transporters.

Citrate synthase CS activity was used as an index for mitochondrial mass Larsen et al. Oxygen consumption ex vivo was measured with a sequential substrate protocol, with state 2 respiration complex I; malate and glutamate followed by state 3 respiration with increasing concentrations of ADP complex I and dual electron input to complex I and II glutamate, malate, succinate, and ADP , ending with uncoupled respiration FCCP as protonophore Supplementary Figure S2A.

First of all, there were no differences in respiration between T2DM and OB, and no significant effect of ADF was observed Supplementary Figure S2A. Reactive oxygen species ROS displayed no difference between the groups and no effect of ADF was observed Supplementary Figure S2B.

ADP sensitivity and maximal oxygen flux Vmax was calculated from the oxygen flux during increasing ADP concentrations, and no differences between groups or effects of ADF were seen Supplementary Figures S2C, D.

The present study represents a comprehensive characterization of the effects of alternate-day fasting regimens on the human metabolism, studied in obese patients with and without type 2 diabetes. The energy balance is essential in every attempt to lose bodyweight, and if weight loss is the primary focus it is fundamental to achieve a negative energy balance, no matter how this is brought about.

This was not the primary focus here. With the present study protocol, we aimed to study the metabolic effects of oscillations in energy intake and thus energy balance. An improvement of the insulin secretory capacity in patients with type 2 diabetes is a therapeutic goal that is difficult to achieve non-pharmacologically, and only a few studies in patients with type 2 diabetes have reported improvements in β-cell secretion following physical training Krotkiewski et al.

The purpose of the present study was to mimic the oscillations that occur in energy stores with frequent exercise training, but at the same time avoid the physiological impact on metabolism that takes place with exercise training i.

muscle contractions. ADF would largely accomplish this, but from previous studies, it is known that weight loss often follows ADF. Therefore, we divided the study into two 3-week periods of ADF, where weight loss was allowed in the latter period, as would be the every day practice. The first-phase insulin response in the patients with type 2 diabetes was, however, not restored albeit the insulin response curve displayed a more marked first phase profile compared with baseline Figure 2C.

An indication that time-restricted feeding i. not the same protocol as used in the present study without weight loss may increase ß-cell responsiveness in pre-diabetic people has been published Sutton et al.

The mechanism for improvements in insulin secretion has been attributed to a decrease in intrapancreatic triacylglycerol Lim et al. This is also a likely explanation in the present study, where we observed large decreases in visceral fat Table 1 and intrahepatic triglyceride content Figure 4 where the latter correlated significantly with the improvement in insulin secretion.

The elevated plasma concentrations of FFA, glycerol, and β-hydroxybutyrate during fasting Table 2 testified to an increased lipolytic rate during fasting, contributing to the marked decrease of adipose tissue during the interventions Table 1. An additional mechanism for the improvement in insulin secretion could also be due to an overall reduced glycemic load on the β-cells i.

reduced glucotoxicity. Apart from documenting the adherence to the protocol, the continuous glucose monitoring Figure 3 revealed a lessened glycemic burden, which in itself reduces the stress on the β-cells.

It is important to note that the duration of type 2 diabetes, or at least the time since diagnosis, was short among the included patients 2. This means that the patients had a relatively well-preserved β-cell function, but of course, diminished compared with the obese subjects without type 2 diabetes Figure 2.

We have previously shown that patients with a high pre-operative β-cell function experience a superior outcome to gastric bypass surgery compared with those patients with the lowest pre-operative β-cell function Lund et al.

Most likely, patients with severely reduced insulin secretory capacity which can be easily estimated by a 6 min glucagon test Dela et al. Many studies have shown positive effects of training on insulin-mediated glucose uptake in skeletal muscle in patients with type 2 diabetes Dela et al.

For muscle glycogen Table 2 this aim was achieved, but the oscillations did not translate into an improvement of insulin-mediated glucose clearance with ADF alone, which is in contrast to earlier findings in young, healthy subjects Halberg et al.

However, the data are in line with findings in obese people, using a calculated index for insulin sensitivity S I from an intravenous glucose tolerance test Catenacci et al. Insulin action at the hepatic level, i. inhibition of endogenous glucose Ra, did not change with ADF.

This finding is in line with the lack of effect of ADF on peripheral insulin action. It may require an extended period of starvation 3—4 days before a reduction of insulin-induced suppression of hepatic glucose output is seen Fery et al.

This indicates a general improvement in hepatic function elicited by dietary regimen. The lack of increases in insulin sensitivity with ADF is in line with the general lack of increases in proteins relevant for skeletal muscle insulin action, e. GLUT4, hexokinase, glycogen synthase Supplementary Figure S1.

In rodents, a similar lack of change in hexokinase after intermittent fasting has been reported Real-Hohn et al. The amount of intramyocellular lipids is inversely correlated with insulin sensitivity Pan et al.

psoas major fit well with the lack of changes in insulin sensitivity. A similar amount of lipid content in the muscle in T2DM and OB has been shown before Hansen et al.

The latter is in line with earlier findings that demonstrated that it requires prolonged fasting e. Lipolysis increases at the beginning of a fasting period here evidenced by increased FFA and glycerol after 30 h fasting; Table 2.

The more so, because the anti-lipolytic effect of insulin diminishes with fasting Jensen et al. Support for an ADF-induced triglyceride-lowering effect is found in low-calorie refeeding studies that demonstrated increased triglyceride turnover and removal efficiency Streja et al.

During the fasting days in the present ADF protocol, it would be reasonable to assume that a large part of the substrates for energy production comes from lipids. If not from intramuscular stores, of which a decrease could not be detected, then from extramyocellular stores, i.

adipose tissue. The amount of adipose tissue decreased Table 1 during the 6-week intervention, which in turn give rise to the increased availability of fatty acids Table 2 that facilitates an increased fatty acid transport across the sarcolemma. To this end, we measured fatty acid translocase CD36 , fatty acids transport protein 4 FATP4 , and plasma membrane fatty acid binding protein FABPpm which are important players in the transport of fatty acids across the plasma membrane.

A mixed result was seen, with CD36 increasing significantly in the OB group, FATP4 decreasing in T2DM, and decreasing in FABPpm main effect Supplementary Figure S1. The changes were small, and the data cannot support the notion that fatty acid transport was increased.

Most likely, the oscillation of carbohydrate and lipid substrates every other day blurred a potential marked increase in these proteins. Once inside the muscle cell, fatty acids can be stored as triglycerides and the final step in the synthesis is catalyzed by diglyceride acyltransferase 1 DGAT1.

DGAT1 protein expression did not change with the intervention Supplementary Figure S2M , but even though we did not detect a difference in lipid content between the two groups, DGAT1 was significantly higher expressed in T2DM compared with OB.

To our knowledge, DGAT1 protein expression in skeletal muscle of patients with type 2 diabetes has only been measured in one other study, in which no change was found compared with obese people and athletes Bergman et al.

Our data suggest that T2DM have the capacity to synthesize greater amounts of intramuscular lipids. We measured two proteins involved in lipolysis adipose triglyceride lipase ATGL , monoacylglyceride lipase MGLL and in lipid storage Perilipin 2 adipophilin , perilipin 3 TIP47 , and perilipin 5 OXPAT and in line with the unchanged lipid content in the muscles Figure 5 we found no effect of the intervention on these proteins Supplementary Figure S1.

An increase in medium-chain acyl-CoA dehydrogenase MCAD might have been seen because MCAD is involved in medium-chain fatty acid beta-oxidation, which would be expected to increase with increased fatty acid availability but not with increased lipid storage.

However, no change was detected Supplementary Figure S1. The expression of proteins involved in lipid transport, synthesis and storage presented here, are in line with data on gene expression mRNA of many of these proteins in a study on females undergoing an intermittent fasting regimen with the muscle biopsies obtained in the same condition i.

after an overnight h fast Liu et al. However, in that study Liu et al. Others have also found that CD36 mRNA remains unchanged with a zero-calorie ADF regimen Heilbronn et al.

Compared with minor caloric restriction, ADF over 6 months does not bring about superior health benefits in terms of body weight, body composition, or cardiovascular risk factors in patients with obesity Trepanowski et al. This difference in design as well as differences in study cohorts between the two studies makes a direct comparison difficult.

The second part of the present study, where ad libitum diet was allowed on feast days demonstrated that the study participants did not inadvertently compensate the overall caloric deficit, because body weight decreased faster in the latter part of the study.

If weight loss is the purpose of ADF, zero-calorie intake must therefore be recommended on fast days because it will not be compensated on feast days. The length of the fasting may also play a role.

In the study by Trepanowski et al. Trepanowski et al. Varady, personal communication , but since lunch was allowed between and on fasting days, the fasting period was, in fact, two periods of 12 and 10 h.

These relatively short periods of fasting every other day may therefore be the reason that this intervention was not superior in reducing body weight compared to ordinary everyday caloric restriction.

In the present study, each zero-calorie fasting period was 30 h, which is of sufficient length to markedly draw from endogenous energy sources, introducing loss of body weight and also mimicking oscillations in energy stores induced by exercise Dela et al.

In line with previous studies for review see Dela et al. Dela and Helge, and newer studies Lund et al. In the present study, we tested ADP sensitivity of the skeletal muscle mitochondria Supplementary Figure S1 but found no difference between the groups or an effect of ADF.

Previously, in patients with type 2 diabetes, we have demonstrated increased sensitivity for complex I glutamate and complex II succinate substrates Larsen et al. This study has some limitations. We did not randomize patients to a non-intervention control group, because it is a well-known risk that patients assigned to passive control groups may exhibit behavioural changes, especially in studies with a focus on dietary behaviour.

Instead, we performed two baseline experiments that were carried out two to 3 weeks apart Figure 1 to account for any variation in methodology and to avoid a time effect of enrollment into a dietary study per se. We did not include a group that performed conventional caloric restriction, thus we cannot make a direct comparison between ADF and conventional caloric restriction, and this was not the purpose here.

The intervention was well tolerated by all patients. The strict zero-calorie regimen is a quite demanding approach, but the reports from the participants were that the most difficult task was to eat the double diet on non-fasting days during the first 3 weeks.

However, a double diet every other day was only used for mechanistic reasons, and it is not the recommended approach for the general use of ADF. It should also be noted that oral medication, except antihypertensive drugs but including glucose lowering drugs, was discontinued during the entire intervention.

Yet, the patients with type 2 diabetes experienced an improvement in fasting glucose and even HbA1c. This suggests that shorter term 6 weeks ADF is a feasible approach in patients in treatment with oral glucose-lowering therapy that will bring about loss of weight and improved glycemic control.

Longer-term more than 6 weeks effects i. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by The Regional Ethics Committee H Conceptualization, FD, and AI; Methodology, FD, SL, CP. CH, and AI; Investigation, AI, FD, HH, MC, CJ, EN, SL, JH, EC, and CP; Writing—Original Draft, AI, and FD; Writing—Review and Editing, AI, FD, JH, SL, and CP; Funding Acquisition, FD.

The Danish Council for Independent Research grant no: B , Nordea Foundation grant to the Center for Healthy Aging. We thank the participants, and the technical assistance provided by R. Kraunsøe, J. Bach, and T. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.

Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Al-Mrabeh A. Hepatic lipoprotein export and remission of human type 2 diabetes after weight loss. Cell Metab. PubMed Abstract CrossRef Full Text Google Scholar. Arnason T. Effects of intermittent fasting on health markers in those with type 2 diabetes: A pilot study.

World J. Diabetes 8 4 , — Barnosky A. Intermittent fasting vs daily calorie restriction for type 2 diabetes prevention: A review of human findings.

Bergman B. Intramuscular triglyceride synthesis: Importance in muscle lipid partitioning in humans. Bille D. Liver fat content investigated by magnetic resonance spectroscopy in obese children and youths included in multidisciplinary treatment.

Carbajo M.

Muscle glycogen content Table 2 was also measured in the evening after a double diet day and in the morning after a fasting day after 30 h fasting see protocol, Figure 1. No significant difference was seen with the analysis of the OB and T2DM group separately. The purpose was to monitor adherence to the fasting, double diet and ad libitum diet days and all subjects followed the intervention.

Glucose levels were higher in T2DM compared with OB at all times Figure 3. During ADF, CGM data from weeks one to three illustrates that eating on double diet days increased after the first week, probably also due to encouragement to the patients based upon the daily home-based weighing.

No hypoglycemic events were recorded. FIGURE 3. Continuous glucose monitoring CGM traces in patients with obesity OB and type 2 diabetes T2DM. Data are shown as mean values. FIGURE 4. Intrahepatic triglyceride content measured by 1 H-magnetic resonance spectroscopy in eleven subjects with obesity OB and eleven patients with type 2 diabetes T2DM.

Experiments were performed at baseline, after 3 weeks of alternate-day fasting with double diet on non-fasting days ADF , and after 3 weeks of alternate-day fasting with ad libitum diet, i. Triglyceride content in m. psoas major by 1 H-MRS was not different between the groups and did not change with the intervention baseline: 5.

Muscle lipid droplet density, size of droplets, or the fractional area of the droplets did not differ between T2DM and OB, and no change with ADF was seen Figure 5A, B, C, F.

These data were confirmed by analysis of intramuscular triglyceride IMTG content Figure 5D. In addition, IMTG content was also measured in Tru-Cut biopsies obtained in the evening after a double diet day and in the morgen after 30 h fasting Figure 5E.

after an overnight fast , but this could not be detected in OB Figure 5E. No difference in IMTG between double diet day and 30 h fasting could be seen in either group Figure 5E. FIGURE 5. Lipid droplets LD density per area of muscle fibre A.

LD size B. Intramuscular triglyceride IMTG content in muscle at baseline clamp and at the clamp after ADF D. IMTG content in muscle after one double diet day and after 30 h fasting E.

Representative images of Bodipy staining F. OB, Obese subjects, T2DM, patients with type 2 diabetes. In figure f, scale bars represent µm top and 25 µm bottom. The expression of proteins relevant for insulin-mediated glucose metabolism is shown in Supplementary Figure S1A-I. Proteins involved in glucose transport GLUT4 and glycolysis hexokinase and pyruvate kinase and glycogen storage glycogen synthase and phosphorylase were similar between the two groups and did not change with ADF.

Proteins relevant for GLUT4 vesicle formation showed minor changes. Thus, Akt was significantly higher in OB compared with T2DM, and AMPKα1 increased significantly with ADF in OB, but not in T2DM. AS and PKCθ remained unchanged and similar in the two groups. The expression of proteins relevant to lipid metabolism is shown in Supplementary Figure S1J-S.

Proteins involved in fatty acids transport into muscle cells were studied by analysis of proteins located in the plasma membrane. This was carried out to determine if alternate day fasting had any effect on fatty acid transport in healthy or diabetic skeletal muscle tissue.

Fatty acid translocase FAT or CD36 binds long-chain fatty acids and is a key player in fatty acid transport across the plasma membrane.

Fatty acid transport protein 4 FATP4 esterifies long-chain fatty acids and has a role in fatty acid transport across the plasma membrane. Plasma membrane fatty acid binding protein FABPpm is also involved in myocellular uptake of long-chain fatty acids.

No change was observed in the T2DM group or between the two groups for all fatty acid transporters. Citrate synthase CS activity was used as an index for mitochondrial mass Larsen et al. Oxygen consumption ex vivo was measured with a sequential substrate protocol, with state 2 respiration complex I; malate and glutamate followed by state 3 respiration with increasing concentrations of ADP complex I and dual electron input to complex I and II glutamate, malate, succinate, and ADP , ending with uncoupled respiration FCCP as protonophore Supplementary Figure S2A.

First of all, there were no differences in respiration between T2DM and OB, and no significant effect of ADF was observed Supplementary Figure S2A.

Reactive oxygen species ROS displayed no difference between the groups and no effect of ADF was observed Supplementary Figure S2B. ADP sensitivity and maximal oxygen flux Vmax was calculated from the oxygen flux during increasing ADP concentrations, and no differences between groups or effects of ADF were seen Supplementary Figures S2C, D.

The present study represents a comprehensive characterization of the effects of alternate-day fasting regimens on the human metabolism, studied in obese patients with and without type 2 diabetes. The energy balance is essential in every attempt to lose bodyweight, and if weight loss is the primary focus it is fundamental to achieve a negative energy balance, no matter how this is brought about.

This was not the primary focus here. With the present study protocol, we aimed to study the metabolic effects of oscillations in energy intake and thus energy balance. An improvement of the insulin secretory capacity in patients with type 2 diabetes is a therapeutic goal that is difficult to achieve non-pharmacologically, and only a few studies in patients with type 2 diabetes have reported improvements in β-cell secretion following physical training Krotkiewski et al.

The purpose of the present study was to mimic the oscillations that occur in energy stores with frequent exercise training, but at the same time avoid the physiological impact on metabolism that takes place with exercise training i. muscle contractions. ADF would largely accomplish this, but from previous studies, it is known that weight loss often follows ADF.

Therefore, we divided the study into two 3-week periods of ADF, where weight loss was allowed in the latter period, as would be the every day practice. The first-phase insulin response in the patients with type 2 diabetes was, however, not restored albeit the insulin response curve displayed a more marked first phase profile compared with baseline Figure 2C.

An indication that time-restricted feeding i. not the same protocol as used in the present study without weight loss may increase ß-cell responsiveness in pre-diabetic people has been published Sutton et al. The mechanism for improvements in insulin secretion has been attributed to a decrease in intrapancreatic triacylglycerol Lim et al.

This is also a likely explanation in the present study, where we observed large decreases in visceral fat Table 1 and intrahepatic triglyceride content Figure 4 where the latter correlated significantly with the improvement in insulin secretion.

The elevated plasma concentrations of FFA, glycerol, and β-hydroxybutyrate during fasting Table 2 testified to an increased lipolytic rate during fasting, contributing to the marked decrease of adipose tissue during the interventions Table 1.

An additional mechanism for the improvement in insulin secretion could also be due to an overall reduced glycemic load on the β-cells i. reduced glucotoxicity. Apart from documenting the adherence to the protocol, the continuous glucose monitoring Figure 3 revealed a lessened glycemic burden, which in itself reduces the stress on the β-cells.

It is important to note that the duration of type 2 diabetes, or at least the time since diagnosis, was short among the included patients 2. This means that the patients had a relatively well-preserved β-cell function, but of course, diminished compared with the obese subjects without type 2 diabetes Figure 2.

We have previously shown that patients with a high pre-operative β-cell function experience a superior outcome to gastric bypass surgery compared with those patients with the lowest pre-operative β-cell function Lund et al. Most likely, patients with severely reduced insulin secretory capacity which can be easily estimated by a 6 min glucagon test Dela et al.

Many studies have shown positive effects of training on insulin-mediated glucose uptake in skeletal muscle in patients with type 2 diabetes Dela et al. For muscle glycogen Table 2 this aim was achieved, but the oscillations did not translate into an improvement of insulin-mediated glucose clearance with ADF alone, which is in contrast to earlier findings in young, healthy subjects Halberg et al.

However, the data are in line with findings in obese people, using a calculated index for insulin sensitivity S I from an intravenous glucose tolerance test Catenacci et al.

Insulin action at the hepatic level, i. inhibition of endogenous glucose Ra, did not change with ADF. This finding is in line with the lack of effect of ADF on peripheral insulin action.

It may require an extended period of starvation 3—4 days before a reduction of insulin-induced suppression of hepatic glucose output is seen Fery et al. This indicates a general improvement in hepatic function elicited by dietary regimen. The lack of increases in insulin sensitivity with ADF is in line with the general lack of increases in proteins relevant for skeletal muscle insulin action, e.

GLUT4, hexokinase, glycogen synthase Supplementary Figure S1. In rodents, a similar lack of change in hexokinase after intermittent fasting has been reported Real-Hohn et al. The amount of intramyocellular lipids is inversely correlated with insulin sensitivity Pan et al.

psoas major fit well with the lack of changes in insulin sensitivity. A similar amount of lipid content in the muscle in T2DM and OB has been shown before Hansen et al.

The latter is in line with earlier findings that demonstrated that it requires prolonged fasting e. Lipolysis increases at the beginning of a fasting period here evidenced by increased FFA and glycerol after 30 h fasting; Table 2.

The more so, because the anti-lipolytic effect of insulin diminishes with fasting Jensen et al. Support for an ADF-induced triglyceride-lowering effect is found in low-calorie refeeding studies that demonstrated increased triglyceride turnover and removal efficiency Streja et al.

During the fasting days in the present ADF protocol, it would be reasonable to assume that a large part of the substrates for energy production comes from lipids. If not from intramuscular stores, of which a decrease could not be detected, then from extramyocellular stores, i.

adipose tissue. The amount of adipose tissue decreased Table 1 during the 6-week intervention, which in turn give rise to the increased availability of fatty acids Table 2 that facilitates an increased fatty acid transport across the sarcolemma. To this end, we measured fatty acid translocase CD36 , fatty acids transport protein 4 FATP4 , and plasma membrane fatty acid binding protein FABPpm which are important players in the transport of fatty acids across the plasma membrane.

A mixed result was seen, with CD36 increasing significantly in the OB group, FATP4 decreasing in T2DM, and decreasing in FABPpm main effect Supplementary Figure S1. The changes were small, and the data cannot support the notion that fatty acid transport was increased.

Most likely, the oscillation of carbohydrate and lipid substrates every other day blurred a potential marked increase in these proteins. Once inside the muscle cell, fatty acids can be stored as triglycerides and the final step in the synthesis is catalyzed by diglyceride acyltransferase 1 DGAT1.

DGAT1 protein expression did not change with the intervention Supplementary Figure S2M , but even though we did not detect a difference in lipid content between the two groups, DGAT1 was significantly higher expressed in T2DM compared with OB. To our knowledge, DGAT1 protein expression in skeletal muscle of patients with type 2 diabetes has only been measured in one other study, in which no change was found compared with obese people and athletes Bergman et al.

Our data suggest that T2DM have the capacity to synthesize greater amounts of intramuscular lipids. We measured two proteins involved in lipolysis adipose triglyceride lipase ATGL , monoacylglyceride lipase MGLL and in lipid storage Perilipin 2 adipophilin , perilipin 3 TIP47 , and perilipin 5 OXPAT and in line with the unchanged lipid content in the muscles Figure 5 we found no effect of the intervention on these proteins Supplementary Figure S1.

An increase in medium-chain acyl-CoA dehydrogenase MCAD might have been seen because MCAD is involved in medium-chain fatty acid beta-oxidation, which would be expected to increase with increased fatty acid availability but not with increased lipid storage.

However, no change was detected Supplementary Figure S1. The expression of proteins involved in lipid transport, synthesis and storage presented here, are in line with data on gene expression mRNA of many of these proteins in a study on females undergoing an intermittent fasting regimen with the muscle biopsies obtained in the same condition i.

after an overnight h fast Liu et al. However, in that study Liu et al. Others have also found that CD36 mRNA remains unchanged with a zero-calorie ADF regimen Heilbronn et al. Compared with minor caloric restriction, ADF over 6 months does not bring about superior health benefits in terms of body weight, body composition, or cardiovascular risk factors in patients with obesity Trepanowski et al.

This difference in design as well as differences in study cohorts between the two studies makes a direct comparison difficult. The second part of the present study, where ad libitum diet was allowed on feast days demonstrated that the study participants did not inadvertently compensate the overall caloric deficit, because body weight decreased faster in the latter part of the study.

If weight loss is the purpose of ADF, zero-calorie intake must therefore be recommended on fast days because it will not be compensated on feast days. The length of the fasting may also play a role. In the study by Trepanowski et al. Trepanowski et al.

Varady, personal communication , but since lunch was allowed between and on fasting days, the fasting period was, in fact, two periods of 12 and 10 h. These relatively short periods of fasting every other day may therefore be the reason that this intervention was not superior in reducing body weight compared to ordinary everyday caloric restriction.

In the present study, each zero-calorie fasting period was 30 h, which is of sufficient length to markedly draw from endogenous energy sources, introducing loss of body weight and also mimicking oscillations in energy stores induced by exercise Dela et al. In line with previous studies for review see Dela et al.

Dela and Helge, and newer studies Lund et al. In the present study, we tested ADP sensitivity of the skeletal muscle mitochondria Supplementary Figure S1 but found no difference between the groups or an effect of ADF. Previously, in patients with type 2 diabetes, we have demonstrated increased sensitivity for complex I glutamate and complex II succinate substrates Larsen et al.

This study has some limitations. We did not randomize patients to a non-intervention control group, because it is a well-known risk that patients assigned to passive control groups may exhibit behavioural changes, especially in studies with a focus on dietary behaviour.

Instead, we performed two baseline experiments that were carried out two to 3 weeks apart Figure 1 to account for any variation in methodology and to avoid a time effect of enrollment into a dietary study per se.

We did not include a group that performed conventional caloric restriction, thus we cannot make a direct comparison between ADF and conventional caloric restriction, and this was not the purpose here.

The intervention was well tolerated by all patients. The strict zero-calorie regimen is a quite demanding approach, but the reports from the participants were that the most difficult task was to eat the double diet on non-fasting days during the first 3 weeks.

However, a double diet every other day was only used for mechanistic reasons, and it is not the recommended approach for the general use of ADF. It should also be noted that oral medication, except antihypertensive drugs but including glucose lowering drugs, was discontinued during the entire intervention.

Yet, the patients with type 2 diabetes experienced an improvement in fasting glucose and even HbA1c. This suggests that shorter term 6 weeks ADF is a feasible approach in patients in treatment with oral glucose-lowering therapy that will bring about loss of weight and improved glycemic control.

Longer-term more than 6 weeks effects i. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The studies involving human participants were reviewed and approved by The Regional Ethics Committee H Conceptualization, FD, and AI; Methodology, FD, SL, CP.

CH, and AI; Investigation, AI, FD, HH, MC, CJ, EN, SL, JH, EC, and CP; Writing—Original Draft, AI, and FD; Writing—Review and Editing, AI, FD, JH, SL, and CP; Funding Acquisition, FD.

The Danish Council for Independent Research grant no: B , Nordea Foundation grant to the Center for Healthy Aging. We thank the participants, and the technical assistance provided by R.

Kraunsøe, J. Bach, and T. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.

Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Al-Mrabeh A. Hepatic lipoprotein export and remission of human type 2 diabetes after weight loss. Cell Metab.

PubMed Abstract CrossRef Full Text Google Scholar. Arnason T. Effects of intermittent fasting on health markers in those with type 2 diabetes: A pilot study. World J. Diabetes 8 4 , — Barnosky A. Intermittent fasting vs daily calorie restriction for type 2 diabetes prevention: A review of human findings.

Bergman B. Intramuscular triglyceride synthesis: Importance in muscle lipid partitioning in humans. Bille D. Liver fat content investigated by magnetic resonance spectroscopy in obese children and youths included in multidisciplinary treatment.

Carbajo M. Weight loss and improvement of lipid profiles in morbidly obese patients after laparoscopic one-anastomosis gastric bypass: 2-year follow-up. Carter S. Effect of intermittent compared with continuous energy restricted diet on glycemic control in patients with type 2 diabetes: A randomized noninferiority trial.

JAMA Netw. Open 1 3 , e Catenacci V. A randomized pilot study comparing zero-calorie alternate-day fasting to daily caloric restriction in adults with obesity.

Silver Spring 24 9 , — Chabanova E. Dela F. Insulin resistance and mitochondrial function in skeletal muscle. Cell Biol. Effects of one-legged High-intensity Interval Training on insulin-mediated skeletal muscle glucose homeostasis in patients with type 2 diabetes.

Acta Physiol. Insulin-stimulated muscle glucose clearance in patients with NIDDM. Effects of one-legged physical training. Diabetes 44, — Physical training increases muscle GLUT-4 protein and mRNA in patients with NIDDM. Diabetes 43, — Effect of physical training on insulin secretion and action in skeletal muscle and adipose tissue of first-degree relatives of type 2 diabetic patients.

Physical training may enhance beta-cell function in type 2 diabetes. Dohlmann T. High-intensity interval training changes mitochondrial respiratory capacity differently in adipose tissue and skeletal muscle. Fery F. Mechanisms of starvation diabetes: A study with double tracer and indirect calorimetry.

Fonvig C. Muscle fat content and abdominal adipose tissue distribution investigated by magnetic resonance spectroscopy and imaging in obese children and youths. Frossing S. Quantification of visceral adipose tissue in polycystic ovary syndrome: Dual-energy X-ray absorptiometry versus magnetic resonance imaging.

Acta Radiol. Halberg N. Effect of intermittent fasting and refeeding on insulin action in healthy men. Hansen M. The effects of diet- and RYGB-induced weight loss on insulin sensitivity in obese patients with and without type 2 diabetes. Acta Diabetol. The effect of metformin on glucose homeostasis during moderate exercise.

Diabetes Care 38 2 , — Harvie M. Potential benefits and harms of intermittent energy restriction and intermittent fasting amongst obese, overweight and normal weight subjects-A narrative review of human and animal evidence.

Heilbronn L. Glucose tolerance and skeletal muscle gene expression in response to alternate day fasting. Holten M. Strength training increases insulin-mediated glucose uptake, GLUT4 content and insulin signaling in skeletal muscle in patients with Type 2 diabetes.

Diabetes 53 2 , — Jensen M. Lipolysis during fasting. Decreased suppression by insulin and increased stimulation by epinephrine. Kelley D. Fatty liver in type 2 diabetes mellitus: Relation to regional adiposity, fatty acids, and insulin resistance.

Krotkiewski M. The effects of physical training on insulin secretion and effectiveness and on glucose metabolism in obesity and type 2 non-insulin-dependent diabetes mellitus. Diabetologia 28, — Add topic to email alerts. Receive an email when new articles are posted on.

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