Current Diabetes Reports (2020) 20:13

https://doi.org/10.1007/s11892-020-1295-2

LIFESTYLE MANAGEMENT TO REDUCE DIABETES/CARDIOVASCULAR RISK (B CONWAY AND H

KEENAN, SECTION EDITORS)

Intermittent Fasting as Part of the Management for T2DM:

from Animal Models to Human Clinical Studies
Liliana Muñoz-Hernández 1,2 & Ziomara Márquez-López 1 & Roopa Mehta 1 & Carlos Alberto Aguilar-Salinas 1,3,4

# Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
Purpose of Review Diet is a pillar of type 2 diabetes mellitus (T2DM) management. Intermittent fasting (IF) is postulated as a
novel approach, able to improve glucose control and potentially capable of reversing some of the pathophysiological alterations
of this condition. In this review, the molecular and clinical evidence of diets based on intermittent energy restriction (IER) in
laboratory animal models and subjects with type 2 diabetes is discussed. The mechanisms through which IF are thought to
improve glucose homeostasis and reverse β cell failure are also reviewed.
Recent Findings Studies derived from murine models suggest that IER is associated with improvements in β cell function and
insulin resistance. Two main mechanisms have been demonstrated, one derived from the autophagy-lysosome pathway and, the
other from an increase in neurogenin3 (Ngn3) levels (a marker for endocrine progenitor cells like β cells during development).
Notably, IER also promotes reconstruction of gut microbiota. In mice, all effects were independent of weight loss. By contrast, in
human studies, outcomes are widely attributable to weight loss. The more consistent results are reductions in body weight,
visceral fat, and glucose and insulin levels. Increases in HDL cholesterol levels are also frequently reported. The decrease in
insulin levels observed in humans is in opposition with the increase reported in mice, suggesting that the main mechanism in
humans is an improvement in peripheral insulin action.
Summary Recommending diets based on intermittent fasting in humans is based on the promising results found in animal models
where an improvement in β cell function has been recorded. β cell function after IF has not been assessed in human subjects with
T2DM. This review provides information regarding different protocols for the implementation of IF in diabetic persons and also
provides important safety advice in order to avoid adverse effects. Clinical studies do not show an increased risk of hypoglyce-
mia, and a recent case series reported reversal of T2DM.

Keywords Intermittent energy restriction . Intermittent fasting . Alternate-day fasting . Time-restricted feeding .
Fasting-mimicking diets . Type 2 diabetes remission

Abbreviations
This article is part of the Topical Collection on Lifestyle Management to ADF Alternate-day fasting
Reduce Diabetes/Cardiovascular Risk AL Ad libitum
ALT Alanine aminotransferase
* Carlos Alberto Aguilar-Salinas ASUI Asthma Symptom Utility Index
caguilarsalinas@yahoo.com AST Aspartate aminotransferase
1 BDNF Brain-derived neurotrophic factor
Metabolic Diseases Research Unit, Instituto Nacional de Ciencias
Medicas y Nutricion, Mexico City, Mexico BF Body fat
2 BMI Body mass index
Consejo Nacional de Ciencia y Tecnología, Mexico City, Mexico
BW Body weight
3
Escuela de Medicina y Ciencias de la Salud, Tecnológico de BUN Blood urea nitrogen
Monterrey, Monterrey, Mexico
CER Continuous energy restriction
4
Division of Nutrition, Instituto Nacional de Ciencias Medicas y CRP C-reactive protein
Nutricion, Vasco de Quiroga #15, Tlalpan, 14080 Mexico
City, Mexico CV Cardiovascular
 13 Page 2 of 10 Curr Diab Rep (2020) 20:13

DBP Diastolic blood pressure benefits of dietary modifications resulting in weight loss and
FFA Free fatty acid associated positive metabolic effects are irrefutable [1, 2].
FI Food intake Recently, dietary approaches to reverse type 2 diabetes, pre-
FT Fat tissue serve B cell mass, and achieve remission of glucose alterations
FEV1 Forced expiratory volume-1 minute have gained importance [3, 4, 5••].
FGF21 Fibroblast growth factor 21 Caloric restriction in its two main modalities, continuous
FM Fat mass and intermittent, has been shown to reverse alterations in glu-
FFM Fat-free mass cose homeostasis in animal and human studies [6–8, 9•]. In
GH Growth hormone human protocols, mechanisms are tightly associated with
GLP-1 Glucose-like peptide type 1 weight loss, but animal studies suggest that the beneficial
GSK-3 Glycogen synthase kinase 3 effects of intermittent energy restriction (IER) go beyond
HDL-C High-density lipoprotein cholesterol weight loss, involving mechanisms that permit β cell
HOMA-IR Homeostasis model assessment insulin neogenesis [9•]. The objective of this review is to describe
resistance studies of intermittent fasting (IF) in both animal models and
HADS Hospital Anxiety and Depression Scale humans with diabetes. All animal studies presented here are
HbA1c Glycated hemoglobin derived from murine models where type 2 diabetes was pro-
HR Heart rate voked through either a high-fat diet or using a leptin receptor
IER Intermittent energy restriction knockout mouse model (micedb/db). In these studies, IER was
IER-PF Intermittent energy restriction plus protein and induced with alternated day fasting (ADF) or fasting-
fat ad libitum mimicking diet (FMD) [10]–[12]. In contrast, the human stud-
IF Intermittent fasting ies in subjects with type 2 diabetes (T2DM) [13]–[17] use
IFCR-F Intermittent fasting + caloric restriction + food fasting protocols such as time-restricted feeding (TRF),
based ADF, and 5:2 fasting ratio.
IFCR-L Intermittent fasting + caloric restriction + liquid These different modalities of IER are detailed in this re-
meals view. Special mention is made of a case series recently pub-
IGF Insulin-like growth factor lished where diabetes remission was achieved after an inter-
IL-6 Interleukin 6 mittent fasting period of 6 to 9 months [6••]. Finally, the po-
LBM Lean body mass tential risks of intermittent fasting are addressed, and manage-
LDL-C Low-density lipoprotein cholesterol ment issues are discussed. Figure 1 shows the outcomes re-
MiniAQLQ Mini Asthma Quality of Life Questionnaire ported from animal and human studies.
mTOR Mammalian target of rapamycin
PEF Peak expiratory flow Animal Models of Type 2 Diabetes and IER
PPAR Peroxisome proliferator-activated receptors
REE Respiratory energy expenditure We will review 3 studies with murine models of T2DM. In
SBP Systolic blood pressure one study, obesity was induced with a hypercaloric high-fat
SHBG Sex hormone–binding globulin diet, resulting in insulin resistance and T2DM In the re-
SMBG Self-monitoring blood glucose maining 2 studies, the leptin receptor knockout model was
CSN Central nervous system used. These leptin-resistant mice show obesity and insulin
T2DM Type 2 diabetes mellitus resistance early in life and subsequently develop β cell
TC Total cholesterol failure, hyperglycemia, and T2DM later in life (after 10–
TCA Tricarboxylic acid 12 weeks).
TG Triglycerides Cheng et al exposed micedb/db to FMD for 90 days. The
TNF-α Tumoral necrosis factor-alpha FMD protocol consisted of 4 days of a very low calorie diet
VLDL-C Very low-density lipoprotein cholesterol (low protein and carbohydrate content) alternating with 7 days
vs Versus of normal feed. The authors reported a temporary reduction in
WHO-5 World Health Organization 5 Well-Being Index β cell number, followed by a return to normal levels after re-
feeding. The net result was a significant improvement in β cell
proliferation and function. The investigators speculate that this
Introduction is due to in vivo lineage reprograming, through the activation
of the protein neurogenin 3 (Ngn3). Ngn3+ cells within pan-
In order to reduce cardiometabolic risk in subjects with type 2 creatic islets have been described as progenitor cells able to
diabetes (T2DM) and obesity, the promotion of moderate and generate α and β cells. With this model, a reduction in glucose
sustained weight loss is a primary goal. In this regard, the levels was observed after day 30 and was associated with an
Curr Diab Rep (2020) 20:13 Page 3 of 10 13

Animal models Human Sttudies

Intermittent Energy Restriction

Fastt-mimicking d iet: Very low

Be
enefits: prote
ein and carboh ydrate Bene
efits:
-C
Cell Neogenesiis nt based or not )
(plan HDDL-C
% -cell mass Inssulin Sensitivity
Ins
sulin secretion Quuality of life
HDDL-C Alte
ernated days: Fasting HOOMA-IR
everry other day or 5:2 ratio Faasting Insulin
-C
Cell apoptosis Weight
Fasting glucose Lip
pid profile
Ne
eutral weight Tim
me restricted feeeding:
Fasting most of da y, daily
basiis (18-20 hours )

Fig. 1 Modalities and outcomes from intermittent energy restriction protocols applied in mice and humans. HDL-C, high-density lipoprotein-cholesterol

increase in insulin secretion and HOMA-β, and a proliferation
of β cells. Between days 30 and 90, improvements in insulin
and glucose tolerance test results were documented. These
findings suggest that the two pathophysiological mechanisms
of T2DM, insulin secretion and insulin resistance, were
reverted [11••]. Liu et al. exposed a murine model of
obesity-induced diabetes (through a high-fat diet), to an IF
protocol. They reported that this regimen restored the autoph-
agic flux in islets and is associated with an improvement in
glucose tolerance by enhancing glucose-stimulated insulin se-
cretion, β cell survival, and nuclear expression of NgN3. They
also demonstrated that mice with lysosomal dysfunction sec-
ondary to deficiency of the lysosomal membrane protein do
not exhibit these positive effects on β cells, establishing the
participation of the autophagy pathway in this process [12••].
More recently, Wei et al. conducted a study with micedb/db
with an IF protocol based on a modified version of the previ-
ously reported FMD regimen implemented by Liu et al. They
subjected the mice to the FMD regimen (low protein and
carbohydrate content) every other week for 8 weeks. This
regimen normalized blood glucose levels, and improved insu-
lin sensitivity and β cell function. As in Liu’s study, the mice
demonstrated an increment in the expression of β cell progen-
itor Ngn3. Additionally, the mice showed improvement in
hepatic steatosis. One of the main contributions of this study
was to show that FMD led to an improvement in the dysbiosis
of T2DM mice. The diet increased the genera of
Parabacteroides and Blautia while reducing Prevotellaceae,
Alistipes, and Ruminococcaceae [10••].
This evidence shows that IER can effectively influence the
progression of diabetes in mice. Interestingly, in the three
studies, the effects were independent of weight loss. In fact,
at the end of the intervention period, weight was comparable
between groups (intervention vs control; see Table 1).
Intermittent Energy Restriction in Humans
As a result of experimental IER research, certain intermittent
fasting methods have been translated into human clinical re-
search and medical practice. Alternate-day fasting consists of
calorie restriction every other day or calorie restriction 2 days
per week (either two continuous days or two non-continuous
days (5:2)). Another fasting subtype, time-restricted feeding
(TRF), consists of daily fasting during most of the day, with
food intake times varying from between 4 and 6 h (and fasting
times from between 18 and 20 h). Normally, the time of day
for eating is chosen by the subject (this type of fasting resem-
bles Ramadan fasting).
IER is considered a weight management strategy to pre-
serve metabolic health and the beneficial effects on cardiomet-
abolic outcomes have been the object of numerous reviews
[18–21]. The effects on subjects with T2DM will be discussed
later in this review.
Prescription Modalities
Fasting Periods
During these periods, intake can vary from 25% of daily en-
ergy requirements to the consumption of only calorie-free
beverages, coffee, and water. Other authors have shown that
IER may be feasible with caloric restriction of about 50 to
90% of daily required intake during the restricted period [21,
22]. This means an average intake of 500 kcal for women and
600 kcal for men or between 400 and 800 kcals/day.
Depending on the chosen regimen, fasting may range from
16 to 20 h daily (time-restricted feeding) to 24–48 h per week
(alternate fasting days).
 13
Page 4 of 10

Table 1 Studies evaluating intermittent energy restriction in animal models with type 2 diabetes or insulin resistance

Date, author Mice type, age Exposure Objective Outcome

2007 Tikoo Sprague-Dawley mice, 4 groups: IF: Alternate-day fasting, Fast day: no Effect of IF in progression of diabetic No significant change in weight between groups.
[49] Diabetic IF food allowed Feeding day: AL nephropathy, through Sir2 and p53 Diabetic rats in IF: significant improvement in BUN,
Diabetic AL pathways creatinine, albumin, and HDL-C. Improvement in
Control IF onset of hypertension.
Control AL ↓ Lipid peroxidation, ↑ SOD activity, IF protected
glomerular damage and prevented an increase in
p38 protein. ↓p53 expression and ↑ Sir2 activitya
2017 Cheng Ten-week-old mice Leprdb/db IF: Fasting-mimicking diet (FMD, Effect on β cell regeneration and insulin ↓ Glucosea, ↑ Insulina, ↑ steady-state β cell function
[11••] Two groups: low-protein, low-sugar) 4 days/week resistance. β cell neogenesis through (%B)a. Improvement in insulin and glucose
IF and control alternated with 7 days of regular dog increase of neurogenin-3 (progenitor tolerance testa
chow AL pancreatic cells) Mice in FMD gained less weight than AL mice, but
Control: AL had a 22% greater weight than wild-type mice
2017 Liu Eight-week-old C57bL76J mice. Two (1) group: IF 6 week Effect of IF in glucose metabolism, B cell IF restored autophagic flux in islets and improved
[12••] groups: High-fat diet (12 week), for IF: alternate-day fasting, Fast day: no food mass, and function through glucose tolerance
induction of obesity and glucose allowed, Feeding day: AL autophagy-lysosome pathway ↑ Insulin secretion, β cell survival, islet insulin
intolerance (2) Group AL content, nuclear expression of neurogenin 3a
2018 Wei Six-week-old C57BL/ksJdb/db (1) Fasting-mimicking diet 1 week and Effect on β cell function and contribution of No differences in weight
[10••] Two groups AL 1 week microbiota ↓ Fasting glucosea, ↓glucose during GTT and ITTa, ↓
(2) Standard chow AL HOMA-IRa, ↑% Beta-cell function. Hepatic
steatosis was reduced in FMDa
↑ β cell and α cella ↑ of neurogenin 3a
Phyla: ↑Bacteroidetes and ↓ Firmicutes and
Saccharibacteriaa
Genera: ↑ Parabacteroides, Blautia and
↓Prevotellaceae, Alistipes, Ruminococcaceae
Species: ↑Bacteroidales S24-7, Parabacteroides
distasonis
Curr Diab Rep

a
p = <0.05
ADF alternate-day fasting, BUN blood urea nitrogen, FMD fasting-mimicking diet, GTT glucose tolerance test, HDL-C high-density lipoprotein cholesterol, HOMA-IR homeostatic model assessment of
insulin resistance, ITT insulin tolerance test, IER intermittent energy restriction, Leprdb/db leptin receptor–deficient mice, T2DM type 2 diabetes mellitus, IF intermittent fasting, AL ad libitum, CR calorie
restriction, SOD superoxide dismutase, Sir2 sirtuin type 2
(2020) 20:13
Curr Diab Rep (2020) 20:13
Feeding Periods
Caloric intake during non-restricted times can range from ad
libitum, hypocaloric (15–30% of energy restriction),
normocaloric, or hypercaloric (approximately 125% of energy
requirements) [21].
Macronutrient Distribution
IER does not have a pre-defined percentage of macronutrients,
neither in the fasting nor feeding periods. Different authors
propose diverse schedules.
Some studies have used the Mediterranean diets (45% en-
ergy from low glycemic load carbohydrates, 30% fat; 15%
MUFA, 8% polyunsaturated fatty acids, and 7% saturated fat-
ty acids) during fasting and feeding periods with
hypoenergetic and isoenergetic intake, respectively. Other in-
terventions significantly reduce the carbohydrate consump-
tion during fasting times (less than 50 g/day) or increase pro-
tein intake during feeding. Diet protocols are heterogeneous
and should be individualized according to the target popula-
tion (i.e., obesity, type 2 diabetes, aging).
Although information is abundant regarding obesity, insu-
lin resistance, and other components of the metabolic syn-
drome associated with obesity, the next section only describes
those studies where the subjects had established T2DM.
IER in Type 2 Diabetes Mellitus
Owing to the benefits in insulin response and glucose regula-
tion, interest has grown for the implementation of these re-
gimes in patients with type 2 diabetes mellitus (T2DM). All
modalities of IER (previously described) have been evaluated
in this population.
Saada et al. studied a population that normally fasts for
religious motives. They studied 66 overweight women with
type 2 diabetes mellitus. The participants were studied the
week before and during the third week of Ramadan. Even
though no changes in body mass index (BMI) were observed
during Ramadan or during the non-fasting days, a significant
decrease in glycated hemoglobin (HbA1c) was reported (9.52
vs 8.78%, p = 0.007). The glucose levels were significantly
higher during the fasting period (pre-Ramadan 1.84 ± 0.20
vs Ramadan 1.98 ± 0.17 g/L, p < 0.05); this could be second-
ary to the significant decrease (14.62 ± 2.79 vs 11.97 ±
2.48 μU/mL, p < 0.05) in insulin levels. The high-density li-
poprotein cholesterol (HDL-C) levels increased significantly
(p < 0.05) demonstrating the beneficial effect of Ramadan on
lipid profile. The augmentation in HDL-C can be explained by
the improvement in insulin resistance (suggested by the reduc-
tion in insulin levels) [13].
Kahleova et al. studied 54 patients between 30 and 70 years,
in a parallel group crossover study design comparing the time-
Page 5 of 10 13
restricted feeding (TRF) method with a regimen of 6 meals per
day. Participants in the TRF phase took breakfast and lunch
(hypocaloric: resting energy expenditure REE * 1.5) while
those in the control phase took 6 meals per day; each crossover
period lasting 12 weeks. Patients were overweight or obesity
and had an HbA1c between 6 and 11.8%. The weight de-
creased in both groups but was greater during TRF (− 2.3;
95% CI − 2.7, − 2, kg vs − 3.7; 95% CI − 4.1, − 3.4 kg,
p < 0.001). The hepatic fat content (HFC) decrease in both
groups, but again, significantly more so in the TRF group
(− 0.03%; 95% CI − 0.033%, 10.027% vs − 0.04%; 95% CI
− 0.041%, − 0.035%, p 0.009). The blood glucose and C pep-
tide concentrations decreased in both groups, again to a great-
er degree during TRF (p = 0.004 and 0.04, respectively).
Glucagon decreased in TRF and increased during the 6 meal
periods (p 0.001). Interestingly, both regimens were isocalo-
ric, but the consumption of calories in two meals/day instead
of six meals/day was associated with better metabolic im-
provement [14].
Carter et al. published a randomized controlled trial (RCT)
with 63 overweight or obese T2DM patients and tested the
fasting protocol 5:2 ratio (non-continuous days), described
earlier, versus continuous hypocaloric diet. They demonstrat-
ed similar reductions in weight (− 5.9 ± 4%, p < 0.001),
HbA1c (− 0.7 ± 0.9%, p < 0.001), and appetite over time, in
favor of the non-continuous method. Furthermore, the in-
creases in the sensation of fullness and satisfaction were sim-
ilar [15].
Arnason et al. carried out a pilot study with 10 T2DM
patients between the ages of 40 and 60 years with a before-
after design, including 3 phases: 2 weeks (wk) of normal con-
sumption, 2 wk of TRF (18–20 h) and finally 2 wk of normal
consumption. They were interested in evaluating the clinical
tolerability and biochemical effects of TRF. They found a
reduction in BMI (− 0.517, p = 0.013), morning fasting glu-
cose measured by self-monitoring blood glucose ((SMBG),
the increased fasting duration improved at goal, < 7 mmol/L,
morning SMBG to 34.1%, from a baseline of 13.8%) during
the TRF phase. There was no hypoglycemia or serious side
effects. Interestingly, although not a study requirement, all
participants preferentially chose eating hours starting in the
midafternoon [16]. The last study in IF in patients with
T2DM was published by Li et al., a RCT with 46 T2DM
patients between 25 and 75 years. The regimen had duration
of 4 months and consisted of 1 week of fasting and 4 months
of follow-up. They used the Buchinger method plus 3 weeks
of Mediterranean diet. This was compared with a control
group assigned to 4 months of a Mediterranean diet. They
found a significant reduction in body weight (3.5 ± 4.5 vs 2
± 4.8 kg p = 0.03), waist size (− 4.4 ± 4.3 vs − 0.3 ± 2 p =
0.001), systolic blood pressure (SBP, − 13.9 ± 15.3 vs 0.4 ±
15 p = 0.02), and diastolic blood pressure (DBP, − 9 ± 12.3 vs
3.2 ± 11). Results also included a significant improvement in
 13 Page 6 of 10
quality of life (WHO-5 well-being index, WHO-5 p = 0.04).
No differences in HbA1c were documented and no hypogly-
cemia or serious adverse effects were informed.
The most consistent finding among the 5 studies was a
reduction in insulin levels after IF, reflected in a reduction in
HOMA index, suggesting an improvement in peripheral insu-
lin action in and/or suppression of insulin secretion in order to
avoid hypoglycemia and/or activate lipolysis as a source of
energy in food deprivation conditions. Special mention must
be made of a recently published case series of three Canadian
patients who achieved T2DM remission after a protocol of
intermittent fasting based on ADF for 7–11 months. Two of
the patients were insulin-dependent, suggesting late-stage
T2DM. The effect was widely attributable to weight loss re-
duction [5••]. The previously discussed studies are summa-
rized in Table 2.
Risks of IER
Careful monitoring of blood glucose levels during the fasting
period is the most important aspect for patients taking phar-
macological agents that can cause hypoglycemia. Fasting by
T2DM Muslim patients during Ramadan has motivated the
development of guidelines for diabetes management during
fasting [23, 24]. Typically, patients have been stratified ac-
cording to hypoglycemic risk in four categories. Patients in
very high and high risk of hypoglycemia should not follow the
Ramadan fast, but studies demonstrate that most of the pa-
tients decide to take the risk [25, 26]. Therefore, careful mon-
itoring is advised in order to avoid hypoglycemia, hypergly-
cemia, or ketoacidosis. Ramadan fasting differs from clinical
fasting because Muslims must abstain from eating and drink-
ing during the daylight hours of dawn to sunset. Meanwhile,
in clinical fasting, the hour of fasting can be chosen for the
person, and liquids are permitted, thus avoiding the risk of
dehydration. Guidelines emphasize a structured education that
includes risk assessment, use of SMBG, indications on when
to break the fast, when to exercise, fluid intake, and meal
planning and information on medication adjustment during
fasting. In addition, other authors have discussed the manage-
ment of T2DM during other types of fasting highlighting the
selection and adjustment of oral hypoglycemic agents, accord-
ing to HbA1c levels [24].
Short-acting secretagogues, such as nateglinide, can be
used safely before meals without an increased risk of hypo-
glycemia. Dosing of sulphonylureas with a longer duration of
action (for example, glyburide) should be reduced or time-
changed to include the largest dose before the evening meal.
In patients using insulin and sulphonylureas, the discontinua-
tion of the sulfonyl urea is suggested if baseline HbA1c is <
7%. If HbA1c is between 7 and 10%, discontinuation of
sulphonylureas and rapid-action insulin is recommended only
during the fasting periods. If HbA1c is > 10%, medications
Curr Diab Rep (2020) 20:13
should remain unchanged. Blood glucose level monitoring at
least twice daily is essential during fasting. Patients should
break the IER diet if dangerously low glycemic levels occur
[27, 28].
In addition to the hypoglycemia risk described above, there
is concern that there may be a loss of fat-free mass (FFM) with
the use of IER. One study reported a FFM loss of up to 27%
on IER despite an intake of 0.7 g of protein/kg [21]. In order to
limit this loss, exercise should be included in the regimen of
patients who are on an IER diet [29]. Other reports suggest
that increasing protein intake to 1.2 g/kg can reduce FFM loss
to < 20%. Short-term studies of 3–4 months follow-up using
diets with 20–25% protein report higher satiety and a better
preservation of FFM, compared with low-protein diets
[30–32]. IER may be of greater risk in malnourished patients,
where it could lead to severe vitamin deficiencies as well as
FFM loss. This may be compensated by supplementation with
multivitamins and the prescription of strength exercises [32].
Heilbronn et al. suggest that IER may not be feasible as a
general public health intervention; they observed that self-
reported hunger on fasting days was considerable and did
not decrease over time [33]. Dorighello et al. showed that
IER is not beneficial in the context of genetic hypercholester-
olemia [34]. In young women with normal weight, IER pre-
scribed with 70% of caloric restriction in consecutive days and
3 days of ad libitum eating increased feelings of hunger, wors-
ened mood, heightened irritability, impaired ability to focus,
increased fatigue, increased eating-related thoughts, and pro-
voked a fear of loss of control and overeating during non-
restricted days [21].
Differences between Animal Models and Clinical
Studies in the Study of Glucose Homeostasis
Animal models have focused on β cell function, while clinical
studies have explored peripheral insulin resistance as the main
outcome to elucidate whether intermittent fasting promotes
improvements in glucose homeostasis. Methods in mice have
included combinations of in vivo and in vitro techniques. In
humans, in vitro techniques would implicate obtaining pan-
creatic tissue which is not feasible from a clinical and ethical
viewpoint. Although testing for β-function using in vivo ex-
periments is feasible in humans, to the best of our knowledge,
this has not been done in currently published IF literature.
There is no gold standard for measuring β cell function and
mass; in vitro approaches utilizing isolated human islets are
currently the method of choice for most researchers [35]. The
study of pancreatic islets can provide information regarding
the different stages of disease. For example, obesity and insu-
lin resistance are characterized by an increased size and num-
ber of β cells, whereas in T1DM and late-stage T2DM, an
increase in apoptosis is seen. Β cell proliferation, apoptosis,
hormone secretion, signal transduction pathway, protein
Table 2 Studies evaluating intermittent energy restriction in human T2DM subjects

Date, author Population Exposure Main outcomes Effects

Curr Diab Rep

design

2009 Saada 66 overweight Ramadan fasting: Two meals between Effect of Ramadan fasting on glucose Significant ↓ HbA1c, TC, TG, and LDL-C. Significant ↑ fasting glucose and HDL-C. No
[13] women sunset and dawn, without restrictions control, lipids, and weight difference in weight
48 ± 2 years
Before and after
2014 N = 54 Time-restricted feeding: Breakfast and Effect of TRF on weight, hepatic fat BW decrease in both groups, more for TRFa
(2020) 20:13

Kahleova 30–70 years lunch (hypocaloric REE*1.5) content, insulin resistance, and β HFC decrease in both groups, more in TRFa
[14] Parallel groups Control group: 6 smaller meals function Fasting glucose and C-peptide ↓ in both groups, more in TRFa glucagon ↓ in TRFa and ↑
RCT (hypocaloric REE*1.5) in control groupa. OGIS ↑ in both, more in TRF
12 weeks
2016 Carter 63 overweight 5:2 intermittent fasting: IER vs CER in HbA1c ↓ HbA1c, BW, and appetite over time similar in both groupsa
[15] or obese IER: 2 days of 1670–2500 kj/day with ↑ Feeling of fullness and satisfaction over time similar in both groupsa
patients 5 days of AL
61 ± 9 years CER: 7 days of 5000–6500 kj/day
2 parallel group
RCT
12 weeks
2017 10 patients Time-restricted fasting: IER biochemical effects and clinical ↓ BW, BMI, SMBG in IER phasea
Arnason 53.8 ± 9.1 years 3 phases: tolerability Change in HFD correlated with SMBG morning readings (x2 likelihood ratio = 8.36)a but
[16] Before-after (1) 2 weeks normal dietary habits not for afternoon or evening SMBG. Good tolerability
design (2) 2 weeks 18–20 h fasting goal per day
6 weeks (3) 2 weeks normal dietary habits
2017 Li [17] 46 Modified intermittent fasting: 1 week fasting vs usual care in T2DM Significant ↓ in BW (3.5 ± 4.5 kg) in fasting groupa vs (2.0 ± 4.8 kg) in control group
25–75 years Two groups: Significant ↓ waist, SBP, and DBP
2 parallel group Control: 4 months of Mediterranean diet Significant ↑ WHO-5 in fasting groupa
RCT Fasting: 1 week of fasting (Buchinger ↓ BMIa, glucose, insulin, and HOMA-IR
4 months method) plus 3 weeks of No difference in HbA1c
Mediterranean diet
2018 Furmli Case series Patient 1: 3 days/week fasting for Effects of IF in control glucose Two patients with reversal of T2DM (control without insulin or medications). One patient
[5••] 3 patients 7 months with control with oral medication. Significant reduction in weight and waist
11 months Patient 2: 3 days/week fasting for circumference
11 months
Patient 3: Alternate-day fasting
11 months

a
p = <0.05
ADF alternate-day fasting, ALT alanine aminotransferase, AST aspartate aminotransferase, BDNF brain-derived neurotrophic factor, BF body fat, BMI body mass index, BW body weight, BUN blood urea
nitrogen, CER continuous energy restriction, CRP C-reactive protein, CV cardiovascular, DBP diastolic blood pressure, FFA free fatty acid, FM fat mass, FFM fat-free mass, HDL-C high-density
lipoprotein cholesterol, HFC hepatic fat content, HFD hour fasting difference, HOMA-IR homeostasis model assessment insulin resistance, HbA1c glycated hemoglobin, HR heart rate, IER intermittent
energy restriction, LDL-C low-density lipoprotein cholesterol, OGIS oral glucose insulin sensitivity, RCT randomized controlled trial, REE resting energy expenditure, SBP systolic blood pressure, SMBG
Page 7 of 10

self-monitoring blood glucose, T2DM type 2 diabetes mellitus, TC total cholesterol, TG triglycerides, TRF time-restricted feeding, WHO-5 World Health Organization 5 Well-Being Index, vs versus, IF
13

intermittent fasting, AL ad libitum, CR calorie restriction, SD standard diet, WL weight loss, BPL blood pressure levels, VLDL-C very low-density lipoprotein cholesterol
 13 Page 8 of 10
localization (by immunohistochemical), and protein expres-
sion (by western blot) are some of the biological activities that
can be investigated in in vitro models. Transcriptomics and
single-cell analysis have permitted the identification of new
transcriptional regulators and specific cellular changes and
subpopulations that would be undetectable when analyzed in
whole islets, respectively. With some of these techniques, a
de-differentiated phenotype has been observed, supporting the
possible contribution of the de-differentiation of β and α cells
in T2DM [36].
In vivo, β cell function can be evaluated measuring
fasting insulin, oral glucose tolerance test (OGTT), mixed
meal tolerance test, intravenous glucose tolerance test
(IVGTT), and the hyperglycemic clamp. The β cell mass
and function can also be evaluated with non-invasive imag-
ing techniques; positron emission tomography (PET), single-
photon emission computed tomography (SPECT), and mag-
netic resonance imaging (MRI) are considered the most
promising imaging methods to achieve this purpose [35].
Both the evaluation of function and mass is feasible in hu-
man clinical studies.
Discussion
Recently, there has been a great deal of interest in fasting
regimes, in particular, forms of IER. However, this is not
new; in the late nineteenth century, Edward Hooker Dewey,
the American physician who pioneered cyclic caloric restric-
tion, advocated fasting every day until late noon. Similarly, in
the early twentieth century, the French physician Guillaume
Guelpa practiced continuing cycles of fasting for 2–5 days,
followed by vegetarian food for 12–15 days [37]. The molec-
ular mechanisms of IER have been demonstrated in animal
models and involve adaptive cellular responses that reduce
oxidative damage and inflammation, optimizing energy me-
tabolism and favor cellular protection. The main pathways
studied involve an increase in autophagy mediated through
an increase in the sirtuin (SIRT-1), a NAD+-dependent
deacetylase [38]. In laboratory murine models, IER has pro-
found beneficial effects on many different indices of health
and can prevent some disease processes and improve func-
tional outcomes in experimental models of aging and a wide
range of age-related diseases such as diabetes, cardiovascular
disease, cancers, and neurological conditions (Alzheimer’s
disease and stroke) [39].
This review has aimed to describe the current evidence
regarding diets based on intermittent fasting, focusing on
diabetes-induced animal models and human studies in persons
with T2DM. In the animal models, mechanisms for the rever-
sion of glucose alterations have been studied and authors have
described the neogenesis of β cells, a reduction in β cell
apoptosis and improvements in insulin islet β cell content.
Curr Diab Rep (2020) 20:13
These phenomena are associated with an increased expression
of Ngn3 in islet cells. Ngn3 is a transcription factor expressed
transiently in developing pancreatic islets in utero that is re-
quired for the development of β and α cells. This benefit is
abolished if autophagy is suppressed suggesting that fasting-
induced autophagy is a key mediator. There is reason to sus-
pect that autophagy suppresses the Nothc1 signaling pathway,
and this promotes Ngn3 expression and β cell neogenesis [9•].
The Notch signaling pathway plays a crucial role at different
stages of cell development, such as proliferation, growth, dif-
ferentiation, and apoptosis.
In contrast to animal models, clinical studies have focused
on body weight and parameters associated with insulin action,
more than insulin secretion.
Clinical studies including T2DM consistently demonstrate
a reduction in fasting insulin levels and HOMA index, an
effect that is widely associated with weight loss, with the
exception of the findings of Saada et al. who reported a reduc-
tion in insulin levels without loss weight [13]. We believe that
hypoinsulinemia results from weight loss consequently reduc-
ing insulin resistance. Additionally, low levels of insulin are a
defense mechanism for the avoidance of hypoglycemia.
Hypoinsulinemia also activates lipolysis amid food depriva-
tion in order to utilize free fatty acids as a source of energy.
Low concentrations of insulin levels after IF can be seen in
insulin-resistant patients without T2DM [40]. The increase in
C-HDL levels observed in studies where this parameter has
been measured also supports our theory of a reduction in pe-
ripheral and hepatic insulin resistance. To our knowledge,
there are no clinical studies that attempt to investigate the
possible recovery of β cell number and function. However,
studies including the hyperglycemic clamp, IVGTT, OGTT,
and mixed meal tolerance test could help to answer some
questions regarding β cell function. Functional studies can
be complemented by evaluating β cell mass with non-
invasive imaging techniques like PCT, SPECT, and MRI.
Several physiological responses to fasting are similar to those
caused by regular aerobic exercise, which include an improve-
ment in insulin sensitivity and cellular stress, and reduced
resting blood pressure and heart rate [41]. All IER modalities
have shown a positive impact on obesity, but few studies have
evaluated the additive effects of exercise and other interven-
tions. Hence, more integrative studies of IER which include
lifestyle interventions are needed. We recommend that health
providers choose protocols that are individualized to patient
preferences and complement the nutritional approach with
exercise in all cases.
In T2DM, time-restricted feeding protocols are more con-
venient and safer, since patients eat every day and the fasting
periods are not long enough to significantly increase hypogly-
cemia risk. Hunger can be a barrier for therapy adherence, so
we recommend the systematic evaluation of hunger and satiety
in patients with IER diets.
Curr Diab Rep (2020) 20:13
IER diets are not risk-free. Beyond hypoglycemia, the main
adverse effects involve psychologic and anatomical/
physiological disturbances like anxiety and muscle loss.
Additionally, in women, it is important to consider the impact
of IER on menstrual cycles. Multidisciplinary treatment can
mitigate most of the deleterious effects, including psychologic
distress imposed by diets. Approaches to manage risks depend
on diet and patient characteristics. Proteins have a positive
effect on satiety and muscle mass, and supplementation with
essential amino acids and vitamins is encouraged when low
protein diets are prescribed.
Independent to promising evidence regarding β cell func-
tion derived from murine models, numerous evidence support
that a weight loss of 7–10% is accompanied by improvements
in several metabolic parameters such as insulin action, lipid
profile and blood pressure, diminution of progression from
pre-diabetes to diabetes, and diminution on diabetes progres-
sion among others [1, 42–46]. Changes in lifestyle behaviors
oriented to weigh loss are recommended for all patients with
diabetes or at increased risk to develop it.
In conclusion, this report supports the use of strictly super-
vised IER in persons with T2DM; this technique aids in gly-
cemic control and helps to minimize the use of insulin and oral
hypoglycemic agents (OHA). The main concern during
fasting is the incidence of hypoglycemia, especially when reg-
imens include insulin or insulin secretagogues. Fortunately,
there are other OHAs with a very low risk of hypoglycemia,
such as SGLT2 inhibitors and DPP-IV inhibitors. Finally, clin-
ical studies are needed which evaluate the functional capacity
and mass of β cells in order to corroborate the findings in
animal models. The techniques to evaluate in vivo function
and the new methods to evaluate mass are promising in the
quest to answer these questions.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflicts of
interest.
Human and Animal Rights and Informed Consent This article does not
contain any studies with human or animal subjects performed by any of
the authors.
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