Comparison of a Carbohydrate-Free Diet vs. Fasting on Plasma Glucose, Insulin and Glucagon in Type 2 Diabetes

Full text

Comparison of a carbohydrate-free diet vs. fasting
on plasma glucose, insulin and glucagon in
type 2 diabetes☆
,
☆☆
Frank Q. Nuttall
a,b,
⁎, Rami M. Almokayyad
a,b,1
, Mary C. Gannon
a,b,c
a
Section of Endocrinology, Metabolism & Nutrition, and the Metabolic Research Laboratory, Minneapolis VA Health Care System,
Minneapolis/St Paul, MN, USA
b
Department of Medicine, University of Minnesota, Minneapolis/St Paul, MN, USA
c
Department of Food Science & Nutrition, University of Minnesota, Minneapolis/St Paul, MN, USA
ARTICLE INFO ABSTRACT
Article history:
Received 4 June 2014
Accepted 5 October 2014
Objective. Hyperglycemia improves when patients with type 2 diabetes are placed on a
weight-loss diet. Improvement typically occurs soon after diet implementation. This rapid
response could result from low fuel supply (calories), lower carbohydrate content of the
weight-loss diet, and/or weight loss per se. To differentiate these effects, glucose, insulin,
C-peptide and glucagon were determined during the last 24 h of a 3-day period without
food (severe calorie restriction) and a calorie-sufficient, carbohydrate-free diet.
Research design. Seven subjects with untreated type 2 diabetes were studied. A
randomized-crossover design with a 4-week washout period between arms was used.
Methods. Results from both the calorie-sufficient, carbohydrate-free diet and the 3-day
fast were compared with the initial standard diet consisting of 55% carbohydrate, 15%
protein and 30% fat.
Results. The overnight fasting glucose concentration decreased from 196 (standard diet)
to 160 (carbohydrate-free diet) to 127 mg/dl (fasting). The 24 h glucose and insulin area
responses decreased by 35% and 48% on day 3 of the carbohydrate-free diet, and by 49% and
69% after fasting. Overnight basal insulin and glucagon remained unchanged.
Conclusions. Short-term fasting dramatically lowered overnight fasting and 24 h
integrated glucose concentrations. Carbohydrate restriction per se could account for 71%
of the reduction. Insulin could not entirely explain the glucose responses. In the absence of
carbohydrate, the net insulin response was 28% of the standard diet. Glucagon did not
contribute to the metabolic adaptations observed.
Published by Elsevier Inc.
Keywords:
Carbohydrate
Diet
Diabetes
Fasting
Circadian rhythm
METABOLISM CLINICAL AND EXPERIMENTAL 64 (2015) 253–262
Abbreviations: T2DM, type 2 diabetes mellitus; CHO, carbohydrate; IRB, Institutional Review Board; h, hour; kcal, kilocalorie; kg,
kilogram; DPC, Diagnostic Product Corporation; DSL, Diagnostic Systems Laboratories, Incorporated; SDTU, Special Diagnostic &
Treatment Unit; AM, ante meridiem; STD, standard; SEM, Standard Error of the Mean; M.T., Medical Technologist.
☆
Supported in partby Merit Review Funds fromthe Departmentof Veterans Affairs. TheDepartment ofVeterans Affairs had no involvement
in study design, collection, analysis and interpretation of data, writing of the report or decision to submit the article for publication.
☆☆
ClinicalTrials.gov Identifier: NCT01469104.
⁎Corresponding author at: Section of Endocrinology, Metabolism and Nutrition, Minneapolis VA Health Care System (111G), One Veterans
Drive, Minneapolis, MN 55417 USA. Tel.: +1 612 467 4424; fax: +1 612 725 2273.
E-mail address: nutta001@umn.edu (F.Q. Nuttall).
1
Dr. Almokayyad was a Fellow in Endocrinology when these studies were done. His current address is Medical Arts Building, 1015 Duff
Avenue, Ames, IA 50010.
http://dx.doi.org/10.1016/j.metabol.2014.10.004
0026-0495/Published by Elsevier Inc.
Available online at www.sciencedirect.com
Metabolism
www.metabolismjournal.com

1. Introduction
In people with type 2 diabetes it has been demonstrated that a
low food energy diet [1], short-term fasting [2], a low CHO-diet
[3] as well as weight loss result in a rapid decrease in glucose
concentration. Thus, various iterations of these have been
advocated as a means of improving the hyperglycemia
present in these patients. However, to our knowledge a direct
comparison of the results of each of these approaches has not
been reported. In order to address these issues, we have
determined the metabolic effects of fasting for 3 days, a time
duration in which loss of fat mass should be minimal, but the
glucose and insulin concentrations have reached a new
quasi-steady-state [4]. This is compared with the effects of
an energy-sufficient, carbohydrate-free diet of the same
duration. Both were compared with data obtained when the
subjects ingested a standard, mixed diet. Twenty-four hour
glucose, insulin, C-peptide and glucagon data were obtained
while on each diet or when the subjects were fasting. Part of
these data was published previously in abstract form [5].
2. Materials and methods
2.1. Subjects
Seven male subjects with type 2 diabetes were studied. One
subject was untreated; 3 subjects had been receiving metformin.
Three had been receiving a sulfonylurea. These medications
were discontinued for 24 or more days before the study. Patient
characteristics are listed in Table 1.
Inclusion criteria were: 45–75 years of age, with type 2
diabetes, currently not on any oral diabetic medications,
never having been on insulin, and a C-peptide > 1.5 ng/ml
(reference range 0.8–3.1 ng/ml). Patients may have been on
metformin and or a sulfonylurea, but would be taken off these
oral agents for the time required to stabilize their HbA1c, with
prior approval of their primary provider. Subjects remained
off these medications until the end of the study. This was
done with the patient consent, and with the approval of and
coordination with the primary care physician. As a result of
the dietary intervention of this study, no adverse health
effects were noted during the study, nor were there adverse
health effects due to the study after its completion.
Exclusion Criteria were: hematologic abnormalities, liver
disease, kidney disease, macroalbuminuria (300 mg albumin/
24 h), untreated thyroid disease, congestive heart failure,
angina, myocardial infarction within 6 months, life threatening
malignancies, proliferative retinopathy, severe diabetic neu-
ropathy, peripheral vascular disease, serious psychiatric disor-
ders (preventing the patient from competently signing the
informed consent), fasting triglyceride > 400 mg/dl, fasting
blood glucose > 250 mg/dl or HbA1c > 11%.
Written informed consent was obtained from all subjects,
and the study was approved by the Department of Veterans
Affairs Medical CenterInstitutional Review Board (IRB). All work
was conducted in compliance with the principles of the
Declaration of Helsinki. The study is registered at clinicaltrials.
gov: NCT01469104.
Table 1 –Subject characteristics.
Subject Age Years of
Diabetes
Diabetes
Med
Days off
Med
Weight
(kg)
Height
(cm)
BMI
(kg/m
2
FPG
mg/dl
Concomitant
Diseases
Medications
1 55 2 Metformin 54 93 183 28 160 hypogonadism,
dyslipidemia
testosterone, lisinopril,
rosuvastatin
2 60 4 Metformin 43 93 183 29 228 CVD, HTN atenolol, gemfibrozil,
lisinopril, simvastatin
3 60 2 Glyburide 47 81 155 34 171 dyslipidemia simvastatin,
omeprazole
4 62 18 Metformin 32 130 170 38 219 dyslipidemia,
CVD, HTN
losartan, omeprazole,
rosuvastatin,
ezetimibe
5 64 10 Glipizide 45 108 174 36 168 dyslipidemia,
HTN
hydrochlorothiazide,
lisinopril, simvastatin
6 72 5 None 88 188 25 118 COPD, CVD,
HTN
lisinopril, amlodipine,
atenolol, simvastatin,
omeprazole
7 49 3 Glipizide 24 88 170 27 225 HTN hydrochlorothiazide,
lisinopril
Average ± SEM 60 ± 3 6 ± 2 41 ± 4 97 ± 6 175 ± 4 31 ± 2 184 ± 16
Range 49–72 2–18 24–54 81–130 155–188 28–38 118–228
Abbreviations:
Med = medication
BMI = body mass index
FPG = overnight fasting plasma glucose
CVD = cardiovascular disease
HTN = hypertension
COPD = chronic obstructive pulmonary disease
254 METABOLISM CLINICAL AND EXPERIMENTAL 64 (2015) 253–262

2.2. Study design
This is a randomized crossover study design with a four-week
washout period between study arms. On one occasion
subjects received a calorie-sufficient, carbohydrate-free diet
for 72 h. This diet contained the same amount of protein as
the standard diet. Dietary fat replaced the carbohydrate
content. On another occasion, the subjects fasted for 72 h.
All subjects participated in both study arms.
The day before admission, each was provided with a
standardized dinner (composed of 55% carbohydrate, 15%
protein, and 30% fat), to be ingested at 1800 h at home. This
was based on 25 kcal/kg body weight. The subjects were
admitted to a clinical research unit (Special Diagnostic and
Treatment Unit, SDTU) for 4 days.
On the day of admission, subjects reported to the SDTU at
0700 h. An indwelling intravenous catheter was inserted, and
blood samples were obtained at 0730 h, 0745 h and 0800 h for
baseline determinations. Subjects received a standard break-
fast, lunch and dinner at 0800 h, 1200 h and 1800 h, respec-
tively. Standard meals were composed of 55% carbohydrate,
15% protein, and 30% fat. Caloric distribution was: breakfast
32%, lunch 40%, dinner 28%. Blood samples were obtained
every 15 min after each meal for the first hour, every 30 min
for the second and third hour and hourly thereafter until the
next meal or until 0800 h the next morning. The urine was
collected on that day for 24 h as well.
On the second day, subjects were provided with the
carbohydrate-free meals at 0800 h, 1200 h and 1800 h daily
for 3 days (72 h), or the 72-h fast was begun. The carbohy-
drate-free meals were composed of <3% carbohydrate, 15%
protein, 82% fat. The amount of food energy provided in the
meals was based on 25–30 kcal/kg body weight. Ingestion of
water was encouraged. Black coffee, tea without sugar or
cream, and calorie-free beverages were allowed when receiv-
ing the carbohydrate-free meals or while fasting. Activity was
limited to quiet diversions such as reading or watching TV.
The subjects were under nearly constant observation in the
SDTU including during meals. Blood samples were obtained
during last 24 h of the 72-h intervention, with the same
time intervals as the first day (i.e. pre-study, Day 1). Urine
was collected during the last 24 h of the 72-h intervention
as well.
Plasma or serum was analyzed for glucose, insulin, C-
peptide and glucagon, during two 24-h periods (days 1–2
and 4–5).
Urine volume and glucose were quantified on days 1–2
and 4–5.
2.3. Assays
Plasma glucose was determined by an automated method on
an Abbott Architect ci 8200 analyzer; serum immunoreactive
insulin using an automated chemiluminescent assay on
DPC’s IMMULITE platform (Diagnostic Products Corp, Los
Angeles CA); serum C-peptide by a radioimmunoassay
method using a kit manufactured by DSL, Webster, TX;
glucagonbyaradioimmunoassaymethodusingakit
purchased from Linco Research (purchased by Millipore,
Billerica, MA).
2.4. Area determination
The net integrated 24-h area responses were calculated using
the overnight fasting concentration as baseline. For net
glucose area response, we also used 85 mg/dl as baseline.
This glucose concentration is typical for a person without
diabetes in the AM after an overnight fast, i.e. it could
represent an ideal goal. Total integrated 24-h area responses
were calculated using zero as baseline.
2.5. Statistical analysis
Statistics were determined using Prism 5 software for the iMac
by Graphpad (LaJolla, CA). First, the three diets were compared
using Friedman’s non-parametric test. If the Friedman’s test
indicated that there were significant differences among the 3
diets, planned pairwise comparisons were made using
Wilcoxon’s signed ranks test with a Bonferroni adjustment for
3 comparisons. Student’s t test was used to analyze the
difference in weight loss between the two arms of the study. A
p-value less than 0.05 was the criterion for significance. Data
are presented as the mean ± SEM.
3. Results
Data for the first day of each arm of the study on which
standard meals were ingested, i.e. pre-CHO-free diet and pre-
72 h fast, were similar and have been combined for analysis
and presentation of all of the data and for comparison with
the CHO-free diet and fasting data.
3.1. Glucose
3.1.1. Standard diet
The mean AM plasma glucose concentration before ingesting
the standard diet was 196 ± 18 mg/dl (Fig. 1). Following
ingestion of the standard diet, it increased after meals, as
expected, reaching a maximum at 1.5 h after breakfast, at 1 h
after lunch, and at 2 h after dinner (Fig. 1). The nadir of 158 ±
19 mg/dl was reached at ~10 h after the start of breakfast. At
the end of the 24 h the concentration was 188 ± 15 mg/dl.
3.1.2. CHO-free diet
On day 4 of the study following ingestion of the CHO-free diet
for 2 days the mean glucose concentration was 160 ± 15 mg/dl
at 0800 h. Small but distinct increases in glucose concentration
were detected after the meals at essentially the same times
noted for the standard meals. A nadir of 118 ± 9 mg/dl was
observed 10 h after the start of breakfast (at 1800 h). The final
mean glucose concentration at the end of the 72 h of CHO-free
meals was 149 ± 15 mg/dl.
3.1.3. Fasting (zero food energy)
The mean glucose concentration was 127 ± 10 mg/dl at 0800 h
on day 4 of the fasting arm of the study. During the last 24 h of
the 72-h fast, a nadir of 103 ± 8 mg/dl was observedat 10 h after
the start of breakfast (at 1800 h) and again during the night at
0200, 0300, and 0400 h. The mean glucose concentration at the
end of the 72-h fast was 114 ±11 mg/dl.
255METABOLISM CLINICAL AND EXPERIMENTAL 64 (2015) 253–262

3.1.4. Net area
(Fig. 1, left insert) The net glucose area, using the fasting
glucose concentration as baseline for each intervention was
548 ± 163, −494 ± 106 and −389 ± 66 mg h/dl for the standard
diet, the CHO-free diet, and with fasting, respectively.
When 85 mg/dl was used as a baseline, the net areas were
3031 ± 665 mg h/dl, 1368 ± 291 and 621 ± 221 for the standard
diet, the CHO-free diet, and fasting, respectively.
3.1.5. Total area
(Fig. 1, right insert) The mean total 24 h integrated glucose area
response, using zero as baseline was 5248 ± 601 mg h/dl, 3408 ±
291, and 2661 ± 221 mg h/dl, for the standard diet, the CHO-free
diet, and with fasting, respectively. Thus, in the absence of
carbohydrate, the total glucose area response decreased by 35%,
compared to a 49% decrease with fasting. Of the decrease in total
integrated glucose area observed with fasting, 71% could be
attributed to the removal of carbohydrate from the diet.
3.2. Insulin
3.2.1. Standard diet
The mean AM serum concentration before ingesting the
standard diet was 18 ± 1.5 μU/ml (Fig. 2). Following ingestion
of the standard diet, the insulin concentration reached a
maximum 1 h after breakfast, 1 h after lunch, and 2 h after
dinner (Fig. 2). The concentration returned to the fasting
baseline during the night at ~ 0400 h.
3.2.2. CHO-free diet
After ingestion of the CHO-free diet, the AM insulin
concentration also was 18 ± 3 μU/ml. The insulin con-
centration modestly increased after meals, but the
increase was markedly attenuated compared to that
observed with the standard meals. At the end of the
72 h on a carbohydrate-free diet the concentration was
21 ± 4 μU/ml.
Fig. 1 –24-h glucose response.The open circle–solid line represents the mean glucose concentration at several time points
during the first 24 h of both days during which the standard diet was ingested (i.e. day 1 of each arm of the study). The
triangle–dotted line represents the mean glucose concentration during the last 24 h on a carbohydrate-free diet. The closed
circle–solid line represents the mean glucose concentration during the last 24 h of the fast (energy-free) diet. B, L, D, indicate
the times at which breakfast, lunch, and dinner were ingested.The net area response (Left Insert) indicates the area under the
curve using the fasting concentration as baseline. Different letters on bars indicate statistically significant differences
(Friedman P = < 0.0012).The total area response (Right Insert) indicates the area under the curve, using zero as baseline.
Different letters on bars indicate statistically significant differences (Friedman: P = <0.0001).
256 METABOLISM CLINICAL AND EXPERIMENTAL 64 (2015) 253–262

3.2.3. Fasting (zero food energy)
At 0800 h on day 4 of the study, the mean insulin concentra-
tion was 14 ± 3 μU/ml, and it changed very little during the
subsequent 24 h. At the end of the 72-h fast the insulin
concentration was 14 ± 2 μU/ml. It is noteworthy that there
was little change in the AM insulin concentration or final
concentrations the next morning, regardless of diet.
3.2.4. Net area
(Fig. 2, left insert) Using the respective individual mean overnight
AM values as baseline, the mean net 24 h insulin area response
was 573 ± 93, 159 ± 34, and −29 ± 30 μUh/ml following the
standard diet, the CHO-free diet, and fasting, respectively.
3.2.5. Total area
(Fig. 2 right insert) The mean total 24 h insulin area responses
were 1005 ± 133, 533 ± 81, and 309 ± 51 μU h/ml, following
the standard diet, the CHO-free diet, and fasting, respectively.
3.3. C-peptide
The respective C-peptide dynamic responses are very
similar to the insulin responses (Fig. 3). The statistical
significance was modestly different between the insulin
area results and the C-peptide area results (Figs. 2 and 3).
Neither the C-peptide net or total area responses were
statistically significantly different between fasting, the
CHO-free or the standard diet. Although some significance
was noted with the Friedman analysis a post hoc analysis
of pairwise comparisons did not reach the Bonferroni
p value.
3.4. Glucagon
The 0800 h plasma glucagon concentration and the 24-h
glucagon concentration profiles were similar following the
standard diet, the CHO-free diet, or fasting (Fig. 4). During the
last 12 h of the study the average glucagon concentration
was ~25% lower than during the first 12 h for the standard
diet and the carbohydrate-free diet. It was only 10% lower
with fasting.
3.4.1. Net area
(Fig. 4, left insert) The 24 h integrated glucagon net area responses
were −89 ± 125, 268 ± 124 and −9 ± 112 pg h/ml following the
standard diet, CHO-free diet, and fasting, respectively.
Fig. 2 –24-h insulin response.The open circle–solid line represents the mean insulin concentration at several time points
during the first 24 h of both days during which the standard diet was ingested (i.e. day 1 of each arm of the study). The
triangle–dotted line represents the mean insulin concentration during the last 24 h on a carbohydrate-free diet. The closed
circle–solid line represents the mean insulin concentration during the last 24 h of the fast (energy-free) diet. B, L, D, indicate the
times at which breakfast, lunch, and dinner were ingested.The net area response (Left Insert) indicates the area under the
curve using the fasting concentration as baseline. Different letters on bars indicate statistically significant differences
(Friedman: P = < 0.0001).The total area response (Right Insert) indicates the area under the curve, using zero as baseline.
Different letters on bars indicate statistically significant differences. (Friedman: P = <0.0001).
257METABOLISM CLINICAL AND EXPERIMENTAL 64 (2015) 253–262

3.4.2. Total area
(Fig. 4, right insert) the 24 h integrated total glucagon area
responses were not different. The total area responses were
1787 ± 175, 1748 ± 73 and 2034 ± 331 pg h/ml following the
standard diet, CHO-free diet, and fasting, respectively.
3.5. Body weight
The body weight decreased from 213 ± 9 lb to 210 ± 6 lb
following ingestion of the calorie-sufficient, carbohydrate-
free diet and from 212 ± 6 lb to 205 ± 6 lb after fasting
(Student’s t test, P = 0.0006).
3.6. Urine
The urine volume decreased from 3247 ± 425 ml on the
standard diet to 3012 ± 311 ml and 2399 ± 297 ml on the
carbohydrate-free and energy-free diets, respectively. The
urinary glucose excretion decreased from 26.5 ± 12 g on the
standard diet to 0.26 ± 0.1 g and to 0.07 ± 0.01 g on the
carbohydrate-free and energy-free diets, respectively.
4. Discussion
4.1. Glucose
The effect of short-term fasting on the circulating glucose
concentration has been reported previously by us [2],by
others reviewed therein and by others [6,7]. However, as
indicated in the introduction, a direct comparison of fasting
(zero food energy intake), with a carbohydrate-free, food
energy-adequate diet over the same time and in the same
individuals, has not been reported to our knowledge either in
ng/ml
-
30
0
30
60
90
120
150
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0
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20
30
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ng • hr/ml
nmol • hr/L
Net Areas
Sta ndard
Diet
CHO
Free
Fasting
a
a
a
0
30
60
90
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150
180
210
240
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20
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nmol • hr/L
Tot al Ar ea s
Standard
Diet
CHO
Free
Fasting
a
a
a
Fast ing
CHO Free
Standard Diet
nmol/L
Time (Hours)
BL D
0
5
10
15
0.0
1.0
2.0
3.0
4.0
5.0
0 2 4 6 8 10 12 14 16 18 20 22 24
C-Peptide Response
Fig. 3 –24-h C-peptide response.The open circle–solid line represents the mean C-peptide concentration at several time points
during the first 24 h of both days during which the standard diet was ingested (i.e. day 1 of each arm of the study). The
triangle–dotted line represents the mean C-peptide concentration during the last 24 h on a carbohydrate-free diet. The closed
circle–solid line represents the mean C-peptide concentration during the last 24 h of the fast (energy-free) diet. B, L, D, indicate
the times at which breakfast, lunch, and dinner were ingested.The net area response (Left Insert) indicates the area under
the curve using the fasting concentration as baseline. Although some significance was noted with the Friedman analysis
(P = < 0.0017), a post hoc analysis of pairwise comparisons did not reach the Bonferroni p value. Identical letter on bars
indicates that results were not statistically different. The total area response (Right Insert) indicates the area under the
curve, using zero as baseline Although some significance was noted with the Friedman analysis (P = < 0.0120), a post hoc
analysis of pairwise comparisons did not reach the Bonferroni p value. Identical letter on bars indicates that results were
not statistically different.
258 METABOLISM CLINICAL AND EXPERIMENTAL 64 (2015) 253–262

people with or without diabetes. A correlation with the
insulin and glucagon concentrations also has not been
reported. In addition, 24-h data relating metabolic responses
to food intake rarely are reported.
4.1.1. Overnight fasting glucose
In a review of the literature we noted, and our data confirmed,
that short-term fasting lowers the morning glucose by ~ 20%–
40% when compared to when subjects were ingesting a
regular mixed diet [2]. This was regardless of the initial
morning (overnight fasting) glucose concentration, i.e. the
higher the preceding morning glucose concentration the
greater the absolute glucose decrease with fasting [2]. The
current data also are compatible with this concept. In the
present study, a 3-day fast resulted in a mean 36% decrease in
the morning glucose. After three days on a carbohydrate-free,
energy-sufficient diet it decreased 18% when compared to the
standard diet. Thus, eliminating carbohydrate from the diet
could account for 50% of the reduction in AM glucose
concentration observed in the absence of food intake. If the
overnight fasting glucose baseline is set at 85 mg/dl, a typical
glucose concentration for an individual without type 2
diabetes, who is eating a regular, food-energy sufficient diet,
then the decrease with no food intake is 80%. With eliminat-
ing carbohydrate from the diet it is 55%.
Our previous long-term data also suggest that in people
with type 2 diabetes the overnight fasting glucose is largely
dependent on the carbohydrate content of the diet. In
addition, in those studies, there was a critical threshold for
maintenance of the typical high AM fasting values. This was
between 30% and 40% of total food energy [8]. The glucose
concentration also was independent of the fasting insulin and
glucagon concentrations.
4.1.2. 24 h integrated glucose area
The 24 h integrated total glucose area response after fasting
for 3 days was decreased 49%, after the CHO-free diet it was
35%, when compared with the standard diet. Thus ~ 70% of
the reduction in integrated glucose area response can be
attributed to elimination of carbohydrate.
Using the respective 0800 h values as baseline, the 24 h
integrated net glucose areas resulting from elimination of all
food energy intake, or eliminating carbohydrate from the diet,
were negative and similar as a percentage when compared to
their respective 0800 h fasting values even though those
fasting values were quite different (Fig. 1).
0
20
40
60
80
100
120
140
0
20
40
60
80
100
120
140
0 2 4 6 8 1012141618202224
ng/L
pg/ml
BL D
Time (Hours)
Fast ing
CHO Free
Standard Diet
a
a
aaa
a
0
500
1000
1500
2000
2500
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ng • hr/L
pg • hr/ml
Tot al A re a s
Sta ndard
Diet
CHO
Free
Fasting
-300
-100
100
300
500
-300
-100
100
300
500
ng • hr/L
pg • hr/ml
Net Areas
Sta ndard
Diet
CHO
Free
Fasting
Glucagon Response
Fig. 4 –24-h glucagon response.The open circle–solid line represents the mean glucagon concentration at several time points
during the first 24 h of both days during which the standard diet was ingested (i.e. day 1 of each arm of the study). The
triangle–dotted line represents the mean glucagon concentration during the last 24 h on a carbohydrate-free diet. The closed
circle–solid line represents the mean glucagon concentration during the last 24 h of the fast (energy-free) diet. B, L., D. indicate
the times at which breakfast, lunch, and dinner were ingested.The net area response (Left Insert) indicates the area under the
curve using the fasting concentration as baseline (Friedman: P = 0.0845). Identical letter on bars indicates that results were not
statistically different.The total area response (Right Insert) indicates the area under the curve, using zero as baseline
(Freidman, P = 0.4861).
259METABOLISM CLINICAL AND EXPERIMENTAL 64 (2015) 253–262

If the baseline is set at 85 mg/dl, the 24-h integrated net
area decreased by 80% with elimination of food energy and
55% by the CHO-free diet. Compared to the standard diet
response, the decrease was 171% and 190%, respectively.
Typical of people with type 2 diabetes, the lowest glucose
concentrations occurred late in the afternoon whether on the
standard or carbohydrate-free diet or with no food energy
intake (Fig. 1). That is, they have a prominent glucose
circadian rhythm [2,9]. This circadian rhythm can explain at
least in part, the net 24-h negative area responses described
above. It occurs without a significant change in insulin or
glucagon concentration when food is not being ingested
(Figs. 2 and 4), but is modified by food ingestion (Fig. 1). The
mechanism remains unexplained but may be mediated by a
local circadian entrainment in the liver and/or to neural input
to the liver through a hypothalamic control system [10–14].
In summary, a lack of carbohydrate played a dominant
role in reducing the 24 h integrated glucose concentration
just as it did with the 0800 h fasting concentration. Presum-
ably this is mediated by a hormone-independent reduction
in glycogenolysis [8,15].
4.2. Insulin
4.2.1. Overnight fasting insulin
The current dietary manipulations did not result in a
significant change in the AM fasting insulin concentration
whether on the standard or carbohydrate-free diet, or with no
fuel consumption (18, 18 and 14 μU/ml, respectively, Fig. 2).
The stability in insulin concentration occurred even though
there was a considerable change in the fasting glucose
concentration (196, 160, and 127 mg/dl, respectively). Thus,
the glucose concentrations per se could not explain the
insulin concentrations obtained. Also, contrariwise, the
difference in glucose concentration cannot be attributed to a
change in insulin concentration. Clearly the insulin concen-
tration is dissociated from the glucose concentration.
These dissociations have been noted previously after short-
term fasting in people with type 2 diabetes [16] and without
fasting [17], although not commented on. It also was observed
in people with or without type 2 diabetes by others [18].
These data suggest an abnormality, or more likely an
adjustment, in βcell glucose sensing such that the phase 2
insulin secretion rate maintains the glucose concentration at
a particular level, i.e. there is a new fasting glucose concen-
tration being maintained to which the insulin concentration
adapts, as suggested by Goodner and associates [18]. The 24-h
basal glucose and insulin concentrations also remained
essentially unchanged by the following morning.
Clearly as pointed out by Goodner et al. [18], and as
demonstrated here, the post-meal insulin secretory response
is vigorous and responsive to increases in the usually ingested
fuel secretagogues. Thus, the total insulin secretory capacity
is large (Fig. 2), and not an issue.
Alternatively, the glucose concentration is being regulated
independent of the insulin concentration, but the insulin
change accommodates to these independent regulators such
that the metabolic disposition of the ingested macronutrients
occurs completely within 24 h, regardless of type and amount
ingested. If so, this regulation occurs in people with type 2
diabetes at an elevated glucose concentration and is depen-
dent on the type of fuel ingested, but also to just fasting.
The glucose concentration in the absence of concurrent
carbohydrate intake correlates very well with the glucose
production rate both in people with [19–21] or without [21–23]
diabetes. Thus, the observed decreases in AM fasting glucose
in the present study likely represent a decreased glucose
production rate. If so, the differences in production rate again
suggest an insulin-independent dietary effect on liver func-
tion associated with an adjustable beta cell insulin secretory
response that maintains that concentration of glucose [18] as
indicated above.
The insulin concentration did not change throughout 24 h
when the subjects were not ingesting fuel. The net increased
response in insulin when the subjects ingested the carbohy-
drate-free diet was 28% as great as with the standard diet.
This is due to the increased fat content as well as the
unchanged protein content. Protein is known to stimulate
insulin secretion [24]. In subjects with type 2 diabetes it is
just as potent as glucose on a weight basis but has little effect
on the glucose concentration [25]. Dietary fats stimulate a
rise in insulin concentration [26–29] but this has not been
extensively studied.
In the present study, in spite of the clear increase in insulin
stimulated by the protein and fat ingested in the carbohy-
drate-free diet, the net glucose area response was similar
to that observed without food energy intake (Fig. 1). In both
cases it was actually negative. Thus, again there was
dissociation between the insulin response and the glucose
response to these dietary manipulations.
4.3. C-peptide
The C-peptide response was similar to the insulin response
indicating that a major change in insulin turnover rate was
not present with any of the dietary manipulations (Fig. 3).
Although statistical significance was not obtained with
comparisons of the C-peptide area responses, unlike the
statistical significance for all comparisons with the insulin
area responses, both insulin and C-peptide area response
results to the dietary interventions were nearly identical.
4.4. Glucagon
The 0800 h fasting glucagon concentrations were similar
regardless of the diet composition or without food energy
intake (standard, 74 ± 5, CHO-free, 71 ± 11, fasting, 83 ±
14 pg/ml) (Fig. 4).
The glucagon concentrations also were rather stable
throughout the 24-h period of the study regardless of the
diet composition or with just fasting (Fig. 4). A clear meal-
related change was not observed. Overall, these data suggest
that changes in overnight fasting glucagon or absolute 24-h
integrated glucagon concentrations do not contribute mech-
anistically to the differences in glucose concentrations
observed in each different nutritional state.
In people without diabetes, an increase in glucagon during
a 72 h fast has been reported [7,30–33]. This could be
attributed to a decrease in insulin concentration [34] and an
increase in non-esterified fatty acids (NEFAs) [35]. Also in all
260 METABOLISM CLINICAL AND EXPERIMENTAL 64 (2015) 253–262

nutritional states including during states of dietary carbohy-
drate deprivation, a reciprocal relationship between insulin
and glucagon is considered to have major effects on glucose
production and thus the glucose concentration in people
without diabetes [34]. Insulin secretion is considered to play
the major role both by a direct inhibition of glucose
production but also by an indirect effect due to its inhibitory
effect on glucagon secretion [36]. Insulin and perhaps zinc
secreted by the β-cell can inhibit glucagon secretion directly
through a proposed paracrine mechanism [37].
The current data are not compatible with this concept.
There was a considerable change in 24 h integrated insulin
area response but essentially no change in glucagon area
response. Thus, the current data and other data in the
literature [38] suggest that this inhibition can be regulated.
4.5. Strengths and limitations
The strength of this report is that it represents complete 24-h
data obtained in people with type 2 diabetes ingesting real
world foods of carefully designed macronutrient composition.
The limitations are 1) the relatively small number of subjects
studied, and 2) all were males. Our primary outcome was
glucose area response. Based on previous data in subjects
with type 2 diabetes from our laboratory, which showed a 29%
decrease in 0800 h glucose concentration after 48 h of
starvation, and a 27% decrease in glucose concentration
after 5 weeks on a 20% carbohydrate diet, the power analysis
indicated that we would have to study 2 subjects to test our
current hypothesis. We studied 7 subjects to provide clinical
significance to the data. Thus, although the number of
subjects is small, it should be noted that such studies require
very dedicated subjects and are very labor intensive. It is not
practical to do such studies in a large population of people
with type 2 diabetes. It also would be difficult to obtain an
equal male:female ratio when recruiting subjects.
It should be noted that our results may only be valid in
the early stages of type 2 diabetes. It is not known whether
similar results would be obtained in subjects with advanced
stages of diabetes, i.e. when beta-cell function is expected to
be diminished.
Nevertheless, these data demonstrate a dramatic and
rapid improvement in blood glucose without weight loss and
without a change in insulin. This suggests that the rapid
improvement in glycemic control following low food energy
diets as well as surgeries for weight loss at least in part, can be
attributes to the diet per se rather than just a weight loss.
That is, it is due to an intrinsic metabolic adjustment to a
change in fuel consumption. Whether these data have
translational potential in other areas of human physiology
and therapeutics remains to be determined.
5. Conclusion
Short-term fasting dramatically lowers the plasma glucose
concentration. A lack of carbohydrate could explain approx-
imately 50% of the decrease in the overnight fasting glucose
and 70% of the 24 h integrated glucose concentration ob-
served with short-term fasting.
Thus, changes in the amount and macronutrient compo-
sition of the diet not only strongly affect the postprandial
glucose but also the basal glucose concentration in people
with type 2 diabetes. The latter occurs without a significant
change in insulin and glucagon concentrations. The mecha-
nism or mechanisms which maintain the different but
abnormally elevated glucose concentrations remain to be
determined. Most likely, it is multifactorial. Nevertheless, it is
clear that a limitation in insulin secretory capacity and/or a
static state of insulin resistance cannot explain the hypergly-
cemia in these subjects.
Author Contributions
Dr. R.M. Almokayyad was an Endocrine Fellow in training at
the time this study was done. Dr. Almokayyad applied for and
obtained IRB approval for the study, recruited the subjects,
obtained the blood specimens, and contributed to the data
analysis and preliminary draft of the manuscript. Drs. F.Q.
Nuttall and M.C. Gannon obtained the funding, formulated
and designed the study, performed the final analysis of the
data and wrote the final manuscript. Dr. Nuttall is responsible
for the contents.
Funding
Supported in part by funds from the Department of Veterans
Affairs. The Department of Veterans Affairs had no involve-
ment in the study design, the collection, analysis and
interpretation of data, in the writing of the manuscript or in
the decision to submit the manuscript for publication.
Disclosure statement
The authors have no conflicts of interest and have nothing
to disclose.
Acknowledgments
The authors thank the volunteers for participating in the
study, the Staff of the Special Diagnostic and Treatment Unit,
Heidi Hoover, R.D., M.S., Research Dietitian, the Staff of the
Clinical Chemistry Laboratory, Linda Hartich, M.T. for labora-
tory assistance, David Prentiss for preparing the figures for
publication and Rachel Anderson for preparing the manu-
script for electronic submission.
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