Normocaloric Diet Restores Weight Gain and Insulin Sensitivity in

May 2016 | Volume 7 | Article 491

ORIGINAL RESEARCH

published: 27 May 2016

doi: 10.3389/fendo.2016.00049

Frontiers in Endocrinology

| www.frontiersin.org

Edited by:

Nicholas Michael Morton,

University of Edinburgh, Scotland

Reviewed by:

Maximilian Zeyda,

Medical University of Vienna, Austria

Matthew Brook,

University of Edinburgh, UK

*Correspondence:

Antonio Brunetti

[email protected];

Diego Russo

[email protected]

Specialty section:

This article was submitted

to Genomic Endocrinology,

a section of the journal

Frontiers in Endocrinology

Received: 24March2016

Accepted: 09May2016

Published: 27May2016

Citation:

LombardoGE, ArcidiaconoB,

DeRoseRF, LeporeSM, CostaN,

MontalciniT, BrunettiA, RussoD,

DeSarroG and CelanoM (2016)

Normocaloric Diet Restores Weight

Gain and Insulin Sensitivity

in Obese Mice.

Front. Endocrinol. 7:49.

doi: 10.3389/fendo.2016.00049

Normocaloric Diet Restores Weight

Gain and Insulin Sensitivity

in Obese Mice

Giovanni Enrico Lombardo

, Biagio Arcidiacono

, Roberta Francesca De Rose

Saverio Massimo Lepore

, Nicola Costa

, Tiziana Montalcini

, Antonio Brunetti

Diego Russo

*, Giovambattista De Sarro

and Marilena Celano

Department of Health Sciences, University “Magna Graecia” of Catanzaro, Catanzaro, Italy,

Department of Medical and

Surgical Sciences, University “Magna Graecia” of Catanzaro, Catanzaro, Italy

An increased incidence of obesity is registered worldwide, and its association with insulin

resistance and type 2 diabetes is closely related with increased morbidity and mortality

for cardiovascular diseases. A major clinical problem in the management of obesity is the

non-adherence or low adherence of patients to a hypocaloric dietetic restriction. In this

study, we evaluated in obese mice the effects of shifting from high-calorie foods to nor-

mal diet on insulin sensitivity. Male C57BL/6JOlaHsd mice (n=20) were fed with high fat

diet (HFD) for a 24-week period. Afterward, body weight, energy, and food intake were

measured in all animals, together with parameters of insulin sensitivity by homeostatic

model assessment of insulin resistance and plasma glucose levels in response to insulin

administration. Moreover, in half of these mice, Glut4 mRNA levels were measured in

muscle at the end of the high fat treatment, whereas the rest of the animals (n=10) were

shifted to normocaloric diet (NCD) for 10weeks, after which the same analyses were

carried out. A signicant reduction of body weight was found after the transition from

high to normal fat diet, and this decrease correlated well with an improvement in insulin

sensitivity. In fact, we found a reduction in serum insulin levels and the recovery of insulin

responsiveness in terms of glucose disposal measured by insulin tolerance test and

Glut4 mRNA and protein expression. These results indicate that obesity-related insulin

resistance may be rescued by shifting from HFD to NCD.

Keywords: insulin resistance, obesity, Glut4, diet, glucose

INTRODUCTION

Modern lifestyle is oen characterized by sedentary activities and overeating. As a consequence,

in the last decades, this has been responsible for the increased incidence and prevalence of obesity

and obesity-induced comorbidities, such as insulin resistance and metabolic syndrome (1, 2) that

may contribute to type 2 diabetes mellitus (T2DM) and cardiovascular disease (3). Several studies

have demonstrated that a healthy lifestyle can lead to weight loss and improve insulin sensitivity

(4–7). In this regard, a crucial role is played by the nutrient composition of the diet, both in terms

of total caloric intake and the variety of its components, with particular attention to the dierent

types of fatty acids (8, 9). Unfortunately, most of anti-obesity interventions are oen limited by

the diculty to maintain a low-calorie dietary regimen, especially when long-term treatments are

required (10,11). us, few anti-obesity programs have been found to be helpful.

FIGURE 1 | Study design. A schematic representation of the study protocol

and experimental plan is shown.

Lombardo et al.

Normocaloric Diet in Obese Mice

Frontiers in Endocrinology | www.frontiersin.org May 2016 | Volume 7 | Article 49

To date, several animal models have been used to evaluate the

eects of various dietetic regimens on body weight and meta-

bolic parameters. A validated experimental model is represented

by mice fed with a high fat diet (HFD), which develop obesity,

insulin resistance, and dyslipidemia (8–13).

In the present study, we evaluated the eects of the transition

from HFD to normocaloric diet (NCD) (regular food with no

additive agents or nutraceutical compounds) on body weight and

insulin responsiveness in C57BL/6JOlaHsd mice, a strain of mice

genetically prone to develop obesity and insulin resistance (14).

MATERIALS AND METHODS

Animals and Study Design

Five-week-old male C57BL/6JOlaHsd mice (n=20), NCD and

HFD, were purchased from Harlan Laboratories S.r.l (Udine,

Italy). Mice were housed in individual cages and maintained on

12-h light/dark cycle at 21±1°C and 50±5% humidity with

free access to water and food adlibitum. Animals were fed with

HFD containing 60.3% kcal fat, 21.3% kcal carbohydrate, and

18.4% kcal protein (HFD group) for 24weeks. Aer this period,

10 mice were euthanized by cervical dislocation and the other

10 were fed with NCD only (Teklad Global 18% kcal fat, 58%

kcal carbohydrate, and 24% kcal protein) (NCD group) for the

subsequent 10weeks. A schematic representation of the study

design is shown in Figure1. Body weight, girth waist, and food

intake were recorded at weekly interval for all animals (15). Liver,

skeletal muscle, and abdominal fat were excised, weighted, and

stored in liquid nitrogen. is study was performed following the

Italian (D.M. 116/92) and ECC regulations (O.J. of E.C.L 358/1

12/18/1986), in accordance with the guide for the care and use of

laboratory animals and approved by the local ethical committee.

Biochemical Analysis

Blood samples were collected aer 12h of fasting. Serum was sep-

arated by centrifugation at 1700g for 10min at room temperature

and stored at −20°C, until use. Total cholesterol and triglycerides

were measured using commercial reagents (Siemens Healthcare

Diagnostics, Milano, Italy) and an automated biochemistry ana-

lyzer (Dimension EXL, Siemens Healthcare Diagnostics). Insulin

levels were measured using ELISA kit (Rat/Mouse Insulin ELISA

Kit, EMD Millipore Corporation, Darmstadt, Germany), accord-

ing to the manufacturers’ instructions.

Insulin Tolerance Test

Insulin tolerance test (ITT) was performed in both HFD and

NCD groups, as previously described (16). Animals were fasted

for 12 h, weighed, and injected intraperitoneally with insulin

(1U/kg body weight Regular

, Novorapid, Novonordisk, Roma,

Italy). Blood glucose levels were measured aer 0, 15, 30, 60, and

90 min using an automatic glucometer (Glucocard, Menarini

Diagnostics, Firenze, Italy).

Expression of Glucose Transporter Type 4

Total RNA was isolated from quadriceps skeletal muscle using

TRIzol reagent (Life Technologies, Monza, Italy), following the

manufacturer’s recommended protocol and quantied with a

NanoDrop Spectrophotometer (ermo Fisher Scientic, Inc.,

Waltham, MA, USA). RNA levels were normalized against 18S

ribosomal RNA in each sample, and cDNAs were synthesized

from 1μg of total RNA using the High Capacity cDNA Reverse

Transcription Kit (Life Technologies). Primers for mouse Glut4

and ribosomal protein S9 (RPS9) were designed according to

sequences from the GenBank database. Relative quantication was

made using a real-time thermocycler (Eppendorf Mastercycler ep

realplex, Milano, Italy). In a 20-μl nal volume, 1μl of cDNA solu-

tion was mixed with SYBR Green RealMasterMix (Eppendorf)

and 0.2μM of each sense and antisense primers. SYBR Green

uorescence was measured, and relative quantication was made

against either RPS9 or Gapdh cDNAs, used as internal standards.

All PCR reactions were carried out in triplicates. Glut4 protein

expression was measured in quadriceps muscle from six to eight

mice of each group, using a rabbit anti-Glut4 polyclonal antibody

as previously described (17).

Statistical Analysis

Results are expressed as mean±SD. e independent t-test was

used to evaluate intergroup dierences. All statistical analyses

were performed using GraphPad Prism version 5.0 statistical

soware (GraphPad Soware Inc., San Diego, CA, USA). p values

lower than 0.05 were considered statistically signicant.

RESULTS

Effects of Normocaloric Diet on Body

Weight and Biochemical Parameters

Twenty mice were fed with HFD (HFD group) for 24weeks, reach-

ing a weight of approximately 43g, with fasting plasma glucose

levels between 90.5 and 117.7mg/dL, which were consistent with

a condition of impaired fasting glucose. Aer the 24-week period,

half of the mice were fed with NCD for the following 10weeks

(NCD group). A signicant decrease of body weight was observed

in the NCD group compared to the HFD group (27%, p<0.001),

TABLE 1 | Weight and waist in HFD and NCD mice.

HFD NCD p value

Liver (g)

1.24±1.09 1.09±0.09 <0.05

White adipose (g) 1.54±0.31 1.29±0.08 <0.05

Epididymis (g) 0.60±0.16 0.33±0.08 <0.01

Girth waist (cm) 10.55±0.42 9.54±0.17 <0.01

Values are expressed as mean±SD.

FIGURE 2 | Effects on body weight, food, and energy intake in mice fed with high fat diet (HFD) for 24weeks and with normocaloric diet (NCD) for

other 10weeks. A signicant reduction of body weight and energy intake was observed in NCD mice (

A,B), whereas no signicant difference was detected in food

intake (

C). Body weight over the time is shown in the inset. Values are expressed as mean±SD. *p<0.001.

Lombardo et al.

Normocaloric Diet in Obese Mice

Frontiers in Endocrinology | www.frontiersin.org May 2016 | Volume 7 | Article 49

as a result of the decrease in energy intake due to the less caloric

supply derived from the NCD rather than the dierent food

intake (Figure2). In the NCD group, we also observed a decrease

in liver size, fat depots, and girth waist (Table1). Moreover, shi-

ing to NCD resulted in a signicant decrease in plasma glucose

levels (p<0.05) and serum insulin levels (p<0.01), as well as

triglycerides (p<0.05) and total cholesterol (p<0.05) (Figure3).

Effects on Insulin Sensitivity

Next, we evaluated the eects of NCD on insulin sensitiv-

ity. ITT performed in mice before and aer NCD showed a

better response to insulin in terms of changes in blood glucose

concentrations in the NCD group than in the HFD group.

In fact, the glucose-lowering eect of exogenous insulin was

FIGURE 3 | Biochemical parameters. Blood samples were collected as indicated in Section “Materials and Methods.” After 10weeks of feeding with a

normocaloric diet (NCD), mice showed a signicant reduction of plasma glucose levels and serum insulin levels, as well as a reduction in both triglycerides and total

cholesterol when compared to the HFD. Values are expressed as mean±SD. *p<0.05.

Lombardo et al.

Normocaloric Diet in Obese Mice

Frontiers in Endocrinology | www.frontiersin.org May 2016 | Volume 7 | Article 49

enhanced in NCD mice during ITT and was reduced in HFD

mice (Figure4). From a mechanistic point of view, the improve-

ment in insulin sensitivity in mice in response to NCD was

dependent, at least in part, on an increase in Glut4 expression

induced in skeletal muscle following the transition from HFD

to NCD. To show such a molecular link between restoration

of insulin sensitivity and NCD, total RNA was extracted from

skeletal muscle of animals before and aer shiing to NCD, aer

insulin stimulation, and Glut4 mRNA and protein levels were

measured. As shown in Figure5, both insulin-stimulated Glut4

mRNA and protein expression were signicantly increased in

skeletal muscle of NCD mice as compared with that of HFD

mice (p<0.05).

DISCUSSION

Obesity is a chronic disorder that can cause other health prob-

lems, such as diabetes, hypertension, hepatic steatosis, obstruc-

tive sleep apnea, and atherosclerosis (18). e association of

obesity with T2DM is well established, due to the negative

inuence of excessive body fat on peripheral insulin action and

hepatic function, leading to insulin resistance (19). Treatment

of obesity includes hypocaloric diet, exercise, and lifestyle

modications, with dietary manipulation still representing the

rst-line therapeutic approach for this common disorder (20,

21). However, it is still debated which is the more appropriated

dietetic regimen to obtain a weight loss, which may be at the

same time rapid, well tolerated, and sustainable for a long period

of time. Although the importance of calorie restriction in this

condition is well recognized, also for the positive psychological

benet for the patient and the family, there is no doubt that

a major problem in treating obesity is still represented by the

relatively low level of adherence of aected subjects to low/very

low-calorie diets (22–24). us, many dietary strategies have

been proposed to overcome such obstacles, but the results are

not satisfactory enough in most of obese patients (25, 26). In

these individuals, we hypothesized that shiing to normoca-

loric balanced diet, formulated to avoid excess fat, rather than

hypocaloric diet – which would obtain a better compliance

especially in view of long-term treatment–might be sucient,

in addition to physical exercise and lifestyle change, to get

more satisfactory results in terms of weight loss and conse-

quent improvement in obesity-related insulin resistance. is

hypothesis is well supported by the present nding in our mouse

model of obesity and obesity-induced insulin resistance. In fact,

shiing from HFD to NCD for 10weeks, caused a signicant

reduction of body weight mainly due to the reduction of vis-

ceral fat, together with the overall reduction of triglycerides,

total cholesterol, and, most importantly, restoration of insulin

sensitivity, as reected by the decline in fasting insulin levels. A

similar approach treating obese mice with NCD has also been

FIGURE 5 | Expression of Glut4. Glut4 mRNA levels were measured by

qRT-PCR in skeletal muscle from HFD and NCD mice, after insulin

stimulation. Results are the mean±SD for six animals per group. *p<0.05

versus HFD mice. A representative Western blot (WB) of Glut4 in quadriceps

muscle from six to eight mice of each group is shown in duplicate in the

autoradiogram. Gapdh, control of protein loading.

FIGURE 4 | Insulin sensitivity. HFD and NCD mice fasted for 12h were injected intraperitoneally with insulin (1U/kg). Blood glucose levels were measured with a

glucometer, as reported in Section “Materials and Methods.” Values are expressed as mean±SD. *p<0.05.

Lombardo et al.

Normocaloric Diet in Obese Mice

Frontiers in Endocrinology | www.frontiersin.org May 2016 | Volume 7 | Article 49

used in a few other studies where, however, some nutraceutical

compounds or other ingredients were added to regular food

(27–30). is is slightly dierent than what we did in our study,

in which NCD itself, without any additive agent, was able to

improve insulin sensitivity and Glut4 expression.

Glut4 is the major insulin-dependent glucose transporter in

muscle. Abnormalities at this level are a hallmark of peripheral

insulin resistance (31). In the present study, the improvement

in insulin sensitivity associated with increased Glut4 mRNA

expression in NCD mice provides a possible mechanistic expla-

nation as to how the normal calorie diet can improve insulin

responsiveness and supports the hypothesis that rescue from

insulin resistance and diabetes can be reached without the adop-

tion of a low-calorie diet. If conrmed in obese humans, such

an approach, in association with adequate and individualized

physical exercise programs, might be able to contribute to coun-

teract the long-term failure of the current therapeutic approaches

adopted in these individuals, and this would conrm further the

appropriateness of mouse models for studying human obesity.

However, on the other hand, it is also known that marked inter-

species dierences exist between human and mouse with respect

to behavioral control of food uptake, tissue energy disposal and

storage, weight, and weight loss, which emphasize the inuence

of non-genetic environmental factors and genetic modiers in

determining the phenotypic variations observed in humans and

animal models of obesity. us, caution is required in general-

izing these ndings. As a limitation of the present work, the fact

is that mice of dierent ages were compared in our study.

In conclusion, numerous anti-obesity initiatives have been

adopted up to now, which include lifestyle changes, drug treat-

ments, and surgery. However, because of the limited ecacy

and the occurrence of adverse events in aected treated patients,

alternative and complementary therapies for weight loss have

been investigated, including acupuncture, dietary supplements,

etc. Our ndings in the current work provide valuable informa-

tion about the ecacy of shiing to NCD in restoring weight and

insulin sensitivity in HFD-induced obese mice. Similar studies in

obese humans would reveal whether this strategy, probably better

accepted by patients, may be successful in correcting weight gain

and obesity-related insulin resistance.

Lombardo et al.

Normocaloric Diet in Obese Mice

Frontiers in Endocrinology | www.frontiersin.org May 2016 | Volume 7 | Article 49

AUTHOR CONTRIBUTIONS

GEL contributed to animal testing and draing of the manu-

script; RFDR elaborated gures and tables and contributed to

the analysis of the results; SML contributed to animal testing

and draing of the manuscript; BA performed the molecular

analysis; NC performed the operation on the animals and

supervised the animals’ maintenance during the treatment

period; TM and GDS reviewed the nal version of the manu-

script; AB contributed to the conception of the idea and critically

reviewed the manuscript; DR contributed to the conception of

the idea, draed the manuscript, and critically reviewed the nal

manuscript; and MC contributed to animal testing, analysis of

the results, and editing of the manuscript. All authors read and

approved the submitted version.

ACKNOWLEDGMENTS

We thank Professor D. Britti and Dr. F. Trimboli for assistance in

the management of animals.

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construed as a potential conict of interest.

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