Fondecyt 1160792

Proposal Title: Interaction between supplemental zinc and muscle-strength

training as a key element to improve type-2 diabetes therapy

IR: Manuel Ruz Ortíz

Departamento de Nutrición,
Facultad de Medicina
[email protected]
anexo 86134

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THE PROBLEM AND ITS RELEVANCE AS RESEARCH TOPIC

Type-2 diabetes is highly prevalent. In Chile this disease affects almost one-tenth of adult population. It is a
chronic condition responsible for long term-severe dysfunction of several organs. Besides, it is a heavy
burden to the health system as result of the continuous care and treatment these patients need. Because
the complexities of this pathology, therapeutical approaches usually include several components, such as
pharmacologic products to treat some of the most prominent features of this disease, that is insulin
resistance and insulin secretion, promotion of healthy life-style, including increasing physical activity and
appropriate nutrition. Nevertheless, there still a number of issues –and how they interact- that need to
be studied/specified, in order to improve results leading to optimization of therapy. For instance, at what
extent the combination of a given type of exercise and nutrient supplementation can promote a more
efficient glucose management in these patients is far from being known.

This research project will address this issue by looking at the effects of the interaction of a defined
scheme of muscle-strength training and supplementation with zinc on diabetes management in a
randomized clinical trial; also, an animal (murine) model will be implemented to perform determinations
in muscle, pancreas, liver, and adipose tissue to understand the mechanisms involved.

BACKGROUND

Diabetes mellitus comprises a variety of syndromes with distinct etiologies characterized mainly by
hyperglycemia. This feature can result from insulin secretion impairment and/or alterations of hormone
activity at the target tissues. The consequences of diabetes mellitus include long-term damage,
dysfunction and failure of several organs, especially eyes, kidneys, heart and blood vessels. Less than
10% of the cases falls into the category of insulin-dependent diabetes mellitus (IDDM or type-1), which
generally appears in childhood and adolescence, and results from autoimmune destruction of insulin-
producing cells within the pancreas. Far more common is non-insulin-dependent diabetes mellitus (NIDDM
or type-2). Unlike type-1, type-2 diabetes is often associated with obesity (American Diabetes Association
2015). Type-2 diabetes affects about 347 million individuals worldwide, (WHO 2015). In Chile, a rapid
increase in recent years has been noted, from 6.3% in 2003 to 9.4% in 2009 according to the latest
National Health Survey (ENS 2009-2010).

Metabolic and molecular aspects:

Insulin resistance along with compensatory hyperinsulinemia is the earliest stage of the disease. Later,
insulin secretion would be altered leading first to post prandial hyperglycemia and a further fasting
hyperglycemia. Regarding to β-cell function, a distinctive defect in type-2 diabetes is the loss of the first
phase of glucose-induced insulin secretion (Kahn SE 2001). Insulin resistance and β-cell dysfunction are
complex processes and a host of molecular mechanisms are involved (see Figures 1 and 2 in ANNEX
FIGURES). Among the factors related to insulin action at the skeletal muscle are mentioned: impaired
insulin signaling mediated by reduced IRS-1 phosphorylation and PI3–kinase activity, lipotoxicity,
mitochondrial dysfunction, increased inflammation mediated by increased lk-B-β/NF-kB pathway activity.
Insulin signaling is also affected at the liver, brain and hypothalamus (Biddinger 2006). Adipose tissue -and
the adipocytes contained within- are key determinants either in the instauration or the perpetuation of
insulin-resistance features. On one hand, it participates in insulin resistance through adipokines secretion:
TNF-α, resistin and interleukin-6 decrease insulin sensitivity and adiponectin improves it (Tripathy 2010).
Type-2 diabetes is highly related to obesity development. Obesity is often associated with the presence of
low-grade chronic inflammation in white adipose tissue. When adipocyte hypertrophy occurs, endoplasmic
reticulum stress, hypoxia, and oxidative stress arise in this tissue. These in turn, induce the release of
inflammatory signals, which initiate the infiltration of monocyte into this tissue (Suganami 2010). Adipose
tissue inflammation is triggered by this macrophages infiltration; the subsequent crosstalk between these
cells and resident adipocytes highlights as a key factor for the development of associated co-morbidities
(Xu 2003), especially insulin-resistance. In this sense, several inflammatory products produced by this
interaction, such as TNF-α, MCP-1 and nitric oxide (NO), correlates with increased body adiposity and
appear to participate in the induction and maintenance of the chronic inflammatory state associated with
obesity (Ferrante 2007)). In addition, white adipose tissue overgrowth leads to downregulation of several
anti-inflammatory products, e.g., adiponectin (Maeda 2002).

In terms of insulin secretion-related mechanisms, decreased β-cell mass, mainly associated to

dysregulation of β cell apoptosis, lipotoxicity as result of chronic FFA exposure, endoplasmic reticulum

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stress, decreased GLP-1 secretion and increased GIP resistance are involved (Marchetti 2010). Progression
to type-2 diabetes is determined by β-cell failure that leads to impaired insulin secretion. β-cell dysfunction
is exacerbated by insulin resistance, whereas the development of the disease is delayed by improvement
of insulin sensitivity (Buchanan 2002). Another crucial factor involved in diabetes pathophysiology is the
presence of an oxidative stress condition. Hyperglycemia is associated to increased production of reactive
oxygen species (ROS) as well as depletion of antioxidant defense system components with the
concomitant accumulation of oxidative-end products. Oxidative stress has been implicated in a number of
consequences of diabetes, such as increased risk of cardiovascular disease, nephropathy, cataract,
retinopathy, and neuropathy, among others (Pan 2010).

Epigenetics aspects. Two new areas of interest regarding epigenetic regulators in diabetes research
have emerged. These are the role of microRNAs (miRNAs) and Sirtuins (SirTs). MicroRNAs are single-
stranded non-coding RNA molecules which play a crucial role in the regulation of protein-encoded genes.
Among the roles of selected miRNAs in diabetes are: participation in insulin synthesis and exocitosis from
pancreatic β-cells, and also in insulin resistance at liver, adipocytes and skeletal muscle (Chen 2014). A
recent meta-analysis identified that 40 miRNAs are significantly dysregulated in type-2 diabetes. The top
upregulated was miRNA-142-3p (OR=6.5), and the top downregulated was miRNA-126a (OR=7.5). Besides
these, miRNA-29a, miRNA-34a, miRNA-103, miRNA-107, miRNA 132, and miRNA-144 are suggested as
potential circulating biomarkers, whereas miRNA-199a-3p and miRNA-223 as potential tissue biomarkers of
diabetes (Zhu 2015).

Sirtuins are a class of NAD-dependent histone and non-histone deacetylases. Mammals have 7 sirtuins,
which show a high degree of functional diversification including a wide range of substrates and diverse
cellular localization patterns. Activity of these proteins is regulated by the NAD/NADH ratio, acting as
sensors the energy status of the cell (Turkmen 2014). Sirtuins function is associated with a regulatory role
in the structure, expression and maintenance of the integrity of the genome. The epigenetic mechanism
based on histone deacetylation is not the only resort for the Sirt complex, they also present important
functions involving regulation of transcription factors such as NF-kB, p53, FOXO, PGC-1α, and Myc, among
others (Kauppinen 2013, Caron 2014). Sirt1 stimulates insulin secretion and insulin signaling in peripheral
tissues (Liang 2009) and attenuates insulin resistance by reduction of mitochondrial dysfunction in skeletal
muscle cells (Zhang 2015). It also regulates adipogenesis and myogenesis (Turkmen 2014). Sirt2 is highly
expressed in adipocytes, while Sirt3, Sirt4, and Sirt5, located in mitochondria are involved in
mitochondrial biogenesis and metabolism (Parihar 2015). Interestingly, Sirt1 is closely related to the
adjustments muscle tissue needs to go through during exercise, in fact, it could be the primary
regulator of such changes (Pucci 2013). On the other hand, Sirt1 also has a key role inhibiting protein
tyrosine phosphatase 1B (PTP1B) resulting in improvement of insulin sensitivity. Zinc performs a similar
action as Sirt1 on PTP1B (Vardatsikos 2013). Thus, information available in the field of nutrition-related
diseases suggests that modulation of sirtuins may be key in the development/evolution of diabetes. The
epigenetic aspects of diabetes are a relevant component of our research proposal.

Clinical features
Diabetic patients can present hyperglycemia during long periods of time, leading to functional defects in a
variety of organs with no obvious manifestations. Diabetes is associated to increased morbidity and
mortality as results of its associated complications which are mainly related to the accumulation of
advanced glycosylation-end products. Among the complications can be mentioned: retinopathy,
angiopathy, neuropathy, musculoskeletal problems, and nephropathy. Diabetic patients usually present
hypertension, alterations of lipoproteins, tryglicerides, and increased risks of cerebrovascular and
cardiovascular disease (American Diabetes Association 2015).

Treatment
The treatment of type-2 diabetes is oriented to correct hyperglycemia by enhancing insulin secretion
and/or insulin sensitivity. It includes changes of feeding patterns and life-style, as well as
pharmacologic therapy, including exogenous insulin (or insulin analogues) administration when there is
minimal or null insulin secretion (American Diabetes Association 2015).

Pharmacological:
Regarding pharmacologic treatment, there are insulin-sensitizing drugs (biguanides, thiazolidinediones),
insulin secretagogues (sulfonylureas, meglitinides), inhibitors of carbohydrates digestion and intestinal

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absorption (α-glucosidase inhibitors), glucagon-like peptide-1 (GLP-1) analogues, dipeptidyl peptidase-4
(DPP-4) inhibitors, amylin agonists, glucosuric agents, and insulin therapy. When diabetes is diagnosed, it
is recommended to initiate the pharmacologic treatment with metformin, diet and life-style modifications,
further use of additional pharmacological preparations will depend on the observed metabolic control of the
patient (American Diabetes Association 2015).

Exercise and diabetes:

Physical activity promotion and exercise has been proposed as a cornerstone in the management of
diabetic patients, together with medication and diet (AACE/ACE 2015). The skeletal muscle adaptations
associated with exercise training are specific to the mode of exercise performed (i.e., resistance or muscle-
strength vs. endurance or aerobic exercise), along with the frequency, intensity, and duration of the
exercise stimulus (Fyfe JJ 2014, Stanford 2015). Unless supervised, physical training presents low
adherence (Saleh 2014), in part because patients believes that it is less useful than drugs (Broadbent
2011). Although both, endurance and muscle-strength training improve insulin action, and glucose
control, the latter has the advantage of increasing skeletal muscle mass optimizing and
prolonging the beneficial responses (Colberg 2010). Besides, results can be obtained in a shorter
period of time, and with minimal or no equipment needed. Interestingly, advantageous effects of a
training program on glycemic indices can be maintained for several weeks after finished such protocol
(Park 2015).

During exercise, contracting skeletal muscle increases glucose uptake in an intensity-dependent manner to
sustain the energy demand caused by increased ATP turnover. This increase is driven by increased capillary
recruitment as well as translocation of glucose transporter type 4 (GLUT4) to the plasma membrane
(Mounier R 2015). A major -although not exclusive- mechanism through which exercise improves insulin
action is mediated by activation of AMPK (Friedrichsen 2013) (See Figure 3 in ANNEX FIGURES). The
leading anti-diabetic drug, metformin, is hypothesized to mediate its effect also by activating AMPK,
however there are interesting observations in this regard. For instance, metformin did not accentuate the
effects of exercise training on whole-body insulin, suggesting exercise would be a more potent stimulus
(Malin 2012). AMPK activation seems not to be needed in response to exercise when only low
‘‘physiological stress’’ is imposed on the cell. Indeed, AMPK activation is observed at exercise intensities
demanding more than 60% of VO2peak or with low intensity exercise performed until exhaustion
(Friedrichsen et al, 2013), that would explain the need of using muscle-strength exercises with an
individual progression to avoid this adaptation, and in turn, be able to optimize glucose
utilization.

Additional mechanisms apart from AMPK are involved in determining the level of exercise-mediated
improvements in insulin sensitivity (Friedrichsen 2013). Thus, there is evidence indicating that exercise is
able to prevent cardiac injury in diabetic mice by activation of Akt signaling as well as peroxisome
proliferator-activated receptor γ co-activator 1 α (PGC -1α); exercise also was able to reduce expression of
pro-apoptotic genes (Wang 2015). In addition, exercise training, increases the appearance of beige
adipocytes interspersed within subcutaneous adipose tissue and concomitantly inducing enhancement of
glucose uptake in skeletal muscle and brown fat tissue (Wallberg-Henriksson 2015).

The effects of exercise do not seem to be limited to glucose uptake and utilization by peripheral tissues,
there is information indicating that insulin secretion is suppressed during exercise but increased
immediately after exercise (Knudsen 2014)

Micronutrients as co-adjuvants:
An interesting approach in diabetes treatment has been the use of micronutients as co-adjuvants. Results
have been controversial, For instance, antioxidant micronutrient supplementation in diabetic patients
without underlying deficiency does not have enough support, furthermore, some data showed potential
adverse effects of vitamin E, C, A, and selenium, making it advisable no to use (Chehade 2009). On the
other hand, the multiple roles of zinc in a number of relevant cellular and systemic functions, some of them
closely related to diabetes features, have called the attention as a potential natural adjuvant in the
management of this pathology.

Zinc
Zinc (Zn) is essential for all forms of life. Zn is required for virtually all aspects of cell metabolism, i.e.,
DNA synthesis and DNA transcription, translation of mRNA into proteins, structure and stabilization of

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proteins. It participates in metabolism through its catalytic, structural, and regulatory roles. Zn is required
for the function of more than 300 enzymes of all classes, and it is involved in the regulation of a large
number of genes (Ruz 2003, Foster 2010). Zn participates in some hormone-receptor interactions and also in
intracellular signaling. Particularly relevant are its involvement in insulin receptor and insulin-growth factor 1
(IGF-1) activation (Vardatsikos 2013). Zinc is also related to increased IGF-1concentrations (Ruz 2006).
Despite Zn is redox-inert, has a number of relevant indirect antioxidant effects (Foster 2010). Thus, Zn is
involved in growth and a number of relevant functions, such as immunity, cellular signaling, tissue repair,
protection against oxidative damage, apoptosis, vitamin A metabolism, neuropsychological functions, and
hormone action, including insulin (Ruz 2003, Vardatsikos 2013). Zn is also involved in both endocrine and
exocrine function of pancreas (Kelleher 2011).

Zn homeostasis is highly complex and needs compartmentalization of this element into cellular organelles.
Twenty four proteins (Zn transporters) have been identified to be involved, and the knowledge regarding
specific aspects of their participation is only partial. There are 14 members of the family gene solute-linked
carrier SLC39 (ZIP 1-14), that transport Zn into the cytoplasm. Ten members of the family gene solute-
linked carrier SLC30 (ZnT 1-10) transport Zn from the cytoplasm. Zn transporter expression is regulated by
cytokines, hormones and Zn itself, among others (Kelleher 2011).

Zinc and diabetes

As mentioned earlier, type-2 diabetes is a complex entity presenting insulin resistance along with decreased
insulin secretion capacity (American Diabetes Association 2015). Zn participates in a number of processes
related to both conditions. Zn is essential for the correct processing, storage and secretion of insulin (Emdin
1980). Zn is co-secreted along with insulin. Zn actions in pancreas are not limited to the β-cell, this element
regulates α-cell response to hypoglycemia (Zhou 2007). Studies in animal models have shown that Zn was
able to reduce both fasting glucose and insulin and increase pancreatic Zn in db/db mice (Simon 2001),
impaired oxidative changes in the retina of diabetic rats (Moustafa 2004), and that Zn supplementation in rats
before treated with the pancreatic toxic agents alloxan or dithiozone, prevented hyperglycemia and islets
destruction (Jansen 2009).

The mechanisms explaining the effects of Zn on diabetes are only partially known. At the pancreatic β-cell
level, the most relevant finding has been the identification of the role of the Zn transporter ZnT8 by Chimienti
(2004). ZnT8 was initially described as pancreatic β-cell specific. Subsequent studies showed it can also be
expressed in subcutaneous fat tissue, pancreatic α-cells and peripheral blood mononuclear cells (Foster
2011). Overexpression of ZnT8 in cultured cells is associated to increased intracellular Zn (Wijesekara
2009). On the other hand, deletion of ZnT8 caused dramatic defects in insulin processing and secretion at
the β-cell (Wijesekara 2010). A single nucleotide polymorphism in the ZnT8 encoding gene has shown to
increase risk of type-2 diabetes (Sladek 2007, Maruthur 2014).

Inflammatory cytokines play a major role in β-cell destruction in both type-1 and type-2 diabetes;
apparently such effects are mediated by the activation of NF-kB. Zn has relevant effects on cytokine
synthesis and activity. In her review, Jansen concludes that Zn may have protective effect in diabetics by
suppressing IL-1β release and suppression of NF-kB activation. In type-2 diabetes increased apoptosis
seems to be a crucial process determining disease’s course. Zinc has shown anti-apoptotic effect in a
number of cells and tissues (Jansen 2009).

Type-2 diabetes is associated with increased oxidative stress (Pan 2010). Despite Zn cannot undergo to
direct redox reactions it shows several antioxidant functions. Zn is able to induce metallothionein (MT) and
glutathione synthesis, which play protecting roles against reactive oxygen species (ROS) effects.
Overexpression of metallothionein, and SOD demonstrated to be β-cell cytoprotective (Chen 2001).
Overexpression of MT has also been related as a protective agent against cardiomyopathy, one of many
diabetes complications. In addition to the roles described above, Zn is able to modulate protein-protein
interactions of redox-sensitive proteins that are part of signaling processes.

Some authors have regarded a crucial role of Zn in insulin-sensitive tissues acting as a signaling ion itself
(see Figure 4 in ANNEX FIGURES). Thus, it has been suggested its involvement in the activation of insulin
receptor by increasing its β subunit phosphorylation, and activation of several key components of insulin
pathways, which include the extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphoinositide 3’-kinase
(PI3K)/Akt signaling pathways (Jansen 2009, Vardatsikos 2013). The zinc transporter Zip7 has been
implicated as key facilitator of intracellular zinc availability in skeletal muscle cells contributing to glycemic

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control (Myers 2013). Zip14 is highly relevant for Zn trafficking at the liver and its alteration is associated to
glucose homeostasis defects (Beker Aydemir 2012).

Our group is carrying out the final year of the Fondecyt 1120323 project on zinc and progression of
diabetes. This project evaluated the effects of additional zinc on cultured β-pancreatic cells, and
myoblasts, as well as a long term zinc supplementation in humans. β-pancreatic cells showed a critical
dependence of selected interleukine presence in the medium modulating ZnT8 and ZnT1 expression and, in
turn, insulin secretion. Myoblasts subjected to increased amount of Zn in the medium, presented increased
expression of IRS1, IRS2 and GLUT4 genes, increased anti-apoptotic genes expression and reduced pro-
apoptotic genes expression (see Figure 5 in ANNEX FIGURES). Thus, despite conditions for better
glucose utilization, total uptake was not increased under these conditions. The question whether exercise
could contribute to overcome such situation becomes highly relevant.

Zinc supplementation and diabetes in human studies.

Despite the literature show a number of processes in which Zn may have a beneficial effect on diabetes
treatment (discussed above), and the report of promising results in animal models, well-designed
randomized Zn supplementation trials carried out in humans are limited. Nevertheless, two meta-analyses
considering the information from 12-14 studies have been made available (Jayawardena 2012, Capdor
2013). Both studies conclude that Zn supplementation (mostly in physiological amounts) may have
beneficial effects on glycemic control, as indicated by a modest but significant reduction of fasting glucose
and a trend toward decreased glycated hemoglobin. It is worth mentioning that among the studies
considered, the vast majority lasted for few months or less. Also, a number of them included other
micronutrients as co-supplements, making it difficult to identify the exact cause of the effects observed. In
terms of diabetes prevention however, information is even more limited and supplementation may not
have such beneficial effects as those observed after the pathology has been diagnosed (El Dib 2015).

With respect to observations in our ongoing Fondecyt 1120323 project on zinc and progression of diabetes,
a double-blind zinc supplementation study was implemented. Despite code has not yet been broken, partial
analysis of data after 1 year of treatment show that the group with the greatest Zn status improvement
presents a trend towards increased acute insulin secretion (AIRg), with modest changes in insulin
resistance. These are just preliminary observations and final conclusion cannot be drawn until the follow up
is complete in all subjects. Nevertheless, how exercise could modify such observations becomes highly
relevant and that is the focus of the current proposal.

The interaction between zinc supplementation and exercise in diabetes has not been appropriately tested in
randomly controlled studies. There are observations however in experimental models that deserve further
exploration. For instance, there is evidence about the relationship with Zinc supplementation and the
activation and proliferation of myogenic cells in experimental animals (Gao 2014;) and cultured myoblasts
(Ohashi 2015) mediated by increased activation of Akt-mediated signal pathways. We believe that
supplementing with Zinc to patients meanwhile they are doing a muscle-strength training program, would
improve the adaptation capacity of the muscle tissue (Jessen 2014, Jinno 2014) resulting on a better
glycemic control on type-2 diabetic patients.

RESEARCH GAPS AND PROPOSED RESEARCH

Therapeutic approaches to diabetes need improvements. While there are drugs directed to improve insulin
resistance and/or insulin secretion enhancers, clearly this is not enough to an appropriate management of this
chronic condition. There is evidence demonstrating beneficial effects of exercise on glycemic control mediated
mainly –but not exclusively- by its participation in AMPK activation, which in turn increases GLUT4
translocation leading to greater glucose uptake by peripheral tissues (discussed in more detail in preceding
section). On the other hand, there is information showing some positive effects of supplemental Zn on
diabetes management. Zn presents roles in insulin packing and secretion in the pancreas, insulin-mimetic
actions in peripheral tissues by participating in insulin signaling pathways, and non insulin-mimetic actions
related to gene expression, protein synthesis, antioxidant functions and inflammation -all of them potentially
beneficial in glucose uptake and utilization (discussed in more detail in preceding section). Since it is crucial
to optimize the role of major co-adjuvants, bringing up the question: do exercise and Zn

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supplementation interact in a synergistic way? becomes matter of interest.

By analyzing potential points of interaction between supplemental Zn and a specific type of training (muscle-
strength), it is possible to identify at least:
a) Downstream signal pathway mediated by AMPK (as stimulated by exercise) also has effects on Akt, a
key component of insulin signal pathway where Zn has shown to have an effect increasing its activation.
This in turn, mediates activation of other signal pathways such as FOXO1, mTOR, and GSK3β, related to
glucose transport, glycogen synthesis, and protein synthesis among others.
b) Increased adequate phosphorylation of insulin receptor (as stimulated by Zn) not only leads to the
classical Akt activation and posterior GLUT 4 translocation, but also to stimulated Ras signal pathway
involved in cellular growth
c) Contribution of Zn to increased muscle mass, which would translate into a greater surface of peripheral
tissue to utilize glucose helping to glycemic control. This will be mediated by increased IGF-1
concentration and/or enhanced IGF-1 function, and in turn, to a greater protein synthesis associated to
increased Zn.
d) The main body of information on the beneficial effect of physical training is on glucose utilization. Thus,
by having muscle cells with less inflammation and a less apoptotic condition as those resulting from Zn
supplementation, glucose management may be more efficient.
e) Increased early insulin secretion after exercise has been recently reported. By having β-pancreatic cells
with less inflammation and a less apoptotic condition as those resulting from Zn supplementation, it may
enhance insulin secretion.

It is worthy to mention that after searching Pubmed website using the terms “Zn” and “exercise” and
“diabetes”, a total of 40 articles were found reporting fragmentary information on such topic, but none of
them evaluating the magnitude of the combined effect of exercise and Zn supplementation on
insulin sensitivity, insulin secretion or glycemic control in type-2 diabetes patients.

The current proposal expands, deepen and complement observations obtained in our ongoing Fondecyt project
1120323 (now in its final year), which is a double blind study directed to evaluate Zn supplementation on
insulin secretion and activity in mild forms of type-2 diabetes. That study had as a main focus the
progression of diabetes but particularly on preservation of pancreatic β-cell function (partial results
mentioned earlier). In the current proposal, the main research question is whether a combined
effect of muscle-strength training and supplemental Zn produces a greater effect on insulin
sensitivity than the simple addition of each component. Besides, in the current proposal -compared
with our 1120323 project- subjects with more severe forms (although still non insulin dependent) of
diabetes are included; in addition, we have increased Zn dose trying to optimize effects.

In this study we propose to use the modified frequently sampled intravenous glucose tolerance test
(FSIVGTT), which enables us to calculate first-phase insulin secretion or acute insulin response (AIRg),
insulin sensitivity (Si), glucose effectiveness (Sg), fractional metabolic clearance rate of insulin (kI) and
disposition index (DI). We have successfully implemented this methodology in our ongoing Fondecyt
1120323 project. In addition, a complete battery of metabolic control tests that include HbA1c, fasting
glucose, lipid profile, oxidative stress and inflammation parameters, will be evaluated. Also, in addition to
plasma Zn as an index of Zn status, we will include the determination of the size of the rapidly
exchangeable for this purpose, a more sensitive of Zn status. Our group has been pioneer in Latin America
implementing this methodology. The study also contemplates to assess gene expression of Zn
transporters, and key modulators of epigenetic mechanisms such as selected miRNAs and sirtuins in
peripheral blood mononuclear cells. In terms of the physical exercise component, a specially designed
training program consisting of a muscle-strength training program to be applied on individual basis with
workloads regulated session by session in order to reach the maximal metabolic impact with minimal
muscle skeletal damage risk and maximizing adherence of the patients.

In order to understand the underlying mechanisms responsible for the changes induced by the treatments
it is proposed to develop a murine model with similar components as the human model (muscle-strength
training, supplemental zinc, and a combination of them). This will allow us to perform biochemical and
molecular-related determinations in selected tissues such as: pancreas, adipose tissue, muscle, and liver.

Feasibility
In terms of feasibility of the Zn supplementation study in human, the rate of diabetic patients is 9.4% of adult

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population, a rather large figure, which would facilitate enrollment. Besides, the group from the Department
of Nutrition, Faculty of Medicine has a significant record of studies related to nutritional aspects of Zn in
morbid obesity (Ruz et al, Fondecyt 1040765, 1080576) and Diabetes (Ruz et al, Fondecyt 1120323). They
have previous experience not only in clinical control of diabetic patients but also with hands-on experience in
controlled clinical research. To carry out the biochemical and molecular components, are taking part in this
endeavor researchers with large experience in the use of cell and animal models to study the participation of
micronutrients in metabolism: in the area of nutrigenomics of diabetes (Perez et al, Fondecyt 1060790,
1100075, 1130240), obesity (García et al Fondecyt 11110219) and on mineral metabolism from the
micronutrient unit from INTA University of Chile (Arredondo et al, Fondecyt 1085173). To cover the physical
activity component a specialist from the Department of Physical Therapy of the Faculty of Medicine with wide
experience in research and clinical aspects of this area is also part of the research team.

Relevance of results
Successful testing the use of a supplementation with Zn plus an appropriate muscle-strength training protocol
resulting in an optimized intervention strategy reaching improved effects would be a major contribution to
diabetes therapy. By conducting the present study as planned, not only major clinical effects will be
documented, but new and more solid information leading to a better understanding of the mechanisms
involved will be available.

HIPOTHESES

Zinc supplementation plus muscle-strength training in type-2 diabetic individuals will decrease insulin
resistance –and consequently will improve clinical and metabolic condition of diabetes- and enhance
glucose-stimulated insulin secretion, when compared with type-2 diabetic subjects treated with zinc alone,
or muscle-strength training alone, or those receiving the regular diabetes-control advice alone.

A combined treatment with supplemental zinc and muscle-strength training in type-2 diabetic animals,
compared with each of these components by separate will induce: a greater glucose uptake and utilization
in muscle and adipose tissue, less apoptosis rate in pancreas, liver, muscle and adipose tissues, improved
insulin secretion by pancreas, an anti-inflammatory circulation profile of interleukins, and a less oxidative
stress condition.

Comments on hypotheses: We expect to demonstrate that a combination of a muscle-strength protocol

plus supplemental zinc will produce synergistic beneficial effects, more than a simple additional effect of
each component. Among the mechanisms explaining such effect (some determined in the human model
and others in a rat model) we postulate that individuals with zinc supplementation plus physical training
will present: enhanced resistance to inflammatory agents, less oxidative stress, up-regulation of tissue
anti-apoptotic genes, down-regulation of pro-apoptotic genes, increased glucose utilization by peripheral
tissues, and a more favorable epigenetic-related condition leading to up-regulation of insulin–signaling
activating genes.

OBJECTIVES

General Objective

To study the effects and underlying mechanisms of a supplementation with zinc plus muscle-strength
physical training compared to zinc supplementation alone, muscle-strength physical training alone and the
regular diabetes-control advise alone (placebo) on insulin resistance, insulin secretion and clinical and
metabolic parameters of control in individuals with type-2 diabetes.

Specific Aims:

Objectives 1-4 are carried out in a human model. Type-2 diabetic individuals will be treated during 24
weeks with supplemental Zn plus a muscle-strength physical training protocol, supplemental Zn alone, a
muscle-strength physical training protocol alone, and the regular diabetes-control advise alone (placebo).
In these we propose:

1.- To evaluate the effects on parameters related to insulin sensitivity and insulin secretion.

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2.- To evaluate the effects on a series of parameters related to the metabolic control (fasting glycated
hemoglobin, plasma glucose and lipid profile), and on muscle mass size and muscle functionality.

3.- To assess the effects on circulating IGF-1, oxidative stress parameters (plasma F2-isoprostanes,
glutathione and TBARS), inflammation markers (plasma high sensitive c-reactive protein, adiponectin, IL-6,
TNF-α), and Zn status indices (EZP, plasma Zn).

4.- To evaluate the effects on gene expression of selected Zn transporters (ZnT1, ZnT8, ZIP4, ZIP7, and
ZIP14), miRNAs (miRNA 142-3p, miRNA 126a) and Sirts (Sirt 1, 2, 4, 7) in peripheral blood mononuclear
cells (PBMNC).

Specific objectives 5-9 are carried out in a rat model. Type-2 diabetic animals will be treated –combined or
separately- with supplemental zinc and a muscle-strength physical training protocol. In these we propose:

5.- To evaluate the effects on whole body insulin resistance and parameters related to metabolic control
(fasting glycated hemoglobin, plasma glucose, lipid profile).

6.- To evaluate in muscle and adipose tissues the effects on expression of pro-apoptotic (Bax) and anti-
apoptotic (Bcl2), Zn transporters (ZIP4, and ZIP7), miRNAs (miRNA 142-3p, miRNA 126a), and Sirts (Sirt
1, 2, 4, 7) genes.

7- To evaluate in muscle and adipose tissues the effects on selected components of insulin signaling
pathway (ERK1/2 and Akt phosphorylation, GLUT4 translocation)

8.- To evaluate in liver tissue the effects on expression of pro-apoptotic (Bax) and anti-apoptotic (Bcl2), Zn
transporters (ZIP4, and ZIP14), miRNAs (miRNA 142-3p, miRNA 126), and Sirts (Sirt 1, 2, 4, 7) genes,
phosphorylation of GSK-3β and glycogen synthase activity.

9.- To evaluate in pancreatic tissue the effects on expression of pro-apoptotic (Bax) and anti-apoptotic
(Bcl2), Zn transporters (ZnT8, ZIP4), miRNAs (miRNA 142-3p, miRNA 126a, miRNA-92a), and Sirts (Sirt 1,
2, 4, 7) genes.

METHODOLOGY:

I) RANDOMIZED ZINC SUPPLEMENTATION-MUSCLE-STRENGTH TRAINING TRIAL: HUMAN STUDY

SUBJECTS: Type-2 diabetic patients will be contacted at primary health units and through press
advertising. Potential volunteers fulfilling the inclusion criteria and not presenting any of the exclusion
criteria will be invited to participate. The patients will be informed of the purposes, benefits and risks of
the study. The enrolled patients will be treated according to the standard criteria as suggested by the
American Diabetes Association (2015), and they will be controlled during the entire study at the
Department of Nutrition, Faculty of Medicine, University of Chile.
Inclusion criteria: Men and women with type-2 diabetes. Thirty-five to 60 years old, sedentary (defined
as: less than 3 sessions per week of 30 minutes of physical activity and / or sport as defined by the
Encuesta Nacional de Hábitos de Actividad Física y Deportes 2012 (Instituto Nacional de Deportes 2012),
BMI 20-40 kg/m2, stable body weight (weight variation <5%) for at least 3 months prior to screening,
glycated hemoglobin (HbA1c) 6.5-9.9% and/or fasting glycemia <180 mg/dL.
Exclusion criteria Insulin therapy. History of ketoacidosis or hyperosmolar hyperglycemic non-ketotic
syndrome in the previous 6 months. Estimated glomerular filtration rate <60 mL/min by the MDRD
equation (Levey 2006). Alanine aminotransferase or aspartate aminotransferase >2.5 times the upper
normal limit. Congestive heart failure (grade III-IV according to the New York Heart Association criteria,
1994). Uncontrolled hypertension. Diabetic polyneuropathy, peripheral macrovascular pathology or
diabetic foot. Proliferative diabetic retinopathy or severe nonproliferative diabetic retinopathy. History of
stroke, transient ischemic attack or acute myocardial infarction (previous 5 years). Recent surgery or
acute infection (previous 3 months). Major psychiatric disorder affecting compliance. Use of antipsychotic
medications. Systemic use of glucocorticoid steroids within previous 6 weeks. Alcohol intake >2
drinks/day. Cancer diagnosis or treatment in the past 5 years, with the exception of cancers that have
been cured, and carry a good prognosis. HIV positivity. Pregnant or lactating women. Having taken

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vitamin-mineral supplements during the previous 6 months.

SAMPLE SIZE: We used the relative change in insulin sensitivity indices (ISI) after intervention with zinc
or exercise as the critical variable to calculate sample size. Power calculation was based on studies in type-
2 diabetic (Meex 2010) and obese subjects (Kim 2012; Marreiro 2006), showing an increase of ∼60% and
∼20% in insulin sensitivity indices, after 8-12 weeks of exercise and zinc supplementation, respectively.
Our primary endpoint is the effect of the combination of supplemental zinc plus exercise, which we
estimated would reach 100% increase in ISI. Thus, twenty subjects per group will allow us to detect a
40% of difference in the change in ISI between exercise plus zinc supplementation vs. exercise group, and
also between exercise vs. zinc supplementation group, after intervention (α=0.05 and β=0.20).
Considering a drop-out rate by 20%, 25 individuals will be enrolled in each group.
All subjects will sign an informed consent form before any procedure takes place. The study protocols will
fully comply with the directions of the Human Research Ethics Committee of the Faculty of Medicine,
University of Chile.

EXPERIMENTAL DESIGN: Participants will be matched by BMI, age, sex and duration of diabetes, then,
allocated into one of the four experimental groups using a randomized approach. A researcher unrelated to
the study will carry out the randomization procedure and assignment to each group. All patients will
undergo routine medical controls by physicians of the Department of Nutrition every 3 months.

Experimental groups: (see Figure 6 in ANNEX FIGURES)

Control Group (CG): The individuals in this group will be treated according to the standard criteria as
suggested by the American Diabetes Association (American Diabetes Association 2015). In addition, they
will receive one capsule/d of similar appearance to the Zn group but containing a placebo.
Zn Group (ZnG): Subjects will be treated according to the standard criteria as suggested by the
American Diabetes Association plus 40 mg/day of elemental zinc contained in one capsule, which is ∼4-5
times the recommended intakes, but unlikely to produce any risk of excessive intake (FNB-IOM 2001).
Muscle-Strength Training group (MSTG): Subjects will be treated according to the standard criteria as
suggested by the American Diabetes Association plus a muscle-strength training plan consisting in: 3 times
a week during 12 weeks of an individually -and at home- supervised strength training approach using body
weight with moderate loads and enough repetitions to cause large muscle exhaustion. This is followed by 12
weeks with a similar approach but with one session per week. This is accompanied by 1 placebo capsule/d.
Zinc plus Muscle-Strength Training group (ZnMSTG): Subjects will be treated according to the
standard criteria as suggested by the American Diabetes Association plus the Training plan as describe
above plus 40 mg/day of elemental zinc contained in one capsule.

Compliance: The patients will receive a container with 30 capsules (Zn or placebo) to be taken once a day.
This container will be replaced monthly. Personnel from the Department of Nutrition will visit their homes
once a month to check the number of capsules consumed and replace the container.

EVALUATIONS: All patients will be evaluated on 3 occasions: before and 12, and 24 weeks after receiving
their first container with the treatment (Zn or placebo).
Clinical, anthropometric and dietary assessment, including medical control of diabetes (blood
pressure, skin examination and comprehensive foot examination carried out by a Diabetes-Nutrition
specialist physician; anthropometric measurements (weight, height and waist circumference by using
standardized methods (Gibson 2005); body composition by DXA (Wang 2010) in a Lunar Prodigy Advance
equipment (only at baseline and after 24 weeks) and food and nutrient intakes by food history and three-
day record questionnaires (Gibson 2005).

Physical activity and physical fitness assessment: Physical activity will be evaluated using 3 axis
accelerometers Actigraph GT3X (Actigraph, Pensacola, USA) recording during 7 days (Vanhelst et al, 2012)
at times 0, 12 and 24 weeks. Muscle mass functionality will be determined using the 30 second sit to
stand up test (Jones et al, 1999) and measurement of hand grip strength (Cetinus et al, 2005).

Metabolic status under overnight fasting conditions: A catheter connected to a three-way stopcock
will be installed in the forearm. Thirty mL of blood will be drawn for determination of plasma HbA1c,
glucose, hemoglobin, and lipid profile according to clinical lab routine procedures. Additional assessment
includes determination of plasma inflammation and oxidative stress markers. Peripheral blood mononuclear
cells (PBMNC) will be also isolated for later Zn-related gene expression analysis (see details below).

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Measurement of insulin secretion and insulin sensitivity: These parameters will be assessed by a
modified frequently sampled intravenous glucose tolerance test (FSIVGTT). Subjects will be admitted after
12 hour overnight fast. Blood samples will be drawn at −15 and −5 min. At time 0, a glucose bolus (0.3
g/kg body weight as 50% glucose in saline solution) will be administered over 2 min. Blood samples will be
drawn at 2, 3, 4, 5, 6, 8, 10, 12, 14, 19, 22, 24, 27, 30, 40, 50, 70, 90, 120, 150, and 180 min after the
bolus for the measurement of glucose (Dialab Gmbh, Neudorf, Austria), insulin by RIA (DPC Diagnostic
Corporation, Bergenfield, NJ USA). Insulin (0.05 U/kg body weight) will be infused over 5 min beginning 20
min after the glucose bolus. The data will be analyzed using MINMOD program to insulin sensitivity (Si),
acute insulin response to glucose (AINg), glucose effectiveness (Sg), fractional metabolic clearance rate of
insulin (kI) and disposition index (DI) (Muniyappa 2008).

Zn status assessment and IGF-1: After the last blood sample of the FSIVGTT has been drawn, 0.5 mg
of the stable isotope 70Zn in about 1 mL of sterile saline solution will be infused to determine the size of the
rapidly exchangeable zinc pool (EZP). Spot urine samples will be collected from day 3 to 8 after infusion
and 70Zn abundance will be determined by ICP-MS. Plasma Zn content will be determined by atomic
absorption spectrophotometry; plasma IGF-1 will be determined by ELISA (Enzo Life Sciences Inc,
Farmingdale, New York, USA).

Inflammation markers: Plasma high-sensitive c-reactive protein, adiponectin, TNFα, and IL-6, will be
determined by ELISA (R&D Systems, Inc. Minneapolis, MN, USA).

Oxidative stress markers: plasma F2- isoprostane (8-epi-prostaglandin F2α) by EIA (Cayman Chem Co,
Ann Arbor, MI, USA), plasma TBARS (Cell Biolab Inc, San Diego, CA, USA), and red blood cell glutathione
using the kit by Cayman Chem Co (Ann Arbor, MI, USA).

Peripheral blood mononuclear cells (PBMNC) isolation and gene expression analysis: PBMNC will
be isolated from blood by Ficoll-Hystopaque gradient. An aliquot will be removed for protein content (using
BCA reagent in a Shimadzu spectrophotometer at 600 nm) and Zn concentration analysis by atomic
absorption spectrophotometry with graphite furnace (Simaa 6100, Perkin Elmer) analysis. Another aliquot
will be used for RNA extraction to carry out further gene expression analyses. Total RNA will be isolated
from PBMNC using Trizol reagent and stored at -80º C. Ten µL of RNA will be used for cDNA synthesis.
Gene expression will be quantified with RT-PCR, using a Billiant II Syber Green qRT-PCR kit, in a Light
cycler equipment. MicroRNAs proposed in this study will be determined by means of TaqMan probes and
normalized using RNU48 a small RNA as an internal control.

STATISTICAL ANALYSES: Variables will be tested for normality according to the Shapiro-Wilks test. If
normally distributed, two-factor repeated measures ANOVA will be used to assess main effects (treatment and
time) as well as interactions, otherwise, non-parametric tests will be conducted. Associations among variables
will be performed through simple and multiple regression analyses. Statistical analyses for gene expression
pattern (see next section) will be carried out using the Kruskal-Wallis and Friedman tests. A probability of
an α error <0.05 will be considered as significant.

II) RANDOMIZED ZINC SUPPLEMENTATION-MUSCLE-STRENGTH TRAINING TRIAL: RAT MODEL

STUDY.
Adult two-month old Wistar rats from the Department of Nutrition’s bioterio will be used. Type-2 diabetes
will be induced according to the procedures suggested by Skovso (2014) and Holmes (2015) by feeding the
animals during two months with a high fat diet as described by García-Díaz (2009) followed by the slow
injection (during 10 min) of a very small amount of streptozotocin (10 mg/kg body wt) intraperitoneally. In
our lab we have successfully implemented the referred cafeteria diet producing insulin resistance in a two-
month period. The rats will be were kept during 12 weeks in individual cages with the experimental diet
and water ad libitum in a temperature (22±2.5 °C) and light-controlled (12:12 h light–dark cycle)
environment. The study protocols will fully comply with the directions of the Animal Care Ethical Committee
of the Faculty of Medicine, University of Chile.

EXPERIMENTAL DESIGN: Rats will be randomly allocated into four experimental groups, and a series of
determinations will be carried out at baseline and during the 12-week experimental period
Sample size: Since biological variability in laboratory animals is less than human subjects, it customary

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the use of 5-9 subjects/group to detect differences in variables similar to those evaluated here (Wang
2015, Talebi-Garakani 2013, Luo 2013), we decided to use 10 animals/group.

Experimental groups: (see Figure 7 in ANNEX FIGURES)

Control Group (CG): The rats will continue receiving the cafeteria diet as describe earlier.
Zn Group (ZnG): The rats will receive the cafeteria diet supplemented with 175 mg of Zn/kg diet. This
represents 5 times the recommended Zn content of control diet as suggested by the American Institute of
Nutriton.
Muscle-Strength Training group (MSTG): Muscle-strength training was adapted from Talebi-Garakani
2013 and Luo 2013. Briefly, it consists of climbing a vertical ladder (1.1 m, 2-cm grid, 80 ° incline) with a
load secured to their back by a backpack. The initial weight attached will be 10% of
their body weight and gradually will be increased in the weeks of training period. Ten
percent of body weight will be added to the prior weight every week. Rats will be trained
3 days/week, 10 climbing repetitions each day during a total period of 12 weeks. Rats
will receive the cafeteria diet during this period.
Zinc plus Muscle- Strength Training group (ZnMSTG): Rats will receive the cafeteria
diet supplemented with 175 mg of Zn/kg diet and will be subjected to the muscle-
strength training protocol as describe above.

Determinations:
Body weight and diet intake will be assessed weekly throughout the experimental period. At baseline, 8
animals will be sacrificed and a series of determinations in blood and tissues will be carried out. Same
procedures and determinations will be performed after 12 weeks:
Intraperitoneal glucose tolerance test: After 6h of fasting, a fasting glucose level will be is obtained from
venous blood from a small tail clip. 1 mg/g body glucose will be injected intraperitoneally and blood
glucose and insulin concentration are obtained at 5, 15 30, 60, and 120 min. Plasma HbA1c, glucose,
hemoglobin, and lipid profile will be determined according to lab routine procedures.

Tissues: Leg muscles (gastrocnemius, soleus, and extensor digitorum long); adipose tissue (epididymal,
subcutanoeus, retroperitoneal, and mesenteric), liver and pancreas will be obtained, weighed and
homogenized. An aliquout of gastronecmius muscle, epididymal adipose tissue, liver, and pancreas will be
analyzed for zinc content by atomic absorption spectrophotometry with graphite furnace, another aliquot
will be analyzed by Western blot to evaluate specific components of cellular signaling pathways
(phosphorylated proteins) as those identified in specific objectives 7 and 8, GLUT4 translocation will be
measured according to Li et al (2001); another aliquot will be transferred to TRIZOL for RNA extraction,
later cDNA will be synthesized and gene expression of Zn transporters, Sirts and apoptosis-related genes
(as identified in objectives 6, 8 and 9) will be quantified with RT-PCR, using a Billiant II Syber Green qRT-
PCR kit, in a Light cycler equipment. MicroRNAs proposed in this study (identified in objectives 6, 8, 9) will
be determined by means of TaqMan probes and normalized using RNU48 a small RNA as an internal control.

WORK PLAN:

As explained in the preceding section the human study considers individualized 24-week training at home
of each participant, which limits the number of simultaneous individuals under control. Thus, we plan to
perform 4 consecutive studies (one each year) to complete total sample: 20% of cases in year 1, 30% in
year 2, 30% in year 3 and 20% in year 4. Similarly, availability of cages
and manipulation facilities limit the number of animals potentially able to
be studied simultaneously, therefore, the animal protocol will be carried
out in two consecutive studies (50% cases in year 1 and 50% cases in
year 2). Details are provided in a Gantt chart.

See full size Gantt chart in ANNEX FIGURES

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