Type 2 Diabetes Mellitus and Zinc Dysregulation: From Basic and Epidemiological Evidence to Interventions

Abstract

Background:

Complex cellular and systemic changes in zinc (Zn) levels have been reported through different stages of Type 2 Diabetes (T2DM) onset and progression.

Methods:

We summarize available evidence on Zn and T2DM, including mechanistic and epidemiological/clinical studies with a focus on Zn pathophysiology, interpretation of Zn biomarkers, and associations of Zn status and T2DM across different populations.

Results:

Misdistribution of Zn in insulin-producing ß-cells are likely key contributors to ß-cell failure in T2DM, with genetic variants in ZnT8 transporters playing an important role. Epidemiological evidence has documented increased urinary Zn and decreased plasma and blood Zn in persons with established T2DM. Changes in Zn biomarkers have been prospectively associated with increased risk of T2DM in pre-diabetes and healthy adults before hyperglycemia occurs. Some studies suggest that Zn supplementation could modify glycemic endpoints in T2DM participants, but evidence is insufficient in healthy adults. Zn biomarkers, including isotopes, can provide novel approaches for T2DM risk assessment and management at different disease stages.

Conclusions:

Dysregulation of Zn metabolism occurs early in T2DM development with ß-cell failure playing a central role. Additional research is needed to connect mechanistic evidence and pathophysiological changes associated with various stages of T2DM progression.

Graphical Abstract

Introduction

Essential for the function of around 3,000 proteins in all mammalian cells [1–3], zinc (Zn) is one of the most abundant metals in mammalian cells and metalloproteins.[4] Zinc is a divalent metal that determines the catalytic and structural role of proteins and regulates the expression and distribution of zinc transporters.[5,6] Despite its importance for various cell functions, excess free Zn exerts toxic effects. [7–9] Therefore, most of intracellular Zn is bound to Zn dependent and Zn buffering proteins [8–11]. Mammalian cells have evolved intricate regulatory mechanisms of Zn concentrations within a narrow physiologic range in each cellular compartment.[1,5,12,13].

The relationship between Zn and type 2 diabetes mellitus (T2DM) endpoints has been studied for decades. Yet, multiple questions remain unanswered. Genetic and lifestyle determinants of Zn status and distribution, molecular mechanisms of Zn dysregulation at the tissue level, genetic variants of Zn transporters, and the temporality of the impact of Zn dysregulation on T2DM development are not fully understood. The use of Zn biomarkers in population-based studies to assess T2DM risk is a growing research area, including the clinical implications of different Zn biomarkers for the assessment and management of T2DM.

In this narrative review, we summarize existing literature assessing biomarkers of Zn status, their relationship with T2DM and related-outcomes in humans. We also describe published evidence on pathophysiologic mechanisms with a special focus on the role of Zn in insulin secreting beta(ß)-cells and on clinical interventions of Zn supplementation in healthy adults and in persons with T2DM through the following sections: A) Determinants of zinc status, B) Pathophysiological aspects of Zn status in T2DM, C) Epidemiological evidence on the relationship between Zinc status and T2DM, and D) Interventional Zn supplementation studies. The goal is to summarize mechanistic and epidemiological evidence connecting Zn status and metabolism to T2DM risk assessment, pathophysiology, and clinical management, and to identify evidence strengths, research needs and knowledge gaps to develop Zn-related interventions for T2DM prevention and treatment.

A. Determinants of zinc status.

This section examines the primary determinants of Zn status in humans including sources of dietary Zn intake and environmental exposure, and the assessment of Zn status through biomarkers including blood, serum, urine, and novel assessment methods such as Zn isotopes.

A.1. Sources of zinc

The human body contains about 1.5 g of Zn in women and 2.5 g in men, primarily stored in the muscles, bones, liver and pancreas.[14] Zn is tightly regulated in the body and there is need for a regular dietary supply. The Recommended Dietary Allowance (RDA) of Zn in the United States (US) ranges from 11 mg to 8 mg in adult men and women, respectively, and increases during pregnancy and 84.[15] Interestingly, dietary Zn intake recommendations vary across countries, as a result of different assessment methods and heterogeneity of approaches used by expert panels.[16–19] Zn is naturally present in animal-based foods such as meat, fish and shellfish. Plant-based foods like nuts, seeds, rice and legumes contain Zn, but its absorption is lower due to phytates, which lowers body absorption of Zn. [14,15] Dietary supplements are another key source of Zn. [20] When Zn intake is reduced, multiple homeostatic mechanisms are activated and respond by upregulating gastrointestinal absorption and reducing elimination through the feces and urine [21] Although Zn is also present in airborne particles and occupational settings, this pathway is not considered for maintaining Zn status and there is substantial evidence that airborne Zn exposure can induce respiratory and other health risks.[22] The assessment of Zn status in the body and monitoring of changes in Zn status in response to dietary intake, supplementation or depletion, remains an ongoing challenge, as there are no sensitive and specific biomarker for Zn exposure.

A.2. Zinc biomarkers

Given the challenges in quantifying zinc intake and exposure, we next explore the biomarkers used to assess zinc status in clinical and epidemiological studies and discuss their strengths and limitations.

Serum (or plasma) Zn concentrations is the most commonly used biomarker in clinical studies.[14,17,19] Some expert panels such as the Biomarkers of Nutrition for Development Zn Expert Panel have set plasma deficiency cutoffs at 66 mcg/dL for females age 10 and older, and 70 mcg/dL for males age 10 and older. These cutoffs are derived from the 2.5th percentile of Zn concentrations in fasting morning samples in NHANES, a nationally representative sample of the US population [23] Two meta-analyses in adults and children identified positive correlations between Zn supplementation and changes in serum/plasma Zn levels. [16,24,25] In children, pooled data from 18 randomized controlled trials (RCT) including N=1,722 participants aged 1 to 17 years receiving daily doses ranging from 3 to 70mg/day, showed a ~9% increase in serum/plasma Zn levels per doubling of the Zn supplement dose. [25] Similar findings have been observed in adults after Zn supplementation. A meta-analysis of ten RCT of adults receiving supplements with doses ranging from 15 to 135.5 mg/day, identified a 6% increase in serum/plasma zinc concentration for every doubling of zinc intake, based on a pooled regression coefficient (β) of 0.08 (95% CI: 0.05, 0.11) using a log-transformed dose–response model [16] While Zn supplementation induce noticeable although relative small changes in serum/plasma Zn levels, studies assessing dietary Zn intakes from natural foods found no association between dietary Zn intake and serum/plasma Zn. [16] The low correlation between Zn intake from food and serum/plasma Zn concentration could be attributed to challenges to estimate bioavailable Zn from food. Furthermore, serum/plasma Zn might not be an accurate marker for changes in Zn dietary intake from foods due to a tight regulation of Zn absorption to maintain Zn homeostasis.[26]

Importantly, serum/plasma Zn concentrations can be influenced by multiple factors other than dietary intake including fasting status, inflammation, pregnancy, and use of medications such as steroids or hormonal contraceptives, as well as genetics, among others, limiting the validity of a single serum/plasma Zn measurement to assess Zn body status. [27–29] Gene variants associated with plasma Zn levels in relation to dietary intake include SNP rs1047626 SNP in ZnT9, a Zn transporter, which can impact intracellular Zn distribution, and SNP rs11558471 in ZnT8, but their effects are not yet well understood.[30–32]

Whole blood is not a sensitive biomarker of Zn absorption or status, as concentrations in the erythrocytes remain stable over time.[33] Although blood Zn is not a sensitive biomarker of Zn status, red blood cells are rich in Zn, and previous studies have suggested that changes in the concentration of Zn in whole blood may reflect alterations in red blood cell Zn transport and body burden. [34]

Urine Zn is increasingly being assessed in epidemiological studies with the increased availability of multielement metal panels.[35–38] It is estimated that the daily excretion of Zn in the urine is >7mmol (0.5 mg).[39] Yet the interpretation, reference dosages, potential diurnal variations, and population ranges for this biomarker, as well as the relationship to serum/plasma Zn and whole blood levels, require further study. It is estimated that urine Zn accounts for ~15% of the daily Zn elimination and biliary excretion is primary Zn elimination route.[14] While the number of studies evaluating the association between dietary intake and urinary Zn is limited, a systematic review and meta-analysis of 5 studies documented increases in urinary Zn levels after Zn supplementation. [24] The association, however, was only significant after intakes larger than 15 mg Zn per day, [24] suggesting a limited sensitivity of urinary Zn as a biomarker of dietary intake within the RDA. More studies are needed to understand the potential of urinary Zn to capture dietary variability as well as other potential sources and routes of exposure. Similarly to plasma and serum, the interpretation of urinary Zn as a biomarker presents challenges. Factors such as renal function, hydration status and comorbidities, particularly diabetes as we discuss in the following sections, can impact urinary Zn levels. Increase in plasma glucose levels can increase renal Zn excretion, resulting in elevated levels of Zn in the urine in individuals with T2DM.[16,34]

Other biomarkers such as hair, nails and saliva have also been proposed as potential biomarkers for Zn status and intake with mixed results. Data from 3 studies including 93 adult participants identified significant associations between Zn supplementation and hair Zn levels.[40] However, hair Zn is influenced by external factors such as hair treatments, external environmental contamination, and variations in hair growth rates. Zn levels in toenails were not associated with dietary Zn or supplement intake, and saliva may correlate with intake, but more studies are needed. [40–42]

Zinc isotopes (δ⁶⁶/⁶⁴Zn) in biological samples can serve as tracers for metabolic changes linked to shifts in metalloprotein expression and ligand coordination. In T2DM, disruptions in Zn homeostasis—particularly in relation to the Zn transporters of the ZIP and ZnT class—may alter Zn isotope distribution across biological compartments.[43–45]

Lighter Zn isotopes have lower bond energy, leading to preferential binding of heavy Zn isotopes in stronger ligand bonds under equilibrium conditions. Cysteine (S-ligands) preferentially binds light Zn isotope (⁶⁴Zn), while histidine (N-ligands) and aspartate (O-ligands) favor isotopically heavy Zn (⁶⁶Zn).[46] Metabolic disruptions affecting Zn-binding proteins or transporters may alter Zn isotope composition in tissues, blood serum, and urine. For instance, high-precision Zn isotope analysis has revealed measurable metabolic changes of Zn for pancreatic cancer. [47]

Given the above, the assessment of Zn status remains complex, as there is no single biomarker highly sensitive to dietary intake or changes in body reserves. Different biomarkers may reflect different aspects of Zn intake, metabolism, accumulation, and elimination. For instance, serum/plasma Zn are more likely to be sensitive to recent dietary intake, urinary Zn may serve as a biomarker of intake only at high doses, and as a potential biomarker of Zn losses in the context of metabolic dysregulation as we discuss in the sections below. Yet, these require further validation.

B. Pathophysiological aspects of Zn status in Type 2 Diabetes Mellitus:

With the strengths and limitations of zinc biomarkers outlined, we will now discuss the current state of knowledge regarding how alterations in Zn homeostasis interacts with the pathophysiology of T2DM.

B.1. A primer on the Pathophysiology of T2DM

T2DM is a progressive metabolic disorder driven by two fundamental pathophysiological changes: insulin resistance and beta(ß)-cell dysfunction.

It is well established that progressive failure of insulin secreting ß-cells to meet the increased demand for insulin is ultimately the pivotal event resulting in the development of T2DM [48]. This ß-cell failure usually occurs in several stages that predate the development of clinically significant hyperglycemia. Initially, compensatory mechanisms result in increased ß-cell insulin secretion in the majority of affected individuals.[49] Over time, ongoing insulin resistance results in ß-cell failure, leading to T2DM in predisposed individuals. [48] Recent evidence also suggest that early ß-cell dysfunction preceding insulin resistance may play a key role in the development of T2DM.[50,51] Given this sequence of events during the development of T2DM, tissues most relevant to the development of T2DM are insulin responsive tissues and insulin producing ß-cells.[48,52] The main insulin responsive cell types involved in developing insulin resistance include the hepatocytes (liver), adipocytes (fat tissue), and myocytes (muscle).[48]The rate and timing of ß-cell failure and therefore, the development of T2DM can vary significantly between individuals. The degree of insulin resistance also varies among persons with T2DM, leading to subclassification of T2DM into several subtypes based on the degree of insulin resistance and serum insulin levels.[53,54] Underlying ß-cell failure to meet insulin requirements, however, is a common mechanism to all T2DM subtypes.[55] The main factors influencing inter-individual variations in the development of insulin resistance and ß-cell failure is a combination of genetic, lifestyle, and environmental risk factors and their effects on insulin responsive tissues and /or ß-cells.[56] Additionally, generalized inflammation, which arises with increased insulin resistance, can further exacerbate both insulin resistance and ß-cell failure, thereby initiating a vicious cycle that accelerates the development of T2DM. [57]

Of note, type 1 diabetes mellitus is a distinct disease process, where insulin producing ß-cells are the target of T-cell mediated autoimmune destruction, leading to absolute insulin deficiency.[58] This process is beyond the scope of this review.

B.2. Role of zinc in ß-cell physiology and pathophysiology

Building on this overview of T2DM mechanisms, we next focus on what is known the roles that zinc plays within insulin-secreting β-cells and what aspects remain to be explored.

Given the large number of Zn dependent proteins and cellular processes, including ß-cells, insulin responsive cells, and immune cells; changes in Zn status have the potential to induce diverse effects in this array of diverse tissue types relevant to T2DM risk. These changes in Zn homeostasis at a cellular level can be induced by altered supply, increased loss, or altered trafficking. As outlined below, changes in Zn levels in biomarkers such as urine and plasma, and homeostasis can occur before the development of hyperglycemia and T2DM, and also continue to evolve and change during the disease onset and progression. [34,36,37,59–62] This will likely result in tissue specific changes in Zn status at various stages in T2DM development and should be taken into account when interpreting results from studies aimed at exploring this relationship.

ß-cells in most mammals––including mice, rats, pigs, primates (including humans)––contain Zn that is seven to ten times higher compared to most other cells. [63–65] Most of ß-cell Zn is contained in insulin secretory vesicles, where it crystalizes with six insulin molecules into insulin hexameres.[66,67] When insulin is released from ß-cells, Zn present in insulin vesicles is co-secreted with insulin in its ionic form. [68]

Zn that is co-released during insulin secretion has been reported to induce various effects, including a paracrine effect on alpha-cells that secrete the insulin counterregulatory hormone glucagon, [69] as well as a modulating effect on hepatic insulin clearance [70]. Additionally, ß-cells express GPR39, a G-Protein Coupled Receptor (GPCR) that is reported to enhance ß-cell insulin secretion in response to Zn binding [71–73]. This raises the possibility of Zn exerting an autocrine effect in ß-cells.[74] In ß-cells, the Zn transporter ZnT8 is the main transporter of Zn from the cytoplasm into insulin granules. and is the main driver for achieving this exceptionally high concentration of Zn in ß-cells.[75–77] In ZnT8 null mice, the concentration of overall islet Zn is reduced by about 78% compared to ZnT8 expressing islets as shown by Pound et al. [78] Similarly, our own results showed a decrease in whole islet Zn from 29326 ± 3515 nmol/g protein in wild type mice to 3659 ± 421 nmol/g protein in ZnT8 null mice (mean ± SEM).[79]The relevance of ß-cell ZnT8 for the development of ß-cell dysfunction and T2DM is illustrated by the fact that a widely prevalent variant of the ZnT8 encoding gene SLC30A8 -the C variant at Single Nucleotide Polymorphism (SNP) rs13266634- is associated with an increased risk for T2DM.[80,81]. The C allele variant results in a tryptophan-to-arginine switch at position 325 in the protein’s intracellular carboxy-terminal domain. [65] The mechanism for the increased T2DM risk associated with SNP rs13266634 is not fully understood despite significant efforts by us and others. [65,70,79,82–84] However, experimental evidence available so far points to a misdistribution of Zn in ß-cells as a contributor to ß-cell dysfunction and T2DM risk. This evidence includes our previously published analysis of Zn levels in isolated human insulin secreting islets.[79] There, we found that the T2DM risk variant of ZnT8 at SNP rs13266634 was associated with a higher islet Zn concentration compared to the non-risk variant. [79] Similarly, downregulation of ZnT8 in cell models conferred a protective effect on primary islets or stem cell derived ß-cells. [85–87] In line with these findings, several rare loss of function mutations in ZnT8 -not related to SNP rs13266634- that result in markedly decreased ß-cell Zn levels, are associated with a significantly lower T2DM risk.[88,89], supporting the hypothesis that lower islet Zn levels likely confer a protective effect. Studies that examined the correlation of SNP rs13266634 of ZnT8 with islet function in humans showed variable results. One in vivo study showed that carriers of the risk allele C had a lower insulin response to intravenous glucose infusion - supporting the notion of disturbed islet function in carriers of the risk allele.[90] However, a meta-analysis of ex-vivo studies in isolated human islets found that the risk allele C does not affect ex-vivo insulin secretion, though there were significant methodological differences between included studies. [91] There are also additional variants of ZnT8 that were found to be associated with an increased risk for T2DM in some, but not other GWAS studies. [92,93] These include SNP rs3802177 and SNP rs11558471. Both variants are synonymous variants, mapping to the non-coding 3’UTR region of SLC30A8. [92,93]

Epidemiological evidence further supports that the association between Zn status and incident T2DM risk is modified by ZnT8 SNP rs13266634 alleles. In a population-based study of 1,796 participants, a 10 μg/dL increase in plasma Zn was associated with 22% (OR 0.78 [0.72–0.85]) lower odds of T2DM in TT genotype carriers, 17% (0.83 [0.80–0.87]) lower odds in CT genotype carriers, and 7% (0.93 [0.90–0.97]) lower odds in CC genotype carriers at SNP rs13266634 (P for interaction = 0.01).[62] Similarly, a meta-analysis on Zn intake and genetic interactions suggests that another SNP in ZnT8 - rs11558471- may modify fasting glucose responses to dietary Zn.[32] This study identified a stronger inverse association between total Zn intake and fasting glucose in individuals carrying the glucose-raising A allele at rs11558471 compared with individuals who did not carry it, suggesting the existence of a gene-nutrient interaction.[32] A more recent analysis of a clinical cohort of 3,445 participants showed a modifying effect of three additional SNPs of ZnT8, namely 8_118252314, 8_118252435, and rs16889462, on the relationship between Zn intake and T2DM risk. [94] A similar modifying effect of ZnT8 genotype at SNP rs13266634 on the relationship between urinary Zn levels and HOMA2-β (a proxy for ß-cell function).[95] Several unknowns remain in the mechanisms underlying ZnT8 related genetic variants and T2DM risk. However, combined, the results from ZnT8 related experimental and epidemiological studies provide evidence for a possible role of ß-cell Zn misdistribution and/or overload in insulin secreting ß-cells as a contributor to islet dysfunction and T2DM, which is particularly relevant in carriers of the T2DM risk allele C of ZnT8, as it has been associated with higher islet Zn levels. While more research is needed, a potential hypothesis is that the relationship between ZnT8 activity, Zn status, and T2DM risk may follow a complex nonlinear dose-response relationship. [62,65,89]

Given the high frequency of risk alleles in the SLC30A8 gene (encoding ZnT8) observed across diverse populations—especially the SNP rs13266634 C allele, for which 84.8% to 93% of the general population carry at least one risk allele C [29,79,80,90,96–98] it is plausible that this allele conferred a degree of evolutionary advantage under nutritional and/ or environmental conditions prevalent during early human evolution that no longer apply in our current environment. These conditions could include lower Zn supply, as well as scarcer and/ or more intermittent alimentation compared to contemporary conditions. However, direct evidence for this potential selective advantage that is now reversed remains limited.

B.3. Role of zinc in insulin resistance

Zinc also modulates insulin signaling and oxidative stress in peripheral tissues, influencing whole-body insulin sensitivity. Zn stimulates phosphorylation of the β-subunit of the insulin receptor, leading to activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt), key regulators of insulin signaling.[99] In addition, Zn activates the glucose transporter 4 (GLUT4) translocation, facilitating glucose uptake in insulin-sensitive tissues.[61,99,100] Zn has antioxidant properties acting as a co-factor for superoxide dismutase (SOD) and inhibiting the NADPH oxidase, which reduces the production of reactive oxygen species (ROS). [101] There is some evidence to suggest that changes in Zn status due to dietary intake, Zn supplementation, or in the setting of hyperglycemia are sufficient to alter cellular Zn levels in insulin responsive tissue to a degree that modifies insulin sensitivity. Specifically, population based studies in participants free of T2DM have shown that serum Zn levels are inversely correlated with insulin resistance. [102] In addition, lower Zn status has been linked to upregulation of inflammatory cytokines, such as TNF-α and IL-6, which can interfere with insulin receptor signaling and promote insulin resistance [99,102,103]. These associations suggest that lower Zn status may contribute to insulin resistance. However, additional experimental and clinical evidence is needed to investigate the directionality and causality of these observational associations. In this context, interventional trials are needed to examine whether Zn supplementation can reduce insulin resistance.

C. Epidemiological evidence on the relationship between Zinc status and type 2 diabetes:

C.1. Observational studies in persons with T2DM and pre-diabetes:

Following the discussion of pathophysiological mechanisms underlying the relationship between Zn status and T2DM, we now discuss changes in Zn biomarkers and its relationship with T2DM endpoints in population-based and clinical studies.

The relationship between Zn dysregulation and T2DM in humans has been studied for decades.[104–107] For example, increased levels of Zn in the urine have been consistently documented in patients with T2DM compared to healthy controls.[34,108–110] It is increasingly accepted that T2DM results in increased renal Zn loss leading to functional Zn deficiency.[37,59,60,107,111] Changes in blood Zn concentrations have been also documented in prospective studies including a meta-analysis of 11 studies that reported progressive declines in blood Zn levels over time in persons with T2DM compared to healthy participants.[34] Meta-regression analyses estimated that, for each year since the diagnosis of T2DM, the concentration of Zn in whole blood decreased by: mean difference (concentration of Zn in blood) = 732.61 + (−77.88303) × (duration of diabetes in years), and observed that the declines were independent of Zn intake. [34] Similarly, lower levels of serum Zn have been documented in persons with established T2DM compared to healthy individuals.[112–114] Even though studies assessing multiple biomarkers of Zn simultaneously are very limited, a study of 82 individuals with T2DM aged 29–59 years in the US that assessed dietary, plasma, whole blood and urinary Zn at a single timepoint identified a significant inverse correlation between plasma Zn levels and glycated hemoglobin percentage (r=−0.325, p=0.003), and a positive correlation of urinary Zn excretion with plasma glycemia (r=0.269, p=0.016), glycated hemoglobin percentage (r=0.318, p=0.004) and HOMA-IR -a calculated proxy for insulin resistance (r=0.289, p=0.009).[115]

Altogether, these findings support the impact of glucose dysregulation on the potential loss of Zn as identified by higher levels of urinary Zn, likely indicating elimination from the body, and lower levels of plasma and whole blood Zn following the development of T2DM. Interestingly, some cross-sectional studies have identified higher serum Zn levels in persons with newly diagnosed T2DM [116,117]. These findings point out at a potential serum Zn increase in early stages of T2DM that is further eliminated through the urine. Yet, studies are needed to further investigate this in a prospective manner.

Some of the proposed mechanisms potentially underlying renal Zn loss in persons with T2DM include declines in the tubular reabsorption of Zn in the kidney induced by increased fasting glucose in plasma.[118] This mechanism is supported by previous studies that showed higher Zn clearance/creatinine clearance ratio in persons with T2DM compared to controls.[119] This is further supported by a handful studies that identified increased urinary Zn excretion and lower plasma Zn in persons with chronic kidney disease (CKD).[120–122] Together, growing evidence points at a likely role of kidney function and renal Zn elimination in the context of T2DM and pre-diabetes on altering Zn homeostasis in persons with hyperglycemia. [119] In this context, the interpretation of Zn biomarkers in persons with T2DM should be different compared to healthy participants, and the potential for reverse causality should be considered when interpreting urinary Zn levels in the presence of T2DM or pre-diabetes.

A sustained loss of Zn through urine without compensatory mechanisms could lead to a deficient or marginal Zn status in persons with T2DM which could potentially contribute to development of diabetes-related complications through the dysregulation of Zn-dependent antioxidant enzymes. This could explain some of the associations found between urinary Zn levels and diabetes related complications.[105,106,119] Prospective studies have identified associations between higher urinary Zn levels and increased risk of T2DM related complications, including peripheral artery disease, and other cardiovascular disease endpoints such as heart failure and coronary calcification. [38,123–125] These studies identified associations between urinary Zn levels and cardiovascular disease risk that remained significant after accounting for T2DM status and indicators of glycemic control such as fasting plasma glucose, suggesting the potential use of urinary Zn as an independent indicator of risk for diabetes-related cardiovascular complications. Additionally, higher serum Zn levels and lower urinary Zn levels have been documented in T2DM patients with good glycemic control compared to those with poor glycemic control, further suggesting a close correlation between hyperglycemia and elimination of Zn through the urine [104,126–128]. These findings could also suggest that Zn metabolism plays a key role in diabetes-related health effects, although the findings could also reflect residual confounding or measurement error compared to other T2DM markers.

C. 2. Observational studies in healthy adults and individuals with pre-diabetes

Studies in populations free of clinical T2DM offer insight into zinc changes that may precede hyperglycemia. These will be discussed in this section.

While most Zn research has been conducted in the context of T2DM progression and management, some prospective studies have shown that altered Zn homeostasis, can occur in early stages of diabetes development.[34,37,112] Some studies suggest that these changes can occur even before fasting plasma glucose levels are altered.[36,37] These studies constitute a novel body of evidence pointing out at the potential role of urinary and other Zn biomarkers as biomarker of T2DM risk among disease-free populations. This also provides evidence for changes in Zn metabolism that precede the appearance of clinical disease.

In the Strong Heart Study, a large epidemiological cohort of American Indian adults in the US with a prevalence of T2DM, baseline urinary Zn levels were significantly associated with increased risk of T2DM over 10 years of follow up among disease free participants.[37] The identified hazard ratio (HR) of T2DM incidence comparing the 75th vs. 25th percentiles (0.63 vs. 0.34 mg/g) of urinary Zn distribution after adjustment for sex, site, education, smoking status, BMI, and eGFR. [37] In models further adjusted for HOMA-IR and fasting plasma glucose, and models excluding participants with prediabetes, the association of urinary Zn with incident diabetes risk was attenuated but remained significant, suggesting that changes in Zn metabolism are detected in early stages of T2DM pathogenesis and could even play a key role in its progression.[37] Interestingly, dietary Zn intake was not associated with incident T2DM risk, further supporting the role of changes in Zn metabolism, rather than changes in Zn intake, as the main drivers of the identified associations with T2DM risk. [37] Similarly, higher urinary Zn levels were associated with markers of early hyperglycemia in mothers and offsprings enrolled in the Hyperglycemia and Adverse Pregnancy Outcomes Follow Up Study (HAPO-FUS).[36]

The association between higher urinary Zn that either precede the development of hyperglycemia, or are found in very early stages of hyperglycemia can be hypothesized to be a result of one or more of the following processes: a) conditions such as early metabolic changes or genetic predisposition that precede hyperglycemia and T2DM, including ß-cell dysfunction, b) a primary increase in renal Zn loss that results in Zn deficiency, which in turn results in higher risk for T2DM, or c) higher urinary Zn indicates a higher Zn intake or absorption (i.e. there is a relative Zn excess).

Some observational evidence supports that elevated levels of other Zn biomarkers are associated with increased risk of T2DM with some differences among healthy vs. prediabetes adults, and cross sectional and prospective evidence. Some prospective studies have found associations between increased serum Zn levels and incident T2DM in adults with pre-diabetes (HR of incident T2DM for serum Zn > 106 μg/dL: 2.27, 95% CI = 1.01–5.10) [129], and healthy adults (HR=1.39, 95% CI 1.04–1.85)[130] Cross-sectional results from the National Health and Examination Survey (NHANES) identified associations between higher serum Zn levels and T2DM prevalence (OR when comparing the highest to the lowest quartile: 3.24 (2.33–4.52))[131]. A prospective study in the Coronary Artery Risk Development in Young Adults Study (CARDIA) cohort, showed an association between higher baseline Zn levels in nails and increased risk for T2DM among disease free participants.[132] These observations raise the possibility that changes in other Zn biomarkers such as blood, nails and hair, may be associated with increased risk for developing T2DM in healthy adults and adults with pre-diabetes similarly to previous findings in urine. For example, despite the robust evidence identifying lower serum and blood Zn in persons with established T2DM, lower serum Zn is not necessarily associated with a higher risk for developing T2DM in the future. Additional prospective studies are critically needed and ruling out temporality, the underlying pathophysiology, and causal mechanisms require further study.

D. Interventional Zn supplementation studies:

Informed by mechanistic and observational evidence, several studies have evaluated the potential of Zn supplementation as a therapeutic or preventive approach for T2DM. Below we discuss the findings from these studies in different population groups. Given the likely differences in Zn homeostasis in persons with and without dysglycemia described above, we will discuss supplementation studies in persons with and without preexisting dysglycemia separately.

D.1. Intervention studies in individuals with established T2DM

The identified relationship between dysregulation of Zn homeostasis and T2DM has motivated a series of Zn supplementation studies in persons with established T2DM. These studies aimed at correcting Zn marginal deficiency through external supplementation with the goal of improving glycemic control. [133,134] The results of these intervention studies have been analyzed in multiple meta-analyses as follows: The first meta-analysis examining this question was published in 2012 and included 12 supplementation studies conducted in persons with T2DM that used highly variable Zn doses which ranged from 10 mg Zn/day (within the RDA of 15–20 mg/day) to 660 mg Zn/day .[134] The study found lower levels of fasting blood glucose (mean difference:−18.13 mg/dl (95%CI:−33.85,−2.41)) and HbA1c (mean difference −0.54 % (95%CI:−0.86;−0.21)) comparing Zn supplementation vs. placebo participants, but the heterogeneity across studies was high (I² = 99%).[134] A more recent dose-response meta-analysis conducted in 2020 included a total of ~2,000 participants (with and without T2DM) across 27 studies. This study found lower levels of fasting blood glucose (mean difference −19.69 mg/dl (−33.64, −5.75)) and lower levels of HbA1c (mean difference −0.37 % (−0.71, −0.03)) comparing Zn supplementation vs. placebo. [135] An additional umbrella meta-analysis conducted in 2024 that analyzed a total of 10 meta analyses of Zn supplementation trials that included mostly patients at various stages of hyperglycemia (9 meta-analyses) and on persons with obesity and insulin resistance (1 meta-analysis) showed similar findings with benefits of Zn supplementation in lowering glycemia and identified participant’s age, dosage, and comorbidities as the main sources of heterogeneity.[136]

Given the evidence of a functional Zn deficiency in persons with established T2DM, it was hypothesized that Zn supplementation may reduce the risk for T2DM related complications. However, there are few studies examining this aspect of Zn supplementation in T2DM, and results are limited by small sample size. For example, a study in persons with T2DM (n=40) showed that high dose Zn supplementation with 240 mg Zn/day does not improve markers of oxidative damage, levels of hydroxyeicosatetraenoic acid products, and vascular indices during a four-month study period.[137] In another study, lipoprotein oxidation, a measure of oxidative stress, were not altered by 30 mg Zn/day of supplementation over 3 weeks. [138,139] Yet, in vivo studies have documented an impact of Zn supplementation on reduced oxidative stress, cell apoptosis and autophagy and inflammation in tissues such as the heart and the kidney [140], warranting the need for more epidemiological interventions to assess the potential effects of Zn supplementation on diabetes complications..

While the evidence supports that in patients with diabetes, Zn supplementation contributes to lower glycemia, clinical guidelines have not yet incorporated Zn supplementation as standard of care in patients with diabetes, potentially because no large clinical trial with hard clinical endpoints are available.

D.2. Intervention studies in individuals with pre-diabetes:

Epidemiological studies have shown that higher Zn levels in urine during pre-diabetes stages are associated with incident diabetes, supporting that restoring Zn metabolism before diabetes could be beneficial.[37] Some but limited clinical trials have investigated the impact of Zn supplementation in prediabetes. In a randomized clinical trial of 200 participants, those who received 20mg/day of Zn supplements were less likely to develop incident diabetes (11% vs. 25%, respectively) after a year of follow-up [141]. Another randomized clinical trial with 20 participants with prediabetes identified significant reductions of fasting plasma glucose levels after 6 months of Zn supplementation, compared with baseline and with a placebo group.[142] The possibility that Zn supplementation could delay or even prevent diabetes in persons with prediabetes is intriguing and deserves additional investigation.

An important, and sometimes overlooked issue in supplementation studies is the large heterogeneity in Zn doses and Zn chemical forms administered in clinical trials. Previous clinical trials have used multiple Zn chemical forms such as Zn sulfate, Zn acetate, or Zn gluconate. These chemical forms translate into different elemental Zn doses (e.g., Zn sulfate consists of 23% elemental Zn, Zn gluconate is 14.3% Zn, and Zn oxide is 80% Zn. Evaluating the effect of different doses and chemical forms on glycemic outcomes could provide further insights into the underlying biological mechanisms, and into identifying cutoffs for potential adverse effects.

D.3. Intervention studies in healthy adults

Finally, to assess prevention potential, some trials in normoglycemic adults have evaluated whether zinc supplementation can modulate early insulin resistance.

While Zn supplementation among persons with T2DM and prediabetes showed improved glycemic markers in some studies, as described above, the role of Zn supplementation for T2DM prevention on healthy individuals is less clear. A handful of previous studies have identified associations of higher dietary Zn intake with a reduced risk for T2DM. [143] Two additional meta-analyses showed a beneficial short term effect of Zn supplementation on markers of hyperglycemia among participants who were overweight or obese. However, most of this effect was observed in participants who also had preexisting prediabetes or established T2DM.[133,144] No effects of Zn supplementation study in the US on insulin markers was identified by previous supplementation study in children 9–11 years old over four weeks.[145]

Regarding dietary Zn intake, a meta-analysis of observational studies identified lower odds of T2DM when comparing the highest versus lowest Zn intakes from diet assessed through food frequency questionnaires in eight studies(OR: 0.87 (95% CI: 0.78–0.98)), but no associations were identified for overall Zn intake (diet + supplements) or Zn supplementation alone assessed in two additional studies.[146] This points out at a potential protective role of Zn-rich diets from natural sources in observational evidence. Yet, measurement error, heterogeneity and residual confounding may be present in these findings.

To our knowledge, there are no intermediate- or long-term randomized, controlled supplementation studies that have yet examined the influence of Zn supplementation or a high Zn diet on the risk of developing T2DM in persons at risk for T2DM without preexisting hyperglycemia, and evidence remains insufficient to recommend Zn supplementation in healthy persons.

Conclusions

Evidence points to complex changes in Zn metabolism occurring throughout the development of prediabetes and T2DM. At the molecular level Zn cellular transporters are altered in insulin producing ß-cells during early stages of T2DM, with some changes in ß-cell Zn levels being genetically determined. In vitro evidence shows that genotypic variations of the islet specific Zn transporter ZnT8 result in higher islet Zn levels and higher T2DM risk. It is unclear, however, whether blood, serum, or urine Zn levels influence islet Zn status and whether changes in urinary Zn excretion preceding the development of T2DM are causally linked to these changes in ß-cells or islets.

At the clinical level, the assessment of Zn status remains an ongoing challenge as there is no single established biomarker of Zn status, and different Zn biomarkers (i.e., plasma, whole blood, urine) reflect different stages of Zn metabolism and regulation.

At the epidemiological level, population-based studies have consistently reported higher levels of urinary zinc and lower levels of blood and serum Zn in persons with T2DM and pre-diabetes. Higher serum and urinary Zn levels preceding hyperglycemia have been documented in a handful of studies of healthy participants as well, suggesting both serum and urinary zinc as a potential biomarker of early T2DM risk. Yet, there is a need to establish causal mechanisms and identify sensitive population groups who may benefit from Zn biomarker screening or monitoring. For example, people with prediabetes or poor glycemic control may benefit from the assessment of urinary Zn levels as an additional, non invasive indicator of short to mid-term T2DM onset and progression, whereas the clinical use of the assessment of urinary Zn levels in the general population is less clear.

In this context, the use of novel biomarkers such as Zn isotopes, may provide a valuable tool to establish the connection between documented molecular changes in cellular Zn and changes in Zn levels in human biomarkers.

Randomized trials show that zinc supplementation can improve glycemic endpoints in participants with established T2DM, but there is insufficient evidence to recommend Zn supplementation in healthy participants and the number of studies in pre-diabetes is small. Dosage, formulation of Zn supplements, and potential underlying risks of high dose supplementation should not be overlooked. Beyond randomized trials with diabetes-related clinical endpoints, molecular and population-level longitudinal studies are needed to identify mechanisms underlying the relationship between Zn metabolism and diabetes.

Table 1.

Summary of Zinc biomarkers and considerations.

Biomarker	Commonly used	Sensitivity to dietary changes	Sensitivity to supplem entation	Other considerations
Plasma/Serum	Yes	Low	High	Most commonly used. Influenced by fasting status, pregnancy, inflammation, use of steroids. [14,17,19,23–26]
Whole blood	No	Low	Low	Tightly regulated Zn in erythrocytes [14,34]
Urine	Yes*	Low	Moderate	Marker of elimination. Influenced by renal filtration, diabetes. Requires adjustment for urine dilution. [24,34,35,38,39]
Hair	No	Low	Moderate	Influenced by contamination and growth rates [40]
Saliva	No	Low	Moderate	Requires further validation [41,42]
Isotopes	No	Yes	Yes	Zn isotope distribution can be affected by changes in cellular Zn transporters [43–45,47]

Table 2.

Descriptive summary of observational epidemiological evidence on zinc and T2DM across populations, study types, and biomarkers

Population	Temporality	Biomarker	Association with T2DM status	Amount of Evidence
Persons with T2DM	Cross-sectional	Plasma/Serum	Lower levels in established T2DM	Moderate [112–115]
		Urine	Higher levels in established T2DM	Moderate [37,59,60,107–111]
	Prospective	Urine	Higher levels associated with T2DM complications	Moderate [38,105,119,123–125]
		Whole blood	Lower levels after T2DM diagnosis	Strong [34]
Healthy participants and participants with pre-diabetes	Cross-sectional	Plasma/Serum	Higher levels in T2DM	Suggestive [34,112,131]
	Prospective	Urine	Higher levels associated with T2DM risk	Suggestive [36,37]
		Whole Blood	Higher levels associated with T2DM risk	Suggestive [147]
		Plasma/Serum	Higher levels in T2DM	Suggestive [129,131]

Suggestive (animal, in vitro, and/or < 3 observational studies).

Moderate ( >3 observational studies, 1 or more randomized controlled trials).

Strong (1 or more Meta-analysis).

Table 3.

Summary of evidence from Zn supplementation and T2DM studies.

Population	Study type and year	Number of studies/sample size	Zn dosage and duration	Results in glycemic endpoints
Persons with T2DM	Meta-Analysis, 2012 [134]	22 studies	10mg – 660 mg/day	Reduced FPG Reduced HbA1c
	Meta-Analysis, 2020 [135]	27 studies	9.8 mg – 75 mg/day up to 24 weeks	Reduced FPG Reduced HbA1c
	Umbrella of interventional meta-analyses, 2024 [136]		18.04 mg - 210 mg/day from 6.6 to 18 weeks	Reduced FPG Reduced HbA1c Reduced insulin and HOMA-IR
Participants with pre-diabetes	RCT, 2018 [141].	N= 200	20mg/day for a year	Reduced FPG Reduced insulin Reduced risk of incident T2DM
	RCT, 2016 [142]	N= 20	6 months	Reduced FPG Reduced HOMA-IR
	Meta-analysis [144]	12 RCT Obese/overweight participants	≥30 mg, variable duration	Reduced FPG Reduced HOMA-IR

Highlights.

• Zinc metabolism dysregulation is tightly connected to type 2 diabetes development.

• Zinc biomarkers can reflect different zinc and diabetes metabolic states.

• Decreased serum and increased urinary zinc was observed in persons with established type 2 diabetes, likely through increased urinary Zn loss.

• Zinc supplementation may reduce hyperglycemia in persons with pre-diabetes and type 2 diabetes.

• Evaluation of the causal mechanisms underlying zinc dysregulation and type 2 diabetes is needed.