INTRODUCTION
Type 2 diabetes mellitus (T2DM) is a multifactorial metabolic disease which is characterized by chronic hyperglycemia. elevated glucose levels, inflammation and oxidative stress are the major factors for development and progression of T2DM[1]. In the human body, the persistent hyperglycemia affecting microvascular and macrovascular system and thereby increasing the risk of diabetes and associated complication like diabetes nephropathy[2,3]. Zinc is a trace and essential element involved in various biological processes, like enzymatic reactions, antioxidant defense mechanisms, immune system regulation, and intracellular and extracellular signaling. In glucose metabolism, zinc plays a crucial role in pancreatic β-cell function and insulin associated pathways[4]. In cells, Insulin is stored in form of secretory granules as zinc-stabilized hexamers, which are regulated by various zinc transporters. ZnT8 (Zinc transporter) which is encoded by the SLC30A8 gene, is majorly expressed in pancreatic β-cells and localized to the membrane of the insulin secretory granules[5]. ZnT8 regulates the transportation of zinc ions from the cytosol into insulin granules and regulates insulin secretion by facilitating insulin crystallization and its storage. ZnT8 also contributes to maintenance of intracellular zinc homeostasis, β-cell survival and protection against oxidative stress[4]. Studies suggested SLC30A8 as a susceptibility locus for T2DM, with both gain-of-function and loss-of-function variants and increasing disease risk[6]. Evidence indicates that in response to metabolic stress, miRNA-based regulation of SLC30A8 plays a critical role in modulating SLC30A8 expression. It is suggested miRNA miR-375/miR-765 regulate SLC30A8 by targeting regulatory networks and contributing to ZnT8 dysregulation in diabetic associated complication like diabetic nephropathy (DN)[7,8]. The aim of this review is to evaluate the mechanism of microRNA-based regulation of SLC30A8 (ZnT8) in T2DM.
MicroRNAs as epigenetic regulators in metabolic disease
MicroRNAs are endogenous, non-coding small RNAs (approximately 18-25 nucleotides in length) which regulate gene expression at the post-transcriptional stage by binding to complementary sequences in target mRNAs, specifically in 3′-untranslated regions. This association degrades/repress mRNA translation thereby regulating protein expression[9,10]. Dysregulation of miRNA expression has been implicated in various pathological processes like insulin resistance, β-cell dysfunction, lipid metabolism, inflammation, fibrosis, and vascular injury all of which contribute to the development and progression of T2DM and its complication[11].
The miR-375/miR-765–PDX-1 axis and SLC30A8 transcription
A defining feature of miRNA-mediated epigenetic silencing of SLC30A8 is the synergistic action of multiple miRNAs. Rather than acting independently, these miRNAs form coordinated regulatory networks that amplify ZnT8 repression in response to hyperglycemia, lipotoxicity, oxidative stress, and inflammatory cytokines. Among these, miR-375 are the most abundant and islet-enriched miRNAs, extensively implicated in β-cell function and dysfunction (Figure 1). Accumulating evidence suggests that dysregulation of miR-375 contributes to the pathogenesis of T2DM[12]. The expression levels of miR-375 and miR-765 are significantly upregulated under diabetic conditions, consistent with previous reports linking their elevated expression to impaired insulin secretion and pancreatic β-cell stress[13].
Figure 1 MiRNA-mediated suppression of PDX-1 disrupts slc30a8/znt8 regulation and insulin granule zinc homeostasis under hyperglycemic conditions schematic representation of the molecular interplay between hyperglycemia-induced microRNAs and PDX-1–ZnT8 signaling in pancreatic β-cells.
Under hyperglycemic conditions, upregulation of specific microRNAs (e.g., miR-375, miR-765) leads to translational repression of PDX-1 mRNA in the cytoplasm, resulting in reduced nuclear PDX-1 levels. Attenuated PDX-1 binding to Enhancer A, a regulatory element associated with the SLC30A8 locus compromises transcriptional regulation of ZnT8. Dysregulated ZnT8 expression alters zinc transport into insulin granules, affecting zinc-stabilized insulin hexamer formation and contributing to β-cell dysfunction. This pathway highlights miRNA-driven epigenetic control as a critical mechanism linking chronic hyperglycemia to impaired insulin storage and secretion in diabetes.
PDX-1 is a master islet-enriched transcription factor, indispensable for pancreatic development, β-cell maturation, and maintenance of insulin gene transcription. Alterations in PDX-1 expression or activity are strongly associated with β-cell dysfunction and diabetes progression[14]. Mechanistically, elevated miR-375 and miR-765 exert inhibitory effects on the expression of PDX-1, either directly or indirectly, thereby perturbing transcriptional networks essential for β-cell identity and function[15,16].
This reduction has profound downstream consequences, particularly for genes under direct PDX-1 transcriptional control[17]. Experimental studies have clearly demonstrated that PDX-1 directly regulates SLC30A8 transcription. Specifically, PDX-1 binds to enhancer A of the SLC30A8 gene both in vitro and in situ. Functional analyses have shown that mutation of two PDX-1 binding sites within enhancer A markedly reduces reporter (fusion gene) expression, underscoring the importance of PDX-1 in driving ZnT8 expression in β-cells[18]. Developmental evidence further supports this regulatory relationship. During pancreatic organogenesis, the restriction of PDX-1 expression to pancreatic islet β-cells temporally correlates with the induction of SLC30A8 gene expression and ZnT8 protein accumulation[18].
Downstream consequences of ZnT8 silencing on β-cell failure and fibrosis
Zinc plays a critical role in protecting β-cells from oxidative stress by stabilizing antioxidant enzymes and inhibiting pro-apoptotic signaling pathways. ZnT8 repression increases β-cell vulnerability to oxidative damage, endoplasmic reticulum stress, and inflammatory cytokines, accelerating β-cell apoptosis which ultimately leads to albuminuria, enhances the epithelial-to-mesenchymal transition (EMT), and contribute to kidney fibrosis[19]. In contrast, increasing ZnT8 levels reduces high-glucose–induced inflammation and EMT in kidney cells. ZnT8 protects the kidney mainly by inhibiting the TGF-β1/Smad signaling pathway[20].
Translating pancreatic ZnT8 loss to diabetic nephropathy
The pathophysiological consequences of ZnT8 silencing extend far beyond the pancreatic islets, reaching into the kidney and contributing directly to the development and progression of DN. Chronic hyperglycemia and the resulting systemic zinc dysregulation trigger inflammatory and fibrotic cascades that specifically target the renal parenchyma. Understanding how ZnT8 deficiency in the pancreas translates into kidney damage is critical for developing therapies that address both the primary metabolic defect and its devastating complications.
Although current evidence suggests that ZnT8 deficiency contributes to metabolic dysregulation and renal injury, the existence of a direct pancreas-to-kidney axis remains hypothetical. The study by Zhang et al[19] utilized a global ZnT8 knockout model and therefore could not distinguish between the systemic consequences of pancreatic ZnT8 loss and the local renal effects of ZnT8 deficiency. Future studies employing β-cell-specific and kidney-specific ZnT8 knockout models are needed to clarify these tissue-specific mechanisms[19].
ZnT8 deficiency as a driver of oxidative stress and β-cell apoptosis
The pancreatic β-cell’s protection against the chronic hyperglycemia-imposed oxidative stress is enormously dependent on zinc’s indispensable role. Pancreatic islets contain some of the most metabolically active tissues in human beings; β-cells in the pancreas are especially reliant on oxidative metabolism to generate ATP[21]. Antioxidant defense enzymes are present at low concentrations within β-cells, which makes these cells extremely susceptible to oxidative damage and more vulnerable to damage[21].
Zinc ions are critical cofactors of antioxidant enzymes, such as superoxide dismutase and catalase, and stabilize protein structures in response to oxidative stress. Inhibition of ZnT8 expression by miRNA, promoter hypermethylation or genetic difference suppresses the zinc transport into insulin secretory granules, which is due to the SLC30A8 gene defect discovered by genome-wide association studies that can influence the cytosolic Zn2+ concentrations and, consequently, predisposition to hypoxia and oxidative stress[22-24]. Clinical significance of this mechanism is indicated by the observation that T2DM patients have lower levels of plasma zinc and higher levels of urinary zinc lost[25]. The changes in zinc level are linked to a higher risk of T2DM, although hyperglycemia has not yet occurred, and therefore zinc dysregulation occurs in the early disease pathogenesis. It was observed that pancreatic zinc concentration in diabetic participants was half that in non-diabetic participants[26].
Breaking the TGF-β1/Smad pathway: ZnT8 as a renal protector
The effects of ZnT8 silencing extend beyond the pancreas. Accumulating evidence confirmes that, ZnT8 has a direct protective effect on the kidney, and with loss causes worsening the fibrotic events that characterize DN. The groundbreaking research of Zhang et al[19] presented the first direct indication that ZnT8 could prevent EMT, as well as tubulointerstitial fibrosis, by inhibiting the TGF-β1/Smad signaling. The investigators evaluated, db/db mice, ZnT8-KO mice, double-mutant ZnT8-KO-STZ and ZnT8-KO-db/db mice and reveal that ZnT8 deficiency has a devastating impact on diabetic kidney injury. ZnT8-KO-STZ and ZnT8-KO-db/db mice had a significantly increased urine albumin-to-creatinine ratio (UACR), a clinical manifestation of DN, in comparison to diabetic mice whose ZnT8 was intact. Additionally, the TGF-β1, interleukin-6, and TNF-α levels in serum samples of ZnT8-deficient diabetic mice were elevated, which suggests the presence of increased systemic and renal inflammation[19]. High glucose-induced stimulation of renal tubular epithelial cells increased the expression of phosphorylated Smad2 and Smad3 the canonical downstream effectors of TGF-β1 signal transduction. Notably, this Smad activation was further increased by ZnT8 silencing by siRNA, and also decreased by ZnT8 overexpression through transfection of hZnT8-EGFP suggested strong connection between ZnT8 and TGF-β1 1/Smad pathway[19].
TGF-β1/Smad is the controller of tissue fibrosis, which stimulates the transformation of fibroblasts to myofibroblasts, the excessive extracellular fibrosis with collagen and fibronectin, and progressive obliteration of normal tissue architecture[27]. Under DN conditions, the chronic TGF-β1/Smad signaling within the kidney results in glomerulosclerosis and tubulointerstitial fibrosis, which result in the loss of renal functions and subsequent end-stage renal disease. Therefore, ZnT8 turns out to be an endogenous reno-protective factor whose silencing is a direct contributor to the fibrotic diabetic complications[28].
EMT and albuminuria in DN consequence
EMT plays a key role in the initial pathogenesis of renal fibrosis in diabetic kidney disease. Mature renal tubular epithelial cells undergo massive cytoskeletal reorganization, lose epithelial markers (e.g., E-cadherin) and become mesenchymal-like, characterized by the expression of mesenchymal proteins (vimentin and alpha-smooth muscle actin) during EMT. The changed cells further proliferate into the interstitium, where they become part of the growing pool of matrix-producing myofibroblasts and lead to tubulointerstitial fibrosis[29,30]. Zhang et al[19] demonstrated that lack of ZnT8 aggravates the pathological progression of kidney in diabetes. Immunofluorescence staining of the kidneys from ZnT8-KO-STZ and ZnT8-KO-db/db mice showed upregulation of the mesenchymal marker vimentin and suppression of the epithelial marker E-cadherin as compared to diabetic control mice, indicative of an acceleration of EMT. In addition, increased UACR was observed, thus associating the exacerbated EMT with increased albuminuria and renal injury[19].
Therapeutic implications and restoring the broken axis
The miRNA-induced silencing, epigenetic repression, and ZnT8 deficiency delineate an essential pathological axis of T2DM and DN. Reinstating this axis either by antagonizing pathogenic miRNAs, modifying epigenetic regulatory elements, increasing ZnT8 levels, or administering zinc provides various therapeutic options for maintaining cell function and preventing kidney damage[31].
Antagomirs against miR-375 and miR-765 restoring PDX-1 expression
The miR-375 and miR-765 in regard to diabetes. MiR-375 is the highest concentration islet specific miRNA and is a critical negative regulator of insulin release. Early data indicated that miR-375 overexpression decreases insulin secretion upon glucose stimulation, whereas suppression of endogenous miR-375 results in an increased amount of insulin released through actions mediated by its target myotrophin[32]. Upregulation of miR-375 and miR-765 has been observed affecting the PDX-1 expression directly or indirectly, thereby altering the transcriptional networks essential for β-cell identity and function[12,32].
CRISPR-based epigenetic editing-permanent restoration of SLC30A8 expression
Antagomirs are a reversible and short-to-medium-term strategy for miRNA inhibition, whereas CRISPR-based epigenetic editing can permanently restore the expression of SLC30A8. SLC30A8 gene locus found that T2DM-associated risk variants are located in an islet-selective super-enhancer cluster that controls not only SLC30A8 but also neighboring genes. Epigenomic profiling uncovered a complex enhancer domain at the SLC30A8 locus that harbors multiple T2DM risk and candidate cis-acting expression associations. This super-enhancer cluster is not only physically linked to the SLC30A8 gene promoter but also to other nearby genes, such as UTP23, RAD21, and MED30. Interestingly, knockout of variant-carrying enhancer segments via CRISPR-Cas9 in human-derived EndoC-H3 cells reduces SLC30A8 expression but, at the same time, increases glucose-stimulated insulin secretion[31,33].
ZnT8 overexpression and direct rescue of zinc homeostasis in β-cells and kidney
Delivery of the SLC30A8 coding sequence using viral vector under β-celll-selective promoter (like insulin or PDX-1 promoters) could overcome transcriptional repression yet maintain cell-type specificity[18]. ZnT8 deficiency worsens diabetic renal damage by activating the TGF-β1/Smad-mediated EMT and tubulointerstitial fibrosis. Some studies have shown that transfection with hZnT8-EGFP, leading to ZnT8 overexpression, markedly counteracts high glucose-induced EMT modifications of renal tubular epithelial cells and lowers Smad2/3 phosphorylation. Therefore, ZnT8 expression restoration in pancreatic cells and renal tissues could be a two-edged sword[19].
Zinc supplementation as simple adjunct to restore insulin granule function
Zinc supplementation stands out as the most straightforward and easily implementable method among these therapeutic strategies. Zinc is an indispensable element in the insulin storage and crystallization processes, and zinc ions are co-secreted with insulin to perform important functions, such as preventing the monomeric insulin from forming amyloidogenic aggregates[34]. However, clinical investigations into zinc supplementation for diabetes have shown a combination of positive and negative results. One recent randomized, double-blind, placebo-controlled study testing pre-diabetic adults using 30 mg zinc gluconate per day for six months found that it did not make any significant difference in HbA1c or fasting blood glucose when compared with the placebo. The researchers concluded that their data do not justify supplemental zinc use in individuals consuming a Western diet, where zinc deficiency at baseline might be rare[35]. Newly diagnosed T2DM patients receiving 50 mg/day zinc supplementation for a period of 12 months with standard metformin treatment not only resulted in a significant decrease in fasting blood glucose (by 21.52 mg/dL), postprandial blood glucose (by 47.53 mg/dL), but also HbA1c was lowered (by 0.79%) as compared with the placebo group. A good influence on the lipid profile was noticed as well, comprising the drop in low-density lipoprotein cholesterol, triglycerides, and total cholesterol[34]. Zinc supplementation may synergize with other approaches that also target the miRNA-PDX-1-ZnT8 axis. For instance, antagomir and zinc combination may bring the most significant impact: Antagomirs would reverse the repression of endogenous ZnT8, while zinc supplementation would provide the substrate for the crystallization of insulin in granules where there still exists some ZnT8 function. Likewise, zinc could potentiate the effectiveness of CRISPR-mediated approaches by providing adequate zinc ions for the newly produced ZnT8 proteins to transport[36]. Since zinc at moderate doses has good safety among the population with verified zinc shortage or in the early stages of diabetes, where residual β-cell mass is still preserved. Nevertheless, the varying results from clinical trials point to the necessity for patient classification according to their initial zinc levels and disease stages before putting forward the general use of zinc (Figure 2)[37].
Figure 2
Restoring PDX-1 and ZnT8 homeostasis in diabetes and nephropathy through novel therapeutic approaches.
However, the genetic evidence for SLC30A8 is more nuanced and suggests a context-dependent role in diabetes. Genetic studies indicate that the role of SLC30A8/ZnT8 in diabetes is context dependent rather than uniformly harmful or beneficial. Although some loss-of-function variants and enhancer deletions are associated with improved glycemic phenotypes and reduced T2DM risk, these findings likely reflect partial reduction of ZnT8 activity, compensatory adaptations, or effects specific to certain cellular and metabolic contexts. Therefore, therapeutic strategies targeting ZnT8 should not be interpreted as simple restoration in all settings, but rather as approaches that aim to normalize zinc homeostasis in a manner that is appropriate to disease stage and biological context.
Although several studies report beneficial effects of zinc supplementation, other investigations have shown limited or no effect, indicating that the therapeutic response is context dependent. These discrepancies may reflect differences in baseline zinc status, supplementation dose and duration, disease severity, and study design. Therefore, zinc supplementation should be interpreted as a potentially useful intervention in selected settings rather than as a universally effective strategy.
Biomarker potential of circulating miRNAs as early warning signals for T2DM and DN
Identifying reliable, non-invasive biomarkers for the early detection of T2DM and DN remains an urgent clinical priority. Current diagnostic approaches, including fasting plasma glucose, HbA1c, and urinary albumin excretion, detect disease only after significant β-cell dysfunction or renal damage has already occurred[38]. Circulating miRNAs, particularly those derived from pancreatic islets and released into the bloodstream, offer a transformative opportunity for early warning, risk stratification, and monitoring of therapeutic response[39-41].
MiR-375, and miR-765 as probes of β-cell health
The most promising candidate biomarkers that have been identified so far are miR-375 and miR-765. Both these miRNAs are greatly enriched in the pancreatic islets and show a significant increase in diabetic conditions. miR-375 is a dominant miRNA found in pancreatic cells since it makes up nearly 40% of the total islet miRNAs. Besides, its level of expression changes depending on the metabolic stress[12]. In hyperglycemia and dyslipidemia, increased levels of miR-375 and miR-765 inhibit the expression of PDX-1, disrupting transcriptional networks that are crucial for maintaining cell identity and function[32]. Similarly, miR-375 is primarily produced and secreted by β-cells, and under stress or damage conditions, it is released into the blood circulation. Hence, taking a sample from the blood and analyzing it can be used as an indicative marker[12]. The levels of miR-375 and miR-765 are greatly increased when cells are exposed to an environment that mimics diabetes. This finding agrees well with previous studies that reported that high levels of miR-375 and miR-765 are associated with reduced insulin secretion and cell dysfunction could be a marker[12]. Elevated circulating miR-375 levels have been consistently found in patients with T2DM. The levels have also been found to correlate with the extent of hyperglycemia and cell dysfunction. Third, alterations in circulating miR-375 occur before manifest hyperglycemia in individuals at risk, implicating its potential for early risk prediction[42]. Combining several miRNAs might be able to achieve better diagnostic accuracy than one miRNA alone since each one reflects different facets of cell pathophysiology[43].
Circulating miRNAs as liquid biopsies for DN
Circulating miRNAs have potential uses beyond the context of pancreatic health; specifically, there is an opportunity to utilize circulating miRNAs as markers for the early identification of DN. During the progression of DN, damaged renal cells such as podocytes, tubular epithelial and glomerular endothelial cells, will release miRNA into the bloodstream and urine. These miRNAs will therefore provide a molecular representation of renal pathology that can be detected prior to the presence of albuminuria[44]. There have been several studies done in which they found circulating miRNA signatures associated with DN. There have been studies where they found that levels of miR-21, miR-29a, and miR-126 are elevated within the circulation of patients with DN compared to a patient who is diabetic without nephropathy or DN. The elevated levels of those biomarkers correlated with how much albuminuria was present in a patient and whether the patient’s estimated glomerular filtration rate was declining. Lastly, there have been some promising studies showing that urinary exosomal miRNAs (miR-15a, miR-34a, and miR-146a) may also provide the means to distinguish early stages of DN vs established DN[45-47].
The miRNAs, particularly miR-375 that are responsible for inducing β-cell dysfunction may also be responsible for causing renal disease. While miR-375 is primarily known to be expressed at high levels in islet tissues, it is now emerging that there is also a possibility that miR-375 is expressed in the kidney when there are pathologies or that β-cell-derived miR-375 can be transported from the blood into the kidney cells to activate inflammatory and fibrotic pathways. Therefore, miR-375 may serve as an integrator of pancreatic and renal damage, which supports the idea of a converging axis as discussed in this review[12,48].
Combining miRNA signatures with epigenetic and clinical markers
Circulating miRNAs is one of the most promising biomarkers for diabetes when coupled with various measurable parameters such as the SLC30A8 promoter methylation state, serum zinc, and the traditional Clinical Markers. With these factors taken into consideration, we will achieve a better representation of the miRNA-PDX-1-ZnT8 pathway, thus enhancing our prediction efficiency. In specific, a biomarker panel would involve[32,49]. First, the promoter methylation state of the SLC30A8 gene in circulating cell-free DNA in the plasma and peripheral blood mononuclear cell will inform us of the epigenetic factor in down regulation of ZnT8 mRNA in diabetic patients as well as zinc serum or plasma levels to measure general zinc balance[50]. Urinary ZnT8 protein concentration measurements will show the presence of renal ZnT8 protein and the status of the tubular systems[50]. Finally, there is clinical correlation using the traditional markers including the fasting blood glucose, HbA1c, and albumin creatinine ratios of the urine samples[50-53]. Long-term studies evaluating these biomarkers from pre-diabetes through overt T2DM to DN will be crucial in determining their prognostic significance. Longitudinal studies will help determine whether variations in serum miRNAs precede or occur after variations in glycemic control and whether miRNA variations can independently predict renal function decline (Figure 3).
Figure 3 Circulating miR-375 and miR-765 as promising biomarkers for early detection of type 2 diabetes mellitus and diabetic nephropathy, integrated with epigenetic and clinical markers.
T2DM: Type 2 diabetes mellitus; DN: Diabetic nephropathy; FBG: Fasting blood glucose.
