Dysregulation of macroautophagy/autophagy contributes to the delay of wound healing in diabetic skin. N⁶-methyladenosine (m⁶A) RNA modification is known to play a critical role in regulating autophagy. In this study, it was found that SQSTM1/p62 (sequestosome 1), an autophagy receptor, was significantly downregulated in two human keratinocyte cells lines with short-term high-glucose treatment, as well as in the epidermis of diabetic patients and a db/db mouse model with long-term hyperglycemia. Knockdown of SQSTM1 led to the impairment of autophagic flux, which was consistent with the results of high-glucose treatment in keratinocytes. Moreover, the m⁶A reader protein YTHDC1 (YTH domain containing 1), which interacted with SQSTM1 mRNA, was downregulated in keratinocytes under both the acute and chronic effects of hyperglycemia. Knockdown of YTHDC1 affected biological functions of keratinocytes, which included increased apoptosis rates and impaired wound-healing capacity. In addition, knockdown of endogenous YTHDC1 resulted in a blockade of autophagic flux in keratinocytes, while overexpression of YTHDC1 rescued the blockade of autophagic flux induced by high glucose. In vivo, knockdown of endogenous Ythdc1 or Sqstm1 inhibited autophagy in the epidermis and delayed wound healing. Interestingly, we found that a decrease of YTHDC1 drove SQSTM1 mRNA degradation in the nucleus. Furthermore, the results revealed that YTHDC1 interacted and cooperated with ELAVL1/HuR (ELAV like RNA binding protein 1) in modulating the expression of SQSTM1. Collectively, this study uncovered a previously unrecognized function for YTHDC1 in modulating autophagy via regulating the stability of SQSTM1 nuclear mRNA in diabetic keratinocytes. Abbreviations: ACTB: actin beta; AGEs: glycation end products; AL: autolysosome; AP: autophagosome; ATG: autophagy related; AKT: AKT serine/threonine kinase; ANOVA: analysis of variance; BECN1: beclin 1; Co-IP: co-immunoprecipitation; DEGs: differentially expressed genes; DM: diabetes mellitus; ELAVL1: ELAV like RNA binding protein 1; FTO: FTO alpha-ketoglutarate dependent dioxygenase; G: glucose; HaCaT: human keratinocyte; GO: Gene Ontology; GSEA: Gene Set Enrichment Analysis; HE: hematoxylin-eosin; IHC: immunohistochemical; IRS: immunoreactive score; KEAP1: kelch like ECH associated protein 1; KEGG: Kyoto Encyclopedia of Genes and Genomes; m⁶A: N⁶-methyladenosine; M: mannitol; MANOVA: multivariate analysis of variance; MAP1LC3: microtubule associated protein 1 light chain 3; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MeRIP: methylated RNA immunoprecipitation; METTL3: methyltransferase 3, N6-adenosine-methytransferase complex catalytic subunit; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin complex 1; NBR1: NBR1 autophagy cargo receptor; NFE2L2: nuclear factor, erythroid 2 like 2; NG: normal glucose; NHEK: normal human epithelial keratinocyte; OE: overexpressing; p-: phospho-; PI: propidium iodide; PPIN: Protein-Protein Interaction Network; RBPs: RNA binding proteins; RIP: RNA immunoprecipitation; RNA-seq: RNA-sequence; RNU6–1: RNA, U6 small nuclear 1; ROS: reactive oxygen species; siRNAs: small interfering RNAs; SQSTM1: sequestosome 1; SRSF: serine and arginine rich splicing factor; T2DM: type 2 diabetes mellitus; TEM: transmission electron microscopy; TUBB: tubulin beta class I; WT: wild-type; YTHDC1: YTH domain containing 1.

Diabetic wounds are recalcitrant to healing. However, the mechanism causing this dysfunction is not fully understood. High expression of matrix metalloproteinase-9 (MMP-9) is indicative of poor wound healing. In this study, we show that specificity protein-1 (Sp1), a regulator of MMP-9, binds directly to its promoter and enhances its expression. Additionally, we demonstrated that Sp1 is the direct target of two microRNAs (miRNAs), miR-129 and -335, which are significantly downregulated in diabetic skin tissues. In vitro experiments confirmed that miR-129 or -335 overexpression inhibits MMP-9 promoter activity and protein expression by targeting Sp1, whereas the inhibition of these miRNAs has the opposite effect. The beneficial role of miR-129 or miR-335 in diabetic wound healing was confirmed by the topical administration of miRNA agomirs in diabetic animals. This treatment downregulated Sp1-mediated MMP-9 expression, increased keratinocyte migration, and recovered skin thickness and collagen content. The combined treatment with miR-129 and miR-335 induced a synergistic effect on Sp1 repression and MMP-9 downregulation both in vitro and in vivo. This study demonstrates the regulatory mechanism of Sp1-mediated MMP-9 expression in diabetic wound healing and highlights the potential therapeutic benefits of miR-129 and -335 in delayed wound healing in diabetes.

OBJECTIVE: To investigate whether the association between insulin resistance and cardiovascular disease (CVD) differs by glucose tolerance status. RESEARCH DESIGN AND METHODS: We analyzed a nationwide sample of 111,576 adults without CVD at baseline, using data from the China Cardiometabolic Disease and Cancer Cohort Study. Insulin resistance was estimated by sex-specific HOMA of insulin resistance (HOMA-IR) quartiles for participants with normal glucose tolerance, prediabetes, or diabetes, separately, and by 1 SD of HOMA-IR for the overall study participants. We used Cox proportional hazards models to examine the association between insulin resistance and incident CVD according to glucose tolerance status and evaluate the CVD risk associated with the combined categories of insulin resistance and obesity in prediabetes and diabetes, as compared with normal glucose tolerance. Models were adjusted for age, sex, education attainment, alcohol drinking, smoking, physical activity, and diet quality. RESULTS: In participants with normal glucose tolerance, prediabetes, and diabetes defined by three glucose parameters, multivariable-adjusted hazard ratios (95% CIs) for incident CVD associated with the highest versus the lowest quartile of HOMA-IR were 1.03 (0.82–1.30), 1.23 (1.07–1.42), and 1.61 (1.30–2.00), respectively; the corresponding values for CVD per 1-SD increase in HOMA-IR were 1.04 (0.92–1.18), 1.12 (1.06–1.18), and 1.15 (1.09–1.21), respectively (P for interaction = 0.011). Compared with participants with normal glucose tolerance, in participants with prediabetes, the combination of the highest HOMA-IR quartile and obesity showed 17% (95% CI 2–34%) higher risk of CVD, while the combination of the lowest two HOMA-IR quartiles and nonobesity showed 15–17% lower risk of CVD. In participants with diabetes, the upper two HOMA-IR quartiles exhibited 44–77% higher risk of CVD, regardless of obesity status. Consistent findings were observed for glucose tolerance status defined by different combinations of glycemic parameters. CONCLUSIONS: Glucose intolerance status exacerbated the association between insulin resistance and CVD risk. Compared with adults with normal glucose tolerance, adults with prediabetes who were both insulin resistant and obese exhibited higher risks of CVD, while in adults with diabetes, the CVD risk related to insulin resistance remained, regardless of obesity.

Diabetic foot ulcer (DFU) caused by impaired wound healing is a common vascular complication of diabetes. The current study revealed that plasma levels of pigment epithelium–derived factor (PEDF) were elevated in type 2 diabetic patients with DFU and in db/db mice. To test whether elevated PEDF levels contribute to skin wound-healing delay in diabetes, endogenous PEDF was neutralized with an anti-PEDF antibody in db/db mice. Our results showed that neutralization of PEDF accelerated wound healing, increased angiogenesis in the wound skin, and improved the functions and numbers of endothelial progenitor cells (EPCs) in the diabetic mice. Further, PEDF-deficient mice showed higher baseline blood flow in the skin, higher density of cutaneous microvessels, increased skin thickness, improved numbers and functions of circulating EPCs, and accelerated wound healing compared with wild-type mice. Overexpression of PEDF suppressed the Wnt signaling pathway in the wound skin. Lithium chloride–induced Wnt signaling activation downstream of the PEDF interaction site attenuated the inhibitory effect of PEDF on EPCs and rescued the wound-healing deficiency in diabetic mice. Taken together, these results suggest that elevated circulating PEDF levels contribute to impaired wound healing in the process of angiogenesis and vasculogenesis through the inhibition of Wnt/β-catenin signaling.

Diabetic foot ulceration is receiving more attention because of its high amputation and mortality rate. It is essential to establish the frequency of amputations in people with diabetes after any change to the management of diabetic foot care. The present study aim to compare the frequency of lower-extremity amputations in patients with diabetes foot ulcer over a ten-year period. Six hundred forty eight patients with diabetes foot ulcer were retrospectively studied from 2004 to 2013. The clinical features, laboratory results and the lower-extremity amputations were recorded. Major amputation was defined as amputations above the ankle while minor amputation was amputations below the ankle in the present study. Patients with diabetic foot ulcer were old (age 66.96 ± 11.96 years), with a long duration of diabetes (10.30 ± 6.94 years), high HbA1c (9.19 ± 2.62 %), SBP (144.05 ± 24.18 mmHg), DBP (79.53 ± 11.88 mmHg), LDL-C (2.71 ± 0.93 mmol/L) and had great frequency of neuropathy (62.7 %), retinopathy (45.0 %), nephropathy (39.5 %) and PAD (33.2 %). From 2004 to 2013, the frequency of all lower-extremity amputations is 12.0 % (5.2 % major amputation, 6.8 % minor amputation). The frequency of major amputations decreased from 9.5 % in 2004 and 14.5 % in 2005 to less than 5.0 % after 2006. In particular, there was a significant decline in major amputations of diabetic foot patient with Wagner 3 to 4 wounds. The frequency rate of major amputations in diabetic foot patient with Wagner 3 to 4 wounds fell from 35.7 % in 2004 to 4.4 % after 2007. The change in frequency of minor amputations was fluctuation. This study demonstrates that the introduction of a multidisciplinary team, coordinated by an endocrinologist and a podiatrist, for managing diabetic foot disease is associated with a reduction in the frequency of major amputations in patients with diabetes.

Background Education attainment can improve life expectancy and guide healthy behaviours throughout an entire lifetime. A nationwide longitudinal study of the association of education status with the risk of hypertension and its control in China is lacking. Methods The China Cardiometabolic Disease and Cancer Cohort Study is a multicentre, population-based, prospective cohort study. We performed the baseline survey from 2011 to 2012. A follow-up visit was conducted during 2014–2016. 101 959 subjects were included in the final data analyses. Cox proportional hazards regression was used to examine the associations of education levels with the risk of hypertension and uncontrolled hypertension. Results During follow-up, 11 189 (19.9%) participants had developed hypertension among subjects without hypertension at baseline. Among the participants with hypertension at baseline, only 40.6% had controlled hypertension. Compared with the participants’ education level at elementary school and below, the multivariable-adjusted HR for incident hypertension was 0.76 (95% CI, 0.72 to 0.80) in those with a middle school education level and 0.67 (95% CI, 0.63 to 0.70) in those with a high school degree or above. Correspondingly, multivariable-adjusted HRs associated with uncontrolled hypertension were 0.90 (95% CI, 0.87 to 0.92) in participants with a middle school education level and 0.85 (95% CI, 0.82 to 0.88) in participants with a high school degree or above level. Conclusion Participants with education attainment at elementary school and below exhibited excess risks of newly diagnosed hypertension and worse blood pressure control compared with individuals with education attainment at middle school or above.