Lupine Publishers| Identification of The Downregulation of TPD52-Like3 Gene and NKX2-1 Gene in Type 2 Diabetes Mellitus Via RNA Sequencing

 Lupine Publishers| Journal of Diabetes and Obesity

Abstract

A recent study using next-generation RNA sequencing was reported on genome-wide changes in gene expressing in the skin between patients with type 2 diabetes mellitus, compared to non-diabetic patients. Ex-post review, based in part on both the existence of lipid droplets, peridroplet mitochondria and cytoplasmic mitochondria, selected in the gene metabolism category the most downregulated gene TPD52L3, and in the gene regulation category the most downregulated gene NKX2-1. There is strong evidence that these two genes are involved in the disease process of type 2 diabetes mellitus.

Keywords: Gene Expression; Lipid Droplets; Mitochondria; RNA Sequencing; Type 2 Diabetes Mellitus

Opinion

In an earlier study, it was proposed that the final consequence of hereditary anomaly results in the development of type 2 diabetes, which already emerges in the prediabetic phase. It was thought to occur due to an increased flux, as compared to the healthy controls where protons (H+-ions) from the mitochondrial intermembranespace re-enter the matrix via uncoupling protein-1 (UCP1). This causes hyperthermia in and around the mitochondria [1].
But the key question that remains to be answered here is for the connection between the increased flow of protons and type 2 diabetes mellitus. In the past decade, a study reported on the visual documentation of the possible interaction of lipid droplets with mitochondria. This interaction was found to be quite intimate with the involvement of membrane attached receptor proteins such as SNAP23 [2]. Also, the cellular population of mitochondria in brown adipocytes tissue could be divided into two subpopulations; i.e. mitochondrial population having physical evidence of adherence to a lipid droplet or peridroplet mitochondria, and a non-lipid droplet-bound cytoplasmic mitochondrial population without any adherence to lipid droplets [3,4].
Although both the peridroplet mitochondria and cytoplasmic mitochondria are similar in their cell membrane composition they differ in other fundamental respects [3,4]. A comparison of the purified peridroplet mitochondria to cytoplasmic mitochondria suggests that peridroplet mitochondria are more elongated, whereas cytoplasmic mitochondria tend to be smaller. Also, peridroplet mitochondria have enhanced oxidative phosphorylation capacity, TCA cycle activity, ATP synthesis, as well as increased ATP-dependent triglyceride synthesis compared to cytoplasmic mitochondria. The measured fatty acid-driven respiration and UCP1 content in the isolated mitochondria suggests that for thermogenic fat oxidation peridroplet mitochondria are not specialized compared to cytoplasmic mitochondria [3]. This signifies that, under healthy conditions, in the peridroplet mitochondria the protons derived from free fatty acids (FFAs) and generated by the electron-transport chain during the oxidation process of FA are used for the production of ATP without any escape of protons via UCP1 to produce heat. On the other hand, the protons generated by the oxidation of cytoplasmic mitochondrial FA are mainly used for the production of heat. So, peridroplet mitochondria have an increased coupled respiration, while cytoplasmic mitochondria have an increased uncoupling activity. The existence of peridroplet mitochondria demonstrates that the essential processes of fat metabolism can be selectively confined to exclusive and segregated subsets of mitochondria. The fatty acids intended for storage undergo synthesis of triacylglycols followed by their storage in the lipid droplets.

Most eukaryotic cells can store lipids in the form of droplets [5]. Lipid droplets are cytosolic storage organelles at the center of the lipid and energy homeostasis. They have a unique architecture consisting of a hydrophobic core of neutral lipids, mostly triacylglycerol and sterol esters and are enclosed by a phospholipid monolayer membrane. This single layer is derived from the endoplasmic reticulum, whereby triacylglycerols are synthesized between the two leaflets of the endoplasmic reticulum membrane. Associated with the monolayer is a specific set of proteins, which decorates the surface of the lipid droplet but is absent from the hydrophobic core [6]. These proteins associate with the membrane through hydrophobic hairpins, amphipathic helices and fatty acid modifications, and are also thought to control lipid droplet positioning inside the cell and association with other organelles.
In 2016, researchers demonstrated that the exogenous expression of human tumor protein D52 (TPD52) in the cultured 3T3 cells result in a significant increase in the numbers of lipid droplets [7]. Starting with the bulging of a triglyceride lens within the endoplasmic reticulum bilayer, lipid droplet biogenesis factors including TPD52 are recruited to the lens structure and facilitate the growth of the nascent lipid droplet [8,9]. Moreover TPD52- expressing 3T3 cells form more lipid droplets following oleic acid supplementation, which contributes to the lens formation [10]. As a previous study has shown, an increase in carbon-carbon double bonds in the acyl chains of phospholipids promotes the flexibility of cellular membranes [11]. So, TPD52 expression increases lipid storage, co-distributes with lipid droplets and is recruited to lipid droplets to stabilize lipid droplets [12]. Moreover, it is interesting to note that TPD52 knockdown decreased both lipid droplet sizes and numbers [12].
Tumor protein D52 is the founding member of the TPD52-like protein family representing four paralogous mammalian genes, i.e. TPD52, TPD52L1, TPD52L2, and TPD52L3 [7,13,14]. The group of Cao demonstrated that human TPD52L3 interacted with itself and with TPD52, TPD52L1, and TPD52L2 [14]. The four human linear proteins of this family are TPD52, TPD52L1, TPD52L2, and TPD52L3, which consist of 184, 204, 206, and 140 amino acid residues, respectively. Their first exon-coded protein located at the N-terminal side is unique to each isoform, and all the members contain a highly conserved coiled-coil motif located towards the N-terminus which is required for homo- and heteromeric interaction with other TPD52-like proteins [14,15] and share a sequence identity of ~50% [7]. Byrne et al. proposed that TPD52 may exert and/or regulate its activities through interaction with itself and its related proteins [15]. The coiled-coils were predicted by use of pairwise residue correlations and were not based on their crystal structures [16].

Figure 1: Alignment of the human TPD52 (upper row) and the human TPD52L3 (lower row) protein sequences. Amino acid residues are indicated by single letters. Vertical lines indicate identical residues and colons/dots indicate highly/weakly conserved residues.

The analysis was focused on the sequences of TPD52 and TPD52L3. The two sequences revealed an obvious similarity, they share 63 identical positions and 42 similar positions resulting in an overall homology of 57.1% (Figure 1). The coiled-coil motif near the N-terminus was found in all TPD52-like proteins. Uniprot predicts a coiled domain for residues 28-57 of TPD52L3, and also for residues 21-70 of TPD52 [17]. The common part of these two coiled-coil protein sequences has an overall homology of 67.9% (Figure 1). The information regarding the amino acid sequences of human TPD52 (P55327-2) and TPD52L3 (Q96J77-1) was retrieved from the UniProtKB/Swiss-Prot databank. Interestingly, although the coiled-coil motif is very important for the interactions mediated by TPD52-like proteins [15,18], the TPD52L3 shortened coiled-coil motif successfully interacted with TPD52-like proteins.

A recent study using next-generation RNA sequencing was reported on genome-wide changes in gene expression in the skin between patients with type 2 diabetes mellitus, compared to nondiabetic patients [19]. The most downregulated gene of patients with type 2 diabetes in the gene metabolism category is TPD52L3 with a “log2 fold change” value of -28, compared to skin samples from non-diabetic patients. So far, this gene has not been linked to type 2 diabetes or wound healing.

The fact that the exogenous expression of human TPD52 increases the number of lipid droplets [7], TPD52 knockdown decreases the number of lipid droplets [12], and the activity of TPD52 depends on the interaction with TPD52L3 [14], support the idea that the major function of TPD52L3 is the lipid storage at the center of the lipid and energy homeostasis [8,9]. In other words, in brown adipocytes tissue, it seems likely that the significant downregulation of TPD52L3 causes a reduction in the number of lipid droplets in the skin samples of type 2 diabetes mellitus patients.

This indicates that an essential reduction in the lipid droplets suggests a substantial decrease in the peridroplet mitochondria for patients with type 2 diabetes and consequently an increase in the saturated plasma FFAs. As the unsaturation index (UI; number of carbon-carbon double bonds per 100 fatty acyl-chains) of FFAs from human white fat cells is substantially lower compared to the UI of serum FFAs in the healthy controls (85.5 and 191.9, respectively), these events force a shift from unsaturated to saturated acyl chains in the phospholipids of both the erythrocyte and vascular membranes [20]. This reduction in UI translates into an increase in the attractive forces between the mutual membrane phospholipid acyl chains, which redistributes the lateral pressure profile of the cell membrane [21]. This redistribution reduces the pore diameter of the transmembrane glucose transport channels of all Class I glucose transporter proteins, leading to a marked reduction in the transmembrane glucose transport [22].

On the other hand, the increased uncoupling activity of the cytoplasmic mitochondria [4] takes up the remaining fatty acid oxidation, including the formation of protons. The rationale is that the overall balance between the number of protons which re-enter the matrix through ATP synthase on the one hand, and the number of protons which re-enter the matrix through UCP1 on the other hand, might shift to the latter side, which in turn promotes an increase in the production of heat. To keep a narrow range of mitochondrial temperature compatible with life, the slow-down principle enters into force, which also results in an essential reduction in UI [1]. This chain of events is a blueprint of the development of type 2 diabetes mellitus.

A second result of the earlier mentioned genome-wide analysis study is the most downregulated gene in the gene regulation category, NKX2-1, of type 2 diabetes patients with a “log2 fold change” value of -28 compared to skin samples from non-diabetic patients [19]. Notably, a study also reported a novel heterozygous mutation in exon 3 of the NKX2-1 gene, which is related to a reduction in the muscle mitochondrial respiratory chain complex activity, a characteristic of type 2 diabetes [23]. It is to be noted that the reduced mitochondrial activity is one of the characteristics of type 2 diabetes [24]. This may be in advance of the patients with type 2 diabetes mellitus as the reduced mitochondrial activity implies a reduction in heat production.

Finally, it is worth considering about the potential benefit of the use of (modified) synthetic TPD52L3 for combating the adverse effects of type 2 diabetes mellitus.

Briefly, the idea is that two genes are pertinently involved in the disease process of type 2 diabetes mellitus: one concerns the downregulation of human TPD52L3 gene expression, which yields a significant reduction in the lipid droplets, whereas the second one relates to the downregulation of human NKX2-1 gene expression which reduces the mitochondrial respiratory chain activity (Figure 2).

Figure 2: Although the results of genome-wide screen for type 2 diabetes susceptibility genes are still under debate, a refined working hypothesis proposes that the primary effect of the downregulation of the human genes TPD52L3 and NKX2-1 generates an increased flux of mitochondrial intermembrane-space protons through UCP1 into the matrix, which causes an increase of extra heat. This process initiates the slow-down principle. UCP: Uncoupling protein; FFA: Free fatty acid; GLUT: Glucose transporter.  

Read More Lupine Publishers Diabetes and Obesity Journal Articles: https://diabetes-obesity-lupine-publishers.blogspot.com/

Lupine Publishers| Properties of Mitochondrial-Derived Peptides (Mdps), Type 2 Diabetes, and Relationship with Oxidative Stress

 Lupine Publishers| Journal of Diabetes and Obesity

Abstract

Objective: In addition to its role in energy production and metabolism, mitochondria play a major role in apoptosis, oxidative stress, and calcium homeostasis. This review highlights the intricate role of mitochondria derived peptides (MPs), oxidative stress, and age-related disease such as diabetes.

Key Findings: The mitochondria produce MDPs: specific peptides that mediate transcriptional stress response by its translocation into the nucleus and interaction with DNA. MDPs are regulators of metabolism with cytoprotective effects through anti-oxidative stress, anti-inflammatory responses and anti-apoptosis. This class of peptides comprises: humanin (HN), MOTS-c, Small HN-like peptides. HN inhibits mitochondrial complex 1 activity and limits oxidative stress level in the cell. HN has been shown to prevent apoptosis by decreasing the reactive oxygen species production. Mitochondrial dysfunction and oxidative stress are implicated in the pathogenesis of diabetes. Data suggested that MDPs had a role in improving type 2 diabetes (T2D).

Summary: The goal of this review is to discuss the newly emerging functions of MDPs and their biological role in ageing and age-related diseases such as T2D.

Keywords:Mitochondrial-Derived-Peptides; Humanin; Oxidative Stress; Diabetes

Introduction

Mitochondria play a critical role in maintaining cellular function by ATP production. In addition to its role in energy production and metabolism, mitochondria play a major role in apoptosis, oxidative stress, and calcium homeostasis. A mitochondrial stress signal, or a ‘mitokine’, could confer protection and promote survival, while priming the cell’s readiness for subsequent insults with increasing severity. The term ‘mitohormesis’ for such a phenomenon has been created [1]. The mitochondrial unfolded protein response (UPRmt) is a central part of the “mitohormetic” response. The UPRmt may be an alternative way in relationship with mitochondria signal in the cell. The mitochondria produce some specific peptides that mediate transcriptional stress response by the translocation into the nucleus and interaction with DNA. Mitochondrial derived peptides (MDPs) are regulators of metabolism and various studies have shown that MDPs exerted cytoprotective effects through anti-oxidative stress, anti-inflammatory responses and anti-apoptosis [2,3]. The goal of this review is to discuss the newly emerging functions of MDPs and their biological role in ageing and metabolic diseases such as T2D.

Mitochondrial Metabolism Modulation

Functions in the Mitonuclear Communication Pathways

Mitochondria booked a portion of the original bacterial genomes that co-evolved with nuclear genome. However, mitochondria import over a thousand proteins encoded in the nuclear genome to maintain their diverse functions, reflecting their adjacent relationship [4].

The mitochondrial genome inherits bacterial-like traits: the DNA molecules (mtDNA) are circular, double stranded, small (16,569 nucleotides in humans) and compact. mtDNA contains 37 genes, including 22 tRNAs, 2 rRNAs (12S and 16S rRNA) and 13 mRNAs encoding the proteins of the electron transport chain [5]. The mtDNA has no introns but a few non-coding nucleotides between adjacent genes and small open reading frames that encode functional MDPs. This class of peptides comprises humanin (HN) and mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) and expands the expression of mitochondrial proteome [6]. It has been established that mitochondria can export peptides and also import cytosolic peptides. It is the class of “cell-penetrating peptides” designed also as “mitochondrial cell-penetrating peptides” [7]. Many age-induced processes and degenerative diseases are related to mitochondrial dysfunction, further highlighting the critical importance of this organelle [8]. Complex human diseases, including diabetes, obesity, fatty liver disease and aging-related degenerative diseases are associated with alterations in mitochondrial oxidative phosphorylation (OXPHOS) function.

Overview on Concepts of Retrograde Signaling and Unfolded Protein

Numerous implications of these anterograde and retrograde signaling pathways between the mitochondria and the nucleus are appropriate for therapeutic exploitation with bioactive molecules.

Concept of Retrograde Signaling

The hallmark of mitochondrial retrograde signaling is the modification of the expression of nuclear genes induced by a signal from mitochondria [9]. Retrograde signaling must be triggered by a mitochondrial signal that in turn is relayed via molecules that finally reach the nucleus. In mammalian cells, altered nuclear expression in response to mitochondrial dysfunction is reported; a number of signaling pathways being implicated in this retrograde communication [10]. Mitochondrial retrograde signaling is a signaling pathway connecting mitochondria and the nucleus. Signal transducers in the yeast retrograde response are Rtg1p, Rtg2p, and Rtg3p proteins [11]. The outcomes of mitochondrial retrograde signaling go far beyond the maintenance or biogenesis of the organelle, affecting the homeostasis of the whole organism through body weight or immunity.

Concept of Unfolded Protein

Mitochondrial protein homeostasis is maintained through proper folding and assembly of newly translated polypeptides. Several factors challenge the mitochondrial protein-folding environment including reactive oxygen species (ROS) that are generated within mitochondria, as well as environmental situations such as exposure to toxic compounds. To promote efficient mitochondrial protein folding mitochondria possess molecular chaperones located in both the intermembrane space and matrix [12].

UPRmt is a mitochondria-to-nuclear communication mechanism that promotes adaptive regulation of nuclear genes related to mitochondrial response, and metabolism, implicated in the cellular homeostasis [13].

Mitochondrial-Derived Peptides: Classification

MDPs are a series of peptides encoded by mitochondrial DNA. This class of peptides comprises HN, MOTS-c, Small HN-like peptides (SHLPs) and expands the expression of mitochondrial proteome [6].

Humanin

The first MDP discovered back in 2001 was HN; the term based on the potential of this peptide for restoring the “humanity” of Alzheimer’s disease (AD) patients. HN promotes cell survival in response to a variety of insults.
It is a small, secreted, 24 or 21 amino acid peptide, depending on cytoplasmic or mitochondrial translation, respectively. If HN is translated within the mitochondria, the peptide will be 21 amino acids; and if it is translated in the cytoplasm, then the result is a 24 amino acid peptide [14]. HN is encoded by an HN open reading frame (ORF) within the gene for the 16S ribosomal subunit within the mitochondrial genome [15]. HN was discovered during a search for survival factors in unaffected areas of an AD patient’s brain. The initial studies were first performed in cell culture and then followed by in vivo studies using both pharmacological mimetics of AD as well as mutant gene: amyloid-β precursor protein. The most recent studies used transgenic models of AD. As HN is a relatively short peptide, exhaustive mutational analysis of the importance of each amino acid has been possible. Interestingly, single amino acid substitutions of HN can lead to significant alterations in its potency and biologic functions. S14G-HN in which the serine at position 14 is replaced by glycine, is a highly potent analogue of HN.
Finally, HN may be the first small peptide of its kind representing a putative set of MDPs, a novel concept that modifies the established concept about retrograde mitochondrial signaling as well as mitochondrial gene expression. HN is a neuroprotective peptide and a cytoprotective factor against oxidative stress [16].

Mitochondrial Open Reading Frame of the 12S rRNA-c ( MOTS-c)

In addition to HN, an in-silico search of the mitochondrial genome revealed additional potential MDPs. MOTS-c is expressed in various tissues and in circulation (plasma) in rodents and humans, suggesting both a cell-autonomous and hormonal role. Its primary target organ appears to be skeletal muscle and fat. The mitochondrially derived peptide MOTS-c was recently discovered. It is a 16 amino acid peptide located in the 12S rRNA gene. The first 11 amino acid residues of MOTS-c are highly conserved in 14 mammalian species [17]. MOTS-c has been identified as a gene expression regulator in the nucleus, leading to retrograde signaling via its interaction with transcription factors. MOTS-c polymorphism has been found to be associated with human longevity [18]. MOTS-c can prevent insulin resistance, dietmediated obesity, and ameliorate diabetes. MOTS-c oxidizes fatty acids and inhibits oxidative respiration [19]. MOTS-c increased the levels of carnitine metabolism, which transport activated fatty acids into the mitochondria for β-oxidation, increased the level of a β-oxidation intermediate. MOTS-c inhibited the folate cycle at the level of 5Me-THF, resulting in an accumulation of 5-aminoimidazole- 4-carboxamide ribonucleotide, an AMP-activated protein kinase (AMPK) activator. MOTS-c also increased cellular NAD+ levels, which are nucleotide precursors [17,20]. MOTS-c regulated a broad range of genes in response to glucose restriction, including those with antioxidant response elements (ARE), and interacted with ARE-regulating stress-responsive transcription factors [21].

Small HN-like Peptides

Recently, six additional peptides encoded within the mitochondrial 16S rRNA region of mtDNA have been discovered and designed as SHLP1-6. SHLP2 and SHLP3 share similar biological effects with HN. The circulating levels of MOTS-c and SHLP2 decline with age. Various studies suggest that SHLP2 and SHLP3 may participate in the pathogenesis of age-related neurodegenerative diseases. The anti-oxidative stress function of SHLP2, and its neuroprotective effect indicate that SHLP2 has a role in the regulation of aging and age-related diseases [5].

Ageing and Plasma MDPs Levels

Ageing and longevity are or are not characterized by high levels of MDPs? It is speculated that MDPs production turns from protective to detrimental adaptive response; in these conditions, the levels increasing during aging. In some studies, HN levels significantly decline with age in humans. Plasma HN level was significantly lower in the older group (1.3 ± 0.2 ng/mL) than that of the younger group (1.7 ± 0.1 ng/mL) [22]. In other studies, it is reported that HN levels significantly decline with age in humans and animals. HN levels in plasma were measured in young and old mice and across age in humans. HN levels decreased with age in both mice and human (Human plasma levels: 45-65 years: 1400 pg/mL; 80-110 years: 1000 pg/mL) [5].

New results are in contrast with these data. HN plasma levels are evaluated in 693 subjects aged from 21 to 113 years. HN levels increased in old age (>500 pg/mL), with the highest levels found in centenarians (> 1000 pg/mL). The plasmatic levels of HN are significantly positively correlated with age. No gender differences were observed for HN. HN plasma level is associated negatively with body mass index in elderly patients [23]. Concerning the other MDPs, it is reported that MOTS-c and SHLP2 circulating levels decline with age. The circulating SHLP2 levels significantly decreased with age in both male and female C57BL/6 mice (young, 3 months old: 3000 pg/mL; aged, 18 months old: 2500 pg/mL). Male mice had higher SHLP2 levels than female mice in both the young and old groups [5]. The results of these studies should be interpreted considering the following limitations. First, the relatively small sample size in each group represents a potential limitation. Second, mitochondrial diseases are an expanding group of disorders with many metabolic deficiencies. In the ideal case, the used patient cohort should display a homogeneous phenotype, disease stage, and organ specificity. Moreover, the discovery of ageing-related biomarkers is supported by the development of advanced proteomics technology. Changes in the circulating concentrations of human proteins can serve as predictive measures of health and disease [24].

Read More Lupine Publishers Diabetes and Obesity Journal Articles: https://diabetes-obesity-lupine-publishers.blogspot.com/