Main Research Findings

1) Administration of Humanin has been shown to reduce hepatic lipid accumulation and insulin resistance through suppression of the mTOR pathway.

2) When encoded and translated in mitochondria, Humanin was found to localize to the mitochondrial compartment and regulate production of reactive oxygen species.

Selected Data
1) This study performed by Kwon et al investigated the role of the mitochondrial-derived peptide Humanin in regulating hepatocyte metabolism, survival, and signaling under stress conditions, with an emphasis on pathways related to insulin sensitivity, lipid accumulation, and cellular apoptosis. To explore these mechanisms, researchers used cultured human primary hepatocytes, as well as human primary myocytes. The hepatocytes were first plated on collagen type-1-coated dishes in a specialized plating medium before being maintained in hepatocyte maintenance medium containing fetal bovine serum, penicillin, and streptomycin. Importantly, the hepatocyte cultures were confirmed to be free of mycoplasma contamination, ensuring experimental reliability. Treatments included sodium palmitate conjugated to bovine serum albumin to induce lipotoxic stress, recombinant Humanin peptide dissolved in dimethyl sulfoxide, and human insulin to stimulate insulin signaling. Inhibitors of specific signaling pathways were also incorporated: Compound C, an AMPK inhibitor, and MK-2206, an Akt1/2 inhibitor, both applied for 24 hours to determine the involvement of these pathways in the protective actions of Humanin [1].
To evaluate protein expression levels and post-translational modifications, hepatocytes were lysed and subjected to SDS-PAGE followed by immunoblotting. Proteins were transferred to nitrocellulose membranes and probed with primary and secondary antibodies, with detection carried out using chemiluminescence. The analysis included markers of insulin signaling such as IRS-1 and its phosphorylated form, phosphorylated and total Akt, as well as phosphorylated and total AMPK. Apoptotic markers Bax and cleaved caspase-3 were also assessed, alongside phosphorylated and total mTOR and β-actin as a loading control. These assays provided insights into the molecular mechanisms by which humanin modulates hepatocyte responses to metabolic stress.
To specifically examine the role of AMPK signaling, gene silencing techniques were applied. Small interfering RNAs (siRNAs) targeting AMPK were transfected into hepatocytes using Lipofectamine Gold, following standard protocols. This allowed for the dissection of whether AMPK activity was essential for humanin’s beneficial effects on lipid metabolism and cell survival. Subsequent experiments compared hepatocytes treated with humanin in the presence or absence of AMPK silencing, providing important evidence regarding pathway dependency [1].
Several functional assays were conducted to measure the impact of treatments on hepatocyte physiology. Lipid accumulation was quantified using Oil Red O staining, in which hepatocytes were stained and then microscopically examined for intracellular lipid droplets. This assay enabled the evaluation of whether humanin could reduce lipid deposition induced by palmitate, a key marker of hepatic steatosis. Cell viability was determined using the MTT assay, which relies on the conversion of MTT to purple formazan crystals by metabolically active cells; the intensity of color provided a quantitative measure of viable cells. Additionally, caspase-3 activity assays were performed using a colorimetric kit to directly assess apoptosis, given that caspase-3 is a central executioner protease in programmed cell death [1].
To evaluate metabolic function more directly, glucose uptake assays were conducted using a colorimetric kit, allowing researchers to assess whether humanin improved cellular glucose handling under stress conditions. Intracellular ATP levels were measured using a luminescence-based assay kit, providing further insight into mitochondrial function and overall energy homeostasis within hepatocytes. Together, these assays created a comprehensive picture of how humanin influences hepatocyte survival, metabolism, and stress resistance.
Beyond cell culture studies, the secretion of endogenous Humanin from human primary skeletal muscle cells was measured. Skeletal muscle cells were cultured in specialized growth medium, and levels of Humanin in the culture supernatants were quantified using an ELISA kit. These measurements were crucial in establishing muscle as a potential source of circulating humanin and linking its secretion to metabolic health.
In addition to the experimental work on cultured cells, the study also incorporated transcriptomic data analysis. Expression profiling of mitochondrial genes in human skeletal muscle tissue was examined using publicly available RNA sequencing data that included 66 samples derived from 90 skeletal muscle tissue samples. The samples were divided into three groups: normal with 18 samples total, obese with 24 samples total, and type 2 diabetes mellitus with 24 samples total. On average, 68 million paired-end reads were generated per sample using Illumina HiSeq instruments. Reads were trimmed to remove adapter sequences and aligned to the human reference genome. BAM files were then indexed and sorted using Samtools, and gene counts were generated. This dataset allowed the researchers to investigate differential expression of mitochondrial-related genes across normal, obese, and diabetic samples, thereby contextualizing their in vitro findings with human disease states [1].
The methodology outlined in this passage demonstrates a multifaceted approach to exploring the protective role of Humanin in hepatocytes and its broader relevance to metabolic health. By combining in vitro experiments with inhibitors, gene silencing, functional assays, and transcriptomic data analysis, the researchers established a framework to uncover how Humanin might regulate insulin signaling, lipid metabolism, apoptosis, and mitochondrial function. Additionally, by integrating both cellular and molecular assays with bioinformatics analysis of human muscle tissue, the collected datasets provide a basis for understanding Humanin’s therapeutic potential in obesity, insulin resistance, and type 2 diabetes [1].
2) The study performed by Paharkova et al utilized the rat insulinoma cell line (INS-1) to investigate mitochondrial function, protein expression, and the cellular response to experimental manipulations. These cells were cultured according to established protocols, with most reagents obtained from Sigma. A central aspect of the work involved the preparation of subcellular fractions. To isolate membrane-associated fractions, researchers adopted a combined approach of differential centrifugation and self-generating Percoll gradient centrifugation. This provided a means to separate cellular compartments with high purity and allowed detailed study of mitochondrial and membrane proteins in subsequent assays [2].
Western blotting was used extensively to characterize protein expression and assess the effects of treatments on INS-1 cells. For these experiments, cells were first treated with 50 µg/mL and 100 µg/mL of the protein synthesis inhibitors cycloheximide and chloramphenicol, respectively, for defined periods. Following treatment, the cell monolayer was washed, lysed in RIPA buffer with protease inhibitors, and protein concentrations determined by Bio-Rad assay. Proteins were resolved using NuPage Bis-Tris gels, transferred onto PVDF membranes, and probed with a variety of primary antibodies. These included antibodies against rattin, cytochrome c oxidase subunit I, and UQCRC2. Secondary antibodies conjugated to peroxidase enabled chemiluminescent detection with enhanced substrates. This combination of protein inhibitors, fractionation, and immunodetection facilitated a detailed look at mitochondrial protein integrity and turnover [2].
To further probe mitochondrial function, researchers generated mitochondrial DNA-depleted INS-1 cells, known as Rho-0 (ρ0) cells. These were produced by chronic exposure of INS-1 cells to 0.4 µg/mL ethidium bromide in medium supplemented with sodium pyruvate and uridine, which are required to compensate for impaired mitochondrial metabolism. Cultures were maintained under these conditions for four weeks. During this period, the depletion of mitochondrial DNA and the ρ0 phenotype were verified by western blot analysis for COX I, a mitochondrial DNA-encoded protein. This model provided an experimental system to examine the contribution of mitochondria to overall cellular processes in insulinoma cells.
Immunohistochemistry complemented the in vitro experiments by exploring protein localization in tissue. Cryosections of testes from 40-day-old mice were used for histological analysis. Following fixation in buffered formalin, antigen retrieval was performed at near-boiling temperatures with Tris-EDTA buffer. Sections were blocked with a serum-free buffer to minimize nonspecific staining and incubated overnight with a purified polyclonal rabbit anti-human Humanin (HN) antibody at 7 µg/mL. Visualization was achieved using the ABC kit with DAB as a chromogen, and nuclei were counterstained with hematoxylin. This procedure allowed for detailed localization of HN within tissue structures, adding a spatial dimension to the biochemical assays [2].
In addition to protein detection and tissue staining, the study investigated mitochondrial oxidative stress by measuring H₂O₂ release. Mitochondria were first isolated from INS-1 cells using a Mitochondria Isolation Kit. To maintain integrity, all samples were kept at 4 °C, and assays were performed within five hours of isolation. Mitochondrial protein content was quantified via the Bradford method. H₂O₂ production was then assessed using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit, a sensitive colorimetric method. Isolated mitochondria were incubated with 1 µM HNG, followed by the Amplex Red reagent, and the assay was carried out for 30 minutes at 37 °C in 96-well plate format. Hydrogen peroxide generated during the assay was detected spectrophotometrically at 560 nm, with a positive control provided by direct addition of H₂O₂. These experiments enabled precise quantification of mitochondrial oxidative output under controlled conditions.
This framework provided a platform to study the role of mitochondria, particularly in the context of insulinoma cells and Humanin peptide biology. The combination of subcellular fractionation, protein detection, mitochondrial DNA depletion, immunohistochemistry, and oxidative stress assays offered a multi-layered perspective. By applying both in vitro cellular models and ex vivo tissue analysis, the researchers could evaluate mitochondrial protein expression, oxidative balance, and peptide localization. Such insights are essential for understanding how mitochondrial function intersects with insulin secretion, oxidative stress, and potential therapeutic roles of mitochondrial peptides [2].

Discussion
1) The study performed by Kwon et al investigates the role of Humanin, a mitochondrial-derived peptide, in skeletal muscle and its potential effects on hepatic metabolism under obesity-associated conditions. Exercise is widely recognized as a first-line therapeutic approach for non-alcoholic fatty liver disease (NAFLD), and identifying muscle-derived intermediators that mediate this benefit is an important area of research. To determine whether Humanin could function as such a myokine, RNA sequencing was performed on skeletal muscle tissue from obese individuals and obese patients with type 2 diabetes. Results showed that Humanin mRNA expression was significantly elevated in the skeletal muscle of obese patients compared to healthy individuals, while no difference was observed in type 2 diabetes patients relative to controls. Furthermore, obese patients exhibited higher Humanin expression than those with type 2 diabetes, suggesting that obesity specifically upregulates Humanin in muscle. Supporting this observation, in vitro treatment of primary human skeletal muscle cells with palmitate induced a dose-dependent increase in Humanin secretion. These findings led to the hypothesis that muscle-secreted Humanin could influence hepatic lipid metabolism and insulin signaling, thereby connecting exercise, obesity, and NAFLD outcomes [1].
To explore the role of Humanin in hepatic lipid accumulation, the researchers established an in vitro steatosis model using primary human hepatocytes treated with palmitate. Palmitate exposure induced significant lipid accumulation, as visualized by Oil red-O staining. When Humanin was applied to hepatocytes, it prevented this lipid accumulation in a concentration-dependent manner. In addition, Humanin suppressed the expression of key lipogenic genes such as processed sterol regulatory element-binding protein 1 (SREBP1), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD1). Because fatty acid oxidation is known to reduce lipogenesis, the researchers indirectly assessed Humanin’s effect on fatty acid metabolism by measuring intracellular ATP levels. They found that Humanin modestly but consistently elevated ATP production in a dose-dependent manner, suggesting enhanced fatty acid oxidation alongside the suppression of lipid synthesis [1].
Another hallmark of NAFLD is hepatocyte apoptosis, which contributes to disease progression. To test whether Humanin could protect against lipotoxicity-induced cell death, hepatocytes were treated with palmitate in the presence or absence of Humanin. Palmitate reduced cell viability, but Humanin treatment significantly rescued viability, as shown in the MTT assay. Moreover, Humanin attenuated palmitate-induced increases in apoptotic markers, including caspase 3 activity, cleaved caspase 3, and Bax expression. These results indicate that Humanin protects hepatocytes from lipotoxic apoptosis in a dose-dependent fashion. Since NAFLD is also strongly associated with insulin resistance, the researchers next examined insulin signaling. Palmitate impaired insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) and Akt, which are key steps in insulin signaling. Humanin treatment restored both IRS-1 and Akt phosphorylation under lipotoxic conditions. Functionally, this translated to improved glucose uptake in hepatocytes treated with Humanin, demonstrating that the peptide not only protected against apoptosis but also preserved insulin sensitivity.
Mechanistically, the study probed whether AMPK and mTOR signaling pathways mediate the observed effects of Humanin. AMPK and mTOR are central regulators of energy metabolism and cell survival. Humanin treatment increased phosphorylation of AMPK, indicating activation, while simultaneously suppressing palmitate-induced mTOR phosphorylation, suggesting inhibition of the lipogenic and growth-promoting pathway. To confirm these pathways’ roles, AMPK expression was silenced using siRNA. Knockdown of AMPK abolished HN’s protective effects on palmitate-induced lipid accumulation, lipogenic gene expression, and mTOR phosphorylation. Furthermore, the beneficial effects of Humanin on hepatocyte apoptosis and insulin resistance were reversed by AMPK silencing. In addition, pharmacological inhibition of either AMPK or Akt prevented HN from enhancing glucose uptake, reinforcing that both pathways are necessary for HN’s metabolic actions [1].
Together, these findings demonstrate that Humanin acts as a muscle-derived myokine that is upregulated in obesity and secreted in response to lipid overload. In hepatocytes, HN counteracts lipotoxic effects by reducing lipid accumulation, suppressing lipogenic gene expression, preventing apoptosis, and restoring insulin sensitivity. These effects are mediated largely through activation of AMPK and suppression of mTOR signaling, with Akt also contributing to improved glucose uptake. By linking skeletal muscle-derived HN to improvements in hepatic metabolism, the study provides evidence for a muscle–liver communication axis in obesity and NAFLD. This highlights Humanin as a potential therapeutic candidate for conditions characterized by lipid accumulation, hepatocyte injury, and insulin resistance [1].
2) This study conducted by Paharkova et al investigates the origin, localization, and function of rat Humanin (rHN), a mitochondrial-derived peptide, with a particular focus on its role in oxidative stress regulation and potential therapeutic implications. Earlier analysis of rodent genomes suggested that rats lack nuclear-encoded Humanin genes and only contain a mitochondrially-encoded version. To confirm the site of origin and translation, INS-1 rat insulinoma cells were depleted of mitochondrial DNA (mtDNA) using ethidium bromide for four weeks. These mtDNA-depleted (Rho-0) cells showed the expected reduction in COX I, a mitochondrial genome-encoded protein, and also lacked detectable rHN peptide, strongly supporting its mitochondrial origin [2].
To further pinpoint the translation site, translation inhibitors were used. Chloramphenicol, which blocks mitochondrial translation, reduced rHN peptide levels over time, whereas cycloheximide, a cytoplasmic translation inhibitor, did not affect rHN levels. This indicated that rHN translation occurs specifically within mitochondria. Subcellular fractionation experiments confirmed this localization, showing that rHN was not present in microsomal fractions but was found in mitochondrial fractions alongside markers such as Hsp60 and COX I. rHN also co-localized with Bax, a pro-apoptotic protein and known HN-binding partner, suggesting functional interactions within mitochondria. Differences observed between endogenous rHN and synthetic peptides in molecular weight were attributed to potential post-translational modifications, highlighting the need for peptide sequencing across species for clarity.
Supporting these findings, prior work demonstrated that Humanin localizes to the mitochondrial-rich midpiece of human sperm, where it may protect the DNA-rich head from oxidative damage. Similar mitochondrial localization was found for mouse Humanin (mHN) in sperm midpieces, as well as in testicular cells such as Leydig cells, spermatocytes, and spermatids. Immunohistochemistry revealed that mHN is also present in Sertoli and spermatogonia cells, underscoring its broad expression in reproductive tissues [2].
The functional role of Humanin in regulating oxidative stress was further explored through studies of HNG, a potent Humanin analog. HNG activates antioxidant defense systems and reduces oxidative stress in rat cardiac myoblasts exposed to hydrogen peroxide. Although pancreatic beta cells normally exhibit low antioxidant activity, HNG was shown to enhance glucose oxidation, increase ATP levels, and elevate reactive oxygen species (ROS) without damaging mitochondrial membranes. To isolate HN’s direct effect on mitochondria, H2O2 production was measured in isolated INS-1 mitochondria. HNG treatment reduced H2O2 production by 55%, demonstrating a direct role for Humanin in attenuating mitochondrial ROS generation and reducing oxidative stress [2].
These results align with growing evidence that mitochondria are central players in metabolic diseases, particularly diabetes. In type 1 diabetes, mitochondrial ROS production during immune attack amplifies apoptotic signaling, while in type 2 diabetes, chronic metabolic stressors such as hyperglycemia and hyperlipidemia also increase ROS and damage mitochondria in insulin-sensitive tissues. Humanin has been reported to improve insulin sensitivity and enhance insulin secretion in beta cells, suggesting that its protective effects are mediated, at least in part, by reducing oxidative stress. The localization of Humanin to the sperm midpiece, a mitochondria-rich region with high oxidative phosphorylation activity, further supports its role as a mitochondrial protector against ROS.
Despite this strong evidence, questions remain about the genetic origin of Humanin. The peptide sequence aligns with a region of the mitochondrial genome coding for 16S rRNA, but until now there was no convincing proof that it is actually translated from this site. While some studies propose the existence of nuclear-encoded short open reading frames (sORFs) that might generate Humanin-like peptides, their biological significance remains under investigation. The current study demonstrates that rHN is indeed mitochondrial in origin and localized within mitochondria, but it does not exclude the possibility that Humanin could also function in other organelles. Mass spectrometry-based sequencing of endogenous Humanin will be necessary to definitively resolve questions of its nuclear versus mitochondrial origin [2].
The findings also place Humanin within the broader context of mitochondrial-derived peptides (MDPs), which have emerged as important signaling molecules. For example, MOTS-c, another MDP encoded within the mitochondrial 12S rRNA, regulates insulin sensitivity and metabolic homeostasis. Unlike rHN, rat MOTS-c is exclusively mitochondrial, while in humans, translation may occur in the cytoplasm due to codon differences. These discoveries highlight the versatility of mitochondria in producing bioactive peptides that act locally and systemically.
In conclusion, rHN originates from mitochondrial DNA and localizes to mitochondria in rat cells, where it functions as a novel antioxidant. By directly reducing ROS production, it protects cells from oxidative stress, a process implicated in both type 1 and type 2 diabetes as well as other pathophysiological conditions. These findings expand the understanding of mitochondrial-derived peptides as regulators of cellular homeostasis and suggest that Humanin and its analogs could have therapeutic potential in treating diseases characterized by oxidative stress and mitochondrial dysfunction [2]

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Kwon C, Sun JL, Jeong JH, Jung TW. Humanin attenuates palmitate-induced hepatic lipid accumulation and insulin resistance via AMPK-mediated suppression of the mTOR pathway. Biochem Biophys Res Commun. 2020;526(2):539-545. doi:10.1016/j.bbrc.2020.03.128

[2] Paharkova V, Alvarez G, Nakamura H, Cohen P, Lee KW. Rat Humanin is encoded and translated in mitochondria and is localized to the mitochondrial compartment where it regulates ROS production. Mol Cell Endocrinol. 2015;413:96-100. doi:10.1016/j.mce.2015.06.015

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