Peniocereus greggii sterols. Insulin, glycaemia and cellular respiration

The root as research material

Peniocereus greggii develops a large tuberous root from which relatively inconspicuous stems emerge. R. Jenifer Muñoz-Gómez and colleagues started from traditional use of these roots by communities in northern Mexico and the southwestern United States. They converted this ethnobotanical context into a series of testable questions about chemical composition and metabolic responses. The account of traditional use justified selection of the plant, whereas experimental answers came from separate animal experiments and cell cultures. [Muñoz-Gómez et al. 2022]

Material came from northern Chihuahua and included four batches collected at different times between 2018 and 2020. The plant was botanically authenticated and a voucher specimen was deposited in the MEXU herbarium. Roots were used to prepare an aqueous decoction and extracts separated with organic solvents. These preparations had different compositions and different roles in the investigation. The decoction was used in metabolic experiments in mice, while chemical separations enabled isolation of substances for identification and subsequent tests of individual compounds. [Muñoz-Gómez et al. 2022]

A mixture and individual sterols

Identified substances included peniocerol, desoxyviperidone, viperidone and viperidinone. These are steroids with a cholestane skeleton. Spectroscopic and spectrometric methods were used for identification, together with crystal X-ray analysis for peniocerol. Establishing structure makes it possible to assign a result to a specific compound. In the publication, desoxyviperidone was numbered 3 and selected for further animal experiments because it was among the major constituents of the analysed fraction derived from the root decoction. [Muñoz-Gómez et al. 2022]

Compound 6, described as a new chemical entity, occupied a separate position. It was obtained from a chloroform extract prepared after alkalinization of the material. The authors questioned whether the substance might have formed during this processing. Its cellular activity can therefore be described as the activity of an isolated and characterized compound. Presence in that extract does not establish its contribution to the aqueous decoction or its occurrence in unprocessed root. Preparation of the material consequently becomes part of the biological interpretation of the result. [Muñoz-Gómez et al. 2022]

Glycaemia in the animal model

The initial tests compared mice with normal glycaemia and mice with chemically induced hyperglycaemia. Nicotinamide and streptozotocin were used in part of the experiments. Decoction doses were 31.6, 100 and 316 mg/kg body weight, with six animals per group for blood glucose measurements. Blood was sampled at successive times after administration. This design enabled assessment of the response over time and comparison with a vehicle group and an appropriate reference compound. [Muñoz-Gómez et al. 2022]

In the acute test, the decoction lowered glycaemia in hyperglycaemic animals, whereas an analogous effect was not found in mice with normal starting values. Following an oral glucose challenge, the response also depended on animal condition and preparation dose. In normoglycaemic mice, a significant effect was described for the highest dose, while all doses were effective in hyperglycaemic animals. The result therefore retains the context of the particular model and challenge procedure, rather than defining a single universal magnitude of decoction activity. [Muñoz-Gómez et al. 2022]

Two carbohydrate challenges

Glucose and sucrose tests addressed related but different questions about the response following carbohydrate administration. Animals received a preparation thirty minutes before the challenge, after which blood glucose changes were followed for two hours. Administered glucose enters subsequent absorption and metabolic processes differently from sucrose, which requires breakdown. The decoction had no significant effect in the sucrose test in normoglycaemic mice. This finding limits support for inhibition of intestinal enzymes breaking down that sugar under the experimental conditions. Effects on other stages of glycaemic regulation remain possible, explaining the separate studies of insulin secretion and responsiveness to the hormone. [Muñoz-Gómez et al. 2022]

A separate question about insulin responsiveness

Desoxyviperidone was then evaluated as a single substance. In the oral glucose tolerance test, it reduced the glycaemic response in both normoglycaemic and hyperglycaemic animals. Doses of 10, 31.6 and 56 mg/kg were examined. This result alone left several possible mechanisms. The compound could affect insulin secretion or tissue responsiveness to insulin. Distinguishing these possibilities required another test in which insulin was introduced at a defined dose. [Muñoz-Gómez et al. 2022]

In the insulin tolerance test, hyperglycaemic animals received desoxyviperidone at 31.6 mg/kg, followed thirty minutes later by insulin at 0.5 units/kg. The compound alone did not significantly lower glycaemia in this procedure. Combining it with insulin produced a clearer response than the control group. The authors described this behaviour as consistent with insulin sensitization. This is a functional interpretation of blood glucose measurements. The test did not identify a single directly confirmed molecular target of the sterol. [Muñoz-Gómez et al. 2022]

Insulin-secreting cells

To examine insulin secretion separately, the investigators used the rat insulinoma line INS-1E. Cells were exposed to selected sterols for two hours, and insulin released into the medium was expressed relative to released insulin plus the amount remaining in the cells. The readout therefore described the fraction of released hormone within the total measured pool. Conditions with lower and higher glucose concentrations were also compared. This distinction allowed examination of whether the compound response depended on the metabolic environment of the cell. [Muñoz-Gómez et al. 2022]

Peniocerol and compound 6 increased insulin secretion at the lower glucose concentration. Desoxyviperidone did not show this effect. At the higher glucose concentration, the authors described only a tendency towards increased secretion with compound 6. Differences between sterols help interpret the plant mixture as a collection of substances with distinct response profiles. The cellular result is consistent with a possible contribution of peniocerol to decoction activity. For compound 6, questions about formation during extraction and actual presence in the aqueous preparation additionally remain. [Muñoz-Gómez et al. 2022]

Myotube respiration and proton flow

The next stage involved mouse C2C12 myotubes, cells differentiated towards a muscle phenotype. After an eighteen-hour exposure to sterols, oxygen consumption and medium acidification were measured using an extracellular flux analyser. Concentrations of 5, 10 and 20 µg/mL were used. Sequential additions of inhibitors and an uncoupling agent separated components of the respiratory response. The authors analysed basal respiration, the component associated with ATP synthesis, proton leak and the maximal respiratory capacity of the cells, among other parameters. [Muñoz-Gómez et al. 2022]

Desoxyviperidone increased basal respiration and proton leak in a concentration-dependent manner. Compound 6 also altered these parameters. At the same time, no increase was found in the respiratory component linked to ATP production. This distinction matters because higher oxygen consumption can reflect different uses of the mitochondrial proton gradient. Connecting proton leak with the observed insulin response remains the authors' mechanistic hypothesis. The myotube experiment and the glycaemia test provide different kinds of evidence that together form the proposed explanation. [Muñoz-Gómez et al. 2022]

Composition control is part of the experiment

The study also developed a chromatographic method for measuring desoxyviperidone and viperidone. Ultra-performance liquid chromatographic separation was combined with mass spectrometric detection. Linearity, precision, accuracy and detection and quantification limits were assessed. The method was applied to four extract batches. Compositional differences between batches demonstrated why the species name alone is insufficient to reproduce chemical exposure. Comparing preparation activity requires information about the content of the substances actually measured and the method used to obtain them. [Muñoz-Gómez et al. 2022]

In a separate acute toxicity experiment, animals were observed for fourteen days after receiving the decoction. No mortality occurred under the conditions tested, even at the highest dose examined. This observation concerns a specified preparation, administration route and short period. It does not cover chronic use, effects on every organ or relationships with other substances. Combined with the metabolic findings, it provides an initial description of the material that requires further development through precise toxicological and pharmacological experiments. [Muñoz-Gómez et al. 2022]

From observation to mechanism

The most informative feature of this work is separation of responses at several levels. The decoction altered blood glucose in mice, desoxyviperidone enhanced the response in the presence of insulin, and peniocerol affected hormone secretion in culture. Respiration measurements added a proposed cellular mechanism, while chromatography characterized material composition. Each finding has its own scope. Together they document the interesting pharmacology of Peniocereus greggii root sterols while retaining the boundary between a controlled experiment and clinical efficacy, which this publication did not examine. [Muñoz-Gómez et al. 2022]